LWT - Food Science and Technology 74 (2016) 242e250

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LWT - Food Science and Technology

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Optimizing the initial moromi fermentation conditions to improve the

quality of soy sauce
Nguyen Xuan Hoang a, Sophia Ferng a, Ching-Hua Ting b, Wei-Hua Huang a,
Robin Yih-Yuan Chiou a, Cheng-Kuang Hsu a, *
a
Department of Food Science, National Chiayi University, Chiayi City 60004, Taiwan, ROC
b
Department of Mechanical and Energy Engineering, National Chiayi University, Chiayi City 60004, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Soy sauce is a traditional condiment consumed by the people in China, Korea, Japan and Taiwan. The
Received 17 April 2016 annual production is about 5 million metric tons in China, accounting for over 50% of the world pro-
Received in revised form duction. The objective was to optimize the process parameters for preparation of initial moromi
9 July 2016
fermentation of soy sauce, which was considered as an enzymatic hydrolysis for the further fermentation
Accepted 21 July 2016
Available online 25 July 2016
stage. Temperature (35, 40 and 45
C), fermentation time (1, 3 and 5 days) and brine content (5 and 10%
w/w) were studied as independent variables. The responses evaluated for deciding the optimum con-
ditions were biochemical properties of total nitrogen, amino nitrogen, reducing sugar and total acid
Keywords:
Soy sauce
contents, as well as microbial properties of yeast and total bacterial counts. Experimental data were fitted
Optimization well into second-order polynomial models. The simultaneously optimal conditions were 40.7
C tem-
Fermentation perature, 4.6 days fermentation time and 10% w/w brine content. The optimized response values for total
nitrogen, amino nitrogen, reducing sugar and total acid contents and pH were 1.43, 0.65, 2.79, and 1.28%
and 5.48, respectively. These conditions could obtain total nitrogen (>1.4%) and amino nitrogen (>0.56%)
contents satisfying the requirement of first-grade soy sauce within 5 days fermentation.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction manufacturing. Such a long aging time could be explained by two

Of the many Oriental fermented products, soy sauce is the one
most widely consumed in Asiatic countries as a condiment and
coloring agent in preparation of foods (Luh, 1995). In the
manufacturing process, soy sauce koji is traditionally prepared by a
mixture of steam-cooked soybean and roasted wheat with the koji
mold such as Aspergillus oryzae or A. sojae. After a couple of days of
this so-called solid-state fermentation, the resultant koji is mixed
with brine solution and the formed mash, called moromi, is fer-
mented to various periods of aging (Zhu & Tramper, 2013). During
the aging period, the proteins and polysaccharides of the starting
materials are hydrolyzed by the fungal protease and amylase, and
the unique soy sauce flavor is artfully developed (Su, Wang, Kwok,
& Lee, 2005). However, the fermentation time generally takes from
six months to one year (Huang & Teng, 2004; Lee, Rho, Kim, Lee, &
Lee, 2013; Zhu & Tramper, 2013), this is a drawback in soy sauce
* Corresponding author. Department of Food Science, National Chiayi University,
No.300 Syuefu Rd., Chiayi City 60004, Taiwan, ROC.
E-mail address: ckhsu@mail.ncyu.edu.tw (C.-K. Hsu).
major reasons, which are fermentation temperature and salt
concentration.
Fermentation temperature is an important factor in defining the
aging period and qualities of soy sauce. However, in regular pro-
duction of soy sauce, the fermentation temperature is usually
dependent on the outdoors weather (Ko, Ahn, Van Den Berg, Lee, &
Hong, 2009; Wu, Kan, Siow, & Palniandy, 2010; Yokotsuka, 1986),
thus fluctuated temperature occurs during fermentation period, is
one of the attributions to prolongs the time required for the for-
mations of taste and flavor components. In order to shorten the
fermentation time, as well as improve the qualities of soy sauce, the
applications of various range of temperature have been investi-
gated. For example, Yong and Wood (1974) indicated that
fermentation time of soy sauce would reduce to only 3e4 months if
the fermentation temperature was controlled at about 35e40
C. It
could be seen that the stability of warm temperature (28e37
C) is
suitable for the metabolism of microorganisms in moromi, espe-
cially lactic acid bacterial and yeast (Fukushima, 2004; Sugiyama,
1984; Van Der Sluis, Tramper, & Wijffels, 2001; Wu et al., 2010;
Yokotsuka, 1986; Yong & Wood, 1974) and thus reduce the

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0023-6438/© 2016 Elsevier Ltd. All rights reserved.
 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250
fermentation time. Therefore, practical experiences have shown
that warm temperature has been widely applied not only in the
process of commercial soy sauce (Feng et al., 2015, 2014; Luh, 1995;
Xu, 1990) but also in many researches (Feng et al., 2012; Song,
Jeong, & Baik, 2015; Van Der Sluis et al., 2001; Yan, Qian, Ji, Chen,
& Han, 2013). On the other hand, it was proposed that for the
good qualities of soy sauce, the temperature was controlled at 15
C
during the first month of fermentation and then gradually raised to
30
C on the remaining period either without or with the starter
(Chou & Ling, 1998; Huang & Teng, 2004; Iwasaki, Nakajima, &
Sasahara, 1994; Luh, 1995; Wei et al., 2013; Yokotsuka, 1986).
Although fermentation at lower temperature gives better products
because the rate of enzyme inactivation was slow and the enzymes
remain active longer (Huang & Teng, 2004; Yokotsuka, 1986), but
the long fermentation time required seems to be only suitable for
special products able to achieve a premium price.
The special attentions have been devoted to the application of
elevated temperature on soy sauce fermentation, which led to the
product development of low-salt solid-state fermentation soy
sauce (LSFSS) employed pure cultures at relatively high tempera-
ture (40e55
C) and low brine concentration (13e15%) within very
short aging period (15e30 days) in China (Fukushima, 2004; Gao
et al., 2010; Huang & Teng, 2004; Sun, Jiang, & Zhao, 2010;
Yanfang & Wenyi, 2009). Related studies have included investi-
gating biochemical changes in LSFSS (Yanfang, Lijuan, & Wenyi,
2009), evaluating aroma differences between high-salt liquid-
state fermentation soy sauce (HLFSS, traditional soy sauce) and
LSFSS from China (Feng et al., 2014), while others focused on flavor
and taste compounds analysis in LSFSS (Yanfang & Wenyi, 2009).
According to their studies, the rapid fermentation (in LSFSS) at high
temperature and low brine concentration was effective in produc-
ing biochemical indices in very short period, which could satisfy the
national standards of China. Nevertheless, its flavor and taste
quality were generally lower than that of the traditional product-
HLFSS (Feng et al., 2014; Yanfang & Wenyi, 2009; Yanfang et al.,
2009). Salt plays an important ingredient for soy sauce fermenta-
tion because it controls organoleptic (flavor and texture) and mi-
crobial properties (Song et al., 2015), consequently, salt reduction
employed can cause not only conservation problems but also
inferior qualities (Feng et al., 2014; Kremer, Mojet, & Shimojo,
2009). Su et al. (2005) also indicated that the combination of
high temperature (45
C) and low salt content (5%) was the best
way to promote enzymes' action during moromi fermentation.
However, they only focused on the initial moromi fermentation
within couple days.
Like the previous point, in soy sauce products, high salt con-
centration has saltiness and cause osmotically high pressure, which
permit the exclusive development of sensory properties and pres-
ervation (Kremer et al., 2009; Song et al., 2015; Yokotsuka, 1986;
Yong & Wood, 1974). Nevertheless, the disadvantages are because
prolong fermentation time (Osaki, Okamoto, Akao, Nagata, &
Takamatsu, 1985; Su et al., 2005; Van Der Sluis et al., 2001; Xu,
1990) and increase the risk to human health such as hyperten-
sion, as well as to be directly related to the development of car-
diovascular disease (Law, 1997; Weinsier, 1976). Therefore, the
reduction of salt content in soy sauce has been recent attempts. For
instance, the utilization of osmotically equivalent substitutes
separately or in combination, namely sugars, polyols, and ethanol
(Chiou, 1999; Chiou, Ferng, & Beuchat, 1999), finding out that
positive results of reduced salt content while still limit growth to
undesirable types of microorganisms. However, these methods
might reduce the original biochemical indices and also inhibit the
growth of some beneficial microorganisms, as a result in lower
sensory properties compare to the traditional method. Especially,
by adding yeast starters, Song et al. (2015) had first successes in
243
compensating undesirable changes (biochemical and microbial
characteristics) caused by reducing the salt content in soy sauce
fermentation.
So far, to the best of our knowledge, there are indeed no stan-
dard applications of temperature and salt concentration used, so
that the sustainability of such above effects have still to be inves-
tigated. Our attempts are to shorten the fermentation period and
reduce salt content used while retain the originally biochemical,
microbial, and sensory characteristics. In our previous study, we
have evaluated a two-stages soy sauce fermentation process and
compared the results with traditional fermentation method.
Particularly, at the first stage, the koji was mixed with 5% brine
solution at 1:2 ratio (w/w) and incubated at 45
C, for 2 days. And
then at the second stage, the fermentation temperature was
controlled at 30
C either without or with the addition of Zygo-
saccharomyces rouxii, and the brine concentrations were increased
from 5% to 14%, 18%, and 22%. As a result, the preparation of
enzymatic hydrolysis at the first stage was effective in producing
high total nitrogen and amino nitrogen contents, however, the
drawbacks were due to low reducing sugar content. On the other
hand, the results of further fermentation (after rapid fermentation
at the first stage) showed that these drawbacks need to be solved,
as the addition of Z. rouxii or reduction of brine concentration did
not improve sensory properties effectively. Hence, being in prog-
ress, the aim of this study was to optimize the process conditions
for enzymatic hydrolysis prepared at the first stage, based on three
main factors, namely temperature, fermentation time and brine
content.
2. Materials and methods
2.1. Raw materials and chemicals
Soybean and wheat were purchased from a local farmer in
Chiayi City, Taiwan and immediately stored in a refrigerator at 4
C
prior to use. The koji mold (Aspergillus oryzae) was purchased from
a supplier (Chuan Feng Microbe Co., Taichung City, Taiwan). All of
the chemicals used in the analysis were of analytical grade.
2.2. Koji fermentation
For koji production, raw soybeans were first washed and soaked
in water for 24 h at 4
C and then steamed at 121
C for 25 min.
Wheat were roasted for 45 min at 140
C, follow by cracking into 4
or 5 pieces per kernel accompanied by smaller particles of wheat
flour. Roasted wheat and steamed soybeans were mixed at a ratio of
1:2 (w/w). The mixture was then inoculated with 0.1% (w/w) of
A. oryzae (approximately 108 spores g1), and subsequently
dispersed onto the perforated stainless steel trays (68  44  3 cm).
Each tray was loaded with the mixture to a ~3 cm thickness and
incubated at 30 ± 3
C. The preparation of koji was completed in
~60e72 h when the culture began turning greenish yellow in color.
2.3. Moromi fermentation
Moromi fermentation was performed in individual cylindrical
mash tanks (20 L). In each tank sample, 3 kg of koji was mixed with
brine solution at 1:2 ratio (w/w) and then fermented at three
different temperatures (35, 40 and 45
C) and two different brine
contents (5 and 10% w/w) based on a 2 factor factorial design with a
total of 6 different treatments. For each treatment, sampling was
conducted at 1, 3 and 5 days. During the fermentation period, the
tanks were located in an oven (98  82  133 cm) to controlled
temperatures (±0.5
C). The mash was stirred for 3 min once every
day.
244 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250
2.4. Sampling and analytical methods
At each sampling, about 300 ml of samples were collected after
being well stirred. The samples were filtered through a muslin cloth
and then centrifuged at 5000g for 15 min. The supernatants,
regarded as raw soy sauce, were kept at 4
C for further analysis.
Five ml of each sample was collected in sterilized tubes and then
immediately analyzed for microbial properties. For each analysis,
three replicates tests were performed.
Determination of pH: the pH values of the soy sauce samples
were measured with a PB-10 pH meter (Sartorius, Go € ttingen,
Germany).
Determination of total nitrogen (TN): TN content was deter-
mined according to the AOAC (2000) with a little modification.
Three milliliters of the sample, 1 g of catalyst (K2SO4/
CuSO4$5H2O ¼ 10:1), and 20 mL of concentrated H2SO4 were added
into a digestion tube and then heated for 6 h at 450
C for digestion.
When the digestion was completed, 25 ml of diluted sample was
mixed with 25 mL of 30% NaOH and subjected to distillation. The
distillate was absorbed with 25 mL of 0.1 N H2SO4. The TN was
determined by back-titrating H2SO4 with 0.1 N NaOH.
Determination of reducing sugar (RS): RS content in the sample
was determined by the 3,5-dinitrosalicylic acid (DNS) method
(Miller, 1959) with a little modification. DNS reagent, which con-
tained (w/v) 1% DNS, 2% NaOH and 30% sodium potassium tartrate,
was prepared by dissolving each component in distilled water. One
milliliter of the sample diluents were mixed with 1 ml of DNS re-
agent and then kept in a boiling water bath for 5 min to colorize.
After cooling with cold water, 5 ml of distilled water was added and
the OD540nm was measured by using distilled water as a blank
control. The standard glucose solutions were used to make a cali-
brated curve for quantitative analysis.
Determination of total acidity (TA): TA content as lactic acid was
determined by alkali titration. To determine total acidity as lactic
acid, 25 ml of properly diluted sample was titrated with 0.01 N
NaOH to pH 8.1.
Determination of amino nitrogen (AN): Firstly, formol nitrogen
(FN) and ammonium nitrogen were determined according to CNS
(Chinese National Standard (CNS), 2002). To determine FN, diluted
sample and formalin solution were adjusted to pH 8.1, then 20 ml of
formalin solution was mixed with 25 ml of diluted samples for
3 min, and titrated to pH 8.1 with 0.01 N NaOH. Then, amino ni-
trogen was determined by subtraction of ammonium nitrogen from
FN according to CNS (Chinese National Standard (CNS), 2002).
Determination of microbial changes: The 10-fold-concentrated
cells were serially diluted (100 to 106) in 0.85% sterile saline so-
lution, and 100 ml of properly diluted samples were spread on YM
agar and plate count agar plates (BD Difco, Franklin Lakes, NJ, USA),
then incubated for 1e2 days at 30
C for the counting of yeast and
total bacteria, respectively. The colonies formed (25e250) were
calculated as log CFU per milliliter of soy sauce (Kang, Kim, Park, &
Shibamoto, 2012).
2.5. Experimental design and statistical analysis
The independent variables applied in experimental design were
temperature (35, 40 and 45
C), time (1, 3 and 5 days) and brine
content (5 and 10% w/w), corresponding to coded levels (1, 0 and
1), (1, 0 and 1) and (1 and 1), respectively. Response factors were
the main biochemical and microbial indices, namely total nitrogen
(TN, g/100 ml), amino nitrogen (AN, g/100 ml), reducing sugar (RS,
g/100 ml) and total acid (TA, g/100 ml) contents, pH, yeast count
(log CFU/ml) and total bacteria count (TBa, log CFU/ml). The
following second order quadratic equation was used to express the
responses as a function of the independent variables (using coded
levels).
Quality response functions:
Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b12 X1 X2 þ b13 X1 X3 þ b23 X2 X3
þ b11 X12 þ b22 X22
(1)
where Y was response variable, YTN: total nitrogen, YAN: amino
nitrogen, YRS: reducing sugar, YTA: total acid, YpH: pH, YYeast: yeast
count and YTBa: total bacteria count; X1, X2 and X3 represented the
independent variables for temperature (
C), time (day) and brine
content (% w/w), respectively. b0: the intercept; b1e3 and b11e22
were the linear and quadratic regression coefficients, respectively;
b12, b13 and b23 were the interactive regression coefficients. Anal-
ysis of variance (ANOVA) was used to evaluate model adequacy and
statistical significance. The adequacy of model was evaluated by the
determination coefficient (R2) and model p-value. To indicate the
interactive effects of the independent variables on the responses, 3-
D response surface plots were drawn by using Design-Expert
software 10.0.1 (Stat-Ease, Minneapolis, MN, USA).
A commercial optimization software, AMPL (The Scientific Press,
San Francisco, CA, USA), was used to search the optimum temper-
ature (X1), time (X2), and brine content (X3) to maximize YTN while
YAN, YRS, YTA and YpH were constrained within certain acceptable
ranges.
3. Results and discussion
3.1. Fitting the model
Table 1 shows experimental data of investigated responses (TN,
AN, RS, TA, pH, yeast and TBa) observed under different fermen-
tation conditions (temperature, time and brine content). The
complete data were fitted to second-order polynomial models as
the quality response functions described in Eq. (1). The resulting
model parameters are presented in Table 2. According to particu-
larly low p-values associated with F test for all models (p < 0.0001
for TN, AN, RS and pH; p < 0.001 for TA and TBa; p < 0.05 for yeast
count) indicated that the models were highly significant. The ad-
equacy of fit of the models were confirmed by the high R2 for all
responses (R2  0.90), except of yeast (R2 ¼ 0.89). Moreover, the
relatively low CV (<10%) with exception of TA (13.2%) suggested
sufficient repeatability of experimental results.
3.2. Biochemical properties of raw soy sauce fermentation
3.2.1. TN and AN
TN and AN are two of the most important quality indices of soy
sauce products, which contribute not only nutritional values but
also sensory characteristics (Fukushima, 2004; Gao et al., 2010;
Huang & Teng, 2004; Kobayashi & Hayashi, 1998; Nakahara,
Yamaguchi, & Uchida, 2012; Su et al., 2005; Xu, 1990). The pres-
ence of TN and AN during the soy sauce fermentation could be
explained by the protease and peptidase activities, which hydro-
lyzed the proteins of raw materials into small fragments of pep-
tides, amino acids and ammonia. However, the activities of protease
and peptidase are generally low due to enzyme denaturation in
high brine content. Besides, the metabolic activities of microor-
ganisms, especially salt tolerant lactic acid bacteria and yeast, thus
consequently exhibited low TN and AN contents, as well as prolong
the fermentation time required (Chen, Feng, Cui, Zhao, & Zhao,
2015; Feng et al., 2012; Huang & Teng, 2004; Van Der Sluis et al.,
2001; Wu et al., 2010). Recently, rapid fermentation at elevated
temperature has been tried out with some promising results in
 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250 245

Table 1
The treatment combinations and their responses.

Run Independent variable Response

Temperature Time Brine content TN AN (g/100 ml) RS TA pH Yeast TBa

(
C) (day) (%, w/w) (g/100 ml) (g/100 ml) (g/100 ml) (log CFU/ml) (log CFU/ml)

1 35 1 5 0.80 0.28 0.18 0.65 6.17 9.06 9.17

2 35 1 10 0.78 0.28 1.49 0.54 6.23 7.25 7.72
3 35 3 5 1.24 0.52 0.14 1.03 5.62 8.85 9.57
4 35 3 10 1.23 0.50 2.30 0.78 5.96 7.50 7.75
5 35 5 5 1.31 0.57 0.17 1.57 5.13 8.75 9.90
6 35 5 10 1.30 0.56 2.23 1.56 4.99 6.70 7.09
7 40 1 5 0.99 0.40 0.32 0.78 6.03 9.07 9.15
8 40 1 10 0.97 0.39 1.86 0.62 6.18 6.26 7.21
9 40 3 5 1.31 0.56 0.51 1.64 5.23 9.02 9.11
10 40 3 10 1.34 0.58 2.51 1.02 5.86 7.81 7.99
11 40 5 5 1.35 0.64 0.78 1.88 4.89 8.87 8.97
12 40 5 10 1.43 0.64 2.71 1.26 5.24 7.88 8.16
13 45 1 5 1.02 0.44 0.96 1.20 5.31 8.92 9.00
14 45 1 10 0.99 0.42 2.09 0.80 6.01 6.03 7.16
15 45 3 5 1.21 0.55 1.59 1.62 4.98 8.53 8.79
16 45 3 10 1.29 0.58 3.01 1.02 5.81 7.64 7.81
17 45 5 5 1.31 0.61 1.95 1.73 4.80 8.15 8.69
18 45 5 10 1.37 0.67 3.52 1.15 5.73 7.10 7.31

Table 2
Summary of regression analysis.

Regression coefficienta Response

TN AN RS TA pH Yeast TBa

b0 1.321* 0.574* 1.554* 1.229* 5.582* 8.408* 8.573*

Linear
b1 0.045* 0.047* 0.551* 0.116* 0.121* 0.146 0.203
b2 0.210* 0.124* 0.372* 0.380* 0.429* 0.071 0.059
b3 0.008 0.003 0.840* 0.186* 0.213* 0.836* 0.786*
Cross product
b12 0.044* 0.019* 0.211* 0.134* 0.186* 0.145 0.034
b13 0.012 0.007 0.118* 0.100* 0.185* 0.030 0.156
b23 0.017 0.007 0.131* 0.046 0.021 0.285 0.020
Quadratic
b11 0.077* 0.038* 0.186* 0.064 0.010 0.278 0.104
b22 0.135* 0.058* 0.156 0.040 0.015 0.386 0.208

Model p < 0.0001 p < 0.0001 p < 0.0001 p < 0.001 p < 0.0001 p < 0.05 p < 0.001
R2,b 0.98 0.99 0.99 0.93 0.95 0.89 0.90
CVc, % 2.89 3.77 9.10 13.22 2.83 5.78 4.56

*Significant at 0.05 level.

a
Coded values.
b
Coefficient of determination.
c
Coefficient of variance, %.

producing high TN and AN contents within short fermentation time
(around 2 weeks) (Feng et al., 2014; Gao et al., 2010; Sun et al., 2010;
Yanfang & Wenyi, 2009; Yanfang et al., 2009). Moreover, in the
work of Fukushima (2004) that the antimicrobial effect of soy sauce
due to the high brine concentration could be created by combina-
tion of high TN content and lower corresponding brine concen-
tration. Because the contribution of increased nitrogen compounds
(about the antimicrobial effect of soy sauce) to the decrease of
water activity compensates for the increase of water activity which
occurs as a result of the decrease of salt content. Therefore, the
production of high TN and AN contents at the first stage-rapid
fermentation, are promising targets, contributing the high con-
tent of substrates for further fermentation, as well as might
reducing salt content used, hence shortening the fermentation
period required.
Table 2 shows that linear terms of temperature and time
exhibited significant influence on TN which was positive in both
cases. On the other hand, temperature-time interaction and both of
quadratic terms of temperature and time exhibited negative effect
on TN, which means that TN increased with their increase, reached
maximum and then started to decrease with continuous increase of
temperature and time (Fig. 1). This phenomenon is not clearly
understood as the protease activity caused the present of TN might
be affected by the interaction of many factors, for example tem-
perature, brine content, pH. Thus, this assumption still needs
further investigation. It is important to note that brine content has
no influence on TN, and interaction term with another variables as
well (Table 2 and Fig. 1), which might be attributed to the narrow
range of brine content from 5% to 10%. These results were fairly
agreed with the finding of Su et al. (2005) that only little difference
was observed between TN contents of soy sauce fermentation
prepared from 5% to 10% brine contents at various ranges of tem-
perature applied. This might be owing to the differences of soy
sauce preparation processes employed, Su et al. (2005) particularly
used defatted soybean, ratio of defatted soybean to wheat was 1:1
w/w and ratio of koji to brine solution was 1:1.5 w/v.
246 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250

Fig. 1. The response surface plots of total nitrogen contents of soy sauce influenced by the fermentation temperature, time and brine content using coded values.

Comparing to TN, same phenomenon was observed in AN
(Table 2). In detail, linear terms of both temperature and time
exhibited positive influence on AN, while brine content showed no
effect. Negative effects of temperature-time interaction and
quadratic terms of temperature and time were also obtained.
Moreover, 3-D response surfaces had similar shape (response sur-
face plot for AN not shown). These results could be explained by
good correlation between TN and AN contents, which has signifi-
cant coefficient of correlation r ¼ 0.98 (Fig. 2).
3.2.2. RS
The biochemical and microbial changes during soy sauce
fermentation are very complex, in which RS is a vital part in
providing carbon source for the formations of taste and flavor
compounds. Based on our previous study, the remarkable effective
of high TN and AN contents were observed in very short time of
rapid fermentation (fermentation condition: 45
C, 5% brine con-
tent and 2 days fermentation time), but the low RS content
(<1.00 g/100 ml) has demonstrated some disadvantages. Therefore,
it is ideal to find out the optimal conditions with having not only
high TN and AN but also high RS content.
The result shows all linear terms of temperature, time and salt
content had positive influence on RS (Table 2). Generally, the
increased RS content in soy sauce fermentation was attributed to
the action of fungal amylase hydrolyzed of starch in raw materials,
and like protease and peptidase, the action of these enzymes was
significantly inhibited in the high salt environment (Chen et al.,
2015; Fukushima, 2004; Huang & Teng, 2004; Luh, 1995; Su et al.,

Fig. 2. Correlations between independent variables and responses.

 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250
2005; Yanfang et al., 2009; Yokotsuka, 1986; Yong & Wood, 1974).
For this reason, and comparing to the results from TN and AN, we
suggested that in the range investigated of brine content (5%e10%)
and temperature (35
Ce45
C), the amylase are more sensitive to
brine content as compare to the protease. Apart from the positive
linear of temperature, its quadratic term also exhibited positive
effect on RS, meaning that RS increased with increased temperature
(Fig. 3). Furthermore, significant effects of all interactions between
there variables indicated that RS content would increase if two of
three variables both increased. It could be especially seen that the
amylase activity are generally inhibited by the present of salt,
however, the results showed that lower RS content was observed at
lower brine content, which might be caused by the rapid con-
sumption of glucose by bacterial metabolism. Even though at high
temperature, as Yong and Wood (1974) reported that some lacto-
bacilli could grow in soy sauce medium at temperatures up to 47
C
and high brine solution. It might be suggested that at the low range
of brine content (less than 10%), salt plays a most important factor
affects RS content. This could be partly seen from Fig. 1, where brine
content could significantly explain 66% (0.812) total variance of RS,
was much higher compared to another variables, namely 19%
(0.442) and 8.4% (0.292) for temperature and time, respectively.
3.2.3. TA
TA is also one of the most important quality indicators of soy
sauce products. In general, under fermentation conditions used in
this study, TA content continuously increased during the soy sauce
fermentation period, owing to the microbial fermentation of car-
bohydrates (Chou & Ling, 1998; Cui, Zheng, Wu, & Zhou, 2014;
Yanfang et al., 2009) and free amino acids as well (Feng et al.,
2014, 2015). Furthermore, the relative content of TA depends on
soy sauce manufacturing process employed and its presence in
product has a major impact on customer acceptance. For example,
the commercial soy sauce product of LSFSS in China, which have
lower flavor and taste properties than those of the traditional
product-HLFSS (Feng et al., 2014; Fukushima, 1981; Sun et al., 2010;
Yanfang & Wenyi, 2009; Yanfang et al., 2009), might be partly
explained by strong acerbity due to high TA content in LSFSS fer-
mented at elevated temperature and low brine content. The
fermentation time is not enough for the formations of other flavor
Fig. 3. The response surface plots of reducing sugar contents of soy sauce influenced by the fermentation temperature, time and brine content using coded values.
247
components, such as alcohols, aldehydes and esters (Feng et al.,
2014). We suggested that TA content in soy sauce product should
not exceed 2.00 g/100 ml, and less than 1.4 g/100 ml based on some
previously reported data (Cui et al., 2014; Feng et al., 2013; Gao
et al., 2010).
As seen from Table 1, basically, TA content increased with
fermentation time and fermentation temperature. According to
results from Table 2, all linear terms of variables had significant
influence on TA, which was positive for temperature and time. The
significant negative interaction of temperature-time showed that
TA content started to decrease slightly at high temperature over
approximately 40
C (Fig. 4), which might be due to the inhibition
of bacterial metabolism (e.g. lactic acid bacteria and yeast) at high
temperature (Fukushima, 2004; Huang & Teng, 2004; Sugiyama,
1984; Yokotsuka, 1986; Yong & Wood, 1974). Brine content
showed reverse effect on TA and the interaction of brine content
and temperature was also negative (Table 2). At low brine content
(around 5%), TA content increased with temperature, however, at
higher brine content (around 10%), TA increased with temperature
up to 40
C and then TA started to decrease as temperature was over
about 40
C.
3.2.4. pH
It is well known that pH plays a vital role on the progress of
moromi fermentation. At first, lactic acid bacteria fermentation
took place and it resulted a decrease in the pH from 7.0 to below 5.0,
and then yeast fermentation accelerated the formation of alcohol
compounds. (Ko et al., 2009; Liu et al., 2015; Luh, 1995; Sugiyama,
1984; Van Der Sluis et al., 2001; Zhu & Tramper, 2013). These
metabolism with biochemical reactions contribute to the unique
characteristic of soy sauce product. Yokotsuka (1986) found that
rapid decrease of the pH value due to rapid lactic fermentation was
undesirable, thus the method with low temperature (<15
C) in
initial fermentation stage was applied, but the disadvantage was it
required long fermentation time to product good quality soy sauce.
Additionally, in consideration of our previous study, pH has drop-
ped sharply to below 4.5 in only 2 days, adversely affected
fermentation progress and sensory properties as well. Therefore, in
the present study, it might be ideal that pH value of optimization
point is more than 5.00.
248 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250

Fig. 4. The response surface plots of total acid contents of soy sauce influenced by the fermentation temperature, time and brine content using coded values.

All linear terms of variables exhibited significant influence on
the pH, which was negative influence for the fermentation tem-
perature and time but positive influence for the brine content the
interactive effect of temperature and time on the pH was positive
(Table 2). So the pH decreased with fermentation temperature and
time from around 1 to 4 days; however, at about 5 days, the pH
slightly increased with temperature (Fig. 5). The interactive term of
fermentation temperature and the brine content exhibited positive
effect on the pH, indicating that at lower brine content, the pH
decreased with temperature, oppositely, at higher brine content,
pH increased slightly with temperature. In general, the decline of
the pH might be because the accumulation of free fatty acids, amino
acids, and peptides containing carbonylic side chains as the results
of hydrolysis of the raw materials, the autolysis of microbial cells, as
well as the microbial fermentation of carbohydrates (Chou & Ling,
1998; Huang & Teng, 2004; Van Der Sluis et al., 2001; Yokotsuka,
1986; Yong & Wood, 1974). Thus, we suggested that at low brine
content (about 5%), there were significant microbial contamination,
which accelerated the microbial fermentation of carbohydrates and
consequently lowering the pH. By contrast, the increase of the
fermentation temperature and brine content (10%) might create
significant stress on microbial metabolism, and thus resulted in the
higher pH values. These results confirmed that TA content signifi-
cantly decreased as both of fermentation temperature and brine
content were at their high levels (45
C and 10% brine content), as
well as a good correlation between TA and pH (r ¼ 0.97 in Fig. 1).
3.3. Microbial properties of raw soy sauce fermentation
The microbial metabolism plays a crucial role in the formations
of taste and flavor compounds during the fermentation period of
soy sauce. In which, yeast (e.g. Zygosaccharomyces rouxii, Candida
versatilis and Candida etchellsii) is predominance in the moromi
fermentation (Fukushima, 2004; Huang & Teng, 2004; Luh, 1995;
Sugiyama, 1984; Yokotsuka, 1986; Yong & Wood, 1974). As shown
in Table 2, only the brine content showed significant influence on
the microbial growth, which the linear coefficient terms were
negative in both cases of the total yeast and TBa counts. This means
that the increased brine content would cause the decrease of total
yeast and TBa counts, which was agreed with the common

Fig. 5. The response surface plots of pH of soy sauce influenced by the fermentation temperature, time and brine content using coded values.
 N.X. Hoang et al. / LWT - Food Science and Technology 74 (2016) 242e250
Table 3
Optimization models including the objective function and constrain functions for searching the optimum fermentation condition of soy sauce.
Objective function
YTN 1.321 þ 0.045X1 þ 0.210X2 þ 0.008X3  0.044X1X2 þ 0.012X1X3 þ 0.017X2X3  0.077X21  0.135X22
Constrain functions
YAN 0.574 þ 0.047X1 þ 0.124X2 þ 0.003X3  0.019X1X2 þ 0.007X1X3 þ 0.007X2X3  0.038X21  0.058X22
YRS 1.554 þ 0.551X1 þ 0.372X þ 0.840X þ 0.211X1X2  0.118X1X3 þ 0.131X2X3 þ 0.186X21  0.156X22
YTA 1.229 þ 0.116X1 þ 0.380X2  0.186X3  0.134X1X2  0.100X1X3  0.046X2X3  0.064X21  0.04X22
YpH 5.582  0.121X1  0.429X2 þ 0.213X3 þ 0.186X1X2 þ 0.185X1X3 þ 0.021X2X3  0.01X21  0.015X22
Constrain of variables
X1, X2, X3
knowledge that salt was one of the main inhibitory factors on the
growth of microorganism in soy moromi (Fukushima, 1981;
Sugiyama, 1984). The results also confirmed that the low brine
content (about 5%) adversely affected on tested biochemical
changes, namely lower RS content and higher TA content due to the
rapid metabolism of contaminated microorganism. On the con-
trary, high brine content (10%) showed the advantages in inhibiting
the growth of contaminated microorganism, thus higher RS and
lower TA contents were obtained at 10% brine content while no
significant effect on TN and AN. Besides, the main effect of the brine
content on the growth of microorganism could be understood by
correlation coefficient given in Fig. 1, where the brine content could
explain 74% ((-0.86)2) and 83% ((-0.91)2) total variances of total
yeast and total bacteria counts, respectively.
3.4. Optimization
A nonlinear programming solver, AMPL, was used to search the
optimum temperature, time, and brine content to maximize TN
content, while AN content, RS content, TA content and pH value
were constrained within the desired targets ranges presented in
Table 3. As a result in the optimal conditions were coded values of
0.136, 0.818 and 1 for temperature, time and brine content,
respectively, which were corresponding actual values of 40.7
C, 4.6
days and 10%, respectively. At this optimal condition, the predicted
values for TN, AN, RS and TA contents and pH were 1.43, 0.65, 2.79
and 1.28 g/100 ml and 5.48, respectively. It could be seen that these
values were compared with the experimental values of 1.43, 0.64,
2.71 and 1.26 g/100 ml and 5.24, respectively at 40
C, 5 days and
10% brine content. Therefore, the mathematical models proposed
here were useful for the prediction of optimal condition of rapid
fermentation at the first stage.
4. Conclusions
The results have demonstrated the second-order polynomial
models were sufficient to predict and describe the biochemical
responses of TN, AN, RS, TA and pH in initial soy sauce moromi
fermentation. The optimized fermentation conditions were tem-
perature of 40.7
C, fermentation time of 4.6 days, and brine con-
tent of 10%. These conditions obtained total nitrogen (>1.4%) and
amino nitrogen (>0.56%) contents satisfying the requirement of
first-grade soy sauce of Taiwan within 5 days. The data of present
study provided the better understanding of effects of temperature,
fermentation time and brine content on biochemical and microbial
characteristics of soy sauce, which would be very useful for the
preparation of enzymatic hydrolysis within short period of soy
sauce fermentation and for further study to be able to shorten the
fermentation time and enhance the qualities of soy sauce.
249
To maximize
Constrain ranges
0.60
2.50
1.40
5.00
1 and 1
Acknowledgements
This research was partially supported by the Ministry of Science
and Technology in Taiwan, R.O.C. (MOST 103-2221-E-415 -016 -)
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