SUPPLEMENTARY MATERIAL
Characterization of Aroma Profiles of Commercial Sufus by Odour
Activity Value, Gas Chromatography-Olfactometry, Aroma
Recombination, and Omission Studies
Zuobing Xiaoa, Yi Shanga, Feng Chenb, YunWei Niua, Yongbo Gua, Shengjiang Liua and
Jiancai Zhua*
a
Department of Perfume and Aroma Technology, Shanghai Institute of Technology,
Shanghai, China
b
Department of Food, Nutrition, and Packaging Sciences, Clemson University,
Clemson, USA 29634
Abstract
Sufu is a solid-state fermented product made from soybeans. For the sake of quality control and
regulation purposes, it is essential to be able to identify key odorants of various commercial sufus.
To identify the aroma-active compounds in sufus, gas chromatography-olfactometry/aroma extract
dilution analysis (GC-O/AEDA) was performed, and odor activity value (OAV) was estimated. The
correlations between aroma profiles and identified aroma-active compounds were also investigated
by principal component analysis. Results showed that thirty-five aroma-active compounds were
detected through OAV calculation, while twenty-eight compounds were identified by using
GC-O/AEDA. Quantitative descriptive analysis (QDA) revealed that aroma recombination model
based on OAV calculation was more similar to original sufu in terms of aroma comparing to aroma
recombination model based on GC-O/AEDA. Omission experiments further confirmed that the
aroma compounds, such as ethyl butanoate, ethyl 2-methylbutanoate, ethyl hexanoate,
(E,E)-2,4-decadienal and 2,6-dimethylpyrazine, contributed most significantly to the characteristic
aroma of a commercial sufu.
Keywords: Commercial sufu, SDE-GC-MS, OAV, GC-O/AEDA, aroma recombination and
omission
Experimental
Sufu samples
A total of 9 different kinds of sufu samples were analysed in the present study. Each kind of
1
sufu sample was randomly purchased three times from the local markets in Shanghai, China. The
details of these samples were summarized in Table S1. All sufu samples (five red and four white)
were kept in their own packages and stored in a refrigerator (4 °C) until analysed. The dressing
mixture of each sample was separated from the sufu cubes before extraction.
Chemicals
Authentic standards were obtained from the following sources: ethanol, 2-butanol, 1-propanol,
2-methyl-1-propanol, 1-butanol, 3-methyl-1-butanol, 1-pentanol, 1-hexnaol, 2-heptanol, 3-octanol,
1-octen-3-ol, phenylmethanol, 2-phenylethanol, 2-methylpropanal, 1-hexanal, (E)-2-heptenal,
1-nonanal,
3-(methylthio)-1-propanal,
(E,E)-2,4-decadienal,
benzaldehyde,
2-Phenylethanal,
(E)-2-nonenal,
4-methoxybenzaldehyde,
(E,E)-2,4-nonadienal,
ethyl
butanoate,
ethyl
2-methylbutanoate, ethyl hexanoate, ethyl heptanoate, ethyl 2-hydroxypropanoate, ethyl octanoate,
ethyl nonanoate, ethyl decanoate, diethyl succinate, ethyl phenylacetate, ethyl dodecanoate, ethyl
tetradecanoate, ethyl hexadecanoate, ethyl octadecanoate, ethyl (Z)-9-octadecenoate, ethyl
(Z,Z)-9,12-octadecadienoate, 3-penten-2-one, 2-heptanone, 3-octanone, 3-hydroxy-2-butanone,
2-nonanone, hexanoic acid, octanoic acid, (Z,Z)-9,12-octadecadienoic acid, hexadecanoic acid,
(Z,Z,Z)-9,12,15-octadecatrienoic
acid,
2-methylpyrazine,
2,3-dimethylpyrazine,
2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2-pentylfuran, furfural, furfurol, 5-methylfurfural,
phenol, 4-ethyl-2-methoxyphenol, 2-methoxy-4-(2-propenyl)phenol,
2-methoxy-4-vinylphenol,
1,8-cineole, styrene, (E)-anethole, 2-acetylpyrrole, 2,4,6-trimethylpyridine (internal standard, IS)
were purchased from Sigma-Aldrich Corporation (Shanghai, China). Linalool, 2-ethyl-1-hexanol,
3-(methylthio)-1-propanol,
(E)-2-hexenal,
2-ethyl-3,5-dimethylpyrazine
came
from
(E)-2-decenal,
Alfa
Aesar
ethyl
Corporation
3-phenylpropionate,
(Tianjin,
China).
(E,E)-2,4-Heptadienal and ethyl heptadecanoate were obtained from TCI Corporation (Shanghai,
China). A C6-C30 n-alkane standard was purchased from Sigma-Aldrich Corporation (Shanghai,
China). All of the chemical standards used above were of chromatography quality.
Anhydrous sodium sulfate (Na2SO4), absolute ethanol and dichloromethane were supplied by
the China National Pharmaceutical Group Corporation (Shanghai, China). Water was obtained from
a Milli-Q purification system (Millipore, Bedford, MA).
Extraction of volatile compounds
A Likens-Nickerson type SDE apparatus (Huake Experimental Equipment Co., Ltd, Shanghai,
2
China) was employed to extract the volatile compounds. 100 g of sufu (muddy mixture), 500 mL of
distilled water, with 10 μL of IS (2,4,6-trimethylpyridine) were loaded in a 1 L flask. 50 mL of
redistilled dichloromethane was used as the extraction solvent and placed in another flask. Both
flasks were loaded onto the SDE apparatus. The temperature of the solvent flask was maintained at
50 °C by a water bath, and the temperature of the sample flask was maintained at 105 °C by an oil
bath. The distillation extraction was carried out for 2 h after the 2 arms started to reflux. The
collected extraction was dried over anhydrous sodium sulphate and filtered at room temperature, and
further condensed to 1 mL under a gentle stream of high-purity nitrogen. The condensed extract was
preserved at -20 °C until further analysis. Each SDE extraction was carried out in triplicate.
GC-MS analysis
GC-MS analysis was performed using an Agilent 6890 GC equipped with an Agilent 5973 mass
selective detector (Agilent Technologies, Inc., Palo Alto, CA, U.S.A.). 1 μL of extract was injected at
250 °C in splitless mode. Helium (purity 99.99 %) was used as the carrier gas with a constant flow
of 1 mL/min. The separation was conducted using a HP-5MS column (60 m × 0.25mm i.d. × 0.25
μm, Agilent Technologies) and a HP-INNOWAX column (60 m × 0.25mm i.d. × 0.25 μm, Agilent
Technologies). The oven temperature was initially kept at 50 °C for 5 min, then reached 230 °C at a
rate of 6 °C/min and was finally maintained at 230 °C for 30 min. The mass selective detector was
operated in the electron ionization mode with ionization energy of 70 eV, a scan range of 30-450 m/z.
The temperature of MSD transfer line was 280 °C and that of ion source was 230 °C.
Aroma Extract Dilution Analysis (AEDA)
GC-O analysis was carried out on an Agilent 6890 GC equipped with flame ionization detection
(FID) system and an olfactometer. The HP-INNOWAX column (60 m × 0.25mm i.d. × 0.25 μm,
Agilent Technologies) was used with a 1μL splitless injection. The column effluent was split 1:1 into
the FID and the heated sniffing port (230 °C) using two deactivated and uncoated fused silica
capillaries. The operating condition was same as those established in GC-MS analysis.
The flavor dilution (FD) factors of aroma compounds were determined by AEDA (Schieberle
1995). The aroma extract was diluted stepwise with dichloromethane at 1:1 ratio, and each dilution
was then analysed in 1 μL aliquots by GC-O until no odorant could be perceived. The FD factors of
the volatile compounds were determined as the maximum dilutions at which the odorants could be
detected. Five experienced panelists (three females and two males, 23 years old on average) were
3
recruited for GC-O analysis. The panelists were trained for 2 months in GC-O using at least 30
aroma-active standard compounds in a concentration 2 times above their odour thresholds in air.
Whenever the aroma was detected by a panelist at the sniffing port in a GC run, then it was
considered as “active” and the aroma descriptor was recorded. The sniffing time of each run was not
more than 40 min. Determination of the odour descriptors detected by sniffing was carried out in
triplicate experiments for each sample. For the purpose of screening the potent aroma compounds of
all the commercial sufus, the average values of the FD factors of each compound detected in the
sufus were calculated. The compounds with high mean values were regarded as the aroma active
compounds of the sufu samples.
Qualitative and quantitative analysis
The volatile compounds were confirmed by comparing their retention indices (RI) and mass
spectra with those of authentic standards. Retention indices of the volatiles were calculated from the
retention times of n-alkanes (C6-C30) under the same chromatographic conditions. RI is given by the
following equation:
RIx =100 × (
log tr(x) - log tr(z)
log tr(z+1) - log tr(z)
+ z)
(1)
Here, RIx is the retention indices of unknown compound x, z is the number of carbon atoms in the
n-alkane eluted before the unknown compound x, z + 1 is the number of carbon atoms in the
n-alkane eluted after the unknown compound x, tr is the retention time.
Selective ion monitoring (SIM) mass spectrometry was operated to quantitate the volatile
compounds (Table S3). Standard solutions were prepared by mixing standards of the volatile
compounds and IS (2,4,6-trimethylpyridine) in the dichloromethane. These solutions at six
concentration levels simulated the concentration ranges of the volatile compounds detected in
commercial sufus (Feng et al. 2015). Triplicate GC-MS analyses were accomplished for each
standard solution. The ion monitored of 2,4,6-trimethylpyridine in the SIM run was m/z 79. For
individual volatile compound, calibration curve was established by plotting the response ratio of
compounds and IS against the volatile compound concentration (Fan et al. 2010).
Sensory evaluation
Quantitative descriptive analyses (QDA) were performed for evaluating nine sufu samples by
12 assessors (6 females and 6 males, 30 years old on average), and 5 of them were from the GC-O
4
analysis. All the assessors were previously trained the ability to recognize, describe and discriminate
different aroma qualities. Each of them completed 150 h of specific training with commercial sufus
and reference standards to clarify definitions, as described in Table S2. Nine sensory attributes were
commonly selected to describe the overall aroma by the panelists: alcoholic, edible oil, fermented,
floral, herbs, Maillard, fruity, sweet spices and vegetable. The respective sufu samples (10 g) were
placed in capped glasses with different three–digit numbers for the sensory assessors. Each assessor
rated the intensity of the nine attributes on a ten-point scale (0, none; 5, moderate; 10, very strong),
and the results were averaged for each sensory note and plotted in a radar diagram. The sensory
evaluations of each sample were performed in triplicate at room temperature (20 ± 1 °C).
Aroma recombination and omission experiments
For each sufu sample, two aqueous aroma models were prepared and then compared with the
original aroma. It was conducted to verify the results obtained from OAV and GC-O/AEDA and
compare the effectiveness of these two methods in characterizing the sufu aroma. Aroma models
were prepared by blending volatile compounds in their natural concentrations shown in Table S3.
The two recombinant aroma models and corresponding sufu sample (10g each) were placed in
capped glasses at room temperature, and evaluated by the panel on the basis of the same scale used
for sensory evaluation.
Triangle tests were carried out to study the significance of certain odorants that were identified
as aroma-active volatile compounds by OAV or GC-O/AEDA analysis. As shown in Table S6, 21
aroma omission models were prepared by omitting one or a group of selected odorants from the
complete recombinant aroma model. Each omission model was evaluated against two complete
recombination models prepared by mixing the standard aroma compounds at the concentrations in
Table S3. Three different testing samples (10g each) were randomly assigned to the panelists for
sensory evaluation. The test series were repeated 2 times. On the basis of the method described
previously (Feng, Su, Zhao, Cai, Cui, Sun-Waterhouse and Zhao 2015), the significance of
difference among the results was calculated.
Statistical analysis
Statistical analysis was conducted by using Principal Component Analysis (PCA) module in
XLSTAT 2010 (Addinsoft, New York, U.S.A.). The purpose was to reveal the correlation among the
5
sufu samples as well as to correlate the sensory attributes to the sufu samples. One-way analysis of
variance (ANOVA) and Tukey test at the p=0.05 level of significance was carried out to determine
significant differences between the different models in each sensory attribute as well as in their
overall aroma by using the SPSS 17.0 software package.
6
Table S1. Details of commercial sufu samples utilized in the present study.
Code
TypeA
Production area
Raw materials
CS1
Red sufu
Guangdong province
Soy, flour, salt, sugar, edible alcohol, red koji
CS2
White sufu
Guangdong province
Soy, salt, edible alcohol
CS3
White sufu
Guangdong province
Soy, salt, edible alcohol, chilies
CS4
Red sufu
Beijing
Soy, flour, salt, sugar, edible alcohol, red koji, spices
CS5
White sufu
Beijing
Soy, salt, edible alcohol, spices
CS6
Red sufu
Beijing
Soy, flour, salt, sugar, edible alcohol, red koji, red rose petals
CS7
Red sufu
Shanghai
Soy, flour, salt, sugar, edible alcohol, red koji, red rose petals, rice, spices
CS8
White sufu
Shanghai
Soy, salt, edible alcohol, chilies
CS9
White sufu
Shanghai
Soy, salt, edible alcohol
A Sufu
samples were classified according to their color and flavor reported by (Han et al. 2001).
7
Table S2. Definition of sensory attributes and training standard.
Sensory attribute
Definition
Reference standardA
Alcoholic
Pungent and minty aroma
1mL 2-methylpropanal
Edible oil
Fatty and fried note
1mL each of (E,E)-2,4-decadienal
Fermented
Yeast fermentation odour
1mL 3-methylthio-1-propanal
Floral
Flowery and honey-like note
1mL 2-phenyl ethanol
Fruity
Smelling strongly of kinds of fruit
1mL each of 2-methylbutanoate
Herbs
Odour of fresh, grassy and unripe fruits
1mL hexanal
Maillard
Roasted and nutty aroma
1mL each of 2,3,5-trimethylpyrazine
Sweet spices
Sweet, anise- and clove-like note
1mL each of 4-methoxybenzaldehyde
Vegetable
Musty and mushroom-like note
1mL each of 1-octen-3-ol
A All
the reference standard were pure compounds.
8
Table S3. Concentrations of 75 volatile compounds in nine commercial sufus obtained by SDE-GC-MS.
Concentration (μg/kg)C
RIB
CompoundA
No.
Quantify ion (m/z)
HP-INNOWAX
HP-5ms
CS1
CS2
CS3
CS4
CS5
CS6
CS7
CS8
CS9
Alcohols (16)
1
Ethanol
31
928
< 600
9.30±0.140
9.40±0.350
15.1±1.02
27.5±0.430
9.70±0.320
29.2±1.21
7.20±0.180
41.4±1.96
350±14.2
2
2-Butanol
45
1008
< 600
124±10.7
66.2±5.48
37.1±1.01
167±10.4
39.2±1.67
33.7±2.01
10.3±1.21
50.2±3.36
45.6±3.16
3
1-Propanol
31
1027
< 600
1.20e3±11.2
327±19.3
397±15.1
334±20.0
140±10.1
125±10.2
249±20.5
371±28.1
127±10.1
4
2-Methyl-1-propanol
43
1103
647
978±30.3
47.9±1.15
67.3±3.91
88.4±4.58
91.6±5.01
590±12.1
39.6±5.24
18.2±1.34
7.60±0.340
5
1-Butanol
56
1142
675
64.9±4.67
21.2±1.53
23.8±2.15
46.1±2.31
32.1±1.15
14.4±1.12
31.5±2.59
80.8±6.16
59.5±3.12
6
3-Methyl-1-butanol
75
1208
738
4.48e3±22.0
120±9.98
161±10.1
641±16.0
99.2±3.76
36.6±2.55
310±19.2
225±12.6
114±10.9
7
1-Pentanol
55
1244
763
66.5±2.02
27.4±1.76
17.9±1.02
19.3±0.750
15.8±0.420
11.6±0.150
13.7±1.05
90.5±3.47
48.9±2.45
8
2-Heptanol
45
1299
902
253±15.1
10.8±0.980
18.3±1.39
19.4±1.24
5.30±0.450
30.1±2.89
103±9.24
37.6±4.26
18.5±1.14
9
1-Hexanol
56
1350
865
434±17.2
98.7±5.03
84.2±2.56
80.5±5.14
6.70±0.540
114±10.1
146±15.2
31.5±1.16
350±12.5
10
3-Octanol
59
1394
1007
268±18.2
30.4±3.15
24.6±2.03
21.3±2.15
14.6±1.28
17.1±1.55
55.2±4.16
41.5±3.15
23.9±2.05
11
1-Octen-3-ol
57
1469
994
410±32.1
897±60.6
573±22.0
155±12.3
122±11.2
137±12.6
149±12.6
297±10.2
351±12.5
12
2-Ethyl-1-hexanol
57
1487
1032
ND
ND
ND
15.3±0.320
23.3±1.03
90.5±3.16
93.5±5.87
ND
ND
13
Linalool
73
1554
1105
ND
ND
ND
ND
ND
60.6±2.29
31.1±2.21
ND
ND
14
Phenylmethanol
79
1864
1026
ND
9.30±0.150
10.2±0.950
ND
11.9±0.250
ND
ND
68.2±3.14
21.3±2.19
9
15
3-(Methylthio)-1-propanol
106
1897
1104
ND
27.2±1.32
28.1±1.33
ND
ND
ND
ND
38.7±2.68
21.3±2.05
16
2-Phenylethanol
91
1902
1132
889±75.3
147±12.9
111±10.4
176±12.1
43.9±1.79
1.15e3±91.4
796±35.3
284±10.8
247±15.9
6.17
2.01
1.67
1.54
0.49
1.81
0.74
1.33
0.98
Subtotal (%)
Aldehydes (14)
17
2-Methylpropanal
43
870
< 600
ND
113±9.23
484±28.5
ND
23.8±1.39
ND
31.3±1.19
70.1±5.95
106±9.12
18
1-Hexanal
56
1080
803
41.1±2.64
73.5±5.73
53.8±2.61
69.7±5.36
91.6±5.95
33.3±2.29
78.1±5.87
341±12.8
213±17.8
19
(E)-2-Hexenal
69
1222
915
ND
12.5±1.15
13.6±1.52
17.7±1.27
12.4±1.02
ND
ND
35.2±2.65
42.6±3.62
20
(E)-2-Heptenal
55
1312
932
ND
13.2±0.530
18.6±1.21
ND
13.2±1.04
ND
ND
299±18.5
63.8±4.91
21
1-Nonanal
57
1361
1121
ND
ND
ND
ND
ND
ND
ND
106±4.78
42.6±2.11
22
Benzaldehyde
77
1497
978
83.7±4.36
18.4±0.960
13.5±0.370
23.8±1.94
8.50±0.820
18.6±1.36
57.1±2.73
74.0±4.58
28.3±1.61
23
(E,E)-2,4-Heptadienal
81
1503
996
ND
13.6±1.37
14.4±1.38
ND
22.5±2.12
ND
ND
106±9.76
42.6±2.47
24
(E)-2-Nonenal
70
1538
1195
ND
ND
ND
ND
ND
ND
ND
70.2±6.12
21.3±1.13
25
(E)-2-Decenal
70
1601
1260
ND
ND
ND
ND
ND
ND
ND
87.7±5.46
21.3±1.45
26
3-(Methylthio)-1-propanal
48
1620
1120
74.9±5.85
57.9±4.66
6.80±0.440
96.5±6.58
8.90±0.620
28.3±1.68
121±11.5
18.1±1.22
27.2±0.940
27
2-Phenylethanal
91
1625
1036
2.69e3±47.4
55.2±2.51
107±8.95
1.11e3±87.2
31.4±2.76
394±18.3
1.20e3±58.7
74.6±5.18
119±9.53
28
(E,E)-2,4-Nonadienal
81
1710
1185
ND
ND
ND
ND
ND
ND
ND
35.4±1.81
21.3±1.23
29
(E,E)-2,4-Decadienal
81
1775
1269
238±2.71
57.6±1.27
38.2±2.17
119±4.14
74.8±2.47
45.1±1.51
35.3±1.47
1.02e3±69.0
152±8.40
30
4-Methoxybenzaldehyde
77
2049
1457
204±12.3
160±10.4
13.7±1.63
ND
83.3±7.19
106±8.34
ND
31.3±1.35
ND
10
Subtotal (%)
2.24
0.62
0.81
1.23
0.27
0.46
0.55
1.87
0.53
Ester (18)
31
Ethyl butanoate
71
1028
817
893±67.4
109±4.87
88.3±6.81
375±26.2
141±9.89
1.64e3±44.0
31.2±2.61
140±11.3
21.2±1.09
32
Ethyl 2-methylbutanoate
102
1112
868
135.1±9.31
ND
ND
92.8±5.90
ND
137±11.2
31.2±2.72
17.5±1.06
ND
33
Ethyl hexanoate
88
1239
1028
2.49e3±122
153±10.9
137±9.12
650±35.3
345±21.7
439±21.7
688±31.6
929±56.3
617±23.5
34
Ethyl heptanoate
88
1304
1115
65.1±4.88
19.1±0.850
26.4±1.59
35.4±2.82
25.8±1.15
30.3±2.34
31.8±2.87
123±10.7
45.3±2.46
35
Ethyl 2-hydroxypropanoate
45
1328
823
392±22.3
84.3±6.53
140±9.69
275±19.9
224±17.1
60.3±4.54
127±9.41
312±21.2
257±12.6
36
Ethyl octanoate
88
1413
1203
2.6e3±18.9
224±8.49
238±16.8
866±58.6
369±16.2
530±29.0
1.41e3±34.8
1.54e3±88.1
447±24.8
37
Ethyl nonanoate
88
1512
1289
ND
ND
ND
33.1±1.75
23.8±1.36
34.1±2.43
62.9±4.98
20.1±1.05
42.3±3.23
38
Ethyl decanoate
88
1602
1398
122±10.9
28.6±1.15
17.3±1.35
72.5±5.51
11.9±1.81
75.7±5.84
125±10.8
263±12.7
21.4±1.13
39
Diethyl succinate
101
1657
1170
34.6±2.56
ND
ND
30.6±2.44
ND
ND
62.5±3.39
ND
ND
40
Ethyl phenylacetate
91
1810
1271
141±12.4
ND
ND
61.3±4.47
ND
45.4±2.29
62.5±4.66
ND
ND
41
Ethyl dodecanoate
88
1819
1589
80.8±6.62
28.6±1.57
17.2±1.43
105±9.47
42.5±2.60
66.2±4.95
136±11.8
1.27e3±49.3
41.6±2.53
42
Ethyl 3-phenylpropionate
104
1868
1348
583±33.2
25.1±1.74
20.4±1.32
338±25.8
11.9±0.520
15.1±1.12
31.2±2.98
211±18.4
19.6±1.42
43
Ethyl tetradecanoate
88
2047
1790
398±21.3
355±15.4
261±18.5
785±26.8
488±25.5
545±24.8
1.84e3±18.5
1.39e3±15.3
447±26.7
44
Ethyl hexadecanoate
88
2252
1946
3.25e4±116
2.13e4±227
2.30e4±78.5
4.34e4±54.9
5.86e4±61.4
3.49e4±49.3
7.91e4±68.7
3.96e4±44.9
3.75e45±54.1
45
Ethyl heptadecanoate
88
2310
2108
262±18.6
205±11.7
287±18.7
120±9.23
20.9±1.41
333±21.2
294±16.6
141±11.6
362±21.4
46
Ethyl octadecanoate
88
2426
2216
3.25e4±28.4
2.74e3±69.9
4.22e3±50.8
3.71e3±45.9
3.43e3±28.6
4.53e3±31.3
7.09e3±58.4
4.12e3±26.9
5.77e3±29.0
11
47
Ethyl (Z)-9-octadecenoate
264
2440
2301
1.38e3±12.6
1.67e3±19.1
74.9±3.77
1.38e3±18.9
1.70e4±88.1
2.37e4±75.8
6.31e4±46.3
1.88e3±16.67
3.83e4±86.2
48
Ethyl (Z,Z)-9,12-octadecadienoate
67
2499
2387
5.71e4±44.6
5.89e4±64.5
6.04e4±69.9
5.58e4±86.3
4.60e4±65.2
5.15e4±73.7
1.11e5±53.5
6.67e4±57.6
9.22e4±128
88.5
94.0
94.9
93.4
94.6
88.3
96.8
94.4
97.1
Subtotal (%)
Ketones (5)
49
3-Penten-2-one
69
1123
735
253±18.3
ND
ND
76.9±3.17
ND
45.5±2.17
93.7±6.05
ND
ND
50
2-Heptanone
58
1185
776
298±15.5
92.3±6.94
73.6±4.12
46.1±2.68
35.7±2.05
45.5±2.59
93.7±5.56
246±12.0
63.3±3.47
51
3-Octanone
57
1258
835
57.8±3.37
11.8±1.04
12.1±1.05
15.8±1.08
11.9±1.04
15.5±0.630
21.2±1.08
12.5±1.03
18.7±1.02
52
3-Hydroxy-2-butanone
45
1295
754
155±12.7
133±9.04
177±10.5
108±7.87
6.90±0.0800
90.1±5.09
93.7±6.05
140±11.1
149±9.03
53
2-Nonanone
58
1376
1087
268±15.8
17.0±0.910
24.2±0.790
30.7±2.24
13.5±0.970
15.6±1.05
62.5±3.84
26.3±1.41
21.1±1.06
0.69
0.27
0.31
0.24
0.05
0.16
0.13
0.34
0.14
Subtotal (%)
Acid (5)
54
Hexanoic acid
60
1841
984
23.1±1.74
30.1±2.63
18.1±1.03
17.7±1.18
25.4±1.74
20.8±1.31
15.5±0.830
19.1±0.790
30.5±1.64
55
Octanoic acid
60
2051
1179
12.5±1.03
15.6±1.04
10.2±0.580
16.2±1.04
20.8±1.05
11.6±0.680
18.5±1.02
9.90±0.0700
17.1±0.840
56
Hexadecanoic acid
73
2931
1975
141±9.75
1.23e3±18.6
645±24.6
801±21.2
322±18.4
125±9.14
719±54.0
140±10.1
1.23e3±26.8
57
(Z,Z)-9,12-Octadecadienoic acid
67
3067
2170
70.7±4.04
38.3±2.08
48.3±2.08
46.1±2.23
35.1±2.07
121±9.37
93.7±5.04
70.1±3.06
38.4±2.09
58
(Z,Z,Z)-9,12,15-Octadecatrienoic acid
79
3276
2325
ND
82.2±5.20
30.7±1.39
ND
ND
ND
121±9.80
31.2±2.08
ND
0.17
1.53
0.81
0.76
0.3
0.21
0.35
0.22
0.73
Subtotal (%)
Pyrazines (5)
12
59
2-Methylpyrazine
94
1267
831
167±12.6
18.7±0.870
27.3±1.17
59.9±3.08
20.6±1.04
30.3±2.06
126±11.7
35.0±2.18
13.2±1.01
60
2,6-Dimethylpyrazine
108
1327
906
186±9.69
13.0±1.08
13.2±1.07
41.2±2.24
9.40±0.480
34.3±1.43
93.5±4.55
12.3±0.980
16.2±1.17
61
2,3-Dimethylpyrazine
67
1345
928
88.3±4.05
65.6±3.76
27.6±1.39
20.4±1.05
13.1±1.08
45.4±2.01
37.3±2.17
31.5±2.01
12.6±0.940
62
2,3,5-Trimethylpyrazine
122
1401
1018
ND
24.7±1.14
20.6±1.08
ND
47.9±2.95
75.7±4.37
ND
ND
ND
63
2-Ethyl-3,5-dimethylpyrazine
135
1456
1085
ND
897±24.9
573±15.8
ND
ND
ND
ND
ND
ND
0.3
1.11
0.71
0.11
0.07
0.14
0.09
0.06
0.02
Subtotal (%)
Furans (4)
64
2-Pentylfuran
81
1236
977
ND
160±9.08
133±10.1
ND
ND
ND
ND
ND
ND
65
Furfural
96
1459
852
213±15.1
ND
12.4±1.01
246±15.7
21.6±1.03
60.6±4.85
250±11.8
ND
21.3±1.16
66
5-Methylfurfural
110
1572
984
ND
ND
ND
16.9±1.06
ND
24.3±1.08
52.8±3.37
26.1±1.59
ND
67
Furfurol
96
1684
851
431±21.2
76.4±3.12
159±9.36
692±25.4
80.6±3.78
167±11.8
532±25.3
290±14.1
448±21.0
0.43
0.26
0.33
0.83
0.08
0.19
0.31
0.25
0.26
Subtotal (%)
Phenols (4)
68
Phenol
94
2014
991
573±34.6
26.4±1.34
19.5±1.23
249±24.3
86.3±5.71
52.2±3.92
10.2±0.610
18.5±1.64
31.7±2.12
69
4-Ethyl-2-methoxyphenol
137
2041
1297
204±16.6
32.3±2.82
259±16.5
81.5±2.94
488±16.7
101±8.58
41.6±2.19
178±12.5
95.7±6.42
70
2-Methoxy-4-(2-propenyl)phenol
103
2064
1465
259±90.2
35.5±2.45
31.6±2.87
734±21.9
216±18.3
2.94e3±189
1.41e3±112
144±11.9
68.0±5.89
71
2-Methoxy-4-vinylphenol
135
2188
1345
67.2±16.5
19.5±6.38
26.9±2.61
810±35.4
41.1±3.96
98.3±7.43
838±56.8
148±11.6
49.1±2.31
0.74
0.12
0.36
1.62
0.62
2.38
0.84
0.39
0.14
Subtotal (%)
13
Miscellaneous (4)
72
1,8-Cineole
81
1212
1031
ND
ND
ND
61.5±4.37
47.6±2.77
107±9.52
147±11.0
ND
ND
73
Styrene
78
1261
875
125±10.3
28.1±1.07
26.1±1.72
46.1±2.26
23.8±1.08
30.3±2.59
93.5±4.71
141±8.53
45.5±2.96
74
(E)-Anethole
148
1835
1024
363±12.2
13.1±1.4
15.2±1.03
120±9.49
4.68e3±23.4
8.27e3±57.1
66.8±1.01
1.21e3±67.5
21.1±1.13
75
2-Acetylpyrrole
94
1978
1051
574±23.0
25.6±2.21
11.6±1.05
30.7±2.93
14.2±0.950
45.4±2.25
81.8±6.80
52.3±2.24
21.2±1.02
0.71
0.07
0.06
0.22
3.56
6.3
0.14
1.12
0.05
Subtotal (%)
A Identification
B Retention
C Mean
method: MS, mass spectrum; Std, confirmation by authentic standards; RI, retention index.
indices determined on HP-5ms and HP-INNOWAX capillary column, respectively.
± standard deviation (average of the triplicate). ND: not detected.
14
Table S4. Odour Activity Value (OAV) of Aroma-Active Compounds in Commercial Sufus.
OAVB
No.
Compound
OTH (μg/L)
A
CS1
CS2
CS3
CS4
CS5
CS6
CS7
CS8
CS9
1.5C
ND
75.5
322.3
ND
15.9
ND
20.9
46.7
70.9
17
2-Methylpropanal
18
1-Hexanal
5C
8.2
14.7
10.8
13.9
18.3
6.7
15.6
68.1
42.6
21
1-Nonanal
1.1C
ND
ND
ND
ND
ND
ND
ND
96
38.7
23
(E,E)-2,4-Heptadienal
15.4C
ND
<1
<1
ND
1.5
ND
ND
6.9
2.8
24
(E)-2-Nonenal
0.69D
ND
ND
ND
ND
ND
ND
ND
101.7
30.9
25
(E)-2-Decenal
0.4D
ND
ND
ND
ND
ND
ND
ND
219.3
53.4
26
3-(Methylthio)-1-propanal
0.45D
166.4
128.7
15.1
214.4
19.8
62.9
268
40.2
60.4
28
(E,E)-2,4-Nonadienal
0.19D
ND
ND
ND
ND
ND
ND
ND
186.3
112.1
29
(E,E)-2,4-Decadienal
0.077D
3092.2
748.1
496.1
1539
971.4
585.7
458.4
13253.2
1972.7
30
4-Methoxybenzaldehyde
47D
4.3
3.4
<1
ND
1.8
2.3
ND
<1
ND
31
Ethyl butanoate
0.9E
991.7
120.6
98.1
416.2
156.1
1818.1
34.7
155.9
23.6
32
Ethyl 2-methylbutanoate
0.2C
675.5
ND
ND
464
ND
683
156
87.5
ND
33
Ethyl hexanoate
2.2E
1131
69.7
62.2
295.6
157
199.7
312.5
422.4
280.5
34
Ethyl heptanoate
1.9E
34.3
10.1
13.9
18.6
13.6
15.9
16.7
64.6
23.8
36
Ethyl octanoate
19.3E
134.7
11.6
12.4
44.9
19.1
27.5
72.9
80
23.2
15
5E
24.3
5.7
3.5
14.5
2.4
15.1
25.1
52.6
4.3
Ethyl (Z)-9-octadecenoate
870F
1.6
1.9
<1
1.6
19.6
27.2
72.5
2.2
44
48
Ethyl (Z,Z)-9,12-octadecadienoate
450F
126.9
130.9
134.3
124.1
102.1
114.5
247.8
148.2
204.9
6
3-Methyl-1-butanol
4E
1118.8
30.1
40.3
160.2
24.8
9.2
77.6
56.4
28.6
8
2-Heptanol
65.235E
3.9
<1
<1
<1
<1
<1
1.6
<1
<1
9
1-Hexnaol
5.6E
77.4
17.6
15
14.4
1.2
20.3
26.1
5.6
62.6
10
3-Octanol
35C
7.7
<1
<1
<1
<1
<1
1.6
1.2
<1
11
1-Octen-3-ol
1.5E
273.2
597.9
382.1
103.5
81.5
91.3
99.1
197.7
234.1
49
3-Penten-2-one
1.5G
168.7
ND
ND
51.3
ND
30.3
62.5
ND
ND
51
3-Octanone
21.4E
2.7
<1
<1
<1
<1
<1
1
<1
<1
60
2,6-Dimethylpyrazine
157.6C
1.2
<1
<1
<1
<1
<1
<1
<1
<1
63
2-Ethyl-3,5-dimethylpyrazine
7.5D
ND
119.5
76.4
ND
ND
ND
ND
ND
ND
69
4-Ethyl-2-methoxyphenol
89.25E
2.3
<1
2.9
<1
5.5
1.1
<1
2
1.1
70
2-Methoxy-4-(2-propenyl)phenol
19D
13.6
1.9
1.7
38.6
11.4
154.9
74.1
7.6
3.6
71
2-Methoxy-4-vinylphenol
12.02E
5.6
1.6
2.2
67.4
3.4
8.2
69.7
12.3
4.1
54
Hexanoic acid
3H
7.7
10
6
5.9
8.5
6.9
5.2
6.4
10.2
55
Octanoic acid
3H
5.2
3.4
5.4
6.9
3.9
6.2
3.3
5.7
5.2
64
2-Pentylfuran
5.8E
ND
27.6
22.9
ND
ND
ND
ND
ND
ND
38
Ethyl decanoate
47
16
74
(E)-Anethole
75D
4.8
<1
<1
1.6
62.4
110.3
<1
16.1
<1
72
1,8-Cineole
4.6D
ND
ND
ND
13.4
10.3
23.2
31.8
ND
ND
A Odour
thresholds value in water were taken from references.
B
OAV was calculated by dividing the concentration by the respective odour threshold in water.
C
(Giri, Osako, Okamoto, et al. 2010)
D
(Czerny et al. 2008)
E
(Giri, Osako and Ohshima 2010)
F (Chung
G (Pino
H
et al. 2005)
2014)
(Buttery et al. 1999)
ND: not detected
17
Table S5. Aroma-active volatile compounds (FD ≥ 1) detected in nine commercial sufus.
FD factor
No.
CompoundA
Odour descriptorsB
Average FDC
CS1
CS2
CS3
CS4
CS5
CS6
CS7
CS8
CS9
74
(E)-Anethole
Aniseed-like
48.0
8
1
1
4
128
256
1
32
1
48
Ethyl (Z,Z)-9,12-octadecadienoate
Fatty
39.1
32
64
64
32
32
32
32
32
32
70
2-Methoxy-4-(2-propenyl)phenol
Clove-like
26.4
16
2
2
32
16
128
32
8
2
27
2-Phenylethanal
Honey-like, floral
22.9
128
4
8
8
2
16
32
4
4
36
Ethyl octanoate
Sweet, apple-like
14.2
32
8
8
16
8
8
8
32
8
29
(E,E)-2,4-Decadienal
Fatty, deep-fried
12.3
16
4
4
8
4
2
1
64
8
33
Ethyl hexanoate
Fruity, sweet
11.6
32
4
4
16
8
8
8
16
8
63
2-Ethyl-3,5-dimethylpyrazine
Roasted
10.7
ND
64
32
ND
ND
ND
ND
ND
ND
11
1-Octen-3-ol
Mushroom-like, grassy, fatty
9.00
8
32
16
4
2
2
1
8
8
16
2-Phenylethanol
Floral, honey-like
8.33
16
4
2
4
1
32
8
4
4
71
2-Methoxy-4-vinylphenol
Clove-like, smoky
7.44
2
1
1
32
2
4
16
8
1
18
69
4-Ethyl-2-methoxyphenol
Smoky, gammon-like
3.44
4
1
8
2
8
2
ND
4
2
65
Furfural
Roasted
2.67
8
ND
1
8
1
2
4
ND
ND
26
3-(Methylthio)-1-propanal
Cooked potato-like
2.67
4
4
ND
8
ND
2
4
1
1
6
3-Methyl-1-butanol
Malty
2.67
16
1
1
4
ND
ND
1
1
ND
31
Ethyl butanoate
Fruity, apple-like
2.00
4
1
1
2
1
8
ND
1
ND
41
Ethyl dodecanoate
Fruity, floral
1.89
ND
ND
ND
1
ND
ND
ND
16
ND
59
2-Methylpyrazine
Roasted, popcorn-like
1.89
8
1
1
2
1
1
2
1
ND
23
(E,E)-2,4-Heptadienal
Moldy, mushroom-like
1.67
ND
1
1
ND
1
ND
ND
8
4
34
Ethyl heptanoate
Fruity, pineapple-like
1.44
2
1
1
1
1
1
1
4
1
35
Ethyl 2-hydroxypropanoate
Fruity, cream-like
1.11
2
1
1
2
1
ND
ND
2
1
53
2-Nonanone
Green, floral
1.00
4
1
1
1
ND
ND
1
1
ND
60
2,6-Dimethylpyrazine
Roasted, nutty
0.778
4
ND
ND
1
ND
1
1
ND
ND
42
Ethyl 3-phenylpropionate
Fruity, floral
0.778
4
ND
ND
2
ND
ND
ND
1
ND
30
4-Methoxybenzaldehyde
Aniseed-like
0.667
2
2
ND
ND
1
1
ND
ND
ND
19
17
2-Methylpropanal
Rancid
0.556
ND
1
4
ND
ND
ND
ND
ND
ND
38
Ethyl decanoate
Fruity, coconut-like
0.444
1
ND
ND
1
ND
ND
ND
2
ND
28
(E,E)-2,4-Nonadienal
Fatty, green
0.333
ND
ND
ND
ND
ND
ND
ND
2
1
A
The compound was detected based on the result of GC-MS identification analysis.
B
Odour descriptors were perceived by panelists during olfactometry.
C
Mean values of determined FD factors of odorants in nine commercial sufus.
ND: not detected
20
Table S6. Omission experiments from the complete aroma model of sample CS1.
No.
1
Odorants omitted
SignificanceA
All esters except ethyl (Z,Z)-9,12-octadecadienoate
***
1A
Ethyl butanoate
**
1B
Ethyl 2-methylbutanoate
**
1C
Ethyl hexanoate
**
1D
Ethyl octanoate
*
1E
Ethyl (Z,Z)-9,12-octadecadienoate
*
All alcohols
**
2A
3-Methyl-1-butanol
NS
2B
1-Hexanol
*
2C
1-Octen-3-ol
*
3
All aldehydes
**
3A
3-(Methylthio)-1-propanal
*
3B
(E,E)-2,4-Decadienal
**
3C
1-Hexanal
*
3-Penten-2-one and 3-octanone
*
3-Penten-2-one
*
All phenols
**
5A
2-Methoxy-4-(2-propenyl)phenol
*
6
Hexanoic acid and octanoic acid
NS
2
4
4A
5
21
A
7
2,6-Dimethylpyrazine
**
8
(E)-Anethole
*
Significance: ***, 0.001 significant level; **, 0.01 significant level; *, 0.05 significant
level; NS: no significant difference.
22
alcoholic
10
vegetable
edible oil
8
6
4
2
sweet spices
fermented
0
Maillard
floral
herbs
fruity
original sample
model 1
model 2
Figure S1. Sensory evaluation of CS1 and aroma recombination model based on OAV calculation and GC-O/AEDA analysis. Model 1 was OAV-based model and
model 2 was GC-O/AEDA-based model.
23
(A)
24
(B)
Figure S2. PCA plots for the 35 key odorants (Table S4) in 9 different sufu samples on PC 1-PC 2 (A) and PC 1-PC 3 (B). The sample codes were defined in Table S1.
The GC-MS data and codes of aroma-active compounds were both shown in Table S3.
25
Reference
Buttery RG, Orts WJ, Takeoka GR, Nam Y. 1999. Volatile flavor components of rice cakes. J Agric
Food Chem. 47:4353-4356.
Chung HY, Fung PK, Kim JS. 2005. Aroma Impact Components in Commercial Plain Sufu. J Agric
Food Chem. 53:1684-1691.
Czerny M, Christlbauer M, Christlbauer M, Fischer A, Granvogl M, Hammer M, Hartl C, Hernandez
N, Schieberle P. 2008. 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 Technol. 228:265-273.
Fan W, Xu Y, Jiang W, Li J. 2010. Identification and quantification of impact aroma compounds in 4
nonfloral Vitis vinifera varieties grapes. J Food Sci. 75:S81-88.
Feng Y, Su G, Zhao H, Cai Y, Cui C, Sun-Waterhouse D, Zhao M. 2015. Characterisation of aroma
profiles of commercial soy sauce by odour activity value and omission test. Food Chem.
167:220-228.
Giri A, Osako K, Ohshima T. 2010. Identification and characterisation of headspace volatiles of fish
miso, a Japanese fish meat based fermented paste, with special emphasis on effect of fish species and
meat washing. Food Chem.120:621-631.
Giri A, Osako K, Okamoto A, Ohshima T. 2010. Olfactometric characterization of aroma active
compounds in fermented fish paste in comparison with fish sauce, fermented soy paste and sauce
products. Food Res Int.43:1027-1040.
Han B-Z, Rombouts FM, Nout MJR. 2001. A Chinese fermented soybean food. Int J Food
Microbiol.65:1-10.
Pino JA. 2014. Odour-active compounds in papaya fruit cv. Red Maradol. Food Chem. 146:120-126.
Schieberle P. 1995. Characterization of food: emerging methods. Amsterdam: Elsevier Science B.V.
Chapter 17, New developments in methods for analysis of volatile flavor compounds and their
precursors; p. 403-431.
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