Article pubs.acs.org/JAFC
Distribution and Organoleptic Impact of Ethyl 3‑Hydroxybutanoate Enantiomers in Wine Georgia Lytra,†,‡ Margaux Cameleyre,†,‡ Sophie Tempere,†,‡ and Jean-Christophe Barbe*,†,‡ †
Univ. Bordeaux, ISVV, EA 4577, Unité de recherche Œnologie, 33882 Villenave d’Ornon, France INRA, ISVV, USC 1366 Œnologie, 33882 Villenave d’Ornon, France
‡
ABSTRACT: Enantiomers of ethyl 3-hydroxybutanoate were assayed in 87 commercial wines from various vintages and origins, using chiral gas chromatography (β-cyclodextrin). Generally, ethyl 3-hydroxybutanoate levels were higher in red than in white wines of the same age. The average S/R enantiomeric ratio of this compound in red wine was approximately 75:25 (±13), with an average total concentration of ∼450 (±150) μg/L. In red wines, R-form levels increased gradually during aging, but no variations were observed in S-form concentrations. To our knowledge, no previous research had determined the enantiomeric distribution of this compound in wine. The olfactory threshold of the S-form in dilute alcohol solution was 21 mg/L, one-third that of the R-form: 63 mg/L. The S- and R-forms had different aromatic nuances. The olfactory threshold of their mixture (85:15, m/m) was 14 mg/L, indicating a simple additive effect in this binary mixture. Furthermore, the concentrations found in red wines were considerably below the olfactory threshold under the same experimental conditions. Sensory analysis revealed that ethyl 3-hydroxybutanoate (S/R, 85:15, m/m) had an enhancing effect on the perception of fruity aromas in the matrices studied. Sensory profiles highlighted the contribution of ethyl 3-hydroxybutanoate to red-berry and fresh-fruit descriptors, despite its subthreshold concentrations. KEYWORDS: ethyl 3-hydroxybutanoate, enantiomers, chiral GC, red wine, fruity aroma, perceptive interactions, enhancing effect
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INTRODUCTION Ethyl 3-hydroxybutanoate (1) occurs widely in the aroma volatiles of fruits and beverages. In wine, 1 was found in highly variable concentrations, between 0.1 and 1 mg/L.1 Its relatively high olfactory threshold, about 3 mg/L in dilute alcohol solution,2 practically excludes any direct impact on wine aroma. However, it may participate in red wine fruity notes via perceptual interactions.2,3 1 has been identified in several fruits, particularly as a major volatile component of various tropical fruits such as wood apple (Feronia limonia),4 papaya,5 passion fruit,6 mango,7 naranjilla fruit,8 caja fruit (Spondias lutea L.),9 pineapple,10 Melón de olor,11 and champa (Campomanesia lineatifolia R. and P.),12 as well as fresh kiwi fruit puree.13 1 has also been identified in aromatic beverages, such as black tea,14 as well as in alcoholic beverages, such as apple cider15 and cherry wine.16 Its presence in several types of grape wine1 has been confirmed, including white Riesling17 and Chardonnay wines;18 red Tannat wine;19 Madeira wines;20 and Fino Sherry.21,22 Various organic extracts were analyzed by gas chromatography−olfactometry to characterize its odor. Several aromatic descriptions have been reported for 1 in the literature, such as “fresh, fruity”16,23 and “artificial strawberry, banana”,3 in wine studies, whereas, in others dealing with fruits, this compound has mainly been described as “sweet, aromatic, nutmeg”,4 “tarry”,8 “sweet-fatty”,11 and “fruity, herbal, green”.12 1 has one asymmetrical carbon atom, indicating the possible occurrence of two different enantiomers. Previous works investigated this enantiomeric distribution in tropical fruits. The (R)-enantiomer predominates in some of them, such as caja fruit (S/R = 46:54, m/m),9 as well as mango and purple © XXXX American Chemical Society
passion fruit (S/R = 22:78, m/m, and S/R = 31:69, m/m, respectively),24,25 whereas the (S)-configuration is prevalent in others, such as yellow passion fruit. To our knowledge, this enantiomeric distribution had never previously been investigated in wine. As the olfactory threshold and descriptors of an odoriferous compound may differ according to the stereoisomer considered,26−28 it was important to separate the two enantiomers to obtain an accurate assessment of their organoleptic impact. The goal of this work was to separate and assay ethyl (3S)-3hydroxybutanoate (1a) and ethyl (3R)-3-hydroxybutanoate (1b) in wines from various vintages and origins and evaluate their organoleptic impact on red wines.
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MATERIALS AND METHODS
Chemicals and Odorant Stimuli. Absolute ethanol (analytical grade, 99.97%) and sodium sulfate (99%) were provided by Scharlau Chemie S.A., Barcelona, Spain. Microfiltered water was obtained using a Milli-Q Plus water system (resistivity = 18.2 MΩ cm, Millipore, Saint-Quentin-en-Yvelines, France). Dichloromethane was provided by Carlo Erba (Pestipur quality, Carlo Erba, SDS, Italy). Standard grade compounds were obtained from commercial sources as follows: ethyl propanoate, ethyl 2-methylpropanoate, ethyl 3-methylbutanoate, ethyl butanoate, ethyl 2-methylbutanoate (racemic mixture 50:50, m/ m), ethyl hexanoate, ethyl octanoate, ethyl 3-hydroxybutanoate (racemic mixture 50:50, m/m) (1), ethyl (3S)-3-hydroxybutanoate (1a), ethyl (3R)-3-hydroxybutanoate (1b), 2-methylpropyl acetate, butyl acetate, and hexyl acetate from Sigma-Aldrich, Saint-QuentinReceived: September 4, 2015 Revised: November 11, 2015 Accepted: November 14, 2015
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DOI: 10.1021/acs.jafc.5b04332 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
splitless time, 30 s; split flow, 50 mL/min). The column was a BP20 (50 m × 0.22 mm i.d.; film thickness, 0.25 μm; SGE). The oven was programmed at 40 °C for the first minute and then increased at a rate of 10 °C/min to a final isotherm at 220 °C for 10 min. The carrier gas was hydrogen 5.5 (Linde, France) with a column head pressure of 15 psi. All compounds used in this work were olfactively pure, and any olfactory impurities were detected by the three judges who performed this analysis. Moreover, FID analysis confirmed the products’ very high purity. Quantitation and Separation of 1. Sample Preparation. A 100 mL wine sample was spiked with 100 μg/L octan-3-ol as an internal standard. It was then extracted using 8, 4, and 4 mL of dichloromethane, with magnetic stirring (700 rpm), for 5 min each and separated in a separating funnel for 5 min. The organic phases were blended, dried over sodium sulfate, and concentrated under nitrogen flow (100 mL/min) to obtain 250 μL of wine extract. Chromatographic Conditions. GC analyses were carried out on an HP 5890 GC system coupled to an HP 5972 quadrupole mass spectrometer (Hewlett-Packard), equipped with a Gerstel MPS2 autosampler. Injections were in split mode (split ratio, 30:1), using a 2 mm i.d. nondeactivated direct linear transfer (injector temperature, 200 °C; interface temperature, 280 °C). A Chiraldex B-DM column (30 m × 0.25 mm i.d.; film thickness, 0.12 μm; Astec, Whippany, NJ, USA) was used for quantitation and separation of the ester. The oven temperature was programmed at 40 °C for 1 min, then increased at a rate of 1 °C/min to 70 °C, and finally raised by 3 °C/min to a final isotherm at 200 °C, maintained for 5 min. The carrier gas was helium 5.5 (Linde, France) with a constant flow of 1 mL/min. The mass spectrometer was operated in electron impact mode at 70 eV with selected-ion monitoring (SIM), using six characteristic ions: m/z 87 as quantifier and m/z 88, 60, 117, 71, and 85 as qualifiers. 1a and 1b were characterized by comparing their linear retention indices and mass spectra with those of standards. For the internal standard three characteristic ions were selected: m/z 59 as quantifier and m/z 83 and 101 as qualifiers. Calibration curves were evaluated in dilute alcohol solution (12%, v/v), using a representative range of average ester concentrations found in wines (linear range, 1.1−1120 μg/L). These samples were then extracted under the conditions described above and then analyzed by GC-MS in SIM mode. The calibration curves were plotted as the relative peak areas (analyte versus internal standard) as a function of concentration. The functions were linear over the concentration range, presenting correlation coefficients of 0.9977 and 0.9987 for 1a and 1b, respectively. Repeatability was evaluated by relative standard deviation of 10 independent assays performed under the same analytical conditions over a short period of time. The relative standard deviation of the area ratios was
