Evaluation of the key aroma compounds in beef and pork vegetable gravies a la chef by stable isotope dilution assays and aroma recombination experiments

Article abstract of DOI:10.1021/jf203340a

Although the aroma compounds of meat processed as such have been studied previously, data on complete homemade dishes containing beef and pork meat were scarcely studied. Recently, 38 odor-active compounds were characterized in beef and pork vegetable gravies using GC-olfactometry. In the present investigation, the most odor-active compounds were quantitated in a freshly prepared stewed beef vegetable gravy (BVG) as well as a stewed pork vegetable gravy (PVG) by means of stable isotope dilution assays. Calculation of odor activity values (OAVs; ratio of concentration to odor threshold) revealed 3-mercapto-2- methylpentan-1-ol, (E,E)-2,4-decadienal, (E,Z)-2,6-nonadienal, (E)-2-decenal, (E)-2-undecanal, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone as the most potent odorants in both gravies. However, significantly different OAVs were found for 12-methyltridecanal, which was much higher in the BVG, whereas (E,Z)-2,4-decadienal showed a clearly higher OAV in the PVG. Aroma recombination experiments performed on the basis of the actual concentrations of the odorants in both gravies revealed a good similarity of the aromas of both model mixtures containing all odorants with OAVs > 1 with those of the original gravies.

Full text of DOI:10.1021/jf203340a

ARTICLE
pubs.acs.org/JAFC
Evaluation of the Key Aroma Compounds in Beef and Pork Vegetable
Gravies a la Chef by Stable Isotope Dilution Assays and Aroma
Recombination Experiments
Monika Christlbauer and Peter Schieberle*
Lehrstuhl fuer Lebensmittelchemie, Technische Universitaet Muenchen, Lise-Meitner-Straße 34, 85354 Freising, Germany
ABSTRACT: Although the aroma compounds of meat processed as such have been studied previously, data on complete
homemade dishes containing beef and pork meat were scarcely studied. Recently, 38 odor-active compounds were characterized in
beef and pork vegetable gravies using GCÀolfactometry. In the present investigation, the most odor-active compounds were
quantitated in a freshly prepared stewed beef vegetable gravy (BVG) as well as a stewed pork vegetable gravy (PVG) by means of
stable isotope dilution assays. Calculation of odor activity values (OAVs; ratio of concentration to odor threshold) revealed
3
-mercapto-2-methylpentan-1-ol, (E,E)-2,4-decadienal, (E,Z)-2,6-nonadienal, (E)-2-decenal, (E)-2-undecanal, and 3-hydroxy-4,5-
dimethyl-2(5H)-furanone as the most potent odorants in both gravies. However, significantly different OAVs were found for 12-
methyltridecanal, which was much higher in the BVG, whereas (E,Z)-2,4-decadienal showed a clearly higher OAV in the PVG.
Aroma recombination experiments performed on the basis of the actual concentrations of the odorants in both gravies revealed a
good similarity of the aromas of both model mixtures containing all odorants with OAVs > 1 with those of the original gravies.
KEYWORDS: 3-mercapto-2-methylpentan-1-ol, 12-methyltridecanal, (E,Z)-2,4-decadienal, meat vegetable gravy, beef, pork,
stable isotope dilution assay
’
INTRODUCTION
’ MATERIALS AND METHODS
By application of aroma extract dilution analyses (AEDA), we
Material. Beef and pork meats (top round) were purchased at a local
butcher’s shop. The vegetables, namely, carrots, leeks, celery roots, and
onions, as well as lard and iodized salt were obtained from a local market.
could recently identify 3-mercapto-2-methylpentan-1-ol, 3-
methylthio)propanal, (E,E)-2,4-decadienal, 3-hydroxy-4,5-di-
methyl-2(5H)-furanone, vanillin, (E,E)-2,4-nonadienal, and (E)-
-undecenal as key aroma compounds of freshly prepared stewed
(
1
The meat vegetable gravies were prepared as described recently.
2
Chemicals. Chemicals. Allyl magnesium bromide, butyl lithium
beef and stewed pork vegetable gravies on the basis of high flavor
2
(
2.5 M in hexane), [ H
3
]-ethyl magnesium iodide, lithium aluminum
1
dilution (FD) factors. Among the 38 aroma compounds identi-
deuteride, 2-(propargyloxy)tetrahydropyrane, and tris(triphenylphosphine)-
rhodium(I) chloride were from Aldrich (Taufkirchen, Germany). 1-Bromo-
octane, 3,4-dihydro-2H-pyrane, 12-hydroxylauric acid, and p-toluene-
sulfonic acid monohydrate were from Fluka (Taufkirchen, Germany).
Ammonium chloride, dimethyl sulfoxide, hydrochloric acid (37%), sodium
carbonate, sodium chloride, sodium hydrogen sulfite, sodium sulfate,
sodium thiosulfate, sulfuric acid, and tert-butyl methyl ether were from
Merck (Darmstadt, Germany). 1,1,1-Triacetoxy-1,1-dihydro-1,2-benzio-
doxol-3(1H)-one was from Lancaster (M €u hlheim/Main, Germany).
Reference Odorants. The reference compounds were purchased from
fied, (E)-2-undecenal was reported for the first time in beef and
pork as well as in the four vegetables (carrot, celery root, leek,
onion) used for its preparation. Furthermore, 12-methyltridecanal
was detected as a potent odorant only in stewed beef vegetable
gravy (BVG), whereas (E,Z)-2,4-decadienal was only found with
1
a high FD factor in the stewed pork vegetable gravy (PVG).
AEDA is a suitable tool to select odor-active compounds from
the bulk of odorless volatiles; however, because for example,
losses of odorants during the isolation procedure are not taken
into account, reliable quantitation experiments should always
follow this approach, if the characterization of key odorants is
intended. Moreover, because the gravy matrix mainly consisted
of water containing 2% fat, the volatility of the odorants may be
influenced by the matrix.
Although the aroma compounds of processed beef and pork
meat have previously been investigated in depth, no information
was available on the aroma compounds of beef or pork gravies
processed in the presence of vegetables as done by chefs when
this study was initiated.
1
commercial sources or were synthesized as recently reported.
Isotopically Labeled Internal Standards. The isotopically labeled
internal standards, labeled with either deuterium or carbon-13, were
2
synthesized as described in the references given: [ H2À5]-2-acetyl-1-
3
2
4 2
2 6
pyrroline, [ H ]-3-mercapto-2-methylpentan-1-ol, [ H ]-bis(2-methyl-
5
2
6
2
2 6
7
3
[
-furyl) disulfide, [ H ]-butanoic acid, [ H ]-(E)-β-damascenone,
2
8
2
9 2
H ]-(E,E)-2,4-decadienal, [ H ]-(E)-2-decenal, [ H ]-dimethyl tri-
4 2 6
1
0
2
6
2
11 2
sulfide, [ H
2
]-(Z)-6-dodeceno-γ-lactone, [ H
2
]-3-ethylphenol, [ H
4
]-
1
2
13
13 13
2 2
hexanal, [ C ]-3-hydroxy-4,5-dimethyl-5(2H)-furanone, [ C ]-4-hydro-
xy-2,5-dimethyl-3(2H)-furanone, [ H ]-2-isopropyl-3-methoxypyrazine,
14
2
15
3
Therefore, the aim was to corroborate the results obtained
1
recently by quantitative studies using stable isotope dilution
Received: August 19, 2011
assays, followed by a calculation of odor activity values (OAVs;
Revised:
November 11, 2011
ratio of concentration to odor threshold) and, finally, aroma
recombination experiments.
Accepted: November 12, 2011
Published: November 12, 2011
2
r 2011 American Chemical Society
13122
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Journal of Agricultural and Food Chemistry
ARTICLE
2
16
2
17
2
[
2 8 3
H ]-3-methylbutanoic acid, [ H ]-3-methylindole, [ H ]-3-
5
2
18 2
(
methylthio)propanal, [ H ]-(E,E)-2,4-nonadienal, [ H ]-(E,Z)-
2
2
8
2
19
2
8 2
2,6-nonadienal, [ H
2
2
]-γ-nonalactone, [ H
2
]-(E)-2-nonenal, [ H
2
]-
]-
2
0
21
2
8
13
4 2
γ-octalactone, [ H ]-octanal, [ H ]-1-octen-3-one, [ C
phenylethanal, [ H ]-2-(sec-butyl)-3-methoxypyrazine, [ H ]-vanillin
and [ H ]-4-vinyl-2-methoxyphenol. [ H ]-Acetic acid, [ H ]-3-
methylphenol, and [ C
2
9
2
22 2
3
3
2
23
2
2
3
3
7
1
3
2
]-phenylacetic acid were purchased from
Aldrich (Taufkirchen, Germany).
2
Syntheses. [ H
2
2 2
]-(E)-2-Undecenal. [ H ]-(E)-2-Undecenal was
synthesized by reduction of 2-undecyn-1-ol with lithium aluminum
2
2
deuteride followed by oxidation of the [ H ]-(E)-undecen-1-ol formed
with DessÀMartin periodinane (1,1,1-triacetoxy-1,1-dihydro-1,2-diben-
ziodoxol-3(1H)-one).
(
a) 2-Undecyn-1-ol. Butyl lithium (2.5 mol/L in hexane, 9 mL;
2.2 mmol) was added to a solution of 2-(propargyloxy)tetrahydro-
pyrane (2.6 g; 18.5 mmol) in dry tetrahydrofuran at 0 °C. After 30 min,
-bromo-octane (3.55 mL; 20.4 mmol) and dimethyl sulfoxide (50 mL)
2
1
were added at 0 °C, and the mixture was stirred at room temperature.
After 24 h, the reaction was terminated by the addition of water (50 mL).
After extraction with tert-butyl methyl ether (2 Â 50 mL), the organic
layer was dried over sodium sulfate, and the solvent was distilled off to
receive crude 1-(2-tetrahydropyranyloxy-2-dodecyne) (3.7 g; 14.6 mmol).
After the addition of methanol (20 mL), p-toluenesulfonic acid mono-
hydrate (1 g; 5.13 mmol) in methanol (80 mL) was added, and the
mixture was stirred at room temperature for 2 h. After the addition of
water (150 mL) and diethyl ether (150 mL), the ethereal layer was
2
Figure 1. Mass spectrum (MS-EI) of [ H ]-12-methyltridecanal.
8
(
a) 12-Hydroxylauric Acid Ethyl Ester. 12-Hydroxylauric acid (4.3 g;
0 mmol), ethanol (60 mL; 1.03 mol), and concentrated sulfuric acid
210 μL; 4 mmol) were refluxed for 5 h. After the mixture had cooled to
room temperature, solid sodium hydrogencarbonate was added, and the
mixture was filtered. The solvent was evaporated, and the residue was
dissolved in diethyl ether (50 mL). The ethereal layer was washed three
times with aqueous sodium hydrogencarbonate solution (0.5 mol/L,
total volume = 150 mL) and then with an aqeuous brine (50 mL), and
2
(
isolated and dried over sodium sulfate, and the solvent was distilled off.
2
(
b) [ H ]-(E)-2-Undecen-1-ol. Lithium aluminum deuteride (1.0 g;
2
2
2
4 mmol) was suspended in anhydrous tetrahydrofuran (30 mL), and the
-undecyn-1-ol (2.0 g; 12.0 mmol) obtained in the previous step, dissolved
finally, the organic layer was dried over sodium sulfate.
]-12-Methyltridecane-1,12-diol. Under an atmosphere of
nitrogen, a solution of 12-hydroxylauric acid ethyl ester (3.05 g; 12.5 mmol)
2
b) [ H
in anhydrous tetrahydrofuran (30 mL), was slowly added to the stirred
solution. The mixture was refluxed for 1 h and stored overnight at
room temperature. After cooling to 0 °C, deuterium oxide (15 mL) was
dropwise added, followed by aqueous sulfuric acid (100 mL; 4 mol/L).
The organic layer was separated, and the aqueous solution was extracted
with diethyl ether (total volume = 150 mL). The combined organic
layers were washed successively with saturated aqueous solutions of
sodium hydrogencarbonate (2 Â 20 mL) and sodium chloride (2 Â
(
6
in dry diethyl ether (50 mL) was added dropwise to an ethereal solution
2
of [ H
3
]-ethyl magnesium iodide (50 mL; 1 mol/L) kept at room
temperature. After the mixture had been refluxed for 2 h and cooled,
hydrochloric acid (2 mol/L) was added until the precipitate was
dissolved. The ethereal layer was separated from the aqueous layer,
which was re-extracted three times with diethyl ether (total volume =
00 mL). The combined organic layers were washed successively with an
aqueous sodium hydrogen sulfite solution (39%, 50 mL), saturated
sodium hydrogencarbonate solution (50 mL), and finally distilled water
2
0 mL). After drying over sodium sulfate, the solvent was evaporated.
2
2
2
(
c) [ H
2 2
]-(E)-2-Undecenal. [ H ]-(E)-2-Undecen-1-ol (1.9 g;
11.2 mmol), dissolved in dichloromethane (50 mL), was added dropwise
to a solution of DessÀMartin periodinane (5.0 g; 11.7 mmol) in dichloro-
methane (60 mL). After overnight stirring at room temperature, the
suspension was diluted with diethyl ether (100 mL). Then, sodium thio-
sulfate (50 mL; 1 mol/L, saturated with sodium hydrogen carbonate)
was added, and the mixture was shaken until the solvent layer was clear.
The aqueous layer was separated, and the organic layer was washed
successively with a solution of sodium thiosulfate (50 mL; 1 mol/L,
saturated with sodium hydrogencarbonate), a saturated solution of sodium
hydrogencarbonate (100 mL), and finally distilled water (100 mL). After
drying over sodium sulfate, the solvent was removed by distillation,
yielding the target compound: MS-EI, m/z (%) 43 (100), 41 (91), 57
(
50 mL). The ethereal layer was dried over sodium sulfate and finally
concentrated to about 50 mL.
2
2
(
c) [ H ]-12-Methyltridec-11-en-1-ol. [ H ]-12-Methyltridecane-
6 6
1
,12-diol (1.05 g; 4.5 mmol) was dissolved in toluene (60 mL), and
phosphoric acid (85%, 60 mL) was added. After 6 h of refluxing, the
aqueous was separated from the organic layer and extracted three times
with saturated sodium hydrogencarbonate (total volume = 210 mL),
then washed with brine, and dried over sodium sulfate. The organic layer
was finally concentrated to 15 mL.
2
(d) [ H ]-12-Methyltridecan-1-ol. To a suspension of Wilkinson’s
8
catalyst (tris(triphenylphosphin)rhodium(I) chloride) (500 mg; 0.54 mmol)
in toluene (15 mL) saturated with deuterium, [ H ]-12-methyltridecane-
2
6
(
(
(
85), 72 (82), 55 (75), 85 (65), 69 (57), 56 (50), 71 (50), 59 (43), 82
41), 83 (40), 70 (40), 42 (36), 86 (30), 68 (27), 99 (26), 100 (26), 123
20), 96 (20), 81 (19), 39 (19), 67 (17), 113 (15), 58 (15), 95 (15); MS-
1,12-diol (700 mg; 3.2 mmol), dissolved in toluene (15 mL), was added.
After 24 h, the product was purified by column chromatography using a
water-cooled column (26 cm  2 cm i.d.) filled with silica gel 60 (25 g)
CI, m/z (%) 171 (100), 172 (12), 153 (6), 170 (5).
2
2
[
H ]-12-Methyltridecanal. [ H ]-12-Methyltridecanal was prepared
in pentane. After the column had been flushed with pentane (200 mL),
8
8
2
in a five-step synthesis starting with 12-hydroxylauric acid, which was
the target compound [ H
pentane/diethyl ether (50:50, v/v, 200 mL). The alcohol was isolated
by SAFE distillation.
8
]-12-methyltridecan-1-ol was eluted with
2
esterified and submitted to a Grignard reaction with [ H
3
]-ethyl
2
24
magnesium iodide to yield [ H ]-12-methyltridecane-1,12-diol. Dehydro-
6
2
2
2
genation by means of phosphoric acid released [ H ]-12-methyltridec-
8 8
(e) [ H ]-12-Methyltridecanal. [ H ]-12-Methyltridecan-1-ol(100mg;
6
1
1-en-1-ol, which was deuterated using Wilkinson’s catalyst (tris(triphenyl-
0.45 mmol) was dissolved in dichloromethane (10 mL) and added
dropwise to a solution of DessÀMartin periodinane (500 mg; 1.12 mmol)
dissolved in dichloromethane (10 mL) under an argon atmosphere.
2
phosphin)rhodium(I) chloride) into [ H
was finally oxidized to obtain [ H ]-12-methyltridecanal.
8
]-12-methyltridecan-1-ol, which
2
8
1
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Journal of Agricultural and Food Chemistry
ARTICLE
2
Figure 2. Mass spectra (MS-CI) of 12-methyltridecanal (A) and [ H ]-12-methyltridecanal (B).
8
The mixture was stirred for 1 h and, after the addition of diethyl ether
(50 mL), treated with a solution of sodium thiosulfate (50 mL; 1 mol/L,
saturated with sodium hydrogencarbonate) for 10 min until the organic
layer was clear. The aqueous layer was separated from the organic layer,
which was washed with a solution of sodium thiosulfate (50 mL; 1 mol/L,
saturated with sodium hydrogencarbonate). After treatment with a
solution of saturated sodium hydrogencarbonate (50 mL) and distilled
water (50 mL), the organic layer was dried over sodium sulfate and
concentrated to about 25 mL.
2
The mass spectrum of [ H ]-12-methyltridecanal (MS-EI) is shown
8
in Figure 1, and the MS-CI is contrasted to that of unlabeled 12-
methyltridecanal in Figure 2.
2
Figure 3. Synthetic route used in the preparation of [ H
octalactone.
2
]-δ-
2
2
[
H
2
]-δ-Octalactone. [ H
step synthesis (Figure 3) starting with the acid-catalyzed hydration of
,4-dihydro-2H-pyran. The tetrahydropyran-2-ol formed was converted
2
]-δ-Octalactone was synthesized in a four-
3
hydrogencarbonate (50 mL), and finally brine (50 mL). The organic
layer was dried over sodium sulfate and concentrated.
into 7-octene-1,5-diol in a Grignard reaction with allyl magnesium
bromide. Then, the diol obtained was oxidized using 1,1,1-triacetoxy-
(
c) 7-Octeno-δ-lactone. DessÀMartin periodinane (4.7 g; 11 mmol)
1,1-dihydro-1,2-dibenziodoxol-3(1H)-one yielding 7-octeno-δ-lactone,
was dissolved in dichloromethane (100 mL), and the solution was added
dropwise to 7-octene-1,5-diol (1.44 g, 10 mmol) dissolved in dichloro-
methane (50 mL). The mixture was stirred for 1 h at room temperature
and then diluted with diethyl ether (100 mL). After the addition of an
aqueous solution of sodium thiosulfate (1 mol/L, saturated with sodium
hydrogencarbonate, 200 mL), the mixture was shaken for ∼10 min until
the solvent layer was clear. The aqueous layer was separated from the
organic layer, and this was washed again with a solution of sodium thio-
sulfate (1 mol/L, saturated with sodium hydrogencarbonate, 100 mL),
then with a saturated solution of sodium hydrogencarbonate (100 mL),
and finally brine (100 mL). The organic layer was dried over sodium
which was finally deuterated in the presence of Wilkinson’s catalyst to
2
yield [ H
a) Tetrahydropyran-2-ol. A solution of 3,4-dihydro-2H-pyrane
23.5 g; 0.25 mol) in hydrochloric acid (100 mL; 0.2 mol/L) was
2
]-δ-octalactone.
(
(
refluxed for 1 h. After cooling to room temperature, the brown solution
was neutralized with sodium hydroxide (1 mol/L). The product was
extracted with diethyl ether (total volume = 250 mL), the extract was
dried over sodium sulfate, and the solvent was removed. An aliquot of
the raw product was purified by column chromatography using a water-
cooled (10À12 °C) column (40 cm  4 cm i.d.) filled with silica gel 60.
Stepwise elution was performed with a pentane/diethyl ether mixture
sulfate and concentrated.
2
(
800 mL; 85:15, v/v) followed by diethyl ether (800 mL). The analyte
(d) [ H ]-δ-Octalactone. Wilkinson’s catalyst (185 mg; 0.2 mmol)
2
24
was isolated by SAFE distillation and the solvent was distilled off
resulting in a colorless viscous liquid.
was suspended in toluene (15 mL), and, after saturation with deuterium,
7-octeno-δ-lactone (0.7 g; 5 mmol), dissolved in toluene (15 mL), was
added. When the hydrogenation was finished (checked by gas chromato-
graphy), the target compound was isolated by column chromatography
(
b) 7-Octene-1,5-diol. To a solution of allyl magnesium bromide
150 mL; 1 mol/L) was added dropwise tetrahydropyran-2-ol (5.11 g;
.05 mol) dissolved in anhydrous diethyl ether (100 mL). The solution
(
0
using a water-cooled (10À12 °C) column (26 cm  2 cm i.d.) filled with
2
was refluxed for 1 h, and, after cooling, a saturated aqueous solution of
ammonium chloride was added until the precipitate was dissolved. After
separation, the aqueous layer was extracted twice with diethyl ether
silica gel 60 (20 g) in pentane. After flushing with pentane, [ H
2
]-δ-
octalactone was eluted with diethyl ether (200 mL). The analyte was
isolated from the orange-brown solution by SAFE distillation, and the
distillate was dried over sodium sulfate: MS-EI, m/z (%) 99 (100), 71
(41), 42 (38), 70 (31), 55 (25), 43 (22), 44 (13), 41 (11), 72 (10), 39
(total volume = 200 mL), and the combined ethereal layers were washed
subsequently with sodium hydrogen sulfite (50 mL), saturated sodium
1
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Journal of Agricultural and Food Chemistry
ARTICLE
Table 1. Selected Ions and Response Factors Used in the Stable Isotope Dilution Assays for the Quantitation of the 35 Aroma
Compounds in the Gravies
a
b
odorant
ion (m/z)
internal standard
ion (m/z)
RF
2
acetic acid
-acetyl-1-pyrroline
61
112
227
89
[ H
3
]-acetic acid
64
0.95
0.98
1.05
0.88
0.94
0.74
0.80
0.95
0.95
0.88
0.93
0.80
0.86
1.00
1.00
0.79
0.92
0.91
0.95
1.07
1.05
0.72
0.99
0.96
0.68
0.91
1.00
0.93
0.65
0.97
1.00
1.00
0.84
1.00
0.99
2
2
[ H2À5]-2-acetyl-1-pyrroline
114À117
233
2
bis(2-methyl-3-furyl) disulfide
[ H
6
2
3
]-bis(2-methyl-3-furyl) disulfide
2
butanoic acid
[ H
]-butanoic acid
91
2
2
4
-(sec-butyl)-3-methoxypyrazine
167
109
191
153
153
155
127
179
123
129
129
193
153
117
103
132
105
213
139
139
157
141
143
143
127
103
121
137
169
153
151
[ H
]-2-(sec-butyl)-3-methoxypyrazine
170
2
-methylphenol
[ H6À7]-4-methylphenol
115À116
197
2
(E)-β-damascenone
(E,E)-2,4-decadienal
(E,Z)-2,4-decadienal
(E)-2-decenal
[ H
6
]-(E)-β-damascenone
2
[ H3À4]-(E,E)-2,4-decadienal
156À157
156À157
157
2
[ H3À4]-(E,E)-2,4-decadienal
2
[ H
2
6
2
2
]-(E)-2-decenal
2
dimethyl trisulfide
Z)-6-dodeceno-γ-lactone
[ H
]-dimethyl trisulfide
]-(Z)-6-dodeceno-γ-lactone
]-3-ethylphenol
133
2
(
[ H
181
2
3
3
4
-ethylphenol
[ H
125
1
3
-hydroxy-4,5-dimethyl-5(2H)-furanone
-hydroxy-2,5-dimethyl-3(2H)-furanone
[ C
2
]-3-hydroxy-4,5-dimethyl-5(2H)-furanone
]-4-hydroxy-2,5-dimethyl-3(2H)-furanone
131
1
3
[ C
2
131
2
β-ionone
[ H
6
3
2
2
]-(E)-β-damascenone
196À197
156
2
2
3
2
3
3
1
-isopropyl-3-methoxypyrazine
-mercapto-2-methylpentan-1-ol
-/3-methylbutanoic acid
-methylindole
[ H
]-2-isopropyl-3-methoxypyrazine
]-3-mercapto-2-methylpentan-1-ol
]-3-methylbutanoic acid
2
[ H
119
2
[ H
105
2
[ H7À8]-3-methylindole
139À140
108
2
-(methylthio)propanal
2-methyltridecanal
3-([ H
3
]-methylthio)propanal
2
[ H7À8]-12-methyltridecanal
220À221
141
2
(
E,E)-2,4-nonadienal
E,Z)-2,6-nonadienal
[ H
2
2
2
2
2
2
2
2
]-(E,E)-2,4-nonadienal
]-(E,Z)-2,6-nonadienal
]-γ-nonalactone
2
(
[ H
141
2
γ-nonalactone
E)-2-nonenal
[ H
159
2
(
[ H
]-(E)-2-nonenal
143
2
δ-octalactone
γ-octalactone
[ H
]-δ-octalactone
145
2
[ H
]-γ-octalactone
145
2
1-octen-3-one
[ H
]-1-octen-3-one
129
2
pentanoic acid
phenylethanal
phenylacetic acid
[ H
]-3-methylbutanoic acid
105
1
3
[ C
2
]-phenylethanal
123
1
3
[ C
2
]-phenylacetic acid
139
2
(
E)-2-undecenal
vanillin
-vinyl-2-methoxyphenol
[ H
2
3
3
]-(E)-2-undecenal
171
2
[ H
]-vanillin
156
2
4
[ H
]-4-vinyl-2-methoxyphenol
154
a
b
7
Compounds were quantified by mass spectrometry in the CI mode. The response factor (RF) was determined as reported previously.
(
1
9), 56 (9), 57 (9), 116 (8), 73 (7), 100 (7); MS-CI, m/z (%) 145 (100),
27 (28), 146 (8).
in mixtures of known amounts of unlabeled odorants and the corre-
sponding labeled standards in ratios of 3:1 to 1:3.
2
5
Quantitation of Odorants. Depending on the amount of the
High-Resolution Gas ChromatographyÀMass Spectrome-
try (HRGC-MS). Quantitation of acetic acid, butanoic acid, 2- and
3-methylbutanoic acid, and pentanoic acid was carried out by means of a
Varian 3800 gas chromatograph coupled to a Varian ion trap mass
spectrometer Saturn 2000 (Darmstadt, Germany) using a DB-FFAP
(30 m  0.32 mm i.d.; 0.25 μm film thickness) (J&W Scientific, Folsom,
CA). Monitoring of the selected ions was carried out in the MS-CI mode
using methanol as reactant gas.
Two-Dimensional High-Resolution Gas ChromatographyÀ
Mass Spectrometry (TD-HRGC-MS). Quantitation of all other
odorants was performed by TD-HRGC using a Trace 2000 series gas chro-
matograph (Thermo Quest, Egelsbach, Germany) equipped with a
Moving Column Stream Switching System (MCSS) (Fisons Instruments,
Mainz-Kastel, Germany) in tandem with a Varian GC CP 3800 gas
chromatograph. The analyte and its isotopologue were cut out from the
effluent using the MCSS system and then transferred via a cold trap onto
respective analyte present in the gravy, which was estimated in a pre-
liminary experiment, aliquots of the material (5À350 g, respectively)
were spiked with known amounts of the labeled internal standards
(
Table 1), dissolved in diethyl ether. The gravy was stirred overnight in
diethyl ether (20À500 mL depending on the amount of sample used)
and filtered over a paper filter, and the volatile material was isolated from
24
the suspension by SAFE distillation. Separation into acidic and
neutral/basic compounds and concentration to about 250 μL at 40 °C
using a Vigreux column followed by a microdistillation apparatus were
1
performed as described previously.
The resulting extracts were analyzed by either HRGC-MS or two-
dimensional HRGC-MS monitoring the intensities of the respective ions
given in Table 1. The concentrations were calculated from the relative
abundances of ions selected for the analyte and the internal standards,
and the data obtained were corrected by calibration factors, determined
1
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Journal of Agricultural and Food Chemistry
ARTICLE
the second column. Analyses were performed with a Saturn 2000 ion
trap mass detector (Varian, Walnut Creek, CA) running in the CI mode
with methanol as the reagent gas (chemical ionization energy = 70 eV).
The following fused silica capillaries were used: DB-FFAP (30 m Â
Table 2. Concentrations of 38 Odorants in Stewed Beef
Vegetable Gravy (BVG) and Stewed Pork Vegetable Gravy
(PVG)
a
0
.32 mm i.d.; 0.25 μm film thickness) (J&W Scientific) in combination
concn (μg/kg) in
with a DB-1701 (30 m  0.32 mm i.d.; 0.25 μm film thickness, J&W
Scientific). The samples were applied by the cold on-column injection
technique at 40 °C. After 2 min, the oven temperature was raised at
odorant
BVG
PVG
acetic acid
-hydroxy-2,5-dimethyl-3(2H)-furanone
234600
4990
3780
516
360
323
292
112
100
34
224700
3680
3950
275
10 °C/min to 50 °C, held for 2 min isothermally, then raised at 6 °C/min
4
to 200 °C, and finally raised at 10 °C/min to 230 °C and then held
butanoic acid
isothermally for 10 min. The cut time intervals on the main column were
2
5
(E,E)-2,4-decadienal
determined by injection of the respective reference compounds.
b
1
2-methyltridecanal
<0.09
Differentiation between 2- and 3-Methylbutanoic Acid.
Differentiation was carried out in the acidic fractions by means of
HRGC-MS running in the EI mode. In reference solutions containing
known amounts of 2- and 3-methylbutanoic acid, the characteristic mass
traces of 2-methylbutanoic acid (m/z 74) and 3-methylbutanoic acid
(
E)-2-undecenal
101
124
36
45
21
47
47
64
25
14
53
27
15
13
pentanoic acid
hexanal
(E)-2-decenal
(
m/z 60) were monitored. After calculation of the intensities of both
2
3
3
4
-methylbutanoic acid
-methylbutanoic acid
-(methylthio)propanal
-vinyl-2-methoxyphenol
mass traces, the share of 3-methylbutanoic acid (in the total amount
determined by the isotope dilution assay) was calculated.
61
60
Sensory Evaluation. Aroma Profile Analyses. Sensory analyses
were performed in a sensory panel room at 21 ( 1 °C equipped with
single booths. The sensory panel consisted of 16 experienced assessors,
48
vanillin
39
26
who were trained regularly as previously described. Eight aroma
descriptors were selected for the evaluation of the aroma profiles of
stewed beef gravy and both gravies as well as of the corresponding model
mixtures in preliminary experiments. Then, panelists were trained to
recognize the chosen aroma descriptors using aqueous solutions of the
following reference compounds (50-fold above the odor threshold):
seasoning-like (3-hydroxy-4,5-dimethyl-2(5H)-furanone), fatty ((E,E)-2,4-
decadienal), roasty (2-acetyl-1-pyrroline), tallowy (12-methyltridecanal),
cooked vegetable-like (3-(methylthio)propanal), meaty (bis(2-methyl-
octanal
36
phenylacetic acid
phenylethanal
33
27
(E)-2-nonenal
27
(
Z)-6-dodeceno-γ-lactone
27
3
-mercapto-2-methylpentan-1-ol
7.7
7.5
(
E,Z)-2,6-nonadienal
5.7
5.5
2.8
3-hydroxy-4,5-dimethyl-2(5H)-furanone
γ-nonalactone
3.4
3-furyl) disulfide), caramel-like (4-hydroxy-2,5-dimethyl-3(2H)-furanone),
5.2
2.7
and sweaty (butanoic acid). The freshly prepared gravies (20 g) were
presented to the panelists in covered glass vessels (i.d. = 40 mm, total
volume = 45 mL) equilibrated at 60 °C for 30 min. Panelists were asked
to rate each odor quality using a seven-point linear scale from 0.0 to 3.0.
The results of the aroma profile analyses obtained at three different
sessions were averaged for each odor note and plotted in a spider web
diagram. The values judged by the single assessors differed by not more
than one scale point of 0.5.
Aroma Recombination Experiments. A stock solution of all
odorants occurring in minor amounts was prepared in ethanol for the
aroma models. One milliliter of this solution was dissolved in approxi-
mately 800 mL of tap water, and then aroma compounds occurring in
high amounts, such as acetic acid, butanoic acid, and 4-hydroxy-2,5-
dimethyl-3(2H)-furanone, were added in ethanol solution; after the
addition of deodorized sunflower oil (20 g), the mixture was filled to
(
E,E)-2,4-nonadienal
γ-octalactone
-octen-3-one
5.1
3.6
2.3
1.1
1
2.0
1.4
β-ionone
1.9
1.1
δ-octalactone
1.1
0.90
0.58
0.25
0.81
0.31
2
-acetyl-1-pyrroline
0.67
0.67
0.39
0.28
0.18
0.16
0.09
0.06
0.06
<0.02
dimethyl trisulfide
4-methylphenol
3-methylindole
(
E,Z)-2,4-decadienal
E)-β-damascenone
39
(
0.16
0.16
2
3
2
-(sec-butyl)-3-methoxypyrazine
-ethylphenol
0.07
-isopropyl-3-methoxypyrazine
0.07
1000 mL with tap water. The fat content was adjusted to that present in
b
b
the gravies (2% by weight).
bis(2-methyl-3-furyl) disulfide
<0.02
a
b
The model solutions (recombinate of either BVG or PVG) were
equilibrated at 60 °C for 30 min, and the samples were presented to the
sensory panel. The overall aroma profile of each model mixture was
determined in the same way as described above for the gravies.
In separate sessions, the overall similarity of the aroma of each freshly
prepared gravy and the respective model mixture, both equilibrated at
Mean values of triplicates, differing not more than (10%. Limit of
detection.
methyltridecanal). The two models were presented to the panelists in
triangle tests for sensory evaluation in comparison to the respective
entire aroma model. Panelists were asked whether the model solutions
were different or not. The significance α of the differences was calculated
as described in ref 27.
2
6
6
0 °C for 30 min, was compared. The similarity was estimated using a
26
seven-point linear scale from 0 to 3.
Omission Experiment. Model solutions were prepared by omit-
ting all aroma compounds from the entire aroma models of BVG and
PVG, respectively, that showed OAVs <1 (butanoic acid, pentanoic acid,
’
RESULTS AND DISCUSSION
2- and 3-methylbutanoic acid, (E)-β-damascenone, vanillin, phenylace-
tic acid, 4-methylphenol, 3-methylindole, δ-octalactone, γ-octalactone,
and γ-nonalactone; in the recombinate of PVG, additionally 12-
Quantitative Analysis. In our previous study, totals of 52 or
53 odor-active compounds, respectively, were characterized in a
1
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Journal of Agricultural and Food Chemistry
ARTICLE
a
Table 3. Odor Activity Values (OAVs) and Orthonasal Odor
factors, were selected for quantitative measurements using stable
isotope dilution assays. The analytical data used are summarized
in Table 1. Although most of the labeled compounds were
Thresholds of 38 Odorants in Stewed Beef Vegetable Gravy
BVG) and Stewed Pork Vegetable Gravy (PVG)
(
available from previous studies done in our group, three labeled
2
OAV in
standards were newly synthesized, namely, [ H ]-(E)-undecenal,
2
2
2
8 2
b
[ H ]-12-methyltridecenal, and [ H ]-δ-octalactone. The re-
odor threshold
sponse factors determined from a calibration curve monitored
by mass spectrometry were 0.88 for (E)-2-undecenal, 0.72 for 12-
methyltridecanal, and 1.0 for δ-octalactone (Table 1).
Application of the assays to the freshly prepared gravies
revealed acetic acid as by far the most abundant odorant in both
samples, with BVG containing 235 mg/kg and PVG, 225 mg/kg
(Table 2). Other compounds also occurring in the mg/kg range
in both gravies were the caramel-like smelling 4-hydroxy-2,5-
dimethyl-3(2H)-furanone (5 mg/kg in BVG and 3.7 mg/kg in
PVG) and the rancid butanoic acid (3.8 mg/kg in BVG and
odorant
(μg/L in water) BVG PVG
3
1
-mercapto-2-methylpentan-1-ol
2-methyltridecanal
0.0016
4800 4700
3600 <1
2600 1400
c
0.1
c
(
(
(
(
E,E)-2,4-decadienal
E,Z)-2,6-nonadienal
E)-2-decenal
0.2
c
0.02
285
250
230
69
56
43
42
39
31
30
27
11
10
7
140
105
72
43
39
34
16
22
23
53
19
4
c
0.4
d
E)-2-undecenal
1.4
c
3
1
3
-hydroxy-4,5-dimethyl-2(5H)-furanone
0.08
d
-octen-3-one
0.036
3
.9 mg/kg in PVG).
On the other hand, some components were present in only
d
-(methylthio)propanal
1.4
d
dimethyl trisulfide
E)-2-nonenal
0.016
trace amounts (<1 μg/kg) in both gravies, such as 2-acetyl-1-
pyrroline, dimethyl trisulfide, 4-methylphenol, 3-methylindole,
(E)-β-damascenone, 2-(sec-butyl)-3-methoxypyrazine, 3-ethyl-
phenol, and 2-isopropyl-3-methoxypyrazine. The concentration
of the meaty-smelling bis(2-methyl-3-furyl) disulfide was below
its detection limit in both gravies.
Clear differences in the concentrations were only determined
for the tallowy-smelling 12-methyltridecanal, which showed a
concentration of 360 μg/kg in the BVG but could not be
detected in the PVG (<0.09 μg/kg), and (E,Z)-2,4-decadienal
d
(
0.69
d
4
2
-hydroxy-2,5-dimethyl-3(2H)-furanone
-(sec-butyl)-3-methoxypyrazine
160
c
0.003
d
(
E,E)-2,4-nonadienal
0.19
d
hexanal
10
d
β-ionone
0.20
6
d
2
-acetyl-1-pyrroline
phenylethanal
E,Z)-2,4-decadienal
octanal
0.1
6
c
4.0
7
7
e
(
0.03
6
1300
2
(
deep-fried, fatty), which, compared to BVG, occurred in a 200-
d
6.9
5
fold higher concentration in PVG. Less pronounced differences
were found for the metallic-smelling (E)-2-undecenal, which was
found in a higher amount in BVG (323 μg/kg) than in PVG
(101 μg/kg), and for the fatty, green-smelling (E)-2-decenal,
which was also higher in BVG (100 μg/kg) compared to PVG
(45 μg/kg).
Calculation of OAVs. To objectify the sensory contribution of
each of the 38 odorants to the overall aroma of both meat
vegetable gravies, their OAVs were calculated on the basis of odor
thresholds in water, the main ingredient of both gravies (Table 3).
By far the highest OAVs of all compounds under investigation
were calculated for the typical onion, gravy-like smelling 3-mer-
capto-2-methylpentan-1-ol (4800 in BVG and 4700 in PVG),
followed by the fatty, deep-fried-smelling (E,E)-2,4-decadienal
(2600 in BVG and 1400 in PVG) (Figure 4). In addition,
relatively high OAVs above 100 were also calculated for (E,Z)-
d
2
4
-isopropyl-3-methoxypyrazine
-vinyl-2-methoxyphenol
0.013
5
5
d
19
3
3
(
Z)-6-dodeceno-γ-lactone
acetic acid
-ethylphenol
13
2
1
d
180000
1
1
f
3
0.05
1
1
d
phenylacetic acid
vanillin
12000
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<39
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<26
d
d
d
d
d
c
210
5800
1200
7700
17000
2
3
-methylbutanoic acid
-methylbutanoic acid
butanoic acid
pentanoic acid
δ-octalactone
γ-nonalactone
γ-octalactone
400
d
27
24
d
d
d
2
,6-nonadienal, (E)-2-decenal, and (E)-2-undecenal, exhibiting
(
E)-β-damascenone
0.43
d
cucumber-like, fatty-green, and soapy-metallic aroma notes
(Figure 4). 3-Hydroxy-4,5-dimethyl-2(5H)-furanone, 1-octen-3-one,
3-(methylthio)propanal, dimethyl trisulfide, (E)-2-nonenal,
4
3
-methylphenol
-methylindole
1.0
0.41
d
bis(2-methyl-3-furyl) disulfide
0.0008
4
-hydroxy-2,5-dimethyl-3(2H)-furanone, 2-(sec-butyl)-3-methoxy-
a
OAVs calculated by dividing the concentrations of the odorants by
their thresholds in water. Odor thresholds in water newly determined
in this study if not stated otherwise. Orthonasal odor threshold in
b
pyrazine, and (E,E)-2,4-nonadienal showed somewhat lower
c
OAVs between 10 and 100 (Table 3).
2
8 d
By contrast, significant differences in the OAVs were obtained
for two compounds: the tallowy-smelling 12-methyltridecanal,
reaching an OAV of 3600 in BVG compared to an OAV of <1 in
PVG, and the fatty-smelling odorant (E,Z)-2,4-decadienal, show-
ing an OAV of 1300 in PVG compared to an OAV of 6 in BVG
(Figure 3). These results indicate a strong influence of 12-
methyltridecanal with its tallowy odor note on the overall aroma
of the gravy made with beef meat. On the other hand, (E,Z)-2,4-
decadienal with its fatty aroma note should contribute more to
the gravy cooked with pork meat.
water as reported previously. Orthonasal odor threshold in water as
2
6 e
reported previously.
Orthonasal odor threshold in water as re-
2
9 f
ported previously. Orthonasal odor threshold in water as reported
12
previously.
BVG and a PVG by application of AEDA and subsequent
identification of the odor-active compounds. To gain a more
detailed insight into the compounds contributing to the aroma of
both gravies and to establish the observed differences in FD
factors, 38 odorants, which had been detected with high FD
1
1
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Journal of Agricultural and Food Chemistry
ARTICLE
Figure 4. Structures of aroma compounds showing the highest odor activity values in beef (B) or pork (P) vegetable gravies.
Figure 5. Comparative aroma profiles of stewed beef vegetable gravy (A) and the respective aroma model mixture (B).
Figure 6. Comparative aroma profiles of stewed pork vegetable gravy (A) and the respective aroma model mixture (B).
Altogether 26 or 25 compounds (taking bis(2-methyl-3-furyl)-
disulfide tentatively into account), respectively, were present in
concentrations above their odor thresholds in BVG as well as in
PVG and are, therefore, likely to contribute to the overall aroma
of the two meat vegetable gravies.
By contrast, in both gravies, the concentrations of 12 com-
pounds, namely, phenylacetic acid, vanillin, 2- and 3-methylbu-
tanoic acid, butanoic acid, pentanoic acid, δ- and γ-octalactone,
γ-nonalactone, (E)-β-damascenone, 4-methylphenol, and
In two separate sessions, a trained sensory panel evaluated
either the similarity of the overall aroma of each aroma model
solution in comparison to the freshly prepared meat vegetable
gravy or the intensity of eight aroma qualities in the respective
gravy and the recombinate.
The aroma of the model containing all odorants quantified in
the beef vegetable gravy revealed a very good similarity to the
aroma of the BVG itself. Furthermore, the aroma profile analysis
showed that both the gravy and the recombinate elicited the same
intensities for the odor qualities fatty and caramel-like and nearly
identical intensities for the odor qualities spicy, roasty, and
tallowy (Figure 5). Only the odor descriptor “meaty” was rated
slightly higher in the gravy compared to the model solution,
whereas the opposite was true for the vegetable-like odor note.
The results of the sensory evaluation of PVG compared to its
aroma model solution are displayed in Figure 6. The odor
3
-methylindole, did not reach their odor thresholds in water and
should, thus, have no influence on the aroma.
Aroma Recombination Experiments. To confirm the role of
the key odorants in the overall aroma, sensory evaluations of gravy
aroma recombinates were performed. Two model solutions were
prepared in an aqueous matrix (containing 2% sunflower oil) and
containing all 38 odorants in their natural amounts.
1
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Journal of Agricultural and Food Chemistry
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qualities meaty, spicy, and roasty were rated somewhat higher in
the gravy than in the model solution, whereas the opposite was
found for the odor qualities fatty and vegetable-like. The overall
aroma of the model solution, however, was judged to be in good
similarity to that of PVG.
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Lebensm. Wiss. Technol. 1990, 23, 513–522.
To evaluate the role of odorants appearing with OAVs <1, the
following sensory experiments were performed: All odorants
showing OAVs <1 in the respective BVG were omitted from the
recombinates. Each of the reduced recombinates was presented
to the sensory panel in triangle tests in comparison with the
respective entire model. The differences in the aroma profiles
were statistically insignificant (α > 5%), suggesting that all aroma
compounds with an OAV <1 were of no importance for the
overall aroma of both BVG and PVG (data not shown).
The results clearly indicate that, in particular, 3-mercapto-2-
methylpentan-1-ol, a compound generated from precursors in
(
9) Pfn €u r, P. Investigations on the aroma of chocolate. Ph.D. Thesis,
Technical University Munich, Germany, 1998 (in German).
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(
and cod as affected by the storage of the raw material. J. Agric. Food Chem.
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(
1
(
4
onions and leek, has the most important influence on the overall
odor of meat/vegetable gravies. If beef meat is used in the
preparation of the gravy, the tallowy aroma note of 12-methyl-
tridecanal will certainly contribute to the overall sensory differ-
ences in the gravy aroma compared to pork gravies. The
branched aldehyde was previously shown to be generated from
plasmalogens (ether phospholipids) present in beef, but not in
1
(
tion of 2,5-dimethyl-4-hydroxy-3(2H)-furanone and its methyl ether
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24, 364–369.
(
15) Semmelroch, P.; Grosch, W. Studies on character impact
odorants of coffee brews. J. Agric. Food Chem. 1996, 44, 537–543.
16) Guth, H.; Grosch, W. Identification of the character impact
3
0
pork, fat. Because aroma compounds known to be derived
(
from Maillard-type reactions, such as 2-acetyl-1-pyrroline, 3-
odorants of stewed beef juice by instrumental analyses and sensory
studies. J. Agric. Food Chem. 1994, 42, 2862–2866.
(methylthio)propanal, or 4-hydroxy-2,5-dimethyl-3(2H)-fura-
none, showed quite low OAVs and no alkylpyrazine was detected
among the key odorants, obviously the “low-cooking” applied in
the preparation of both gravies leads to lipid oxidation processes
and, thus, a more dominant influence of fatty-smelling aroma
compounds such as (E,E)- and (E,Z)-2,4-decadienal, (E,Z)-2,4-
nonadienal, (E)-2-decenal, or (E)-2-undecenal. The results are
the basis for the development of signature convenience foods,
such as soups or sauces, but may also help cooks interested in
molecular gastronomy.
(
17) Steinhaus, M.; Schieberle, P. Role of the fermentation process
in off-odorant formation in white pepper: on-site trial in Thailand.
J. Agric. Food Chem. 2005, 53, 6056–6060.
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pounds of Criollo cocoa beans during roasting. J. Agric. Food Chem.
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(
19) Poisson, L.; Schieberle, P. Characterization of the key aroma
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(
’
AUTHOR INFORMATION
(
carbonyl compounds leading to a facile synthesis of γ-lactone. J. Chem.
Soc., Perkin Trans. 1 1988, 1669–1675.
Corresponding Author
*
Phone: +49 8161 71 2932. Fax: +49 8161 71 2970. E-mail:
(
21) Blekas, G.; Guth, H. Evaluation and quantitation of potent
odorants of Greek virgin olive oils. Dev. Food Sci. 1995, 37, 419–427.
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odorants of coffee brews. J. Agric. Food Chem. 1996, 44, 537–543.
23) Semmelroch, P.; Laskawy, G.; Blank, I.; Grosch, W. Determina-
Peter.Schieberle@Lrz.tum.de.
(
’
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