Enantiomeric differentiation of three key volatile compounds in three different palm wines (Elaeis guineensis, Borassus flabellifer and Nypa fruticans)

ABSTRACT

The contents and enantiomeric distributions of three chiral compounds, linalool, phenylethanol and acetoin, were investigated in three different palm wines (i.e. Elaeis guineensis, Borassus flabellifer, and Nypa fruticans). While N. fruticans and B. flabellifer wines were predominated with the (S)-enantiomers of linalool, phenylethanol and acetoin, respectively, E. guineensis wine contained acetoin primarily as (R)-enantiomers in addition to the (S)-forms of linalool and phenylethanol. Interestingly, results revealed a high level of acetoin in all wines with concentrations ranging from 2437 to 6611 µg/L and an average ratio of S/R of 4:96–100:0. Moreover, noticeable differences occurred in the enantiomeric ratios and concentrations of enantiomers of the chiral compounds during storage. In all the wines, concentration of the (S)-form decreased during storage, whereas those of the (R)-form increased.

RESUMEN

En este estudio se investigaron los contenidos y las distribuciones enantioméricas de tres compuestos quirales (linalol, feniletanol y acetoína) en tres vinos de palma distintos (Elaeis guineensis, Borassus flabellifer y Nypa fruticans). Mientras que los (S)-enantiómeros de linalol, feniletanol y acetoína predominan en los vinos N. fruticans y B. flabellifer, el vino E. guineensis contiene principalmente acetoína como (R)-enantiómeros, además de las formas (S) de linalol y feniletanol. Los resultados dan cuenta de la presencia de un elevado nivel de acetoína en todos los vinos, comprobándose que sus concentraciones oscilan en un rango de 2437 a 6611 µg/L y una ratio promedio de S/R de 4:96 a 100:0. Por otra parte, se constató que durante su almacenamiento se presentaron diferencias notables en las ratios enantioméricas y las concentraciones de enantiómeros de los compuestos quirales. En todos los vinos disminuyó la concentración de la forma (S), aumentando la de la forma (R).

KEYWORDS:

• Palm wine
• enantiomer differentiation
• linalool
• phenylethanol
• acetoin

PALABRAS CLAVE:

• Vino de palma
• diferenciación enantiomérica
• linalol
• feniletanol
• acetoína

Introduction

Palm wine is consumed throughout the tropics and appears as a whitish liquid produced by natural fermentation of the sap of Elaeis guineensis and Raphia hooker (Mbuagbaw & Noorduyn, 2012). Palm sap contains abundant sucrose and polar side chain amino acids especially asparagine and glutamine. The sap is oyster-white and translucent, with a nearly neutral pH (Aimi, AbuBakar, & Dzulkifly, 2013). However, it is highly susceptible to spontaneous fermentation by natural microbial floral. Fermentation brings about rapid decreases in sugar level as it is converted to alcohol and other products (Obire, 2005). The sap becomes milky-white (i.e. palm wine) due to the increased microbial suspension resulting from the prolific growth of the fermenting organisms (Amoa-Awua, Sampson, & Tano-Debrak, 2007). Sources of the fermenting organisms are the gourds, tapping implements and air.

The composition, flavour and quality of palm wine have been reported to vary with the time, duration and place of tapping. For instance, the flavour of palm wine thereof is not only dependent on the type, but majorly determined by microbial reactions. Several numbers of volatile compounds have been identified in different types of palm wines (Aimi et al., 2013; Jirovetz, Buchbaucer, Fleischhacker, & Ngassoum, 2001). In recent years, studies have been focused on the chemical basis of palm wine aroma (Lasekan, Buettner, & Christlebauer, 2007, 2009; Lasekan & Otto, 2009). Recent study has shown that acetoin, linalool and 2-phenyl ethanol (all chiral compounds) are important components of palm wine, and they contributed to the moody, citrusy aroma of palm wine (Lasekan, 2013). The enantiomeric compositions of chiral compounds have been well documented in sweet white wine (Takatoshi, Niclass, Frerot, & Dubourdieu, 2006), red wine (Lytra, Tempere, de Revel, & Barbe, 2012), fruit beverages (Castillo, Caja, & Herraiz, 2003) and fruit juices (Weber, Maas, & Mosandl, 1995). The enantiomeric compositions of chiral compounds of palm wine varieties are yet to receive attention from researchers. Therefore, a better knowledge of the enantiomer differentiation of the chiral compounds in palm wine would help in the improvement of the flavouring and inhibition of off-flavours through technological processes. Enantiomer differentiation allows a more accurate evaluation of flavours and fragrances (Armstrong, Chang, & Li, 1990). In addition, each enantiomer of many chiral compounds is known to evoke different neural responses in consumers (Barba, Flores, & Herraiz, 2010).

In order to fulfil the lack of study in the area of enantiomeric distribution of chiral compounds in palm wine, this study was carried out to establish the enantiomeric composition and distribution of three key compounds in three palm wine varieties.

Materials and methods

Raw materials

Three bottles (4.5 L) of each wine obtained from three different palm trees (E. guineensis, Borassus flabellifer and Nypa fruticans) were freshly obtained from the production farm in a sterilized container encrusted in ice. The samples were bottled, pasteurized and dispensed into 45 mL glass-tubes. A set was stored (3 months) at ambient temperature (28 ± 2°C), and the others were stored at −18°C prior to analysis. Accordingly, there was no extended storage of samples at elevated temperatures prior to analysis. The alcohol contents of the palm wines were 3.5% (E. guineensis), 3.3% (B. flabellifer) and 3.0% (N. fruticans). Dilute alcohol solution was prepared as described by Lytra, Cameleyre, Tempere, and Barbe (2015) with high-purity ethanol and micro filtered water, to produce an ethanol content of 12% (v/v) and 5 g/L tartaric acid (pH adjusted to 3.5 with sodium hydroxide.

Chemicals

The following reference compounds were obtained from the suppliers given in parentheses: ethyl acetate, 98%; methyl butanoate, 99%; 2-methylbutanal, 98%; ethyl-2-methylbutanoate, 99%; ethylpentanoate, 97%; 2-heptanone, 98%; 3-methylbutanol, 99%; ethylhexanoate, 97%; acetoin, 97%; ethyl lactate, 98%; 3-hydroxy-2-butanone, 98%; (Z)-1,5-octadien-3-one, 97%; 1-hexanol, 99%; ethyl octanoate, 98%; hexyl-3-methylbutanoate, 98%; methional, 98%; 3-isobutyl-2-methoxypyrazine, 70%; 3-methylbutyl acetate, 80%; linalool, 98%; 2-methyl propionic acid, 97%; butanoic acid, 97%; 3-methyl butanoic acid, 80%; (E,E)-2,4-nonadienal, 97%; 3-methylthiol-1,1-propanal, 98%; 3-mercapto-2-methylpentanone, 98%; β-damascenone, 97%; 2-methoxyphenol, 98%; 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 99%; homofuraneol, 98%; ethyl cinnamate, 98%; sotolone, 98%; and phenylacetic acid, 97% (Aldrich, Steinheim, Germany); while, 2,3-butandione, 99%; 2-acetylpyridine, 99%; and pentanoic acid, 97% were purchased from Fluka, Neu-Ulm, Germany. Others such as acetic acid, 99%; 2-ethyl-3,5-dimethylpyrazine, 98%; 2-phenyl ethanol, 98%; and vanillin were from Acros Organics, Morris Plain, NJ, USA & Merck Darmstadt, Germany, respectively. For chiral analysis, the following reference compounds were used: (R, S)-linalool and (R, S)-phenylethanol were from Fluka, Buchs, Switzerland. Absolute ethanol (analytical grade, 99.97%, Scharlau Chemie S.A., Barcelona Spain) was distilled before use. Micro filtered water was obtained from a Milli-Q Plus water system (resistivity = 18 Ω cm, Millipore, Saint-Quentin-en-Yvelines, France)

Synthesis

(R)-acetoin was synthesized by monobenzylation of commercially available (2R, 3R)-butanediol (99% purity) (Sigma-Aldrich, Steinheim, Germany), with sodium hydride and benzyl bromide as described by Crout, Littlechild, and Morrey (1986). Oxidation of the product with pyridinium dichromate was followed by debenzylation of the resulting ketone (H₂/Pd-C). Careful workup yielded >98% (R)-acetoin in chemical purity as determined by (Chiral) Gas chromatography (GC). Due to its high racemization rate, it was stored at −70°C until use.

Separation of enantiomers from the commercial racemic mixtures (i.e. linalool phenylethanol)

High performance liquid chromatography (HPLC) separation was performed on a Merck L-7100 pump connected to a Merck variable-wavelength UV detector. The preparative column was a Chiralpak AS-H model (250 × 20 mm). The eluent was made up of 10% isopropanol in n-heptane with a flow rate of 20 mL/min at a constant temperature of 25°C. The two compounds were collected after HPLC purification. Each enantiomer of linalool and phenylethanol yielded approximately 47 mg/L.

Chiral identity

The configuration of linalool and phenylethanol was determined by measuring the specific rotation values of the optically active compounds. Optical rotation was determined on a Perkin-Elmer 341 polarimeter with a 1 dm cell. The following specific rotation values were obtained for (R)-linalool: [α]_D²⁰ = −15.1° and (S)-linalool: [α]_D²⁰ =  +17.4°, while the (R/S) – (±)-linalool: [α]_D²⁰ = 0° and contains 50.9% of the (R)-form and 49.1% of the (S)-form, respectively. (R)-1-phenylethanol has specific rotation of −55.15° while the (S)-1-phenylethanol has +45°.

Isolation and identification of wines volatile compounds

The isolation of the palm wines’ volatile compounds was performed by extracting 300 mL of each palm wine with diethyl ether (200 mL), followed by distillation in vacuum (Buettner & Schieberle, 2001). A similar workup procedure reported earlier (Lasekan & Ng, 2015) was carried out on both palm wines to produce 400 µL extract. The flavour compounds were identified by comparison with reference compounds on the basis of the following criteria: retention index (RI) on two stationary phases of different polarities; mass spectra; and odour notes perceived at the sniffing port.

Enrichment and fractionation of compounds

The solvent-assisted flavour extraction (SAFE) distillate (Engel, Bahr, & Schieberle, 1999) obtained from (500 mL) of palm wine was concentrated to 100 mL and extracted three times with an aqueous sodium carbonate solution (0.5 M) to remove the acidic volatiles (Lasekan & Ng, 2015). The organic phase containing the neutral and basic volatiles fraction (NBF) was dried over anhydrous sodium sulphate and concentrated to 1 mL. The acidic fraction obtained as described previously (Lasekan & Ng, 2015) was also concentrated to 1 mL. A portion (1 mL) of the NBF was applied onto a water-cooled glass column (25 × 1 cm i.d., 12°C) filled with a slurry of silica gel 60 in n-pentane, and separated into five fractions by applying the following n-pentane/diethyl ether mixtures: first fraction (100 mL; n-pentane); second fraction (100 mL; 95 + 5 mL); third fraction (100 mL; 90 + 10 mL); fourth fraction (100 mL; 80 + 20 mL); and fifth fraction (100 mL; 50 + 50 mL). The eluate was collected and concentrated to 1 mL, and the concentrate was used for the gas chromatography olfactometry (GC-O) and gas chromatography-mass spectrometry (GC–MS) analyses.

GC-O and GC–MS

The GC-O was performed with an Agilent 6890N instrument equipped with a split–splitless injector, a flame ionization detector (FID) and an ODP2 (Olfactory Detector Port; Gerstel, Mulheim, Germany) (Lasekan & Ng, 2015). The following fused silica columns were used: DB-FFAP (30 m × 0.32 mm, film thickness, 0.25 µm; Chrompack, Mulheim, Germany) and DB5 (30 m × 0.32 mm, film thickness 0.25 µm; J & W Scientific, Folsom, CA, USA). An aliquot (2 µL) of the wine extracts was applied by the on-column injection method at 35°C. After 2 min, the temperature of the oven was raised at 40°C min⁻¹ to 50°C and held for 2 min isothermally. It was also raised at 6°C min⁻¹ to 180°C, and it was finally raised at 10°C min⁻¹ to 230°C and held for 10 min. Helium (2.5 mL min⁻¹) was used as the carrier gas. At the end of the capillary, the effluent was split 1:1 (by vol.) into an FID and a sniffing port. The FID and the sniffing port were held at 220°C and 240°C, respectively. Linear RIs were calculated according to Kovats method using a mixture of normal paraffin C₆– C₂₈ as external references.

MS analysis was performed with a MS-95S (Finnigan Bremen, Germany) in tandem with the capillaries described above. Mass spectra in the electron impact mode (MS/EI) were generated at ionisation energy of 70 eV; relative electron multiplier voltage (EM) of 400 V, with a resulting voltage of 1553 V, was used. The detector temperature was maintained at 280 C, with the actual temperature in the MS source reaching 180°C (Guen, Prost, & Demaimay, 2001).

Chiral analysis

The wine fractions 3 and 4 (Table 1) were used for the chiral analysis. Fraction 3 was used for the chiral analyses of acetoin and phenyl ethanol, respectively; while chiral analysis of linalool was conducted with fraction 4. Chiral analyses were performed with an Agilent 6890N gas chromatograph (Agilent Technologies, Deutschland, Gmbh, Waldronn, Germany) by using the following capillaries: Chirasil-β-Dex (25 m × 0.25 mm i.d. fused silica capillary column coated with 0.25 µm film thickness of permethylated β-cyclodextrin; Varian, Middelburg, The Netherlands) for acetoin and BGB-176 (30 m × 0.25 mm, 2,3-dimethyl-6-tert-butyldimethylsilyl-β-cyclodextrin, film thickness, 0.25 µm; BGB Analytik AG, Rothenfluh, Germany) for linalool and phenylethanol, respectively. The wine fractions were injected at 35°C while maintaining the following conditions: the temperature of the oven was initially raised at 40°C min⁻¹ to 50°C, and then raised at 6°C min⁻¹ to 180°C. The flow rate of the helium was 2.5 mL min⁻¹. The compounds were separated into their enantiomers without derivatization, and the order of elution was assigned by employing optically pure reference compounds.

Table 1. Odorants obtained in the SAFE extracts of palm wines (E. guineensis, B. flabellifer and N. fruticans).

Tabla 1. Odorantes obtenidos de las extracciones SAFE de los vinos de palma E. guineensis, B. flabellifer y N. fruticans.

Fraction Retention index
No.	Odorant	Odour quality		FFAP	DB5
1	Ethyl acetate	Fruity	1	889	624
2	2-Methylbutanal	Malty	3	912	663
3	Methylbutanoate^a	Sweet fruity	2	981	723
4	2,3-Butandione	Buttery	3	990	592
5	Ethyl-2-methylbutanoate	Fruity	2	1040	852
6	Ethylpentanoate	Sweet fruity	2	1067	900
7	2-Heptanone	Soapy, fruity	3	1181	891
8	3-Methylbutanol	Malty	5	1214	739
9	Ethyl hexanoate	Fruity	2	1226	1001
10	Acetoin	Buttery	3	1275	nd
11	Ethyl lactate	Phenolic, smoky	3	1323	nd
12	3-Hdroxy-2-butanone	Buttery	3	nd	718
13	(Z)-1,5-Octadien-3-one	Geranium-like	3	1367	982
14	1-Hexanol	Rubbery	3	1356	872
15	Acetic acid	Sour, pungent	Acidic	1428	600
16	Ethyl octanoate	Fruity	2	1429	1199
17	Hexyl-3-methylbutanoate*	Fruity	5	1430	nd
18	2-Ethyl-3,5-dimethylpyrazine*	Roasty	3	1451	1083
19	Methional	Cooked potato	3	1469	nd
20	3-Isobutyl-2-methoxypyrazine*	Earthy	3	1517	1175
21	3-Methylbutyl acetate	Banana	2	1527	878
22	2-Acetylpyridine	Popcorn	4	1532	nd
23	Linalool	Fresh-blooming	4	1540	1103
24	2-Methylpropanoic acid	Sweaty	Acidic	1563	nd
25	Butanoic acid	Sweaty-buttery	Acidic	1619	821
26	2/3-Methyl butanoic acid^a	Sweaty	Acidic	1661	875
27	(E,E)-2,4-Nonadienal	Fatty	3	1718	1215
28	Pentanoic acid	Sweaty	Acidic	1720	911
29	3-Methylthiol-1,1-propanal	Broth-like	3	1723	903
30	3-Mercapto-2-methylpentanone*	Gravy-meaty	3	1742	883
31	β-Damascenone	Flowery	4	1801	1389
32	2-Methoxyphenol	Smoky	3	1842	1089
33	2-Phenylethanol	Honey	3	1911	1117
34	4-Hydroxy-2,5-dimethyl-3(2H)-furanone^a	Caramel-like	Acidic	2038	1070
35	3-Methylpentanoic acid	Sweaty	Acidic	2040	nd
36	Homofuraneol	Apple-like	3	2095	nd
37	Ethyl cinnamate	Sweet	2	2167	1469
38	Sotolone*	Spicy	3	2190	1110
39	Diethyl succinate*	Sweet-pineapple	5	2390	nd
40	Phenylacetic acid	Honey	Acidic	2577	1262
41	Vanillin^a	Vanilla	5	2601	1404

*Not detectable in Nypa fruticans wine. ^a Not detectable in B. flabellifer. nd, not detected.

*No detectado en el vino Nypa fruticans. ^a No detectado en el vino B. flabellifer. nd, No detectado.

Quantification

Quantification was performed by GC–MS, and triplicate calibration graphs at five concentrations level were constructed by least square linear regression using the results for the standard solution (12% hydro-alcoholic solution) submitted to the same procedure as described above. The concentration ranges employed for the relevant palm wine compounds were: linalool (4.4–68.7 µg/L), ethylbutanoate (3.0–18.1 µg/L), ethylacetate (0.3–19.2 µg/L), 2-phenylethanol (1.5–14.9 µg/L), β-damascenone (1.4–10.5 µg/L), 2,3-butandione (0.9–20.8 µg/L), ethylhexanoate (0.8–12.4 µg/L), geraniol (1.4–17.0 µg/L), 3-methylbutanol (1.9–15.1 µg/L) and acetoin (2.4–20.5 µg/L). The calibration curves were linear with r² values between 0.974 (linalool) and 0.998 (2-phenylethanol). Student’s t-test was used to evaluate the differences. The statistically significant level was 5% (XLSTAT software, P < 0.05).

Results and discussion

Table 1 shows the results of odour qualities and the retention indices of the SAFE extracts from three different palm wines (E. guineensis, B. flabellifer and N. fruticans). While a total of 41 odorants were detected in E. guineensis wine, N. fruticans and B. flabellifer wines produced a total of 35 and 37 odorants, respectively. All the compounds identified are well-known constituents of the organic fractions of wine and have previously been described in numerous publications and reviews (Fretz, Kanel, Luisier, & Amado, 2005; Guen et al., 2001; Lasekan & Abbas, 2010; Lasekan et al., 2007, 2009). Most of the compounds are mainly formed during alcoholic fermentation. In the current study, five fractions were obtained by column chromatography of the NBF fraction and were subsequently analysed by GC-O and GC–MS. Acetic acid, 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 3-methylpentanoic acid and phenylacetic acid were detected in the acidic fraction. However, the chiral compounds of interest (i.e. acetoin, linalool and phenylethanol) (Figure 1) were identified and isolated from fractions 3 and 4, respectively.

Figure 1. Enantiomeric separations of racemic mixtures of: (a) phenylethanol, (b) acetoin and (c) linalool.

Figura 1. Separaciones enantioméricas de mezclas racémicas de A) feniletanol, B) acetoína y C) linalol.

The analysis of the chiral compounds is shown in Table 2. Chiral GC analysis of the three palm wine varieties revealed three types of distribution patterns, namely the racemic form, an excess of (R) and an excess of (S). The results showed a high excess of laevorotatory enantiomers over the dextrorotatory enantiomers in all wines. Generally, the results revealed a high level of acetoin in all the wine varieties with concentrations ranging from 2437 to 6611 µg/L and an average ratio of S/R of 4:96–100:0. While the (R)-acetoin predominates in E. guineensis wine, the (S)-configuration was prevalent in the N. fruticans and B. flabellifer wines, respectively. The probable explanation for this observation might be due to the type of yeast strain responsible for the wine fermentation or the enzyme-catalysed processes that occurred during fermentation. While studies have shown that fermentation can induce stereo selective synthesis of some chiral compounds (Romano et al., 2000), other studies have reported the role of enzyme-catalysed processes in stereo selective synthesis of chiral compounds as well (Crout et al., 1986; Luddeke & Harder, 2011). For instance, Romano et al. (2000) reported the prevalence of (R)-acetoin in wines obtained by Saccharomyces cerevisiae as compared to those obtained by other yeast strains. However, in an earlier study, Crout et al. (1986) reported the production of acetoin by Klebsiella aerogenes during the condensation of pyruvate to α-acetolactate in a reaction catalysed by acetolactate synthase, followed by decarboxylation of the (S)-isomer in a reaction catalysed by acetolactate decarboxylase.

Table 2. Enantiomeric concentrations (µg/L) and enantiomeric ratios of some selected chiral compounds in E. guineensis, B. flabellifer and N. fruticans wines.

Tabla 2. Concentraciones enantioméricas (µg/L) y ratios enantioméricas de algunos compuestos quirales seleccionados de los vinos E. guineensis, B. flabellifer y N. fruticans.

Palm wine variety	Chiral compound	Racemic mixture	(S)-enantiomer	(R)-enantiomer	S/R
E. guineensis	Acetoin	6611 ± 123^A	264 ± 11^C	6346 ± 105^A	4:96
	Linalool	11.2 ± 0.1^B	10.6 ± 0.1^B	0.6 ± 0.0^A	95:5
	Phenylethanol	588 ± 25^A	588 ± 26^A	–	100:0
N. fruticans	Acetoin	3073 ± 55^B	3073 ± 55^A	–	100:0
	Linalool	12.6 ± 0.2^A	12.5 ± 0.1^A	0.1 ± 0.0^B	99:1
	Phenylethanol	547 ± 35^B	519 ± 56^B	274 ± 12^A	95:5
B. flabellifer	Acetoin	2437 ± 97^C	2193 ± 40^B	244 ± 10^B	90:10
	Linalool	10.7 ± 0.1^B	10.7 ± 0.1^B	–	100:0
	Phenylethanol	536 ± 34^C	429 ± 24^C	107 ± 15^B	80:20

Data are reported as mean ± SD (n = 3).

Values followed by different superscript letters between different varieties are significant (P < 0.05).

Los datos reportados corresponden a las medias ± DE (n = 3).

Los valores indicados con distintas letras superíndice entre las distintas variedades son significativos (P < 0,05).

Apart from the influence of the type of fermentation yeast and enzyme-catalysed processes that occurred during fermentation, there was a noticeable impact of varietal differences on the S/R ratio in the palm wine samples. Contrary to the results obtained for chiral acetoin in E. guineensiswine, the (S)-forms predominate in all wine varieties (Table 2). However, (S)-acetoin exhibited higher concentrations in N. fruticans and B. flabellifer wine varieties, while lower concentrations were found in (S)-linalool. The maximum enantiomeric (S)-form of acetoin was found in N. fruticans wine with an S/R average ratio of 100:0 and a concentration of 3073 µg/L. On the other hand, the least (S)-acetoin was obtained in E. guineensis wine with an enantiomeric ratio S/R of 4:96 (Table 2). In the case of linalool, the maximum enantiomeric (S)-form was produced in B. flabellifer wine with an S/R average ratio of 100:0 and a concentration of 10.7 µg/L. For phenylethanol, the maximum (S)-form was obtained in E. guineensis wine with an enantiomeric ratio of 100:0 and a concentration of 588 µg/L. Previous studies have shown that the enantiomeric ratio of chiral compounds can be used as important indicators of varietal differences and quality in wines (Askari, Hener, Schmarr, Rapp, & Mosandl, 1991; Chiavaro, Caligiani, & Palla, 1998). The enantiomeric ratio of acetoin is considered important in distinguishing between traditionally aged and common vinegars (Chiavaro et al., 1998). Similarly, linalool as well as phenylethanol has been used as an important indicator of good quality in wines (Askari et al., 1991; Carrau et al., 2005). In addition, the chiral compounds are known to exhibit clear odour distinctiveness between enantiomers. For instance, while the (S)-form of linalool has been described as sweet and petigrain-like, and the (R)-form as having a lavender-like note (Brenna, Fuganti, & Serra, 2003), the (S)-form of phenylethanol and its (R)-form are noted for their delicate hyacinth, strawberry and honey-like nuances, respectively (Szaleniec, 2007). On the other hand, (R)-acetoin is characterized by buttery note, while the (S)-form has been described as almond-like (Crout et al., 1986).

Generally, the three chiral compounds currently being studied are produced by the yeast metabolism during alcoholic fermentation (Loscos, Hernandez-Orte, Cacho, & Ferreira, 2007). However, the origins of these compounds are quite different and complex. In addition, each of their enantiomers also exhibited clearly different pathways. For instance, Luddeke and Harder (2011) studied the reaction of linalool dehydratase-isomerase (LDI) on the si-face of the prochiral β-myrcene, and they observed a high enantiospecific hydration reaction to (S)-linalool with an enantiomeric excess (ee) value of 95.4%. Similarly, Hummel (1990) demonstrated that the (R)-form of phenylethanol was predominantly produced by the dehydrogenase catalysing the NADPH-dependent reduction of acetophenone.

The effect of three-month storage on the concentration and the enantiomeric ratio of the chiral compounds are shown in Table 3. Noticeable differences were observed in enantiomeric ratio S/R and concentration of the chiral compounds. In all the wines, concentration of the (S)-form decreased during storage, whereas those of the (R)-form increased when compared with the fresh wines (Table 2). Similar trend was reported by Lytra et al. (2015) for white wines. In this study, results have shown that the maximum (R)-form concentrations were found in the chiral acetoin of all wines. For example, in E. guineensis wine, the maximum (R)-form of 5289 µg/L was obtained in acetoin with an enantiomeric ratio of 20:80. Similar trend was obtained in N. fruticans wine and B. flabellifer wine, respectively. Also, the maximum (S)-form concentration followed the same trend. The probable explanation for this might be due to the high initial concentrations of acetoin in the three wines. Moreover, the significant differences in the enantiomeric ratios of the chiral compounds after 3 months of storage might be due to racemization. Racemization of some natural flavour compounds such as 4-hydroxyl-2,5-dimethyl-3(2H)-furanone (HDMF, Furaneol®) and sotolone has been attributed to keto-enol-tautomerism (Pons, Lavigne, Landais, Darriet, & Dubourdieu, 2008; Raab et al., 2003). Under mild acidic medium of palm wine (pH 3–3.5), racemization occurs probably via a keto-enol tautomerism, catalysed by the presence of an acid (Raab et al., 2003). This phenomenon results in the decrease in the enantiomeric ratio. Under extreme acidic conditions (pH <3), the racemization will increase significantly and the ee value would decrease significantly.

Table 3. Enantiomeric concentrations (µg/L) and enantiomeric ratios of some selected chiral compounds in stored (3 months) palm wines (E. guineensis, B. flabellifer and N. fruticans).

Tabla 3. Concentraciones enantioméricas (µg/L) y ratios enantioméricas de algunos compuestos quirales seleccionados y almacenados durante 3 meses de los vinos de palma E. guineensis, B. flabellifer y N. fruticans.

Palm wine variety	Chiral compound	(S)-enantiomer	(R)-enantiomer	S/R
E. guineensis	Acetoin	1322 ± 31^C	5289 ± 115^A	20:80
	Linalool	9.97 ± 0.1^B	1.23 ± 0.0^C	89:11
	Phenylethanol	529 ± 36^A	1.12 ± 0.0^C	90:10
N. fruticans	Acetoin	2151 ± 75^A	922 ± 26^C	70:30
	Linalool	10.7 ± 0.1^A	1.89 ± 0.1^A	85:15
	Phenylethanol	475 ± 96^B	71 ± 5.2^B	87:13
B. flabellifer	Acetoin	1462 ± 40^B	975 ± 45^B	60:40
	Linalool	8.9 ± 0.1^C	1.8 ± 0.0^A	83:17
	Phenylethanol	428 ± 24^C	107 ± 15^A	80:20

Data are reported as mean ± SD (n = 3).

Values followed by different superscript letters between different varieties are significant (P < 0.05).

Los datos reportados corresponden a las medias ± SD (n = 3).

Los valores indicados con distintas letras superíndice entre las distintas variedades son significativos (P < 0,05)

Conclusion

The analysis by GC–MS/GC-O of the SAFE extracts from three palm wine varieties allowed the identification of 41 odorants. Of this, 37 odorants were identified in B. flabellifer wine, while E. guineensis and N. fruticans wines produced 41 and 35 odorants, respectively. Chiral GC analysis of the chiral compounds of interest (i.e. acetoin, linalool and phenylethanol) revealed different distributions and concentrations of their enantiomers. With the exception of E. guineensiswine, the other wines contained both (S)-and (R)-forms of the chiral compounds in various ratios. Generally, results revealed a high level of acetoin in all the wines with concentrations ranging from 2437 to 6611 µg/L and an average ratio of S/R of 4:96–100:0. Moreover, noticeable differences were observed in enantiomeric ratios and concentrations of the enantiomers of the compounds during storage. In all the wines, concentration of the (S)-form decreased during storage, whereas those of the (R)-form increased.

Acknowledgements

The author is grateful for the extensive financial support of the Fundamental Research grant scheme (No. 5524558) at the Univ. Putra Malaysia, Malaysia.

Disclosure statement

No potential conflict of interest was reported by the author.

Additional information

Funding

The author is grateful for the extensive financial support of The Malaysian higher education under fundamental research grant scheme (No. 5524558) at the Univ. Putra Malaysia, Malaysia.