Enantiomeric Distribution of Ethyl 2-Hydroxy-4 ... - ACS Publications

Chapter 6

Downloaded by RMIT UNIV on March 11, 2016 | http://pubs.acs.org Publication Date (Web): June 15, 2015 | doi: 10.1021/bk-2015-1191.ch006

Enantiomeric Distribution of Ethyl 2-Hydroxy-4-methylpentanoate in Wine, A Natural Enhancer of Fruity Aroma Georgia Lytra,1,2 Sophie Tempere,1,2 Gilles de Revel,1,2 and Jean-Christophe Barbe*,1,2 1Univ.

Bordeaux, ISVV, EA 4577 Œnologie, F-33140 Villenave d’Ornon, France 2INRA, ISVV, USC 1366 Œnologie, F-33140 Villenave d’Ornon, France *E-mail [email protected].

Ethyl 2-hydroxy-4-methylpentanoate enantiomers were assayed in 55 commercial wines using chiral gas chromatography. White wines presented only the R form, whereas red wines contained both enantiomers, in various ratios according to aging (average ratio: 95:5, m/m) with an average total concentration of about 400 μg/L. The olfactory threshold of ethyl (2R)-2-hydroxy-4-methylpentanoate (126 μg/L) was almost twice that of the S- form (55 μg/L). The olfactory threshold of the mixture of ethyl (2R)-2-hydroxy-4-methylpentanoate and ethyl (2S)-2-hydroxy-4-methylpentanoate (95:5, m/m) was 51 μg/L, indicating a synergistic effect. Sensory analysis revealed that fruity character was perceived at concentrations 2.2, 4.5, and 2.5 times lower, when the matrix was supplemented with ethyl (2R)-2-hydroxy-4-methylpentanoate, ethyl (2S)-2-hydroxy-4-methylpentanoate, and the mixture of ethyl (2R)-2-hydroxy-4-methylpentanoate and ethyl (2S)-2-hydroxy-4-methylpentanoate (95:5, m/m), respectively, at their average concentrations in red wines, demonstrating a synergistic effect of this ester on fruity aroma perception. Sensory profiles of aromatic reconstitutions highlighted the contribution of this compound to black-berry and fresh fruit descriptors.

© 2015 American Chemical Society Guthrie et al.; The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Introduction Ethyl 2-hydroxy-4-methylpentanoate (1) is a compound used in flavor chemistry. 1 has been identified in several distillates, such us grape brandies (1), as well as freshly-distilled Calvados and Cognac (2). Concerning wine, 1 was first characterized in dry white wines made from the Romanian cultivar, Feteasca Regala (3), and later in dry white Chardonnay wines (4). This ethyl ester was also found in aged Madeira wines and some types of sherry (5), as well as in dry red wines, at an average concentration of about 400 μg/L (6). Its enhancer effect was reported by Luccarelli, Mookherjee, Wilson, Zampino and Bowen (7) who demonstrated that, when 1 was mixed with C4-C10 alkanoic acids, it enhanced natural, ripe, tropical fruit flavors in food. Although 1 clearly has an asymmetrical carbon atom in position 2 (Figure 1), to our knowledge, no previous work investigated the possibility of two enantiomers.

Figure 1. (a): Ethyl (2R)-2-hydroxy-4-methylpentanoate (CAS number =60856-83-9) and (b): ethyl (2S)-2-hydroxy-4-methylpentanoate (CAS number =60856-85-1). This paper reports the separation, distribution, and concentrations of 1 enantiomers in wines from various vintages and origins and evaluates the organoleptic impact of this compound in red wines, by determining their olfactory thresholds and studying their perceptive interactions.

Materials and Methods Samples. 1 was assayed in wines from several vintages and origins: 42 red (vintages 1981-2010) and 13 white wines (vintages 1989-2008). Pays d’Oc Merlot was used to evaluate the organoleptic impact of 1 on quantitative odor perception, while Margaux wine (vintage 2000) was used to evaluate the organoleptic impact of 1 on qualitative odor perception. Wine samples from the 2010 vintage were collected and analyzed 3 months after alcoholic fermentation. Dilute alcohol solution was prepared using double-distilled ethanol and microfiltered water (12%, v/v). Aromatic Reconstitution. Sample preparation was as described by Lytra, Tempere, de Revel and Barbe (8) using liquid–phase extraction technique. Reversed-phase (RP) HPLC was performed on this raw extract, under the 68 Guthrie et al.; The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


chromatographic conditions optimized by Pineau, Barbe, Van Leeuwen and Dubourdieu (9). The 25 fractions obtained in dilute alcohol solution were then directly evaluated by three trained assessors. 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 content of 12% (v/v). Ethyl 2-Hydroxy-4-methylpentanoate Enantiomer Quantification. Chromatographic conditions and sample preparation were as optimized by Lytra, Tempere, de Revel and Barbe (8) using liquid–phase extraction technique. The enantiomers of 1 were separated by chiral gas chromatography on a γ-cyclodextrin phase. Gas chromatography analyses were carried out on an HP 6890 GC system coupled to an HP 5973i quadrupole mass spectrometer. The mass spectrometer was operated in electron ionization mode at 70 eV with selected-ion-monitoring (SIM) mode. Ester and Acetate Analyses in HPLC Fractions. Chromatographic conditions and sample preparation were as optimized by Antalick, Perello and de Revel (10) using the Solid-phase microextraction technique (SPME). Gas chromatography analyses were carried out on an HP 5890 GC system coupled to an HP 5972 quadrupole mass spectrometer. The mass spectrometer was operated in electron ionization mode at 70 eV with selected-ion-monitoring (SIM) mode.

Sensory Analyses General Conditions. Sensory analyses were performed as described by Martin and de Revel (11). Judges were all research laboratory staff at ISVV, Bordeaux University, selected for their experience in assessing fruity aromas in red wines. Olfactory Thresholds. The olfactory thresholds of ethyl (2R)-2-hydroxy4-methylpentanoate (1a), ethyl (2S)-2-hydroxy-4-methylpentanoate (1b), and the mixture of 1a and 1b (95:5, m/m) were determined by 15 judges in a three-alternative, forced-choice presentation (3-AFC) (12). The impact of a mixture of 1a and 1b (95:5, m/m) in dilute alcohol solution was evaluated using an additive model (13), as developed by Miyazawa, Gallagher, Preti and Wise (14). Particular "olfactory thresholds" of fruity HPLC fractions (18 to 22), corresponding to an initial wine volume of 0.3, 0.6, 1.3, 2.5, 10, 20, 40, 80, 160 ml, diluted in 50 mL matrix, were determined by 19 judges, using four different matrices (dilute alcohol solution and dilute alcohol solution supplemented with 400 µg/L 1a, 20 µg/L 1b or 420 µg/L of the mixture of 1a and 1b (95:5, m/m), in a three-alternative, forced-choice presentation (3-AFC) (12). Data Analysis. The results of the three-alternative, forced-choice tests were statistically interpreted and the olfactory threshold value was determined using an adaptation of the ASTM – E1432 method (15). Sigma Plot 8 (SYSTAT) software was used for graphic resolution and ANOVA transform for non-linear regression (16). 69 Guthrie et al.; The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Sensory Profiles for red-berry, black-berry, fresh-, and jammy-fruit aromas were evaluated by 15 judges on a 0-7 point intensity scale. Two samples of aromatic reconstitutions in dilute alcohol solution were presented. The first consisted of HPLC fruity fractions (18 to 22) and the second contained the same HPLC fruity fractions, supplemented with 550 µg/L of the mixture of 1a and 1b (95:5, m/m). Statistical data were analyzed using R analysis of variance (ANOVA) software (R v2.15.0 - R Development Core Team 2009, Vienna, Austria, R Foundation for Statistical Computing): the homogeneity of variance was tested using Levene’s and the normality of residuals was tested using Shapiro-Wilk Test. All descriptors are mean-centered per panelist and scaled to unit variance. The statistically significant level was at 5% (p

Enantiomeric Distribution of Ethyl 2-Hydroxy-4 ... - ACS Publications

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