Studies on the key aroma compounds in raw (unheated) and heated japanese soy sauce

Article abstract of DOI:10.1021/jf400353h

An investigation using the aroma extract dilution analysis (AEDA) technique of the aroma concentrate from a raw Japanese soy sauce and the heated soy sauce revealed 40 key aroma compounds including 7 newly identified compounds. Among them, 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone and 3-hydroxy-4,5-dimethyl-2(5H)-furanone exhibited the highest flavor dilution (FD) factor of 2048, followed by 3-(methylthio)propanal, 4-ethyl-2-methoxyphenol, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone having FD factors from 128 to 512 in the raw soy sauce. Furthermore, comparative AEDAs, a quantitative analysis, and a sensory analysis demonstrated that whereas most of the key aroma compounds in the raw soy sauce were common in the heated soy sauce, some of the Strecker aldehydes and 4-vinylphenols contributed less to the raw soy sauce aroma. The model decarboxylation reactions of the phenolic acids during heating of the raw soy sauce revealed that although all reactions resulted in low yields, the hydroxycinnamic acid derivatives were much more reactive than the hydroxybenzoic acid derivatives due to the stable reaction intermediates. Besides the quantitative analyses of the soy sauces, the estimation of the reaction yields of the phenolic compounds in the heated soy sauce revealed that although only the 4-vinylphenols increased during heating of the raw soy sauce, they might not mainly be formed as decarboxylation products from the corresponding hydroxycinnamic acids but from the other proposed precursors, such as lignin, shakuchirin, and esters with arabinoxylan.

Full text of DOI:10.1021/jf400353h

Article
pubs.acs.org/JAFC
Studies on the Key Aroma Compounds in Raw (Unheated) and
Heated Japanese Soy Sauce
Shu Kaneko,* Kenji Kumazawa, and Osamu Nishimura
Ogawa & Company Ltd., Chidori 15-7, 279-0032 Urayasu, Chiba, Japan
ABSTRACT: An investigation using the aroma extract dilution analysis (AEDA) technique of the aroma concentrate from a raw
Japanese soy sauce and the heated soy sauce revealed 40 key aroma compounds including 7 newly identified compounds. Among
them, 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone and 3-hydroxy-4,5-dimethyl-2(5H)-furanone exhibited the
highest flavor dilution (FD) factor of 2048, followed by 3-(methylthio)propanal, 4-ethyl-2-methoxyphenol, and 4-hydroxy-2,5-
dimethyl-3(2H)-furanone having FD factors from 128 to 512 in the raw soy sauce. Furthermore, comparative AEDAs, a
quantitative analysis, and a sensory analysis demonstrated that whereas most of the key aroma compounds in the raw soy sauce
were common in the heated soy sauce, some of the Strecker aldehydes and 4-vinylphenols contributed less to the raw soy sauce
aroma. The model decarboxylation reactions of the phenolic acids during heating of the raw soy sauce revealed that although all
reactions resulted in low yields, the hydroxycinnamic acid derivatives were much more reactive than the hydroxybenzoic acid
derivatives due to the stable reaction intermediates. Besides the quantitative analyses of the soy sauces, the estimation of the
reaction yields of the phenolic compounds in the heated soy sauce revealed that although only the 4-vinylphenols increased
during heating of the raw soy sauce, they might not mainly be formed as decarboxylation products from the corresponding
hydroxycinnamic acids but from the other proposed precursors, such as lignin, shakuchirin, and esters with arabinoxylan.
KEYWORDS: raw soy sauce, aroma extract dilution analysis, standard addition method, heating process, phenolic compounds
of the key aroma compounds exhibiting high flavor dilution
(FD) factors by a stable isotope dilution assay, and the sensory
evaluation of their reconstituted solution.2 This study suggested
that 3-methylbutanal, 3-hydroxy-4,5-dimethyl-2(5H)-furanone,
5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone, 2-
methylbutanal, and 3-(methylthio)propanal were the most
important aroma components in the soy sauce. More recently,
the key aroma compounds in five different types of Japanese
soy sauces categorized according to the Japan Agricultural
Standard were investigated by AEDA.3 This study suggested
that the contributions of 5(or 2)-ethyl-4-hydroxy-2(or 5)-
methyl-3(2H)-furanone and 4-hydroxy-2,5-dimethyl-3(2H)-
furanone to the respective soy sauce aromas were widely
different among the varieties despite the significant contribu-
tions of sotolon and methional in all of the varieties. On the
contrary, the key aroma compounds in the raw soy sauce are
thought to be slightly different from those in the common soy
sauce because of the lack of the final heating process, which is
generally conducted at 80−85 °C for 30 min or at 110−130 °C
for several minutes and at 60−65 °C for several days.4,5
Although previous studies demonstrated that furanones, such as
4-hydroxy-2,5-dimethyl-3(2H)-furanone and 4-hydroxy-5-
methyl-3(2H)-furanone, Strecker aldehydes, such as 3-methyl-
butanal and phenylacetaldehyde, α-dicarbonyl compounds,
such as 2,3-butanedione, pyrazines, and sulfur compounds,
such as dimethyl sulfide and dimethyl disulfide, increased
during this process,4,6−9 the key aroma compounds contribu-
INTRODUCTION
■
Traditionally brewed soy sauce has been one of the most
popular seasonings in Japan for hundreds of years. Especially
the Koikuchi-type soy sauce that accounts for about 85% of
total soy sauce consumption in Japan is widely consumed all
over the world.1 Soy sauce is used not only as a sauce for food
but as a seasoning in cooking. Nearly an equal amount of
soybeans boiled to denature the protein and wheat
comminuted after roasting are used as starting materials for
brewing the soy sauce, followed by the addition of a small
amount of seed mold of Aspergillus oryzae or Aspergillus sojae for
cultivation over several days under high-humidity conditions.1
This cultured mold is then mixed in an approximate 20% saline
solution, and the obtained mash is traditionally fermented for
6−8 months. At first, a salt-tolerant lactic acid bacterium
(Tetragenococcus halophius) and salt-tolerant yeasts (Candida
versatilis and Candida etchellsii) grow in the mash, followed by
growth of the main fermentation yeast (Zygosaccharomyces
rouxii). After maturation, the brew is compressed to separate
the filtrate, called the raw soy sauce, and the residue. Soy sauce
is generally produced as a dark-red liquid by heating the raw
soy sauce to deactivate the enzymes and to bring about a
complex flavor. On the contrary, the raw soy sauce has been
widely marketed in recent years as a new type of soy sauce
having fresh aroma characteristics with a short expiration date.
Many investigations concerning the many volatile com-
pounds in soy sauce have been reported, and most of them
were generated during the manufacturing processes as
described above.1 In recent years, the key aroma compounds
of the Koiokuchi-type soy sauce were reported by combining
the screening of the soy sauce aroma concentrate with the
aroma extract dilution analysis (AEDA), a quantitative analysis
Received: January 24, 2013
Revised: March 18, 2013
Accepted: March 23, 2013
Published: March 23, 2013
© 2013 American Chemical Society
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Journal of Agricultural and Food Chemistry
Article
°C, <5.0 × 10−3 Pa).14 The soy sauce aroma concentrate was obtained
by concentrating the dichloromethane layer by rotary evaporation,
followed by nitrogen steam evaporation to about 100 μL. The
respective aroma concentrates were used in gas chromatography−
olfactometry (GC-O) and AEDA.
Identification of the Key Aroma Compounds in the Aroma
Concentrates of the Raw Soy Sauce and the Heated Soy
Sauce. Each key aroma compound in the respective aroma
concentrates was identified by comparing its Kovats GC retention
index (RI) and mass spectrum with the authentic compound by gas
chromatography−mass spectrometry (GC-MS), in addition to
comparison of its RI and odor quality with the authentic compound
by GC-O.
ting to the raw soy sauce aroma had not yet been fully
identified.
The objectives of the present investigation were to clarify the
key aroma compounds in the raw Japanese soy sauce by AEDA;
to clarify the differences in the aroma characteristics of the raw
soy sauce and the heated soy sauce by the combination of
AEDA, quantitative analysis, and sensory analysis; and to
propose formation mechanisms of the phenolic compounds by
the model reactions of hydroxybenzoic acid derivatives and
hydroxycinnamic acid derivatives under the same heating
conditions of the raw soy sauce.
Quantitative Analysis of the Key Aroma Compounds in the
Raw and the Heated Soy Sauces by Standard Addition
Method. An aliquot (5 mL) of the soy sauce with added 2-octanol
(4150 μg/L) and the standard solution (0, 100, or 200 μL) containing
the chemicals listed in Table 1 was concentrated to about 100 μL using
MATERIALS AND METHODS
■
Materials. Soy Sauce Sample. The Koikuchi-type raw soy sauce
was purchased from Kikkoman Co., Ltd. (Noda, Japan). The heated
soy sauce was prepared by heating the vacuum-packed raw soy sauce in
a water bath at 80 °C for 30 min.
Table 1. Selected Ions and the Concentration of the
Standard Solution of the Key Aroma Compounds for
Quantitative Analysis by the Standard Addition Method
Chemicals. Ethyl acetate, 2-methylbutanal, 3-methylbutanal, ethyl
2-methylpropanoate, 2,3-butanedione, ethyl 2-methylbutanoate, ethyl
3-methylbutanoate, dimethyl disulfide, 3-hydroxy-2-butanone, 2-
methyl-3-furanthiol, 2-ethyl-3,5-dimethylpyrazine, dimethyl trisulfide,
2-isopropyl-3-methoxypyrazine, 3-(methylthio)propanal, 2-isobutyl-3-
methoxypyrazine, phenylacetaldehyde, 3-methylbutanoic acid, methyl
2-methyl-3-furyl disulfide, 3-(methylthio)propanol, 3-methyl-2(5H)-
furanone, 3-methyl-1,2-cyclopentanedione, 2-methoxyphenol, 3-ethyl-
1,2-cyclopentanedione, 3-hydroxy-2-methyl-4-pyrone, 4-ethyl-2-me-
thoxyphenol, 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone,
4-hydroxy-5-methyl-3(2H)-furanone, 4-decanolide, 5-decanolide, 2′-
aminoacetophenone, 2,6-dimethoxyphenol, 3-methylindole, 4-hy-
droxy-3-methoxybenzaldehyde, 3,5-dimethoxy-4-hydroxybenzalde-
hyde, malonic acid, piperidine, vanillic acid, syringic acid, ferulic
acid, and sinapic acid were purchased from Tokyo Chemical Industry
Co., Ltd. (Tokyo, Japan). 1-Octen-3-one, 4-hydroxy-2,5-dimethyl-
3(2H)-furanone, 2-methoxy-4-vinylphenol, 3-hydroxy-4,5-dimethyl-
2(5H)-furanone, and phenylacetic acid were purchased from Sigma-
Aldrich Japan Co., Ltd. (Tokyo, Japan). trans-4,5-Epoxy-(E)-2-decenal
was synthesized according to the literature.10 2,6-Dimethoxy-4-
vinylphenol was synthesized according to the literature with a slight
modification.11
concentration, mg/L
selected
for the raw for the heated
compound
3-methylbutanal
ion m/z
soy sauce
soy sauce
44
57
94.6
92.8
20.4
0.602
479
94.6
92.8
20.4
0.602
479
2-methylbutanal
3-(methylthio)propanal
2-ethyl-3,5-dimethylpyraizine
phenylacetaldehyde
48
135
91
3-methylbutanoic acid
3-(methylthio)propanol
2-methoxyphenol
60
97.4
101
97.4
101
106
109
137
128
1.91
5.32
103
1.91
5.32
103
4-ethyl-2-methoxyphenol
4-hydroxy-2,5-dimethyl-3(2H)-
furanone
5(or 2)-ethyl-4-hydroxy-2(or 5)-
methyl-3(2H)-furanone
142
1370
1370
2-methoxy-4-vinylphenol
150
83
3.37
4.02
40.4
4.02
3-hydroxy-4,5-dimethyl-2(5H)-
furanone
2,6-Dimethoxy-4-vinylphenol. Malonic acid (2.00 g, 19.2 mmol)
was dissolved in 20 mL of pyridine in a 50 mL two-neck round-bottom
flask. 3,5-Dimethoxy-4-hydroxybenzaldehyde (2.34 g, 12.8 mmol) and
piperidine (0.48 mL, 4.8 mmol) were added to the flask, and the
solution was then stirred at 70 °C for 60 min. After cooling to room
temperature, the reaction mixture was added to cold water, and then
the pH was adjusted to 3.0. The mixture was extracted with ethyl
acetate (20 mL × 5) and dried with an excess amount of anhydrous
sodium sulfate. After replacement of the solvent by a n-hexane/ethyl
acetate (8:2) mixture, the mixture was separated by silica gel
chromatography using a glass column (500 × 20 mm i.d.) filled
with a n-hexane slurry of 50 g of Wakogel C-300 (40−64 μm, Wako
Pure Chemical Industries, Ltd., Osaka, Japan). Separation was
performed with 500 mL of a n-hexane/ethyl acetate (8:2) mixture
(fractions I−V). The eluates of fractions III and IV were combined and
2,6-dimethoxyphenol
phenylacetic acid
154
91
3.06
98.1
3.06
98.1
9.30
2,6-dimethoxy-4-vinylphenol
180
0.511
the same concentration method described above. The respective
aroma concentrates were analyzed by GC-MS in the synchronous
selected ion monitoring and full scan (SIM/scan) mode. On the basis
of the relationship between the ratio of the total area count of each key
aroma compound to that of 2-octanol and the added concentration of
each compound, the quantitative analysis of the key aroma compounds
was performed by calculation from the approximated curve using the
linear least-squares method. The respective quantitative values of the
key aroma compounds were determined by averaging the triplicate
experiments.
Model Decarboxylation Reactions of Phenolic Acids. An
aliquot (100.0 ppm) of phenolic acid was dissolved in an aqueous
solution with the pH adjusted to 4.7 with citric acid and sodium citrate
and then packed in a glass bottle under a nitrogen atmosphere. The
packed solution was heated in a water bath at 80 °C for 30 min. After
the heating, the sample was rapidly cooled to lower than room
temperature. Quantitative analysis of the generated phenolic
compounds was performed by reverse phase high-performance liquid
chromatography (RP-HPLC). The quantitative values of the respective
compounds were determined by the absolute calibration method by
averaging the triplicate experiments.
dried in vacuo. The isolated yield was 19%. H and 13C {1H} NMR
1
data agreed with the data reported in the literature.12,13 MS-EI: m/z
(%) 180 (100), 165 (40), 137 (27), 77 (15), 122 (12), 91 (12).
Preparation of the Aroma Concentrates of the Raw Soy
Sauce and the Heated Soy Sauce for GC-O and AEDA. The
aroma concentrates of the raw soy sauce and the heated soy sauce
were obtained according to the concentrating method described in the
literature.3 An aliquot (5 mL) of the soy sauce was passed through the
glass column (100 mm × 10 mm i.d.) filled with 5 mL of SP 700 resin
(Mitsubishi Chemical Co., Ltd.), which was conditioned with distilled
water before use, followed by washing with distilled water (5 mL × 4)
and eluting with 20 mL of dichloromethane. The dichloromethane
fraction was dried with an excess amount of anhydrous sodium sulfate
and then distilled by solvent-assisted flavor evaporation (SAFE) (40
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Table 2. Key Aroma Compounds in Raw Soy Sauce and Heated Soy Sauce
a
c
RI
FD factor
DB-
b
d
no.
WAX
DB-1
odor qualities
stimulus
compound
raw soy sauce heated soy sauce
ref
1
937
947
559
599
ethyl acetate
1
16
4
1
32
2
22
2
malty
2- and 3-methylbutanal
ethyl 2-methylpropanoate
2,3-butanedione
22, 29
2
3
975
746
fruity
4
979
558
milky
4
4
24
5
1045
1061
1074
1278
1295
1352
1428
1449
1463
1515
1631
1657
1682
1709
1718
1723
1831
1851
1877
1960
1995
2016
2024
2053
2123
2123
2181
2182
2190
2223
2257
2471
2546
2560
2569
819
fruity
ethyl 2-methylbutanoate
ethyl 3-methylbutanoate
dimethyl disulfide
4
4
25
6
831
fruity
4
2
26
7
729
rancid
1
1
27
8
665
milky
3-hydroxy-2-butanone
8
8
22
e
9
849
roasty
2-methyl-3-furanthiol
4
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
952
rancid
dimethyl trisulfide
1
1
23
e
1078
860
earthy, pea-like
cooked potato-like
nutty, roasty
earthy
2-isopropyl-3-methoxypyrazine
3-(methylthio)propanal
2-ethyl-3,5-dimethylpyrazine
1
1
512
64
1
1024
64
1
28
28
1061
1158
1009
833
e
2-isobutyl-3-methoxypyrazine
honey-like
sour, cheese-like
meaty
phenylacetaldehyde
32
64
nd
32
4
32
64
1
29
30
3-methylbutanoic acid
e
f
f
1171
944
methyl 2-methyl-3-furyl disulfide
3-(methylthio)propanol
unknown
cooked potato-like
floral
32
8
31
940
1002
1071
1092
1077
1342
1265
1041
1107
1013
1425
1287
1084
1456
1276
1311
1365
1232
1352
caramel-like
caramel-like
burnt
3-methyl-2(5H)-furanone
3-methyl-1,2-cyclopentanedione
2-methoxyphenol
nd
8
2
32
27
33
3
8
64
8
64
8
caramel-like
caramel-like
metallic
3-ethyl-1,2-cyclopentanedione
3-hydroxy-2-methyl-4-pyranone
16
8
16
8
34
2
e
trans-4,5-epoxy-(E)-2-decenal
spicy, burnt
caramel-like, sweet
caramel-like, sweet
caramel-like, sweet
fatty, milky
spicy, burnt
caramel-like, seasoning-like
milky
4-ethyl-2-methoxyphenol
256
128
2048
4
256
256
2048
8
35
27
36
37
38
39
40
4-hydroxy-2,5-dimethyl-3(2H)-furanone
5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone
4-hydroxy-5-methyl-3(2H)-furanone
4-decanolide
f
1
nd
128
2048
1
2-methoxy-4-vinylphenol
16
2048
8
3-hydroxy-4,5-dimethyl-2(5H)-furanone
5-decanolide
e
grape-like
spicy, burnt
animal-like
honey-like
vanilla-like
spicy
2′-aminoacetophenone
8
16
64
1
3
2,6-dimethoxyphenol
64
1
29
e
3-methylindole
phenylacetic acid
32
16
4
32
16
64
27
41
4-hydroxy-3-methoxybenzaldehyde
2,6-dimethoxy-4-vinylphenol
1525
a
b
RI, retention index. The compound was identified by comparing its RI and mass spectrum with the authentic compound by GC-MS in addition to
c
d
comparison of its RI and odor quality with the authentic compound by GC-O. FD factor, flavor dilution factor. Previous papers identified as
volatile components in soy sauce. The compound was tentatively identified by GC-O analysis using DB-WAX and DB-1 columns by comparison to
the authentic compound, but no unequivocal mass spectrum was available by GC-MS. nd, not detected.
e
f
Quantitative Analysis of Phenolic Acids in Raw Soy Sauce.
Vanillic acid, syringic acid, ferulic acid, and sinapic acid in the raw soy
sauce were quantitated by RP-HPLC. The quantitative values of the
respective compounds were determined by the absolute calibration
method by averaging the triplicate experiments.
GC-O. An Agilent model 6850 series gas chromatograph equipped
with a thermal conductivity detector (TCD) was used for the GC-O
analysis. A fused silica column (30 m × 0.25 mm i.d. coated with a 0.25
μm film of DB-Wax, J&W Scientific; or 30 m × 0.25 mm i.d. coated
with a 0.25 μm film of DB-1, J&W Scientific) was used with a 1.0 μL
splitless injection. Helium was used as the carrier gas at the flow rate of
1 mL/min. The injector temperature was set to 250 °C and the purge
valve time was set to 60 s. The chromatography was performed at the
oven temperature of 40 °C, which was increased to 210 °C at the rate
of 5 °C/min. The detector temperature was set to 230 °C. A glass
sniffing port was connected to the outlet of the TCD and heated to
>210 °C by a ribbon heater. Moist air was pumped into the sniffing
port at 100 mL/min to quickly remove the odorant eluted from the
TCD out of the sniffing port. The soy sauce aroma concentrates
underwent a GC-O analysis by three subjects. Determination of the
odor qualities detected by sniffing was achieved by triplicate
experiments for each subject.
AEDA. The original odor concentrate of the soy sauce aroma
concentrate was stepwise diluted with dichloromethane from 1:2 to
1:4096, and aliquots (1 μL) of each fraction were analyzed by capillary
GC on a DB-WAX column. AEDA was performed three times with
respect to each sample by three subjects.15 The detection of each
compound was defined as not less than two detections by all subjects,
and the FD factor of each compound was determined as the maximum
dilution degree of detection.
GC-MS. An Agilent 7890A gas chromatograph coupled to an
Agilent model 5975C inert XL series mass spectrometer was used. A
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Table 3. Quantitative Analysis of the Key Aroma Compounds in Raw Soy Sauce and Heated Soy Sauce by the Standard Addition
Method
raw soy sauce
heated soy sauce
a
b
a
b
concentration, μg/L (SD,
μg/L)
concentration, μg/L (SD,
c
R2
c
R2
no.
compound
μg/L)
ratio
d
2a 3-methylbutanal
2b 2-methylbutanal
2640 ( 20)
2670 ( 70)
388 ( 4)
0.944−0.995
0.962−0.997
0.994−1.000
0.987−1.000
0.994−0.997
0.998−0.999
0.981−0.995
0.996−1.000
0.995−1.000
0.999−1.000
0.989−0.998
3220 ( 60)
3160 ( 200)
513 ( 16)
0.982−0.988
0.932−0.989
0.968−1.000
0.997−1.000
0.988−0.995
0.990−1.000
0.959−0.997
0.975−0.994
0.965−1.000
0.984−1.000
0.982−1.000
1.22
1.18
1.32
1.08
1.31
0.98
1.09
1.02
0.95
1.48
0.94
d
d
12 3-(methylthio)propanal
13 2-ethyl-3,5-dimethylpyrazine
15 phenylacetaldehyde
15.2 ( 0.7)
10000 ( 700)
2340 ( 120)
3390 ( 120)
51.9 ( 1.6)
280 ( 31)
16.4 ( 0.6)
13100 ( 600)
2300 ( 120)
3690 ( 140)
52.6 ( 3.0)
266 ( 12)
d
16 3-methylbutanoic acid
18 3-(methylthio)propanol
22 2-methoxyphenol
26 4-ethyl-2-methoxyphenol
27 4-hydroxy-2,5-dimethyl-3(2H)-furanone
d
1650 ( 60)
42800 ( 1700)
2440 ( 160)
40400 ( 2400)
28 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-
furanone
d
31 2-methoxy-4-vinylphenol
32 3-hydroxy-4,5-dimethyl-2(5H)-furanone
35 2,6-dimethoxyphenol
98.6 ( 7.5)
105 ( 4)
0.988−0.998
0.991−0.999
0.984−1.000
0.998−1.000
0.985−0.996
1120 ( 62)
113 ( 6)
0.969−0.996
0.985−0.998
0.948−0.998
0.996−0.998
0.989−0.997
11.3
1.08
1.09
104 ( 6)
114 ( 10)
2520 ( 120)
235 ( 11)
37 phenylacetic acid
2480 ( 100)
1.02
d
39 2,6-dimethoxy-4-vinylphenol
13.6 ( 0.8)
17.2
a
b
c
d
Mean value in triplicate experiments. SD, standard deviation. R2, coefficient of determination obtained by the linear least-squares method. There
were significant differences between the raw soy sauce and the heated soy sauce by two-sample t test (p < 0.05).
fused silica column (60 m × 0.25 mm i.d. coated with a 0.25 μm film
of DB-Wax, J&W Scientific; or 60 m × 0.25 mm i.d. coated with a 0.25
μm film of DB-1, J&W Scientific) was used for the analyses. Helium
was used as the carrier gas at the flow rate of 1 mL/min, and the
injector temperature was set to 250 °C. Aliquots (1 μL) of the sample
were injected with the split ratio of 1:30 or splitless. The
chromatography was performed from an oven temperature of 80 to
210 °C at the rate of 3 °C/min for the split injections and from an
oven temperature of 40 to 210 °C at the rate of 3 °C/min for the
splitless injections. The mass spectrometer was used under the
following conditions: ionization voltage, 70 eV (EI); ion source
temperature, 150 °C.
for caramel-like aroma, and 4-ethyl-2-methoxyphenol for spicy/burnt
aroma. A two-way layout analysis of variance without replication was
applied to the sensory evaluation results performed with a seven-point
scoring method.
RESULTS AND DISCUSSION
■
Identification of the Key Aroma Compounds in Raw
Soy Sauce. Raw Japanese soy sauce had caramel-like, burnt/
spicy, and malty aromas, and the aroma concentrate of it had
the same characteristics when it was dipped in a filter paper for
sensory evaluation and sniffed.
RP-HPLC. An Agilent model 1200 series HPLC apparatus equipped
with a photodiode array detector was used for quantitative analysis.
The phenolic acids were analyzed using an ODS RP18 column (10
μm, 250 mm × 4.6 mm i.d., Shiseido Co., Ltd., Tokyo, Japan) under
the following conditions. Monitoring the effluent at 280 nm for vanillic
acid and syringic acid and at 324 nm for ferulic acid and sinapic acid,
chromatography was performed with a 3% acetonitrile solution
containing 0.1% phosphoric acid at the flow rate of 1.0 mL/min,
then increasing the acetonitrile content to 25% over 29 min, and finally
increasing the acetonitrile content to 100% over an additional 1 min.
The phenolic compounds of 2-methoxyphenol, 2-methoxy-4-vinyl-
phenol, 2,6-dimethoxyphenol, and 2,6-dimethoxy-4-vinylphenol were
analyzed using the same ODS column but with a slightly modified
condition. Monitoring the effluent at 280 nm, chromatography was
performed with a 10% acetonitrile solution containing 0.1%
phosphoric acid at the flow rate of 1.0 mL and then increasing the
acetonitrile content to 100% over 25 min.
Sensory Evaluations of Raw Soy Sauce and Heated Soy
Sauce. Eighteen subjects were recruited from Ogawa & Co., Ltd., and
trained for 6 months with in-house programs of recognition,
description, and discrimination tests involving about 300 odorants.
The respective soy sauce samples (5 mL) were poured into a plastic
cup at 25 °C for the sensory panelists, who were asked to score the
strengths of the malty, cooked potato-like, nutty, honey-like, sour,
caramel-like, and spicy/burnt aroma notes on a scale from 1 (weak) to
7 (strong). The evaluation terms were defined as the following aromas:
3-methylbutanal for malty aroma, 3-(methylthio)propanal for cooked
potato-like aroma, 2-ethyl-3,5-dimethylpyrazine for nutty aroma,
phenylacetaldehyde for honey-like aroma, 3-methylbutanoic acid for
sour aroma, 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone
To identify the key aroma compounds in the raw soy sauce,
AEDA was used for the aroma concentrate of the raw soy sauce,
and 37 compounds were detected with FD factors of ≥1 (Table
2). Among them, 2-methyl-3-furanthiol (9), 2-isopropyl-3-
methoxypyrazine (11), 2-isobutyl-3-methoxypyrazine (14), 5-
decanolide (33), 3-methylindole (36), and 2,6-dimethoxy-4-
vinylphenol (39) were identified as the key aroma compounds
in the soy sauce for the first time.
5(or 2)-Ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone
(28), exhibiting the caramel-like/sweet note, and 3-hydroxy-
4,5-dimethyl-2(5H)-furanone (32), exhibiting the caramel-like/
seasoning-like note, were detected as having the highest FD
factor of 2048, followed by 3-(methylthio)propanal (12)
(cooked potato-like), 4-ethyl-2-methoxyphenol (26) (spicy/
burnt), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (27) (caramel-
like), 2-ethyl-3,5-dimethylpyrazine (13) (nutty), 3-methylbuta-
noic acid (16) (sour/cheese-like), 2-methoxyphenol (22)
(spicy/burnt), and 2,6-dimethoxyphenol (35) (spicy/burnt)
as having FD factors from 64 to 512. Because most of these
compounds have already been reported as the key aroma
compounds in the common heated Koikuchi-type Japanese soy
sauce,2,3 they might also be important in the raw soy sauce
aroma.
Characterization of Raw Soy Sauce Aroma by
Comparative AEDAs, Quantitative Analysis, and Sensory
Analysis. To compare the aroma characteristics of the raw soy
sauce with those of the heated soy sauce, AEDA was applied to
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Table 4. Sensory Evaluation of Raw Soy Sauce and Heated Soy Sauce
a
b
aroma intensities (SD )
sample
malty
cooked potato-like
nutty
honey-like
sour
caramel-like
spicy/burnt
raw soy sauce
3.6a ( 0.9)
4.8b ( 0.9)
4.8b ( 1.0)
4.0c ( 0.7)
4.7d ( 1.0)
4.7d ( 0.9)
3.1 ( 1.0)
3.4 ( 0.7)
3.7 ( 0.9)
3.7 ( 1.0)
3.8 ( 1.1)
4.3 ( 1.3)
3.2 ( 1.0)
3.6 ( 1.1)
3.6 ( 1.0)
3.8 ( 0.9)
4.4 ( 0.8)
4.7 ( 1.0)
3.6e ( 0.7)
4.8f ( 0.6)
5.3f ( 1.0)
heated soy sauce
c
raw soy sauce + increased chemicals
a
b
c
Different letters indicate significant differences within the column (p < 0.05). SD, standard deviation. 3-Methylbutanal (580 μg/L), 2-
methylbutanal (490 μg/L), 3-(methylthio)propanal (125 μg/L), phenylacetaldehyde (3100 μg/L), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (790
μg/L), 2-methoxy-4-vinylphenol (1020 μg/L), and 2,6-dimethoxy-4-vinylphenol (221 μg/L) were added to the raw soy sauce.
the aroma concentrate of the heated soy sauce in addition to
the raw soy sauce. The comparative AEDAs revealed that 2-
methoxy-4-vinylphenol (31) and 2,6-dimethoxy-4-vinylphenol
(39) significantly increased and 5-decanolide (33) decreased
during the heating process, in addition to the formation of
methyl 2-methyl-3-furyl disulfide (17) (Table 2). Methyl 2-
methyl-3-furyl disulfide (17) might be formed from 2-methyl-3-
furanthiol (9) and methanethiol. Although 2-methoxy-4-
vinylphenol (31) and 2,6-dimethoxy-4-vinylphenol (39)
might be formed from the corresponding hydroxycinnamic
acids, these glycosides, and/or lignin by decarboxylation
reactions,1,16 there have been no experimental data to support
this claim. Model reactions of the phenolic acids under the
same heating conditions of the raw soy sauce were investigated,
as will be discussed in detail later. Because 5-decanolide (33) is
easy to decompose in an aqueous solution, it might have been
reduced during heating.
Furthermore, to clarify the precise changes in the key aroma
compounds of the raw soy sauce during the heating process, a
quantitative analysis of the key aroma compounds exhibiting
high FD factors of ≥32 in either soy sauce by the standard
addition method was performed (Table 3). A comparison of
the quantitative data of the respective soy sauces revealed that
3-methylbutanal (2a), 2-methylbutanal (2b), 3-(methylthio)-
propanal (12), phenylacetaldehyde (15), 4-hydroxy-2,5-
dimethyl-3(2H)-furanone (27), 2-methoxy-4-vinylphenol
(31), and 2,6-dimethoxy-4-vinylphenol (39) significantly
increased during the heating process. On the basis of previous
studies concerning the formation mechanisms of these
compounds, they all seem to be generated from amino acids,
sugars, and phenolic acids by a thermal treatment.16−18
Additionally, to clarify the changes in the aroma character-
istics during the heating process, a sensory experiment was
performed by comparing both soy sauces as well as the raw soy
sauce with the added significantly increased compounds during
the heating process listed in Table 3. The evaluation terms were
determined according to the aroma descriptions of the key
aroma compounds detected as having the FD factor of ≥32
(Table 4). The raw soy sauce represented moderate cooked
potato-like (4.0) and caramel-like (3.8) notes, followed by
honey-like (3.7), malty (3.6), and spicy/burnt (3.6) notes,
whereas the heated soy sauce was represented as relatively
strong spicy/burnt (4.8), malty (4.8), cooked potato-like (4.7),
and caramel-like (4.4) notes. Furthermore, the heated soy sauce
as well as the raw soy sauce with the increased chemical
compounds exhibited significantly higher scores on malty,
cooked potato-like, and spicy/burnt notes than the raw soy
sauce. On the contrary, although 4-hydroxy-2,5-dimethyl-
3(2H)-furanone (27) exhibiting a caramel-like note increased
during the heating process as shown by quantitative analysis,
there were no significant differences in the caramel-like note
among all of the samples by the sensory analysis. Because the
caramel-like note might also be contributed by other
compounds, such as 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-
3(2H)-furanone (28) and 3-hydroxy-4,5-dimethyl-2(5H)-fur-
anone (32), exhibiting the highest FD factor of 2048, no
significant differences were observed among the samples. There
were also no significant differences in the honey-like note
among them. Although phenylacetaldehyde (15) exhibiting the
honey-like note increased during the heating process, the
relatively low FD factor of 128 might be the cause of no
significant differences in the sensory evaluation result.
In summary, comparative AEDAs, a quantitative analysis, and
a sensory analysis demonstrated that whereas most of the key
aroma compounds in the raw soy sauce were common in the
heated soy sauce, some of the Strecker aldehydes (3-
methylbutanal (2a), 2-methylbutanal (2b), and 3-(methylthio)-
propanal (12)), exhibiting a malty note and a cooked potato-
like note, and 4-vinylphenols (2-methoxy-4-vinylphenol (31)
and 2,6-dimethoxy-4-vinylphenol (39)), exhibiting a spicy/
burnt note, contributed less to the raw soy sauce aroma.
Model Reactions of the Phenolic Acids and the
Proposed Mechanism of the Phenolic Compounds
Formation in Raw Soy Sauce during the Heating
Process. Whereas 2-methoxy-4-vinylphenol (31) and 2,6-
dimethoxy-4-vinylphenol (39) significantly increased during the
heating process in the raw soy sauce by the quantitative
analysis, 2-methoxyphenol (22) and 2,6-dimethoxyphenol (35),
which could be formed as decarboxylation products from the
respective hydroxybenzoic acids, did not increase during the
heating process (Table 3). To verify these differences, the
model decarboxylation reactions of the phenolic acids were
investigated on the basis of the heating conditions of the raw
soy sauce (Table 5). Sinapic acid (43) resulted in the highest
Table 5. Yields of the Model Decarboxylation Reactions of
a
Phenolic Acids by a Thermal Treatment
b
c
no.
compound
decarboxylation product
yield, (SD, %)
40
41
42
43
vanillic acid
syringic acid
ferulic acid
sinapic acid
2-methoxyphenol
0.010 ( 0.000)
0.023 ( 0.001)
0.758 ( 0.019)
2,6-dimethoxyphenol
2-methoxy-4-vinylphenol
2,6-dimethoxy-4-vinylphenol
1.656 ( 0.033)
a
b
c
80 °C × 30 min, pH 4.7. Mean value in triplicate experiments. SD,
standard deviation.
yield (1.656%) of 2,6-dimethoxy-4-vinylphenol (39), followed
by ferulic acid (42), which resulted in the next highest yield
(0.758%) of 2-methoxy-4-vinylphenol (31). On the contrary,
vanillic acid (40) and syringic acid (41) reacted with low yields
of the corresponding phenolic compounds (0.010 and 0.023%,
respectively). These results revealed that although all reactions
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resulted in low yields, the hydroxycinnamic acid derivatives
were more reactive than the hydroxybenzoic acid derivatives.
This might be because the hydroxycinnamic acid derivatives
react through β-carbonyl ions (protonation to the olefin at
first) as the intermediates, but the hydroxybenzoic acid
derivatives react through the unstable carbanions.19,20 The
decarboxylation reaction of the hydroxycinnamic acid deriva-
tives might proceed as a zwitter ion form under the weakly
acidic condition (pH 4.7) of the raw soy sauce.19 Furthermore,
the 2,6-dimethoxy skeleton is more reactive than the 2-methoxy
skeleton on the basis of the model reaction yields (Table 5).
Because the decarboxylation reaction of the phenolic acid is
promoted by the increasing stability of the reaction
intermediate,20 the 2,6-dimethoxy derivatives are thought to
be more reactive due to the stable intermediates provided by
the two methoxy substituents at the meta positions of the
phenyl group, which have an electron-withdrawing effect
(inductive effect).
vinylphenol (39) were comparatively high (150 and 62.9 μg/
L). Furthermore, whereas the ratios of the increased amounts
of 2-methoxyphenol (22) and 2,6-dimethoxyphenol (35) by
the above reactions in the total increased amounts during the
heating process were estimated to be high (84 and 89%,
respectively), those of 2-methoxy-4-vinylphenol (31) and 2,6-
dimethoxy-4-vinylphenol (39) were estimated to be quite low
(15 and 28%, respectively) (Table 7). These results indicated
that although only the 4-vinylphenols increased during the
heating process in the raw soy sauce, they were not mainly
formed from the respective hydroxycinnamic acids, but from
the other precursors. Although some components, such as
lignin, shakuchirin, and esters with arabinoxylan,1,21 are
proposed as precursors, it is still unclear.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-47-305-1408. Fax: +81-47-305-1423. E-mail:
kaneko.shu@ogawa.net.
Furthermore, to verify the increases in the phenolic
compounds during the heating process in the raw soy sauce,
quantitative analysis of the phenolic acids in the raw soy sauce
was performed by RP-HPLC (Table 6). Syringic acid (41) was
Notes
The authors declare no competing financial interest.
REFERENCES
Table 6. Quantitative Analysis of Phenolic Acids in Raw Soy
Sauce
■
(1) Yokotsuka, T.; Sasaki, M. Fermented protein foods in the Orient:
shoyu and miso in Japan. In Microbiology of Fermented Foods; Wood, B.
J., Ed.; Blackie and Academic and Professional: London, UK, 1998;
Vol. 1 and 2, pp 351−415.
(2) Steinhaus, P.; Schieberle, P. Characterization of the key aroma
compounds in soy sauce using approaches of molecular sensory
science. J. Agric. Food Chem. 2007, 55, 6262−6269.
a
b
no.
compound
concentration, mg/L (SD, mg/L)
40
41
42
43
vanillic acid
syringic acid
ferulic acid
sinapic acid
6.1 ( 0.2)
36.7 ( 0.4)
19.7 ( 0.1)
3.8 ( 0.1)
(3) Kaneko, S.; Kumazawa, K.; Nishimura, O. Comparison of the key
aroma compounds in five different types of Japanese soy sauces by
aroma extract dilution analysis (AEDA). J. Agric. Food Chem. 2012, 60,
3831−3836.
a
b
Mean value in triplicate experiments. SD, standard deviation.
contained in the highest amount of 38.4 μg/L in the raw soy
sauce, followed by vanillic acid (40), ferulic acid (42), and
sinapic acid (43) (29.2, 22.1, and 4.1 μg/L, respectively). To
discuss the contribution to the increase in the phenolic
compounds in the heated soy sauce by the respective phenolic
acids, the estimated yields of the phenolic compounds were
calculated from the yields of the model reactions (Table 7).
Although the estimated yields of 2-methoxyphenol (22) and
2,6-dimethoxyphenol (35) were quite low (0.6 and 8.3 μg/L),
those of 2-methoxy-4-vinylphenol (31) and 2,6-dimethoxy-4-
(4) Nakadai, T. Heating of raw shoyu. J. Soy Sauce Res. Technol. 2008,
34, 274−282.
(5) Sasaki, M. Flavor component. Jozobutsu no Seibun; Brewing
Society of Japan: Tokyo, Japan, 1999; pp 476−501.
(6) Yamada, M. Origin of aldehydes in fermentation products. II (in
Japanese). Nippon Nogei Kagaku Kaishi 1928, 4, 1−15.
(7) Asao, Y.; Yokotsuka, T. Flavoring substances in soy sauce. XXIV.
α-Diketones in soybean sauce. 4. Nippon Nogei Kagaku Kaishi 1963,
37, 569−575.
(8) Yokotsuka, T. Flavoring substances in soy sauce. XV. In heated
soy sauce. 2. Nippon Nogei Kagaku Kaishi 1958, 32, 23−26.
(9) Nunomura, N.; Sasaki, M.; Asao, Y.; Yokotsuka, T. Shoyu (soy
sauce) volatile flavor components: basic fraction. Agric. Biol. Chem.
1978, 42, 2123−2128.
(10) Kumazawa, K.; Wada, Y.; Masuda, H. Characterization of
epoxydecenal isomers as potent odorants in black tea (Dimbula)
infusion. J. Agric. Food Chem. 2006, 54, 4795−4801.
(11) Aldabalde, V.; Risso, M.; Derrudi, M. L.; Geymonat, F.; Seoane,
G.; Gamenara, D.; Saenz-Mendez, P. Organocatalyzed decarboxylation
of naturally occurring cinnamic acids: potential role in flavoring
chemicals production. Open J. Phys. Chem. 2011, 1, 85−93.
(12) Bernini, R.; Mincione, E.; Barontini, M.; Provenzano, G.; Setti,
L. Obtaining 4-vinylphenols by decarboxylation of natural 4-
hydroxycinnamic acids under microwave irradiation. Tetrahedron
2007, 63, 9663−9667.
(13) Spielmeyer, A.; Wagner, A.; Jahreis, G. Influence of thermal
treatment of rapeseed on the canolol content. Food Chem. 2009, 112,
944−948.
(14) 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.
Table 7. Estimated Yields of Phenolic Compounds by a
Thermal Treatment from Raw Soy Sauce
concentration, μg/L
increase
during
estimated yield formed
a
b
c
no.
compound
heating
from phenolic acid
ratio
22 2-methoxyphenol
0.7
9
0.6
8.3
84
89
35 2,6-
dimethoxyphenol
31 2-methoxy-4-
1020
121
150
15
28
vinylphenol
39 2,6-dimethoxy-4-
62.9
vinylphenol
a
Difference between the raw soy sauce and the heated soy sauce
b
calculated from Table 3. The multiplication of the concentration of
the corresponding precursor in the raw soy sauce shown in Table 5
and the yield of the corresponding phenolic compound by the thermal
treatment shown in Table 6. The ratio of the estimated yield to the
increased amount during heating of the raw soy sauce.
c
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Article
(15) Schieberle, P. New developments in methods for analysis of
volatile flavor compounds and their precursors. In Characterization of
Food: Emerging Methods; Goankar, A., Ed.; Elsevier: Amsterdam, The
Netherlands, 1995; pp 403−431.
(16) Belitz, H. D.; Grosch, W.; Schieberle, P. Aroma compounds. In
Food Chemistry , 4th revised and extended ed.; Belitz, H. D., Grosch,
W., Schieberle, P., Eds.; Springer-Verlag: Berlin, Germany, 2009; pp
340−402.
(17) Pripis-Nicolau, L.; de Revel, G.; Bertrand, A.; Maujean, A.
Formation of flavor components by the reaction of amino acid and
carbonyl compounds in mild conditions. J. Agric. Food Chem. 2000, 48,
3761−3766.
(18) Blank, I.; Fay, L. B. Formation of 4-hydroxy-2,5-dimethyl-
3(2H)-furanone and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-
furanone through Maillard reaction based on pentose sugars. J. Agric.
Food Chem. 1996, 44, 531−536.
(19) Cohen, L. A.; Jones, W. M. A study of pH dependence in the
decarboxylation of p-hydroxycinnamic acid. J. Am. Chem. Soc. 1960, 82,
1907−1911.
furanone, as a flavor component in shoyu (soy sauce). Agric. Biol.
Chem. 1976, 40, 491−495.
(37) Nunomura, N.; Sasaki, M.; Yokotsuka, T. Isolation of 4-
hydroxy-5-methyl-3(2H)-furanone, a flavor component in shoyu (soy
sauce). Agric. Biol. Chem. 1979, 43, 1361−1363.
(38) Kim, H. J.; Lee, E. J.; Shin, O. S.; Ji, W. D.; Choi, M. R.; Kim, J.
K. Volatile components in the soy sauce manufactured by Bacillus
species and fused yeast. J. Microbiol. Biotechnol. 1996, 6, 194−201.
(39) Sugawara, E. Formation by yeast of the aroma component
specific to miso and soy sauce. Food Ingred. J. Jpn. 2001, 193, 57−65.
(40) Kihara, K. Capillary gas chromatographic pattern of volatile
components in shoyu and their relation to quality. I. Preliminary
experiment and GC-MS. J. Jpn. Soy Sauce Res. Inst. 1986, 12, 12−19.
(41) Yokotsuka, T. Flavoring substances in soy sauce XIII. The
fungistatic substances in brewed soy sauce and the increace of the
fungistatic nature by heating the soy sauce. Nippon Nogei Kagaku
Kaishi 1954, 28, 114−118.
(20) Sykes, P. Carbanions and their reactions. In Mechanism in
Organic Chemistry, 6th ed.; Sykes, P., Ed.; Longman Group: London,
UK, 1986; pp 270−298.
(21) Kato, Y.; Nevins, D. J. Enzymic dissociation of Zea shoot cell
wall polysaccharides. IV. Dissociation of glucuronoarabinoxylan by
purified endo (1 → 4)-β-xylanase from Bacillus subtilis. Plant Physiol.
1984, 75, 759−765.
(22) Akabori, S. Aroma of shoyu (soy sauce) I. J. Chem. Soc. Jpn.
1936, 57, 828−831.
(23) Nunomura, N.; Sasaki, M.; Yokotsuka, T. Shoyu (soy sauce)
flavor components: neutral fraction. Agric. Biol. Chem. 1984, 48, 1753−
1762.
(24) Yokotsuka, T.; Asao, Y. Flavor substances in soy sauce XIX. The
structure of C7H12O5 compound and α-diketone compounds in soy
sauce. Nippon Nogei Kagaku Kaishi 1961, 35, 837−845.
(25) Sasaki, M.; Nunomura, N. Studies of flavor constituents of soy
sauce. 7. Constituents of top note aroma of soy sauce. J. Chem. Soc. Jpn.
1981, 736−745.
(26) Yokotsuka, T. Flavoring substances in soy sauce. VIII. Flavors
with higher boiling points. 3. Benzaldehyde and ethyl benzoate. J.
Agric. Chem. Soc. Jpn. 1951, 25, 446−450.
(27) Nunomura, N.; Sasaki, M.; Yokotsuka, T. Shoyu (soy sauce)
flavor components: acidic fractions and the characteristic flavor
component. Agric. Biol. Chem. 1980, 44, 339−351.
(28) Ishihara, K.; Honma, N.; Matsumoto, I.; Imai, S.; Nakazawa, S.;
Iwafuchi, H. Comparison of volatile components in soy sauce
(koikuchi shoyu) produced using Aspergillus sojae and Aspergillus
oryzae. J. Jpn. Soc. Food Sci. Technol. 1996, 43, 1063−1074.
(29) Nunomura, N.; Sasaki, M.; Asao, Y.; Yokotsuka, T. Identification
of volatile components in shoyu (soy sauce) by gas chromatography-
mass spectrometry. Agric. Biol. Chem. 1976, 40, 485−490.
(30) Yamada, K.; Ino, M. Shoyu flavor by gas chromatography. II.
Flavor components produced by shoyu koji microflora. Chomi Kagaku
1969, 16, 15−20.
(31) Akabori, S.; Kaneko, T. Aroma of shoyu (soyo sauce) II.
Isolation of (γ-methylmercapto)propyl alcohol and its synthesis.
Nippon Kagaku Kaishi 1936, 57, 832−837.
(32) Liardon, R.; Philippossian, G. Volatile components of fermented
soya hydrolyzate. I. Identification of some lactones. Z. Lebensm. Unters.
Forsch. 1978, 167, 180−185.
(33) Asao, T.; Yokotsuka, T. Flavoring substances in soy sauce. XVI.
In raw soy sauce 2. J. Agric. Chem. Soc. Jpn 1958, 32, 617−622.
(34) Kihara, K. The aroma of soy sauce. II. Difficulty volatile
constituents. J. Soc. Chem. Ind. Jpn. 1940, 43 (Suppl. Binding), 403.
(35) Yokotsuka, T. Flavoring substances in soy sauce IX. Flavors with
higher boiling points. 4. Isolation of phenolic flavoring substances. J.
Agric. Chem. Soc. Jpn. 1953, 27, 276−281.
(36) Nunomura, N.; Sasaki, M.; Asao, Y.; Yokotsuka, T. Isolation and
identification of 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-
3402
dx.doi.org/10.1021/jf400353h | J. Agric. Food Chem. 2013, 61, 3396−3402