Identification of a New Lactone Contributing to Overripe Orange

Article pubs.acs.org/JAFC

Identification of a New Lactone Contributing to Overripe Orange Aroma in Bordeaux Dessert Wines via Perceptual Interaction Phenomena Panagiotis Stamatopoulos,†,‡ Eric Frérot,§ Sophie Tempère,†,‡ Alexandre Pons,†,‡ and Philippe Darriet*,†,‡ †

Univ. Bordeaux, ISVV, EA 4577 Œnologie, F-33140 Villenave d’Ornon, France INRA, ISVV, USC 1366 Œnologie, F-33140 Villenave d’Ornon, France § R&D Division, Firmenich SA, 1 Route Des Jeunes, CH-1211 Genève 8, Switzerland ‡

S Supporting Information *

ABSTRACT: Recent studies have demonstrated the existence of a typical sensory concept for Bordeaux dessert wines, including the world famous wines of Sauternes. Volatile compounds from several chemical families (thiols, aldehydes, and lactones) were identified and correlated with aromatic typicality in these wines. However, these studies were unable to indicate “key” aromas of overripe fruits, especially overripe orange. The alternative strategy developed in this research combined both analytical and sensory studies of fractions of dessert wine extracts obtained by semipreparative high-performance liquid chromatography (HPLC). Multidimensional gas chromatography coupled to olfactometry and mass spectrometry (MDGC-O/MS) was applied to some of the HPLC fractions recalling “overripe fruit”, and a new lactone, 2-nonen-4-olide, was identified. Reconstitution and omission tests using the HPLC fractions highlighted the importance of specific compounds, particularly 2-nonen-4-olide, in the expression of overripe orange notes. Although this lactone presents minty and fruity odors, its key contribution to the typical aroma of orange in Bordeaux dessert wines was revealed through perceptual blending. KEYWORDS: 2-nonen-4-olide, HPLC fractionation, multidimensional gas chromatography−olfactometry−mass spectrometry, overripe orange aroma, Bordeaux dessert wines

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INTRODUCTION Bordeaux white dessert wines, including Sauternes wines, are world famous. They have an exceptional range of aromas, evoking citrus and dried fruit in young wines, orange peel in older wines, and honey or waxy notes in wines subjected to oxidative aging.1 These exceptional wines can be produced only under specific conditions in limited quantities. In the vineyard, the Botrytis cinerea fungus develops on perfectly ripe grapes, producing “noble rot”. Furthermore, Bordeaux dessert wines are the result of incomplete fermentation, leaving a certain proportion of grape sugar that has not been transformed into alcohol. The Sauternes-Barsac protected designation of origin is certainly one of the most highly esteemed areas of Bordeaux for noble rot sweet wines, but other Bordeaux dessert wines (Loupiac, Saint-Croix du Mont) or wines from other regions in France (Montbazillac, Anjou, and Alsace), Germany (Rheingau and Moselle), and Hungary (Tokaj) also have an excellent reputation. Wine consists of highly complex mixtures of hundreds of volatiles derived from grapes, fermentation processes, and aging. Even at trace concentrations, some of these compounds have powerful odors and have been demonstrated to contribute directly to wine aroma. They belong to various chemical families (thiols, pyrazines, sesquiterpenes, carbonyls, etc.) and are considered to have a key impact, as they are frequently detected at concentrations far above their olfactory perception thresholds in simple solution.2−8 The perception of these key compounds is also modulated by various phenomena, involving numerous volatile compounds that produce masking or additive © 2014 American Chemical Society

effects, and sometimes synthetic perception at the brain level through combinatorial phenomena.9−13 Various types of sensory interactions between different aromatic compounds in wine have thus been described.14−17 Recent studies have demonstrated the existence of a typical sensory concept for Bordeaux dessert wines. Volatile compounds from several different chemical families (thiols, aldehydes, and lactones) have been identified and correlated with the typicality of these wines.18,19 However, the compounds responsible for the key “overripe orange” aromas in Bordeaux dessert wines had not previously been identified. The goal of this study was to identify compounds involved in the typical ripe-fruit notes in these wines. Aside from gas chromatography−olfactometry (GC-O), an additional strategy was developed, combining both analytical and sensory studies of fractions of wine extracts, obtained by semipreparative highperformance liquid chromatography (HPLC). Multidimensional GC-O-MS was then used to identify the key compounds present in selected HPLC fractions. Reconstitution and omission tests were finally performed to identify and confirm the contribution of such compounds to the perceptual interaction phenomena responsible for the typical “overripe fruit, orange” character of Bordeaux dessert wines. Received: Revised: Accepted: Published: 2469

June 4, 2013 February 17, 2014 February 22, 2014 February 22, 2014 dx.doi.org/10.1021/jf405397c | J. Agric. Food Chem. 2014, 62, 2469−2478

Journal of Agricultural and Food Chemistry

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Article

Wine Extraction for HPLC Fractionation. Seven hundred and fifty milliliter wine samples were extracted at room temperature (20 °C) using 60, 60, and then 40 mL of dichloromethane with magnetic stirring (700 rpm) for 10 min each and separated in a funnel. The organic phases were collected, dried over sodium sulfate, concentrated to around 2 mL using a Buchi R-114 rotary evaporator (Buchi, Rungis, France), and then further concentrated under nitrogen flow (100 mL/ min) in a graduated glass tube (Atelier Jean Prémont, Bordeaux) to obtain a 750 μL wine extract. Semipreparative HPLC on Wine Extracts and Sensory Evaluation. Reversed-phase (RP) HPLC was performed on the raw wine extract, using a Novapak C18 column (300 × 7.8 mm internal diameter (i.d.), 6 μm, Waters, Saint Quentin, France) with a guard column of the same phase. The Ultimate 3000 semipreparative HPLC system was from Dionex (Courtaboeuf, France). The procedure was based on the method described by Ferreira et al.,24 later adapted by Pineau et al.16 Chromatographic conditions were as follows: flow rate, 1 mL/min; injection volume. 250 μL; gradient, eluent A, microfiltered water, eluent B, ethanol; 0−2 min, 0% B, 2−50 min, 0−100% B. Fifty effluent fractions of 1 mL were collected. Each fraction was diluted into 12% ethanol, then poured into normalized glasses from the Association Française de Normes (AFNOR), and evaluated by three experienced assessors aged 38.6 ± 9.8 [mean ± standard deviation (SD)] years old. The repeatability of the quantitation measurements for specific concentrations (65 μg/L for 3-methyl-4-octanolide, 2.5 μg/ L for γ-nonalactone, 2.4 μg/L for eugenol, and 2.0 μg/L for 2-nonen4-olide) of the considered compounds was calculated after HPLC fractionation of the same wine (i.e., 750 mL), initially extracted with dichloromethane, for 3-methyl-4-octanolide at 6%, for eugenol at 2.6%, for γ-nonalactone at 6%, and finally for 2-nonen-4-olide at 2.6%. Extracting HPLC Fractions. Each fraction of interest (1 mL) was diluted with ultrapure water (Milli-Q, Millipore, Bedford, MA, USA) to obtain 12% ethanol (v/v) and then re-extracted three times with 1 mL of dichloromethane each time to mimic the wine extraction. The organic phases were combined and concentrated to 250 μL under nitrogen flow before GC-O analysis and heart-cut multidimensional GC-O-MS. GC-O. GC-O analyses were carried out using a Hewlett-Packard 5890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and a sniffing port (ODO-1 from SGE, Ringwood, Australia). A 1 μL aliquot of concentrated wine extract was injected by a splitless injector (230 °C; purge time, 1 min; purge flow, 50 mL/min) at an oven of temperature of 45 °C into a type-HP5 capillary column (Agilent; 30 m, 0.25 mm i.d., 0.25 μm film thickness) or a type BP20 capillary column (SGE; 50 m, 0.22 mm i.d. 0.25 μm film thickness). The analysis temperature program was as follows: 45 °C for 1 min, then 3 °C/min to 230 °C, with a 20 min isotherm for both types of columns. The carrier gas was hydrogen (Air Liquide, Floirac, France) with a flow rate of 1 mL/min. Each sample was analyzed in triplicate for 30 min each time and was assessed by three well-experienced assessors. Linear retention indices (LRI) were obtained by simultaneous injection of samples and a series of alkanes (C7−C23). Heart-Cut Multidimensional Gas Chromatography−Olfactometry−Mass Spectrometry (MDGC-O-MS). All analyses were performed using a multidimensional gas chromatograph consisting of two independent gas chromatographs (Agilent 6890), interconnected by means of a thermoregulated transfer line kept at 230 °C (West 4400, West Instruments, Gurnee, IL, USA). For chromatograph 1 (GC-1), the 6890 chromatograph (Agilent) was equipped with an FID and an olfactometric port (ODP-3, Gerstel, Mülheim an der Ruhr, Germany), both connected by a flow-splitter to the column exit for simultaneous effluent monitoring. This GC was retrofitted with a pressure-driven switching valve MCS (Multi Column Switching system, Gerstel) to transfer selected heart cuts eluted from the first column directly into the analytical column in the second chromatograph. The carrier gas helium N 60 (Air Liquide) was delivered at a constant flow of 1 mL/min. The pressure ramp program for constant flow was 224 kPa for 1 min, then increased by 1.4 kPa/min to 310 kPa, and maintained at this pressure for 42 min. The column was a 30 m ×

MATERIALS AND METHODS

Chemicals and Reference Compounds. Dichloromethane (99.99%) was supplied by Fischer Scientific (Illkirch, France) and absolute ethanol (99.9%) by Merck (Semoy, France). All of the reference compounds were provided by Sigma-Aldrich (SaintQuentin-Fallavier, France) [3-methyl-4-octanolide (98%), γ-nonalactone (98%), and eugenol (99%)]. Synthesis of 2-Nonen-4-olide. The lactone was prepared by the reaction of a stabilized vinyl Grignard reagent (isopropyl magnesium chloride, iPrMgCl)20 on hexanal, as described.21 A flame-dried 500 mL round-bottom flask, equipped with a Teflon-coated magnetic stirring bar, was loaded with 3-iodobut-2-enoic acid ethyl ester22 (6.9 g, 32.7 mmol) under an argon atmosphere. Tetrahydrofuran (THF; 130 mL) was added and the mixture cooled to −78 °C. iPrMgCl solution (16.4 mL, 2 M in Et2O) was then added dropwise. The mixture was stirred for an additional 2 h at −78 °C. This solution was added through a double-ended needle to a stirred solution of freshly distilled n-hexanal (1 equiv) in THF (130 mL) under argon at −78 °C. The reaction was allowed to warm slowly to 0 °C, then quenched with saturated aqueous ammonium chloride, and left to warm to room temperature. Next the reaction mixture was extracted three times with diethyl ether (Et2O). The combined organic layers were washed with water followed by brine, dried over sodium sulfate (Na2SO4), filtered, and concentrated. After purification by flash chromatography over silica gel, using a PuriFlash SI Std IR-50SI (50 μm) cartridge from Interchim (Montluçon, France), 2.6 g (23%) of 2-nonen-4-olide was obtained (purity > 99%). The product exhibited the same spectral data as described by Dai:23 13C NMR δ 13.5 (C-5′), 22.0 (C-4′), 24.2 (C-2′), 31.0 (C-3′), 32.7 (C-1′), 83.1 (C (4), 120.9 (C-2), 156.2 (C-3), 172.9 (C-1); GC-MS (EI) m/z 154 (4, M+), 125 (72), 84 (100), 83 (19), 55 (15). Wine Samples. All wines selected are listed in Table 1. These wines were used for various analyses, including semipreparative HPLC and GC-O/MS.

Table 1. All Wines Used in Different Essays through This Experiment, Including Vintage, Producer, Codes, and Appellation vintage

wine

code

appellation

2001 2001 2002 2003 2004 2005 2005 2005 2005 2006 2007 2009 2010 2006 2007 2008 2010 2006 2007 2007 2007 2008 2008 2008 2008

Château Lamothe Château Malfourat Château Latour Blanche Château Laville Castelnau de Suduiraut Château Latour Blanche Château Laville Château de Malle Chateau Suduiraut Castelnau de Suduiraut Château Laville Château d’Yquem Château d’Yquem Dauphiné Rondillon Dauphiné Rondillon Dauphiné Rondillon Pavillon de Rouquette Jean Moreau et fils Domaine Laroche Billaud-Simon Château Bonnet Château Moutin Château Thieuley Château Bonnet Domaine Albert Mann

Liq-La Liq-Mal Liq-Tb 2002 Liq-Jcb 2003 Liq-Cds 2004 Liq-Tb 2005 Liq-Jcb 2005 Liq-Ml Liq-Cs 2005 Liq-Cds 2006 Liq-Jcb 2007 Liq-Sg 2009 Liq-Sg 2010 Liq-Phd 2006 Liq-Phd 2007 Liq-Phd 2008 Liq-Phd 2010 Sec-Jmf Sec-Lar Sec-Bs Sec-Bon 2007 Sec-Cm 2008 Sec-Tey Sec-Bon 2008 Sec-Ann

Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Loupiac Loupiac Loupiac Loupiac Chablis Chablis Chablis Bordeaux Graves Bordeaux Entre Deux Mers Alsace 2470

dx.doi.org/10.1021/jf405397c | J. Agric. Food Chem. 2014, 62, 2469−2478


Article

0.32 mm i.d. × 0.5 μm film HP5 (Agilent). An uncoated, deactivated, fused-silica column (40 cm × 0.05 mm i.d.) from Supelco (Bellefonte, PA, USA) was used as an interface between the switching valve MCS to a FID or an olfactometric port. The oven temperature program was as follows: 45 °C for 1 min, then increasing by 5 °C/min to 230 °C, and maintained at 230 °C for 43 min. The injector was maintained at 250 °C (total run time = 83.67 min). A cryotrapping unit (liquid N2) was mounted between the two GCs for cryofocusing (−50 °C) of the heart-cut fraction at the beginning of the second column. The gas chromatograph 2 (GC-2, Agilent 6890) was coupled to an Agilent HP 5973 mass spectrometer (Agilent). Also, an olfactometric port (ODP3, Gerstel) was set up so that the 2D column exit was split between the MS and ODP3 for simultaneous mass spectrometry and sniffing detection (division 1:1). Two types of columns were used alternately in GC-2 with different polarities. The first column was a 50 m × 0.22 mm i.d. × 0.25 μm [BP20 (SGE)] and the second one a 50 m × 0.22 mm i.d. × 1 μm [BP1 (SGE)]. In GC-2, the column (BP20 or BP1) was connected to the MCS switching valve (situated in the first chromatograph) and routed via a transfer line thermostatically controlled at 230 °C. Two minutes after the heart cut, the oven temperature program (45 °C initially, then 3 °C/min increase to 230 °C) of GC-2 was activated. The pressure ramp program was 224 kPa at midpoint for the first 25 min, then increased at a rate of 1.4 kPa/min to 310 kPa, and maintained at that level for 27 min (total run time = 83.67 min). GC-O port parameters were as follows: GC effluent was combined with humidified air [reconstituted air (nitrogen 80, oxygen 20; v/v), Air Liquide] at a rate of 35 mL/min at the bottom of the glass mask (SGE) to avoid nasal dehydration. The MS parameters were as follows: transfer line at 250 °C; source at 230 °C, and quadrupole at 150 °C, in EI mode at 70 eV. The MS was used in full scan mode (m/z 40−300). MS data were recorded and processed using Chemstation software (version B04.03) from Agilent equipped with an NIST 2008 MS library (U.S. National Institute of Standards and Technology, Gaithersburg, MD, USA). For chemical ionization (CI) with methanol, a similar MDGC configuration with two Agilent 7890A GCs was coupled with a time of flight (TOF) mass spectrometer by JEOL (JEOL Europe, Croissy sur Seine, France). Quantitation of Higher Aliphatic Lactones and Eugenol by GC-MS. The extraction protocol for quantifying aliphatic lactones and eugenol in wine or in selected HPLC fractions was applied as described by Ferreira et al.25 Briefly, after solid phase extraction chromatography, the eluent was supplemented with external standard (100 μL of 2-octanol at 10 mg/L in CH2Cl2) and concentrated under nitrogen flow (approximately 100 mL/min) to 100 μL. Each extraction was repeated twice. Then, a 2 μL sample of each concentrated wine extract was injected on an Agilent 6890 gas chromatograph in splitless injector mode (injector temperature, 230 °C; purge time, 1 min; purge flow, 50 mL/min) with a Carbowax 20 M type capillary column [BP20, 50 m, 0.25 mm i.d., 0.22 μm film thickness (SGE)]. The temperature program for all analyses was as follows: 45 °C for 1 min, increased by 3 °C/min to 230 °C, followed by a 20 min isotherm. The carrier gas was helium N 60 (Air Liquide) with a constant flow rate of 1 mL/min. The detector was an Agilent HP 5973 mass spectrometer functioning in electron impact (EI) mode (70 eV) and connected to the GC with a heated transfer line at 230 °C (Agilent). The compounds were quantified using selected ion monitoring mode (SIM) on MSD Chemstation software (version B04.03) from Agilent. The external standard, 2-octanol, was detected with m/z 84 and 97 ions and quantified using m/z 84. 3-Methyl-4-octanolide was detected with m/z 99 and 42 ions and quantified with the m/z 99 ion. γNonalactone was detected using the ions m/z 85 and 56 and quantified with the ion m/z 85, and eugenol was detected with ions m/ z 164 and 149 and quantified with the ion m/z 164. The repeatability of the quantitation measurements was calculated by the extraction and injection of eight spiked samples (50 mL) with known concentrations of the compounds (100 μg/L for 3-methyl-4-octanolide, 25 μg/L for eugenol and γ-nonalactone). The repeatability values were for 3methyl-4-octanolide, 5%; eugenol, 2.5%; and γ-nonalactone, 2.3%. Quantitation of 2-Nonen-4-olide by GC-MS. The extraction protocol for quantifying 2-nonen-4-olide in wine or in selected

fractions was similar to that used for 3-methyl-4-octanolide, eugenol, and γ-nonalactone. All quantitative analyses were carried out using an Agilent 6890 GC. One microliter of each concentrated extract (extraction method described by Ferreira et al.25) was injected in splitless mode (230 °C; purge time, 1 min; purge flow, 50 mL/min) onto a DB1 capillary column (Agilent, 30 m, 0.25 mm i.d., 0.25 μm film thickness). The temperature program was as follows: 45 °C for 1 min, increasing by 3 °C/min to 130 °C, and increasing again at 40 °C/ min until 280 °C, followed by a 10 min isotherm. The carrier gas was helium N 60 (Air Liquide) with a constant flow rate of 1.5 mL/min. The detector was an Agilent HP 5973 mass spectrometer, functioning in EI mode (70 eV), with a transfer line heated to 280 °C. 2-Nonen-4olide was detected in SIM mode by selecting the m/z 84, and 125 ions and quantified using m/z 84. The external standard, 2-octanol, was detected with m/z 84 and 97 ions and quantified using m/z 84. Measurements were linear in a range of concentrations from 0.5 to 20 μg/L [2-nonen-4-olide, μg/L) = 3.5906x − 0.0323 (x = ratio of peak area of 2-nonen-4-olide over the peak area of the external standard); R2 = 0.9975]. Repeatability of the measurement of 2-nonen-4-olide for a series of eight extractions and injection of the same wine was assessed at 1.4%. The quantitation limit (0.5 μg/L) was calculated as the minimum concentration that generated a peak signal 10-fold higher than the background noise signal. Recovery was calculated at >80%. General Conditions for Sensory Analyses. Sensory analyses were performed as described by Martin and de Revel.26 Samples were evaluated in individual booths, at controlled room temperature (19 ± 1 °C), using covered black Association Française des Normes (AFNOR) glasses27 containing 50 mL of liquid, coded with random three-digit numbers. Sessions lasted approximately 10 min. Sensory Panel. The panel consisted of 15 judges, 5 males and 10 females, aged 30.5 ± 4.6 years (mean ± SD). All panelists belong to the enology research laboratory staff at ISVV, Bordeaux University. The judges were selected for their experience in assessing fruity aromas of dessert wines. Olfactory Thresholds. All analyses were performed in a model solution consisting of twice-distilled ethanol (12%, v/v) and tartaric acid (5 g/L) at pH 3.5 (adjusted using NaOH). For all sensory analyses, the glasses were labeled with three-digit random codes and presented to panelists in random order. All experiments were performed in duplicate. The detection threshold of the aroma compound was determined in a model solution with an ascending procedure (0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg/L), using the three-alternative forced-choice presentation method (3-AFC) (ISO 13301 2001).27 For each concentration, subjects received a set of three glasses; two of them were blank samples (model solution), and one contained the odorant at various concentrations (positive sample). Sensory analyses covered the whole series of dilution sets: each assessor was asked to first sniff each of the three glasses in the prescribed order and then choose the spiked sample. The detection threshold was defined as the concentration at which the probability of detection was 50%. This statistical value was determined according to an adaptation of the ASTM-E1432 method.28 The concentration/response function fitted by a sigmoid curve [y = 1/ (1 + e(−λx))] was designated by a psychometric function reflecting behavior during detection. The detection probability was corrected by the chance factor (one-third for 3-AFC): (3p − 1)/2. The software used for graphic resolution and nonlinear regression by ANOVA transform was Sigma Plot version 8 (SYSTAT, San Jose, CA, USA). Reconstitution and Omission Experiments. For the reconstitution and omission tests, two different types of dessert wines were selected. One was a typical Bordeaux dessert wine (Bordeaux dessert wine 1), with a desirable odor, reminiscent of overripe fruit, particularly orange peel, whereas the other was a nontypical Bordeaux dessert wine (Bordeaux dessert wine 2), with a fresh fruity aroma. Orthonasal typicality was measured by the panel on a nonstructured scale, which was anchored by “bad example” and “good example” on the left and the right ends, respectively. The panel had to respond to the question if a presented sample was considered to be a good or a 2471



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Table 2. Concentrations (Micrograms per Liter) of Four Compounds (2-Nonen-4-olide, trans-3-Methyl-4-octanolide, γNonalactone, and Eugenol) in Fractions 37 and 38 of Two Different Bordeaux Dessert Wines (Mean ± SD; n = 2)a HPLC fraction

a

trans-3-methyl-4-octanolide

2-nonen-4-olide

F37 F38

10.7 ± 0.9 tr

F37 F38

nd nd

cis-3-methyl-4-octanolide

γ-nonalactone

eugenol

tr 2.9 ± 0.1

9.1 ± 0.1 tr

tr 0.6 ± 0.2

0.2 ± 0.09 tr

Bordeaux Dessert Wine 1 81.1 ± 0.2 95.1 ± 0.3 1.8 ± 0.1 tr Bordeaux Dessert Wine 2 tr 0.2 ± 0.1 tr tr

nd, not detected; tr, traces.

Table 3. Composition of Different Samples through the Reconstitution and Omission Analysesa HPLC fractions

compounds

sample

1−36

37 + 38

39−50

3-methyl-4-octanolide

eugenol

2-nonen-4-olide

γ-nonalactone

TAR 37 + 38 PAR PAR4C PAR4C-WL PAR4C-E PAR4C-N PAR4C-γN

+ − + + + + + +

+ + − − − − − −

+ − + + + + + +

− − − + − + + +

− − − + + − + +

− − − + + + − +

− − − + + + + −

a

TAR, total aromatic reconstitution; 37 + 38, aromatic reconstitution for fractions 37 and 38; PAR, partial aromatic reconstitution; PAR4C, sample PAR plus the four compounds; +, addition; −, omission; PAR4C-W, sample PAR4C with 3-methyl-4-octanolide omission; PAR4CE, sample PAR4C with eugenol omission; PAR4C-N, sample PAR4C with 2-nonen-4-olide omission; PAR4C-γN, sample PAR4C with γ-nonalactone omission.

Table 4. Sensory Evaluation of HPLC Fractions from Various Bordeaux Dessert Wines and Dry White Wines dessert wines HPLC fraction

dry white wines

Liq-Cs 2005

Liq-Cds 2004

Liq-Cds 2006

Liq-Phd 2010

35 36 37 38 39 40 41

solvent moldy ripe orange ripe orange/moldy mushroom cherry banana

fruity odorless ripe orange ripe orange apricot odorless banana

floral citrus ripe orange ripe orange moldy spicy banana

floral citrus citrus fresh fruit apricot spicy banana

citrus thiols citrus floral green solvent banana

citrus odorless odorless fruity cherry cherry solvent

typicality (/10)

7.2 ± 0.8

5.6 ± 1.5

6.4 ± 1.4

0.9 ± 0.3

0.4 ± 0.2

0.6 ± 0.3

bad example for a wine of the category Bordeaux dessert wine, and no further information was asked regarding a sensory profile.29 After fractionation of each extract by reversed-phase HPLC, the abovementioned volatile compounds were quantified by GC-MS in selected fractions (Table 2) (experimental protocol in quantitation of higher aliphatic lactone and eugenol, 2-nonen-4-olide). Several reconstitution tests were then performed on the basis of the quantitation results. For each sensory session, samples of aromatic reconstitutions in hydroalcoholic solution (12% vol, pH 3.9) were presented. Names and compositions of the samples are presented in Table 3. The fractions of those Bordeaux dessert wines were pooled to form total aromatic reconstitution (TAR) samples, corresponding to the 50 fractions gathered together. Partial aromatic reconstitution (PAR) samples corresponded to the 50 fractions except fractions 37 + 38, PAR supplemented with the four following volatile compounds, 3methyl-4-octanolide, eugenol, γ-nonalactone, and 2-nonen-4-olide, at concentrations assayed in specific Bordeaux dessert wine (PAR4C samples), and PAR with three of the four selected volatile compounds. For each type of dessert wine, a specific sensory session was organized, and each session was performed in duplicate. In practical terms, during a first sensory session, the fractions corresponded to those of Bordeaux dessert wine 1, and the concentrations of the compounds added were related to those assayed in that wine. In a

Sec-Cm 2008

Sec-Bon 2007

second sensory session, the fractions corresponded to those of Bordeaux dessert wine 2, and the concentrations of the compounds added were related to those assayed in that wine. In a third session, HPLC fractions corresponding to Bordeaux dessert wine 2 without fractions 37 + 38 were supplemented with the volatile compounds corresponding to Bordeaux dessert wine 1. Each sensory session was performed in duplicate on different days. The overripe orange aroma intensity was evaluated in a direct orthonasal way on a 0−10 point nonstructured scale. The scale used was 0 = no odor perceived and 10 = high intensity perceived. Concerning typicality experiments, the scale was 0 = bad example of Bordeaux dessert wine and 10 = good example of Bordeaux dessert wine. For aromatic reconstitutions, fractions were retained and added individually or blended together to reproduce the initial concentrations in the original wines, adding double-distilled ethanol and microfiltered water to obtain an ethanol level of 12% (v/v). Statistical data were analyzed using R (R Foundation for Statistical Computing, Vienna, Austria, 2011) version 2.15.1 software. Analysis of variance (ANOVA) was performed, the homogeneity of variance was tested with Levene’s test, and the normality of residuals was tested using the Shapiro−Wilk test. Statistical significance was set at 5% (p < 0.05). 2472



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RESULTS AND DISCUSSION Characterization of Compounds Associated with Key Aromatic Fractions Reminiscent of Overripe Orange

possible to pick out the key odoriferous zones with overripe fruit and orange aromas. However, on HP5, a typical orange aroma lasting 1 min was perceived in a chromatographic zone situated between 1414 and 1443 LRI (Table 5). At this point, MDGC-O-MS was required to analyze this complex matrix and identify the compound(s) associated with this aroma. Identification and Quantitation of 2-Nonen-4-olide. Using MDGC technology, the odoriferous zone was targeted for its olfactory characteristics on the GC1 olfactometry port. Then, an adequate 3 min cut was performed. The separation of the extract on the analytical column (GC2, BP20 capillary), coupled with both the olfactometry port and the mass spectrometry detector, revealed several odoriferous zones potentially related to the overripe orange odor. Odoriferous zones with coconut, spicy clove, and ripe-fruit aromas corresponding to 3-methyl-4-octanolide, eugenol, and γ-nonalactone, respectively, were identified by mass spectrometry. A fourth odoriferous zone with minty and fruity aromas, corresponding to an unknown compound, was also detected. Coupling the capillary to the MS detector produced a chromatographic peak (Figure 1) with a clear mass spectrum at the retention time of this odoriferous zone, (LRI 2068, BP20 capillary, GC-2). The existence of a similar odoriferous zone associated with the same fragmentation pattern was confirmed by repeating MDGC analysis of fraction 37 extract on a nonpolar capillary BP1 (LRI 1368, GC-2). On the basis of the MS data obtained by EI and CI) which indicated a molecular mass of 154.099 ± 0.05 Da, and the associated chromatographic peak, the unknown odoriferous zone was identified as a lactone: 2-nonen-4-olide or 5-pentyl-5H-furan-2-one (CAS Registry No. 21963-26-8). The mass spectra of the 2-nonen4-olide standard and the wine extract presented the same characteristics (Figure 2). To our knowledge, this was the first time that this compound had been identified in wine. Odor Characteristics of 2-Nonen-4-olide and Assay in Wines. The perception threshold of (±)-2-nonen-4-olide was calculated at 4.3 μg/L in model solution and at 10.8 μg/L in a dessert wine (Figure 3). The 2-nonen-4-olide molecule has one asymmetrical carbon, indicating the possibility of various enantiomeric ratios between R and S forms. The perception thresholds and descriptors may change, depending on the ratio of these two isomers, as is the case for other compounds.30−32 2-Nonen-4-olide was then quantified in 15 dessert wines from

Table 5. Distribution of Odoriferous Zones Found in Fraction 37 of a Dessert Wine Extract Analyzed by GC-O (BP20 and HP-5 Columns) BP20 column LRI

time

odoriferous zone

1712 1724 1754 1832 1912

34.6 35 37.3 39.1 44.3

2007 2225 2383

45.3 51.9 52.3

HP5 column LRI

time

citrus vanilla warm citrus coconut

1204 1339 1346 1360 1414−1443

28.1 31.4 34.7 35.3 38.3−38.9

fruity caramel burned sugar

1502 1690

41.4 48.8

odoriferous zone plastic fruity floral citrus overripe orange plastic spicy

Aromas. Semipreparative HPLC of wine extracts resulted in fractions containing increasing proportions of ethanol in water. It was thus possible to describe their aromatic characteristics without toxic or malodorous solvent. Selected dessert and dry white wine extracts were analyzed by HPLC to constitute fractions with different aromatic descriptors. The comparative sensory evaluation of the fractions revealed clear sensory differences between dessert wine and dry white wine extracts, particularly in the ripe fruit character, but also revealed sensory differences for the typicality appreciation (Table 4). Among others, fractions 37 and 38 in some dessert wine extracts (LiqCs 2005, Liq-Cds 2004, and Liq-Cds 2006) were clearly reminiscent of overripe fruits and oranges, which were not detected in the other fractions (Liq-Phd 2010) or in dry white wine extracts (Sec-Cm 2008 and Sec-Bon 2007). To characterize the compounds associated with this aroma, fraction 37, the most significant from a sensory point of view, was re-extracted with dichloromethane, and the concentrated extract was injected on both a polar (BP20) and a nonpolar capillary (HP5) to identify odoriferous zones recalling the abovementioned descriptors. With the BP20 type capillary, it was not

Figure 1. MDGC separation chromatogram from fraction 37 of a dessert wine on a BP1 nonpolar capillary (analysis on GC-2) indicating the peak corresponding to the odoriferous zone. 2473



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Figure 2. Chemical formula and mass spectra of 2-nonen-4-olide in wine (A) and as pure compound (B).

Reconstitution and Omission Experiments. These experiments were conducted using two Bordeaux dessert wines presenting different levels of typicality, that is, 6.4 ± 1.4 for dessert wine 1 and 0.9 ± 0.3 for dessert wine 2 (see Materials and Methods). After fractionation of these wine extracts by HPLC, the four above-mentioned specific compounds (3-methyl-4-octanolide, eugenol, γ-nonalactone, and 2-nonen-4-olide) were assayed in HPLC fractions 37 and 38 of these two different wines (Table 2). Then, recombination and omissions tests were organized using selected HPLC fractions, and their sensory impact on the expression of the overripe fruit was studied. In particular, experiments on total aromatic reconstitution, partial aromatic reconstitution without HPLC fractions 37 + 38, or partial aromatic reconstitution

the Bordeaux region and 8 dry white wines from various appellations in France, including Chablis and Alsace. 2-Nonen4-olide was not detected in dry white wines, but only in dessert wines obtained from botrytized grapes, thus indicating that this compound is perhaps specific to noble rot dessert wines. Its concentration in dessert wines apparently depends on the vintage, ranging from 2.6 to 18.2 μg/L, that is, values frequently over the olfactory detection threshold in model solution (Table 6). The absence of a specific odoriferous zone related to this overripe orange note suggested that it resulted from perceptual interactions among the above-mentioned volatile compounds (3-methyl-4-octanolide, eugenol, γ-nonalactone, and 2-nonen4-olide), so relevant sensory analysis protocols were conducted. 2474



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Figure 3. Detection threshold for 2-nonen-4-olide in model solution and in a dessert wine.

comparison between TAR and PAR supplemented with the four compounds together (PAR4C) at concentrations assayed in Bordeaux dessert wine 1 did not reveal, within two repeated sensory sessions, any significant difference in intensity of the orange descriptor (sensory sessions 1 and 1*, Table 7). The mean values for orange aroma perception in TAR sample were 6.7 ± 0.5 and 6.8 ± 0.9 with repetition, whereas PAR4C presented similar values at 6.2 ± 0.9 and 6.2 ± 0.7 with the repetition, indicating that adding all four compounds produced a sensory appreciation very close to the sample TAR. Thus, clear sensory differences were observed when PAR fractions were supplemented with only one of four selected compounds or with the four compounds together. The 37 + 38 sample presented always the highest values of 7.4 ± 0.4 and 7.5 ± 0.6 for the repetition. Then, sensory tests were conducted to study the impact of each compound omission, with Bordeaux dessert 1 wine, on the overripe aroma register. The mean values for the 37 + 38 sample were 7.6 ± 0.6 and 7.5 ± 0.7 with the repetition, whereas those of TAR were 6.7 ± 0.6 and 6.9 ± 0.6 with the repetition (sensory sessions 1 and 1*, Table 8). Under these conditions, although the mean values for PAR were 3.0 ± 0.6 and 3.3 ± 0.7, the orange aroma was always perceived as much more intense when the sample was supplemented with three of four compounds in PAR fractions. With regard to the omission of the compounds, only sample PAR4C-γN (omitting γ-nonalactone) was not significantly different from the total reconstitution. This contributes to demonstrating the direct effect of 2-nonen-4-olide, eugenol, and 3-methyl-4-octanolide on the overripe orange aroma of Bordeaux dessert wines, whereas γ-nonalactone had only a minor impact on the blend at the concentrations considered. In fact, the association of 2nonen-4-olide with oak compounds is probably responsible for the new aromatic perception, which may be associated with perceptual blending. When the sensory reconstitution and omission tests with Bordeaux dessert wine 2, presenting a lower typicality, on the expression of overripe orange aroma were considered, the results were much less significant. When the PAR sample was supplemented with the four above-mentioned compounds at

Table 6. 2-Nonen-4-olide Concentrations in Various Wines (Dessert Wines and Dry White Wines; Mean ± SD; n = 2) vintage

wine

appellation

2001 2001 2002 2003 2005 2005 2005 2006 2007 2009 2010 2006 2007 2008 2010 2006 2007 2007 2007 2008 2008 2008 2008

Liq-La Liq-Mal Liq-Tb 2002 Liq-Jcb 2003 Liq-Tb 2005 Liq-Jcbc 2005 Liq-Ml Liq-Cds 2006 Liq-Jcb 2007 Liq-Sg 2009 Liq-Sg 2010 Liq-Phd 2006 Liq-Phd 2007 Liq-Phd 2008 Liq-Phd 2010 Sec-Jmf Sec-Lar Sec-Bs Sec-Bon 2007 Sec-Tey Sec-Bon 2008 Sec-Cm Sec-Ann

Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Sauternes Loupiac Loupiac Loupiac Loupiac Chablis Chablis Chablis Entre Deux Mers Bordeaux Entre Deux Mers Graves Alsace

a

concentrationsa (μg/L) 3.7 2.6 18.2 3.6 5.5 6.5 7.7 11.6 7.7 4.1 5.0 5.2 4.0 4.5 2.0

Identification of a New Lactone Contributing to Overripe Orange

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