Contribution of Some Grape-Derived Aromatic Compounds to the Primary Aroma in Red Wines from Cv

Journal of Agricultural Science (2008), 146, 325–332. f 2007 Cambridge University Press 325 doi:10.1017/S0021859607007022 Printed in the United Kingdom

Contribution of some grape-derived aromatic compounds to the primary aroma in red wines from cv. Cainõ Tinto, cv. Cainõ Bravo and cv. Cainõ Longo grapes

M.VILANOVA1*, S. CORTE´S 2, J.L. SANTIAGO1, C.MARTIŃEZ1 AND E.FERNAŃDEZ3 1 Misioń Biolo´gica de Galicia (CSIC), Apdo. de Correos 28, 38080 Pontevedra, Spain 2 Estacioń de Viticultura y Enologıá de Galicia, Ponte San Clodio s/n, 32427 Leiro, Ourense, Spain 3 Departamento de Quı´mica Analı´tica y Alimentaria, A´rea de Quı´mica Analı´tica, Facultad de Ciencias, Universidad de Vigo, As Lagoas s/n, 32004 Ourense, Spain (Revised MS received 9 November 2006; First published online 16 November 2007)

SUMMARY The free volatile compounds of two successive vintages of cv. Cain˜ o Tinto, Cain˜ o Bravo and Cain˜ o Longo red wines, together with the volatile compounds released after the enzymatic hydrolysis of their glycosidically bound forms, were identified and quantified by gas chromatography using a flame ionization detector (GC/FID). All these wines possessed the same free volatile compounds; Cain˜ o Longo wines showed the highest concentrations and Cain˜ o Tinto wines the lowest. In all cases, the release of the bound forms of these compounds may contribute to the final aroma, from both a qualitative standpoint (with the appearance of free 4-terpineol, nerol and geraniol) and quantitative standpoint (notable increases were recorded for most of the compounds detected). The principal component analysis (PCA) showed a good separation of the different wine cultivars and vintages. Cain˜ o Tinto wines were more homogeneous between vintages than the others.

INTRODUCTION The volatile compounds responsible for the primary or ‘grape-derived’ aroma of wines are of great Galicia, a region of northwestern Spain, has a long importance in the current effort to differentiate wines tradition of winemaking. The red cultivar Cain˜ o on the basis of the grape variety used and the pro- Tinto is one of the most appreciated in the most duction area (Arrhenius et al. 1996). These volatile important Appellation d’Origine Controˆleé (AOC) compounds are mainly terpenes and C -noriso- areas of the region (Rıás Baixas and Ribeiro), al- 13 prenoids, which supply fruity or floral notes to the though presently it is not grown in great quantity. aroma (Ong & Acree 1999). They are formed during The cultivars Cain˜ o Bravo and Cain˜ o Longo are the ripening of the grape (Bayonove & Cordonnier also traditional red cultivars of the region (Santiago 1971; Marais 1983; Marais & Van Wyck 1986; et al. 2005); however, they appear in the Lista Carballeira et al. 2001) and their concentration is de Variedades Comerciales y Portainjertos de Vid strongly influenced by the cultivar, the soil, climate (Ministerio de Agricultura 2002) as ‘provisional and viticultural practices (Jackson & Lombard 1993; inscriptions’. They will become ‘definitive inscrip- Razungles et al. 1993, 1998; Agosin et al. 2000). tions’ when more information becomes available on These aromatic compounds exist either in a free their ampelographic and oenological characteristics. or glycosidically bound state (Williams et al. 1981; For a variety to be officially cultivated in any Spanish Sefton et al. 1993; Skouroumounis & Winterhalter winemaking region, or for it to be introduced into an 1994; Skouroumounis et al. 1995; Die´guez et al. AOC area as an authorized or preferred variety, its 2003) and are found in the skin (especially) and pulp name must appear as a definitive inscription in the (Wilson et al. 1986; Park et al. 1991; Go´mez et al. above list. 1994). Certain winemaking techniques, such as skin * To whom all correspondence should be addressed. contact, can increase the aromatic content of a wine Email: [email protected] (Lanaridis et al. 2002). According to Di Stefano 326 M. VILANOVA ET AL.

(1989), although these compounds pass directly into (CSIC) at Salcedo (Pontevedra), which was also home the wine, they can become modified over time de- to a meteorological station. The mean annual tem- pending on the pH of the wine and storage tempera- perature of this region is 14.4 xC and the mean annual ture. In aromatic grape varieties, the bound fraction rainfall is 1586 mm. The soil where the grape cultivars of aromatic compounds is usually the more abundant were grown is a sandy loam with 80 g organic (Dimitriadis & Williams 1984; Gunata et al. 1985; material/kg soil. All the studied plants (10 replicates Rocha et al. 2000). The contribution of these com- per cultivar) were of the same age, had 110-Richter pounds to the final aroma depends on whether their rootstocks, were grown en espalier, were subjected to concentration in the wine is above the perception Sylvoz pruning and received identical crop protection threshold. According to Di Stefano et al. (1995), the treatments. Manual weeding was performed several main aromatic differences between grape varieties times per year. are more quantitative than qualitative. The ratios es- tablished between the different aromatic compounds Fermentation are also important. The presence and concentration of grape-derived aromatic compounds have been used The wines investigated were made from Cain˜ o by several authors for varietal differentiation and Blanco, Cain˜ o Tinto and Cain˜ o Longo grapes characterization (Williams et al. 1981; Gunata et al. harvested in two different seasons (2002 and 2003). 1985; Marais & Van Wyck 1986; Razungles et al. After removing the leaves, stalks, etc., the grapes were 1993; Sefton et al. 1993; Die´guez et al. 2003). crushed by hand to prevent the breakage of the seeds. In aromatic varieties, the concentrations of free SO2 was added at 50 mg/l. Spontaneous fermen- volatile compounds are usually above their percep- tations were performed in 16 litre glass vessels con- tion thresholds. In more neutral wines, the release of taining 10 litres of grape juice of each variety; these the bound forms by chemical or enzymatic hydrolysis were allowed to proceed at 18 xC for 15 days. Sugar (Williams et al. 1981; Gunata et al. 1986; Marais & density was measured daily. When fermentation was Van Wyck 1986; Wilson et al. 1986; Rapp 1998; Ollat finished, the wine was separated from the skins. The et al. 2002) can notably increase the aroma. Among latter were then pressed to extract any wine they the compounds responsible for grape-derived aroma, contained. All of the samples were racked and the free linalool, citronellol and geraniol stand out, with floral SO2 content adjusted to 50 mg/l. After bottling, 1 litre and citrus aromas and very low perception thresholds of each wine was conserved at 10 xC until analysis. (Francis & Newton 2005). In any event, the presence The wines were analysed 3 months after the com- of these compounds in a wine can be detected by their pletion of primary fermentation. All analyses were synergistic effects: the presence of one tends to performed in triplicate. potentiate the perception of the rest. The present work reports the concentration of the Sample preparation major aromatic compounds (both in the free and bound form) responsible for the primary or grape- Free and bound terpenes were fractionated by selec- derived aroma of the wines produced from cv. Cain˜ o tive retention on SepPak Vac C-18 (1 g, purchased Tinto, Cain˜ o Longo and Cain˜ o Bravo grapes. The from Waters), according to the procedure described study forms part of a wider project to differentiate by Di Stefano (1991) with some modifications and classify the minority red grape varieties of (Carballeira et al. 2001). The cartridges were sequen- Galicia. tially conditioned with methanol (5 ml) and distilled water (10 ml). A sample of 100 ml of centrifuged wine diluted with 100 ml of distilled water and containing MATERIALS AND METHODS 1 ml of internal standard (3-octanol at 10 ppm in Reagents ethanol) was passed through the cartridge; the residue was washed with 25 ml of distilled water. The Methanol, distilled azeotropic dichloromethane– free fraction was eluted with 10 ml of pentane– pentane (1:2 v/v) and ethanol were all of analytical dichloromethane (2:1) and the solution was dried grade and supplied by Merck (Germany). Acetate over anhydrous sodium sulphate and, prior to . buffer (acetic acid 0 0595 mol/l) pH 5 was made with gas chromatography (GC) analysis, concentrated to analytical grade reagents from Probus (Spain). The 0.5 ml by evaporation under a nitrogen stream. The corporate governance (CG) standards used were from bound fraction was eluted with 10 ml of methanol Aldrich Chemical (Switzerland). and concentrated to dryness in a rotary evaporator before dissolution in 5 ml of citrate-phosphate buffer (pH 5.0). Enzyme solution 200 ml of with Grape juice b-glycosidase activity (0.5 g of AR-2000 (Gist The vine strains used in the present study belonged to Brocades, France) in 5 ml of the same buffer) was the collection of the Misioń Biolo´gica de Galicia added and the mixture was incubated at 40 xC for Cainõ Tinto, Cainõ Bravo and Cainõ Longo wines 327

18 h to accomplish enzymatic hydrolysis. After the of all aromatic compounds in both vintages. The addition of the same internal standard (3-octanol), total content of compounds varied from 34675.83 the aglycons were extracted on SepPak Vac C-18 to 39342.77 mg/l in 2002 vintage and 10494.54 to (1 g), according to the procedure described pre- 18133.93 mg/l in 2003 vintage (Table 1). viously, to the free forms. Before GC analysis, the organic phase was dried with sodium sulphate and Free forms concentrated to 0.5 ml by evaporation with a stream of nitrogen. The highest concentrations of free volatile compounds were found in the Cain˜ o Longo wines, fol- lowed by the Cain˜ o Tinto wines. Cain˜ o Bravo wines Chromatographic analysis had the lowest concentrations of free terpenes. In To determine the identities and amounts of free and the wines made from all three cultivars, the most bound aromatic compounds present, the extracts abundant free aromatic compound was 2-phenyl- were analysed by GC. ethanol, with levels well over the perception threshold The analyses were carried out using a Hewlett (10 000 mg/l) (Swiegers et al. 2005). Packard 5890 Series II Gas-Chromatograph equip- In the Cain˜ o Bravo wines from 2003, a-terpineol ped with an HP 6890 Automatic Injector and a was the most abundant monoterpene, although it Flame Ionization Detector (hydrogen, 40 ml/min; air, was never above the perception threshold (330 mg/l) 400 ml/min). The compounds were separated on a (Escudero et al. 2004; Table 1). Of the terpene CHROMPACK CP-WAX 57CB (polyethylene glycol alcohols, linalool was the most abundant mono- stationary phase; 50 mr0.25 mm id with 0.25 mm terpene in the Cain˜ o Longo wine from 2003, with film thickness) fused-silica capillary column. a concentration above the perception threshold The instrumental conditions were: column tem- (25 mg/l; Francis & Newton 2005). Apart from the perature, 60 xC for 5 min, rising to 200 xCat3xC/min, benzene alcohols, theaspirene was the compound then 200 xC for 25 min; injector temperature: 250 xC; with the highest concentration in the wines of all three detector temperature: 260 xC; make-up gas: nitrogen varieties in 2003 vintage. The results of statistical 25 ml/min; injection mode, Splitless (30 s); volume analysis show significant differences among varieties injected, 1.0 ml; carrier gas: helium at 1.07 ml/min. in the two vintages. The Cain˜ o Longo wines were those with the highest concentrations of most of the free compounds with Identification and quantification floral notes, linalool and 2-phenylethanol. Aromatic compounds were identified by comparison of their retention times with those of pure standards. Bound forms An internal standard (3-octanol) was used for quantitative purposes. Table 1 shows bound aromatic compounds in Cain˜ o Longo, Cain˜ o Tinto and Cain˜ o Bravo in the 2002 and 2003 vintages. Citronellal was also present, although Statistical analyses it did not form part of the initial composition of the Differences among the wines with respect to their aroma of any wine. aromatic compound contents were assessed by The bound form of 2-phenylethanol was the most one-way analysis of variance (ANOVA). Principal abundant bound aromatic compound in all the wines, component analysis (PCA) was performed using SAS although its concentration was much lower than that Software v8.1 (SAS Institute, Cary, NC). of its free form. The Cain˜ o Longo wines had the highest concentrations of bound linalool in the 2003 vintage, RESULTS again above the perception threshold. The levels of Figure 1 shows climate data for the 2002 and 2003 geraniol and b-ionone in the Cain˜ o Longo wines were growth periods (temperature and total rainfall). much higher than in the other two types of wine. Mean temperature and rainfall were higher during the Geraniol, which did not appear in the free form, may July–August ripening period of 2003 than in 2002. contribute directly to the aroma of Cain˜ o Longo and To assess the aromatic composition of Cain˜ o Cain˜ o Bravo wines since the concentration is above Longo, Cain˜ o Tinto and Cain˜ o Bravo wines, the the perception threshold (30 mg/l; Francis & Newton concentration of free and bound aroma compounds 2005). was determined. Mean concentration and standard The bound benzyl alcohol content of Cain˜ o Bravo deviation of free and bound compounds in Cain˜ o wines from 2002 was very much higher than that of Tinto, Cain˜ o Longo and Cain˜ o Bravo wines in 2002 the free form and well above its perception threshold and 2003 are shown in Table 1. Significant differences (620 mg/l). It therefore may contribute to the aroma were found among wines in the mean concentrations with notes of blackberry (Latrasse 1991). Cain˜ o Tinto 328 M. VILANOVA ET AL.

(a) 25

20 C) ° 15

10 Temperature (

May June July August October September November Month

(b) 450

400

350

300

250

200 Rainfall (mm) 150

100

May June July August October September November Month Fig. 1. Climate data for the 2002 and 2003 growth periods. (a) Temperature and (b) total rainfall. ----^- - - -, Year 2002; ––––&––––, year 2003.

from 2003 had the least quantity of this bound com- citronellol would not allow the free form concen- pound. tration to surpass the perception threshold (100 mg/l; Bound a-terpineol was found in much greater Ribereau-Gayon et al. 1998). concentration than in its free form. However, even Of the two benzenoid compounds detected, if it were all released it would never rise above 2-phenylethanol predominated in its free form the perception threshold (0.4 mg/l; Ribereau-Gayon while benzyl alcohol was most abundant in its bound et al. 1998). Similarly, the release of all the bound form. Cainõ Tinto, Cainõ Bravo and Cainõ Longo wines 329

Table 1. Mean concentration (m) and standard deviation of free and bound volatile compounds in Cainõ Tinto, Cainõ Longo and Cainõ Bravo wines in 2002 and 2003 vintages

2002 2003

Cain˜ o Cain˜ o Cain˜ o Cain˜ o Cain˜ o Cain˜ o Compound Longo Tinto Bravo P Longo Tinto Bravo P

Free form Theaspireno 176¡1.5 134¡3.6 131¡2.3 <0.001 23¡2.020¡1.613¡2.70.003 Linalool 9¡0.59¡0.57¡1.00.006 71¡3.89¡0.99¡1.6 <0.001 a-Terpineol n.d. 4¡0.34¡0.7 <0.001 41¡2.317¡2.255¡2.8 <0.001 Citronellol 2¡0.2 n.d. 3¡0.2 <0.001 13¡1.65¡0.59¡0.8 <0.002 Benzyl 167¡2.2 124¡0.7 163¡2.1 <0.001 132¡4.9 154¡5.0 174¡1.8 <0.001 alcohol 2-Phenyl 37082¡357.4 34914¡49.3 31505¡181.8 <0.001 12037¡340.7 16473¡590.7 8692¡144.8 <0.001 ethanol b-Ionona 18¡0.361¡0.812¡0.5 <0.001 56¡2.312¡3.119¡2.5 <0.001 Bound form Citronellal 36¡6.2 n.d. 76¡1.1 <0.001 20¡1.9 n.d. 18¡2.0 <0.001 Linalool 20¡4.6 n.d. 26¡0.6 <0.001 48¡1.5 n.d. n.d. <0.001 4-Terpineol 145¡3.2 134¡13.7 192¡5.80.001 177¡3.092¡2.8 155¡4.4 <0.001 a-Terpineol 128¡2.3 157¡14.0 141¡3.40.018 162¡4.979¡0.4 101¡3.3 <0.001 Citronellol 16¡0.810¡2.012¡0.50.003 5.8¡1.011¡1.111¡0.1 <0.001 Nerol 28¡1.326¡3.222¡1.20.024 13¡0.535¡3.533¡1.5 <0.001 Geraniol 30¡1.812¡2.630¡2.1 <0.001 105¡13.411¡1.435¡2.4 <0.001 Benzyl 483¡3.5 145¡11.9 1365¡47.5 <0.001 149¡3.39¡2.9 217¡5.6 <0.001 alcohol 2-Phenyl- 784¡6.7 1370¡9.1 934¡23.0 <0.001 1013¡41.5 1096¡64.5 932¡37.30.018 ethanol b-Ionona 218¡2.528¡2.756¡2.6 <0.001 34¡1.426¡0.423¡0.3 <0.001 Total 39342.8 37129.1 34675.8 14100.2 18133.9 10494.5

The data are the mean of three values¡standard deviation; n.d.: not detected.

Figure 2 shows the PCA results. This analysis Since the grapes were all collected from the same identified the compounds that best discriminate experimental plot and treated in the same way in between the wines made from the different grape both years, the differences of vintage must be due to varieties. The first two axes accounted for 0.76 of climate. the variability (0.42 and 0.34, respectively). The first A total of seven free volatile compounds were axis (Prin 1) was characterized by bound citronellal, identified in Cain˜ o Longo, Cain˜ o Tinto and Cain˜ o bound linalool and bound 4-terpineol having positive Bravo wines in 2002 and 2003 vintage. Free 2-phenyl- loadings. In the second axis, free linalool and bound ethanol was the most abundant compound in the geraniol had positive loadings while free benzyl three varieties and in the two vintages. Similar results alcohol had a negative loading. are found of red wines of V. vinifera cv. O¨ku¨zgo¨zu¨ and Bogazkere grown in Turkey (Cabaroglu et al. 2002). This compound seems to be related to the DISCUSSION maturation index and it has a rose odour. Its level in The present paper studies the major aromatic com- wine is also related to the grape variety and to the pounds, free and bound, responsible for the primary yeast metabolism (Gomez Plaza et al. 1999). The or grape-derived aroma of the wines produced from concentrations of linalool and a-terpineol are always Vitis vinifera cv. Cain˜ o Tinto, Cain˜ o Longo and correlated since the latter is obtained from the Cain˜ o Bravo grapes. The wines studied from Galician former by cyclical reactions in acidic environments varieties showed a different volatile profile and all (Arrhenius et al. 1996). In all cases, the concentration volatile compounds showed significant differences of b-ionone, with notes of violet (Francis & Newton in two consecutive vintages. The high number of 2005), was above the perception threshold (0.09 mg/l). significant differences of the aromatic compounds Table 1 also shows a total of ten bound volatile studied reflects the influence of cultivar and vintage. compounds identified. The concentration of bound 330 M. VILANOVA ET AL.

(a)

2 CL03

CT02 0 −2 −10 1 2 3 Prin 2 (34·13%) CT03 CB02 CB03 CL02 −1

−2 Prin 1 (42·30%)

(b) Free linalool

2 Geraniol Linalool 2-Phenyl ethanol

1 4-Terpineol

0 −2 −1 0123

Prin 2 (34·13%) β-Ionone −1 Benzyl alcohol Citronellal

Free benzyl alcohol −2 Prin 1 (42·30%) Fig. 2. PCA of aromatic compounds in Cain˜ o Tinto (CT), Cain˜ o Longo (CL) and Cain˜ o Bravo (CB) wines from 2002 and 2003 vintages (a) wine samples and (b) volatile compounds. Prin 1 and Prin 2 are the first and second principal components respectively and the percentage of variation each accounts for is given in parentheses. compounds in Cain˜ o Longo, Cain˜ o Tinto and Cain˜ o grape varieties and in different years was achieved. Bravo wines were higher in the 2002 vintage than in the The first two principal components accounted for 2003 vintage. The high levels of non-monoterpenes 0.76 of total sensory variability (0.42 and 0.34 re- indicate that these varieties are neutral cultivars. spectively). Cain˜ o Tinto from 2002 and 2003 were Quantitatively, differences in bound compounds were characterized by 2-phenylethanol, and Cain˜ o Bravo found when the variety and the year were considered. from 2002 and 2003 were characterized by free benzyl The total bound composition was higher in 2002 alcohol, bound citronellal and bound 4-terpineol. vintage than in 2003 vintage. Citronellal, 4-terpineol, Cain˜ o Longo was more heterogenic: benzyl alcohol nerol and geraniol have been present only in bound content characterized the 2002 vintage and linalool form. The perception threshold of citronellal is un- and geraniol contents the 2003. known; its contribution to the aroma of these wines The main conclusions from the present work are after its full hydrolytic release cannot be predicted. that several volatile compounds can distinguish the The concentration of bound b-ionone was high in red wines obtained from Cain˜ o Tinto, Cain˜ o Longo Cain˜ o Longo from 2002 vintage. and Cain˜ o Bravo. Significant differences were found CA was performed on the complete data set. When in the levels of all aroma compounds studied in the a two-dimensional plot (Fig. 2) was drawn, a good wines analysed in two consecutive vintages. Many distinction between the wines made from the different factors were found to influence the levels of varietal Cainõ Tinto, Cainõ Bravo and Cainõ Longo wines 331 volatile compounds in wine as cultivar and vintage. This work was supported by the Ministry of The influence of vintage on volatile composition Science and Technology (VIN00-036-C6-3, HP2000- profile would seem to indicate the degree of grape 0032, RF02-004-C5-2), Diputacioń Provincial of maturity. Non-terpenyl compounds were the most Pontevedra and Xunta de Galicia (Spain). Mar abundant aroma substances in the considered vari- Vilanova was supported by an I3P contract financed eties. Cain˜ o Longo was richest in varietal aroma. by the CSIC-European Social Fund.

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