Characterization of the Key Aroma Compounds in Two Commercial

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Characterization of the Key Aroma Compounds in Two Commercial Dark Chocolates with High Cocoa Contents by Means of the Sensomics Approach Carolin Seyfried# and Michael Granvogl*,#,§,&

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Department für Chemie, Lehrstuhl für Lebensmittelchemie, Technische Universität München, Lise-Meitner-Strasse 34, D-85354 Freising, Germany § Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Lehrstuhl für Analytische Lebensmittelchemie, Technische Universität München, Maximus-von-Imhof-Forum 2, D-85354 Freising, Germany & Institut für Lebensmittelchemie, Fachgebiet für Lebensmittelchemie und Analytische Chemie (170a), Fakultät Naturwissenschaften, Universität Hohenheim, Garbenstrasse 28, D-70599 Stuttgart, Germany ABSTRACT: The overall aroma of two orthonasally distinguishable dark chocolates with high cocoa content (90% CC and 99% CC) was decoded using the systematic sensomics approach, including solvent extraction, separation of the volatiles, identification using aroma extract dilution analysis (AEDA) based on gas chromatography−olfactometry (GC-O) combined with gas chromatography−mass spectrometry (GC-MS), quantitation by stable isotope dilution analysis (SIDA), calculation of odor activity values (OAVs), and recombination experiments. Sixty-nine aroma-active compounds were identified and quantitation of 49 compounds revealed 28 odorants in 90% CC and 30 aroma-active compounds in 99% CC with OAVs ≥ 1. Among them, dimethyl trisulfide, acetic acid, 2-methoxyphenol, 3-methylbutanoic acid, phenylacetic acid, vanillin, and linalool showed the highest OAVs. Subsequently, very high similarities of the reconstitution models, containing all odorants with OAVs ≥ 1 in their naturally occurring concentrations in odorless sunflower oil as matrix, proved the correct identification and quantitation of all key odorants in both dark chocolates. KEYWORDS: dark chocolate, aroma extract dilution analysis, stable isotope dilution analysis, odor activity value, aroma recombination, sensory analysis

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INTRODUCTION Cocoa and its product, chocolate, rank among the most consumed luxury foods (about 9 kg/year/person in West European countries).1 Thereby, consumers’ attention is focused on sensory qualities generated along the complex manufacturing process, e.g., aroma, taste, appearance, or melt viscosity. Several steps including drying, fermentation, and roasting have to be performed to obtain cocoa mass, the raw material for chocolate production, from cocoa beans. Chocolate manufacturing itself consists of mixing the ingredients, cocoa mass, sugar, cocoa butter, and lecithin, prior to refining and conching steps followed by a controlled cooling process.1,2 Because the olfactory sensation plays an important role in sensory perception, the first known extraction and analysis of the volatile fraction of cocoa was performed by Bainbridge and Davies already in 1912.3 Up to now, almost 600 volatile compounds have been identified in cocoa.4 Pioneering work in this field was also achieved by van Elzakker and van Zutphen,5 who analyzed a diethyl ether extract of cocoa butter using gas chromatography− flame ionization detection (GC-FID) and, thereby, tried to do a sensory examination of the effluent at the FID. The first work applying parts of the sensomics concept6,7 to cocoa products was performed by Schnermann and Schieberle8 and aimed at the identification of key odorants in milk chocolate and cocoa mass. Using aroma extract dilution analysis (AEDA) on the basis of gas chromatography−olfactometry (GC-O) in combination with gas chromatography−mass spectrometry © XXXX American Chemical Society

(GC-MS), 44 key odorants in milk chocolate and 37 in cocoa mass were identified with 3-methylbutanal, 2-ethyl-3,5dimethylpyrazine, vanillin, 2- and 3-methylbutanoic acid, and 2-methyl-3-(methyldithio)furan showing the highest flavor dilution (FD) factors in both samples. Studies on key odorants in dark chocolate before and after conching were previously performed by Counet et al.,9 applying gas chromatography−olfactometry (GC-O), AEDA, and GC-MS for identification and comparison of key odorants. Quantitation with standard addition revealed methylpropanal, 2-methylbutanal, 3-methylbutanal, phenylacetaldehyde, and linalool as important odorants with concentrations of ∼10 mg/kg. However, the contribution of each odorant to the overall aroma by calculation of odor activity values (OAVs) and recombination experiments was not tested. Studies on cocoa powder revealed 24 odorants with an OAV ≥ 1 and the respective recombinate was in good accordance to the original sample. Key odorants with the highest OAVs ≥ 100 were acetic acid, 3-methylbutanal, 3-methylbutanoic acid, phenylacetaldehyde, and 2-methylbutanal.10 Further investigations were focused on the influence of ingredients on the overall aroma of chocolate. Schmitt11 focused Received: November 8, 2018 Revised: January 29, 2019 Accepted: January 30, 2019

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DOI: 10.1021/acs.jafc.8b06183 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry on crumb chocolate, whose typical flavor is generated by the reduction of odorants originating from ingredients during the production process. The overall aroma of crumb chocolate could be reconstituted with 38 odorants (of which 15 had an OAV ≥ 1) in a deodorized matrix. Weiss12 analyzed key odorants in milk chocolate and its ingredients to monitor their generation or degradation during the manufacturing process. Besides studies on the precursors of Strecker aldehydes (2- and 3-methylbutanal, phenylacetaldehyde) in cocoa, Weigl13 recombined the flavor of Arriba cocoa beans with 31 odorants in a deodorized cocoa matrix. Liu et al.14 analyzed the differences between aroma-active compounds in dark and milk chocolate by means of AEDA and sensory evaluations, which they afterward combined in correlation analysis. Thereby, they were able to convert milk chocolate into dark chocolate and vice versa by addition of odorants with different concentrations to the respective chocolate. However, quantitation was only performed by external calibration and no recombination experiments were performed. Further investigations on the differences between milk and dark chocolate were performed by Schütt,15 revealing a correlation between the amount of lactones and the milky odor impression. However, an influence of the conching temperature on the formation of lactones could not be found. Furthermore, the aroma of milk chocolate was simulated in a deodorized chocolate matrix using 22 odorants, of which 13 had shown an OAV ≥ 1, but not of the dark chocolate. Fricke’s16 investigations were focused on creaminess and aroma recombination of milk crumb chocolate. She constituted, next to some lactones, methanethiol, 4-hydroxy-2,5-dimethyl3(2H)-furanone, 3-hydroxy-2-methyl-4H-pyran-4-one, and phenylacetaldehyde as drivers for creaminess. The aroma recombination of milk crumb chocolate was performed in an odorless chocolate matrix with 18 key aroma compounds (OAV ≥ 1) and several aroma compounds with an OAV < 1 to include possible synergistic effects. Therefore, also omission experiments were performed to prove the importance of aroma compounds with an OAV < 1 for the overall aroma. Thus, to the best of our knowledge, a systematic approach to characterize the overall aroma of dark chocolate using state-ofthe-art methodology is missing up to now. Therefore, the aim of this study was to decode the aroma of two olfactory distinguishable dark chocolates with different cocoa contents, applying the molecular sensory science concept consisting of (i) the identification of the odorants with a high aroma impact by aroma extract dilution analysis (AEDA) based on gas chromatography−olfactometry (GC-O) combined with gas chromatography−mass spectrometry (GC-MS), (ii) quantitation experiments by means of stable isotope dilution analysis (SIDA), (iii) calculation of odor activity values (OAVs), and (iv) recombination experiments.

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phenylacetate, ethyl 3-phenylpropanoate, (Z)-4-heptenal, 3-hydroxy-4,5dimethylfuran-2(5H)-one, indole, β-ionone, 2-isobutyl-3-methoxypyrazine, 2-isopropyl-3-methoxypyrazine, 2-methoxyphenol, 2-methylbutanal, 3-methylbutanal, 2-methylbutanoic acid, 3-methylbutanoic acid, 3-methylbutyl acetate, 3-methylindole, 2-methyl-3-(methyldithio)furan, 4-methylphenol, methylpropanoic acid, (E)-2-nonenal, (E,E)2,4-octadienal, octanal, (E)-2-octenal, 1-octen-3-one, pentanoic acid, 1-penten-3-one, pentyl acetate, phenylacetic acid, 2-phenylethanol, trimethylpyrazine (Sigma-Aldrich, Taufkirchen, Germany); hexyl acetate, 4-hydroxy-2,5-dimethylfuran-3(2H)-one, linalool (racemic mixture) (Fluka, Neu-Ulm, Germany); butanoic acid, 4-hydroxy-3methoxybenzaldehyde (vanillin) (Merck, Darmstadt, Germany); dimethyl trisulfide (Acros Organics, Geel, Belgium); 1-hexen-3-one, 4-vinylphenol (Alfa Aesar, Karlsruhe, Germany). 1-(2,6,6-Trimethyl1,3-cyclohexadien-1-yl)-2-buten1-one ((E)-β-damascenone) was a kind gift from Symrise (Holzminden, Germany). The following compounds were synthesized as previously reported: 2-acetyl-1-pyrroline, 17 3,4-dimethyl-5-pentylfurane, 13 and trans-4,5-epoxy-(E)2-decenal.18 The following chemicals were commercially obtained: liquid nitrogen (Linde, Munich, Germany); dichloromethane, diethyl ether, ethanol, hydrochloric acid (32%), anhydrous sodium carbonate, sodium chloride, anhydrous sodium sulfate (Merck); helium (4.6), hydrogen (5.0), nitrogen (6.0), synthetic air (Westfalen, Muenster, Germany); n-alkanes (C5−C26), methyl octanoate (Sigma-Aldrich). Dichloromethane and diethyl ether were freshly distilled prior to use. All other chemicals were at least of analytical grade. Stable Isotopically Labeled Internal Standards. The following standards were commercially obtained: [13C2]-ethyl phenylacetate, [13C2]-4-hydroxy-2,5-dimethylfuran-3(2H)-one, [13C2]-(E,E)-2,4-octadienal (Aromalab, Planegg, Germany); [2H9]-2-methylbutanoic acid, [2H5]-2-phenylethanol (EQ Laboratories, Augsburg, Germany); [2H3]-acetic acid, [13C2]-phenylacetic acid (Sigma-Aldrich). The following compounds were prepared as previously described: [2H3]-acetylpyrazine,19 [2H2−4]-2-acetylpyridine,20 [13C5]-2-acetyl-1pyrroline,17 [13C4]-2,3-butanedione,21 [2H2]-butanoic acid,22 [2H3]-2sec-butyl-3-methoxypyrazine, 23 [ 2 H 5−7 ]-(E)-β-damascenone, 24 [2H2−4]-(E,E)-2,4-decadienal,25 [2H3]-2,3-diethyl-5-methylpyrazine,26 [2H6]-dimethyl trisulfide,27 [2H3]-ethyl cinnamate,28 [2H3]-2-ethyl3,5-dimethylpyrazine, 26 [ 2 H 3 ]-ethyl hexanoate, 29 [ 2 H 9 ]-ethyl 3-methylbutanoate,30 [2H5]-ethyl methylpropanoate,30 [2H5]-ethyl 3-phenylpropanoate,28 [13C2]-hexyl acetate,31 [13C2]-3-hydroxy-4,5dimethylfuran-2(5H)-one,32 [2H3]-β-ionone,33 [2H3]-2-isobutyl-3methoxypyrazine,23 [2H3]-2-isopropyl-3-methoxypyrazine,23 [2H2]linalool,34 [ 2 H 3 ]-2-methoxyphenol, 26 [ 2 H 2]-3-methylbutanal,35 [2H11]-3-methylbutyl acetate,36 [2H3]-methyl 2-methylbutanoate,36 [2H3]-2-methyl-3-(methyldithio)furan,10 [2H3]-3-methylnonane-2,4dione,25 [2H2]-methylpropanoic acid,10 [2H2]-(E,E)-2,4-nonadienal,37 [13C2]-γ-nonalactone,38 [2H2]-(E)-2-nonenal,28 [2H2−4]-octanal,39 [2H2]-(E)-2-octenal,40 [2H2]-1-octen-3-one,25 [2H5]-2-phenylethyl acetate,15 [2H3]-trimethylpyrazine,41 [2H3]-vanillin.42 The concentrations of the stable isotopically labeled standards were determined prior to use as previously described.43 Isolation of the Volatiles. Separation into Neutral/Basic and Acidic Fractions. Chocolate was manually crushed into small pieces and aliquots (30 g) were extracted with diethyl ether (250 mL) by stirring for 90 min at room temperature (two times). The solvent was decanted between both extractions, and afterward the organic phases were combined. For the separation of the volatile from the nonvolatile compounds, high vacuum distillation using the solvent assisted flavor evaporation (SAFE) technique was applied.44 Due to the high fat content, the distillate obtained was subjected to a second distillation to remove remaining traces of cocoa butter. The final distillate was dried over anhydrous sodium sulfate, filtered, concentrated to about 50 mL using a Vigreux column (50 cm × 1 cm), and extracted with an aqueous sodium bicarbonate solution (0.5 mol/L; three times, 50 mL each). The organic phase containing the neutral-basic fraction (NBF) was washed with a saturated sodium chloride solution (three times, 50 mL each), dried over anhydrous sodium sulfate, filtered, and then concentrated at 42 °C to a volume of ∼500 μL using a Vigreux

MATERIALS AND METHODS

Chocolate Samples. The two chocolates with a cocoa content of 90% (90% CC) and 99% (99% CC) were purchased in a local supermarket. Both samples were stored under vacuum in the dark at 14 °C prior to analysis. Chemicals. The following reference compounds were commercially obtained: acetic acid, acetylpyrazine, 2-acetylpyridine, 2-acetyl2-thiazoline, 2-aminoacetophenone, 2-sec-butyl-3-methoxypyrazine, (E,E)-2,4-decadienal, decanoic acid, 2,3-diethyl-5-methylpyrazine, γ-dodecalactone, δ-dodecalactone, ethyl cinnamate, 2-ethyl-3,5(6)dimethylpyrazine, ethyl hexanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, ethyl methylpropanoate, 3-ethylphenol, ethyl B


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Journal of Agricultural and Food Chemistry column (50 cm × 1 cm) and microdistillation. The aqueous phase containing the acidic fraction (AF) was adjusted to a pH value of 2−3 using hydrochloric acid. Afterward, the odorants were extracted with diethyl ether (four times, 80 mL each). The combined organic phases were washed again with a saturated sodium chloride solution (three times, 50 mL each) and dried over anhydrous sodium sulfate. After filtration, the fraction was concentrated as described above for NBF. High-Resolution Gas Chromatography−Olfactometry (HRGC-O). HRGC-O was performed using a Carlo Erba Instruments type 5160 gas chromatograph (Hofheim, Germany) equipped either with a DB-FFAP (30 m × 0.25 mm i.d., 0.25 μm film thickness; J&W Scientific, Agilent Technologies, Waldbronn, Germany) or a VF-5ms (30 m × 0.25 mm i.d., 0.25 μm film thickness; Crawford Scientific, Lanarkshire, Scotland) capillary column to avoid overlooking of possibly coeluting flavor compounds. Both columns were used with a carrier gas flow rate (helium) of 1.2 mL/min. The injection of the sample (0.5 μL) was manually performed by the cold on-column technique at 40 °C. The column oven was first held at 40 °C for 2 min, raised with 6 °C/min up to 230 °C, and held for 5 min (NBF). Separation of AF was performed using a heating rate of 8 °C/min. At the end of the column, the effluent was split 1:1 using a Y-type quick-seal glass splitter (Chrompack, Frankfurt, Germany) connected to two deactivated fused-silica capillaries (0.32 mm i.d.) with identical length. Thus, a simultaneous detection was enabled at the sniffing port (held at 230 °C) and the flame ionization detector (FID, held at 250 °C; signal was recorded with an analogue writer (Servogor SE 120; BBC Goerz Metrawatt, Nuremberg, Germany)). Comparative Aroma Extract Dilution Analysis (cAEDA). cAEDA was performed to get a first impression of the compounds which may contribute to the overall aroma of the dark chocolates and to detect differences between both samples. Therefore, the same amounts of both chocolate samples were worked up (30 g), the distillates were concentrated to the same volume (500 μL), and identical volumes (1 μL) were used for HRGC-O. The distillates were stepwise diluted with diethyl ether 1 + 1 (by volume), and each dilution was analyzed via HRGC-O. For a better comparison of both chocolates, all dilutions were alternatingly analyzed starting with the concentrated sample downward to the highest dilution but skipping every second dilution (FD 1 of both chocolate samples, FD 4 of both chocolate samples, etc.), until reaching a dilution with no perceivable smell and then beginning again with FD 2 of both chocolate samples, FD 8 of both chocolate samples, etc. cAEDA was performed separately for the neutral/basic and the acidic fractions. Comprehensive Two-Dimensional Gas Chromatography− Time-of-Flight Mass Spectrometry (GC × GC-ToF-MS). GC × GC-ToF-MS was performed for distinct identification and prevention of any overlaying of volatile compounds with a Pegasus 4D GC × GCToF-MS. This system consisted of an Agilent Technologies GC Model 7890 A (Boeblingen, Germany), a dual-stage quad-jet thermal modulator, and a secondary oven coupled to a time-of-flight mass spectrometer (Leco, St. Joseph, MI). Aliquots of the sample (1 μL) were injected by a PAL autosampler (CTC Analytics, Zwingen, Switzerland) using the Gerstel KAS 4 injection system (Muehlheim an der Ruhr, Germany) in splitless mode at 20 °C. Separation in the first dimension was carried out on a DB-FFAP capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness; J&W Scientific) using helium as carrier gas (flow: 2 mL/min). The effluent of the first dimension was completely transferred onto the second column whereupon the thermal modulator (modulation time: 4 s) enabled a high resolution by the generation of two distinct trapping zones. Thereby, the effluent was cryofocused with liquid nitrogen before it was thermodesorbed onto the second column (VF-5ms, 2 m × 0.15 mm, 0.30 μm film thickness; J&W Scientific). Mass spectrometry was performed in the electron ionization (EI) mode at 70 eV. The ion source temperature was set at 230 °C, and the detector voltage was 1750 V. Spectra were acquired within m/z 35−350 at a rate of 100 spectra/s. Data evaluation was performed with GC Image (version 2.1, Lincoln, NE). Quantitation by Stable Isotope Dilution Analysis (SIDA). Manually crushed chocolate (0.2−40 g, amount depending on the analyte) was dissolved in diethyl ether (used volume = sample weight

× 10, but at least 20 mL). Then defined amounts of the stable isotopically labeled internal standards (dissolved in diethyl ether or dichloromethane, amounts depending on the respective analyte determined in previous experiments) were added, and the mixture was extracted twice for 1 h via stirring at room temperature. The combined organic extracts were then subjected to high vacuum distillation (SAFE technique),44 and the distillate obtained was dried over anhydrous sodium sulfate and concentrated to a volume of 0.2− 1 mL (depending on the analyte) using a Vigreux column (60 cm × 1 cm). Differing from the workup for identification, SAFE distillation was only performed once for quantitation, whereupon defatted cotton was used in the distillation apparatus to prevent fat drops in the distillate. Experiments were performed in triplicates for each chocolate. Furthermore, mixtures of the labeled standards and the unlabeled analytes (5 + 1, 3 + 1, 1 + 1, 1 + 3, and 1 + 5; m + m) were analyzed as described below, and the obtained data were used for the calculation of the response factors (Rf; obtained by standard curves, which were not forced to the origin) (Table 1). Quantitation was performed using three different gas chromatography−mass spectrometry systems (Table 1). Organic acids (acetic acid, butanoic acid, 2- and 3-methylbutanoic acid, methylpropanoic acid) were analyzed using GC-MS. Therefore, a Varian 431 gas chromatograph (Darmstadt) equipped with a DB-FFAP column (30 m × 0.25 mm i.d., 0.25 μm film thickness; J&W Scientific) coupled to a Varian 220 ion trap mass spectrometer running in the chemical ionization (CI) mode with methanol as reactant gas at 70 eV was used. Cold on-column sample injection (1 μL) was performed by a CombiPal autosampler (CTC Analytics). The temperature program started at 40 °C for 2 min, raised with 6 °C/min up to 230 °C, and held for 5 min. All other compounds were quantitated by two-dimensional gas chromatography due to their low amounts and possible overlaying by major volatile compounds. Therefore, either GC × GC-ToF-MS in EI mode (see above) or GC/GC-MS in CI mode was used. This system consisted of a Thermo Quest Trace 2000 series gas chromatograph (Egelsbach, Germany) equipped with a DB-FFAP column (30 m × 0.25 mm i.d., 0.25 μm film thickness) and connected to a Varian CP-3800 gas chromatograph equipped with an OV-1701 column (30 m × 0.25 mm i.d., 0.25 μm film thickness; both J&W Scientific). The samples (1 μL) were injected cold on-column with a CombiPal autosampler. Selected parts eluted from the first column (heart cuts) were transferred onto the second column by using the moving capillary stream switching (MCSS) system (Fisons Instruments, Mainz, Germany) and cryogen-focusing with liquid nitrogen. Mass spectra were obtained by a Varian Saturn 2000 ion trap mass spectrometer in CI mode with methanol as ionization gas at 70 eV. Differentiation of 2- and 3-Methylbutanoic acid. The volatile compounds 2- and 3-methylbutanoic could not be separated on both used capillary columns and were therefore analyzed by their different fragmentation patterns in EI mode (2-methylbutanoic acid: m/z 74, 3-methylbutanoic acid: m/z 60). First, a calibration curve was measured (EI mode) with reference compounds of both acids mixed in different known ratios (5 + 1, 3 + 1, 1 + 1, 1 + 3, and 1 + 5; m + m). This calibration was used to determine the ratio of both isomers in each chocolate extract. Later on, the total amount of both acids was quantitated in CI mode via SIDA using [2H9]-2-methylbutanoic acid as internal standard, and the amount of each isomer was calculated with the previously determined ratio. Aroma Profile Analysis (APA). APA was performed by a trained sensory panel evaluating six selected odor attributes (vanilla-like (vanillin), earthy (2-ethyl-3,5(6)-dimethylpyrazine), caramel-/honeylike (4-hydroxy-2,5-dimethylfuran-3(2H)-one + phenylacetic acid, 1 + 1), malty (3-methylbutanal), vinegar-like (acetic acid), and banana-like (3-methylbutyl acetate)) on a scale from 0 (not perceivable) to 3 (strongly perceivable) in steps of 0.5. Reference solutions containing the respective compounds 50 times above their odor thresholds were provided. Prior to analysis, the chocolates were frozen with liquid nitrogen and ground in a mill (DPA141 La Moulinette, Moulinex, Groupe SEB Deutschland, Frankfurt am Main, Germany), and an aliquot (2 g) was filled into a Teflon sensory vessel (40 mm i.d., total volume: 45 mL) covered with a lid. The sensory C


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Journal of Agricultural and Food Chemistry

Table 1. Stable Isotopically Labeled Standards, Selected Ions for Quantitation, Response Factors (Rf), and Systems Used for Stable Isotope Dilution Analysis ion (m/z)a compound acetic acid acetylpyrazine 2-acetylpyridine 2-acetyl-1-pyrroline 2,3-butanedione butanoic acid 2-sec-butyl-3methoxypyrazine (E)-β-damascenone (E,E)-2,4-decadienal 2,3-diethyl-5methylpyrazine dimethyl trisulfide ethyl cinnamate 2-ethyl-3,5dimethylpyrazine 2-ethyl-3,6dimethylpyrazineg ethyl hexanoate ethyl 2methylbutanoateh ethyl 3methylbutanoate ethyl methylpropanoate ethyl phenylacetate ethyl 3phenylpropanoate hexyl acetate 3-hydroxy-4,5dimethylfuran-2 (5H)-one 4-hydroxy-2,5dimethylfuran-3 (2H)-one

isotope label

ion (m/z)a

analyte

internal standard

system

Rf b

compound

H3 H3 2 H2−4 13 C5 13 C4 2 H2 2 H3

61 123 122 112 87 89 167

64 126 124−126e 117 91 91 170

Ic IId IId IId IId Ic IId

0.64 0.70 1.00 0.91 0.89 0.88 1.00

2

191 153 151

196−198e 155−157e 154

IId IId IId

0.90 0.96 1.00

β-ionone 2-isobutyl-3methoxypyrazine 2-isopropyl-3methoxypyrazine linalool 2-methoxyphenol 2-methylbutanali 3-methylbutanal 2-methylbutanoic acidj 3-methylbutyl acetate methyl 2methylbutanoate 2-methyl-3(methyldithio)furan 3-methylnonane-2,4dione methylpropanoic acid (E,E)-2,4-nonadienal γ-nonalactone (E)-2-nonenal (E,E)-2,4-octadienal octanal (E)-2-octenal 1-octen-3-one phenylacetic acid 2-phenylethanol 2-phenylethyl acetate trimethylpyrazine vanillin

2 2

H5−7 H2−4 2 H3 2

2

d

2

H6 H3 2 H3

127 177 137

133 180 140

II IIIf IId

0.82 0.98 0.67

−g

137

142

IId

1.00

2

H3 −h

145 131

148 139−141e

IId IId

1.00 0.84

2

131

140

IId

0.84

2

117

122

IId

0.91

13

164 179

166 184

IIIf IId

1.00 0.82

145 129

147 131

IId IId

0.99 0.93

128

130

IIIf

1.00

H9 H5 C2 H5

2

13

C2 C2

13

13

C2

isotope label

analyte

internal standard

system

Rfb

193 167

196 170

IId IId

0.94 0.87

2

153

156

IId

0.96

2

H2 H3 −i 2 H2 2 H9

137 124 87 87 103

139 127 89 89 112

IId IIIf IId IId Ic

1.00 1.00 0.95 0.95 0.96

2

131 117

142 120

IId IId

1.00 0.83

2

161

164

IId

0.81

2

171

174

IId

0.80

2

89 139 85 141 125 129 127 127 136 91 104 122 151

91 141 87 143 127 131−133e 129 129 138 96 109 125 154

Ic IId IIIf IId IId IId IId IId IIIf IIIf IIIf IIIf IIIf

0.92 0.99 1.00 1.00 0.92 1.00 0.79 0.56 0.82 0.81 0.76 0.90 0.94

2

H3 H3

2

H3

2

H11 H3

2

H3 H3

H2 H2 13 C2 2 H2 13 C2 2 H2−4 2 H2 2 H2 13 C2 2 H5 2 H5 2 H3 2 H3 2

a

Ions used for quantitation. bResponse factor (Rf) determined by analyzing defined mixtures of analyte and internal standard. cSystem I: GC-MS(CI). dSystem II: GC/GC-MS(CI). eQuantitation was performed using isotopologues. fSystem III: GC × GC-ToF-MS(EI). gQuantitation of 2-ethyl-3,6-dimethylpyrazine was performed using [2H5]-2-ethyl-3,5-dimethylpyrazine as internal standard. hQuantitation of ethyl 2-methylbutanaote was performed using [2H9]-ethyl 3-methylbutanaote as internal standard. iQuantitation of 2-methylbutanal was performed using [2H2]-3-methylbutanal as internal standard. jDifferentiation of 2- and 3-methylbutanoic acid was performed in EI mode as described in the experimental part.

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panel consisted of at least 20 trained members (weekly sensory training in describing and recognizing odor qualities) evaluating the samples at room temperature (21 ± 1 °C) in a single booth in a specific sensory room.45 Triangle Test. Triangle tests were performed to prove that there is a difference between both chocolates, which were prepared and presented as described above for APA.45 The triangle test was also performed with both recombinates. Determination of Orthonasal Odor Thresholds (OTs). To calculate OAVs, OTs were determined as previously described using odorless sunflower oil as matrix.46,47 Aroma Recombination. Aroma recombination experiments were performed for both chocolates by preparing a mixture of all quantitated odorants with an OAV ≥ 1 in their naturally occurring concentrations. Therefore, the odorants were dissolved in ethanol and added to odorless sunflower oil, which was used as matrix. Thereby, the added amount of ethanol did not exceed its odor threshold in sunflower oil (850 μg/kg). The recombinates were compared to the original chocolates by the sensory panel as described above for APA.45

RESULTS AND DISCUSSION

First of all, a triangle test, performed to check if there was an orthonasal difference between the overall aroma of the two commercial chocolates (90% and 99% cocoa content, respectively), revealed a significantly different odor perception (significance level α = 0.1%). Thus, comparative aroma profile analysis (cAPA) was performed to get a first hint with which odor attributes these differences might be correlated. cAPA indicated that both chocolates showed earthy, malty, and caramel-/honeylike odor impressions at the same intensities. Differences were found for the vanilla-like odor, which was more pronounced in 90% CC. However, vinegar-like and banana-like attributes were more intense in 99% CC (Figure 1). Identification of Key Aroma Compounds in the 90% Cocoa Chocolate. After volatile isolation by means of diethyl ether extraction, SAFE distillation and separation into neutralbasic (NBF) and acidic fractions (AF) were performed. These D


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Journal of Agricultural and Food Chemistry

fractions were used for AEDA to distinguish between odoractive and nonodor-active compounds. AEDA revealed 77 odor-active regions in an FD factor range from 2 to 4096, with 60 compounds in NBF and 17 in AF. In NBF, the highest FD factor of 2048 was obtained for 35 (cooked meat-like odor), followed by 49 (cinnamon-like, flowery), 59 (fruity, soapy, cinnamon-like) (both FD factor of 1024), 17 (citrus-like, aniseed-like), 22 (earthy), 23 (earthy), 24 (pea-like, roasty), 47 (honey-like, flowery), 50 (flowery, honey-like), 53 (metallic), 60a,b (phenolic, leather-like), and 69 (phenolic, earthy) (all FD factor of 512) (Table 2). In AF, 48 (smoky, gammon-like) and 74 (vanilla-like) were obtained with the highest FD factor of 4098, followed by 33a,b (both fruity, sweaty), 61 (goat-like), 62 (seasoning-like, spicy), and 73 (beeswax-like, honey-like) (all FD factor of 2048) (Table 3). Identification of the odor-active compounds was performed by comparison of retention indices on two capillary columns of different polarities (DF-FFAP and VF-5ms), odor qualities and intensities perceived at the sniffing port, and mass spectra

Figure 1. Comparative aroma profile analysis of 90% CC (broken line) and 99% CC (solid line). The intensities of the orthonasally perceived attributes were scored on a scale from 0 (not perceivable) to 3 (strongly perceivable).

Table 2. Comparison of FD Factors (≥2) Determined in the Neutral/Basic Fraction of Both Chocolates RIa c

d

no.

compound

1a,b 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 23 24 25 26 27 29 30 31 34 35 36 37 38 39 41 42 43 45

2- and 3-methylbutanalh ethyl methylpropanoate 1-penten-3-one ethyl 2-methylbutanoate ethyl 3-methylbutanoate 1-hexen-3-one 3-methylbutyl acetate pentyl acetate ethyl hexanoate (Z)-4-heptenal hexyl acetate octanal 1-octen-3-one 2-acetyl-1-pyrroline dimethyl trisulfide trimethylpyrazine 3,4-dimethyl-2-pentylfuran (E)-2-octenal 2-isopropyl-3-methoxypyrazine 2-ethyl-3,6-dimethylpyrazine 2-ethyl-3,5-dimethylpyrazine 2,3-diethyl-5-methylpyrazine 2-sec-butyl-3-methoxypyrazine 2-isobutyl-3-methoxypyrazine (E)-2-nonenal linalool 3-ethyl-5-methyl-2-vinylpyrazinei (E,E)-2,4-octadienal 2-acetylpyridine acetylpyrazine 2-methyl-3-(methyldithio)furan (E,E)-2,4-nonadienal 3-methylnonane-2,4-dione ethyl phenylacetate dimethyl tetrasulfidei unknown 2-acetyl-2-thiazoline unknown (E,E)-2,4-decadienal

odor quality

e

malty fruity pungent, train oil-like fruity blueberry-like rubber-like, pungent banana-like fruity pineapple-like biscuit-like, fishy pear-like citrus-like, green mushroom-like popcorn-like, roasty cabbage-like earthy citrus-like, aniseed-like nutty, roasty earthy, pea-like earthy earthy earthy pea-like, roasty bell pepper-like fatty, green citrus-like, flowery roasty, popcorn-like fatty nutty, roasty popcorn-like cooked meat-like fatty, green hay-like, aniseed-like, fishy beeswax-like sulfury roasty, popcorn-like roasty, popcorn-like rubber-like, pungent fatty, deep-fried E

FD factorb

DB-FFAP

VF-5ms

90% CCf

99% CCg

933 967 1006 1038 1044 1094 1105 1181 1226 1238 1254 1283 1292 1329 1367 1379 1408 1421 1427 1433 1446 1479 1509 1514 1518 1536 1564 1577 1614 1657 1662 1695 1710 1715 1725 1745 1755 1780 1800

657 ndi 682 847 852 775 879 917 nd 901 nd 1004 879 922 968 1003 1196 1057 1093 1083 1083 1157 1168 1186 1146 1104 nd 1107 nd 1024 1182 1226 1252 1245 nd nd 1106 nd 1328

2 2 2 32 16 64 4 4 2 2 2 8 4 2 128 16 512 64 2 2 512 512 512 2 32 64 8 2 32 128 2048 32 64 64 32 128 2 32 64

2 8 2 32 8 64 4 4 2 2 2 8 4 ndj 256 32 128 128 2 4 1024 2048 512 2 16 128 32 4 32 128 8192 16 128 64 32 64 2 32 16


Article

Journal of Agricultural and Food Chemistry Table 2. continued RIa no.c 46 47 49 50 51 52 53 54 55 57 58 59 60a,b 63 66 68 69 70 72

compoundd (E)-β-damascenone 2-phenylethyl acetate ethyl 3-phenylpropanoate 2-phenylethanol β-ionone unknown trans-4,5-epoxy-(E)-2-decenal δ-octenolactonei γ-nonalactone 4-methylphenol unknown ethyl cinnamate 3- and 4-ethylphenolh 2-aminoacetophenone γ-dodecalactone δ-dodecalactone 4-vinylphenol indole 3-methylindole

FD factorb

odor qualitye

DB-FFAP

VF-5ms

90% CCf

99% CCg

baked apple-like, grape juice-like honey-like, flowery cinnamon-like, flowery flowery, honey-like flowery, violet-like honey-like metallic coconut-like coconut-like fecal, phenolic, horse stable-like mint-like fruity, soapy, cinnamon-like phenolic, leather-like foxy peach-like peach-like, coconut-like phenolic, earthy fecal, mothball-like fecal, mothball-like

1805 1811 1868 1905 1921 1947 1995 1995 2018 2082 2094 2128 2178 2212 2363 2388 2397 2440 2487

1389 1263 1350 1125 1492 nd 1396 1281 1360 1077 nd 1468 1193 nd 1677 1700 nd nd 1390

4 512 1024 512 4 32 512 256 2 32 32 1024 512 4 16 64 512 8 32

4 1024 4096 512 4 32 512 256 2 64 32 2048 512 4 16 64 512 8 32

Retention index determined using homologues series of n-alkanes (C5−C26 on DB-FFAP, C5−C18 on VF-5ms). bFlavor dilution factor determined on DB-FFAP during AEDA. cNumbering according to their retention indices on DB-FFAP. dOdorants were identified by comparison of retention indices on two capillaries with different polarities, the odor quality perceived at the sniffing port, and mass spectra (EI) with reference compounds. e Odor quality detected at the sniffing port. fChocolate with 90% cocoa. gChocolate with 99% cocoa. hFD factors were determined as sum of both isomers. iIdentification was performed by comparison of RI, odor quality, and mass spectra (EI) from database; no reference compound was available. nd: not detected. a

Table 3. Comparison of FD Factors (≥64) Determined in the Acidic Fraction of Both Chocolates RIa no.

c

21 28 32 33a,b 40 44 48 56 61 62 64 65 67 71 73 74

compound

d

acetic acid methylpropanoic acid butanoic acid 2- and 3-methylbutanoic acidh pentanoic acid unknown 2-methoxyphenol 4-hydroxy-2,5-dimethylfuran-3(2H)-one 4-ethyloctanoic acidi 3-hydroxy-4,5-dimethylfuran-2(5H)-one decanoic acid unknown unknown unknown phenylacetic acid vanillin

odor quality

e

vinegar-like sweaty, cheese-like sweaty fruity, sweaty sweaty glue-like, rubber-like smoky, gammon-like caramel-like goat-like seasoning-like, spicy soapy, musty musty musty, goat-like sweaty beeswax-like, honey-like vanilla-like

FD factor in chocolateb

DB-FFAP

VF-5ms

90% CCf

99% CCg

1444 1553 1619 1656 1733 1793 1860 2038 2193 2200 2250 2325 2367 2483 2564 2579

610 807 821 874 910 1007 1090 nd 1328 1108 1396 nd 1488 1222 1285 1446

128 64 1024 2048 1024 64 4096 128 2048 2048 256 64 256 1024 2048 4096

64 64 1024 4096 64 64 1024 128 256 2048 256 64 64 256 2048 128

a Retention index determined using homologues series of n-alkanes (C5−C26 on DB-FFAP, C5−C18 on VF-5ms). bFlavor dilution factor determined on DB-FFAP during AEDA. cNumbering according to their retention indices on DB-FFAP. dOdorants were identified by comparison of retention indices on two capillaries with different polarities, the odor quality perceived at the sniffing port, and mass spectra (EI) with reference compounds. e Odor quality detected at the sniffing port. fChocolate with 90% cocoa. gChocolate with 99% cocoa. hFD factors were determined as sum of both isomers. iIdentification was performed by comparison of RI, odor quality, and mass spectra (EI) from database; no reference compound was available. nd: not detected.

(EI mode) with the data obtained by authentic reference compounds available in an in-house database containing more than 1000 odor-active volatiles. Following this concept, the compounds with the highest FD factors in NBF could be identified as 2-methyl-3-(methyldithio)furan (35; cooked meat-like), ethyl 3-phenylpropanoate (49; cinnamon-like, flowery), and ethyl cinnamate (59; fruity, soapy, cinnamon-like), followed by 3,4-dimethyl-2-pentylfuran

(17; citrus-like, aniseed-like), 2-ethyl-3,5-dimethylpyrazine (22; earthy), 2,3-diethyl-5-methylpyrazine (23; earthy), 2-secbutyl-3-methoxypyrazine (24; pea-like, roasty), 2-phenylethyl acetate (47; honey-like, flowery), 2-phenylethanol (50; flowery, honey-like), trans-4,5-epoxy-(E)-2-decenal, (53; metallic), 3- and 4-ethylphenol (60a,b; phenolic, leather-like), and 4-vinylphenol (69; phenolic, earthy) (Table 2). The highest intensities in AF referred to 2-methoxyphenol (48; smoky, gammon-like), vanillin F


Article

Journal of Agricultural and Food Chemistry (74; vanilla-like), 2- and 3-methylbutanoic acid (33a,b; fruity, sweaty), 4-ethyloctanoic acid (61; goat-like), 3-hydroxy-4,5dimethylfuran-2(5H)-one (62; seasoning-like, spicy), and phenylacetic acid (73; beeswax-like, honey-like) (Table 3). Identification of Key Aroma Compounds in the 99% Cocoa Chocolate. Using the same procedure, 59 odor-active regions were detected in NBF and 17 in AF of 99% CC within an FD factor range from 2 to 8192. The highest FD factors in NBF were found for 2-methyl-3-(methyldithio)furan (35; FD factor of 8192), followed by ethyl 3-phenylpropanoate (49; 4096), 2,3-diethyl-5-methylpyrazine (23), and ethyl cinnamate (59; both 2048) (Table 2). The odor-active region with the highest FD factor of 4096 in AF was a mixture of 2- and 3-methylbutanoic acid (33a,b), followed by 3-hydroxy4,5-dimethylfuran-2(5H)-one (62), phenylacetic acid (73; both 2048), butanoic acid (32), and 2-methoxyphenol (48; both 1024) (Table 3). Quantitation of Key Aroma Compounds in Both Chocolates by Means of SIDA and Calculation of OAVs. AEDA is a screening method, which is a useful tool to get a first information about the compounds of the volatile fraction which may contribute to the overall aroma by interacting with the human olfactory receptors. However, in a next step, these compounds have exactly to be quantitated. Therefore, SIDA was used, which is the most accurate quantitative methodology. Usually, the concept follows the idea that all odorants from the highest FD factor down to the odorants revealing a 100 times lower FD factor than the highest have to be quantitated (e.g., highest FD factor of 8096 leads to quantitation until at least FD ≥ 64).6 However, in a complex matrix, such as chocolate, this rule may not be successful due to differences between odor thresholds in air (used at GC-O during AEDA) and the corresponding odor thresholds in oil (used for calculation of OAVs). Therefore, some additional compounds with low odor thresholds in oil were selected for quantitation. Furthermore, 2,3-butanedione and methyl 2-methylbutanoate were quantitated although they were not detected during AEDA but known to be important for the aroma of cocoa products.11,13,15,28 Concentration of Key Aroma Compounds. Among all 49 quantitated compounds, acetic acid revealed the highest concentration with about 250 mg/kg in 90% CC, followed by vanillin, 2-phenylethanol, phenylacetic acid, and 3-methylbutanoic acid, all in the concentration range of mg/kg. Lower amounts were obtained for 2- and 3-methylbutanal, 2-methoxyphenol, and linalool. Compounds only detectable in traces (≤ 2 μg/kg) were for example 2-methyl-3-(methyldithio)furan, 2-acetyl-1pyrroline, and the methoxypyrazines (Table 4). The 99% CC contained about 250 mg of acetic acid/kg as well, followed by phenylacetic acid, 3-methylbutanoic acid, vanillin, and 2-phenylethanol, all in the range of mg/kg. Lower amounts were found for methylpropanoic acid, 3-methylbutyl acetate, and linalool. 2-Methyl-3-(methyldithio)furan and the methoxypyrazines were only detectable in traces (about 2 μg/kg or lower) (Table 4). Calculation of OAVs. To obtain the OAV for an odorant, the ratio of the determined concentration and the respective odor threshold has to be calculated. Due to the fat content of about 50% in both chocolates, odor thresholds in oil were used. The highest OAVs in 90% CC were obtained for dimethyl trisulfide (840) and acetic acid (720), followed by 2-methoxyphenol (260), 3-methylbutanoic acid (120), phenylacetic acid (110), vanillin (100), linalool (35), 2,3-butanedione

Table 4. Concentrations of the Most Important Odorants in Both Chocolates concentration (μg/kg)a compound

90% CCb

99% CCc

acetic acid vanillin 2-phenylethanol phenylacetic acid 3-methylbutanoic acid 2-methylbutanal 3-methylbutanal 2-methoxyphenol methylpropanoic acid 2-methylbutanoic acid γ-nonalactone trimethylpyrazine 3-methylbutyl acetate 2-phenylethyl acetate linalool ethyl phenylacetate ethyl cinnamate butanoic acid 4-hydroxy-2,5-dimethylfuran-3(2H)-one 2-ethyl-3,6-dimethylpyrazine (E,E)-2,4-decadienal octanal 2,3-butanedioned 2-ethyl-3,5-dimethylpyrazine (E)-2-octenal ethyl hexanoatee dimethyl trisulfide ethyl 3-phenylpropanoate ethyl 3-methylbutanoate hexyl acetate 2-acetylpyridine β-ionone 3-methylnonane-2,4-dione (E,E)-2,4-nonadienal (E)-2-nonenal methyl 2-methylbutanoated,e (E)-β-damascenone (E,E)-2,4-octadienal acetylpyrazine ethyl 2-methylbutanoate 2,3-diethyl-5-methylpyrazine 3-hydroxy-4,5-dimethylfuran-2(5H)-onee 2-methyl-3-(methyldithio)furan ethyl methylpropanoate 1-octen-3-one 2-sec-butyl-3-methoxypyrazine 2-isobutyl-3-methoxypyrazine 2-acetyl-1-pyrroline 2-isopropyl-3-methoxypyrazine

252000 14200 3380 2750 1310 486 479 474 432 391 314 230 216 177 119 113 105 89.4 59.5 57.2 52.2 45.9 29.9 27.3 27.3 26.1 25.2 18.0 9.90 9.15 9.01 8.60 8.39 6.04 4.40 3.98 3.93 3.64 3.30 3.27 2.86 2.73 2.00 1.11 0.75 0.46 0.24 0.08 0.08

255000 1810 1480 3750 2270 92.6 186 20.4 564 747 264 245 500 273 297 135 59.0 130 31.1 55.6 22.8 56.4 13.7 40.1 13.6 23.3 21.9 8.67 3.73 15.7 8.68 8.63 14.6 2.79 1.41 3.63 4.83 6.21 5.35 1.18 11.3 3.96 2.35 5.84 0.60 0.58 0.36 nd 0.01

Mean values of triplicates, differing not more than ±15%. Chocolate with 90% cocoa. cChocolate with 99% cocoa. dNot detected during AEDA. eMean values of duplicates, differing not more than ±15%. nd: not detected. a

b

(33), and 3-methylbutanal (32). All in all, 28 quantitated compounds revealed an OAV ≥ 1 (Table 5). The highest OAVs in 99% CC were obtained as well for acetic acid and dimethyl trisulfide (both 730), followed by 3-methylbutanoic acid (210), phenylacetic acid (140), linalool G


Article

Journal of Agricultural and Food Chemistry Table 5. Odor Thresholds and Odor Activity Values (OAVs) of the Key Odorants in Both Chocolates OAVa compound dimethyl trisulfide acetic acid 2-methoxyphenol 3-methylbutanoic acid phenylacetic acid vanillin linalool 2,3-butanedione 3-methylbutanal 2-ethyl-3,5dimethylpyrazine 2-methylbutanal methyl 2-methylbutanoate 3-hydroxy-4,5dimethylfuran-2(5H)-one 3-methylnonane-2,4-dione ethyl 3-methylbutanoate ethyl 2-methylbutanoate 2-isopropyl-3methoxypyrazine β-ionone 2-phenylethanol 2-isobutyl-3methoxypyrazine 2-methyl-3-(methyldithio) furan 2-methylbutanoic acid 3-methylbutyl acetate butanoic acid

90% CCc

99% CCd

0.03 350 1.8 11 26 140 3.4e 0.9 15 1.7

840 720 260 120 110 100 35 33 32 16

730 730 11 210 140 13 87 15 12 24

34 0.3 0.23

14 13 12

3 12 17

0.78e 0.98 0.37 0.01

11 10 9 8

19 4 3 1

1.3e 490 0.04

7 7 6

7 3 9

0.37e

5

6

110 76 34

4 3 3

7 7 4

odor threshold in sunflower oil (μg/kg)b

OAVa compound 4-hydroxy-2,5dimethylfuran-3(2H)-one 2-acetyl-1-pyrroline trimethylpyrazine 2-sec-butyl-3methoxypyrazine 2,3-diethyl-5methylpyrazine hexyl acetate methylpropanoic acid (E,E)-2,4-decadienal 2-ethyl-3,6dimethylpyrazine (E)-β-damascenone (E,E)-2,4-octadienal ethyl hexanoate ethyl phenylacetate octanal γ-nonalactone (E)-2-octenal acetylpyrazine (E,E)-2,4-nonadienal 2-acetylpyridine ethyl 3-phenylpropanoate ethyl methylpropanoate (E)-2-nonenal ethyl cinnamate 2-phenylethyl acetate 1-octen-3-one

90% CCc

99% CCd

27

2

1

0.053 180 0.46

2 1 1

nd 1 1

7.2

Characterization of the Key Aroma Compounds in Two Commercial

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