Quantitation and Enantiomeric Ratios of Aroma Compounds Formed

Article pubs.acs.org/JAFC

Quantitation and Enantiomeric Ratios of Aroma Compounds Formed by an Ehrlich Degradation of L‑Isoleucine in Fermented Foods Katrin Matheis, Michael Granvogl, and Peter Schieberle* Lehrstuhl für Lebensmittelchemie, Department fuer Chemie, Technische Universitaet Muenchen, Lise-Meitner-Straße 34, D-85354 Freising, Germany ABSTRACT: The conversion of parent free amino acids into alcohols by an enzymatic deamination, decarboxylation, and reduction caused by microbial enzymes was first reported more than 100 years ago and is today known as the Ehrlich pathway. Because the chiral center at the carbon bearing the methyl group in L-isoleucine should not be prone to racemization during the reaction steps, the analysis of the enantiomeric distribution in 2-methylbutanal, 2-methylbutanol, and 2-methylbutanoic acid as well as in the compounds formed by secondary reactions, such as ethyl 2-methylbutanoate and 2-methylbutyl acetate, are an appropriate measure to follow the proposed degradation mechanism in the Ehrlich reaction. On the basis of a newly developed method for quantitation and chiral analysis, the enantiomers of the five metabolites were determined in a great number of fermented foods. Whereas 2-methylbutanol occurred as pure (S)-enantiomer in nearly all samples, a ratio of almost 1:1 of (S)and (R)-2-methylbutanal was found. These data are not in agreement with the literature suggesting the formation of 2methylbutanol by an enzymatic reduction of 2-methylbutanal. Also, the enantiomeric distribution in 2-methylbutanoic acid was closer to that in 2-methylbutanol than to that found in 2-methylbutanal, suggesting that also the acid is probably not formed by oxidation of the aldehyde as previously proposed. Additional model studies with (S)-2-methylbutanal did not show a racemization under the conditions of food production or during workup of the sample for volatile analysis. Therefore, the results establish that different mechanisms might be responsible for the formation of aldehydes and acids from the parent amino acids in the Ehrlich pathway. KEYWORDS: Ehrlich pathway, aroma-active compounds, L-isoleucine, quantitation, yeast, spirits, bread, dairy products

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INTRODUCTION

another metabolism in Ehrlich’s and Neubauer’s experiments, such as glucolysis. The identification of the key enzymatic steps in the Ehrlich pathway started when Sentheshanmuganathan and Elsden6 investigated the metabolism of the amino acid tyrosine to tyrosol, and further studies7 showed that cell extracts of Saccharomyces cerevisiae were able to transfer the amino groups of asparagine, isoleucine, leucine, methionine, norleucine, phenylalanine, tryptophan, and tyrosine to α-ketoglutarate. Thus, as already assumed by Ehrlich, the first step in amino acid catabolism is catalyzed by an aminotransferase activity. In addition, Sentheshanmuganathan7 investigated whether the transamination is followed by a decarboxylation or vice versa. Because tyramine was not found as an intermediate in the conversion of tyrosine into tyrosol, he concluded that the transamination forming the corresponding α-keto acid must be the first step. Consequently, the author7 proposed that tyrosol was formed by a decarboxylase activity and an NADHdependent reduction of the intermediate aldehyde. Decades later, Dickinson et al.8−11 and Perpète et al.12 analyzed the catabolism of the branched-chain amino acids isoleucine, leucine, valine and the aromatic amino acids phenylalanine and tryptophan as well as methionine in vivo by 13C NMR experiments combined with gas chromatography−mass spec-

Food fermentation by yeast strains or lactic acid bacteria is known to generate volatiles from amino acid metabolism, some of which are well-known as fusel alcohols, such as 2- or 3methylbutanol. However, although the name suggests a negative effect, these alcohols positively contribute to the overall aroma of fermented foods, such as spirits, beer, wine, bread, or dairy products. They are known to be formed by amino acid degradation via a pathway first suggested by Ehrlich a century ago.1 After isolation and characterization of the branched-chain amino acid isoleucine, he noticed structural similarities between the amino acid and amyl alcohol as well as between leucine and isoamyl alcohol. By addition of either isoleucine or leucine to fermentation mixtures with yeast, Ehrlich detected increased levels of the respective alcohols and proposed a “hydrating” enzyme responsible for their formation. In 1911, Neubauer and Fromherz2 proved that from 2-amino-2phenylacetic acid, yeast was able to generate α-ketophenylacetic acid and α-hydroxyphenylacetic acid as well as benzaldehyde and phenylmethanol. A transamination to the α-keto acid was proposed as the first step, followed by a decarboxylation to the respective aldehyde. Finally, the aldehyde should be reduced to the corresponding alcohol in parallel to a reduction of the αketo acid forming the α-hydroxy acid as byproduct. Thorne3,4 demonstrated that yeasts form all corresponding alcohols by a degradation of the respective parent amino acid, and Gale5 showed that the active transport of amino acids across the cell membrane is an energy-requiring process, which is provided by © XXXX American Chemical Society

Received: November 13, 2015 Revised: December 30, 2015 Accepted: December 30, 2015

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DOI: 10.1021/acs.jafc.5b05427 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

sodium sulfate. The formation of (S)-2-methylbutanal was followed by HRGC-FID. MS-EI, m/z (%) 57 (100), 41 (94), 46 (69), 58 (54), 43 (18), 45 (17), 74 (13), 86 (12, M+), 53 (7), 59 (6). MS-CI, m/z (%) 87 (100, [M + H]+), 69 (40), 58 (20). Retention index (RI) on OV-1701, 731; RI on BGB-174E, 1061. Ethyl (S)-2-Methylbutanoate. (S)-2-Methylbutanoic acid (2 mmol), ethanol (14 mmol), and sulfuric acid (100 μL) were dissolved in diethyl ether (50 mL) and stirred for 150 min at 30 °C. After cooling to room temperature, the reaction mixture was washed with an aqueous solution of sodium hydrogen carbonate (3 × 50 mL; 1 mol/ L), and the organic phase was dried over anhydrous sodium sulfate. The formation of ethyl (S)-2-methylbutanoate was followed by HRGC-FID. MS-EI, m/z (%) 57 (100), 102 (80), 85 (46), 74 (26), 87 (16), 73 (14), 56 (16), 55 (8), 58 (6), 103 (6). MS-CI, m/z (%) 131 (100, [M + H]+). RI on DB-FFAP, 1016; RI on BGB-176, 1173. Quantitation by Stable Isotope Dilution Assays. To aliquots of liquid food samples (1−100 mL, depending on the amounts of analytes determined in preliminary experiments), dichloromethane (30−300 mL) and the respective isotopically labeled internal standards (1−10 μg; dissolved in dichloromethane) were added and stirred for 30 min at room temperature. Then, the organic layer was subjected to high-vacuum distillation using SAFE.21 The distillate was separated into two fractions by extraction with an aqueous sodium bicarbonate solution (0.5 mol/L; 3 × 40 mL; pH 10) obtaining either the neutral/ basic volatiles or the acidic volatiles. The fractions were washed with brine (0.5 mol/L; 3 × 50 mL), dried over anhydrous Na2SO4, and concentrated to ∼100 μL using a Vigreux column (60 cm × 1 cm i.d.) followed by microdistillation. Solid samples (1−200 g) were frozen with liquid nitrogen and finely ground by a commercial blender. Dichloromethane (10−300 mL) and aliquots of the internal standards (1−10 μg; dissolved in dichloromethane) were added and stirred at room temperature for 90 min. After filtration and SAFE,21 the workup procedure was continued as described above for liquid samples. Two-Dimensional High-Resolution Gas Chromatography− Mass Spectrometry (HRGC/HRGC-MS). HRGC/HRGC-MS was performed with a Trace 2000 gas chromatograph (Thermo Quest, Mainz, Germany) coupled via a moving column stream switching system (Thermo Quest) to a CP 3800 gas chromatograph (Varian, Darmstadt, Germany). The column used in the first dimension was a 30 m × 0.32 mm i.d. DB-FFAP (0.25 μm film thickness) (J&W Scientific, Waldbronn, Germany) and in the second dimension either a 30 m × 0.25 mm i.d., BGB-174E or a BGB-176, respectively (0.25 μm film thickness) (BGB Analytik, Boeckten, Switzerland). Mass spectra were generated using a Varian Saturn 2000 ion trap mass spectrometer running in chemical ionization mode (MS-CI) at 70 eV using methanol as the reagent gas. The peak areas of the analyte and the labeled standard were determined from the mass traces of the respective protonated molecular masses or from selected fragments, respectively (Table 1). MS response factors were determined by measuring defined ratios (5 + 1, 3 + 1, 1 + 1, 1 + 3, 1 + 5) of the analyte and the corresponding stable isotopically labeled standard (Table 1). The degradation products of L-leucine, namely, 3methylbutanal, 3-methylbutanol, and 3-methylbutanoic acid as well as the esters formed therefrom, that is, ethyl 3-methylbutanoate and 3methylbutyl acetate, were eluted close to the respective metabolites from L-isoleucine. The following parameters were found to separate the respective metabolites in the first dimension on an achiral DBFFAP column: 2-methylbutanal and 2-methylbutanol, 35 °C for 10 min, then raised at 6 °C/min to 230 °C; ethyl 2-methylbutanoate, 2methylbutanoic acid, and 2-methylbutyl acetate, 40 °C for 2 min, raised at 1 °C/min to 60 °C, and finally at 10 °C/min to 230 °C. In the second dimension, the following chiral stationary phases and oven parameters were used: BGB-174E, 2-methylbutanal, 35 °C for 2 min, raised at 2 °C/min to 70 °C, and finally at 40 °C/min to 200 °C; 2methylbutanol, 30 °C for 30 min and finally raised at 40 °C/min to 200 °C; BGB-176: 2-methylbutanoic acid, 35 °C for 2 min, raised at 2

trometry (GC-MS). Their results showed that the amino acid catabolism induced by yeasts included three amino transferases, five decarboxylases, and six dehydrogenases. The exact combination of enzymes was found to depend on the respective amino acid, the carbon source, and the stage of growth of the yeast with the decarboxylase possessing the highest specificity. According to a recent review by Hazelwood et al.,13 it is now commonly accepted that the last step in the formation of alcohols in the Ehrlich mechanism is the reduction of the aldehyde to the corresponding alcohol. This reduction easily occurs, for example, when an aldehyde is offered to baker’s yeast.14 The amino acid catabolism of branched-chain, aromatic, and sulfur-containing amino acids by lactic acid bacteria was found to proceed via a transamination as the first step,15 resulting in an α-keto acid, followed by a decarboxylation to the corresponding aldehyde as shown for Streptococcus lactis var. maltigenes strains.16 The conversion of the aldehyde to the corresponding alcohol by an alcohol dehydrogenase of Streptococci was first proposed by Morgan in 1966.17 In contrast to all other proteinogenic amino acids, the degradation products formed from L-isoleucine are all chiral. Until the present, data on the enantiomeric distribution of the metabolites formed in foods are scarcely available. Also, the odor thresholds of the enantiomers of the five metabolites formed from L-isoleucine have not been published yet. Thus, the aim of this study was to develop a method for the separation and quantitation of the three volatile metabolites formed from L-isoleucine, that is, (R)- and (S)-2-methylbutanoic acid and (R)- and (S)-2-methylbutanol as well as (R)- and (S)-2-methylbutanal and the respective esters derived thereof, that is, ethyl (R)- and (S)-2-methylbutanoate as well as (R)and (S)-2-methylbutyl acetate. An additional challenge in method development was the separation of these 10 metabolites from the 5 odorants generated from L-leucine.

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MATERIALS AND METHODS

Fermented Food Samples. All food samples used in the study were purchased at local supermarkets. Chemicals. The following compounds were obtained from commercial sources: (R/S)-2-methylbutanoate (99%), ethyl 3methylbutanoate, 3-methylbutanal, (R/S)-2-methylbutanoic acid, (S)2-methylbutanoic acid, 3-methylbutanoic acid, (R/S)-2-methylbutanol, (S)-2-methylbutanol (≥95%, sum of enantiomers), 3-methylbutanol, 3-methylbutyl acetate (≥99%), oxalyl chloride, and 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (Dess−Martin periodinane) (Sigma-Aldrich, Taufkirchen, Germany); (R/S)-2-methylbutanal (Alfa Aesar, Karlsruhe, Germany); (R/S)-2-methylbutyl acetate (98%) (TCI Europe Fine Chemicals, Eschborn, Germany); dichloromethane, ethanol, sodium carbonate, sodium chloride, sodium hydrogen carbonate, sodium sulfate, sodium thiosulfate, and sulfuric acid (95−98%) (Merck, Darmstadt, Germany). Stable Isotopically Labeled Standards. The following stable isotopically labeled standards were prepared as previously described: [2H2]-ethyl 2-methylbutanoate,18 [2H2]-2-methylbutanal,19 [2H9]-2methylbutanoic acid,14 and [2H2]-3-methylbutanol.20 Syntheses. [2H2]-(S)-2-Methylbutyl acetate was synthesized by reduction of (S)-2-methylbutanoic acid chloride with lithium aluminum deuteride in a first step, followed by esterification of the formed [2H2]-(S)-2-methylbutanol with acetic acid. (S)-2-Methylbutanal. Dess−Martin periodinane (1.4 mmol) was added to (S)-2-methylbutanol (1.2 mmol) dissolved in dichloromethane (20 mL) and stirred for 90 min at room temperature. The mixture was washed with an aqueous solution of NaHSO4 followed by sodium thiosulfate (3 × 35 mL; 1:1:1, v/v/v) by solvent extraction in a separating funnel, and the organic phase was dried over anhydrous B


Article

Journal of Agricultural and Food Chemistry Table 1. Stable Isotopically Labeled Standards, Selected Ions, Response Factors (Rf), and GC Stationary Phases Used in the Stable Isotope Dilution Assays

Table 2. Retention Indices (RI) of Aroma-Active LIsoleucine and L-Leucine Degradation Products RI on

ion (m/z)a

odorant

labeling of internal standard

no.a analyte

standard

Rfb

ethyl 2methylbutanoate 2-methylbutanal

2

131

134

0.83

2

87

89

0.96

2-methylbutanoic acid 2-methylbutanolc

2

103

112

0.69

2

71

73

0.89

2-methylbutyl acetate

2

131

133

0.92

H3 H2 H9 H2 H2

column combination DB-FFAP/ BGB-176 DB-FFAP/ BGB-174E DB-FFAP/ BGB-176 DB-FFAP/ BGB-174E DB-FFAP/ BGB-176

Ions used for quantitation (MS-CI). bResponse factor determined by analyzing defined mixtures of the analyte and the internal standard. c 2 [ H2]-3-Methylbutanol was used for the quantitation of 2methylbutanol. °C/min to 102 °C, and finally at 40 °C/min to 200 °C; ethyl 2methylbutanoate and 2-methylbutyl acetate, 35 °C for 2 min, raised at 2 °C/min to 70 °C, and finally at 40 to 200 °C. Determination of Odor Thresholds in Air. Odor thresholds in air were determined by the following procedure:22 after adding the reference compound (E)-2-decenal (for 2-methylbutanoic acid, 2methylbutanol, ethyl 2-methylbutanoate, and 2-methylbutyl acetate) or octanal (for 2-methylbutanal), for which the odor thresholds in air were determined earlier, gas chromatography−olfactometry (GC-O) was performed on BGB-176 or on BGB-174E (for 2-methylbutanal), respectively. The solution was stepwise diluted (1:1, v/v), and GC-O of an aliquot was performed until no odorant was detectable. The odor thresholds in air were then calculated with the published odor thresholds of (E)-2-decenal and octanal (2.7 ng/L and 15 ng/L, respectively).23 Model Experiments. To check the possibility of racemization of (S)-2-methylbutanal by keto−enol tautomerism, several model experiments were performed using a solution of enantiopure (S)-2methylbutanal in phosphate buffer (KH2PO4/Na2HPO4·2H2O). Thereby, varying parameters such as pH, temperature, and time were applied. After treatment, the aqueous solution was extracted with dichloromethane (3 × 30 mL), and the combined organic layers were subjected to high-vacuum distillation (SAFE). The organic distillate was dried over anhydrous sodium sulfate and concentrated to a volume of ∼100 μL using a Vigreux column (60 cm × 1 cm i.d.) followed by microdistillation prior to HRGC/HRGC-MS. In addition, simultaneous distillation/extraction (SDE) according to Likens and Nickerson24 was performed at pH 4 and 8, respectively. After 120 min, the distillate/extract was dried over anhydrous sodium sulfate and concentrated to a volume of ∼100 μL using a Vigreux column (60 cm × 1 cm i.d.) followed by microdistillation prior to HRGC/HRGC-MS.

DB5MS

BGB176

BGB174E

−b −b −b

860 860 851

1039 1039 1028

1061 1067 1021

1a 1b 2

(R)-2-methylbutanal (S)-2-methylbutanal 3-methylbutanal

3a

1016

1045

1164

1087

1016

1045

1173

1087

4

ethyl (R)-2methylbutanoate ethyl (S)-2methylbutanoate ethyl 3-methylbutanoate

1050

1049

1175

898

5a 5b 6

(S)-2-methylbutyl acetate (R)-2-methylbutyl acetate 3-methylbutyl acetate

1111 1111 1118

1076 1076 1074

1208 1212 1219

1114 1114 929

7a 7b 8

(R)-2-methylbutanol (S)-2-methylbutanol 3-methylbutanol

1197 1197 1202

934 934 930

1227 1233 1231

1105 1109 1083

9a 9b 10

(S)-2-methylbutanoic acid (R)-2-methylbutanoic acid 3-methylbutanoic acid

1659 1659 1659

1435 1454 1191

1448 1448 1091

3b

a

odorant

DBFFAP

−c −c −c

a

Odorants were consecutively numbered according to their RI on the DB-FFAP column. bRI could not be calculated due to solvent overlapping. cRI could not be calculated due to poor elution properties.

leucine could be separated successfully (Table 2). The separation of the two enantiomers of 2-methylbutanal (1a and 1b) and 3-methylbutanal (2) was achieved by twodimensional GC-MS using a column combination of DB-FFAP and BGB-174E (Table 2). The same combination worked for the separation of 3-methylbutanol (8) and the enantiomers of 2-methylbutanol (7a and 7b). To separate 3-methylbutanoic acid (10) from the enantiomers of 2-methylbutanoic acid (9a and 9b), a combination of DB-FFAP with BGB-176 was used (Table 2). The same column combination was found to be appropriate for the separation of ethyl 3-methylbutanoate (4) from ethyl (R)- and ethyl (S)-2-methylbutanoate (3a and 3b) (Figure 1) and 3-methylbutyl acetate (6) and (S)- and (R)-2methylbutyl acetate (5a and 5b). The elution order of the enantiomers was determined on the respective chiral column. Therefore, solutions either of the racemic mixtures or of the (S)-enantiomers were mixed and analyzed. Odor Thresholds in Air. On the basis of the methods developed, the odor thresholds in air were determined using a previously published method.22 Ethyl (R)- and ethyl (S)-2methylbutanoate showed the lowest threshold of 0.33 ng/L (Table 3); however, there was no difference between both enantiomers. Small differences were found for 2-methylbutanal and 2-methylbutanol with the respective (S)-isomer showing the lower threshold (Table 3). The highest odor thresholds were found for (R)-2-methylbutyl acetate (>172 ng/L) and for its (S)-enantiomer (>187 ng/L). (S)-2-Methylbutanoic acid (18.4 ng/L) and (R)-2-methylbutanoic acid (23.4 ng/L) revealed similar odor thresholds to (S)-2-methylbutanol. In summary, the thresholds between the different compounds clearly varied, but the odor thresholds of the respective enantiomers of each compound differed only slightly.

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RESULTS AND DISCUSSION Method Development. First, a method for the separation of the enantiomers of the L-isoleucine degradation products, namely, 2-methylbutanal, 2-methylbutanol, and 2-methylbutanoic acid as well as the esters ethyl 2-methylbutanoate and 2methylbutyl acetate, was developed. In addition, the five corresponding metabolites from L-leucine interfering during GC separation also had to be separated. Therefore, different parameters (stationary phase, carrier gas flow, heating rate, and injection volume) were varied to obtain a separation of all 15 compounds. As shown by the retention indices, the 10 enantiomers from L-isoleucine and the 5 metabolites from LC


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Figure 1. Separation of (R)- and (S)-ethyl 2-methylbutanoate on BGB-176 and quantitation of the enantiomers in Pilsner beer.

Table 3. Odor Qualities and Odor Thresholds in Air for Enantiomers Derived from the Amino Acid L-Isoleucine no. 1a 1b 3a 3b 5a 5b 7a 7b 9a 9b

odorant

odor quality

(R)-2-methylbutanal (S)-2-methylbutanal ethyl (R)-2-methylbutanoate ethyl (S)-2-methylbutanoate (R)-2-methylbutyl acetate (S)-2-methylbutyl acetate (R)-2-methylbutanol (S)-2-methylbutanol (R)-2-methylbutanoic acid (S)-2-methylbutanoic acid

Table 5. Concentrations and Enantiomeric Ratios of (R)and (S)-2-Methylbutyl Acetate in Fermented Foods concna (μg/L)

odor threshold (ng/L) a

malty malty fruity fruity fruity fruity malty malty sweaty sweaty

3.3 1.5a 0.33b 0.33b >172b >187b >37.2b 10.5b 23.4b 18.4b

a

Odor threshold in air determined with octanal as reference compound.23 bOdor threshold in air determined with (E)-2-decenal as reference compound.23

Table 4. Concentrations and Enantiomeric Ratios of (R)and (S)-2-Methylbutanol in Fermented Foods concna (μg/L)

ratio (%)

fermented food

(S)enantiomer

(R)enantiomer

(S)

(R)

apricot brandy bourbon whiskey schnaps mezcal single-malt whiskey tequila Pilsner beer wheat beer wheat beer, alcohol-free red wine white wine black bread (pumpernickel) pretzel French bread rye bread sourdough wheat bread Emmental cheese kefir Parmesan cheese

7710 133000 15500 90000 173000 150000 8170 16000 890 58600 18200 250 118 5340 737 978 110 6330 53.3

nd nd nd nd nd nd nd nd nd nd nd 75.0 11.7 nd 79.2 nd 74.2 807 8.7

100 100 100 100 100 100 100 100 100 100 100 77 91 100 90 100 59 89 86

0 0 0 0 0 0 0 0 0 0 0 23 9 0 10 0 41 11 14

ratio (%)

fermented food

(S)enantiomer

(R)enantiomer

(S)

(R)


187 470 1220 105 765 117 135 492 111 156 202 24.3 33.4 20.9 32.1 33.1 12.5 634 78.7

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 17.7 nd nd

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 43 100 100

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 57 0 0

a Mean values of triplicates. Concentrations in solids are given in μg/ kg. nd, not detected.

the concentrations of the (S)- and (R)-isomers of 2methylbutanol were measured, and the enantiomeric ratio was calculated (Table 4). In all alcoholic beverages, only (S)-2methylbutanol occurred, which was clearly correlated with the (S)-configuration of the methyl group in L-isoleucine. The enantiomeric ratio of 2-methylbutanol in Dornfelder red wine had already been examined by Frank et al., revealing 100% of the (S)-enantiomer,25 which was in good agreement with the data obtained in the present study. Also in the bread samples, kefir, and Parmesan cheese, the (S)-isomer predominated, whereas in Emmental cheese a nearly racemic mixture of the alcohol was present. Compared to the other foods analyzed, this cheese is produced with propionic acid bacteria, which might follow a different metabolic pathway. On the basis of well-established biosynthetic pathways, in microorganisms 2-methylbutanol is enzymatically esterified with activated acetic acid. Consequently, the ester 2methylbutyl acetate should appear in the foods in the same enantiomeric ratio as the alcohol. This was confirmed for all

Mean values of triplicates. Concentrations in solids are given in μg/ kg. nd, not detected. a

Quantitation of the L-Isoleucine Metabolites in Alcoholic Beverages, Bread, and Dairy Products. First, D


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Figure 2. Metabolism of L-isoleucine ((2S,3S)-2-amino-3-methylpentanoic acid) by the Ehrlich mechanism.

Table 6. Concentrations and Enantiomeric Ratios of (R)and (S)-2-Methylbutanal in Fermented Foods concna (μg/L)

Table 7. Influence of pH Value, Heating Conditions, and Treatments during Workup on the Enantiomeric Ratio of 2Methylbutanala

ratio (%)

fermented food

(S)enantiomer

(R)enantiomer

(S)

(R)


26.3 82.5 149 131 163 204 2.06 3.41 5.78 14.3 4.02 24.4 181 48.8 24.4 22.0 53.6 33.3 63.9

18.9 42.5 71.1 80.0 130 72.1 0.79 2.02 2.73 6.11 3.08 9.50 161 45.2 9.40 20.8 27.8 17.5 39.1

58 66 68 62 56 74 73 64 68 69 58 65 53 52 72 51 66 66 63

42 34 32 38 44 26 27 36 32 31 42 35 47 48 28 49 33 34 37

enantiomeric ratio (%) expt

pH

time (min)

temp (°C)

(S)

(R)

1 2 3 4 5 6 7 8 9 10 11 12 13b 14b 15c 16d

3 4 5 6 7 8 9 12 8 8 8 8 4 8 10 6

10 10 10 10 10 10 10 10 10 20 40 60 120 120 2 10

80 80 80 80 80 80 80 80 80 80 80 80 100 100 22 100

99.1 99.1 99.1 97.5 91.5 89.5 56.3 50.5 89.5 83.2 77.4 79.8 98.6 85.9 97.6 93.0

0.9 0.9 0.9 2.5 8.5 10.5 43.7 49.5 10.5 16.8 22.6 20.2 1.4 14.1 2.4 7.0

a

2-Methylbutanal used contained 99.1% of the (S)-enantiomer. Treatment was done in a simultaneous steam distillation/extraction (SDE) device.24 cExtraction at room temperature was done with an aqueous NaHCO3 solution (three times, each 30 s, pH 10). dThe amino acid, dissolved in phosphate buffer (pH 6.0), was reacted for 10 min at 100 °C in the presence of 2-oxopropanal. b

Mean values of triplicates. Concentrations in solids are given in μg/ kg. a

ingly neither in the alcoholic beverages nor in bread or dairy products was pure (S)-2-methylbutanal detected. The relative amount of the (S)-isomer was lowest in the pretzel (52%) and highest in tequila (74%) (Table 6). These results allow the conclusion that the respective alcohols may be formed from a different precursor rather than by a reduction of the aldehyde. Also, if a reductase would specifically reduce (S)-2-methylbutanal, a high amount of the (R)-2-methylbutanal should have been left. Because the methyl group in 2-methylbutanal is in the αposition to the carbonyl group, a racemization might be possible (Figure 3). To study this assumption, (S)-2-

Figure 3. Possible racemization of (S)-2-methylbutanal under alkaline conditions.

alcoholic beverages, for all bread samples, and Emmental cheese (Table 5). On the other hand, kefir and Parmesan cheese showed slight differences in the enantiomeric distribution compared to the respective alcohol (Tables 4 and 5). According to the scheme representing the Ehrlich mechanism (Figure 2), 2-methylbutanol should be formed from 2methylbutanal by an enzymatic reduction. However, surprisE


Article


Table 9. Concentrations and Enantiomeric Ratios of Ethyl (R)- and (S)-2-Methylbutanoate in Fermented Foods concna (μg/L)

Figure 4. Generation of 2-methylbutanal by a Strecker degradation of L-isoleucine.

(S)enantiomer

(R)enantiomer

(S)

(R)


875 159 1800 544 485 690 265 441 439 722 415 110 349 325 161 286 3100 166 494

197 35.8 222 112 24.5 162 11.5 261 257 nd 18.0 62.2 142 24.3 54.7 41.5 nd nd 159

83 89 89 83 95 81 97 69 63 100 98 64 71 93 75 94 100 100 76

17 11 11 17 5 19 3 31 37 0 2 36 29 7 25 6 0 0 24

(S)

(R)


177 39.2 2010 128 249 172 1.11 12.8 22.2 71.6 40.8 4.11 0.29 0.34 1.32 0.86 0.39 0.24 416

nd 1.65 10.6 nd 5.50 nd 0.09 1.35 2.90 nd nd 1.37 0.10 0.22 0.51 0.37 0.13 0.16 111

100 96 99 100 98 100 94 91 88 100 100 75 75 61 72 70 72 59 79

0 4 1 0 2 0 6 9 12 0 0 25 25 39 28 30 28 41 21

2-Methylbutanal can also be generated by a Strecker-type degradation of L-isoleucine initiated by α-dicarbonyls (Figure 4). To test the influence of this reaction on the ratio of the enantiomers, L-isoleucine was thermally degraded in the presence of 2-oxopropanal in a model experiment (expt 16, Table 7). The results showed that even under these conditions only a small racemization occurred, suggesting that a Strecker reaction running in parallel, for example, during food processing, might not influence the enantiomeric ratio. Although also the influence of different microbial strains on the racemization of 2-methylbutanal might be possible, the clear difference between the enantiomeric ratios in 2methylbutanol and 2-methylbutanal suggests a different formation mechanism of both metabolites. The formation of 2-methylbutanoic acid from L-isoleucine via the Ehrlich pathway is postulated to arise either directly from a decarboxylation of 2-oxo-3-methylpentanoic acid or by an oxidation of 2-methylbutanal, respectively (Figure 2). Thus, the enantiomeric distribution in 2-methylbutanoic acid was also determined in a series of fermented foods (Table 8). In all foods, the (S)-enantiomer clearly dominated. However, only in red wine, Emmental cheese, and kefir was the pure (S)-isomer found. In contrast, in both wheat beer samples, black bread, and pretzels, also a significant portion of the (R)-isomer was detected. The data for the Dornfelder red wine and for the whiskey agreed with previous results,25,26 suggesting that both pathways are obviously possible, with an oxidative decarboxylation from the 2-oxo acid being followed by microorganisms in wine, Emmental cheese, or kefir. The activated (S)-2-methylbutanoic acid is commonly esterified with ethanol in microbial fermentation, leading to ethyl (S)-2-methylbutanoate. The ester appeared in quite high concentrations in spirits and Parmesan cheese, but in lower amounts in beer and wine, and the bread samples as well as kefir and Emmental cheese showed only low concentrations

ratio (%)

fermented food

fermented food

(R)enantiomer


Table 8. Concentrations and Enantiomeric Ratios of (R) and (S)-2-Methylbutanoic Acid in Fermented Foods concna (μg/L)

ratio (%)

(S)enantiomer


methylbutanal was treated at different pH values (expts 1−8) (Table 7) and for different reaction times at pH 8 (expts 9− 12). The results showed that only heating under strong alkaline conditions (expts 7 and 8) led to complete racemization of the aldehyde. On the other hand, workup procedures such as steam distillation (expts 13 and 14) did not, if the pH was kept below 8. An extraction of a model solution containing (S)-2methylbutanal with a sodium bicarbonate solution at pH 10 (expt 15) also did not cause a racemization. F


Article


and studies on the formation of key odorants during dough processing. Z. Lebensm.-Unters. Forsch. 1995, 201, 241−248. (15) Jackson, H. W.; Morgan, M. E. Identity and origin of the malty aroma substance from milk cultures of Streptococcus lactis var. maltigenes. J. Dairy Sci. 1954, 37, 1316−1324. (16) Tucker, J. S.; Morgan, M. E. Decarboxylation of α-keto acids by Streptococcus lactis var. maltigenes. Appl. Microbiol. 1967, 15, 694−700. (17) Morgan, M. E.; Lindsay, R. C.; Libbey, L. M.; Pereira, R. L. Identity of additional aroma constituents in milk cultures of Streptococcus lactis var. maltigenes. J. Dairy Sci. 1966, 49, 15−18. (18) Guth, H.; Grosch, W. Quantitation of potent odorants of virgin olive oil by stable isotope dilution assays. J. Am. Oil Chem. Soc. 1993, 70, 513−518. (19) Schieberle, P.; Grosch, W. Changes in the concentrations of potent crust odourants during storage of white bread. Flavour Fragrance J. 1992, 7, 213−218. (20) Guth, H.; Grosch, W. Identification of the character impact odorants of stewed beef juice by instrumental analysis and sensory studies. J. Agric. Food Chem. 1994, 42, 2862−2866. (21) Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation − a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237−241. (22) Buettner, A.; Schieberle, P. Aroma properties of a homologous series of 2,3-epoxyalkanals and trans-4,5-epoxyalk-2-enals. J. Agric. Food Chem. 2001, 49, 3881−3884. (23) Teranishi, R.; Buttery, R. G.; Guadagni, D. G. Odor quality and chemical structure in fruit and vegetable flavors. Ann. N. Y. Acad. Sci. 1974, 237, 209−216. (24) Likens, S. T.; Nickerson, G. B. Detection of certain hop oil constituents in brewing products. Proc. Am. Soc. Brew. Chem. 1964, 5− 13. (25) Frank, S.; Wollmann, N.; Schieberle, P.; Hofmann, T. Reconstitution of the flavor signature of Dornfelder red wine on the basis of the natural concentrations of its key aroma and taste compounds. J. Agric. Food Chem. 2011, 59, 8866−8874. (26) Poisson, L.; Schieberle, P. Characterization of the key aroma compounds in an American bourbon whiskey by quantitative measurements, aroma recombination, and omission studies. J. Agric. Food Chem. 2008, 56, 5820−5826.

(Table 9). In all alcoholic beverages, except the alcohol-free wheat beer, the amount of the (S)-isomer was >90%. Because the enantiomeric ratio in the acid (Table 8) was not in full agreement with that in the ester (Table 9), it can be assumed that during mash fermentation a specific esterase could be involved in the formation of the ester. In conclusion, the reinvestigation of the Ehrlich pathway established that the respective alcohols are obviously not formed by a reduction of the respective aldehyde. Although this reduction easily occurs in model experiments, a direct reductive decarboxylation of the respective α-oxo acid might be more probable.

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AUTHOR INFORMATION

Corresponding Author

*(P.S.) Phone: +49 8161 71 2932. Fax: +49 8161 71 2970. Email: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Quantitation and Enantiomeric Ratios of Aroma Compounds Formed

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