Characterization of the Key Aroma Compounds in a Commercial

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Characterization of the Key Aroma Compounds in a Commercial Amontillado Sherry Wine by Means of the Sensomics Approach Pauline Marcq and Peter Schieberle* Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Straße 34, D-85354 Freising, Germany ABSTRACT: An aroma extract dilution analysis (AEDA) carried out on the volatile fraction isolated by extraction/solventassisted flavor evaporation (SAFE) distillation from a commercial Amontillado sherry wine revealed 37 odor-active compounds with flavor dilution (FD) factors in the range of 16−4096. Among them, 2-phenylethanol (flowery, honey-like) and ethyl methylpropanoate (fruity) showed the highest FD factors, followed by ethyl (2S,3S)-2-hydroxy-3-methylpentanoate (fruity) reported for the first time in sherry wine. A total of 36 aroma-active compounds located by AEDA were then quantitated by a stable isotope dilution assay, and their odor activity values (OAVs; ratio of concentration to odor threshold) were calculated. The highest OAV was displayed by 1,1-diethoxyethane (2475; fruity), followed by 2- and 3-methylbutanals (574; malty) and methylpropanal (369; malty). Aroma reconstitution experiments and a comparative aroma profile analysis revealed that the entire orthonasal aroma profile of the Amontillado sherry wine could be closely mimicked. KEYWORDS: GC−O, AEDA, static headspace−olfactometry, OAV, ethyl (2S,3S)-2-hydroxy-3-methylpentanoate, aroma recombinate

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INTRODUCTION Sherry is a fortified wine produced in the Jerez region in southern Spain. Three different white grape varieties are used in its production, Palomino Fino, Pedro Ximénez, and Moscatel, with the first being the most abundant in dry sherry production, while the others are commonly used to produce sweet sherry wines. Although made from the same grape variety, namely, Palomino Fino, different types of dry sherry wines are manufactured by three different maturation processes: Fino, Amontillado, and Oloroso. Fino is processed by a dynamic biological aging with a layer of yeast, also called f lor, developing on the surface of the wine, thus protecting it from oxidation. Fino is fortified to 15.5% (v/v) ethanol and results in a pale, yellow product with a sharp and delicate bouquet.1 Oloroso, which is dark, brown, and full-bodied,1 is aged under oxidative conditions and fortified to at least 17% ethanol (v/v), hindering the formation of a f lor, thus allowing direct contact between the wine and ambient air. Amontillado is aged by means of two consecutive steps: a biological step and an oxidative step. Its color is in between that of Fino and Oloroso, and it has been reported to possess a complex flavor.2 Amontillado is the oldest and most valued of the three wine styles made from the Palomino Fino grape.3 A particularity of sherry wine is a dynamic aging system called “criaderas and solera”. The wine is stored in American oak casks (500−600 L) stacked in several levels, with the lowest level containing the older wine and the highest level containing the youngest wine. This technique involves many transfers of wine of different ages and vintages, enabling the production of a consistent and homogeneous sherry aroma. A detailed description of the system can be found in various textbooks cited in reviews.3,4 Data on the volatile composition of sherry wine were published as early as 1976,5 and over 130 volatile components were identified. Subsequently, numerous studies mainly involving gas chromatographic analysis were undertaken, attempting to © XXXX American Chemical Society

correlate the entire volatile fraction of sherry wine with its aroma profile.6−8 Thus, today more than 300 volatiles are known in sherry.9 It is well-accepted in state-of-the-art aroma analysis that only a small subset of volatiles cause the aroma perception in the brain initiated by an interaction of odorants with the human olfactory receptors.10−12 Therefore, it can be assumed that only a few of the numerous volatiles identified in sherry wine are probably odor-active. To characterize the key aroma compounds in foods, the sensomics analysis was suggested by Schieberle and Hofmann11 as a molecular sensory analysis approach to differentiate the key aroma compounds from the bulk of odorless volatiles. By combination of sensory-supported analysis, such as gas chromatography−olfactometry (GC−O), with modern instrumental techniques, it is possible to determine the combinatorial food aroma code, also called the “aroma blueprint”, and various aromas have already successfully been decoded by means of the sensomics analysis.12 By application of GC−O, Zea et al.13 have monitored the aroma profile of Fino during aging; however, no quantitative analysis was performed in this study. In a recent work by Moyano et al.,14 Amontillado sherry wine was analyzed by GC−O to identify the odorants generated during the sequential biological and oxidative aging steps. A total of 25 compounds were monitored and based on so-called odor spectrum values, and ethyl octanoate, ethyl butanoate, eugenol, ethyl methylpropanoate, and sotolon were proposed as the most potent odorants in Amontillado sherry wine. Several further studies on sherry wines applied the odor activity value concept (OAV; ratio of concentration to odor threshold), however, without performing a preliminary screening of aroma compounds by Received: March 19, 2015 Revised: April 28, 2015 Accepted: April 28, 2015

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DOI: 10.1021/acs.jafc.5b01418 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

according to a procedure described by Esterbauer.20 For chromatographic separation, water was purifed with a Milli-Q Advantage A10 system (Millipore, Molsheim, France). For sensory reconstitution experiments, Deer Park bottled water was used (Nestlé Water North America, Inc., Stamford, CT). D-Glucose and L-(+)-tartaric acid were supplied by Sigma-Aldrich (St. Louis, MO), and glycerol was supplied by Fisher Scientific (Pittsburgh, PA). Synthesis of Ethyl (2S,3S)-2-Hydroxy-3-methylpentanoate. This was carried out in two steps and was based on previously published procedures,21,22 with some modification. (2S,3S)-2-Hydroxy-3-methylpentanoic Acid. L-Isoleucine (22.9 mmol) was dissolved in aqueous hydrochloric acid (1 mol/L, 22.9 mL), and concentrated acetic acid (45.8 mL) was added with stirring. Then, an aqueous solution of sodium nitrite (23 mmol in 27.6 mL) was added dropwise at 0 °C to the mixture within 30 min with stirring. The solution was stirred for another 20 min at 0 °C and then stirred overnight at room temperature under an argon atmosphere. The solution was concentrated, and the residue was dissolved in water (50 mL) and adjusted to a pH of ∼2 with concentrated hydrochloric acid. After extraction with diethyl ether (3 × 100 mL), the combined organic extracts were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off using a Vigreux column at 40 °C to yield the target compound. Ethyl (2S,3S)-2-hydroxy-3-methylpentanoate. The acid was mixed with ethanol (20 mL) and concentrated hydrochloric acid (50 μL) and was refluxed for 150 min. After the mixture was cooled, diethyl ether (50 mL) was added and the organic phase was consecutively washed with sodium carbonate (0.75 mol/L, 20 mL) and bidistilled water (15 mL). The organic layer was dried over anhydrous sodium sulfate and then concentrated using a Vigreux column (60 × 1 cm). The target compound was purified using a water-cooled glass column (30 × 1 cm) containing 20 g of purified silica gel,20 conditioned in n-pentane. For this purpose, the solution (0.2 mL) was placed on the top of the column and separation was performed using the following n-pentane/ diethyl ether gradient: n-pentane (I; 100 mL), n-pentane/diethyl ether (II; 95:5, 100 mL), n-pentane/diethyl ether (III; 80:20, 100 mL), and finally, diethyl ether (IV; 100 mL). The target compound was detected by GC−O in fraction III and was further purified using flash chromatography (40 g of Lichroprep DIOL phase; particle size of 40−63 μm) under pressure with a flow rate of 2 mL/min and the n-pentane/diethyl ether gradient. MS (EI), m/z (%): 87 (100), 76 (66), 104 (53), 45 (37), 69 (24), 57 (17), 41 (10), 88 (7), 75 (6), 58 (5), 103 (5), 105 (5), 71 (4), 55 (3), 77 (3), 85 (3), 86 (3), 43 (2), 47 (2), 70 (2), 99 (2), 117 (2), 131 (2), 145 (2), 46 (1), 53 (1), 59 (1), 67 (1), 68 (1), 89 (1), 97 (1), 98 (2), 106 (1), 113 (1), 114 (1). MS (CI), m/z (%): 161 (100). The concentration of ethyl (2S,3S)-2-hydroxy-3-methylpentanoate was determined by GC/FID using 1-octanol as the internal standard. Its odor threshold was determined in a 17% (v/v) hydroalcoholic solution according to the procedure described previously,23 with 12 panelists participating in this session (4 males and 8 females, ranging in age from 29 to 51 years old). Isolation of the Volatiles. The sherry (100 mL) was mixed with sodium chloride (10 g) and was extracted with diethyl ether (total volume of 250 mL). The organic layers as well as the emulsion formed were combined, dried over anhydrous sodium sulfate, and concentrated to 100 mL using an automated solvent evaporator system operating at 25 °C, 0.7 bar, and using nitrogen gas vortex shearing (TurboVap II, Biotage, Uppsala, Sweden). The volatiles were then separated from the non-volatile compounds by high-vacuum distillation using the solvent-assisted flavor evaporation (SAFE) apparatus,24 and the distillate was separated into an acidic fraction (AF) and a neutral-basic fraction (NBF) by treatment with an aqueous sodium carbonate solution.25 AF and NBF were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to ∼4 mL using the automated solvent evaporator system described above. The distillate was further concentrated to ∼1 mL under a subtle stream of nitrogen at 37 °C.

GC−O.2,15−17 Moreno et al.18 monitored volatile compounds with OAV > 5 in Fino sherry wine during aging on the basis of GC/flame ionization detector (FID) quantitation. In addition to acetaldehyde, these authors suggested sotolon, 1,1-diethoxyethane, and cis-whiskylactone as markers of aging. Although several studies have been performed aimed at identifying important odorants by either applying GC−O or the OAV concept, up to now, no comprehensive studies employing the full sensomics approach12 have been performed on a sherry wine. Therefore, this research consisted of (i) the identification of the key aroma compounds in an Amontillado sherry wine by means of aroma extract dilution analysis, (ii) the quantitation of these compounds by a stable isotope dilution assay (SIDA), (iii) the determination of the importance of each odorant on the basis of OAVs, and (iv) a validation of the results through aroma reconstitution experiments and sensory evaluation.

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MATERIALS AND METHODS

Sherry Wine. The investigated commercial sherry wine was an Amontillado produced in Spain in the Sanlúcar de Barrameda region (Jerez region). The wine was called Don Benigno and has been produced and bottled by Pedro Rodriguez e Hijos. On the basis of the label, it contained 17.5% (v/v) ethanol and carried the Jerez-XérèsSherry denomination of origin. The wine was purchased at a local spirit shop. This selection was not performed for advertising purposes nor does it imply a research contract with the manufacturer. Chemicals. Reference compounds of the odorants were obtained from the sources given in parentheses: acetaldehyde, acetic acid, 4-allyl-2-methoxyphenol (eugenol), 2,3-butanedione, butanoic acid, ethyl butanoate, ethyl 2-hydroxyhexanoate, ethyl propanoate, ethyl octanoate, 4-ethyl-2-methoxyphenol, ethyl methylpropanoate, 4-ethylphenol, hexanoic acid, 4-hydroxy-2,5-dimethylfuran-3(2H)-one, 4-hydroxy3-methoxyacetophenone (acetovanillone), 4-hydroxy-3-methoxybenzaldehyde (vanillin), 2-methyl-1-butanal, 3-methyl-1-butanal, 2-methylbutanoic acid, 3-methylbutanoic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, methylpropanal, methylpropanol, methylpropanoic acid, 3-(methylthio)propanal, 2-phenylacetic acid, 2-phenylethanol, octanoic acid, and whiskylactone (Sigma-Aldrich, St. Louis, MO); 1,1-diethoxyethane, ethyl hexanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolon) (Fisher Scientific, Pittsburgh, PA); ethyl 2-hydroxy-4-methylpentanoate (TCI America, Portland, OR); and (E)-β-damascenone (Vigon, East Stroudsburg, PA). The isotopically labeled standards were obtained commercially from the sources given in parentheses: [2H3]-acetaldehyde, [2H5]-butanoic acid, [2H4]-1,1-diethoxyethane, [2H3]-ethyl butanoate, [2H11]-ethyl hexanoate, [2H15]-ethyl octanoate, [2H10]-4-ethylphenol, [2H4]-pentanoic acid, [2H2]-3-methylbutanoic acid, [2H2]-3-methyl-1-butanal, [2H4]-3methyl-1-butanol, [2H3]-methylpropanoic acid, [2H5]-2-phenylethanol, and [2H15]-octanoic acid (CDN Isotopes, Inc., Pointe Claire, Canada); [13C2]-acetic acid and [13C2]-2-phenylacetic acid (Isotech Stable Isotopes, Sigma-Aldrich, St. Louis, MO); [2H3]-2,3-pentanedione, [2H2]-ethyl methylpropanoate, [2H2]-methylpropanal, and [2H4]-(E)-β-damascenone (Adesis, Inc., New Castle, DE); and [2H3]-4-allyl-2-methoxyphenol, [2H5]-ethyl propanoate, [2H5]-ethyl (2S,3S)-2-hydroxy-3-methylpentanoate, [2H2]-ethyl 2-methylbutanoate, [13C2]-3-hydroxy-4,5-dimethylfuran-2(5H)-one, [13C2]-4-hydroxy-2,5-dimethylfuran-3(2H)-one, [2H3]-4-hydroxy-3-methoxybenzaldehyde, and [2H3]-3-(methylthio)propanal (AromaLab AG, Freising, Germany). [2H2]-cis-Whiskylactone was synthesized as previously reported.19 Anhydrous sodium carbonate, anhydrous sodium sulfate, and hydrochloric acid (37%) were obtained from Fisher Scientific (Pittsburgh, PA). Sodium chloride, sodium hydroxide, sodium nitrite, L-isoleucine, and concentrated sodium chloride (26%) were obtained from Sigma-Aldrich (St. Louis, MO). Diethyl ether (Acros, Fisher Scientific, Pittsburgh, PA) and n-pentane (Merck, Darmstadt, Germany) were freshly distilled prior to use. Dichloromethane was obtained from Fisher Scientific (Pittsburgh, PA). Silica 60 was purified B

DOI: 10.1021/acs.jafc.5b01418 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Selected Ions (m/z) of Analytes and Isotopically Labeled Standards Used in the SIDA odorant

ion (m/z)

labeled standard

ion (m/z)

Rf

acetaldehyde acetic acid 4-allyl-2-methoxyphenol 2,3-butanedione butanoic acid cis-whiskylactone 1,1-diethoxyethane ethyl butanoate ethyl hexanoate ethyl propanoate ethyl octanoate 4-ethyl-2-methoxyphenol ethyl (2S,3S)-2-hydroxy-3-methylpentanoate ethyl 2-hydroxy-4-methylpentanoate ethyl 2-methylbutanoate ethyl 3-methylbutanoate ethyl methylpropanoate 4-ethylphenol hexanoic acid 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolon) 4-hydroxy-2,5-dimethylfuran-3(2H)-one 4-hydroxy-3-methoxyacetophenone (acetovanillone) 4-hydroxy-3-methoxybenzaldehyde (vanillin) 2- and 3-methylbutanoic acids 2- and 3-methyl-1-butanals 2- and 3-methyl-1-butanols methylpropanal methylpropanol methylpropanoic acid 3-(methylthio)-propanal 2-phenylacetic acid 2-phenylethanol octanoic acid (E)-β-damascenone

43 60 164 86 73 157 73 116 99 102 88 152 104 87 102 88 116 107 60 128 128 151 151 60 71 70 70 43 88 104 136 122 60 190

[2H3]-acetaldehyde [13C2]-acetic acid [2H3]-4-allyl-2-methoxyphenol [2H3]-2,3-pentanedione [2H5]-butanoic acid [2H2]-cis-whiskylactone [2H4]-1,1-diethoxyethane [2H3]-ethyl butanoate [2H11]-ethyl hexanoate [2H5]-ethyl propanoate [2H15]-ethyl octanoate [2H2]-4-ethyl-2-methoxyphenol [2H5]-ethyl (2S,3S)-2-hydroxy-3-methylpentanoate [2H5]-ethyl (2S,3S)-2-hydroxy-3-methylpentanoate ethyl 3-methylbutanoate [2H2]-ethyl 2-methylbutanoate [2H2]-ethyl methylpropanoate [2H10]-4-ethylphenol [2H4]-pentanoic acid [13C2]-3-hydroxy-4,5-dimethylfuran-2(5H)-one [13C2]-4-hydroxy-2,5-dimethylfuran-3(2H)-one [2H3]-4-hydroxy-3-methoxybenzaldehyde [2H3]-4-hydroxy-3-methoxybenzaldehyde [2H2]-3-methylbutanoic acid [2H2]-3-methylbutanal [2H4]-3-methyl-1-butanol [2H2]-methylpropanal [2H4]-3-methyl-1-butanol [2H3]-methylpropanoic acid [2H3]-3-(methylthio)-propanal [13C2]-2-phenylacetic acid [2H5]-2-phenylethanol [2H15]-octanoic acid [2H4]-(E)-β-damascenone

46 62 167 103 75 159 77 119 110 107 91 154 109 77 88 59 118 113 62 130 130 154 154 62 73 74 74 74 91 107 138 127 63 194 + 195

0.70 1.09 0.93 1.24 1.00 0.59 1.33 1.16 1.07 1.10 1.14 1.00 1.10 0.96 0.99 1.03 1.06 1.01 0.98 0.96 1.10 0.82 1.03 1.05 0.78 0.99 1.09 0.94 1.02 0.91 1.03 1.04 0.94 0.89

GC−O. GC−O was performed using an Agilent gas chromatograph type 6890 (Agilent Technologies, Inc., Santa Clara, CA) and the following fused silica capillaries: J&W Scientific DB-FFAP and DB-5 (30 m × 0.32 mm inner diameter, 0.25 μm film thickness) (Folsom, CA). The sample (1 μL) was applied by cool on-column injection at 40 °C using helium as the carrier gas with a flow rate of 2.5 mL/min. The temperature programs were as follow: 40 °C for 2 min, 8 °C/min up to 80 °C, 5 °C/min up to 120 °C, 8 °C/min up to 230 °C, and 230 °C for 15 min (DB-FFAP) and 40 °C for 2 min, 8 °C/min up to 80 °C, 5 °C/min up to 120 °C, 8 °C/min up to 250 °C, and 250 °C for 15 min (DB-5). GC−O was performed with simultaneous flame ionization and olfactometric detection by evenly splitting the effluent using a Y-type glass splitter and two deactivated fused silica glass capillaries of the same length. Both FID and sniffing port temperature were at 220 °C. Linear retention indices (RIs) of the aroma compounds were calculated from the retention times of a series of n-alkanes (C5−C26) as described previously.26 Aroma Extraction Dilution Analysis (AEDA). AEDA was performed on the DB-FFAP column. The original NBF and AF were first sniffed by four panelists to ensure a consistency of detection and aroma language selection. Each fraction was then stepwise-diluted with diethyl ether (1:1, v/v) and subjected to GC−O. The flavor dilution (FD) factor of an odorant represented the last dilution in which it could be perceived. Each panelist was trained on a weekly basis by means of solutions of reference odorants in water using a common aroma language.23 Static Headspace−Olfactometry (SHO) and Aroma Dilution Analysis (ADA). GC of static headspace samples was carried out by

means of a Thermo Trace Ultra gas chromatograph in connection with a cryotrap system type 915 (Braunschweig, Germany), cooled with liquid nitrogen. An aliquot of the sherry wine (10 mL) was equilibrated in a septum-sealed vessel (120 mL) for 15 min at 30 °C. From different samples of the same volume, decreasing headspace volumes (10−0.1 mL) were withdrawn with a gastight syringe, injected by cold on-column injection, and subsequently trapped at −150 °C. The trap was then rapidly heated to 250 °C, and the sample was flushed onto a J&W Scientific DB-5 fused silica column (30 m × 0.25 mm inner diameter, 0.5 μm film thickness) (Folsom, CA). After injection, the temperature of the oven was held at 0 °C for 2 min, then raised at 6 °C/min to 100 °C, followed by 40 °C/min to 240 °C, and held at 240 °C for 2 min. At the end of the column, the effluent was split evenly between an FID and a sniffing port held at 250 and 220 °C, respectively. The flow rate of the carrier gas helium was 0.8 mL/min. The FD factors were calculated by dividing the largest volume analyzed to the lowest volume in which the odorant could be detected. Gas Chromatography/Mass Spectrometry (GC/MS). Dependent upon the concentration of the aroma compound and the presence of co-eluting compounds, several GC/MS systems were used for identification. The majority of the compounds was identified by means of a gas chromatograph type 6890 coupled to a MS type 5975 (Agilent Technologies, Inc., Santa Clara, CA) operated in the electron impact mode (MS−EI) at 70 eV. Separation was performed at a constant flow (average velocity of 37 cm/s), and the scan was between 35 and 350 amu with 4.45 scans/s. Trace analytes were identified using a twodimensional GC/MS instrument consisting of an Agilent GC1 and GC2 system type 7890A and 6890N, respectively (Santa Clara, CA). C

DOI: 10.1021/acs.jafc.5b01418 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry The column combination consisted of a BP-20 (SGE, Inc., Austin, TX) in the first dimension and a J&W Scientific DB-5 (Folsom, CA) in the second dimension (30 m × 0.32 mm inner diameter, 0.25 μm film thicknesses each). For the identification of low boiling substances, the system used for the SHO described above was coupled to a Varian Saturn 2100 ion trap mass spectrometer (Darmstadt, Germany), and mass spectra were generated by MS−EI at 70 eV. Separation was performed at a constant pressure with an average velocity of 45.103 cm/s. The MS was operated with a scan mode between 45 and 400 amu and a scan speed of 3.99 scans/s. Quantitation by a SIDA in Combination with GC/MS. Aliquots of the labeled standard in dichloromethane were added to different amounts of sherry wine (1−100 mL) depending upon the concentration of the target compound estimated in preliminary experiments. The samples were stirred for 30 min and then extracted with diethyl ether. Volatiles were isolated according to the method described above, and quantitation of the aroma compounds in the extracts was performed using both the two-dimensional GC/MS system as described above as well as a one-dimensional GC/MS system with an Agilent 6890 gas chromatograph and an Agilent 5973 mass spectrometer (Santa Clara, CA). Separation was performed using a J&W Scientific DB-FFAP column (30 m × 0.32 mm inner diameter, 0.25 μm film thickness) (Folsom, CA) using the following temperature program: 40 °C for 2 min, 5 °C/min up to 240 °C, and 240 °C for 5 min. The temperature of the injector (splitless mode) was at 180 °C, and the flow rate of the carrier gas helium was 1.5 mL/min. Details of the ions selected for the SIDA and the correction factors are given in Table 1. Separation was performed at a constant flow (average velocity of 44 cm/s), and the mass spectrometer was operated in the selected ion monitoring (SIM) mode with a dwell time of 100 ms. Static Headspace Analysis. The analysis was performed using the GC/MS system described above equipped with an unpolar Restek RT-Q-Bond capillary column (30 m × 0.32 mm inner diameter, 10 μm film thickness) (Bellefonte, PA). The sample (5 mL) was mixed with NaCl (1 g) in a septum-sealed vessel (20 mL), and the labeled standards dissolved in ethanol (17%, v/v) were added to the wine sample (5 mL). To quantitate acetaldehyde, the wine (0.1 mL) was diluted with water to reach a final volume of 5 mL and [2H3]acetaldehyde was added. The headspace was generated for 20 min at 40 °C with automatic agitation at 500 rpm, and 1 mL was injected in the splitless mode using a gastight syringe at 50 °C and an injector temperature of 80 °C. The temperature program was as follows: 40 °C for 0 min, 12 °C/min up to 220 °C, 20 °C/min up to 260 °C, and 260 °C for 5 min. The flow rate of the carrier gas helium was 2 mL/min. A calibration factor was determined for each compound by analyzing mixtures of defined amounts of labeled and unlabeled compounds in ratios of 1:3, 1:1, and 3:1, respectively (Table 1). All quantitative analyses were performed in MS−EI at 70 eV, except for cis-whiskylactone, which was quantitated in chemical ionization (CI) mode as described previously.19 Determination of Ethanol. Ethanol was enzymatically quantitated using a test kit (R-Biopharm, Darmstadt, Germany). Determination of Isomeric Distribution. 2- and 3-methylbutanoic acids were quantitated according to the procedure described by Frank et al.25 Ethyl 2-methylbutanoate and ethyl 3-methylbutanote were quantitated in two steps because of the availability of only one labeled standard (Table 1) and mass fragment interferences between the two esters and the labeled standard. First, ethyl 3-methylbutanoate was quantitated (Table 1). Then, the ion intensities of the mass fragments m/z 102 and 88 for ethyl 2-methylbutanoate and ethyl 3-methylbutanoate, respectively, were monitored by MS−EI, and finally, the concentration of ethyl 2-methylbutanoate was calculated using a calibration curve prepared by analyzing defined mixtures of both isomers. Aroma Profile Analysis. Panelists who participated in the study were all trained for at least 3 months on a weekly basis to recognize and describe the odor qualities of a wide range of odorants and products. In a first step, the wine was evaluated orthonasally by a small panel to generate a list of aroma attributes that were later on ranked on the basis of their frequency of citation as well as their intensity. The following nine aroma attributes were selected for the descriptive

Figure 1. Aroma profile analysis of Amontillado sherry wine.

Figure 2. FD chromatogram of (A) NBF and (B) AF isolated from Amontillado sherry wine (FD factors are given as a function of the linear retention indices). Numbering refers to Table 2. analysis: fruity, cooked apple, dark/dry fruit, honey, caramel, alcohollike, sour/pungent, smoky, and malty. Aqueous solutions of each of the following compounds (OAV = 100) were provided to the panelists as references during each session: ethyl 3-methylbutanoate (fruity), (E)-β-damascenone (cooked apple), 2-phenylethanol (flowery, honeylike), 4-hydroxy-2,5-dimethylfuran-3(2H)-one (caramel), ethanol (alcohol-like), acetic acid (sour/pungent), 2-methoxyphenol (smoky), and 3-methylbutanol (malty). For the dark/dry fruit descriptor, dry figs and raisins were provided. The wine (20 mL) was poured into a glass vessel (45 mL) and analyzed by ranking the intensity of each attribute on a seven-point scale (steps of 0.5) from 0 (not perceivable) to 3 (strongly perceivable). The aroma profile D

DOI: 10.1021/acs.jafc.5b01418 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. Most Odor-Active (FD ≥ 16) Volatile Constituents Identified in Amontillado Sherry Wine RIa b

compound number

odorant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

1,1-diethoxyethane 2- and 3-methylbutanals ethyl propanoate ethyl methylpropanoate 2,3-butanedione ethyl butanoate ethyl 2-methylbutanoate ethyl 3-methylbutanoate methylpropanol 2- and 3-methylbutanols ethyl hexanoate unknown ethyl octanoate acetic acid 3-(methylthio)-propanal ethyl (2S,3S)-2-hydroxy-3-methylpentanoate ethyl 2-hydroxy-4-methylpentanoate methylpropanoic acid butanoic acid 2- and 3-methylbutanoic acids (E)-β-damascenone hexanoic acid 2-methoxyphenol 2-phenylethanol cis-whiskylactone 4-ethyl-2-methoxyphenol octanoic acid p-cresol 4-allyl-2-methoxyphenol 4-ethylphenol 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolon) wine lactonef decanoic acid unknown phenylacetic acid 4-hydroxy-3-methoxybenzaldehyde (vanillin) 4-hydroxy-3-methoxyacetophenone (acetovanillone)

odor quality

c

green/fruity malty fruity/green fruity buttery fruity fruity fruity malty malty fruity fruity fruity sour cooked potato fruity fruity sweaty sweaty sweaty cooked apple sweaty sweet, burnt flowery/honey-like coconut clove-like fatty horse stable clove horse stable seasoning-like coconut fatty woody honey vanilla sweet/vanilla

FFAP

DB5

fractiond

FD factore