Article Cite This: J. Agric. Food Chem. 2017, 65, 9287-9296
pubs.acs.org/JAFC
Characterization of the Key Aroma Compounds in White Alba Truffle (Tuber magnatum pico) and Burgundy Truffle (Tuber uncinatum) by Means of the Sensomics Approach Philipp C. Schmidberger and Peter Schieberle* Lehrstuhl für Lebensmittelchemie, Technische Universität München, Lise-Meitner-Straße 34, 85354 Freising, Germany ABSTRACT: Aroma extract dilution analysis of distillates prepared by solvent extraction and solvent-assisted flavor evaporation distillation from white Alba truffle (WAT; Tuber magnatum pico) and Burgundy truffle (BT; Tuber uncinatum) revealed 20 odoractive regions in the flavor dilution (FD) factor range of 16−4096 in WAT and 25 in BT. The identification experiments in combination with the FD factors showed clear differences in the overall set of key odorants of both fungi. While 3(methylthio)propanal (potato-like) followed by 2- and 3-methylbutanal (malty), 2,3-butanedione (buttery), and bis(methylthio)methane (garlic-like) showed the highest FD factors in WAT, 2,3-butanedione, phenylacetic acid (honey-like), and vanillin (vanilla-like) had the highest FD factors in BT. Odor activity values (OAVs, ratio of concentration to odor thresholds), which were calculated on the basis of quantitative data obtained by stable isotope dilution assays, of >1000 for bis(methylthio)methane, 3-methylbutanal, and 3,4-dihydro-2-(H)pyrrol (1-pyrroline) revealed they are key contributors to the aroma of WAT. In BT, 1pyrroline and 2,3-butanedione showed the highest OAVs of 1530 and 1130, respectively. Aroma recombination experiments successfully mimicked the overall aroma profiles of both fungi when all odorants showing OAVs of >1 were combined. Omission experiments confirmed the amine-like and sperm-like smell of 1-pyrroline, identified for the first time as a key odorant in both truffle species. KEYWORDS: aroma extract dilution analysis, 3,4-dihydro-2H-pyrrol, 2,3-butanedione, bis(methylthio)methane, stable isotope dilution assay, [2H8]bis(methylthio)methane, [13C4]-3,4-dihydro-2H-pyrrol
■
Culleré et al.2 and Paolocci et al.12 were the first to perform a sensory-guided study of the aroma compounds of summer truffles (Tuber aestivum), the same species as Burgundy truffles, but harvested in the summer. In these investigations, volatiles were isolated by means of a purge and trap system for 7.5 h, followed by application of aroma dilution analysis (ADA). Fourteen odor-active compounds were detected, and of these, dimethyl disulfide, p-cresol, 3-ethylphenol, and 3-(methylthio)propanal showed the highest flavor dilution (FD) factors. In quantitative studies using external calibration, only low concentrations were found for, e.g., dimethyl sulfide, 3ethylphenol, and (E,E)-2,4-decadienal in the range of 6−8 ng/kg. The concentrations of 2,3-butanedione, 3-(methylthio)propanal, and 1-octen-3-one were lower (5 ng/kg). Recent studies of the sources of odor-active volatiles in truffles suggest that odorants produced by microorganisms of the soil may contribute to the truffle aroma.13 Buzzini et al.14 were able to isolate 29 different strains of yeast from the ascocarp of black and white truffles. These were cultivated in a medium containing L-methionine, and the volatiles in the headspace were analyzed by SPME with gas chromatography and mass spectrometry (GC−MS). All strains were able to metabolize L-methionine and to synthesize methanethiol, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, 3(methylthio)propanal, and dihydro-3(2H)-thiophenone. How-
INTRODUCTION Truffles are a fungus of the genus Tuber and usually grow beneath the soil surface, in particular in symbiosis with tree roots. Some species are among the most highly priced foods because of their high demand in “haute cuisine”, particularly in France and other European countries. The fruiting bodies of truffles are found in distinct Mediterranean areas in Italy, France, and Spain, and the white Alba truffle (WAT, Tuber pignatum pico) is regarded as the noblest among the truffle species, because it elicits the most intense and pleasant aroma. On the other hand, although its aroma is less intense, Burgundy truffle (BT, Tuber uncinatum), the third most expensive species, is more widespread and more frequently consumed. To date, ∼300 volatiles have been reported in truffle,1 but the isolation of volatiles was mostly performed using different headspace extraction techniques such as long-lasting purge and trap or SPME.2−8 However, such methods are known to discriminate volatiles by their boiling points, and thus, the original composition of the volatiles may be significantly changed because of differences in extraction yields. A known sulfur compound reminiscent of truffle aroma is bis(methylthio)methane, first isolated from white truffles by Fiecchi et al.,9 and later also found in white Alba truffle, Burgundy truffle, and Tuber panniferum.3,7,10 The compound is often added to vegetable oils to mimic truffle aroma. In addition, other sulfur compounds, like dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide, were also detected in the gas phase of different truffle species and were suggested as important volatiles.2,4,5,8,11 Also 2- and 3-methylbutanal are thought to be characteristic aroma volatiles in truffles.1 © 2017 American Chemical Society
Received: Revised: Accepted: Published: 9287
August 31, 2017 September 29, 2017 September 30, 2017 September 30, 2017 DOI: 10.1021/acs.jafc.7b04073 J. Agric. Food Chem. 2017, 65, 9287−9296
Article
Journal of Agricultural and Food Chemistry
Concentrations of the isotopically labeled compounds were calculated by means of GC-FID using methyl octanoate as the internal standard. The FID response factor was determined from a defined mixture of the unlabeled compound and methyl octanoate in a ratio of 1:1. Assuming no differences in the FID response for the unlabeled and the labeled compound, we calculated the concentration of the latter using the same response factor. Analysis was performed on a Thermoquest (Egelsbach, Germany) Trace 2000 gas chromatograph equipped with an FID. Synthesis of [2H8]Bis(methylthio)methane. The synthesis of [2H8]bis(methylthio)methane was performed following a modified procedure of Zaraiskii et al.33 for the unlabeled compound. A mixture of [2H6]dimethyl sulfoxide (1.25 g, 16 mmol) and acetic acid anhydride (2.2 g, 21.6 mmol) was placed in a Pyrex glass tube, and the tube was sealed with a cap and heated at 130 °C for 1 h. After the mixture had cooled, sulfuric acid (51%) was added and the mixture was incubated for 5 min. After the addition of water (2.5 mL) and anhydrous sodium sulfate (1 g), the organic layer was separated and washed twice with brine (2 mL). The yield was 0.86 g (7.9 mmol, 98%): MS-EI [m/z (%)] 66 (100), 116 (81), 46 (18), 48 (12), 118 (8). Synthesis of [13C4]-3,4-Dihydro-2H-pyrrol (1-pyrroline). The isotopically labeled standard was synthesized according to a modified synthesis previously reported.18 [13C5]-L-Proline (0.1 g, 0.83 mmol) was dissolved in water (5 mL) and added to an aqueous solution of sodium periodate (0.3 mol/L, 7 mL). The reaction vessel was equipped with a suction tube cap, and the solution was stirred in the dark for 2 h. The pH was adjusted to 10.5 with an aqueous sodium hydroxide solution (80 g/L); a small portion of sodium chloride (2.5 g) was added, and the solution was extracted with diethyl ether (total volume of 15 mL). The combined organic layers were washed with brine (5 mL) and concentrated to 500 μL on a Vigreux column. The solution was applied to a column (30 cm, 1 cm inside diameter) filled with alumina (neutral, deactivated with 10% water, by weight). After being rinsed with n-pentane (50 mL), the target compound was eluted with n-pentane/diethyl ether [100 mL, 50/50 (v/v)]. The concentration of [13C4]-1-pyrroline was determined with 3-pyrroline as the internal standard measuring a yield of 12.5 mg (0.17 mmol, 20.5%). The structure was confirmed by mass spectrometry: MS-EI [m/z (%)] 73 (100), 72 (86), 43 (50), 44 (47), 40 (100). Synthesis of [2H4]-1,2,4-Trithiolane. [2H4]-1,2,4-Trithiolane was synthesized according to a modified synthesis previously reported.34 Sodium sulfide nonahydrate (7.5 g, 96 mmol) and sulfur (1.5 g, 57 mmol) were mixed with distilled water (25 mL) and stirred for 1 h at room temperature. The orange colored suspension was filtered, and d2-dichloromethane (25 mL, 0.4 mmol) was added. After being vigorously stirred for 7 h, the organic layer was washed with brine (25 mL) followed by distilled water (25 mL). The solution was concentrated on a Vigreux column to ∼200 μL, and finally, the target compound was isolated by flash chromatography on silica with npentane: MS-EI [m/z (%)] 128 (100), 46 (90), 80 (76), 48 (34), 64 (20), 62 (19), 130 (13), 82 (8), 44 (7). Isolation of the Volatiles. Truffles (30 g wet weight) were frozen in liquid nitrogen, crushed into small pieces in a mortar, and then ground by means of a commercial blendor (Grindomix GM 200, Retsch, Haan, Germany). The material was extracted twice with methylene chloride (150 mL each) by being vigorously stirred for 60 min. The suspension was filtered, and the volatile fraction was isolated by solvent-assisted flavor evaporation (SAFE).35 The distillate obtained was dried over anhydrous sodium sulfate and finally concentrated at 45 °C to ∼200 μL using a Vigreux column (50 cm × 1 cm inside diameter) and a microdistillation apparatus.36 Gas Chromatography−Olfactometry (GC−O). GC−O analysis was performed using a Thermo Scientific (Dreieich, Germany) Trace GC Ultra gas chromatograph with the following capillary columns (fused silica): DB-FFAP and DB-5 (both 30 m × 0.25 mm inside diameter, 0.25 μm film thickness) (J&W Scientific, Folsom, CA; Agilent Technologies, Santa Clara, CA). The samples (1.0 μL) were injected by the cold on-column method at 40 °C using helium as the carrier gas (flow rate of 1.2 mL/min). The oven temperature started at
ever, the microorganisms did not generate all key odorants previously reported to be present in the truffle tissue. It is widely accepted in the scientific literature that not all volatile constituents of a food contribute to its overall aroma, and in particular, the sensomics approach has been identified as a valuable tool for identifying those aroma compounds generating the overall aroma impression in the human brain. However, such methods combining analytical and sensory data, including aroma recombination experiments, have not yet been performed for any truffle variety. Thus, the aim of this study was (i) to characterize the key aroma compounds in white Alba and Burgundy truffles by application of aroma extract dilution analysis (AEDA) and ADA in combination with GC−MS, (ii) to quantitate the odor-active compounds appearing with the highest FD factors by means of stable isotope dilution assays followed by calculation of odor activity values (OAVs), and (iii) to simulate the aroma of both fungi by sensory experiments, such as recombination experiments.
■
MATERIALS AND METHODS
Truffles. Both truffle varieties were purchased from an online supplier. Chemicals. Acetic acid anhydride was obtained from Sigma-Aldrich Chemie (Taufkirchen, Germany). Alumina 90 (neutral), anhydrous sodium sulfate, diethyl ether, methylene chloride, n-pentane, silica 60, sodium periodate, and sulfuric acid were obtained from Merck (Darmstadt, Germany). Diethyl ether, methylene chloride, and npentane were freshly distilled before use. Liquid nitrogen was obtained from Linde (Munich, Germany). Reference Odorants. The following reference odorants were obtained from the commercial sources given in parentheses: acetaldehyde, acetic acid, bis(methylthio)methane, 2,3-butanedione, (E,E)-2,4-decadienal, 2,6-dimethoxyphenol, 3,5-dimethyl-2-ethenylpyrazine, dimethyl sulfide, dimethyl trisulfide, δ-dodecalactone, ethyl 3methylbutanoate, ethyl 2-methylpropanoate, geosmine, hexanal, hydrogen sulfide, 3-hydroxy-2-butanone, 3-hydroxy-4,5-dimethyl2(5H)-furanone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, methanethiol, 2-methoxyphenol, 2-methylbutanal, 3-methylbutanal, 2-methylbutanoic acid, 3-methylbutanoic acid, 2-methylbutanol, 3-methylbutanol, 4-methyl-2-methoxyphenol, 3-methylnonan-2,4-dione, 2-methylpropanoic acid, 2-methylpropanal, 2-methylpropanol, methyl 2methylbutanoate, 3-(methylthio)propanal, 3-(methylthio)propanol, (E,E)-2,4-nonadienal, γ-nonalactone, δ-nonalactone, (E)-2-nonenal, (E)-2-octenal, (Z)-1,5-octadien-3-one, 1-octen-3-ol, 2,3-pentanedione, pentanoic acid, phenylacetaldehyde, phenylacetic acid, propanoic acid, 3-propylphenol, and 4-vinylphenol (Sigma-Aldrich Chemie), 3,4methylnonanedione (Chemos GmbH, Regenstauf, Germany), 1octen-3-one (Alfa Aesar, Karlsruhe, Germany), and vanillin (Merck). The following reference odorants were synthesized as previously described: 2-acetyl-1-pyrroline,15 2-ethenyl-3-ethyl-5-methylpyrazine,16 2-propionyl-1-pyrroline,17 3,4-dihydro-2-(H)pyrrol,18 trans4,5-epoxy-(E)-2-decenal,19 and 1,2,4-trithiolane.20 Isotopically Labeled Internal Standards. The isotopically labeled compounds, labeled with deuterium or 13C, were synthesized according to the literature cited: [13C4]-2,3-butanedione,21 [2H4](E,E)-2,4-decadienal, [2H4]-(E,E)-2,4-nonadienal, and [2H2]-(E)-2nonenal,22 [2H6]dimethyl trisulfide,23 [2H4]-trans-4,5-epoxy-(E)-2decenal,24 [2H3]-2-methoxyphenol,25 [2H3]-3-(methylthio)propanal and [2H3]-3-(methylthio)propanol,26 [2H2]-3-methylbutanal,27 [2H2]3-methylbutanoic acid and [ 2 H 2 ]-3-methylbutanol, 28 [ 2 H 2 ]methylpropanal,29 [2H2]-(E)-2-octenal and [2H2−4]-1-octen-3-one,30 [2H2−4]-1-octen-3-ol,31 and [2H3]vanillin.32 [2H6]Dimethyl sulfide and [13C2]phenylacetic acid were purchased from Sigma-Aldrich. [13C4]-3-Hydroxy-2-butanone was purchased from Toronto Research Chemicals (Toronto, ON) and [2H3]pentanoic acid from C/D/N isotopes (Quebec, QC). 9288
DOI: 10.1021/acs.jafc.7b04073 J. Agric. Food Chem. 2017, 65, 9287−9296
Article
Journal of Agricultural and Food Chemistry 40 °C, was held for 2 min, was increased at a rate of 6 °C/min to 230 °C for the DB-FFAP column or to 240 °C for the DB-5 column, and was held for 5 min. The flow of the carrier gas was split at the end of the capillary column by means of a Chrompack (Frankfurt, Germany) Y-type quick-seal glass splitter and two deactivated fused silica capillaries of the same length (25 cm × 0.32 mm inside diameter). One part was directed to an FID (250 °C) and the other to a sniffing port (200 °C). A series of n-alkanes C6−C26 (DB-FFAP) and C6− C18 (DB-5) were used to determine linear retention indices (RI) for each compound. Aroma Extract Dilution Analysis. For the determination of FD factors, first, the concentrated original distillate was subjected to GC− O on the FFAP column to locate and evaluate the odor attributes of all aroma-active areas. Then, the distillate was diluted stepwise 1:1 (by volume) with methylene chloride, and each dilution was analyzed in 1.0 μL aliquots by GC−O. To avoid overlooking odor-active compounds, the concentrated distillate was sensorially analyzed by at least three experienced panelists. Gas Chromatography−Mass Spectrometry (GC−MS). For compound identification, mass spectra were acquired using a HewlettPackard (Waldbronn, Germany) 5890 series II gas chromatograph coupled to a Finnigan (Bremen, Germany) MAT 95 S sector field mass spectrometer. Mass spectra in electron impact mode (MS-EI) were generated at 70 eV and in chemical ionization mode (MS-CI) at 115 eV using isobutane as the reactant gas. Headspace GC−MS (HS-GC−MS) and Aroma Dilution Analysis. For identification of highly volatile compounds, HS-GC− MS analysis was performed using a Varian (Darmstadt, Germany) CP 3800 gas chromatograph and a Varian Saturn 2000 ion trap mass spectrometer. Samples (200 mg) were weighed into a headspace vial, and a small amount of water (1 mL) was added. After the vial had been sealed, the suspension was stirred for 45 min at 35 °C. An aliquot of the headspace was then withdrawn with a gastight syringe and cryofocused in a PTV injector at −150 °C. Volatile compounds were then transferred onto the column by increasing the temperature of the injector quickly to 250 °C. The temperature of the oven started at 0 °C, was held for 5 min, was increased at a rate of 6 °C/min to 120 °C, was then increased at a rate of 20 °C/min to 240 °C, and was held for 5 min. The effluent was split at the end of the capillary column using a Y-type splitter to an FID and a sniffing port in a ratio of 4:6. For ADA, the largest volume of 20 mL headspace was stepwise reduced by analyzing reduced volumes of 10, 5, 2.5, 1.2 mL, etc. The largest volume was assigned as FD = 1. Mass spectra were recorded in MS-CI mode using methanol as the reagent gas. Quantitation of Odorants by Stable Isotope Dilution Assays (SIDAs). First, mixtures of the respective labeled and unlabeled compound were prepared in five different mass ratios (1:5, 1:3, 1:1, 3:1, and 5:1) and analyzed by GC−MS to calculate the MS response factor for each component from the peak areas of the respective mass fragments (Table 1). Quantitation of compounds present at higher concentrations was then performed using a Varian CP 3800 gas chromatograph and a Varian Saturn 2000 ion trap mass spectrometer. The labeled internal standards (0.1−70 μg), dissolved in methylene chloride, were added to a sample (1−30 g) suspended in methylene chloride containing the aroma compounds at similar concentrations as determined in preliminary experiments. After the sample had been stirred for 1 h, the workup procedure followed the protocol described above for the isolation of volatiles. Two microliters of each sample was injected on the DB-FFAP capillary column (60 m × 0.25 mm inside diameter, 0.25 μm film thickness, J&W Scientific) by the cold on-column technique, and mass spectra were recorded in MS-CI mode using methanol as the reagent gas (Table 1). For the quantitation of 2- and 3-methylbutanoic acid, the sum of both acids was determined by SIDA. Then, the ratio of 2- and 3methylbutanoic acid was determined by comparing the intensities of the fragments at m/z 60 (3-methylbutanoic acid) and m/z 74 (2methylbutanoic acid). For calibration, defined mixtures of 2- and 3methylbutanoic acid were analyzed and a calibration line was drawn
Table 1. Isotopically Labeled Standards, Selected Ions, and Accuracy of Calibration Lines Used in the Stable Isotope Dilution Assays ion (m/z)a odorant bis(methylthio) methane 2,3-butanedione (E,E)-2,4decadienal 2,3-diethyl-5methylpyrazine 2,6dimethoxyphenol dimethyl sulfide dimethyl trisulfide trans-4,5-epoxy(E)-2-decenal 3-hydroxy-2butanone 4-hydroxy-2,5dimethyl-3(2H)furanone 3-hydroxy-4,5dimethyl-2(5H)furanone 2-methylbutanal 3-methylbutanal 2-methylbutanole 3-methylbutanol 2- and 3methylbutanoic acid 4-methyl-2methoxyphenol 2-methylpropanal 3-(methylthio) propanal 3-(methylthio) propanol 2-methoxyphenol (E,E)-2,4nonadienal (E)-2-nonenal (E)-2-octenal 1-octen-3-ol 1-octen-3-one pentanoic acid phenylacetic acid 3,4-dihydro-2(H) pyrrol (1pyrroline) 1,2,4-trithiolane vanillin
isotope label 2
H8
analyte
internal standard
R2b
systemc
109
117
1
1
C4 H3−5
87 153
91 156−158d
1 0.9985
2 2
2
H3
151
154
0.9998
2
2
H5−8
154
159−162d
0.9999
3
63 127 167
69 133 171
0.9999 1 0.9998
5 3 4
13 2
2
H6 H6 2 H4 2
13
C4
89
93
0.9998
1
13
C2
128
130
0.998
3
13
C2
128
130
0.9996
3
H2 H2 − 2 H2 2 H3
87 87 71 71 103
89 89 73 73 106
1 1 0.9998 1 0.998
2 2 5 1 1
2
H3
139
142
0.997
3
2
H2 H3
73 105
75 108
0.9996 0.997
5 3
2
H3
107
110
0.997
3
2
H3 H2
124 139
127 141
1 0.9991
2 2
H2 H2 2 H2−4 2 H2 2 H3 13 C2 13 C4
141 127 111 127 103 136 70
143 129 113−115d 129 106 138 74
0.999 0.999 0.995 0.996 0.997 0.9993 0.9996
2 2 1 2 1 3 1
2
125 152
129 155
0.9999 1
2 3
2 2
2
2
2 2
2
H4 H3
a
Ions used for quantitation. bCoefficient describing the accuracy of the calibration line; five calibration points. cLegend: 1, GC−MS; 2, GC×GC−MS; 3, comprehensive GC×GC−TOF-MS; 4, GC−MS (CI neg.); 5, HS-GC−MS. dA mixture of isotopologues was used as the internal standard. e2-Methylbutanol was quantitated using [2H2]-3methylbutanol as the internal standard.
plotting the ratio of m/z 60 to m/z 74 against the amount of 3methylbutanoic acid in the mixture. Quantitation of dimethyl sulfide and 2-methylpropanal was performed using HS-GC−MS by adding the labeled standards to the 9289
DOI: 10.1021/acs.jafc.7b04073 J. Agric. Food Chem. 2017, 65, 9287−9296
Article
Journal of Agricultural and Food Chemistry sample prior to stirring. Analysis was performed under the same conditions that were used for identification. Quantitation of trans-4,5-epoxy-(E)-2-decenal was performed using an Agilent (Waldbronn, Germany) 7890B gas chromatograph and an Agilent ion trap 240 mass spectrometer. Two microliters of the solutions was injected on the DB-FFAP capillary column (60 m × 0.25 mm inside diameter, 0.25 μm film thickness, J&W Scientific) by the cold on-column technique, and mass spectra were recorded in external negative CI mode using methanol as the reagent gas (Table 1). In the case of co-eluting compounds, a TD-GC−MS instrument consisting of a Thermo Scientific Trace GC 2000 series gas chromatograph coupled to a Varian CP 3800 gas chromatograph and a Varian Saturn 2000 ion trap mass spectrometer was used. After cold on-column injection, separation of the distillate in the first dimension was achieved on the DB-FFAP capillary column (30 m × 0.32 mm inside diameter, 0.25 μm film thickness, J&W Scientific). The elution range containing the selected odorant and the internal standard was then transferred into a cold trap by the moving capillary stream switching system (MCSS). After complete trapping, the analyte and the standard were transferred onto the second column, a J&W Scientific DB-1701 column (30 m × 0.25 mm inside diameter, 0.25 μm film thickness), by heating the trap to 200 °C. For compounds present at low concentrations that could not be separated using TD-GC−MS, comprehensive two-dimensional gas chromatography was performed using an Agilent (Bö blingen, Germany ) 6890N gas chromatograph equipped with a Gerstel (Mühlheim an der Ruhr, Germany) PTV 4 injector and a Pal autosampler (CTC-Analytics, Zwingen, Switzerland). After cold oncolumn injection (−20 °C) and a rapid increase of the temperature to 240 °C, the separation of the distillate in the first dimension was achieved on the DB-FFAP capillary column (30 m × 0.32 mm inside diameter, 0.25 μm film thickness, J&W Scientific) with helium as the carrier gas at a flow rate of 1.2 mL/min. The effluent was continuously transferred onto the second column, a DB-5 column (1 m × 0.25 mm inside diameter, 0.25 μm film thickness, J&W Scientific). Mass spectra were recorded using a Pegasus Time-of-Flight mass spectrometer (LECO Corp., St. Joseph, MI). The ionization energy was set to −70 eV and the detector voltage to 1700 V. Mass spectra were recorded with a frequency of 100 scans/s. The modification temperature offset was set to +50 °C. Data were recorded with LECO ChromaTOF software (version 4.50.8.0, LECO Corp.). The autosampler software was Maestro 1 (version 1.4.12.14, Gerstel). Data were treated using GC-Image (version 2.2b4, GCxGC, Lincoln, NE). Determination of Orthonasal Odor Thresholds. Orthonasal odor thresholds were determined using the triangular test and decreasing concentrations of aqueous odorant solutions against water as the control. Odorless glass vessels filled with either 20 mL of purified water or the respective odorant solution were presented to a panel of 15−20 trained assessors, who were asked to identify the different sample in each row and to describe the odor quality. Calculation of odor thresholds was performed as previously described.37 Aroma Profile Analysis. Aroma profile analysis was performed by a trained panel consisting of 20−25 panelists, who participated in weekly sensory sessions to train their ability to recognize and describe different aroma attributes. The following reference compounds were used to define these: bis(methylthio)methane (sulfury, garlic-like), dimethyl sulfide (cabbage-like), 3-methylbutanal (malty), 3(methylthio)propanal (cooked potato-like), (E,E)-2,4-nonadienal (fatty, green), 1-octen-3-one (mushroom-like), and 3,4-dihydro2(H)pyrrol (amine-like, sperm-like). For aroma profile analysis, the intensities of the respective aroma qualities were ranked on a sevenpoint scale from 0 (not perceivable) to 0.5, 1.0, 1.5, ..., to 3.0 (strongly perceivable). The single judgments of the panelists were averaged. As a reference, a sample of either white Alba or Burgundy truffle (10 g) was presented in a glass vessel at room temperature. Aroma Recombination Experiments. An aqueous aroma model was prepared using all quantitated aroma compounds with OAVs of >1 at their actual concentrations as determined in white Alba or Burgundy truffles. The recombinate and a sample of the respective truffle were
each placed in closed glass vessels (10 g each) and presented to the sensory panel at room temperature. The overall aroma was evaluated on the basis of the same scale that was used for aroma profile analysis.
■
RESULTS AND DISCUSSION Aroma Profiles of White Alba (WAT) and Burgundy (BT) Truffles. In the first sensory test, the single aroma
Figure 1. Aroma profiles of white Alba (WAT, gray) and Burgundy (BT, black) truffles.
Figure 2. Structures of key odorants in (A) white Alba truffles and (B) Burgundy truffles showing the highest FD factors (numbering refers to Table 2).
attributes of the truffles described by the panel members were agreed upon and the respective reference odorants eliciting the respective attribute were chosen. Then, the profiles of both fungi were compared. WAT was rated with a high intensity in the attribute sulfury, garlic-like, but with lower intensity in the attributes fatty, green, mushroom-like, and cabbage-like (Figure 1). In BT, in particular, the cabbage- and mushroom-like odors as well as the fatty, green notes were high. In both fungi, an amine-like, sperm-like attribute was clearly detectable. The smell of both truffle varieties was associated with the smell of cooked potato, malt, and amine and sperm. Identification of Odor-Active Compounds. Via AEDA on the distillates obtained by SAFE distillation, a total set of 33 odorants were found in WAT in the FD factor range of 4− 8192, while 43 odor-active regions were detectable in BT. For the identification of the compounds responsible for the 9290
DOI: 10.1021/acs.jafc.7b04073 J. Agric. Food Chem. 2017, 65, 9287−9296
Article
Journal of Agricultural and Food Chemistry
Table 2. Most Odor-Active Compounds (FD ≥ 4) Identified in White Alba Truffle (WAT) and Burgundy Truffle (BT) FDc factor
RI no.
odoranta
odor qualityb
FFAP
DB-5
WAT
BT
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 45 46 47 48 49 50 51 52 53 54 55 56
2-methylpropanal 2- and 3-methylbutanal 2,3-butanedione 3,4-dihydro-2(H)pyrrol 2,3-pentanedione 2-methylpropanol unknown 2- and 3-methylbutanol 3-hydroxy-2-butanone bis(methylthio)methane 1-octen-3-one unknown unknown 2-acetyl-1-pyrroline dimethyl trisulfide (Z)-1,5-octadien-3-one 2-propionyl-1-pyrroline (E)-2-octenal acetic acid 1-octen-3-ol 3-(methylthio)propanal 2,3-diethyl-5-methylpyrazine propanoic acid (E)-2-nonenal 3,5-dimethyl-2-ethenylpyrazine 2-methylpropanoic acid 2-ethenyl-3-ethyl-5-methylpyrazine phenylacetaldehyde unknown 2- and 3-methylbutanoic acid (E,E)-2,4-nonadienal 3-(methylthio)propanol 1,2,4-trithiolane 3-methylnonane-2,4-dione pentanoic acid unknown (E,E)-2,4-decadienal geosmine 2-methoxyphenol unknown 4-methyl-2-methoxyphenol trans-4,5-epoxy-(E)-2-decenal γ-nonalactone 4-hydroxy-2,5-dimethyl-3(2H)-furanone δ-nonalactone 2-propyl-4-methoxyphenol unknown 3-hydroxy-4,5-dimethyl-2(5H)-furanone 3-propylphenol 2,6-dimethoxyphenol δ-dodecalactone 4-vinylphenol unknown unknown phenylacetic acid vanillin
malty malty buttery amine-like, sperm-like buttery malty pungent malty buttery sulfury, garlic-like mushroom-like earthy sulfury popcorn-like, roasty cabbage-like geranium-like, metallic popcorn-like, roasty fatty, nutty vinegar-like mushroom-like cooked potato-like earthy sweaty fatty, green earthy sweaty moldy, earthy flower-like sulfury sweaty fatty, green cooked potato-like sulfury, onion-like anise-like, hay-like, fishy sweaty pungent fatty, fried beet-like, moldy smoky phenolic vanilla-like, clove-like, smoky metallic coconut-like caramel-like coconut-like clove-like, phenolic sufury seasoning-like phenolic, leather-like smoky, sweet, clove-like peach-like phenolic metallic phenolic honey-like vanilla-like
877 965 995 1027 1055 1091 1176 1193 1276 1268 1295 1307 1322 1334 1374 1370 1423 1429 1442 1443 1452 1484 1509 1523 1550 1571 1585 1638 1650 1658 1690 1706 1723 1725 1731 1790 1805 1816 1859 1881 1942 2000 2020 2040 2077 2106 2115 2201 2225 2281 2387 2393 2403 2480 2547 2573
555 688 641 682 708 648 ndd 727 800 898 979 ndd ndd 931 973 984 1025 1053 612 993 917 1151 727 1163 1107 729 ndd 1055 ndd 858/846 1223 998 1109 1238 891 ndd 1327 1430 1090 ndd 1193 1388 1371 1052 1371 1360 1383 1120 1283 1358 1702 1254 ndd ndd 1258 1413
4 1024 1024 64
