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J Sci Food Agric 79 :243–248 (1999)
Journal of Science of Food and Agriculture
Volatile components of the Chinese fermented
soya bean curd as affected by the addition of
ethanol in ageing solution
Chyong-Hs yuan Hwan and Cheng-Chun Chou*
Graduate Ins titute of Food Science and Technology , National Taiwan Univers ity , Taipei , Taiwan
Abstract : Two Chinese fermented soya bean curd products were prepared using Actinomucor taiwanensis and ageing in brine solution with or without ethanol. The volatile compounds in these Sufu
products were identiüed and quantiüed by GC and GC-MS. A total of 61 volatile compounds were
identiüed including 22 esters, 18 alcohols, 7 ketones, 3 aldehydes, 2 pyrazines, 2 phenols and 7 other
compounds from the 75-day aged Sufu products. Of the 61 identiüed compounds, 55 compounds were
found to exist simultaneously in the two Sufu products prepared. Quantitatively and qualitatively, less
volatile components were found in Sufu ageing without ethanol than those ageing with ethanol. In
general, as the ageing period extended, contents of nearly all the volatile fractions in Sufu, particularly ester and alcohol, increased. In addition, a signiücantly higher amount of ester was noted in Sufu
aged with ethanol than in that aged without ethanol.
( 1999 Society of Chemical Industry
Keywords : Chinese fermented soya bean curd ; volatile components ; Actinomucor taiwanensis ; ethanol
INTRODUCTION
The fermented product of soya bean curd, known as
Sufu or tofunyu, has existed in China for many centuries.1 It possesses a characteristic ýavour and a
relatively high protein content. Chinese people
consume it as an appetiser.
It is known that moulds such as Actinomucor spp,
Mucor spp or Rhizopus spp are involved in the fermentation.2,3 The manufacturing process generally
consists of three steps : preparation of soya bean curd
(tofu); moulding, and brining and ageing.1,4 During
the ageing period enzymes produced by moulds act
upon protein and lipid, the main constituents of soya
bean curd, yielding various degradation products.
The characteristic ýavour and aroma of Sufu gradually develop during the brining and ageing process,
although the freshly moulded soya bean curd is
bland in taste. It is a general practice to add rice
wine or ethanol to the brine solution in which the
moulded soya bean curd is soaked for ageing. The
added ethanol is believed to not only add a pleasant
odour to the product but also prevents the growth of
contaminating organisms.4 Sufu prepared in this way
is soft, pale yellow and has a salty and sweet and
meaty taste in addition to the distinctive pleasant
fruit odour and alcoholic fragrance. However, some
commercial Sufu products are supplemented with
other ingredients such as hot pepper, rose essence,
sesame oil, or red mash fermented with Monascus sp,
to enhance or modify the ýavour and/or colour of the
products.
The physicochemical and microbiological characteristics of Sufu have received extensive attention.5h9
However, data regarding the volatile constituents of
Sufu are still quite limited except for the investigation of Liu et al10 who described the aroma components of two Sufu products prepared with Mucor
sp. Since these products contained the added ýavour
additives such as red rice mash and sesame oil and
were collected from the local market, the data
obtained do not precisely reýect the aroma compounds formed during the fermentation. In the
present study, Sufu fermented with A taiwanensis,
the conventional starter in commercial Sufu preparation in Taiwan, were prepared. The volatile constituents formed during the fermentation were
identiüed and quantiüed. In addition, the impact of
added alcohol on the formation of volatile components during ageing was also investigated.
* Corres pondence to : Chen-Chun Chou, Graduate Ins titute of Food
Science and Technology, National Taiwan Univers ity, 59 Lane 144
Keelung Road, Sec. 4, Taipei, Taiwan
Contract/grant s pons or : National Science Council (Republic
of China)
Contract/grant number : NSC78-0409-B002-17
(Received 30 July 1997 ; revis ed vers ion received 15 April 1998 ;
accepted 8 May 1998 )
MATERIALS AND METHODS
Sufu fermentation
In this study, two kinds of Sufu fermented soya bean
curd products were prepared by using A taiwanensis
( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50
243
CH Hwan, CC Chou
as the starter and the ageing was in brine solution
with and without the addition of ethanol. Detailed
procedures for the fermentation are described in a
previous paper.9 Brieýy, tofu was purchased from a
tofu
processing
plant,
cut
into
pieces
(3.0 ] 3.0 ] 2.5 cm) and inoculated with the spore
suspension of the test organism and incubated at
30¡C and about 97% relative humidity for 48 h. At
this time the cubes were covered with white mycelium (pehtze). Twenty-four mould grown pehtzes
were each mixed with brine solution at a ratio of 2 : 3
(w/w) and placed into individual wide-mouthed glass
bottles. The bottles were then kept at 25¡C for 75
days. The brine solution contained 120 g litre~1
NaCl with or without the addition of 100 g litre~1
ethanol.
Isolation of volatiles
Essentially, the method described by Liu et al10 was
followed to isolate the volatiles from the Sufu products. Duplicate samples were taken at predetermined periods during ageing and all results presented
are the average of these. On each sampling day, two
bottles were drawn randomly from each treatment.
The ageing solution was decanted and the Sufu
pieces were homogenised separately with a Stomacher lab-blender 400 (Seward Laboratory, London,
UK) for 2 min to ensure the homogeneity and so
minimise experimental error. A total of 400 g Sufu
and 400 ml of distilled water was then blended for
1 min. The blended mixtures were immediately concentrated for vacuum distillation (45¡C, 20–
25 mmHg). Approximately 400 ml distillate was
collected by passing a 2¡C condenser. The distillate
were combined, saturated with NaCl and extracted
with dichloromethane three times. After the internal
standard (methyl phenyl acetate) was added, the
extract was preconcentrated with a distillate apparatus at 40¡C and carefully reconcentrated to approximately 50 ll by using a 10 cm ] 0.2 cm id Vigreux
column at 40¡C.
Capillary gas chromatography
A
CP-Wax
52
CB
Chrompack
column
(50 m ] 0.32 mm id, df \ 0.22 lm, bonded polyethylene glycol plase) was connected to a ýame injection
detector (FID) in a Varian 3400 gas chromatograph.
The column temperature was programmed as
follows : 60¡C held 4 min, raised to 190¡C at
2¡C min~1, held 21 min. The injector and detector
temperature both were 250¡C. Hydrogen carrier gas
was used at a linear velocity of 30 ml min~1. Sample
injection volume was 0.5 ll with a split ratio of 1/30.
Gas chromatograph/mass spectrometry (GC/MS)
A Hewlett-Packard 5840A gas chromatograph
equipped with the same capillary column as in the
GC analysis was connected directly into a quadrupole mass spectrometer (Hewlett-Packard 5985B MS
system). The column temperature was programmed
244
from 60¡C (4 min isothermal) to 190¡C at 2¡C min~1,
held 21 min. The carrier gas (He) was at the ýow
velocity of 30 ml min~1. The temperature of the ion
source and the connection parts were 200¡C. The
electron multiplier voltage was 2600 V. The electron
energy was 70 eV.
Component identification and quantitation
Retention indices were calculated for separated components relative to a C5-C25 n-alkanes (Alltech
Associates, Inc.) mixture.11 The identiücation was
carried out by comparing both the retention index of
sample and mass spectrum with those reported in the
literature.12,13 Quantitative determination was
accomplished by using an internal standard.
RESULTS AND DISCUSSION
Figure 1 illustrates the capillary gas chromatograms
of volatiles from the 75-day-old Sufu aged with or
without ethanol. Table 1 presents the identiüed compounds and quantitative analysis distribution.
Cumulatively, 61 volatile compounds were identiüed
including 22 esters, 18 alcohols, 7 ketones, 3 aldehydes, 2 pyrazines, 2 phenols and 7 other compounds.
Diþerences in both quantitative analysis and qualitative analysis were observed between Sufu products
aged with and without ethanol. For example, the 61
volatile compounds were identiüed in Sufu aged in
the presence of ethanol while 55 compounds were
identiüed from Sufu aged without ethanol. Compounds including ethyl n-dodecanoate, ethyl 3phenyl propionate, acetoin, butyl dimethyl
bicyclo-hexanone and 2,5 dimethyl pyrazine were
detected in Sufu ageing with ethanol while these
compounds were not present in Sufu ageing without
ethanol. A total content of identiüed compounds of
59.4 mg kg~1 dry weight was found in the former
while 43.3 mg kg~1 were detected in the latter Sufu
product.
In agreement with the investigation of Liu et al,10
esters and alcohols were the major volatile components in the 75-day aged Sufu product regardless
of the ageing solution used (Table 1). Qualitatively,
more alcohols and esters were identiüed in Sufu prepared with A taiwanensis in the present study than
from white sufu fermented with Mucor sp.10 Only 14
alcohols and 16 esters were detected from white Sufu
by Liu et al.10 Among the esters and alcohols
detected in the present study, 10 esters and 11 alcohols were also detected in white Sufu (Table 1).
Among the aroma compounds identiüed, ethyl
acetate, 2-butanone, ethyl propanate, 2-butanol, 2methyl 1-propanol, 2-methyl hexanol, acetoin, 2,5dimethyl pyrazine, ethyl butyrate, benzyl alcohol,
phenols and phenyl ethyl alcohol are commonly
found in the aroma constituent of some oriental fermented products eg soya sauce and miso.14h17
J Sci Food Agric 79 :243–248 (1999)
Volatile components of Chinese fermented soya bean curd
Figure 1. GC profile of volatile components in the 75 day aged products . (A) ageing without ethanol ; (B) ageing with ethanol.
Many of the volatile compounds identiüed possess
the characteristic of a fruit odour described by Arctander.18 For instance, ethyl propionate, 2pentanone, ethyl butyrate, ethyl benzoate exhibited
rum-like, banana-like, pineapple-like and ýoralfruity odour, respectively.
The pyrazines are typical components of the
cooking odour produced by the amino-carbonyl reaction. Limited amounts of alkylpyrazines have been
found in fermented soya cake due to the metabolic
activity of Aspergillus oryzae.19 They are also formed
in large quantities in natto, a J apanese fermented
product of soya bean with Bacillus oralto and were
regarded to contribute to the characteristic odour of
this product.20 However, in Sufu prepared in this
study using A taiwanensis as the starter and that fermented with Mucor sp,10 only small amounts of
pyrazines were detected. The contribution of these
pyrazines to the aroma of Sufu seems to be extremely
limited. Worthy of mention is that more than 12
pyrazine compounds were detected by Liu et al10
from white Sufu, with only 2 pyrazine compounds
were detected in the present study. Some of the
pyrazine compounds were identiüed in the volatile
component of sesame oil.21 Therefore, some of the
pyrazine compounds detected by Liu et al10 originated from sesame oil which was added to white
Sufu for enhancing and modifying the ýavour of
Sufu.
The presence of sulphur-containing compounds in
Sufu was limited both qualitatively and quantitatively although they were another important contributor to the characteristic odour of natto.20 Only one
J Sci Food Agric 79 :243–248 (1999)
sulphur-containing compound, 1-phenyl thio-3acetoxy 2-butanone (peak number 47) was detected
in the Sufu products prepared.
Flavour and aroma of Sufu developed during the
ageing process. The phenomenon is further demonstrated by the quantitative data shown in Fig 2. As
the ageing period extended, concentrations of almost
all the volatile fractions, particularly alcohol and
ester, increased. For instance, concentration of
alcohol and ester in Sufu ageing without ethanol
were 2.26 and 4.68 mg kg~1 dry weight, respectively,
after 15 days ageing. After a 75 day ageing period,
the concentration of alcohol and ester increased to
9.40 and 15.64 mg kg~1 dry weight, respectively. In
addition, during the ageing period, the increase of
ester fraction is also found to be signiücantly higher
in Sufu aged with ethanol than that in the Sufu aged
without alcohol. At the end of the 75 day ageing
period, Sufu aged with ethanol had an ester content
of 20.90 mg kg~1 dry weight, while Sufu aged
without ethanol had an ester content of only
9.40 mg kg~1 dry weight. The high amount of esters
may be attributed to the reactions of fatty acids,
digestion products of soya bean lipid, with the added
alcohol which occurred during the ageing process.4,9
Furthermore, a signiücant increase in the content of
ethyl esters of oleate, palmitate and linoleate, the
main fatty acid components of soya bean, was noted
in Sufu aged with ethanol (Fig 3). The total amount
of these esters was found to be 10.42 mg kg~1 which
contributes about 50% of the total ester detected in
the 75 day aged Sufu. It is, therefore, believed that
the signiücantly higher amount of alcohol and esters,
245
CH Hwan, CC Chou
Table 1. Volatile components of Sufu products a
Peak number
Components
RI c
MW
Amount (lg kg É1)d
ageing
without ethanol
ageing
with ethanol
Es ters
1
6
8
12
13
16
17
25
28
33
36
44
46
48
49
50
52
56
58
59
60
61
ethyl acetate
ethyl butyrate*e
ethyl 2-methyl butyrate*
is o-butyl pivalate
ethyl 2-butenoate
n -amyl formate
ethyl n -hexanoate
ethyl 2-hydroxy propanoate
ethyl heptanoate*
ethyl octanoate*
2-hydroxy 4-methyl pentanoate
ethyl benzoate*
2,4,4-trimethyl pentyl 3,5,5-trimethyl hexanoate
triethyl glycol butyl es ter
ethyl benzeneacetonate
ethyl n -dodecanote
ethyl 3-phenyl propionate
ethyl tetradecanoate*
ethyl palmitate*
ethyl s tearate*
ethyl oleate*
ethyl linoleate*
894
1025
1049
1085
1122
1187
1223
1312
1321
1423
1441
1647
1706
1753
1786
1822
1837
2029
2229
2389
2410
2505
88
116
130
158
114
116
144
118
158
172
146
150
270
206
164
228
178
256
284
312
310
308
149
1422
310
1625
209
1096
230
79
1584
216
81
67
568
500
110
Èf
È
81
89
592
284
109
114
1268
355
1525
1386
1396
318
381
165
317
152
63
915
394
114
890
89
98
3432
533
4703
2287
Alcohols
2
4
5
9
11
15
19
27
30
35
38
40
41
42
43
45
51
53
ethanol*
2-butanol*
n -propanol*
2-methyl 1-propanol*
n -butanol*
is o-amyl alcohol
acetoin*
n -hexanol*
3-octanol*
1-(2-ethoxypropoxy)-2-propanol
2-ethyl hexanol*
2,6-dimethyl 4-heptanol
n -octanol
2-decycloethanol
methyl 2-is opropenyl 4-hexanol
3-chloro-4-methyl 2-pentanol
benzyl alcohol*
phenyl ethyl alcohol*
900
1000
1002
1054
1113
1184
1236
1316
1380
1436
1458
1506
1519
1557
1578
1689
1826
1859
46
88
60
74
102
168
88
102
130
162
130
144
130
202
154
136
108
122
580
338
1354
352
416
1489
È
1015
1760
433
311
595
57
2803
149
2919
609
460
1398
1270
1260
305
394
1589
572
1004
1716
405
105
381
254
2542
165
1907
648
3686
Ketones
3
14
18
20
21
47
57
3-methyl 2-butanone
methyl is o-amyl ketone
6-methyl heptan 3-one
2-nonanone
cyclohexanone
1-phenylthio 3-acetoxy 2-butanone
butyl dimethyl bicyclo-hexanone
956
1140
1231
1244
1258
1727
2144
86
114
128
142
98
238
236
1489
1218
67
1488
1624
609
È
635
1118
1334
1359
1372
508
114
Aldehydes
10
22
39
n -hexanal
2-propyl hexanal
benzaldehyde
1084
1279
1502
100
98
106
31
669
2979
317
648
2796
246
J Sci Food Agric 79 :243–248 (1999)
Volatile components of Chinese fermented soya bean curd
Table 1. Continued
Peak number
Components
RI c
MW
Amount (lg kg É1)d
ageing
without ethanol
ageing
with ethanol
Pyrazines
23
24
2,5-dimethyl pyrazine
2,6-dimethyl pyrazine*
1306
1308
108
108
È
1760
1398
1817
Phenols
54
55
4-methyl di-tert-butyl phenol
phenol*
1878
1932
220
94
31
115
228
165
Mis cellaneous
7
26
29
31
32
34
37
toluene
2-oxabicyclo-octane
triethylene glycol butyl ether
1-butoxy 2-methoxy ethane
3-ethyl 2-methyl hexadiene
2-hexyl thiazolidine
acetic acid*
1031
1316
1345
1401
1415
1427
1450
92
112
206
132
124
160
60
119
3656
1218
568
176
È
392
34
3558
457
419
181
190
330
a Number refers to Fig 1
b Sufu aged for 75 days
c Retention Index us ing n -paraffin C5–C25 as references
d Average of duplicate s amples (by dry bas is )
e Components with as teris k were als o found in white Sufu10
f Not detected
especially ethyl esters, which formed during the
ageing of Sufu in the presence of ethanol may contribute to the relatively pleasant fruit odour and alcoholic fragrance noted in such Sufu compared with
that aged without alcohol.
In conclusion, data obtained in this study provide
qualitative and quantitative information regarding
the volatile compounds formed during the fermentation process of Sufu with A taiwanensis. Besides this,
the data also demonstrate the importance and the
Figure 2. Changes in concentration of various volatile fractions in
Sufu during the ageing proces s ; (A) ageing without ethanol ; (B)
ageing with ethanol. Ageing time : K, 15 days ; =, 45 days ; =, 75
days .
Figure 3. Changes in concentration of s ome es ters in Sufu during
the ageing proces s . (A) ageing without ethanol ; (B) ageing with
ethanol. Ageing time : K, 15 days ; =, 45 days ; =, 75 days .
J Sci Food Agric 79 :243–248 (1999)
247
CH Hwan, CC Chou
impact of added ethanol on the formation of aroma
compounds, particularly esters, during the ageing of
Sufu.
ACKNOWLEDGEMENTS
This research was supported by the National Science
Council (Republic of China) grant number NSC780409-B002-17. We are also indebted to Dr Wu CM,
Food Industry Research and Development Institute,
Hsinchu, Taiwan, for technical assistance.
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J Sci Food Agric 79 :243–248 (1999)
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