Vol.4, No.5B, 116-121 (2013) Agricultural Sciences
The development of soy sauce from organic soy bean
Shoupeng Wan, Yanxiang Wu, Cong Wang, Chunling Wang, Lihua Hou*
Key Laboratory of Food Nutrition and Safety(Tianjin University of Science & Technology), Ministry of Education, Tianjin, China;
*Corresponding Author: lhhou@tju.edu.cn
Received 2013
Soy sauce with a high salt liquid-state fermenta-
tion process was prepared by using organic soy
beans as raw material. Beidahuang organic soy
bean was selected among different raw materi-
als. Here, the best technique was determined.
Firstly, the organic soy beans were soaked for 7
hours under 121, steamed for 15 min and
mixed with the fried wheat (5:5, w/w). After in-
oculated with Aspergillus oryzae 3.042 (0.3%,
w/w) and cultured for 36 hours, the koji was ob-
tained. When the brine (2:1, w/w) was added,
fermentation started. At the end of the fermenta-
tion, is of lavone content of organic soy sauce
was 0.22 mg-1 higher than those in the non-
organic soy beans. In addition, compared to the
control there were a higher unsaturated fatty
acids content, the linoleic acid content in crude
fat of 51.61% and γ-linolenic acid content in crude
fat of 0.55%.
Keywords: Organic Soy Bean; Soy Sauce;
Fermentation; Fatty Acid; Isoflavone
Soy sauce is a necessary seasoning in many Asian
countries. As a condiment and coloring agent, it almost
appears in every meal on the table. The annual produc-
tion of soy sauce in China is more than 5,000,000 tons,
accounting for over 55% of the world production [1].
The raw material of the soy sauce plays a very important
role to the quality of the soy sauce including the flavor,
the safety, the nutrition et al [2]. As the development of
society and the economy, more and more people pay
attention to food nutrition and health problems. There-
fore, organic foods are more and more popular.
Soy sauce is produced by fermentation of steamed
soybean and raw wheat with Aspergillus oryzae [3]. Or-
ganic soybean contains more high quality protein, higher
unsaturated fatty acid content and more balanced nutri-
tion collocation, belongs to the high-end soybean prod-
ucts, can meet the high consumption demand with high
food quality [4]. Soybean isoflavone is an important or-
ganic soybean functional component. Soybean isofla-
vone is the best natural anti-cancer substances [5], and
soybean isoflavone can significantly reduce blood cho-
lesterol level, estrogen acting synergistically [6]. Besides,
for rich in biological active substance, organic soybean
fermentation food had many unique functions, such as
anti-cancer, dissolve thrombus, antioxidant, fall blood
pressure and antibacterial, etc [7]. Organic soy bean had
not been large-scale developed for the valuable resources,
resulting in the decrease of the economic value of the
organic soy beans and the enthusiasm of planting organic
soy beans.
To meet the needs of health, organic soy bean was
used as materials to produce the soy sauce in this study.
Using high-salt diluted state fermentation process, a
high-quality organic soy bean soy sauce was obtained.
2.1. Preparation of Materials and Chemicals
Organic soybean (origin, the northeast China) and the
non-organic soybean (origin, the northeast China) pro-
vided from the marked. Fatty acid standards, include
methyl stearate, methyl oleate, methyl palmitate, methyl
γ-linoleate, methyl linolenate, were purchased from
Sigama-Aldrich (Shanghai) trading Co. Ltd. Isolavone
standard (Genistein), were purchased from Beijing
Dingguo biological Technology Co., Ltd (China). All
other chemicals used were of analytical grade.
2.2. Preparation of Seed Koji
To prepare seed koji, wheat bran was first blend with
100% hot water (w/w) and cooked at 121 for 30 min.
The cooked wheat bran was inoculated with A. oryzae
spores and incubated at 30 for 3 days, then dried in a
drying cabinet at 60and was grounded into powders.
The germination rate and the spore count were deter-
mined according to the Chinese National Standard.
2.3. Preparation of Koji
Cooked Organic soy beans were mixed with fried
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S. P. Wan et al. / Agricultural Sciences 4 (2013) 116-121 117
wheat which had been soaked in water (1:1, w/w) for 30
min previously, quickly blend and cooled to a tempera-
ture of about 35, then inoculated with a certain amount
of A. oryzae seed koji. The initial culture temperature
was 30 ± 2. After a period of time, the culture tem-
perature was adjusted to 25.
2.4. Fermentation Process and Preparation
of the Samples
The resulting koji was fermented in 18% to 20% brine
solution at a ratio of 2:1 (Brine: Raw material, w/w) to
yield soy sauce mash (brine fermentation or soy sauce
mash fermentation process) [8]. At first, the cultivate
temperature was controlled at 15, and then increased
1 per day until to the temperature of 30. After fer-
mentation of 6 month, the ripened soy sauce mash was
pressed to yield soy sauce. At various time intervals of
the fermentation process, samples of 100 g soy sauce
mash were taken from each of the mash tanks containing
the same type of koji. They were stirred individually and
centrifuged at 8000 rpm for 10 min. The supernatants
were filtrated through Whitman no.3 paper. The filtrate,
regarded as raw soy sauce, was placed in brown bottles
and kept at 4 [9]. For each analysis, tests were per-
formed three times and all results presented are the av-
erage. As the control, the soy sauce derived from soy-
bean was produced under the same conditions.
2.5. Parameters Analysis
Determination of the quality parameters of soy sauces
total nitrogen and total solids were determined by AOAC
methods 991.20 and 990.2 (AOAC, 2000), respectively.
Salt-free soluble solids were calculated as TS minus SC
(GB18186-2000, 2001). Amino nitrogen content was
measured as described by the Chinese National Standard.
Determination of the fatty acids contents were by High
Performance Liquid Chromatography.
The fatty acids were detected by Gas chromatograph
[10]. 1.0000 g sample, 100 mL mixed solution of chlo-
roform and methanol (V/V) was added, putted in the
water bath shaker (New Brunswick Scientifics C24, jin-
tan, China) at 38 for 14h, filtered by the chronic filter
paper. After the rotary evaporation (55), add 10 ml 0.5
mol/L NaOH : ethanol, kept in the water bath at 60 for
40 min. Remove the unsaponifiable matter with a sepa-
rating funnel by adding 10 mL aether twice. Then adjust
the pH to 2-3 with 10% solution of hydrochloric acid,
add 5 ml normal saline, extracted by 10 ml hexane, take
the upper, using ammonia stripping method remove the
solvent hexane, so we get the fatty acid. the GC2010
(Shimadzu Co.), FID detector, chromatographic column:
CBP - 20 capillary column (50 m, 0.25 mm, 0.25 um),
Initial temperature: 180, temperature rising to 240
at a speed of 6 /min and keep 40 min, Injection port
temperature: 280, Detector temperature: 280. Car-
rier gas: high purity nitrogen, Velocity 30 ml/min, and
the air velocity: 400 ml/min, Hydrogen flow rate: 47
ml/min, sample size: 1 μl [11].
The production processes of fermented soy sauce con-
sisted of four major steps, including raw material selec-
tion, koji production, brine fermentation and refining
3.1. Selection of Raw Materials
In this study, organic soy bean and non-organic soy
beans were compared: organic soy bean (HO) and non-
organic soy bean (HN). By comparing protein, crude fat,
crude starch, moisture, ash content and fatty acid compo-
sition, the differences of the basic components between
organic soy beans and non-organic soy bean were got.
Figure 1 showed that the higher protein content of or-
ganic soy beans varieties was HO; the higher ash content
of organic soy beans varieties was non-organic soy beans;
the higher crude starch content of organic soy beans va-
rieties was HO. Figure 2 indicated the content of un-
saturated fatty acid of HO was more than HN. In fer-
mentation process of the soy sauce, the level of the pro-
tein content was very important. In addition, the fatty
acids components in the organic soy beans exhibited a
higher unsaturated fatty acids content, the linoleic acid
content in crude fat of 51.61%, γ-linolenic acid content
in crude fat of 0.55%. Linoleic acid, α-Linolenic andγ-
Linolenic acid were the essential fatty acids of the human
body. Thereby, HO had adventages to be the raw materi-
als of fermented soy sauce.
3.2. Determination of the Cooking Process
The purpose of cooking was to make moderately de-
naturing of the raw materials, for the reason that easy to
Figure 1. Comparison of the various components of or-
ganic soy bean and non-organic soy bean.
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S. P. Wan et al. / Agricultural Sciences 4 (2013) 116-121
be used by A. oryzae and provide a basis for the later
enzymatic decomposition [13]. The moderate cooking
were needed due to effect obviously on the utilization of
raw material and quality of soy sauce. Whether the
cooking was appropriate depend on the soaking time,
cooking time and cooking temperature.
As seen from Figure 3(a), with the extension of
cooking time, the digestibility showed a rising trend, but
over 8 hours, digestibility declined. The reason may be
the water-soluble protein dissolution. Therefore, 7 hours
was selected as soaking time (a little different with the
seasons change). Figure 3(b) and Figures 3-7 indicated
that during the initial stage, digestibility was signifi-
cantly increased with the extension of the cooking time
and temperature. However, more than 25 minutes and
123 the digestibility began to decrease, which may be
due to excessive degeneration of the protein. Finally, 25
minutes and 123 was selected as cooking time and
cooking temperature.
Figure 2. Comparison of the fatty acids of the four kinds of or-
ganic soy beans, Peak No.1 respects Palmitic acid; Peak No.3
respects Stearic acid; Peak No.6 respects Linoleic acid; Peak
No.7 respects γ-Linolenic acid.
Soak ing t ime (h)
Digestibilit y (%)
15 20 25 30
Cook ing t ime (min)
Digestibility (%)
110 115121 126
Cooking t emperatu r e
Digestibility (%)
Figure 3. The different soaking beans time (a), cook-
ing beans time (b) and cooking beans temperature
(c), Data presented are the average of duplicate ex-
3.3. Determination of the Koji Process
The koji production process was the most important in
the fermentation of the soy sauce. At this stage, the A.
oryzae grew to a large number, and produced a complex
enzyme system. The proportion of raw materials, koji
duration, the amount of seed koji, moisture, humidity and
temperature were the main factors that affected the pro-
duction of enzymes. This study investigated the rela-
tionship between the enzyme activity and the amount of
seed koji, proportion of raw materials and the duration
time of koji, respectively, in order to determine the best
process [14].
Standard of high-quality of seed koji was germination
rate > 90%, spore count >109 (A·g-1) [15]. The results of
the amount of the seed koji were observed from Figure
4(a). With the increase of the amount of the seed koji, the
protease activity was significantly increased but once
more than a certain numerical, protease activity declined.
This may be because the seed koji was excessively added
to the raw material, resulted in inadequate nutrition and
lack of space. The growth and reproduction of the A.
oryzae was affected, leading to the decline of protease
activity. Therefore, the added amount of the koji was
0.3% (w·w-1). Figure 4(b) demonstrated that when the
ratio of raw material was 5:5, the protease activity
reached a maximum. Meanwhile, the protease activity
attained the highest at 36 hours. Once more than 36
hours, A.oryzae began to pick spores and affect the gen-
eration of the protease.
3.4. Fermentation Process and Chemical
During the fermentation of soy sauce, proteins in the
raw materials were hydrolyzed into small molecular in-
cluding peptides and amino acids by the proteases pro-
duced from A.oryzae. The total nitrogen content was an
important indicator to measure soy sauce grade [16]. As
can be seen from Figure 5(a), the total nitrogen content
showed some fluctuations in the soy sauce fermentation
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S. P. Wan et al. / Agricultural Sciences 4 (2013) 116-121 119
process. Its content had been slow to improve as time
progresses. The total nitrogen content in the organic soy
bean sauce was higher than that in the non-organic soy
sauce. In addition, amino acid nitrogen can increase the
flavor of soy sauce and was an important quality indica-
tor of the soy sauce. Figure 5(b) indicated that the amino
acid nitrogen content increased dramatically and then
increased little. This may be because Maillard reaction
need amino and carboxyl [17]. Amino nitrogen content
was higher in the organic soy sauce than that in the non-
organic soy sauce.
Salt-free solids refer to various decomposition produc-
tions of soluble protein, such as amino acids, peptone,
peptides, dextrin, low molecular weight sugars, organic
acids, alcohols, esters, pigments and other substances. It
was one of the major nutrition matters in soy sauce.
There was a direct relationship between salt-free solids
content and quality of soy sauce. Figure 5(c) revealed
that organic soy sauce had a higher content.
3.5. Isoflavone Results
Isoflavone was a functional ingredient in soy beans.
The isoflavone was a water soluble organic substance
Annotation: 1 for the 5:5, 2 for the 4.5:5.5, 3 for the 6:4.
Figure 4. The relationship of the different inoculation amount
(a), the different ratio and different time of raw materials (Or-
ganic soy bean: wheat) and protease activity (b). Data presented
were the average of duplicate experiment.
with small molecule, have effectively anti-cancer func-
tion, anti-tumor function and prevent the cardiovascular
disease and the osteoporosis disease. Therefore, dietary
intake of soybeans can keep the body health and decrease
the risk of some disease and a number of cancers includ-
ing those of breast, colon and prostate. Figure 6(a)
showed the liquid chromatogram of standards, Peak No.1
respects the Daidzin, Peak No. 2 respects the Genistin
and Peak No.1 respects the Genistein, Figures 6(b)-(e)
showed the liquid chromatogram of different samples.
Figure 7 demonstrated the isoflavone content of the
organic soy beans was higher than that in the non-organic
soy beans, followed with the higher content in the or-
ganic soy sauce. The isoflavone content of organic soy-
bean group in the raw material was 3.81 mg﹒g-1, con-
0 15 30 45 60 75 90 105 120 135 150 165 180
Aging time
sa lt -free so lid mass
content (g/100mL)
Figure 5. Total nitrogen content (a), amino nitrogen con-
tent (b) and salt-free solids content (c). Data presented are
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S. P. Wan et al. / Agricultural Sciences 4 (2013) 116-121
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the average of duplicate experiments.
(a) (b) (c)
(d) (e)
Figure 6. Liquid chromatogram of the isoflavone standards (a), isoflavone in the organic soy beans (b), isoflavone in the non-organic
soy beans (c), isoflavone in the organic soy sauce (d), isoflavone in the non-organic soy sauce (e).
(a) (b)
Figure 7. Isoflavone content in the organic soy bean and non-organic soy bean(a), isoflavone content in the organic soy sauce and
non-organic soy sauce (b). Data presented are the average of duplicate experiments.
of the organic group soy sauce at the end of the fermen-
tation was 0.22 mg﹒g-1.
Openly accessible at
This work was supported by these projects in China (2012AA022108,
31000768, 2012BAD33B04, 2012GB2A100016, 2013AA102106,
IRT1166, 10ZCZDSY07000 and 31171731).
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