Impact odorants and sensory profile of young red wines from four Galician (NW of Spain) traditional cultivars

Research article Received: 21 March 2013 Revised: 13 February 2015 Accepted: 10 July 2015 Published online in Wiley Online Library: 16 September 2015 (wileyonlinelibrary.com) DOI 10.1002/jib.252 Impact odorants and sensory profile of young red wines from four Galician (NW of Spain) traditional cultivars Sandra Cortés-Diéguez, 1,2 * Raquel Rodriguez-Solana, 1,2 José Manuel Domínguez 1,2 and Emilia Díaz 3 Monovarietal red wines from,, and grape varieties were analysed in order to determine the volatile composition in terms of fermentative compounds. The wines were also sensorially evaluated with the aim of defining their sensory profile based on the descriptive attributes provided by a panel. Significant analytical and sensorial differences were found among the four wine cultivars. The wine from the cultivar showed a lower content for the majority of the aromatic compounds, whereas wines from and cultivars were the most aromatic ones with higher contents in ethyl esters and acetates. Odour activity values (OAV) for all volatile compounds identified were calculated and classified into seven odorant series, fruity, floral, sweet and fatty being the series with higher contribution to the aroma profile of the evaluated wines. Based on the individual odour thresholds, wines from were the most aromatic, owing to the higher OAV values found for the majority of the volatiles analysed, mainly ethyl esters. Principal component analysis accounted for 81.19% of the variance, thus showing a good separation of the wine cultivars according to the volatile compounds. Copyright 2015 The Keywords: Caíño grape variety; red wine; volatile composition; odour activity values; sensory profile; aromatic series; PCA analysis 628 Introduction Galicia, a region located in the northwest of Spain, is one of the most important wine-producing areas of the country, with five Appellations of Origin Controlled. In the last few years, there has been a growing interest within the Galician winemaking industry in the recuperation of the denominated traditional or noble varieties, in order to endow differentiated and singular characteristics to the Galician red and white wines. The majority of monovarietal Galician white wines are produced from the Albariño, Treixadura and Godello grape varieties. Numerous studies have been conducted about their chemical composition in grape, must and wine (1 5). Meanwhile, in the case of Galician red wines, Mencia is the principal variety employed, and some studies on its composition can be found in the literature (6,7). Several red varieties have been traditionally used in Galicia; they can be separately vinified to produce monovarietal wines with the characteristics that consumers demand: structure in mouth, high colour intensity and personal aroma. In addition, there is a growing market for red wines elaborated or aged in contact with oak; thus, the variety of grape employed to produce this kind of wine must allow for this vinification process. Among the red grapes cultivated and traditionally used in Galicia, the name Caíño involves a group of several varieties with different agronomic and enological characteristics. The names of these varieties change in the different cultivar areas and this aspect causes many difficulties in ensuring their typification and varietal characterization. The quality of these grapes is appreciated in winemaking but the majority of Caíño wines are blended with other varieties of Galician grapes such as Mencia, Souson or Garnacha, because monovarietal red Caíño wines present a high acidity that requires a blending stage to balance them. From the enological point of view, Vilanova et al. (8,9) published the volatile composition of, Caíño Tinto and wines, with different characteristics among them. Later, Canosa et al. (10) also evaluated the free and bound volatile compounds present in wines from five minority red cultivars, among them,. However, considerable research and development on the viticulture and enology of these varieties is still necessary, in particular in relation to the chemical aroma composition and the sensory properties of the corresponding wines. Recent DNA studies clarify the status of the cultivars responding to the name Caíño. They show the existence in the region of different cultivars known under this name (11 13): (CR1 and CR2), (CL1 and CL2), and. Genetic studies have shown a relationship among CR1, CL1 and, also showing the importance of Caíño Bravo as the progenitor of these and other cultivars considered indigenous to the region of Galicia. is also known * Correspondence to: Sandra Cortés-Diéguez, Chemical Engineering Department, Sciences Faculty, University of Vigo (Ourense Campus), As Lagoas s/n, 32004 Ourense, Spain and Laboratory of Agro-food Biotechnology, CITI- Tecnópole, Parque Tecnológico de Galicia, San Cibrao das Viñas, Ourense, Spain. E-mail: smcortes@uvigo.es 1 Chemical Engineering Department, Sciences Faculty, University of Vigo (Ourense Campus), As Lagoas s/n, 32004 Ourense, Spain 2 Laboratory of Agro-food Biotechnology, CITI-Tecnópole, Parque Tecnológico de Galicia, San Cibrao das Viñas, Ourense, Spain 3 Viticulture and Oenology Research Station from Galicia, Ponte San Clodio s/n, Leiro, 32427 Ourense, Spain J. Inst. Brew. 2015; 121:628 635 Copyright 2015 The

Impact odorants and sensory profile of young red wines under the names of Caíño Tinto and Tinta Femia in the southwest of Galicia and Borraçal in Portugal. is also known under the name of Tinta Femia or Zamarra according to the area in Galicia whereas is called Azal Tinto in Portugal. The present study aims to contribute to the evaluation of the volatile composition and sensory characteristics for a better understanding of the monovarietal wines from, Caíño Longo, and grapes varieties belonging to the germplasm bank of grapevines collected in Galicia, sited at the Viticulture and Oenology Research Station in Galicia (EVEGA). Because the grapes of the four varieties were from the same vineyard and they were vinified according to the same technology and conditions, the differences that might be found in the composition of their wines should only be attributed to the variety of grape. The results obtained will allow for the definition of a sensory and volatile profile for the four red varietal wines produced in a specific viticulture area. Material and methods Wine samples,, and (Vitis vinifera L.) red grapes were harvested in one experimental vineyard of the Viticulture and Oenology Research Station from Galicia. The four cultivars were grafted on the same root-stock 196-17C, grown on espaliers using a simple cordon pruning system, and were subjected to the same phytosanitary treatments. Grape samples were manually harvested, fixing the date as a function of the sugar content, acidity, sanitary state of the grape and phenolic maturation, after monitoring the ripening process with weekly samplings performed after veraison. The grapes were processed in the installations of the EVEGA following the traditional protocol for red winemaking. Briefly, once the grapes had been destemmed and treaded, three equal portions of the juice were introduced in separate stainless steel tanks (35 L) and sulphited at the rate of 40 mg/l of SO 2. In all cases, the same Saccharomyces cerevisiae dried active (LSA) commercial yeast, Excellence FW from Lamothe Abiet, (20 g/hl), was added. Two cycles of pumping-over with aeration each day were performed. As a control of the fermentation kinetics, the density value and temperature were measured daily (always <25 C). When the alcoholic fermentation was considered to have finished, after analysing the corresponding wines, the skins were removed from the vat. The pressing of the resulting grape mass was performed in a small pneumatic press, mixing to the pre-run wine with the first pressing. Malolactic fermentation took place immediately after alcoholic fermentation. Once this process had finished, the wines were racked, corrected for SO 2 (30 mg/l) and allowed to clarify naturally over a period of 6 months at a controlled temperature of <10 C. The fermentations were performed in triplicate. In the final wines, the fermentative volatile compounds were determined, and a sensorial evaluation was carried out. Reagents Ethanol, diethyl ether and hexane, of analytical grade, were purchased from Merck (Germany). 1-Butanol, 1-propanol, 2-methyl- 1-propanol, 4-methyl-2-pentanol, (Z)-3-hexen-1-ol, 3-octanol, acetaldehyde and diethyl succinate were supplied by Aldrich (Aldrich Chemical, Switzerland). 2-Phenyl-ethanol, 2-methyl-1- butanol, hexanol, ethyl butyrate, hexyl acetate, isoamyl acetate, ethyl acetate and 2-phenylethyl acetate were purchased from Merck (Germany). Ethyl hexanoate, ethyl octanoate, ethyl decanoate and 3-methyl-1-butanol were supplied by Fluka (Switzerland) and isobutyric acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid and ethyl lactate were from Sigma (Switzerland). A stock solution of reference standards was prepared in distilled water containing 12% (v/v) of ethanol. The internal standards, 4- methyl-2-pentanol, 3-octanol and heptanoic acid were prepared in absolute ethanol. Volatile compounds analysis For determination of the major volatile compounds (ethyl acetate, acetaldehyde and higher alcohols), 1 ml of an internal standard solution (1 g of 4-methyl-2-pentanol per 1 L of ethanol) was added to 10 ml sample of wine. A 2 μl sample was injected directly (split 1:20) into the chromatograph. The analyses were carried out using an Agilent Technologies 6890 N Gas-Chromatograph equipped with a 7683 Series automatic injector and a flame ionization detector. The compounds were separated on a Chrompack CP-WAX 57CB (polyethylene glycol stationary phase; 50 m 0.25 mm i.d. with 0.25 μm film thickness) fused-silica capillary column. Instrumental conditions were: injector temperature, 250 C; detector temperature, 260 C; carrier gas, H 2 at 1.5 ml min 1 ; and make-up gas, nitrogen 30 ml min 1. The detector gas flow rates were: hydrogen, 50 ml min 1 ; air, 400 ml min 1. The temperature programme of the oven was as follows: initial temperature, 40 C (isothermal for 5 min); ramp, 3 C min 1 to 220 C; post-run, 40 C for 1 min. Total run time was 57.43 min. A 2 ml aliquot of 3-octanol (50 mg/l), 2 ml of heptanoic acid (70 mg/l), both as internal standard, and 1 ml of sulphuric acid (1:3) were added to 50 ml of wine for the determination of organic acids, ethyl esters and acetates. Each sample was extracted three times with 4, 2 and 2 ml of diethyl ether hexane (1:1, v/v). A 1 μl aliquot of the organic extract was injected into the chromatograph in splitless mode (30 s). The analyses were carried out using an HP 5890 gas chromatograph and a flame ionization detector. The compounds were separated on a FFAP (polyethylene glycol stationary phase; 50 m 0.2 mm id with 0.33 μm film thickness) fused-silica capillary column. Instrumental conditions were as follows: injector temperature, 220 C; detector temperature, 250 C; carrier gas, H 2 at 1.3 ml min 1 ; and makeup gas, nitrogen 30 ml min 1. The detector gas flow rates were hydrogen, 50 ml min 1 and air, 400 ml min 1. The temperature programme was as follows: initial Tª, 50 C; ramp, 3 C min 1 to 200 C, after which the temperature was held constant for 25 min. Total run time was 75 min. Volatile compounds were identified by comparing retention times with those of commercial pure standards. The internal standard method was used for quantitative purposes. The concentration of each compound was calculated employing the corresponding response factor (RF) in the reference solution. The response factor of each compound, RF i,was calculated by RF i =(A is /A si ). (C si /C is ), where A is and A si are the peak areas of the chromatographic internal standard and of the chromatographic standard of the compound of interest, respectively, and C is and C si are the concentrations of the chromatographic internal standard and of the chromatographic standard of the compound of interest, respectively. In the quantification, the concentration of each compound of interest, C i, was determined by C i =(A i /A is ). C is.rf i,wherea i is the area of the peak of interest. All determinations were performed in triplicate. 629 J. Inst. Brew. 2015; 121: 628 635 Copyright 2015 The wileyonlinelibrary.com/journal/jib

Odour activity values The odour activity value (OAV) is defined as the ratio between the concentration of a volatile compound in a sample and its odour perception threshold value from the literature. Volatile compounds with an OAV 1 are considered to contribute directly and individually to the aroma and they are commonly appointed the most important volatile compounds or the most active odorants. The rest of the volatiles with an OAV < 1 could increase the aromatic notes of other compounds through synergistic effects and therefore contribute to the global wine aroma. To evaluate the contribution of each volatile compound to wine aroma from Caíño s varieties, the OAV was calculated. Sensory analysis Wine sensory analysis was carried out in two sessions by a panel composed of seven trained tasters, winemakers and laboratory personnel, all of whom had previously participated in wine sensory descriptive analyses. They were selected based on interest and availability. A simple scorecard was used to evaluate the samples of wines for the visual phase, aroma and mouthfeel. They rated each descriptor on a scale from 1 (not present) to 5 (high intensity). Samples of wine (30 ml) coded with two-digit random numbers were served in clear tulip-shaped glasses. Statistical analyses Sensory and instrumental data were analysed using XLstat-Pro (Addinsoft). Statistical analysis of the wine volatile compounds was performed using Analysis of Variance and the least significant difference test to determine statistically different values at a significance level of α 0.05, 0.01 and 0.001. For interpreting the results, principal component analysis (PCA) on volatile compounds of wines was also applied. Results and discussion S. Cortés-Diéguez et al. Volatile composition of wines Twenty-four individual volatile compounds have been identified in Caíño wines, classified in seven different chemical families. Table 1 Table 1. Volatile composition (mg/l) of,, and wines. ANOVA results are also shown Mean SD Mean SD Mean SD Mean SD Significance 630 Ethyl esters of volatile acids Ethyl butyrate 0.71 a 0.19 0.56 a 0.12 0.91 b 0.19 0.69 a 0.08 ** Ethyl hexanoate 0.42 a 0.03 0.28 b 0.01 0.30 b 0.13 0.32ª,b 0.07 * Ethyl octanoate 0.32 a 0.05 0.26 a 0.01 0.32 a 0.05 0.24 a 0.07 n.s. Ethyl decanoate 0.37 a 0.20 0.70 a 0.26 0.39 a 0.13 0.60 a 0.41 n.s. Ethyl esters of fixed acids Ethyl lactate 216.58ª,b 28.06 139.69 b 77.44 324.61 a 41.23 305.54 a 70.31 * Diethyl succinate 2.01 a 0.52 2.45 a 0.16 1.58 a 0.25 2.70 a 1.18 n.s. Acetates Isoamyl acetate 0.82 a 0.37 0.58ª,b 0.19 0.42 b 0.07 0.41 b 0.20 * Hexyl acetate 0.25 a 0.37 0.01 b 0.00 0.02 b 0.02 0.01 b 0.01 * Ethyl acetate 49.42ª,b 18.17 23.48 b 4.60 78.36 a 4.86 49.46ª,b 27.22 * 2-Phenyl ethyl acetate 0.03 a 0.01 0.02 a 0.01 0.02 a 0.01 0.02 a 0.01 n.s. C6 Hexanol 2.61ª,b 0.66 2.07 b 0.03 3.02 a 0.92 2.95ª,b 0.07 * (Z)-3-Hexen-1-ol 0.09ª,b 0.06 0.06 b,c 0.03 0.12 a 0.02 0.01 c 0.01 *** Higher alcohols 1-Propanol 22.72ª,b 8.04 19.48 b 0.71 26.18 a 1.84 30.18ª,b 6.94 * 2-Methyl propanol 99.00 a 24.88 61.57 b 23.39 112.36 a 18.89 115.77 a 2.04 * 1-Butanol 1.25 a 0.13 1.05 a 0.25 1.65 b 0.28 1.75 b 0.23 *** 2-Methyl butanol 71.02 a,b 5.72 53.82 b 18.72 65.72 a,b 13.97 85.03 a 18.11 * 3-Methyl butanol 262.96 a 10.45 178.94 b 68.99 238.41 a 44.51 301.76 a 57.36 ** 2-Phenyl ethanol 42.49ª,b 5.11 57.53 a 8.93 33.13 b 7.23 49.05ª,b 18.67 * Volatile acids Isobutyric acid 3.12 a 0.44 3.62 a 0.36 2.63 b 0.18 3.53 a 0.65 *** Butyric acid 0.18 a 0.14 0.18 a 0.20 0.24 a 0.08 0.10 a 0.10 n.s. Hexanoic acid 1.36 a 0.23 1.43 a 0.02 0.94 b 0.17 1.21 a 0.06 *** Octanoic acid 0.73ª,b 0.25 0.78 a 0.15 0.54 b 0.23 0.54 b 0.03 * Decanoic acid 0.65 a 0.86 0.51 a 0.10 0.41 a 0.25 0.30 a 0.03 n.s. Other compounds Acetaldehyde 8.67 a 3.07 18.90 a 12.06 21.76 a 12.77 23.26 a 12.40 n.s. Statistical significance is given by * p 0.05, ** p 0.01), *** p 0.001. n.s., Not significant. Values, within the same row, with the same letter are not significantly different. wileyonlinelibrary.com/journal/jib Copyright 2015 The J. Inst. Brew. 2015; 121: 628 635

Impact odorants and sensory profile of young red wines shows the mean and standard deviation obtained for each volatile compound in the red wines obtained from the four different cultivars analysed. These compounds are responsible for the secondary aroma in the wine, and the majority are produced during the alcoholic fermentation, so their concentration depends on various factors, such as yeast strain, fermentation temperature, degree of aeration, clarity of the juice and nitrogen and sugar content in the must (3,14 16). Seventeen of the volatile compounds identified showed significant differences among the wines analysed: all higher alcohols, C 6 compounds and the majority of acetates. Ethyl esters. Ethyl butyrate, ethyl hexanoate and ethyl lactate showed significant differences among the wines analysed. The concentration of these important compounds was significantly higher in the monovarietal wines from, whereas there was a lower value in wines from. Ethyl butyrate was significantly higher in wines and no significant differences were found for the concentration of this important compound with fruity notes (17) between the other three wines analysed in this study. Ethyl hexanoate was present in significantly higher concentration in the wines from the variety. However, this compound contributes directly with fruity notes (apple) to the aroma of the four monovarietal wines, owing to it being present in concentrations above the threshold value of 0.014 mg/l (8). The grapes from Caíño varieties had a large content of malic acid with a low ph value. The kinetics of the malolactic fermentation are very slow and ethyl lactate increases in content significantly in the resulting wines. Wines from showed the significantly lowest concentration of ethyl lactate. According to Soufleros et al. (19), diethyl succinate and ethyl lactate are characteristic volatile compounds of the malolactic fermentation in young wines. Diethyl succinate also increases its concentration during wine storage and aging. Acetates. Acetates are produced during fermentation but their content has also been correlated with the variety of grape employed (20). The presence of this group of compounds is positive for the wine aroma; they contribute fruity and floral notes. The results showed that they are more abundant in the Caíño Longo wines. Three of the four acetates identified showed significant differences between Caíño s wines. Isoamyl and hexyl acetate were present in higher concentrations in wines from variety. No significant differences were found for 2-phenyl-ethyl acetate and its concentration was lower than the corresponding threshold value fixed at 0.25 mg/l by Guth (17). Isoamyl acetate, which gives a pleasant banana-like aroma to wine, was reported to exist at a significantly higher concentration in the wines. Ethyl acetate increases its concentration with the malolactic fermentation (8). In this study, wines from grape variety showed a significantly high concentration of ethyl lactate and ethyl acetate, both volatile compounds formed during the malolactic fermentation. C6 Compounds. According to Simpson (20), the concentration of 1-hexanol in wine is dependent primarily on the grape variety, thus the fermentation conditions have little influence on its content. The descriptors employed for these compounds are grass, herbaceous, woody, green, bitter and fatty (21), thus the content of C 6 compounds could be low in a red wine. Significant different concentrations were found with hexanol and (Z)-3-hexen-1-ol among the analysed wines, with higher values in wines and lower values in those from. Higher alcohols. The compounds 1-propanol, 1-butanol, 2- methyl-propanol, 2-methyl-butanol and 3-methyl-butanol were the most abundant higher alcohols and these are very important in wine aroma. The descriptors employed to describe this group of compounds are alcoholic, fusel and chemical, so they tend to be unpleasant at higher concentrations. Rapp and Versini (22) established 350 mg/l as the suitable content for a quality wine, in this case all of the wines analysed showed a higher content, especially the wine from the Caíño Bravo variety, whereas the wines showed a lower content of this group of compounds. VISUAL PHASE AROMA orange glints chemical TASTE PHASE microbiological animal structure astringent colour intensity Fruity toasted Alcohol bitter Floral spicy purple glints Vegetal sweet acid Figure 1. Aroma profiles of,, Caíño Astureses and Caíño da Terra wines. 631 J. Inst. Brew. 2015; 121: 628 635 Copyright 2015 The wileyonlinelibrary.com/journal/jib

The compound 2-phenylethanol is described as rose like, thus it is a very important compound in wine aroma. The concentration of 2-phenylethanol is very high in the wines of the four varieties, especially in and. In all cases its content is above its odour threshold, so these wines will show an important floral note. Acids. Volatile acids have descriptors such as cheese and rancid (23), so it is important to avoid a high content of these. Butyric and isobutyric acids are indicative of spoilage of the wine and usually imply bacterial activity. The wines from and Caíño longo grape varieties showed a higher concentration of this group of compounds, which during the storage or aging could be esterified with the higher alcohols and increase the fruity aroma. Other compounds. No significant differences were observed for the mean concentration of acetaldehyde Acetaldehyde is a carbonyl compound related to wine oxidation during the winemaking process or during the storage. The mean concentration of this compound in the wines analysed was very low. Sensory analysis Figure 1 shows the profile obtained after the sensory evaluation of the wines from the four red grape varieties. Similar characteristics were obtained in the visual phase for the wines from Caíño Redondo, and varieties; however, wine from showed a very different profile, with low colour intensity with high orange glints. In aroma, had a different profile and the descriptors employed to define this wine were dried vegetables (hay, tea, tobacco), olive, asparagus and chemical notes; the intensity of fruity and floral notes was very low for the wine from this variety. The other three monovarietal wines were defined by fruity, vegetal, spicy and floral notes. Caíño Bravo produced the wine with the most intense positive notes, especially the intensity of floral and toasted descriptors. The wines from variety showed an important contribution of pepper, cinnamon, anise, vanillin and liquorice, under the name of spicy notes. In taste, variety produced astringent, bitter and acid wines with high level of structure in the mouth. The wines from variety showed a profile with low intensities for the majority of parameters evaluated in the taste phase; however, they resulted in more alcoholic wines. Caíño bravo and showed similar profiles in this phase. S. Cortés-Diéguez et al. Total volatile compounds Figure 2 shows the differences between the four red wines according with the total concentration of volatiles from each family. Higher alcohols was the group that was quantitatively more abundant in the wines analysed. The compounds with positive notes, ethyl esters and acetates, were present in higher concentrations in the wines from variety and in lower content in, which shows the higher total concentration of volatile acids. The wines from variety were characterized by an important total concentration of higher alcohols and acetaldehyde. Wines from showed an important concentration of acetates, volatile acids and C 6 compounds. Odour activity values and aromatic series In order to evaluate the most active odorants in Caíño wines, the concentration of each volatile was correlated with its threshold value (reported in the literature) and the corresponding results obtained, with the odorant series, are shown in Table 2. The majority of volatile compounds identified in the wines analysed showed OAV > 1: 64% in wines from and and 60% in those from and. Ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl lactate, isoamyl acetate, ethyl lactate, 1-propanol, 2-methyl-1-propanol, isoamyl alcohols, 2- phenyl ethanol, isobutyric, butyric, hexanoic and octanoic acids were the volatile compounds considered as impact odorants in Caíño wines. The highest OAV was obtained for ethyl esters of volatile acids (ethyl butyrate, ethyl hexanoate and ethyl decanoate), contributing favourably to wine aroma with fruity nuances. Isoamyl acetate also contributed to the overall fruity aroma of Caíño s wines, mainly in those from (OAV = 27.33). With regard to higher alcohols, 1-propanol showed a high OAV value and also enhanced the fruity notes. 2-Phenyl ethanol was found to be an active odorant, contributing with floral aroma, showing higher OAV value in wines from variety and lower in those from Caíño da Terra. C6 compounds, diethyl succinate, 2-phenyl ethyl acetate, hexyl acetate, 1-propanol, acetaldehyde and decanoic acid do not appear to contribute individually to wine aroma; with OAVs < 1, however, they can enhance some attributes by synergism effects with other active odorants. To evaluate the global fermentative aroma of the wines, all volatile compounds were grouped into seven different classes Volatile compounds Concentration (mg/l) 90 80 70 60 50 40 30 20 10 0 Ethyl esters of Ethyl esters of volatile fatty fixed acids/10 acids Acetates C6 Higher alcohols/10 Volatile acids acetaldehyde 632 Figure 2. Mean and standard deviation of volatile compounds families. wileyonlinelibrary.com/journal/jib Copyright 2015 The J. Inst. Brew. 2015; 121: 628 635

Impact odorants and sensory profile of young red wines Table 2. Odour descriptors, odorant series, odour threshold (mg/l) for volatile compounds in Caíño wines Compound Odour quality Odorant series a Odour threshold (mg/l) Caíño Longo Odorant activity value (OAV) b Caíño Redondo Caíño da Terra Ethyl butyrate Fruity, apple 1 0.02 c 35.50 28.00 45.50 34.50 Ethyl hexanoate Green apple,fruity 1 0.014 d 30.00 20.00 21.43 22.86 Ethyl octanoate Fruity, fat, sweet soap 1, 2, 4 0.005 d 64.00 52.00 64.00 48.00 Ethyl decanoate Sweet, fruity 1, 4 0.2 e 1.85 3.50 1.95 3.00 Ethyl lactate Buttery, butterscotch, 6 154.6 e 1.40 0.90 2.10 1.98 fruity, medicine Diethyl succinate Wine-like, vinous 7 200 d 0.01 0.01 0.01 0.01 Isoamyl acetate Banana 1 0.03 e 27.33 19.33 14.00 13.67 Hexyl acetate Perfume, green, floral 2, 3 1.5 e 0.17 0.01 0.01 0.01 Ethyl acetate Nail polish, fruity, 1, 6 7.5 c 6.59 3.13 10.45 6.59 pineapple, solvent 2-Phenyl ethyl acetate Flowery, rose, honey 2 0.25 c 0.12 0.08 0.08 0.08 hexanol Resin, flower, green, cut grass 2, 3 8 c 0.33 0.26 0.38 0.37 (Z)-3-Hexen-1-ol Green, cut grass 3 0.4 c 0.23 0.15 0.30 0.03 1-Propanol Pungent, harsh, ripe fruit, alcohol 1, 6 0.830 d 27.37 23.47 31.54 36.36 2-Methyl propanol Wine, solvent, bitter, fusel, 3, 6 40 d 2.48 1.54 2.81 2.89 oily, greem 1-Butanol Fusel, spiritous, medicinal, 6 150 d 0.01 0.01 0.01 0.01 phenolic 2-Methyl butanol Whiskey, malt, burnt, harsh, 4, 6 30 c 2.37 1.79 2.19 2.83 nail polish 3-Methyl butanol Whiskey, malt, burnt, harsh, 4, 6 30 c 8.77 5.96 7.95 10.06 nail polish 2-Phenyl ethanol Honey, spice, rose, lilac, floral 2 10 c 4.25 5.75 3.31 4.91 Isobutyric acid Rancid, butter, cheese 6 2.3 d 1.36 1.57 1.14 1.53 Butyric acid Rancid, cheese, sweat 6 0.173 d 1.04 1.04 1.39 0.58 Hexanoic acid Sweat 6 0.42 d 3.24 3.40 2.24 2.88 Octanoic acid Sweat, cheese 6 0.5 e 1.46 1.56 1.08 1.08 Decanoic acid Rancid fat 6 1 d 0.65 0.51 0.41 0.30 Acetaldehyde Pungent, bruised apple, sherry, nutty 1, 6 100 d 0.09 0.19 0.22 0.23 a 1, fruity; 2, floral; 3, green; 4, sweet; 5, spicy; 6, fatty; 7, others b OAV, Concentration/odour threshold c Guth (17) d Etievant (18) e Ferreira et al. (25) Caíño Bravo (aromatic series), according to their similar odour descriptors, like the approach applied by other authors (24). A compound with several descriptors may belong to different series. The total value in each series results from the sum of individual OAVs of the volatile compounds that are included in each class. Figure 3 shows the mean value of each aromatic series. Based on the synergic effects, compounds with OAV < 1 were also included in the sum. The results showed that fruity was the most important aromatic series to describe wines from all Caíño grapes, showing greater intensity in those from. Fatty, floral and sweet were also aromatic series with a marked influence on the overall aroma. The aroma profile obtained through the sum of individual OAV intensities of volatiles that belong to the same aromatic series (Fig. 3) differed from the corresponding profile obtained with the mean value of aroma sensory descriptors given by the panel fatty others spicy fruity 200 180 160 140 120 100 80 60 40 20 0 Figure 3. sweet floral green Mean value of aromatic series. 633 J. Inst. Brew. 2015; 121: 628 635 Copyright 2015 The wileyonlinelibrary.com/journal/jib

634 (Fig. 1). The main reason for this is the influence of synergic, additive and antagonistic effects that can be produced among the volatile compounds present in a wine. For this reason the aroma profile defined taking only into account the most active odorants (OAV) must be considered as a tentative. PCA of volatile compounds For interpreting the results, PCA was applied. PCA makes the interpretation of multivariate analysis easier and in this study was used to identify the volatile compounds that best discriminated among the samples analysed. A first PCA was performed on the concentration of the 17 volatile compounds in wine samples analysed, with significant differences (Fig. 4A). The two first principal components, PC1 and PC2, accounted for 81.19% of total variance (60.64 and 20.55%, respectively). The first component (PC1) was characterized by major A Variables (axis PC1and PC2: 81,19 %) PC2 (20,55 %) B PC2 (20,55 %) 1 0,75 0,5 0,25 0-0,25-0,5-0,75 Q Ñ O J B D C H Y Z W E T A V -1-1 -0,75-0,5-0,25 0 0,25 0,5 0,75 1 PC1 (60,64 %) 4 3 2 1 0 Observations (axis PC1and PC2: 81,19 %) Caiño Longo Caiño da -1 Terra Caiño Bravo Caiño Redondo -2-5 -4-3 -2-1 0 1 2 3 4 PC1 (60,64 %) Figure 4. Principal component analysis (PCA) store plot of, Caíño Redondo, and wines (A) and volatile compounds (B) variables. (A) Ethyl butyrate; (B) isoamyl acetate; (C) ethyl hexanoate; (D) hexyl acetate; (E) ethyl lactate; (F) hexanol; (H) (Z)-3-hexenol; ( J) isobutyric acid; (Ñ) hexanoic acid; (O) 2- phenyl ethanol; (Q) octanoic acid; (T) ethyl acetate; (V) propanol; (W) 2-methylpropanol; (X) butanol; (Y) 2-methyl-butanol; (Z) 3-methyl-butanol. X F levels of ethyl butyrate (A), ethyl lactate (E), hexanol (F), ethyl acetate (T), propanol (V), 2-methyl-propanol (W) and butanol (X) on the positive side and hexanoic acid (Ñ), 2-phenyl ethanol (O) and octanoic acid (Q) on the negative side. For the second principal component (PC2), isoamyl acetate (B), ethyl hexanoate (C) and hexyl acetate (D) showed high and positive values. The plot of Fig. 4B shows a good separation of Caíño wines, especially for Caíño longo and. Wines from Caíño Longo variety, situated on the positive side of PC2 of the plot, were mainly characterized by isoamyl acetate, ethyl hexanoate and hexyl acetate, all of them with fruity notes. Wines from and varieties, situated in the positive side of PC1 and negative side of PC2, were mainly characterized by the higher alcohols propanol (V) and butanol (X). Finally, wines from Caíño Redondo variety were situated in the negative side of PC1 and PC2 and characterized by isobutyric acid and 2-phenyl ethanol. Conclusions Significant differences were found between the wines obtained from the four cultivars both in volatile compounds and in the sensory profile. The wines from cultivar showed the lower content for the majority of aromatic compounds. Caíño da Terra and wines were the most aromatic with the higher contents in ethyl esters and acetates. Wines from Caiño Bravo showed the highest content of acetaldehyde and higher alcohols; however these wines were the best rated wines for tasters in aroma. Ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl lactate, isoamyl acetate, ethyl lactate, 1- propanol, 2-methyl-1-propanol, isoamyl alcohols, 2-phenyl ethanol, isobutyric, butyric, hexanoic and octanoic acids were the volatile compounds considered as impact odorants in Caíño wines, with OAV > 1, fruity, sweet, fatty and floral being the aromatic series with more influence on the aroma. 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