Changes in the Key Aroma Compounds of Raw Shiitake Mushrooms (Lentinula edodes) Induced by Pan-Frying As Well As by Rehydration of Dry Mushrooms

Article abstract of DOI:10.1021/acs.jafc.0c01101

Application of the aroma extract dilution analysis (AEDA) on an extract/distillate from raw shiitake mushrooms revealed 32 odorants among which 3-(methylthio)propanal (cooked potato), 1-octen-3-one, and 1-octen-3-ol (both mushroom-like) showed the highest flavor dilution (FD) factors. An isotope enrichment experiment with raw shiitake tissue and either ¹³C₁₈-linoleic acid or ²H₄-1-octen-3-ol confirmed that both 1-octen-3-ol and 1-octen-3-one are direct degradation products of the fatty acid, but it could be proven for the first time that the ketone is not formed by an oxidation of the alcohol. After pan-frying, 42 odor-active compounds appeared among which 3-hydroxy-4,5-dimethylfuran-2(5H)-one (savory), 1,2,4,5-tetrathiane (burnt, sulfury), 4-hydroxy-2,5-dimethylfuran-3(2H)-one (caramel-like), phenylacetic acid (honey-like), 3-(methylthio)-propanal, and trans-4,5-epoxy-(E)-2-decenal (metallic) showed the highest FD factors. To get a deeper insight into their aroma contribution, 19 key odorants were quantitated in the raw shiitake and twenty-one in the pan-fried mushrooms by stable isotope dilution assays, and new methods for the quantitation of four sulfur compounds were developed. A calculation of odor activity values (OAV; ratio of concentration to odor threshold) showed that 1-octen-3-one was by far the most important odorant in raw shiitake. During pan-frying, in particular, four aroma compounds were significantly increased, i.e., 4-hydroxy-2,5-dimethylfuran-3(2H)-one, dimethyl trisulfide, 1,2,4,5-tetrathiane, and 1,2,3,5,6-pentathiepane. The overall aroma profile of pan-fried shiitake could very well be mimicked by an aroma recombinate consisting of 15 reference aroma compounds in the concentrations determined in the pan-fried mushrooms. Further results showed that the sulfur compounds were even higher in rehydrated dry shiitake as compared to the pan-fried mushrooms.

Full text of DOI:10.1021/acs.jafc.0c01101

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Chemistry and Biology of Aroma and Taste
Changes in the Key Aroma Compounds of Raw Shiitake
Mushrooms (Lentinula edodes) Induced by Pan-
Frying as well as by Rehydration of Dry Mushrooms
Philipp Schmidberger, and Peter Schieberle
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c01101 • Publication Date (Web): 20 Mar 2020
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Page 1 of 47
Journal of Agricultural and Food Chemistry
Changes in the Key Aroma Compounds of Raw Shiitake
Mushrooms (Lentinula edodes) Induced by Pan-Frying
as well as by Rehydration of Dry Mushrooms
Philipp C. Schmidberger and Peter Schieberle
Technische Universität München, Lehrstuhl für Lebensmittelchemie, Lise-Meitner-
Straße 34, 85354 Freising, Germany
* Corresponding author
phone 004987197698160
e-mail peter.schieberle@ch.tum.de
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ABSTRACT:
Application of the aroma extract dilution analysis (AEDA) on an extract/distillate
from raw shiitake mushrooms revealed 32 odorants among which 3-
(methylthio)propanal (cooked potato), 1-octen-3-one and 1-octen-3-ol (both
mushroom-like) showed the highest flavor dilution (FD) factors. An isotope enrichment
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experiment with raw shiitake tissue and either C18-linoleic acid or H4-1-octen-3-ol
confirmed that both, 1-octen-3-ol and 1-octen-3-one are direct degradation products of
the fatty acid, but it could be proven for the first time that the ketone is not formed by
an oxidation of the alcohol. After pan-frying, 42 odor-active compounds appeared
among which 3-hydroxy-4,5-dimethylfuran-2(5H)-one (savory), 1,2,4,5-tetrathiane
(burnt, sulfury), 4-hydroxy-2,5-dimethylfuran-3(2H)-one (caramel-like), phenylacetic
acid (honey-like), 3-(methylthio)-propanal and trans-4,5-epoxy-(E)-2-decenal (metallic)
showed the highest FD factors. To get a deeper insight into their aroma contribution,
19 key odorants were quantitated in the raw shiitake and twenty-one in the pan-fried
mushrooms by stable isotope dilution assays, and new methods for the quantitation of
four sulfur compounds were developed. A calculation of odor activity values (OAV; ratio
of concentration to odor threshold) showed that 1-octen-3-one was by far the most
important odorant in raw shiitake. During pan-frying, in particular four aroma
compounds were significantly increased, i.e., 4-hydroxy-2,5-dimethylfuran-3(2H)-one,
dimethyl trisulfide, 1,2,4,5-tetrathiane and 1,2,3,5,6-pentathiepane. The overall aroma
profile of pan-fried shiitake could very good be mimicked by an aroma recombinate
consisting of 15 reference aroma compounds in the concentrations determined in the
pan-fried mushrooms. Further results showed that the sulfur compounds were even
higher in rehydrated dry shiitake as compared to the pan-fried mushrooms.
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KEYWORDS: raw shiitake, pan-fried shiitake, dry,rehydrated shiitake, sensomics,
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odor activity value, [2H4]-1,2,3,5,6-pentathiepane, [2H4]-1,2,4,5-tetrathiane, [2H6]-
1,2,4,6-tetrathiepane, stable isotope dilution analysis,
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INTRODUCTION
The global market consumption of mushrooms (edible fungi) is about 12.7 million
tons (2018) and button mushrooms (Agaricus bisporus) hold the highest market share
followed by shiitake mushrooms (Lentinula edodes). The growing demand for foods
produced without meat in particular in Western countries, the low content of fat and
cholesterol in the mushroom tissue, the presence of a lot of micronutrients as well as
the unique aroma are undoubtedly the main drivers for the increase in mushroom
production/consumption in the past decades. Shiitake is mainly used for its flavor
properties in the Asian cuisine, but is today also increasingly sought after in European
countries. Several odorants have been suggested to be important for the shiitake
aroma. Morita and Kobayashi1 were among the first to identify 1,2,3,5,6-pentathiepane
in shiitake suggesting that the sulfur compounds is an impact odorant due to its
“shiitake-like” odor. Later, Chen and Ho2 identified further 18 sulfur compounds, for
example 1,2,4,5-tetrathiane, 1,2,3,5-tetrathiane and 1,2,4-trithiolane in raw shiitake.
Hong et al.3 isolated the volatile fraction from raw and dry shiitake by simultaneous
distillation/extraction (SDE) and reported on carbon disulfide, dimethyl disulfide,
dimethyl trisulfide and 1-(methylthio)-dimethyl disulfide besides 1-octen-3-ol, 1-octen-
3-one, 3-octanol, 3-octanone, cis-2-octenol, cis-2-octenal and n-octanol as further
volatile components. However, application of SDE to raw, enzyme containing foods is
known to cause artifacts either during maceration of the tissue or by the thermal
treatment during distillation.
It is meanwhile accepted in the scientific literature that not all volatiles occurring
in a given food contribute to the overall aroma profile. One reference method to locate,
identify and quantitate the key aroma compounds able to interact with the human
odorant receptors is the Sensomics approach.4 Mushrooms are scarcely used as such,
but are mostly processed either by cooking or pan-frying, and thus, non-volatile
precursors present in the raw tissue may be thermally converted into volatile
compounds. To get an idea on such conversions, the Sensomics approach has
previously4 been used in a study on changes in the key aroma compounds of raw and
pan-fried white mushroom (Agaricus bisporus L.), and 4-hydroxy-2,5-dimethylfuran-
3(2H)-one (caramel-like) followed by 2-propionyl-1-pyrroline (popcorn-like) and 3-
hydroxy-4,5-dimethylfuran-2(5H)-one (seasoning-like) could clearly be assigned as the
key aroma compounds increasing during pan-frying.
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However, systematic investigations on the influence of a thermal treatment on
changes in the aroma compounds of shiitake mushrooms are rather scarce. Therefore,
the aim of the present study was to (i) characterize the key aroma compounds in raw
shiitake mushrooms by application of the AEDA and (ii) to quantitate the odor-active
compounds by means of stable isotope dilution assays (SIDAs) followed by a
calculation of odor activity values. The same approach should then be applied on pan-
fried as well as dry, rehydrated shiitake to elucidate the compounds undergoing
changes during the thermal treatment or the hydration process. To establish the
analytical data, the aroma profile of pan-fried shiitake and the dry,rehydrated
mushrooms should be mimicked by means of an aroma recombinate on the basis of
the quantitative data measured in the mushroom samples. Finally, isotope labeling
experiments should be undertaken to elucidate the formation pathway of 1-octen-3-
one, the key aroma compound in raw shiitake also occurring in nearly all types of raw
edible mushrooms.
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MATERIALS AND METHODS
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Mushrooms. Several batches of fresh shiitake mushrooms were purchased from a
local supplier. The mushrooms contained 0.3 % lipids per 100 g fresh weight. The
main fatty acid was linoleic acid with about 70 % after methanolysis of the total lipids.
Linoleic acid was mainly bound in the phospholipid fraction (data not shown).
For pan-frying, fresh mushrooms were cut into slices, and fried in a hot pan (140 °C)
for 5 min with several turnovers, but without addition of fat.
Dry shiitake mushrooms were purchased from Hawlik Vitalpilze (Straßlach;
Germany). The drying temperature used was 40 °C (as indicated on the label).
Chemicals
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Acetic acid, acetic anhydride, aluminium oxide 90 (neutral), anhydrous sodium sulfate,
deuterated methylene chloride, methylene chloride, sulfur, silica 60, sodium iodide,
sodium sulfide, sodium sulfite, and methylene dithiocyanate were obtained from Merck,
(Darmstadt, Germany). Methylene chloride was freshly distilled before use. Liquid
nitrogen was obtained from Linde (Munich, Germany).
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Reference Odorants.
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The following reference odorants were obtained from the commercial sources given in
parentheses: acetic acid, 4-allyl-2-methoxyphenol, 2,3-butanedione, butanoic acid, δ-
decalactone, γ-decalactone, 2,3-diethyl-5-methylpyrazine, (E,E)-2,4-decadienal,
dimethyl trisulfide, (Z)-6-dodeceno-γ-lactone, 3,5-dimethyl-2-ethenylpyrazine, 3-
ethylphenol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, 4-hydroxy-2,5-dimethyl-3(2H)-
furanone, indole, 2-methoxy-5-methylphenol, 2-methoxyphenol, 3-methylbutanal, 2-
methylbutanoic acid, 3-methylbutanoic acid, 3-(methylthio)propanal, (E,E)-2,4-
nonadienal, γ-nonalactone, δ-nonalactone, (E)-2-nonenal, octanal, (E)-2-octenal, 1-
octen-3-ol, pentanoic acid, phenylacetaldehyde, phenylacetic acid, 3-methylindole and
γ-undecalactone (Sigma Aldrich Chemie, Taufkirchen, Germany); 1-octen-3-one (Alfa
Aesar, Karlsruhe, Germany); 1,2,3,5,6-pentathiepane (Chemos Group, Regenstauf,
Germany); vanillin (Merck); 4-tert-butylphenol, 2-isobutyl-3-methoxypyrazine, (Acros
Organics BVBA, Geel, Belgium).
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Synthesis of 1,2,4-Trithiolane and 1,2,4,6-Tetrathiepane
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The compounds were synthesized following a procedure published earlier5 with some
modifications: Sodium sulfide nonahydrate (100 g; 0.42 mol) and sulfur (20g; 0.62 mol)
were stirred in deionized water (330 mL) until an orange-red color appeared. The
mixture was filtrated and after addition of dichloromethane (330 ml) stirred at room
temperature (RT) for seven h. Then, the organic phase was washed with brine (total
volume 240 mL) and dried over anhydrous sodium sulfate. The solvent was carefully
removed in vacuo, and the residue was re-crystallized from dry dichloromethane.
1,2,4-Trithiolane (1.5 g) and 1,2,4,6-tetrathiepane (5.6 g) were characterized by mass
spectrometry (see spectra in Figure 1A and Figure 2A).
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Synthesis of 1,2,4,5-Tetrathiane
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The odorant was synthesized following a method published previously6 with several
modifications: Methylene dithiocyanate (6.5 g) and sodium disulfate (11.4 g) were
dissolved in demineralized water (50 mL) and refluxed for 15 min. Water was removed
in vacuo and, after addition of potassium iodide (20 g), acetic acid (225 mL) followed
by acetic acid anhydride (25 mL) refluxed for 1 h. After cooling, water (100 mL) was
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added, the excess of iodine was removed by addition of ascorbic acid, and the mixture
was extracted with dichloromethane (total volume 200 mL). The organic phase was
washed with sodium bicarbonate (0.5 mol/l; 100 mL) and after drying over anhydrous
sodium sulfate concentrated in vacuo to about 5 mL. The solution was filtered over
neutral aluminium oxide using dichloromethane, and after concentration of the solution
purified by column chromatography on silica gel flushed with n-pentane. The
tetrathiane was obtained in a yield of 0.58 g and was characterized by mass
spectrometry (Figure 3A).
The following reference compounds were synthesized according to literature
cited: 2-acetyl-1-pyrroline,7 trans-4,5-epoxy-(E)-2-decenal,8 (Z)-1,5-octadien-3-one,9
and 2-propionyl-1-pyrroline.10
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Isotopically Labeled Reference Compounds.
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The compounds, either labeled with deuterium or carbon-13, were synthesized
according to the literature cited: [2H2-5]-2-acetyl-1-pyrroline,7 [2H3]-2,3-diethyl-5-
methylpyrazine,
[2H3]-3,5-dimethyl-2-ethylpyrazine
and
[2H3]-2,5-dimethyl-3-
ethylpyrazine,11 [13C2]-3-hydroxy-4,5-dimethylfuran-2(5H)-one,12 [13C2]-4-hydroxy-2,5-
dimethylfuran-3(2H)-one,13 [2H3]-3-(methylthio)propanal,14 [2H2]-3-methylbutanal,15
[2H2]-3-methylbutanoic acid,16 [2H4]-1-octen-3-ol,17 [2H4]-1-octen-3-one,18 [13C2]-
phenylacetaldehyde,19 [2H2−4]-2-propionyl-1-pyrroline,20 [2H4]-trans-4,5-epoxy-(E)-2-
decenal;21
[2H3]-acetic acid and [13C2]-phenylacetic acid were obtained from Sigma-Aldrich
(Taufkirchen, Germany); [2H3]-pentanoic acid was obtained from C/D/N isotopes
(Quebec, Canada).
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Synthesis of sulfur-containing isotopically labeled internal standards.
[2H4]-1,2,3,5,6-pentathiepane, [2H4]-1,2,4,5-tetrathiane, [2H6]-1,2,4,6-tetrathiepane
and [2H4]-1,2,4-trithiolane were synthesized following a procedure published for the
unlabeled compounds22 with some modifications: Sodium sulfide nonahydrate (7.5 g;
96 mmol) and sulfur (1.5 g; 57 mmol) were dissolved in distilled water (25 mL) and
stirred for 1h at RT. The orange colored solution was filtered and [2H2]-dichloromethane
(25 mL; 0.4 mmol) was added. After vigorous stirring for 7 h, the organic layer was
washed with brine (25 mL) followed by distilled water (25 mL). The solution was
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concentrated to ~200 µL at 52 °C using a Vigreux column. The compound mixture was
purified by flash chromatography on silica with n-pentane. The mass spectra obtained
after separation by gas chromatography are displayed in Figure 1B ([2H4]-1,2,4-
trithiolane; Yield 89 mg, Figure 2B ([2H6]-1,2,4,6-tetrathiepane; yield 35,1 mg), Figure
3B ([2H4]-1,2,4,5-tetrathiane; yield 20,3 mg) and Figure 4B ([2H4]-1,2,3,5,6-
pentathiepane; yield 3,9 mg).
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The concentration of each compound was determined with methyl octanoate as the
internal standard using a calibration curve determined with mixtures of methyl
octanoate and the respective unlabeled compound.
Isolation of the Volatiles. Either fresh (cut into small slices), pan-fried or dry,
rehydrated shiitake mushrooms (on the basis of 30 g fresh weight) were frozen in liquid
nitrogen and ground by means of a commercial blender after addition of anhydrous
sodium sulfate to bind water. For volatile isolation, the material was extracted with
dichloromethane (total volume 300 mL) by vigorous stirring for 60 min. The mixture
was filtered and the volatile fraction was isolated using the solvent assisted flavor
evaporation (SAFE) technique.23 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 i.d.) and a microdistillation apparatus.24
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Fractionation of the Volatiles. For identification experiments, the mushroom material
(300 g) was extracted and worked up as described above. To separate the acidic from
the neutral and basic volatiles, the SAFE distillate was treated with an aqueous sodium
bicarbonate solution (0.5 mol/L, total volume 300 mL) to isolate the neutral/basic
volatile fraction in the solvent phase (NBF).20 The combined aqueous layers were
adjusted to pH 2 with hydrochloric acid, and the acidic volatiles (acidic fraction, AF)
were extracted with methylene chloride (total volume 300 mL). Both fractions were
dried over anhydrous sodium sulfate, and after filtration each fraction was concentrated
to ~500 μL. The NBF was further fractionated by column chromatography using a
water-cooled glass column (30 cm × 1.8 cm) filled with a slurry of 30 g of acid-washed
silica (G60, 7% water) in n-pentane and separated into six fractions using n-pentane/
diethyl ether mixtures of increasing polarity (fraction I, 100:0, 75 mL; fraction II, 95:5,
80 mL; fraction III, 90:10, 80 mL; fraction IV, 75:25, 80 mL; fraction V, 50:50, 80 mL;
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fraction VI, 0:100, 110 mL)20 plus a seventh fraction using methylene chloride
(100 mL). Each fraction was dried over anhydrous sodium sulfate, concentrated to
~200 μL as described above, and analyzed by means of HRGC-O and HRGC-MS.
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High-Resolution Gas Chromatography−Olfactometry (GC-O). GC-O analysis was
performed using a Thermo Electron Trace Ultra gas chromatograph (Dreieich,
Germany) with the following J&W Scientific fused silica capillary columns: DB-FFAP
and DB-5 (both 30 m × 0.25 mm i.d.; 0.25 μm film thickness) (Folsom, CA, USA). The
sample (1.0 µL) was injected cold on-column at 40 °C using helium as carrier gas at a
flow rate of 1.2 mL/min. The oven temperature was programmed from 40 °C, held for
2 min, increased with 6 °C/min to 230 °C (for the DB-FFAP column or to 240 °C for the
DB-5 column) and held for 5 min. The flow of the carrier gas was split at the end of the
capillary column by a Chrompack Y-type quick-seal glass splitter (Frankfurt, Germany)
and two deactivated fused-silica capillaries (50 cm × 0.32 mm i.d.). One part was
directed to an FID (250 °C) and the other to a sniffing port (190 °C). A series of n-
alkanes C6−C26 for the DB-FFAP and C6−C18 for the DB-5 was used to determine
linear retention indices (RI).
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Aroma Extract Dilution Analysis. For the determination of FD factors the
concentrated SAFE distillate was subjected to GC-O on the FFAP column to detect
and describe the odors of all aroma-active areas. To avoid overlooking of odor-active
compounds, the original distillate was sensorially analyzed by at least three
experienced panelists. Then, the distillate was diluted stepwise 1:1 (by vol) with
methylene chloride, and each dilution was analyzed in 1.0 μL aliquots by HRGC-O.
High-Resolution Gas Chromatography−Mass Spectrometry (HRGC-MS). For
compound identification, mass spectra were acquired using a Hewlett-Packard gas
chromatograph 5890 series II (Waldbronn, Germany) coupled to a Finnigan MAT 95 S
sector field mass spectrometer (Bremen, Germany). Mass spectra in the electron
impact (MS-EI) mode were generated at 70 eV, and chemical ionization (MS-CI) was
performed at 115 eV using isobutane as the reactant gas.
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Quantitation of Odorants by Stable Isotope Dilution Assays (SIDA). Depending on
the amounts of the odorants estimated in preliminary experiments, isotopically labeled
internal standards (0.1−70 μg), dissolved in methylene chloride, were added to the
mushroom material (1−50 g) suspended in methylene chloride. After 1 h of stirring for
equilibration, the work-up procedure was performed as described above.
Quantitation of compounds present in higher concentrations was performed using a
Varian CP 3800 gas chromatograph and a Varian Saturn 2000 ion trap mass
spectrometer (Darmstadt, Germany) (system 1; Table 1). The sample was injected on
the J&W Scientific DB-FFAP capillary column (60 m × 0.25 mm i.d.; 0.25 μm film
thickness) by the cold on-column technique, and mass spectra of the separated
compounds were recorded for the respective ions in the MS-CI mode using methanol
as reagent gas (system 1; Table 1).
For the separation of co-eluting compounds, a two dimensional-GC-MS system (TD-
GC-MS) consisting of a Thermo Scientific Trace GC 2000 series (Dreieich, Germany)
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 J&W Scientific DB-FFAP capillary
column (30 m × 0.32 mm i.d.; 0.25 μm film thickness). The elution range containing the
selected odorant and the internal standard was then transferred into a cold trap by a
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 (30 m × 0.25 mm i.d.; 0.25 μm film thickness) by heating the trap to 200 °C.
Mass spectra were recorded in the MS- CI mode using methanol as reagent gas
(system 2; Table 1).
For compounds present in low concentrations comprehensive two-dimensional gas
chromatography (GC x GC) was performed using an Agilent 6890N gas
chromatograph (Böblingen, Deutschland) equipped with a Gerstel PTV 4 injector
(Mühlheim an der Ruhr, Deutschland) and a CTC-analytics PAL autosampler
(Zwingen, Schweiz) (system 3; Table1). After cold on-column injection (-20 °C) and
immediate increase of the temperature to 240 °C, separation of the distillate in the first
dimension was achieved on the J&W Scientific DB-FFAP capillary column (30 m × 0.32
mm i.d.; 0.25 μm film thickness) with helium as carrier gas at a flow rate of 1.2 mL/min.
The effluent was transferred onto the second column, a J&W Scientific DB-5 (1 m ×
0.25 mm i.d.; 0.25 μm film thickness). Mass spectra were recorded using a Leco
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Pegasus 4 time-of-flight mass spectrometer (St. Joseph, USA). Ionization energy was
set to -70 eV, detector voltage to 1700 V. Mass spectra were recorded with a frequency
of 100 scans/s. Mod. temperature offset was set to +50 °C. Data were recorded with
the LECO ChromaTOF software (v. 4.50.8.0, LECO Corporation, Michigan, USA). The
autosampler software was Gerstel Maestro 1 (v. 1.4.12.14) (Mülheim an der Ruhr).
Data were treated using GC-Image (v. 2.2b4 GCxGC) (Lincoln, NE, USA). Response
curves were prepared from defined mixtures of the labeled and the unlabeled
compound as detailed for the four sulfur compounds below.
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Quantitation of trans-4,5-epoxy-(E)-2-decenal was performed using an Agilent 7890B
gas chromatograph and an Agilent ion trap 240 mass spectrometer (Waldbronn,
Germany). The sample was injected on the J&W Scientific DB-FFAP capillary column
(60 m × 0.25 mm i.d.; 0.25 μm film thickness) by the cold on-column technique, and
mass spectra of the separated compounds were recorded in the negative MS-CI mode
using methanol as reagent gas (system 4;Table 1).
For the quantitation of 2- and 3-methylbutanoic acid, the sum of both acids coeluting
on the respective gc stationary phase was determined by SIDA. Then, the ratio of 2-
and 3-methylbutanoic acid in the samples was determined by HRGC-MS by calculating
the intensities of the fragments m/z 60 (3-methylbutanoic acid) and m/z 74
(2-methylbutanoic acid). For method standardization, defined mixtures of 2- and 3-
methylbutanoic acid were analyzed under the same conditions, and a calibration line
was drawn plotting the intensity ratio of the ions against the percentage of 3-
methylbutanoic acid in the mixture.
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Response curves for the quantitation of [2H4]-1,2,3,5,6-pentathiepane, [2H4]-
1,2,4,5-tetrathiane, [2H6]-1,2,4,6-tetrathiepane and [2H4]-1,2,4-trithiolane
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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 HRGC-MS to
calculate the response factor (RF) from the intensities of the mass fragments. The
response curves obtained are shown in the supplementary information (Figures S1 to
S4).
Determination of Orthonasal Odor Thresholds. Orthonasal odor thresholds were
determined using the triangular test with decreasing concentrations of aqueous
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odorant solutions against odorless water as the control. Glass vessels either filled with
the odorless water (Volvic; 20 mL) or with the respective aqueous odorant solution (20
mL) were presented to a panel of 15−20 trained assessors, who were asked to identify
the different sample in each row and describe the odor quality. Calculation of odor
thresholds was performed as described before.25
Aroma Profile Analysis. Aroma profiles were determined 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 qualities. The following reference
compounds were used to define the aroma attributes: 2-acetyl-1-pyrroline (popcorn-
like, roasty), (E,E)-2,4-decadienal (fatty; fried), 2,3-diethyl-5-methylpyrazine (earthy),
trans-4,5-epoxy-(E)-2-decenal (metallic), 3-hydroxy-4,5-dimethylfuran-2(5H)-one
(seasoning-like), 1-octen-3-one (mushroom-like), 3- (methylthio)propanal (cooked
potato-like), phenylacetaldehyde (flowery), and 1,2,4,5-tetrathiane (sulfury, burnt). The
intensities of the respective aroma qualities were ranked on a seven-point scale from
0 (not perceivable) over 0.5, 1.0, 1.5, ..., to 3.0 (strongly perceivable). The judgements
of the panelists were averaged. Samples (20 g) were presented in glass vessels at
room temperature (RT).
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Aroma Recombination Experiments. An aqueous aroma model was prepared using
all quantitated reference aroma compounds with OAVs >1 in their actual
concentrations determined in the pan-fried mushrooms. The recombinate and the pan-
fried mushrooms were each placed in closed glass vessels (20 g each) and presented
to the sensory panel at RT. The overall aroma was evaluated on the basis of the same
scale as used for aroma profile analysis.
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Isotopologue Enrichment Study with Carbon-13 Labeled Linoleic Acid and
Deuterium labeled 1-Octen-3-ol
Either [13C18]-linoleic acid or [2H4]-1-octen-3-ol were dissolved in phosphate buffer (18
mL; pH 6.5, 0.1M, according to Sørensen) with the aid of Tween 80 (0.1%). In
separate experiments, the amount of each labeled compound was used in an
equimolar concentration compared to the respective unlabeled compound measured
in the mushroom homogenate. Mushrooms were added and homogenized with an
ultraturrax homogenizer for 30 s. To stop enzymatic reactions, glass vials containing
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hydrochloric acid (1 mL; 2mol/l) were prepared. In the experiment with labeled
linoleic acid, 1 mL (n=3) of the homogenate was transferred after 1, 2, 4, 8, 15 and
30 min into these vials followed by the internal standards 2-nonanone, [2H4]-1-octen-
3-ol, [2H4]-1-octen-3-one and [2H2]-(E)-2-nonenal. The formation of 1-octen-3-ol, 1-
octen-3-one, 3-octanone and (E)-2-octenal as well as the four eight-fold carbon-13
labeled isotopologues (formed from [13C18]-linoleic acid) was measured by solid
phase micro extraction (SPME) after 15 min equilibration. The experiment with [2H4]-
1-octen-3-ol followed the same approach, but only 2-nonanone was added as the
internal standard.
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RESULTS AND DISCUSSION
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Identification of Odor-Active Compounds in Raw Shiitake.
Freshly macerated raw shiitake mushrooms showed an intense mushroom-like,
metallic aroma, and the volatile fraction isolated by extraction and SAFE distillation
elicited the same overall aroma profile when evaluated on a strip of filter paper.
Application of the AEDA resulted in 34 odorants in the FD factor range of 4 to 4096
(Figure 5), among which 13 exhibiting a potato-like odor showed the highest FD factor
of 4096, followed by 5 and 12, both with a mushroom-like odor attribute. With
somewhat lower FD factors, 30 with a metallic odor quality and 52 exhibiting a vanilla-
like odor were detected. The odor qualities, retention indices and mass spectra (MS-
EI and MS-CI) were first compared with data available in an in-house database
established by the analysis of more than 1000 odor-active compounds as recently
reported for a group of sulfur containing odorants.26 By GC/O and GC/MS analysis of
the respective reference compound, the following aroma compounds were
subsequently identified: 13 (3-(methylthio)propanal), 5 (1-octen-3-one), 12 (1-octen-3-
ol), 30 (trans-4,5-epoxy-(E)-2-decenal) and 52 (vanillin)(Figure 6). Besides these five
odorants, 3-methylbutanal (1, malty), (E)-2-octenal (10, fatty, nutty),
phenylacetaldehyde (19, flowery), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (32,
caramel-like), and 3-hydroxy-4,5-dimethyl-2(5H)-furanone (40, seasoning-like) also
reached quite high FD factors among the twenty-eight odor-active compounds
identified (Table 2). Only four out of the compounds identified in this study (5,12,41,53)
have previously been described as volatiles in shiitake. Among them, the sulfury and
burnt smelling compounds 1,2,3,5,6-pentathiepane (41) and 1,2,4,5-tetrathiane (53)
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have earlier been identified in a steam distillate of fresh or dried shiitake
mushroom1,2,27-30 and were reported to be enzymatically formed in raw shiitake by a
degradation of the precursor lentinic acid.31-33
Development of stable isotope dilution assays for the quantitation of 1,2,3,5,6-
pentathiepane, 1,2,4,5-tetrathiane, 1,2,4,6-tetrathiepane and 1,2,4-trithiolane
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During GC/O the entire amount of a single component present in an extract is
vaporized and is, thus, available for the odorant receptors during GC/O.34 However, in
a food only the relative proportion present in the headspace above a food can be used
to evaluate the aroma contribution of an odorant. To approach this challenge, a
quantitation followed by the calculation of odor activity values (OAV; ratio of
concentration to odor threshold) is an appropriate method, because the influence of
the food matrix on the volatility of a given aroma compound is taken into account.
However, while the isotopically labeled internal standards for 15 out of the 19
odorants selected or quantitation were available in our group (see experimental part),
except for the unlabeled pentathiepane, neither the other three sulfur containing
unlabeled compounds nor the four deuterium labeled isotopologues were commercially
available. Following the synthetic route described above, the three unlabeled sulfur
compounds could successfully be synthesized as confirmed by the mass spectra
displayed in Figures 1A to 3A. The deuterium labeled isotopologues [2H4]-1,2,3,5,6-
pentathiepane, [2H4]-1,2,4,5-tetrathiane, [2H6]-1,2,4,6-tetrathiepane and [2H4]-1,2,4-
trithiolane were synthesized in a one pot reaction (Figure 7) using [2H2]-
dichloromethane to introduce the labeling into the four target compounds. Their mass
spectra are contrasted in Figures 1B to 4B to the spectra of the unlabeled analytes.
The successful introduction of either four or six deuterium atoms is confirmed by a
comparison of the respective molecular ions. The response curves obtained by
monitoring the molecular ions in defined mixtures of the respective unlabeled and
labeled compound by mass chromatography are shown in Figures S1 to S4 in the
supporting information. To verify that no deuterium/hydrogen exchange occurred
during the work-up procedure, a 1:1 mixture of all eight compounds was added to a
raw mushroom homogenate, and the volatile fraction was isolated as described for the
samples.
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Quantitation of Selected Odorants in Raw Shiitake Mushrooms and
Calculation of Odor Activity Values.
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Quantitation of 19 odorants in the raw shiitake revealed by far the highest
concentrations for 1-octen-3-ol, followed by phenylacetaldehyde, phenylacetic acid
and 1-octen-3-one (Table 3). The sulfur compounds 1,2,3,5,6-pentathiepane, 1,2,4,5-
tetrathiane and 1,2,4,6-tetrathiepane were only present in quite low amounts
For OAV calculations, the odor thresholds of trans-4,5-epoxy-(E)-2-decenal, 2,3-
diethyl-5-methylpyrazine and of the four sulfur compounds were newly determined in
this study (Table 4). The results showed that only 14 of the 19 odorants quantitated
exceeded their odor threshold in water. By far the highest OAV of 2913 was calculated
for 1-octen-3-one, followed by 3-(methylthio)propanal, 3-methylbutanal and 1-octen-3-
ol with OAVs >75, while 1,2,3,5,6-pentathiepane, 1,2,4,5-tetrathiane and 1,2,4,6-
tetrathiepane showed only low OAVs between 9 and 12. The importance of 1-octen-3-
one for the typical mushroom-like odor is well in line with data on the high OAV in raw
white mushrooms.4 However, aldehydes known as degradation products of amino
acids, i.e. phenylacetaldehyde, 3-(methylthio)propanal and 3-methylbutanal (Table 4)
were previously not reported as constituents of raw shiitake, but the same observation
was reported for aroma compounds in white mushrooms.4 However, while these
aldehydes are well-known to be formed in a thermal degradation reaction of amino
acids with alpha dicarbonyls (Strecker reaction), obviously these can also be formed
at lower temperatures, probably catalyzed by pyridoxamine, a well-known catalyst in
the enzymatically induced amino acid metabolism.
Isotopologue enrichment study on the formation of C-8 aroma compounds
in raw shiitake
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Most mushrooms contain linoleic acid as the main fatty acid, and it has been
reported for Boletus edulis many years ago35 that the fatty acid is enzymatically
converted via the 10-hydroperoxide into the mushroom-like smelling 1-octen-3-ol.
Furthermore, several other C-8 compounds, such as 1-octen-3-one, 3-octanone and
(E)-2-octenal were found in shiitake36 and could also be formed from linoleic acid as
precursor. In particular the most odor-active mushroom aroma compound 1-octen-3-
one is assumed to be formed by an oxidation of the alcohol. However, this reaction
step has not yet been proven by a systematic experiment. An isotope enrichment
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approach is the most reliable way to identify a non-volatile precursor of an aroma
compound directly in the food itself. This idea is explained in the following: The food,
here the mushrooms, are administered with the same amount of an isotopically labeled
2
precursor (13C or H labeled) compared to the assumed, unlabeled precursor
quantitated in the food itself. In this case, the isotope ratio in the precursor for either
carbon-12 to carbon-13 or deuterium to hydrogen is 1:1. Thus, any reaction product
only formed from this assumed precursor during thermal treatment should
consequently appear in the same isotope ratio. But, if additional precursors would be
present, the ratio should be shifted to a higher number for 12C or 1H, respectively.
13
The experiments were first done with C18-linoleic acid, which was added to a
shiitake homogenate, and the formation of four C-8 degradation products was
monitored in a time span between 20 s and 30 min. The data indicated that the
unlabeled 1-octen-3-ol showed by far the highest concentration.as compared to 1-
octen-3-one, 3-octanone and (E)-2-octenal, respectively (Table 5). For all four
compounds the maximum concentration was reached after 8 min. The enzymatic
conversion of the 13C18 linoleic acid was a bit slower compared to the natural linoleic
acid, but a nearly 1:1 ratio for 1-octen-3-ol and [2H4]-1-octen-3-ol was reached after
about 8 to 15 min. This result clearly corroborated that this odorant was exclusively
formed from linoleic acid. A similar trend was observed for 1-octen-3-one and (E)-2-
octenal showing a delayed formation from the labeled linoleic acid, but reaching a ratio
close to 1:1 ratio in the unlabeled to labeled compound after about 15 min. The quite
13
late formation of the C8 3-octanone suggests that this compound is probably not
directly formed from linoleic acid, but obviously by an enzymatic hydrogenation of 1-
octen-3-one.
In the next experiment, the role of 1-octen-3-ol in the formation of 1-octen-3-one
was studied (Table 6). The results showed that the amount of [2H4] -1-octen-3-ol added
to the mushroom homogenate remained nearly unchanged within an incubation time
of 60 min, while the unlabeled alcohol was generated from linoleic acid. However, the
data clearly showed that no [2H4]-1-octen-3-one was formed by conversion of the
labeled alcohol (Table 6), while labeled 3-octanone as well as labeled (E)-2-octenal
were formed, however, in very small amounts. These results suggest that a direct
formation pathway of 1-octen-3-one from linoleic acid does exist, but not involving 1-
octen-3-ol as the intermediate.
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Identification of Odor-Active Compounds in Pan-fried Shiitake Mushrooms.
To get an insight into changes induced by a thermal treatment, the same batch of
shiitake was pan-fried, and the volatile fraction was isolated by extraction/SAFE
distillation. In the FD factor region of 8 to 8192 fifty-three odor-active areas were
detected by GC/O (Figure 8) among which 40 exhibiting a seasoning-like odor showed
the highest FD factor of 8192, followed by 41 and 51 with a sulfury, burnt and a honey-
like odor quality, respectively, as well as 32 with a caramel-like aroma. The
identification experiments in combination with the FD factors revealed 40 as 3-hydroxy-
4,5-dimethyl-2(5H)-furanone, 41 as 1,2,4,5-tetrathiane, 51 as phenylacetic acid and
32. 4-hydroxy-2,5-dimethylfuran-3(2H)-one (Table 7). Among the further forty-eight
odorants identified in the pan-fried mushrooms, 3-(methylthio)propanal (13) and trans-
4,5-epoxy-(E)-2-decenal (30) also showed high FD factors. However, although a few
compounds, in particular the sulfur containing odorants and 1-octen-3-one, have
previously been reported in heat treated shiitake, sensory studies on their impact on
the aroma have been performed for the first time in the present study. In addition, 33
compounds were newly reported as odorants of shiitake (Table 7).
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Quantitation of Selected Odorants in Pan-Fried Shiitake and Calculation of Odor
Activity Values. Twenty-one aroma compounds detected with high FD factors in the
pan-fried shiitake were quantitated by stable isotope dilution assays. The highest
concentrations within these odorants were measured for 1-octen-3-ol, 4-hydroxy-2,5-
dimethyl-3(2H)-furanone, 2,3-butanedione, 1,2,3,5,6-pentathiepane, 2-methylbutanoic
acid and 1,2,4,5-tetrathiane (Table 8). However, a calculation of odor activity values
revealed that only 14 aroma compounds were present above their odor threshold
(Table 9). The highest OAVs of 981 and 891 were calculated for 1,2,3,5,6-
pentathiepane and 1,2,4,5-tetrathiane. High OAVs were also found for 2,3-
butanedione, 3-(methylthio)propanal, (E,E)-2,4-decadienal, 3-methylbutanal, 1-octen-
3-one and dimethyl trisulfide.
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Aroma Simulation. To study the aroma interactions among the key odorants in
a mixture, an aroma recombinate was prepared in an aqueous solution containing all
15 aroma compounds odorants with OAVs ≥1 in their actual concentration in the pan-
fried shiitake mushrooms (Table 8). The aroma profile was evaluated by a descriptive
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aroma profile analysis and also compared to the overall odor elicited by a powder made
from freshly prepared pan-fried mushrooms. Eight aroma descriptors agreed in
preliminary sessions of the panel were used: mushroom-like, sulfury/burnt, fatty/fried,
seasoning-like, metallic, earthy, popcorn-like/roasty, and cooked potato-like. The
sensory panel agreed that the recombinate elicited the typical aroma of pan-fried
shiitake mushrooms with small differences in the attributes mushroom-like and metallic
(Figure 9). The similarity of the aqueous aroma recombinate was judged to be 2.6 of
3.0, thus corroborating the quantitative data obtained.
Changes induced by the thermal treatment
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Besides a remarkable overall difference in the aroma profiles of the raw and pan-
fried shiitake (data not shown), also clear changes in nearly all important odorants were
observed, and either a formation or a degradation occurred (Table 10). In particular, 4-
hydroxy-2,5-dimethyl-3(2H) furanone (4-HDF) was increased by a factor of over 150
in the pan-fried mushrooms followed by dimethyl sulfide, which was increased by a
factor of over 50. The caramel-like smelling 4-HDF is known to be formed by a
degradation of carbohydrates in processed foods, and hexose phosphates as well
acetylformoin were previously confirmed as important precursors.37
Also 1,2,3,5,6-pentathiepane and 1,2,4,5-tetrathiane were much higher in the
pan-fried material, obviously due to a thermal degradation of the precursor lentinic
acid. Although both sulfur compounds have previously been reported as constituents
of a steam distillate of shiitake,30 this is the first sensory study confirming the impact of
these two compounds in the aroma of heat-treated shiitake.
Nine aroma compounds were lost/degraded during pan-frying, and in particular
the mushroom like smelling 1-octen-3-one and 1-octen-3-ol were lower by a factor of
100 or 10, respectively, compared to the amounts in the raw mushroom (Table 3). As
it was recently shown for the homologue of 1-octen-3-one, the 1-penten-3-one, such
alpha, beta unsaturated ketones with a double bond at the end may easily undergo
aldol-type reactions during thermal treatment of foods and can also bind to thiols.38
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Changes induced by rehydration of dry shiitake
To increase their shelf-life and to maintain the availability over the year,
mushrooms are often dried. When such mushrooms are rehydrated for use in the
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preparation of dishes, it is common knowledge that an intense aroma is quickly
released, which is, however, quite different from the aroma of the fresh mushroom. To
get a first insight into changes in key aroma compounds, dry mushrooms from the trade
were rehydrated and the concentrations of 28 odorants detected with high FD factors
in the dry/rehydrated mushrooms (Table S1; supporting information) were quantitated
in the dry as well as in the dry/rehydrated mushrooms to see the influence of water
addition on aroma compound generation (Table S2; supporting information). In Table
11 the amounts of ten aroma compounds showing an increase during rehydration by
at least a factor of 5 are contrasted. In particular the sulfur compound 1,2,4-trithiolane
showed the highest increase during rehydration, while 1,2,4,5-tetrathiane and
1,2,3,5,6-pentathiepane showed the highest concentrations among the sulfur
compounds formed by rehydration. The increase in 1,2,3,5,6-pentathiepane
corroborates data previously published by Hirade et al.38 However, the quantitative
results confirm for the first time that the latter two compounds are more effectively
formed by rehydration of dry shiitake compared to thermal processing and both sulfur
compounds reached odor activity values of 11800 and 7500, respectively in the
rehydrated mushroom tissue ( Table S2; supporting information). Aroma profile
analyses (data not shown) of the dry/rehydrated mushrooms as well as an aroma
recombinate prepared on the basis of the quantitative data in Table S2 (supporting
information) confirmed that the overall aroma of the dry, rehydrated shiitake was mainly
influenced by the sulfury, burnt odor attributes of both key sulfur aroma compounds.
On the other hand, the mushroom-like odor note was rated lower by the sensory panel
as compared to the pan-fried shiitake.
In conclusion, the data showed for the first time that 1-octen-3-one, a long-known
key odorant of many edible mushrooms, is also the key odorant in raw shiitake. As
confirmed by isotope labeling experiments with U-13C-linoleic acid, the ketone as well
as the corresponding alcohol 1-octen-3-ol are rapidly formed during storage of a
shiitake homogenate at room temperature. However, a second labeling experiment
clearly showed that, against the literature view35, the ketone is not formed by an
enzymatic oxidation of the alcohol suggesting a yet unknown intermediate in its
generation from linoleic acid.
A thermal treatment by heating in a pan led to a significant change in the
concentrations of aroma compounds leading to over 30 odorants newly characterized
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as aroma compounds of pan-fried shiitake. While in particular the caramel-like smelling
4-HDF, a carbohydrate degradation product, was significantly increased during pan-
frying, the typical mushroom-like smelling compounds 1-octen-3-one and 1-octen-3-ol
were drastically decreased. As it was recently shown for the homolog of 1-octen-3-
one, the 1-penten-3-one, such alpha, beta unsaturated ketones with a double bond at
the end may easily undergo aldol-type reactions during thermal treatment of foods and
can also bind to thiols.39
Also 1,2,3,5,6-pentathiepane and 1,2,4,5-tetrathiane were generated during pan-
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frying of shiitake confirming previous data on their formation during steam distillation.30
However, simply the addition of water to dry shiitake, led to a much more effective
generation of both sulfur compounds. In general, these results can be used to develop
special shiitake products for use in food flavoring.
Supporting information
Table S1: Most Odor-Active Compounds (FD ≥ 8) Identified in dry, rehydrated
shiitake
Figures S1 to S4: Response curves for the quantitation of [2H4]-1,2,3,5,6-
pentathiepane, [2H4]-1,2,4,5-tetrathiane, [2H6]-1,2,4,6-tetrathiepane, [2H4]-1,2,4-
trithiolane by stable isotope dilution assays
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REFERENCES
(1)
substance of shiitake, an edible mushroom. Tetrahedron Letters 1966, 6, 537-577.
(2) Chen, C. C.; Ho, C. T., Identification of sulfurous compounds of shiitake mushroom
Morita, K.; Kobayashi, S., Isolation and synthesis of lenthionine, an odorous
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
(Lentinus edodes Sing.). J. Agric. Food Chem. 1986, 34, 830-833.
(3) Hong J.; Lee, K.; Kim, Y.; Kim, D.; Kim, M.; Kim, Y.; Yeo, K., Volatile flavor compounds of
Korean shiitake mushroom (Lentinus edodes). Kor. J. Food Sci. Technol. 1988, 20.
(4)
Grosshauser, S.; Schieberle, P., Characterization of the key odorants in pan-fried
white mushrooms (Agaricus bisporus L.) by means of molecular sensory science:
comparison with the raw mushroom tissue. J. Agric. Food Chem. 2013, 61, 3804-3813.
(5)
Chem. Ber. 1965, 98, 1455-1462.
(6) Gil, V.; MacLeod, A. J., Synthesis and assessment of three compounds suspected as
egg aroma volatiles. J. Agric. Food Chem. 1981, 29, 484-488.
(7) Schieberle, P.; Grosch, W., Quantitative analysis of aroma compounds in wheat and
Böhme, H.; Müller, O., Über Bis-acylmercapto-methane und ihre Spaltung mit Chlor.
rye bread crusts using a stable isotope dilution assay. J. Agric. Food Chem. 1987, 35, 252-
257.
(8)
Schieberle, P.; Grosch, W., Potent odorants of the wheat bread crumb Differences to
the crust and effect of a longer dough fermentation. Z Lebensm. Unters. Forsch. 1991, 192,
130-135.
(9)
Schieberle, P.; Gassenmeier, K.; Guth, H.; Sen, A.; Grosch, W., Character impact
odour compounds of different kinds of butter. LWT-Food Sci. Techn. 1993, 26, 347-356.
(10) Schieberle, P., Primary odorants in popcorn. J. Agric. Food Chem. 1991, 39, 1141-
1144.
(11) Cerny, C.; Grosch, W., Quantification of character-impact odour compounds of
roasted beef. Z. Lebensm. Unters. Forsch. 1993, 196, 417-422.
(12) Blank, I.; Schieberle, P.; Grosch, W., Quantification of the flavor compounds 3-
hydroxy-4,5-dimethyl-2(5H)-furanone and 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanon by a
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607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
stable isotope dilution assay. Prog. Flavour Precursor Stud. Proc. Int. Conf. 1993 (Meeting
Date 1992) 1993, 103-109.
(13) Sen, A.; Schieberle, P.; Grosch, W., Quantitative determination of 2,5-dimethyl-4-
hydroxy-3(2H)-furanone and its methyl ether using a stable isotope dilution assay. Lebensm.
Wiss. Techn. 1991, 24, 364-369.
(14) Sen, A.; Grosch, W., Synthesis of six deuterated sulfur-containing odorants for use as
internal standards in quantification assays. Z. Lebensm Unters. Forsch. 1991, 192, 541-547.
(15) Schieberle, P.; Grosch, W., Changes in the concentrations of potent crust odorants
during storage of white bread. Flavour Fragrance J. 1992, 7, 213-218.
(16) Guth, H.; Grosch, W., Identification of character impact odorants of stewed beef juice
by instrumental analyses and sensory studies. J. Agric. Food Chem. 1994, 42.
(17) Kubickova, J.; Grosch, W., Quantification of potent odorants in Camembert cheese
and calculation of their odour activity values. Int. Dairy J. 1998, 8, 17-13.
(18) Lin, J.; Welti, D. H.; Vera, F. A.; Fay, L. B.; Blank, I., Synthesis of deuterated volatile
lipid degradation products to be used as internal standards in isotope dilution assays. J.
Agric. Food Chem. 1999, 47.
(19) Schuh, C.; Schieberle, P., Characterization of the key aroma compounds in the
beverage prepared from Darjeeling black tea: Quantitative differences between tea leaves
and infusion. J. Agric. Food Chem. 2006, 54, 916-924.
(20) Schieberle, P., Quantitation of important roast-smelling odorants in popcorn by stable
isotope dilution assays and model studies on flavor formation during popping. J. Agric. Food
Chem. 1995, 43, 2442-2448.
(21) Guth, H.; Grosch, W., Deterioration of soya-bean oil: quantification of primary flavor
compunds using a stable isotope dilution assay. Lebensm. Wiss. Techn. 1990, 23, 513-522.
(22) Morita, K.; Kobayashi, S., Isolation, structure, and synthesis of lenthionine and its
analogs. Chem. Pharm. Bull. 1967, 15, 988-993.
21
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Page 22 of 47
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
(23) Engel, W.; Bahr, W.; Schieberle, P., Solvent assisted flavour evaporation–a new and
versatile technique for the careful and direct isolation of aroma compounds from complex
food matrices. Eur. Food Res. Technol. 1999, 209, 237-241.
(24) Schieberle, P., New developments in methods for analysis of volatile flavor
compounds and their precursors. In: Elsevier Science: Amsterdam, The Netherlands: 1995;
403-431.
(25) Czerny, M.; Christlbauer, M.; Christlbauer, M.; Fischer, A.; Granvogl, M.; Hammer, M.;
Hartl, C.; Hernandez, N. M.; Schieberle, P., Re-investigation on odour thresholds of key food
aroma compounds and development of an aroma language based on odour qualities of
defined aqueous odorant solutions. Eur. Food Res. Techn. 2008, 228, 265-273.
(26) Schönauer, S.; Schieberle, P. Screening for Novel Mercaptans in 26 Fruits and 20
Wines Using a Thiol-Selective Isolation Procedure in Combination with Three
Detection Methods. J.Agric. Food Chem. 2019, 67, 4553-4559
(27) Wu, C.-M.; Wang, Z., Volatile compounds in fresh and processed shiitake mushrooms
(Lentinus edodes Sing.). Food Sci. Technol. Research 2000, 6, 166-170.
(28) Chen, C. C.; Chen, S. D.; Chen, J. J.; Wu, C. M., Effects of pH value on the formation
of volatiles of Shiitake (Lentinus edodes), an edible mushroom. J. Agric. Food Chem. 1984,
32, 999-1001.
(29) Hong, J.; Lee, K.; Kim, Y.; Kim, D.; Kim, M.; Kim, Y.; Yeo, K., Volatile flavor
compounds of Korean shiitake mushroom (Lentinus edodes). Kor. J. Food Sci. Technol.
1988, 20.
(30) Da Costa, N. C.; Eri, S., Identification of volatile compounds in shiitake mushrooms
using modern extraction techniques. In Modern Extraction Techniques, American Chemical
Society: 2006; Vol. 926, 163-175.
(31) Kyoden, Y.; Iwami, K.; Mitsuda, H., Enzyme-catalyzed evolution of lenthionine from
lentinic acid. Agr. Biol. Chem. 1971, 35, 2070-2080.
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660
661
662
663
664
665
666
667
668
669
670
(32) Chen, C. C.; Liu, S. E.; Wu, C. M.; Ho, C. T., Enzymic formation of volatile
compounds in shiitake mushroom (Lentinus edodes Sing.). ACS Symp. Ser. 1986, 317, 176-
183.
(33) Yasumoto, K.; Iwami, K.; Mitsuda, H., Enzymic formation of shiitake aroma from non-
volatile precursors (S) - lenthionine from lentinic acid. Mushroom Sci. 1976, 9, Pt. 1, 371-383.
(34) Schieberle, P.; Hofmann,T. Mapping the Combinatorial Code of Food Flavors by
Means of Molecular Sensory Science Approach. In: Food Flavors: Chemical,. Sensory and
Technological Properties, Jelen, H: (Ed.) 2013 CRC press, Taylor and Francis, 411-436.
(35) Wurzenberger, M.; Grosch,W.The formation of 1-octen-3-ol from the 10-
hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms.
Biochim.Biophys. Acta, 1984, 794,25-30.
671
672
(36) Rapior,S.;Cavalie,S.;Croze,P.;Andary,C.;Pelissier,Y.;Bessiere, J.M. Volatile
components of ten frozen mushrooms (Basidiomycetes) J.Essent. Oil Res. 1996, 8, 63-66.
673
674
675
676
(37) Hofmann, T.; Schieberle, P. Acetylformoin - an Important Progenitor of 4-hydroxy-2,5-
dimethyl-3(2H)-furanone and 2-Acetyltetrahydropyridine during Thermal Food Processing, In:
Proceedings of the 6th Wartburg Aroma Symposium, 2001; Rothe, M., Ed. Eigenverlag:
Bergholz-Rehbrücke: 2001; 311-322.
677
678
679
(38) Hiraide,M.; Kato,A.;Nakashima, T.The smell and odorous components of dried
shiitake mushroom (Lentinula edodesV):Changes in lenthionine and lentinic acid contents
during the drying process. J.Wood Sci.2010,56,477-482.
680
681
682
(39) Mall,V.;Sellami, I.; Schieberle,P. New degradation pathways of the key aroma
compound 1‑penten-3-one during storage of not-from-concentrate orange juice. J. Agric.
Food Chem. 2018, 66, 11083-11091
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Figure captions
Figure 1. Mass spectra of 1,2,4-trithiolane (Figure 1 A) and [2H4]-1,2,4-trithiolane
(Figure 1B
Figure 2 Mass spectra of 1,2,4,6 tetrathiepane (Figure 2 A) and [2H6]- tetrathiepane
(Figure 2B).
Figure 3 Mass spectra of 1,2,4,5-tetrathiane (Figure 3 A) and [2H4]- 1,2,4,5-
tetrathiane (Figure 3B)
Figure 4 Mass spectra of 1,2,3,5,6-pentathiepane (Figure 4 A) and [2H4]- 1,2,3,5,6-
pentathiepane (Figure 4B)
Figure 5. Flavor dilution (FD) chromatogram of the volatile fraction isolated from raw
shiitake mushrooms.
Figure 6. Structures of five key odorants in raw shiitake mushrooms showing the
highest FD factors (numbering refers to Table 2).
Figure 7. Synthetic route used in the preparation of deuterium labeled [2H4]-1,2,4-
trithiolane, [2H6]- tetrathiepane, [2H4]- 1,2,4,5-tetrathiane and [2H4]- 1,2,4,5-tetrathiane
Figure 8. Flavor dilution (FD) chromatogram of the volatile fraction isolated from pan-
fried shiitake mushrooms.
Figure 9. Aroma profile analysis of pan-fried shiitake (grey line) and the aroma
recombinate (black line)
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Table 1. Isotopically Labeled Standards, Selected Ions, and Accuracy of
Calibration Lines Used in the Quantitation of 32 Shiitake Aroma Compounds by
Stable Isotope Dilution Assays.
ion (m/z) a
isotope
label
internal
standard
odorant
analyte
R2 b
System c
acetic acid
2-acetyl-1-pyrroline
2,3-butanedione
butanoic acid
(E,E)-2,4-decadienal
δ-decalactone
2,3-diethyl-5-methylpyrazine
2,6-dimethoxyphenol
3,5-dimethyl-2-ethenylpyrazine
dimethyl trisulfide
2H3
61
112
87
64
114-117d
91
0.9999
1
1
3
2
1
2
2
2
3
3
2
4
3
3
3
2
2
1
2
2
2
2
2
1
2
1
1
3
3
2
1
1
3
2H2-5
13C4
2H2
1
1
89
91
2H3-5
2H2
153
170
151
154
134
127
167
122
128
128
165
87
103
105
124
167
139
127
111
127
103
189
120
136
125
157
171
152
156-158 d
172
0.9985
1
0.9998
0.9999
0.9999
1
0.9998
0.998
0.998
0.9996
0.9997
1
0.998
0.997
1
2H3
154
2H5-8
2H2
159-162 d
136
2H6
133
171
124
130
130
168
89
105
trans-4,5-epoxy-(E)-2-decenal
3-ethylphenol
2H4
2H2
4-hydroxy-2,5-dimethyl-3(2H)-furanone 13C2
3-hydroxy-4,5-dimethyl-2(5H)-furanone 13C2
2-methoxy-4-(prop-1-en-1-yl)phenol
3-methylbutanal
2- and 3-methylbutanoic acid
3-(methylthio)propanal
2-methoxyphenol
2-methoxy-4-propylphenol
(E,E)-2,4-nonadienal
(E)-2-octenal
1-octen-3-ol
1-octen-3-one
pentanoic acid
1,2,3,5,6-pentathiepane
phenylacetaldehyde
phenylacetic acid
2H3
2H2
2H3
2H3
2H3
2H2-4
2H2
2H2
2H2-4
2H2
2H3
2H4
13C2
13C2
2H4
2H4
2H6
2H3
108
127
169-171 d
141
1
0.9991
0.999
0.995
0.996
0.997
0.9999
0.996
0.9993
0.9999
0.9997
1
129
113-116 d
131
106
193
122
138
129
161
176
155
1,2,4-trithiolane
1,2,4,5-tetrathiane
1,2,4,6-tetrathiepane
vanillin
1
a Ions used for quantitation by either MS-CI or MS-EI.
b Coefficient describing the accuracy of the calibration line; number of calibration points: n =
5.
c The different equipments used in the quantitation.
GC-MS (1)
GCxGC-MS; (2)
Comprehensive GCxGC-TOF-MS (3);
(4) GC-MS (MS-CI neg.)
d Internal standard was used as a mixture of isotopologues.
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Table 2. Most Odor-Active Compounds (FD ≥ 8) Identified in Raw Shiitake Mushrooms.
RI on
previously
no.
odorant a
odor quality b
FD c
identified d
FFAP
927
DB-5
640
<600
n.b.
1006
976
926
983
1062
613
982
910
1 3-methylbutanal
2 2,3-butanedione
3 unknown
malty
128
16
16
16
1024
16
-
-
-
buttery
garlic-like
citrus-like
983
1256
1279
1293
1329
1364
1423
1443
1445
1452
1477
1514
1530
1630
1638
4 octanal
-
27-30
5 1-octen-3-one
6 2-acetyl-1-pyrroline
7 (Z)-1,5-octadien-3-one
mushroom-like
popcorn-like, roasty
geranium, metallic
fatty, nutty
vinegar-like
mushroom-like
cooked potato-like
earthy
earthy, green bell pepper
fatty, green
sweaty
flower-like
sweaty
-
-
-
32
10 (E)-2-octenal
11 acetic acid
12 1-octen-3-ol
13 3-(methylthio)propanal
14 2,3-diethyl-5-methylpyrazine
15 2-isobutyl-3-methoxypyrazine
16 (E)-2-nonenal
128
128
512
4096
64
64
32
8
128
64
8
32
64
16
256
128
8
8
32
-
27-30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1154
1182
1164
807
18 butanoic acid
19 phenylacetaldehyde
20 2- and 3-methylbutanoic acid
26 (E,E)-2,4-decadienal
27 unknown
1049
1658 858/846
fatty, fried
sweaty
phenolic
phenolic
1808
1824
1861
1944
2000
2029
2109
2142
2182
2213
1319
n.b.
28 2-methoxyphenole
1086
1191
1380
1049
1366
n.b.
29 2-methoxy-5-methylphenole
30 trans-4,5-epoxy-(E)-2-decenal
32 4-hydroxy-2,5-dimethyl-3(2H)-furanone
34 2-methoxy-4-propylphenol
35 unknown
metallic
caramel-like
caramel-like
seasoning-like
clove-like
39 3-ethylphenol
40 3-hydroxy-4,5-dimethyl-2(5H)-furanone
1165
1105
seasoning-like
128
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Table 2. continued
2
41 1,2,4,5-tetrathiane
47 unknown
51 phenylacetic acid
52 vanillin
sulfury, burnt
burnt
honey-like
vanilla-like
sulfury, burnt
2226
2361
2561
2586
>2600
1484
n.b.
1240
1409
1679
32
64
32
-
-
256
-
1
53 1,2,3,5,6-pentathiepane
64 g
a Identification was performed by comparing retention indices (RI) on capillaries FFAP and DB-5, odor quality, and mass spectra (MS-EI) with data
of reference compounds.
b Odor quality perceived at the sniffing-port.
c Flavor dilution (FD) factor determined by application of the AEDA.
d Reference reporting the respective compound as volatile compound in raw Shiitake mushroom.
e No unequivocal mass spectra could be obtained. Identification is based on the remaining criteria given in footnote a.
f nd, not determined.
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Table 3. Concentrations of Nineteen Key Aroma Compounds in Raw Shiitake.
concn
odorant
(µg/kg fresh
weight)
3380
97
RSD a (%)
1-octen-3-ol
phenylacetaldehyde
phenylacetic acid
1-octen-3-one
3-methylbutanal
3-(methylthio)propanal
2-methoxyphenol
3-ethylphenol
3-methylbutanoic acid
vanillin
trans-4,5-epoxy-(E)-2-decenal
1,2,3,5,6-pentathiepane
1,2,4,5-tetrathiane
2-methylbutanoic acid
4-hydroxy-2,5-dimethyl-3(2H)-furanone
3-hydroxy-4,5-dimethyl-2(5H)-furanone
1,2,4,6-tetrathiepane
2,3-diethyl-5-methylpyrazine
2-isobutyl-3-methoxypyrazine
2.9
5.1
2.1
1.8
1.9
3.5
0.2
0.5
0.5
0.8
10.2
2.0
9.3
7.4
8.1
0.8
9.9
7.4
3.0
51
47
38
34
24
15
15
7.9
4.9
2.8
2.8
0.95
0.87
0.85
0.65
0.1
0.02
a number of replicates: n = 3.
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Table 4. Orthonasal Odor Thresholds and Odor Activity Values (OAVs) of
Nineteen Aroma Compounds in Raw Shiitake.
odor
threshold in
odorant
OAVd
2913
watera
(µg/kg)
0.016a
0.43a
0.5a
1-octen-3-one
3-(methylthio)propanal
3-methylbutanal
80
76
75
23
19
18
17
12
10
10
9
1-octen-3-ol
45a
0.22c
5.2b
0.85a
0.43c
0.23c
0.27c
2.5a
trans-4,5-epoxy-(E)-2-decenal
phenylacetaldehyde
3-ethylphenol
2,3-diethyl-5-methylpyrazine
1,2,4,5-tetrathiane
1,2,3,5,6-pentathiepane
2-methoxyphenol
1,2,4,6-tetrathiepane
2-isobutyl-3-methoxypyrazine
3-hydroxy-4,5-dimethyl-2(5H)-furanone
4-hydroxy-2,5-dimethyl-3(2H)-furanone
vanillin
2-methylbutanoic acid
3-methylbutanoic acid
phenylacetic acid
1.16c
0.0052
0.49a
40a
5
2
<1
<1
<1
<1
<1
53a
2200a
490a
6100a
a Czerny et al. 25, bin-house data base, cnewly determined in this study.
dOdor activity value (ratio of odor concentration to odor threshold).
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