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Characterization of Aroma-Active Compounds in Italian Tomatoes with Emphasis on New Odorants Johanna Kreissl and Peter Schieberle* Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Str. 34, D-85354 Freising, Germany S Supporting Information *

ABSTRACT: An aroma distillate was prepared by solvent extraction and subsequent SAFE distillation from Italian vine-ripe tomatoes eliciting an intense overall aroma. Application of gc/olfactometry and the aroma extract dilution analysis revealed 44 odor-active compounds, 42 of which could be identified. The highest odor activity value of 2048 was established for the green, grassy (Z)-3-hexenal, the metallic smelling trans-4,5-epoxy-(E)-2-decenal, the potato-like 3-(methylthio)propanal, and the caramel-like 4-hydroxy-2,5-dimethyl-3(2H)-furanone. Of the further odorants, 13 compounds have previously not been reported as tomato odorants. Although most of these showed lower FD-factors, in particular, the coconut/dill-like smelling wine lactone ((3S,3aS,7aR)-3a,4,5,7a-tetrahydro-3,6-dimethylbenzofuran-2(3H)-one) appeared with a quite high FD factor. In addition, a fruity, almond-like odorant (6) with an FD factor of 1024 was detected. By application of high resolution mass spectrometry and polarity considerations, the structure of a methyl-2-ethoxytetrahydropyran isomer was suggested for 6. Four of the five possible isomers, the 3-methyl-, 4-methyl-, 5-methyl-, and 6-methyl-2-ethoxytetrahydropyran were synthesized and showed similar mass spectrometric patterns. However, these were excluded by their different retention indices. Although the synthesis of the remaining 2-methyl-2-ethoxytetrahydropyran resulted in only small yields, which were not sufficient for NMR measurements, this structure is very likely for 6. This compound was never reported as a food constituent before. Finally, quantitation of 23 odorants by stable isotope dilution assays allowed for the preparation of an aroma recombinate resembling the overall aroma of the tomatoes. KEYWORDS: stable isotope dilution assay, 2-methyl-2-ethoxytetrahydropyran, 3-methyl-2-ethoxytetrahydropyran, 5-methyl-2-ethoxytetrahydropyran, 6-methyl-2-ethoxytetrahydropyran, wine lactone

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the human nose. Following this approach, Mayer et al.10 previously reported the highest odor activity values for (Z)-3hexenal, trans-4,5-epoxy-(E)-2-decenal, β-damascenone, β-ionone, and 1-octen-3-one in five different unprocessed tomato varieties. Further attempts to characterize odor-active compounds in unprocessed tomatoes with similar results were also reported by other groups.10−14 Compared to investigations on raw, unprocessed tomatoes, studies on the aroma compounds of thermally processed tomato products are rather scarce. Because our group is currently studying chemical changes in key aroma compounds on the way from raw tomatoes to processed tomato products, the need appeared to verify whether the complete set of odoractive compounds of tomatoes is already available in the current literature. Thus, using an Italian vine-ripe tomato from Sicily eliciting a very intense aroma, the aim of this study was to characterize the aroma active compounds by means of the Sensomics concept9 in order to provide a basis for further investigations on chemical changes in tomato odorants during a large-scale commercial process for tomato juice, sauce, and paste.

INTRODUCTION With a total consumption of 21 kg per person/per year,1 tomatoes or tomato products are among the most appreciated vegetable based foods in Germany. While about one-third of the tomatoes is consumed fresh, two-thirds are used commercially in the manufacturing of tomato juices, sauces, or purées. In 2014, the world production of tomatoes was about 171 million tons,2 with one tenth produced in the EU. Tomatoes are much appreciated by consumers due to their characteristic aroma, and therefore, several studies were already undertaken to identify the tomato volatiles and also to address changes in volatile composition during ripening.3−7 Thus, today, more than 400 tomato volatiles are known. Buttery et al.8 were the first to undertake experiments identifying those aroma compounds responsible for the aroma of unprocessed tomatoes. The authors determined the concentrations of selected volatiles and calculated their odor units (ratio of concentration to odor threshold). Sixteen compounds, among them (Z)-3-hexenal, β-ionone, hexanal, βdamascenone, and 1-penten-3-one, were found to either reach or exceed their odor threshold.8 However, because no gas chromatography/olfactometry was used in this study, potent odorants occurring in trace amounts might have been overlooked at that time. By applying the molecular sensory science concept (Sensomics), the complex odor image of a food can be elucidated.9 This bioactivity-guided approach allows one to identify all odorants interacting with the odorant receptors in © 2017 American Chemical Society

Received: Revised: Accepted: Published: 5198

March 10, 2017 May 30, 2017 June 1, 2017 June 2, 2017 DOI: 10.1021/acs.jafc.7b01108 J. Agric. Food Chem. 2017, 65, 5198−5208

Article

Journal of Agricultural and Food Chemistry

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C6), 3.34−3.40 (m, 1H, Hα-C7), 3.41−3.46 (m, 1H, Hα-C2), 3.41− 3.48 (m, 1H, Hβ-C7), 3.69−3.76 (m, 1H, Hβ-C2), 4.74 (t, J = 2.87 Hz, 1H, H−C3). 13 C NMR (HSQC, HMBC, 125 MHz; chloroform-d, 300 K): δ (ppm) 15.1 (CH3, C1), 17.4 (CH3, C8), 26.7 (CH2, C5), 30.0 (CH2, C4), 30.4 (CH, C6), 62.3 (CH2, C2), 66.2 (CH2, C7), 96.2(CH, C3). 6-Methyl-2-ethoxytetrahydropyran (6-Methyl-isomer). Starting from butenone (0.7 g, 10 mmol), the procedure described above for the 3-methyl-isomer was followed. MS-EI; m/z (%): 75 (100), 47 (63), 72 (53), 42 (51), 55 (42), 70 (36), 99 (20), 143 (M+ − 1, 9), 144 (M+,5). Isotopically Labeled Internal Standards. [2H6−7]-4-Methylphenol and [13C2]-phenylacetic acid were supplied by Sigma-Aldrich Chemie. The following isotopically labeled internal standards were synthesized as previously reported: [13C4]-2,3-butanedione,20 [2H2]butanoic acid,21 [2H4−6]-(E)-β-damascenone,22 [2H3−6]-(E,E)-2,4decadienal,23 [2H3−4]-hexanal and [2H2]-hexanoic acid,24 [2H2]-(E)2-hexenal,25 [2H2]-(Z)-3-hexenal,23 [13C2]-3-hydroxy-4,5-dimethyl2(5H)-furanone,26 [13C2]-4-hydroxy-2,5-dimethyl-3(2H)-furanone,27 [2H3]-4-hydroxy-3-methoxybenzaldehyde,28 [2H2]-linalool,29 [2H3]-2methoxyphenol, 30 [ 2 H 2 ]-3-methylbutanoic acid, 31 [ 2 H 3 ]-3(methylthio)propanal, and [2H3]-3-(methylthio)propanol;32 [2H2](E,Z)-2,6-nonadienal, [2H2]-(E)-2-nonenal, and [2H2−4]-1-octen-3one;23 and [2H2]-pentanoic acid,24 [2H2−3]-1-penten-3-one,33 [13C2]phenylacetaldehyde, 34 [2H2−4]-4-propyl-2-methoxyphenol,35 and [2H3]-wine lactone.17 [2H4]-trans-4,5-Epoxy-(E)-2-decenal. The epoxide was prepared in a four-step synthesis as follows: [2H4]-Hexanol. Wilkinson’s catalyst (tris(triphenylphosphine)rhodium(I) chloride, 0.5 g, 0.5 mmol) was suspended in toluene under nitrogen atmosphere. After replacing nitrogen by deuterium, the suspension was stirred for 30 min under a slightly increased pressure until the color changed from dark red to orange. Then, hexyn-1-ol (2.5 g, 25 mmol) dissolved in toluene (15 mL) was added to the activated catalyst. The mixture was flushed with deuterium and stirred for 18 h. To purify the product, pentane (50 mL) was added, and toluene was removed by flushing the reaction mixture over silica gel (G60, 100 g, water-cooled column, diameter 3.5 cm). The [2H4]-hexanol formed was purified by high-vacuum distillation (10 mPa, 45 °C). [2H4]-Hexanal. The oxidation of [2H4]-hexanol to [2H4]-hexanal was performed as described in ref 36 with some modifications. Under argon atmosphere, a suspension of Dess-Martin periodinane (1,1,1triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one, 3.5 g, 8.2 mmol) in dichloromethane was added dropwise to [2H4]-hexanol (0.8 g, 8 mmol) dissolved in dichloromethane (30 mL). After 1 h, the organic layer was washed with a sodium thiosulfate solution saturated with sodium hydrogen carbonate (1 mol/L, 2 × 75 mL), followed by an aqueous sodium hydrogen carbonate solution (100 mL) and finally with tap water (100 mL). The organic layer was dried over anhydrous sodium sulfate to yield [2H4]-hexanal. [2H4]-(E,E)-2,4-Decadienal. [2H4]-(E,E)-2,4-Decadienal was synthesized as previously reported23 with some variations. 1-Methoxy-1buten-3-in (0.84 g, 10 mmol) dissolved in anhydrous tetrahydrofuran (THF, 5 mL) was added dropwise to ethyl magnesium bromide (3 mL, 3.2 M) in anhydrous THF, and the mixture was kept at 40 °C. After stirring for 1 h at room temperature and cooling, [2H4]-hexanal (0.38 g, 3.65 mmol) dissolved in THF (5 mL) was added within 20 min. After 2 h at room temperature, the reaction was cooled and treated with dry ethanol (0.2 mL), and then lithium aluminum hydride (5.3 mL, 2.0 M) was added in small portions. The mixture was stirred for another 2 h and then washed with sulfuric acid (25 mL, 2 mol/L), followed by water (10 mL), sodium carbonate solution (10 mL, 10%), and finally water (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated by means of a Vigreux column. The target compound was purified by chromatography over silica gel (G60, 30 g, column diameter 2 cm). [2H4]-trans-4,5-Epoxy-(E)-2-decenal. The final product was obtained by oxidation of [2H4]-(E,E)-2,4-decadienal with 3-chloroperoxybenzoic acid.23 To a solution of [2H4]-(E,E)-2,4-decadienal (0.36 g,

MATERIALS AND METHODS

Materials. Italian vine-ripe tomatoes (Solanum lycopersicum L.) from Sicily were purchased at a local store. On the basis of their intense red color and intense aroma, tomatoes were considered as fully ripe. Sensory evaluations of six tomato samples from different origins by a sensory panel pointed to the Sicilian tomato as the one showing the most intense overall aroma. Chemicals. The following compounds were obtained from the sources given in parentheses: 4-allyl-2-methoxyphenol (eugenol) and acetic acid (Merck, Darmstadt, Germany); 2,3-butanedione (diacetyl), butanoic acid, (E,E)-2,4-decadienal, decanoic acid, ethyl butanoate, 4ethyloctanoic acid, (E,E)-2,4-heptadienal, heptanoic acid, hexanal, hexanol, hexanoic acid, (E)-2-hexenal, 3-hydroxy-4,5-dimethyl-2(5H)furanone (3-HDF; sotolon), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (4-HDF), 4-hydroxy-3-methoxybenzaldehyde (vanillin), 2-isobutylthiazole, linalool (3,7-dimethylocta-1,6-dien-3-ol), methyl salicylate, 3(methylthio)propanal (methional), 3-(methylthio)propanol (methionol), 3-methylbutanal, 3-methylbutanoic acid, 3-methylindole (skatole), 4-methylphenol (p-cresol), (E,E)-2,4-nonadienal, (E,Z)-2,6nonadienal, nonanal, (E)-2-nonenal, octanal, (E)-2-octenal, 1-penten3-one, phenylacetaldehyde, and phenylacetic acid (Sigma-Aldrich Chemie, Taufkirchen, Germany); (E)-β-damascenone, cis-4,5-epoxy(E)-2-decenal, and (Z)-4-heptenal were gifts from Symrise (Holzminden, Germany); 2-methoxyphenol (guaiacol) (Serva, Heidelberg, Germany); 1-octen-3-one (Lancaster, Mühlheim/Main, Germany); and 3-methyl-2,4-nonanedione (Chemos GmbH, Ratisbon, Germany). The following odorants were synthesized as previously reported: trans-4,5-epoxy-(E)-2-decenal,15 (Z)-1,5-octadien-3-one,16 and wine lactone ((3S,3aS,7aR)-3a,4,5,7a-tetrahydro-3,6-dimethylbenzofuran2(3H)-one).17 (Z)-3-Hexenal. The compound was synthesized following closely an approach previously reported.18 Dess-Martin periodinane (520 mg) (Sigma-Aldrich Chemie) was added to dichloromethane (15 mL, distilled, Merck), flushed with argon, and sealed with a septum. (Z)-3Hexenol (100 mg, 1 mmol) (Sigma-Aldrich Chemie) was dissolved in dichloromethane (5 mL), then added to the suspension, and stirred for 20 h at room temperature. The organic phase was subsequently washed with an aqueous sodium thiosulfate-solution (1 M, 50 mL) saturated with sodium hydrogen carbonate, followed by an aqueous sodium hydrogen carbonate solution (0.5 M, 50 mL), and finally with distilled water (50 mL). The organic phase was dried over sodium sulfate and contained 30% of the target compound. MS (EI): m/z (%) 41 (100), 39 (58), 42 (49), 55 (46), 69 (42), 83 (36), 57 (32), 98 (M+, 10). Syntheses of Methyl-2-ethoxytetrahydropyran Isomers. 3Methyl-2-ethoxytetrahydropyran (3-Methyl-isomer). Synthesis was performed as reported previously19 with some modifications. 2Propenal (acrolein, 0.56 g, 10 mmol) was added to ethyl propenyl ether (1.08 g, 12.5 mmol). After the addition of hydroquinone (11.0 mg, 0.1 mmol) and heating for 2 h at 185 °C in a sealed glass vessel, platinum(IV) oxide was added, and hydrogen was introduced. The mixture was stirred for 4 days under normal pressure until the reduction was complete. EI-MS: m/z (%): 75 (100), 42 (68), 47 (53), 55 (50), 99 (44), 70 (40), 144 (M+, 6), 143 (M+ − 1, 4). 4-Methyl-2-ethoxytetrahydropyran (4-Methyl-isomer). 2-Butenal (0.7 g, 10 mmol) was added to ethyl vinyl ether (0.9 g, 12.5 mmol). Further procedures were performed as described above for the 3methyl-isomer. EI-MS: m/z (%): 99 (100), 55 (59), 75 (49), 42 (46), 47 (34), 43 (31), 70 (26), 143 (M+ − 1, 13), 144 (M+, 2). 5-Methyl-2-ethoxytetrahydropyran (5-Methyl-isomer). Synthesis followed the procedure for the 4-methyl-isomer but replacing 2butenal by 2-methyl-2-propenal (0.7 g, 10 mmol). MS-EI; m/z (%): 99 (100), 55 (90), 75 (85), 47 (63), 42 (56), 72 (55), 81 (52), 70 (48), 143 (M+ − 1, 15), 144 (M+, 5). 1 H NMR (COSY, 500 MHz; chloroform-d, 300 K): δ (ppm) 0.82 (d, J = 6.8 Hz, 3H, H−C8), 1.22 (t, 7.2 Hz, 3H, H−C1), 1.41−1.59 (m, 2H, H−C5), 1.66−1.73 (m, 2H, H−C4), 1.70−1.77 (m, 1H, H− 5199

DOI: 10.1021/acs.jafc.7b01108 J. Agric. Food Chem. 2017, 65, 5198−5208

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Table 1. Isotopically Labeled Internal Standards, Selected Ions, and Response Factors Used in the Stable Isotope Dilution Assays odorant 4-allyl-2-methoxyphenol 2,3-butanedione butanoic acid 4-methylphenol (E)-β-damascenone (E,E)-2,4-decadienal trans-4,5-epoxy-(E)2-decenal hexanal hexanoic acid (E)-2-hexenal (Z)-3-hexenal 3-hydroxy-4,5-dimethyl-2(5H)-furanone 4-hydroxy-2,5-dimethyl-3(2H)-furanone 4-hydroxy-3-methoxybenzaldehyde linalool 2-methoxyphenol 3-methylbutanoic acid 3-(methylthio)propanal 3-(methylthio)propanol (E,Z)-2,6-nonadienal (E)-2-nonenal 1-octen-3-one pentanoic acid 1-penten-3-one phenylacetaldehyde phenylacetic acid wine lactone a

ion (m/z)a 165 87 103 109 191 153 139 83 131 99 81 129 129 153 137 125 117 105 89 139 123 127 117 85 121 137 167

labeled standard 2

[ H2−4]-propyl-2-methoxyphenol [13C4]-2,3-butanedione [2H2]-butanoic acid [2H6−7]-4-methylphenol [2H4−6]-(E)-β-damascenone [2H3−6]-(E,E)-2,4-decadienal [2H4]-trans-4,5-epoxy-(E)2-decenal [2H3−4]-hexanal [2H3]-hexanoic acid [2H2]-(E)-2-hexenal [2H2]-(Z)-3-hexenal [13C2]-3-hydroxy-4,5-dimethyl-2(5H)-furanone [13C2]-4-hydroxy-2,5-dimethyl-3(2H)-furanone [2H3]-4-hydroxy-3-methoxybenzaldehyde [2H2]-linalool [2H3]-2-methoxyphenol [2H2]-3-methylbutanoic acid [2H3]-3-(methylthio)propanal [2H3]-3-(methylthio)propanol [2H2]-(E,Z)-2,6-nonadienal [2H2]-(E)-2-nonenal [2H2−4]-1-octen-3-one [2H3]-pentanoic acid [2H2−3]-1-penten-3-one [13C2]-phenylacetaldehyde [13C2]-phenylacetic acid [2H3]-wine lactone

ion (m/z)a c

169−171 91 105 115/116 195−197c 156−159c 143 86/87c 134 101 83 131 131 156 139 128 119 108 92 141 125 129−131c 120 87/88c 123 139 170

Rfb 1.09 0.97 0.99 0.96 0.76 0.88 0.53 0.80 1.03 0.78 0.57 1.12 0.95 0.88 0.80 0.99 0.81 0.99 1.14 0.86 0.71 0.91 0.97 0.99 0.95 0.90 1.0d

Ions used to monitor the analyte or the internal standard (MS-CI). bMS response factor. cInternal standard was used as a mixture of isotopologues. A response factor of 1.0 was assumed.

d

μm (Varian, Darmstadt, Germany), the initial temperature of 40 °C was held for 2 min, then raised at 6 °C/min to 250 °C and held for 5 min. Samples were injected at 40 °C by means of the cold on column technique. For GC/O, the effluent was evenly split at the end of the column between a flame ionization detector (FID, 250 °C) and a heated sniffing port (200 °C) using a Y-shaped glass splitter and two deactivated fused silica capillaries (500 mm × 0.2 mm i.d.). Helium adjusted to a flow rate of 1.5 mL/min served as the carrier gas. Retention indices (RI) of the odorants were calculated from the retention times of n-alkanes by linear interpolation. Aroma Extract Dilution Analysis (AEDA). The undiluted distillate (100 μL) was subjected to GC/O to detect odor-active regions. This was done by three trained panelists. Following this, the flavor dilution factors (FD factors) of the odor-active compounds were assigned by stepwise dilution of the distillate 1:1 (v+v) using dichloromethane as solvent to obtain dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, and up to 1:2048 of the original extract. Sniffing of the dilutions was continued until no odorant could be detected. Each odorant was thus assigned an FD factor representing the last dilution in which the odorant was still detectable. The results of the three panelists, which differed to not more than plus/minus one dilution step, were averaged. Static Headspace−Gaschromatography−Olfactometry/ Mass Spectrometry (SH-GC-O/MS). A 1:1 mixture of tomatoes (30 g) and a saturated CaCl2-solution (30 mL) was placed in a 120 mL flask, sealed, and equilibrated at room temperature for 30 min. Ten milliliters of the headspace were withdrawn by means of a gastight syringe and injected via an on-column injector onto a deactivated capillary (250 mm × 0.53 mm i.d.) placed in a cold trap 915 (Thermo Fisher Scientific, Dreieich, Germany). During the injection, the volatiles were cryofocused at −150 °C, and the air was purged through a flow controlled (20 mL/min) valve. After the injection, the valve was closed, and the trap was immediately heated to 250 °C to transfer the

0.8 mmol) dissolved in dichloromethane (5 mL), small portions of 3chloroperoxybenzoic acid (0.16 mg, 1 mmol) in dichloromethane (5 mL) were added within 30 min. The reaction was stirred for 12 h at room temperature and then washed with an aqueous sodium hydrogen carbonate solution (0.5 mol/L, 2 × 20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated by means of a Vigreux column. Purification of the compound was achieved by chromatography over silica gel (G60, 30 g, diameter 2 cm). EI-MS: m/z (%): 68 (100), 81 (32), 40 (24), 39 (21), 44 (18), 57 (14), 41 (13), 55 (13), 43 (12), 45 (12), 69 (11), 46 (10), 107 (9), 143 (6), 156 (3). MS-CI (methanol); m/z (%): 68 (100), 143 (15), 103 (8), 83 (7), 110 (6), 155 (3), 174 (3). Isolation of Volatiles. Tomatoes (100 g) were puréed using a Multiquick 5 stick blender (Braun, Kronberg, Germany). After 5 min, enzymes were inhibited by adding saturated aqueous calcium chloride solution (100 mL) as suggested by Buttery et al.3 The tomato/CaCl2 mixture was extracted with dichloromethane (100 mL) by stirring for 15 min at room temperature. To break the emulsion, the mixture was centrifuged for 10 min at 4500 rpm. The organic layer was separated, the aqueous phase was extracted twice with dichloromethane (2 × 50 mL), and the combined organic layers were dried over anhydrous sodium sulfate. Volatiles were separated from the nonvolatile material by SAFE distillation,37 and the resulting distillate was concentrated to 200 μL at 45 °C using a microdistillation apparatus. Gas Chromatography/Olfactometry (GC/O). GC/O was performed by means of a GC 8000 gas chromatograph (Carlo Erba Instruments, Hofheim, Germany), using the following fused silica capillary columns and temperature programs. DB-FFAP: 30 m × 0.32 mm i.d., 0.25 μm (J&W Scientific, Folsom, USA), the initial temperature of 40 °C was held for 2 min, then raised at 6 °C/min to 230 °C and held for 5 min. CP Sil 8 CB: 30 m × 0.32 mm i.d., 0.25 5200


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Journal of Agricultural and Food Chemistry compounds onto a fused silica capillary (30 m × 0.25 mm i.d., 1 μm, DB-5) (J&W Scientific). An initial temperature of 0 °C was held for 2 min, then the temperature was raised at 6 °C/min to 230 °C and held for 5 min. Helium was used as the carrier gas. At the end of the column, the effluent was transferred to an FID, a sniffing-port, and a Saturn 2100 T ion trap mass spectrometer (Varian, Darmstadt, Germany). Spectra were generated in the chemical ionization mode (CI-MS) with methanol as the reagent gas. Gas Chromatography/Mass Spectrometry (GC/MS). For the identification and high resolution (HRGC-MS) experiments, mass spectra were recorded using a 5890 series II gas chromatograph (Hewlett-Packard, Waldbronn, Germany) connected to an MAT 95 S sector field mass spectrometer (Finnigan, Bremen, Germany) at 70 eV in the electron impact mode (EI-MS) and at 115 eV in the chemical ionization mode (CI-MS; reagent gas: isobutane) using the capillaries and the oven programs described above. Two Dimensional (Heart Cut) Gas Chromatography− Olfactometry/Mass Spectrometry (GC-GC/O/MS). To obtain unequivocal mass spectra, e.g., in the case of the trace wine lactone, GC-GC/O/MS was performed. An FFAP column (30 m × 0.32 mm i.d., 0.25 μm (J&W Scientific), installed in the first Mega 2 Series GC (Fisons Instruments, Mainz, Germany), was connected to the Moving Column Stream Switching (MCSS) system (Fisons Instruments, Mainz, Germany). This was connected to an FID and the first sniffingport by a Y-type splitter, and to a transfer line via a cold trap with the column in the second Mega 2 Series GC (Carlo Erba, Hofheim, Germany). Volatiles were cryofocused in the trap with liquid nitrogen at −80 °C. The end of the second capillary was coupled via a Y-type splitter to an ITD 800 ion trap mass spectrometer (Finnigan, Bremen, Germany) and a sniffing-port. The trap was quickly heated to 250 °C, and the volatiles were transferred to the second GC equipped with a DB-5 capillary (30 m × 0.25 mm i.d., 0.25 μm) (J&W Scientific). Quantitation by Two Dimensional (Heart Cut) Gas Chromatography/Mass Spectrometry (GC-GC/MS). On a Trace Ultra gas chromatograph (Thermo Fisher Scientific), a Combi PAL autosampler (CTC Analytics) was mounted. The GC was equipped with a cold on-column injector and an FFAP capillary (30 m × 0.32 mm i.d., 0.25 μm) (J&W Scientific). The end of the capillary was connected via the MCSS by a Y-type splitter to an FID, a sniffing-port, or a heated transfer line (250 °C) leading to a cold trap cooled with liquid nitrogen located in the CP-3800 s GC (Varian, Darmstadt, Germany). The transfer line led into a Saturn 2200 ion trap mass spectrometer (Varian) operated in the CI-MS mode with methanol as the reactant gas. Quantitation by Stable Isotope Dilution Assays (SIDA). Volatile Isolation. Several tomatoes were puréed and weighed, and enzymes were inhibited after 5 min by using the same volume of an aqueous saturated calcium chloride solution. Depending on the concentrations of the aroma compounds determined in preliminary experiments, different amounts of the sample (from 2 to 100 g) were extracted to obtain concentrations between 1 and 5 μg/mL of the respective analyte. The internal standards dissolved in dichloromethane were added, and the mixture was equilibrated for 30 min. The isolation followed the procedure described above for GC/O analysis, and the extracts obtained were analyzed by means of GC-GC/MS. Concentrations were calculated from the area counts of selected MS fragments for the analyte and the internal standard (Table 1) as recently reported for linalool.29 Because of the fast degradation of (Z)-3-hexenal during freezing/ thawing, this compound was always analyzed in tomatoes before freezing the fruits. For all other odorants, frozen fruits were crushed, and after 5 min, the saturated CaCl2 solution was added. Further procedures were performed as described above. Quantitation by Mass Spectrometry. A response factor (Table 1) was determined for each compound by analyzing mixtures containing defined amounts of the unlabeled odorants and the respective labeled standard in five different ratios (5:1, 3:1, 1:1, 1:3, and 1:5). Time Course of the Formation of Hexanal and (Z)-3-Hexenal during Storage of Tomato Pureé . After crushing the tomatoes, the purée was stored for 5, 10, 60, 120, 180, or 300 min, respectively, and

10 mL of a saturated calcium chloride solution was added to 10 mg of purée after the storage period. Volatiles were then extracted by adding 20 mL of dichloromethane containing [2H3−4]-hexanal and [2H2]-(Z)3-hexenal and isolated as described above. One intact tomato was immediately puréed with an equal volume of saturated calcium chloride to obtain a zero value. For freezing experiments, several tomatoes were stored at −25 °C for 24 h. The frozen tomatoes were puréed for 5 min and worked up as described above. Odor Thresholds. For the calculation of odor activity values, odor thresholds in water were either determined by means of the triangle test in increasing concentrations or were used from the literature.38 Aroma Recombinate. All odorants showing an OAV ≥ 1 in tomatoes were mixed in an aqueous buffer (5 mmol citric acid, pH 4.4) in the concentrations measured in the tomato. A sensory panel of 20 participants 22−45 in age rated the intensities of selected aroma attributes on a seven-point scale from 0 (not perceivable) to 3 (strongly perceivable). Samples were presented in covered Teflon vessels (15 g each), and solutions of single aroma compounds reflecting each aroma attribute were presented in concentrations about 100-fold above their odor threshold. The following odorants were chosen: (Z)-3-hexenal (grassy and green), 1-penten-3-one (pungent), (E,E)-2,4-decadienal (fatty), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel-like), trans-4,5-epoxy-(E)-2-decenal (metallic), and 2,3butanedione (butter-like). The panel was trained weekly to perform a comparative aroma profile analysis, which showed a quite good similarity in the overall rating of the descriptors during training with model mixtures. The evaluation of the descriptors showed a good day to day variation among the panel members of about 20%. Nuclear Magnetic Resonance Experiments. 1D- and 2D-NMR experiments (1H, 13C, 1H−1H COSY, HSQC, and HMBC) were performed on an Avance III 500 MHz spectrometer with a cryo-TCI probe (Bruker, Rheinstetten, Germany) at 300 K. Benzene-d6, chloroform-d, and methanol-d4 were obtained from Euroiso-Top (Saarbrücken, Germany).

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RESULTS AND DISCUSSION In a preliminary experiment, the overall aroma of seven tomatoes from the trade and from different origins was

Figure 1. Flavor dilution (FD) chromatogram of the volatile fraction isolated from Italian tomatoes.

evaluated, and the panel was asked to rank the samples according to the intensity of the aroma. An Italian wine-ripe tomato was rated to elicit the most intense aroma and was therefore selected for the analyses. Identification of Odor-Active Compounds. A fresh tomato purée was treated with saturated CaCl2 solution39 and 5201


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Journal of Agricultural and Food Chemistry Table 2. Compounds (FD ≥ 4) Identified in a Distillate Prepared from Italian Tomatoes RIc on a

no.

aroma compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

3-methylbutanal 2,3-butanedione 1-penten-3-one hexanal unknown 2-methyl-2-ethoxytetrahydropyran (Z)-3-hexenal (E)-2-hexenal (Z)-4-heptenalf octanal 1-octen-3-one hexanol nonanal (Z)-1,5-octadien-3-onef 2-isobutylthiazol (E)-2-octenal 3-(methylthio)propanal unknown (Z)-2-nonenalf unknown (E)-2-nonenal linalool (E,Z)-2,6-nonadienalf (Z)-2-decenal butanoic acid phenylacetaldehyde (E,Z)-2,4-nonadienalf 2- and 3-methylbutanoic acidg (E,E)-2,4-nonadienalf 3-(methylthio)propanol (E,E)-2,4-decadienal (E)-β-damascenone 2-methoxyphenol cis-4,5-epoxy-(E)-2-decenal trans-4,5-epoxy-(E)-2-decenal 4-hydroxy-2,5-dimethyl-3(2H)-furanone unknown 4-methylphenol 4-allyl-2-methoxyphenol 4-ethyloctanoic acid 3-hydroxy-4,5-dimethyl-2(5H)-furanone wine lactoneg 2-phenylacetic acid 4-hydroxy-3-methoxybenzaldehyd

aroma quality

b

malty buttery pungent grasy, green fruity fruity, almond-like grassy, green grassy, apple-like fishy citrus-like mushroom-like grassy, fruity fatty geranium-like pungent citrus-like, fatty cooked potato-like cheese-like, fruity fatty fatty, moldy, pungent fatty, moldy flowery fatty, cucumber-like fatty sweaty honey-like, beeswax-like fatty sweaty fatty cooked potato-like, tallowy fatty grape juice-like smoky metallic metallic caramel-like metallic horse stable-like, phenolic clove-like fecal, moldy seasoning coconut-like, dill-like honey-like vanilla-like

FFAP

CP Sil C8 CB

FD factord

lite

976 993 1024 1086 1094 1100 1124 1207 1242 1284 1287 1328 1367 1373 1400 1423 1459 1483 1509 1517 1543 1545 1591 1605 1624 1649 1668 1671 1710 1725 1816 1826 1868 1992 2025 2038 2071 2076 2179 2182 2206 2255 2541 2581

n.d. 625 684 800 n.d. 929 800 n.d. 900 n.d. 978 869 n.d. 979 1033 1058 905 n.d. 1147 n.d. 1160 1106 1155 1243 806 n.d. 1191 876 1210 980 1313 1385 1086 1350 1379 1067 n.d. n.d. n.d. 1345 1108 1459 n.d. 1407

8 4 512 32 32 1024 2048 32 16 8 64 4 4 128 4 64 2048 4 4 4 32 16 4 4 4 8 4 64 4 256 32 512 32 4 2048 1024 32 4 8 16 32 128 32 512

39 39 39

39 39 47 10 10 13 39 14 48

10 10 47 8 12 48 10 8 48 49 12 49

12 12

6

a

Identification of the compounds was performed by comparing their mass spectra (MS-EI, MS-CI), retention indices on capillaries FFAP and CP Sil 8 CB, as well as the odor quality and the odor intensity perceived during sniffing with data of reference compounds. bOdor-quality perceived at the sniffing-port. cRetention index. dFlavor dilution factor n.d., not determined (odor-quality of the compound was not detected during HRGC-O on this capillary column). eReference reporting the compound for the first time as a fresh tomato odorant. fNo unequivocal mass spectrum was obtained. Tentative identification is based on the remaining criteria given in footnote a. gStereochemistry was not determined. Odor qualities are given for the racemate.

extracted with dichloromethane, and the nonvolatile components were removed by SAFE distillation.37 By adding a drop of the extract on a strip of filter paper and comparing the smell to the original sample, it could be verified that the total set of aroma compounds was extracted. Application of GC/O on the aroma extract and subsequent application of the AEDA revealed 44 aroma-active regions in the flavor dilution (FD) factor range between 4 and 2048 (Figure 1). Among them,

three odorants 7, 17, and 35 with a grassy/green, a cooked potato-like, and a metallic notes, respectively, showed the highest FD factors. To identify the compounds responsible for the odor-active regions, first the retention indices (RI) and odor qualities were compared to an in-house database containing data of about 1000 food odorants and their specific characteristics. In addition, mass spectra were recorded. Finally, the structure of 5202


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index (Table 3) was clearly different from that of 6. Therefore, it was assumed that 6 is a positional isomer of 4-methyl-2ethoxytetrahydropyran, and thus, three further compounds, i.e., the 3-methyl-, 5-methyl-, and 6-methyl-2-ethoxytetrahydropyran, were synthesized. Because the syntheses followed the same principle, only the NMR data were recorded for the 5methyl-isomer. The NMR clearly confirmed the success of the synthesis (see Materials and Methods). The mass spectra of the three isomers are shown in Figure 3A, C, and D. All isomers showed similar fragments in their mass spectrum compared to those of compound 6, but from all isomers, the intensities of the fragments were different (Figure 3). A spectrum closest to that of 6 was shown by the 6-methyl-isomer (Figure 3D). However, also for these three isomers, the retention indices on two stationary GC phases were different, and in particular, the 6-methyl-isomer showed the largest difference (Table 3). It should be mentioned that with the exception of the 4-methylisomer, the other three isomers were characterized for the first time in the literature. The last possible isomer would be 2-methyl-2-ethoxytetrahydropyran (Figure 4). However, this compound was scarcely mentioned in the literature.40,41 Unfortunately, the synthesis route could not follow an easy 1,4-cycloaddition (Diels−Alder reaction) as used in the synthesis of the other isomers. Thus, alternative routes were thought of, and a possible synthesis is shown in Figure 5. While a nucleophilic addition via Grignard reaction from δ-valerolactone and methylmagnesium bromide did not result in the desired intermediate 2-hydroxy-2methyltetrahydropyran, finally by adding methyllithium to δvalerolactone at −78 °C, a mixture of 2-hydroxy-2-methyltetrahydropyran and 6-hydroxy-2-hexanone was obtained.42 However, the chemical equilibrium of the tautomers always shifted toward the open-chain keto form, in particular under acidic conditions for the substitution of the hydroxyl- by the ethoxy-group. In the NMR spectrum, the equilibrium of both tautomers is illustrated. In benzene-d6 (unipolar solvent), the open-chain tautomer already existed in a ratio of 4:1, but in methanol-d4, this was even fortified toward an excess of 20:1. Therefore, the last step (Figure 5) did not yield the desired 2methyl-2-ethoxytetrahydropyran in great quantities. However, a small peak showing the same mass spectrum, retention indices, and aroma qualities as those of 6 was obtained. However, the amounts were not sufficient for NMR experiments. Because of the very low concentrations in the tomato distillate and close eluting peaks such as (Z)-3-hexenal, it was also not possible to purify the compound from the tomato distillate for NMR analysis. However, taking all data into account, it seems very likely that compound 6 is 2-methyl-2-ethoxytetrahydropyran. Because the amounts were estimated to be about 20 μg per kg, it can be assumed from the high FD factor (Table 2) that the odorant has a very low odor threshold. The compound has never been reported before in tomatoes or any food. There are also no hints on its formation pathway by a known biochemical reaction mechanism. 42 reaching an FD factor of 128 (Table 2) with a coconut/ dill-like scent is another compound previously not reported in tomato. To obtain a clear mass spectrum, a heart-cut GC/GC procedure was applied. During the first injection, the coconut-, dill-like odor was located by GC/O at 26.8 min. In a second GC run, the effluent between 26.2 to 27.2 min was transferred to the second GC and again analyzed by GC/O and mass spectrometry to obtain the mass spectrum shown in Figure 6. A comparison with the literature suggested wine lactone earlier

Figure 2. Mass spectrum (EI-MS) of compound 6.

an odorant was confirmed by comparing the analytical and sensory characteristics with that of the respective reference compound. Thus, the three compounds with the highest FD factors were identified as (Z)-3-hexenal, 7, 3-(methylthio)propanal, 17, and trans-4,5-epoxy-(E)-2-decenal, 35, followed by 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 36, 1-penten-3one, 3, (E)-β-damascenone, 32, and 4-hydroxy-3-methoxybenzaldehyde, 44. Additionally, the cooked potato-like 3(methylthio)propanol, 30, and the geranium-like (Z)-1,5octadien-3-one, 14, contributed with a quite high FD factor to the overall aroma (Table 2). These findings confirmed results of previous studies done on the aroma compounds of tomatoes of other origins.5,8−11 However, as indicated in Table 2, 13 compounds were previously not reported as tomato aroma compounds. In particular, 6 eliciting a fruity, almond-like odor, was of interest because it appeared with a high FD factor of 1024, which was close to the FD factors of the well-known odorants, such as (Z)-3-hexenal or 3-(methylthio)propanal. As a first hint on its structure, it was observed that during chromatography of the tomato distillate on silica gel in an n-pentane/diethyl ether gradient, 6 was eluted with a very low diethyl ether concentration indicating that the compound is quite nonpolar. Its mass spectrum (Figure 2) showed a molecular mass of m/z 144, which was confirmed by CI-MS (data not shown). To gain better insight into its structure, the molecular mass was determined by high resolution mass spectrometry and was calculated as C8H16O2. This molecular formula indicated one double bond equivalent or a ring system. Because of its low polarity and assuming that one oxygen is part of a ring system, this consideration allows only an ether bond for the second oxygen. A comparison of the mass spectrum (Figure 2) with data in the NIST database proposed 4-methyl-2-ethoxytetrahydropyran as a possible structure. This structure would be in agreement with the fragments m/z 100 and m/z 99 in its mass spectrum indicating a loss of the ethoxy group from the molecular ion (Figure 2). Therefore, 4-methyl-2-ethoxytetrahydrofuran was synthesized. Its mass spectrum (Figure 3 B) contained a very similar set of fragments as compared to those of 6, however, with different intensities. Furthermore, although the odor quality during GC/O was in agreement, the retention 5203


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Figure 3. Mass spectra of 3-methyl (A)-, 4-methyl (B)-, 5-methyl (C)-, and 6-methyl-2-ethoxytetrahydropyran (D).

Table 3. Odor Quality and Retention Indices of Compound 6 and Four Methyl-2-ethoxytetrahydropyran Isomers RIb on compound 6 3-methyl-2-ethoxy-isomer 4-methyl-2-ethoxy-isomer 5-methyl-2-ethoxy-isomer 6-methyl-2-ethoxy-isomer a

aroma qualitya fruity, fruity, fruity, fruity, fruity,

almond-like almond-like almond-like almond-like almond-like

FFAP

CP Sil C8 CB

1100 1116 1130 1151 1198

929 926 958 956 967

Figure 4. Structure of 2-methyl-2-ethoxytetrahydropyran.

sweaty), hexanal (4; grassy, green), (E,E)-2,4-decadienal (31; fatty), 2-methoxyphenol (33; smoky), 3-hydroxy-4,5-dimethyl2(5H)-furanone (41; seasoning), and phenylacetic acid (43; honey-like) with somewhat lower FD factors (Table 2). The application of a GC/O-headspace revealed no further odorants (data not shown). As mentioned in the Introduction, only one previous study also applied the molecular sensory science concept on fresh tomatoes before. While 16 odorants identified in our study were also found by Mayer et al.,10 13 odorants, such as 2,3butanedione, linalool, (E)-2-nonenal, 3-hydroxy-4,5-dimethyl-

Odor-quality perceived at the sniffing-port. bRetention index.

identified in white wine.17 With the aid of the reference compound, 42 was identified as one of the eight possible isomers of wine lactone as (3S,3aS,7aR)-3a,4,5,7a-tetrahydro3,6-dimethylbenzofuran-2(3H)-one. The further identification experiments revealed 1-octen-3one (11; mushroom-like), 2- and 3-methylbutanoic acid (28; 5204


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Figure 7. Changes in the concentrations of (Z)-3-hexenal and (E)-2hexenal in a fresh tomato purée stored for 5 h.

Figure 5. Synthetic route proposed for the synthesis of 2-methyl-2ethoxytetrahydropyran from δ-valerolactone via 2-hydroxy-2-methyltetrahydropyran.

to more than 1400 μg/kg during the first 5 min reaching its maximum (1510 μg/kg) after 10 min and finally decreased to its initial value after 3 h. The amount of (E)-2-hexenal peaked after 5 min up to 106 μg/kg and decreased over the next 2 h. Thus, the endogenous enzymes were always inhibited5,8 by CaCl2 after 5 min before the following workups. This procedure compromised the time needed for enzymatic aroma generation but also saved time for the throughput of a larger number of samples. Quantitation of Aroma Compounds. Twenty-four aroma compounds were quantitated by means of stable isotope dilution assays (SIDA). The highest concentrations were determined for hexanal (1,510 μg/kg) and (Z)-3-hexenal (943 μg/kg) (Table 4), followed by 4-hydroxy-2,5-dimethyl3(2H)-furanone (142 μg/kg), 1-penten-3-one (92.6 μg/kg), trans-4,5-epoxy-(E)-2-decenal (61.5 μg/kg), 3-methylbutanoic acid (40.1 μg/kg), 3-(methylthio)propanol (29.3 μg/kg), butanoic acid (17.1 μg/kg), 4-hydroxy-3-methoxybenzaldehyde (12.1 μg/kg), and 3-(methylthio)propanal (11.7 μg/kg). Many compounds, however, were only present in trace amounts, for example, the wine lactone (5.4 μg/kg), (E)-β-damascenone (3.1 μg/kg), 1-octen-3-one (1.1 μg/kg), or 4-allyl-2-methoxyphenol (0.5 μg/kg). The amount of the newly identified 2methyl-2-ethoxytetrahydropyran was estimated to be below 20 μg/kg. Calculation of OAVs. To get further insight into the contribution of the odorants to the overall aroma, OAVs (ratio of odor threshold to concentration) were calculated. Because

2(5H)-furanone, wine lactone, or 2-phenylacetic acid, were not reported in the tomato varieties investigated by this group. This indicates that aroma differences in tomato varieties not only can be explained by odorants with high odor activity but also may be caused by differences in the set of odorants with lower FD factors. Quantitation of Aroma Compounds. Freezing Experiments. Since a batch of the same tomatoes had to be stored at −25 °C prior to analysis, stability experiments on (Z)-3-hexenal at −25 °C were performed, due to the known instability of this compound, i.e., during freezing.43−45 (E)-2-Hexenal was also determined because it is a known isomerization product of (Z)3-hexenal.46 The concentration of (Z)-3-hexenal was 2219 μg/ kg before and 152 μg/kg after the freezing/thawing process. Hence, only 7% of the original amounts were left. A different behavior was found for (E)-2-hexenal, which was not affected by freezing/thawing. However, recently it was reported that even storage temperatures of 4 °C for 30 days led to a clear decrease in (Z)-3-hexenal, hexanal, and (E)-2-hexenal.43 Time Course of Odorant Formation. (Z)-3-Hexenal was earlier shown to be released from its precursor linolenic acid via a hydroperoxide already during the grinding of tomatoes when the cell tissue is destroyed.46 To illustrate the degree of (Z)-3-hexenal generation in the Italian tomatoes, the concentration of both aldehydes was determined over a period of 5 h (Figure 7). For both aldehydes, an immediate formation was observed. (Z)-3-Hexenal increased

Figure 6. (A) Chemical structure of wine lactone and a detailed chomatogram (EI-MS) of the second dimension. (B) EI-MS of the coconut/dill-like smelling region. 5205


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Journal of Agricultural and Food Chemistry Table 4. Concentrations of 24 Important Aroma Compounds in Italian Tomatoes aroma compound

concna (μg/kg)

range (μg/kg)

hexanal (Z)-3-hexenal 4-hydroxy-2,5-dimethyl-3(2H)-furanone 1-penten-3-one trans-4,5-epoxy-(E)-2-decenal 3-methylbutanoic acid 3-(methylthio)propanol butanoic acid 4-hydroxy-3-methoxybenzaldehyde 3-(methylthio)propanal wine lactone (E)-β-damascenone 2,3-butanedione (E)-2-nonenal linalool (E,E)-2,4-decadienal 1-octen-3-one 2-methoxyphenol phenylacetaldehyde 2-phenylacetic acid 4-allyl-2-methoxyphenol p-cresol 3-hydroxy-4,5-dimethyl-2(5H)-furanone (E,Z)-2,6-nonadienal

1514 943 142 92.6 61.5 40.1 29.3 17.1 12.1 11.7 5.4 3.1 2.9 2.3 1.7 1.5 1.1 0.9 0.8 0.6 0.5 0.3 0.1

Characterization of Aroma-Active Compounds in ... - ACS Publications

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