Study of Substituted Ester Formation in Red Wine ... - ACS Publications

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Study of Substituted Ester Formation in Red Wine by the Development of a New Method for Quantitative Determination and Enantiomeric Separation of Their Corresponding Acids Georgia Lytra,†,‡ Celine Franc,†,‡ Margaux Cameleyre,†,‡ 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

‡

S Supporting Information *

ABSTRACT: A new method was developed for quantifying substituted acids including, where applicable, their various unexplored enantiomeric forms. A new step was added to acids’ usual quantification methods, consisting of extraction, derivatization to methyl esters, and gas chromatography analysis: preliminary extraction was performed at basic pH to eliminate ethyl esters, thus avoiding their transesterification during derivatization. Quantitation and enantiomeric distribution of some substituted esters and their corresponding acids were established in 31 commercial Bordeaux red wines from 0 to 20 years old. A strong positive correlation was observed between the age of wine and levels of ethyl 2-methylpropanoate, ethyl 3methylbutanoate, ethyl 2-methylbutanoate, ethyl (3R)-3-hydroxybutanoate, both enantiomeric forms of ethyl 2-hydroxy-3methylbutanoate, and ethyl (2S)-2-hydroxy-4-methylpentanoate, but not ethyl (3S)-3-hydroxybutanoate. However, the standard deviations of average concentrations for the corresponding substituted acids were so large that only few correlations between concentrations and age were observed. Concentrations of (2S)-2-hydroxy-3-methylbutanoic acid and (2S)-2-hydroxy-4methylpentanoic acid increased slightly over time, while (2R)-2-hydroxy-4-methylpentanoic acid levels decreased slightly with the age. Variations in the ratio of substituted ethyl esters to their corresponding acids over time detected thanks to these analytical advances suggested that, in general, acids were continuously esterified during aging. KEYWORDS: enantiomeric distribution, chiral substituted esters, chiral substituted acids

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increase during aging.12−14 Pineau et al.6 demonstrated that two of them, ethyl 2-methylpropanoate and ethyl 2-methylbutanoate, contributed to black-berry fruit expression in red wines via perceptive interaction phenomena that had not previously been described. Other works4,12,15 reported that substituted esters, such as ethyl 2-hydroxy-4-methylpentanoate, ethyl 2methylbutanoate, and ethyl 3-hydroxybutanoate, presenting an asymmetrical carbon atom, played the role of natural enhancers of fruity notes in red wine, even at subthreshold levels. Considering the importance of these substituted esters in the fruity aromas of red wines, the knowledge of their precursors remains indispensable. Few publications had mentioned this topic, and only the esterification path of certain acids during aging had previously been established.16,17 The quantitative determination of alkyl substituted acids was described many years ago and widely applied in wine.18,19 However, hydroxycarboxylic acids had received very little attention. Chemical analysis of these acids is greatly influenced by their high polarity, which prevents their direct detection by gas chromatographic techniques. Consequently, hydroxycarboxylic acid analyses usually rely on their chemical conversion into the corresponding more volatile derivatives, which are suitable for gas chromatographic analysis.20−22 A method for the

INTRODUCTION Among wine volatiles, esters have been studied for a long time, always distinguishing between ethyl esters of nonvolatile acids, which accumulate during aging, and ethyl esters of volatile acids and higher-alcohol acetates, mainly produced during alcoholic fermentation.1,2 More recent studies3−5 reported that some of these ethyl esters and acetates contribute to the fruity character of red wines. Indeed, mainly thanks to perceptual interactions, these compounds enhance the perception of fruity aromas, even at concentrations below their individual olfactory thresholds.6,7 Ethyl esters of fatty acids are generally produced by the yeast metabolism (Ehrlich pathway)8 and then hydrolyzed during wine maturation and bottle aging,9 suggesting that these esters contribute to the fruity aroma of young wines, and their aromatic contribution is attenuated after 2 or 3 years’ aging.9 Ribéreau-Gayon et al.10 highlighted this phenomenon by determining the kinetic equations for the hydrolysis of certain unsubstituted ethyl esters of linear aliphatic acids over 29 months’ storage. However, several studies demonstrated that, among red wine ethyl esters, those derived from short-chain substituted acids constituted a family with a particular behavior and sensory importance. They have been discussed in the literature for over 20 years,10,11 but recent studies have established that some of them were strongly involved in the fruity aroma of red wines.7 Furthermore, in contrast to the levels of most ethyl esters produced during alcoholic fermentation, substituted ester levels © XXXX American Chemical Society

Received: March 2, 2017 Revised: May 21, 2017 Accepted: May 23, 2017

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DOI: 10.1021/acs.jafc.7b00979 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Compounds’ structures. old), Saint-Estèphe (8 wines, 10 to 20 years old), Pomerol (5 wines, 3 to 17 years old), and Saint-Emilion (8 wines, 3 to 20 years old). In addition, 4 red wines from Cadillac Côtes de Bordeaux and 2 red wines from Pessac-Léognan were analyzed just after the end of alcoholic fermentation. Enantiomeric Separation and Quantitation of Substituted Ethyl Esters. Sample Preparation. The extraction procedure was based on the Bertrand method.18 A 50 mL sample was spiked with 300 μg/L ethyl 2-hydroxy-2-methylpropanoate (not naturally present in wine) as an internal standard and 4 drops of H3PO4 (1/3 in H2O). Five grams of sodium chloride was added to the sample, and sample was then extracted using 4, 4, and 2 mL of dichloromethane, with magnetic stirring (700 rpm), for 10 min each, and separated in a separatory funnel, where the phases were allowed to sit for 5 min before separation. The organic phases were combined, dried over sodium sulfate, and concentrated using a Rotavapor (Laborota 4010 digital Rotary Evaporator, Heidolph, Germany), with a 20 °C bath temperature, to obtain 250 μL of final wine extract. Chromatographic Conditions. Gas chromatography 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. non-deactivated direct linear transfer (injector temperature, 200 °C; interface temperature, 200 °C). The column used for enantiomeric separation and quantitation of these compounds was a Chiraldex Gamma-TA (50 m × 0.25 mm i.d., film thickness 0.12 μm, Astec, Whippany, NJ, USA). The oven temperature was programmed at 40 °C for 1 min, then raised at a rate of 1 °C min−1 to 100 °C, and finally raised at 3 °C min−1 to a final isotherm at 180 °C, and maintained for 5 min. The carrier gas was helium N55 (Air Liquide, France) with a constant flow of 2 mL min−1. The mass spectrometer was operated in electron impact ionization at 70 eV in selected-ion-monitoring (SIM) mode. The ions monitored, chosen on the basis of the best measured signal-to-noise ratio, are listed in Table 1. Compounds were identified by comparing their linear retention indices and mass spectra with those of standards. Enantiomeric Separation and Quantitation of Alkyl-Substituted Acids. The sample preparation and chromatographic conditions for alkyl-substituted acid enantiomeric separation and quantitation were as described for ester enantiomeric separation and

quantitative determination of hydroxycarboxylic acids was very recently developed and applied to a set of wines and other alcoholic beverages.17 However, the enantiomeric separation of these hydroxycarboxylic acids in wine remained totally unexplored. The goal of this research was to develop a quantitation method for substituted acids including, where applicable, their various enantiomeric forms, preliminary to investigation into their formation pathways.

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MATERIALS AND METHODS

Reagents and Chemicals. Standard grade compounds were obtained from commercial sources as follows: ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, ethyl (3S)-3hydroxybutanoate, ethyl (3R)-3-hydroxybutanoate, 2-methylpropanoic acid, 3-methylbutanoic acid, 2-methylbutanoic acid, 3-hydroxybutanoic acid, 2-hydroxy-3-methylbutanoic acid, 2-hydroxy-4-methylpentanoic acid, 2-hydroxy-2-methylpropanoic acid, and ethyl 2-hydroxy-2methylpropanoate, from Sigma-Aldrich, Saint-Quentin-Fallavier, France; ethyl (2R)-2-hydroxy-4-methylpentanoate, ethyl (2S)-2hydroxy-4-methylpentanoate, ethyl (2R)-2-hydroxy-3-methylbutanoate, ethyl (2S)-2-hydroxy-3-methylbutanoate, and ethyl (2S)-2methylbutanoate were synthesized by Hangzhou Imaginechem Co., Ltd., Hangzhou, China. All solvents were HPLC grade. Absolute ethanol (purity >99.8%) and methanol were obtained from Merck (Darmstadt, Germany). Milli-Q water was obtained from a Milli-Q Plus water system (Millipore, Saint-Quentin-en-Yvelines, France). Sodium chloride, anhydrous sodium sulfate, sodium hydroxide, sodium bicarbonate, and sulfuric acid were supplied by VWR-Prolabo (Fontenay-sous-bois, France). Dichloromethane was provided by Carlo Erba (Pestipur quality, Carlo Erba, SDS, Italy). For retention index calculation, a mixture of n-alkanes was obtained from SigmaAldrich (Saint-Quentin-Fallavier, France). Compounds’ structures are illustrated in Figure 1. Standard solutions of the esters were prepared in ethanol, whereas standard solutions of the acids were prepared in acetonitrile to avoid esterification. Samples. Substituted esters and their corresponding acids were assayed in 31 red wines, 0 to 20 years old, from different Bordeaux appellations. Four sets of wines were studied, each covering a 17-to 20year period in the same vineyard: Margaux (6 wines, 3 to 20 years B


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before separation. The organic phases were combined, dried over sodium sulfate, and concentrated using a Rotavapor to obtain 1 mL of extract. Derivatization. The hydroxycarboxylic acids were characterized by derivatization. This method for generating methyl esters of the naturally occurring acids was based on the method proposed by Gallart et al. and Sigma-Aldrich,23,24 and modified as follows. Two milliliters of the derivatization reagent (10% H2SO4 in methanol) was added to 1 mL of concentrated wine extract; the mixture was shaken for 30 s with a vortex shaker and left for 30 min at 60 °C. Extraction of the Derivative Compounds (Methyl Esters). Ten milliliters of saturated aqueous sodium bicarbonate solution was added to the sample, which was then extracted using 3, 1, and 1 mL of dichloromethane, with magnetic stirring (700 rpm), for 5 min. The lower layer (dichloromethane), containing methyl esters, was then removed carefully, dried over anhydrous sodium sulfate, and concentrated using a Rotavapor to obtain 250 μL of extract. Chromatographic Conditions. These methyl esters (Table 1) were analyzed by gas chromatography−mass spectrometry using the chromatographic conditions described for ester enantiomeric separation and quantitation on a Chiraldex Gamma-TA column in the paragraph Chromatographic Conditions. Derivative compound concentrations (expressed in μmol/L) were converted to hydroxycarboxylic acid concentrations (μg/L). Parameters of Development and Optimization of Hydroxycarboxylic Acid Enantiomeric Separation and Quantitation Method. Influence of pH on Ester Extraction. To avoid errors due to ethyl ester transesterification, a preliminary extraction of ethyl esters was conducted to remove them from the sample prior to derivatization. The influence of pH on ester elimination was evaluated. A dilute alcohol solution (prepared with high-purity ethanol and microfiltered water to obtain an ethanol content of 12% (v/v), supplemented with 5 g/L tartaric acid) was adjusted to pH 3.0, 7.0, 8.0, and 9.0 by adding pure sodium hydroxide or sulfuric acid solutions and then spiked with the pool of substituted esters. The solutions were then extracted using 10, 10, and 5 mL dichloromethane, as described in the paragraph Ester Elimination. The organic phases were collected, dried over sodium sulfate, and concentrated to 1 mL. Derivatization, extraction of the derivative compounds (methyl esters), and chromatographic analyses were then carried out as described in the paragraphs Derivatization, Extraction of the Derivative Compounds (Methyl Esters), and Chromatographic Conditions, respectively. Influence of pH on Hydroxycarboxylic Acid Preservation during Ester Elimination. The same protocol (Influence of pH on Ester Extraction) was also applied to dilute alcohol solution supplemented with the pool of hydroxycarboxylic acids instead of substituted esters, to verify the effectiveness of hydroxycarboxylic acid retention in the aqueous layer and their possible elimination during sample preparation for ester elimination at pH 8.0. The sample was then extracted using 10, 10, and 5 mL of dichloromethane, as described in the paragraph Ester Elimination. The organic phases were collected, dried over sodium sulfate, and concentrated to 1 mL. Derivatization, extraction of the derivative compounds (methyl esters), and chromatographic analyses were carried out as described in the paragraphs Derivatization, Extraction of the Derivative Compounds (Methyl Esters), and Chromatographic Conditions, respectively. Influence of pH on Hydroxycarboxylic Acid Extraction. The influence of pH was assessed by studying the effectiveness of hydroxycarboxylic acid extraction from the dilute alcohol solution. A 100 mL sample of dilute alcohol solution was first adjusted to pH 0.5, 1.0, 2.0, and 3.0 by adding sulfuric acid and supplemented with the pool of hydroxycarboxylic acids. These solutions were extracted using 10, 5, and 5 mL of dichloromethane, as described in the paragraph Acid Extraction. The organic phases were combined, dried over sodium sulfate, and concentrated to obtain 1 mL of extract. Derivatization, extraction of the derivative compounds (methyl esters), and chromatographic analyses were carried out as described in the paragraphs Derivatization, Extraction of the Derivative Compounds (Methyl Esters), and Chromatographic Conditions, respectively.

Table 1. Ions Used for Identification and Quantitation of the Compounds and of the Derivative Compounds qualitative ions (m/z)

compound

quantitative ion (m/z)

Substituted Ethyl Esters ethyl 2-methylpropanoate (1) 43/88/71 ethyl 3-methylbutanoate (2) 85/57 ethyl (2R)-2-methylbutanoate (R-3) 57/85/74 ethyl (2S)-2-methylbutanoate (S-3) 57/85/74 ethyl (3R)-3-hydroxybutanoate (R-4) 71/88/117 ethyl (3S)-3-hydroxybutanoate (S-4) 71/88/117 ethyl (2R)-2-hydroxy-3-methylbutanoate 76/104 (R-5) ethyl (2S)-2-hydroxy-3-methylbutanoate (S76/104 5) ethyl (2R)-2-hydroxy-4-methylpentanoate 87/104 (R-6) ethyl (2S)-2-hydroxy-4-methylpentanoate 87/104 (S-6) ethyl 2-hydroxy-2-methylpropanoate 43/89/41 (internal standard) Alkyl-Substituted Acids 2-methylpropanoic acid (7) 73 3-methylbutanoic acid (8) 87 (2R)-2-methylbutanoic acid (R-9) 87/57 87/57 (2S)-2-methylbutanoic acid (S-9) qualitative hydroxycarboxylic acids derivative methyl esters ionsa (3R)-3-hydroxybutanoic acid (R-10) (3S)-3-hydroxybutanoic acid (S-10) (2R)-2-hydroxy-3methylbutanoic acid (R11) (2S)-2-hydroxy-3methylbutanoic acid (S11) (2R)-2-hydroxy-4methylpentanoic acid (R-12) (2S)-2-hydroxy-4methylpentanoic acid (S-12)

73 69 69 59

88 60 74 74 quantitative ionsa

45/74/87

103

45/74/87

103

73/55/43

90

methyl (2S)-2-hydroxy3-methylbutanoate

73/55/43

90

methyl (2R)-2-hydroxy4-methylpentanoate

69/90

87

methyl (2S)-2-hydroxy4-methylpentanoate 2-hydroxy-2methyl 2-hydroxy-2methylpropanoic acid methylpropanoate (internal standard)

69/90

87

43/103

59

a

methyl (3R)-3hydroxybutanoate methyl (3S)-3hydroxybutanoate methyl (2R)-2-hydroxy3-methylbutanoate

116 88 102 102 87 87 73

Of derivative compounds.

quantitation in the paragraphs Sample Preparation and Chromatographic Conditions, respectively. Enantiomeric Separation and Quantitation of Hydroxycarboxylic Acids. Sample Preparation (Figure S1). Ester Elimination. A 100 mL wine sample, previously adjusted to pH 8.0 with sodium hydroxide pellets, was spiked with 1000 μg/L 2-hydroxy-2methylpropanoic acid as an internal standard. Ten grams of sodium chloride was added to the sample, which was then extracted using 10, 10, and 5 mL of dichloromethane, with magnetic stirring (700 rpm), for 15 min each. The sample was then centrifuged for 15 min (3000 rpm, High-Speed Refrigerated Centrifuge, CR22N, Hitachi Koki Co.,Ltd.) and poured into a separatory funnel, where the phases were allowed to sit for 5 min before separation. The organic phases were collected and then discarded. Acid Extraction. The pH of the aqueous phase was then readjusted to 0.5 with pure sulfuric acid. Ten grams of sodium chloride was added to the sample, which was then re-extracted using 10, 5, and 5 mL of dichloromethane, with magnetic stirring (700 rpm), for 15 min each. The sample was then centrifuged for 15 min and poured into a separatory funnel, where the phases were allowed to sit for 5 min C


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Journal of Agricultural and Food Chemistry Validation Method for Enantiomeric Separation and Quantitation of Hydroxycarboxylic Acids. Calibration curves were established in dilute alcohol solution (12%, v/v), using a representative range of acid concentrations found in wines (8 points). The calibration curves were plotted as the relative peak areas (analyte versus internal standard) against concentration. The repeatability of the curve’s slope was calculated from three repetitions. Repeatability was evaluated by calculating the standard deviation of 10 independent assays, performed under the same analytical conditions over a short period of time. The limits of detection (LODs) (concentration for signal/noise = 3) and the limits of quantitation (LOQs) (concentration for signal/ noise = 10) were manually calculated from the ratio of the peak heights to the average noise before and after each peak. The accuracy of the analytical method was evaluated by calculating the recoveries with a standard addition technique: by adding each compound to a red wine naturally containing low levels of the corresponding compound. The wine was spiked with six different concentrations of each compound prior to sample preparation. Accuracy is reported as percent recovery by the assay of known added amount of analyte in the sample. The above validation parameters were also evaluated for the adaptation of the method of enantiomeric separation and quantitation of substituted esters, as well as alkyl substituted acids. Statistical Data Analysis. Spearman’s correlation test was used to evaluate the relationship between the age of wine and substituted ester and acid concentrations, as well as between the age of wine and ester to acid ratio. The statistically significant level was 5% (XLSTAT software, p < 0.05).

precisely, extraction at pH 0.5 resulted in the highest absolute peak areas and was thus selected for this method. Method Validation. To validate the method for enantiomeric separation and quantitation of hydroxycarboxylic acids, linearity was established for the concentrations listed in Table 2 Table 2. Hydroxycarboxylic Acid Quantitation Methoda compound (3R)-3-hydroxybutanoic acid (R-10) (3S)-3-hydroxybutanoic acid (S-10) (2R)-2-hydroxy-3methylbutanoic acid (R11) (2S)-2-hydroxy-3methylbutanoic acid (S11) (2R)-2-hydroxy-4methylpentanoic acid (R12) (2S)-2-hydroxy-4methylpentanoic acid (S12) a

LOD (μg/L)

LOQ (μg/L)

linear range (μg/L)

recovery (%)

21

70

102−2052

87 ± 9

7

24

91−1828

117 ± 6

6

20

115−2300

109 ± 5

LOD: limit of detection; LOQ: limit of quantitation.

with coefficients of determination from R2 = 0.9797 to R2 = 0.9869. LODs and LOQs, which varied depending on the chemical structure, are also listed in Table 2. Repeatability for hydroxycarboxylic acids was satisfactory, with coefficients of variation below 10%, and the recovery rate for the method was between 87% and 117%. Concerning the adaptation of the method of enantiomeric separation and quantitation of substituted esters, as well as alkyl substituted acids, our results corroborated those of previous studies.12,15,19 The functions were linear over the concentration range, with coefficients of determination from 0.9943 to 0.9996. Repeatability was evaluated by relative standard deviation. The relative standard deviations of the area ratios of 10 independent assays performed under the same analytical conditions over a short period of time were below 7% for all compounds. The limits of detection and quantitation were considerably lower than the concentrations present in the wines studied. Recovery ranged from 88 to 104% for all substituted esters and alkyl substituted acids. Application to Wine Analysis. Variations in the Content of Substituted Esters over Time. The Spearman correlation test revealed a strong positive correlation between the age of wine and total concentrations of 1, 2, 3, 5, and 6 (Table 3). However, only a slight positive correlation was found between the age of wine and total concentrations of 4, which is consistent with previous observations reported by Lytra et al.15 As previously observed, substituted ester levels were generally higher in the oldest wines than in younger ones (Table 3).6,13,27 In light of these findings, it seems likely that only a few μg/L of these compounds are formed during alcoholic fermentation and that wine aging is marked by an increase in substituted ester concentrations over time. These results corroborated those of other authors4,12,28 who observed a tendency for substituted esters to form during wine aging, possibly resulting from esterification of the corresponding acids. These trends were described in the literature many years ago for esters produced from the main nonvolatile acids (malic, tartaric,

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RESULTS AND DISCUSSION Development and Optimization of Hydroxycarboxylic Acid Quantitation and Enantiomeric Separation. Influence of pH on Ester Elimination. The usual analytical methods for acids25,26 consist of extraction, derivatization to methyl esters, and gas chromatography analysis. During this process, depending on the conditions (reagent, time, and temperature), the ethyl esters may be transesterified to methyl esters. Therefore, in samples containing ethyl esters, methylation resulted in methyl esters produced directly from free acid, as well as from ethyl ester transesterification. Thus, to assay the free acids accurately, it was essential to determine the amount of methyl ester formed from ethyl ester transesterification and apply the suitable correction. To eliminate the need for this correction, esters were extracted prior to derivatization. As described in paragraph Influence of pH on Ester Extraction, different pH values for extraction were evaluated. Analysis of the derivatives (methyl esters) demonstrated that, at basic pH, such as pH 8.0 and 9.0, esters were completely extracted and eliminated from the sample, as no traces of derivative compounds were detected (levels < LODs). However, when the dilute alcohol solution was adjusted to pH 9.0, a thick emulsion appeared, making difficult the separation of aqueous and organic phases. Consequently, pH 8.0 was selected for ester elimination. Influence of pH on Hydroxycarboxylic Acid Preservation during Ester Elimination. Analysis of the organic extract of a hydroxycarboxylic acid solution at basic pH, such as pH 8.0, did not detect any traces (levels < LODs) of these compounds. Thus, under these conditions, preliminary ester extraction did not impact the hydroxycarboxylic acid content, thus validating this step of the methodology. Influence of pH on Hydroxycarboxylic Acid Extraction. Analyses of derivatives (methyl esters), after extraction of the hydroxycarboxylic acid solution at various acidic pH, revealed that acid extractability was enhanced at lower pH. More D


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Journal of Agricultural and Food Chemistry Table 3. Correlations between Aging Time and Substituted Ester Levelsa

a

End AF: analyzed just after the end of alcoholic fermentation.

etc.).1 In 1982, Ribéreau-Gayon et al.29 reported that the presence in wine of numerous acids and high ethanol concentrations led to the formation of esters, which continued throughout aging. They demonstrated that the concentrations of esters thus formed increased by approximately 30% after one year, 50% after 2 to 3 years, and 80% after 50 years. Enantiomeric separation of 3 showed only the S-enantiomer in all wines studied (Table 3). This finding is in agreement with data from previous studies on red wine, where analysis of 3 revealed the presence of the S-enantiomer as a key odorant.12,30 As observed in previous studies,12 S-3 levels are generally higher in older red wines. As observed very recently,15 concentrations of the R-4 form increased gradually over time, while the S-form remained unchanged (Table 3). As observed in previous studies,4,28 concentrations of both forms of 5 and 6 increased gradually over time. Young red wines, analyzed just after the end of alcoholic fermentation, contained only the R-form, thus corroborating the results of recent studies (Table 3).4,31 As previously observed, the ratio of S-5 and S-6 concentrations to total 5 and 6 concentrations, respectively, correlated positively with the age of wine, while S-4 to total 4 concentrations were negatively correlated (Table 3, p < 0.05). Globally, these results highlighted the relatively minor role played by alcoholic fermentation in the direct production of substituted ethyl esters. Indeed, except, perhaps, for 4, ester concentrations at the end of alcoholic fermentation remained at

a relatively low level (generally not exceeding 5%), compared to the maximum concentrations found in older wines. This suggests that aging is the decisive factor in increasing concentrations, probably due to esterification of the corresponding acid by ethanol. In view of the major organeleptic impact of substituted esters as natural enhancers of fruity notes in red wine, better knowledge of these acids is required to understand their influence on the sensory potential of wines. Variations in the Content of Substituted Acids over Time. The Spearman correlation test did not show any correlation between the age of wine and total concentrations of 7, 8, 9, 10, and 11, whereas a slight negative correlation was detected between the age of wine and total 12 concentrations (Table 4). As shown in Table 4, considerable variations in substituted acid levels were observed in wines of the same set of age, which is not totally unanticipated, in view of the many parameters involved.32 For example, the lipolytic activity of wine lactic acid bacteria, which has not been thoroughly investigated, suggests that some lactic acid bacteria may produce lipases; the action of lipases on wine lipids may yield a range of volatile compounds, including fatty acids.33 Enantiomeric separation of 9 showed only the S-enantiomer in all wines studied. This finding is in agreement with data from previous studies on red wine, which detected only the Senantiomer of 9.34 This result is not totally surprising, considering that its corresponding amino acid, L-isoleucine (2S,3S), is the only natural enantiomer. This result supported the hypothesis that this compound was probably formed only E


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Journal of Agricultural and Food Chemistry Table 4. Correlations between the Age of Wine and Substituted Acid Levelsa

a

End AF: analyzed just after the end of alcoholic fermentation.

from the corresponding natural amino acid, L-isoleucine (2S,3S), during alcoholic fermentation by Saccharomyces cerevisiae. This is converted into α-keto acid, which is then decarboxylated to produce an aldehyde, which, in turn, is oxidized into carboxylic acid (Ehrlich pathway).8 Alternatively, these α-keto acids may be generated through a de novo synthesis pathway from glucose−carbohydrate metabolism.35 Correlations between concentrations and age have been observed for only a few acid enantiomers. Concentrations of S11 and S-12 increased slightly over time, while R-12 levels decreased slightly with the age of wine (Table 4). Indeed, the decrease in R-12 concentration over time may be due to esterification, leading to the corresponding ethyl ester. To our knowledge, this is the first study to report the enantiomeric distribution of 10, 11, and 12 in a wine matrix. As observed for total substituted acid levels and some of their enantiomers, the standard deviations of average concentrations for the wines of the same age were too large to conclude to a statistical difference over time. As illustrated in Table 4, the ratio of S-11 and S-12 concentrations to total 11 and 12 concentrations, respectively, was positively correlated with the age of wine (p < 0.05), while no correlation was observed for S- and R-4 to total 4 concentrations. Average ratios of R- and S-11 and R- and S12 enantiomers to their total concentrations were very close to those of the corresponding ethyl esters, but this was not the case for 10. The maximum ratio of an enantiomer to the total corresponding acid concentration was 100% for the R-forms

of 11 and 12, in wines analyzed just after the end of alcoholic fermentation, as was also the case for their corresponding esters. A slight positive correlation between the age of wine and the ratio of S-forms to total acid concentrations was observed (p < 0.0001). In light of these findings, it seems likely that the S-forms of both acids, which have very similar structures, behave in the same way as their corresponding esters: only a few μg/L is formed during alcoholic fermentation, and wine aging is marked by an increase in their concentrations over time. Ratio of Substituted Esters to the Corresponding Acids over Time. In order to elucidate the possible esterification of substituted acids by ethanol, the ratio of substituted esters to the corresponding acids was studied over time. As shown in Table 5, the ratios of 2, S-3, R-4, R-5, S-5, and R-6 to the corresponding substituted acids increased slightly with the age of wine. These results support the hypothesis that esterification of the corresponding substituted acids resulted in the accumulation of these esters. In addition, this esterification may be related to the significant decrease in R-12 observed over time. Diaz-Maroto et al.16 reported that the acid−ester equilibrium was the most effective factor in generating branched fatty acid ethyl esters during wine aging. As shown in Table 5, no difference was observed in the ratios of 1, S-4, or S-6 to the corresponding substituted acids (Spearman’s correlation coefficient < 0.4, p > 0.7). Concerning the ratio of S-4 to S-10, this finding is not surprising; no significant change was observed in S-4 over time. F


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these particular compounds during aging suggests esterification of the corresponding acids. Nevertheless, for the formation of some compounds, new perspectives implicating yeast and lactic acid bacteria metabolism seem to appear. Now, once these analytical tools have been developed, conditions of the terms of formation of these compounds during enological steps, including particularly fermentations and potential biosynthesis occurring from amino acids as suggested by ref 8, could be studied. Thus, an assessment of the overall “aromatic potential” of these esters involved in red wine fruity aroma enhancement may be imagined, thanks to quantitation of the corresponding acids.

Table 5. Correlations between the Age of Wine and Ratio of Substituted Esters to the Corresponding Acids ratio

Spearman’s correlation coefficient

1/7 2/8 S-3/S-9 R-4/R-10 S-4/S-10 R-5/R-11 S-5/S-11 R-6/R-12 S-6/S-12

+0.359 +0.544 +0.593 +0.451 +0.070 +0.609 +0.472 +0.722 +0.306

spearman’s p value p p p p p p p p p

= = = = = = = < =

0.010 0.010 0.004 0.036 0.754 0.003 0.028 0.0001 0.165

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Figure 2 illustrates the ratio of 6 enantiomers to the corresponding substituted acids with the age of wine, highlighting a strong correlation for the R-form and no correlation for the S-form. This absence of correlation for the Sform may be explained by the fact that concentrations of the Sform of the acid increased with the age of wine. This result also suggests that another extra-fermentation formation pathway for the S-enantiomer may exist and that other metabolisms may affect concentrations of some substituted esters and/or acids. Thanks to the development of this analytical method, the quantitation and the enantiomeric separation of 6 substituted esters and their corresponding acids were established in wine. These results clarified current data on these compounds, particularly the relative abundance of their enantiomers. To our knowledge, enantiomeric distributions of 3-hydroxybutanoic, 2hydroxy-3-methylbutanoic, and 2-hydroxy-4-methylpentanoic acids are reported for the first time in wines. The analytical advances of the evolution of the ratio of substituted ethyl esters to their corresponding acid levels over time suggested that acids were esterified continuously during aging for at least approximately 20 years. According to the compound and aging time, the absolute ratio of ester to acid varied from 0 to a maximum of 0.714, validating previous studies which have demonstrated that the accumulation of

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00979.

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Hydroxycarboxylic acid sample preparation (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Unité de Recherche Œnologie, EA4577 INRA/Institut Polytechnique de Bordeaux/Université de Bordeaux ISVV, 210 Chemin de Leysotte, CS 50008 33882 VILLENAVE D’ORNON, France. Tel: +33 (0)5 57 57 58 63. ORCID

Jean-Christophe Barbe: 0000-0001-6013-4770 Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Bertrand, A. Recherches sur l’analyse des vins par chromatographie en phase gazeuse; PhD thesis; Université de Bordeaux: 1975.

Figure 2. Ratio of substituted esters to the corresponding acids over time. 6, ethyl 2-hydroxy-4-methylpentanoate; 12, 2-hydroxy-4-methylpentanoic acid. G


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