African Journal of Biotechnology Vol. 9(5), pp. 702-706, 1 February, 2010
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2010 Academic Journals
Full Length Research Paper
Effect of temperature on moromi fermentation of soy
sauce with intermittent aeration
Ta Yeong Wu1*, Mun Seng Kan2, Lee Fong Siow2 and Lithnes Kalaivani Palniandy1
1Chemical and Sustainable Process Engineering Research Group, School of Engineering, Monash University, Jalan
Lagoon Selatan, Bandar Sunway, 46150, Selangor Darul Ehsan, Malaysia.
2School of Science, Monash University, Jalan Lagoon Selatan, Bandar Sunway, 46150, Selangor Darul Ehsan,
Accepted 14 December, 2009
Soy sauce has been widely used as one of the main seasoning agents in Asian countries. Soy sauce is
produced by two-steps fermentation processes, namely koji fermentation and moromi fermentation. In
this study, different temperatures (25, 35 and 45°C) for moromi fermentation in bioreactor were
investigated for understanding their influences on soya sauce quality, in terms of pH variations, ethanol
concentrations and total nitrogen contents in raw soy sauce during moromi fermentation. It was learned
that as the aging of moromi took place, the pH level was decreased from pH 7 to 4.88. Also, the soy
sauce had lower concentration of ethanol when higher temperature was used in moromi fermentation
but the difference of temperature did not show significantly effect on total nitrogen content in soy
sauce. This study indicated that the temperature used in the moromi fermentation, coupled with
intermittent aeration, imposed significant effects on soy sauce aging and quality. Higher fermentation
temperature of 45°C enhanced the aging of soy sauce, accompanying with lower contents of ethanol
and higher pH level in soy sauce. However, the total nitrogen content in the soy sauce was not
significantly influenced by the fermentation temperature.
Key words: Aspergillus oryzae, bioreactor, moromi, soy sauce, temperature.
Soy sauce is a traditional fermented food in Malaysia that
has been practiced long time ago. Traditionally, soy
sauce has been used in Japan and several oriental coun-
tries and is presently used as a liquid seasoning in
cooking worldwide (Kataoka, 2005; Yokotsuka, 1986).
Soy sauces are the main condiments for foods and table-
top seasoning in the most of the Asian countries.
In Malaysia, there are so many varieties of soy sauces
in the market such as dark soy sauce, light soy sauces,
‘Kicap lemak manis’, ‘Kicap lemak masin’ and others. Soy
sauce in Malaysia is produced in small scale cottage
industries. This production is mainly performed in a
*Corresponding author. E-mail: firstname.lastname@example.org.
edu.my. or email@example.com. Tel: +60 3 55146258. Fax:
+60 3 55146207.
conventional method as compared to the Japanese
production method using high technology machines for
improving the tastes. Soy sauce in Malaysia is naturally
brewed by two step fermentation processes, namely, koji
fermentation and moromi fermentations. The koji fermen-
tation process involves the mixtures of soybean and
wheat flour with the inoculation of Aspergillus oryzae.
During koji fermentation, the addition of A. oryzae
excretes protease, amylase and other enzymes. These
enzymes will hydrolyze the raw materials into simpler
forms. Proteolytic enzymes will convert soy beans pro-
teins into peptides and amino acids while amylase
enzymes will hydrolyze starch into simple sugars. The
hydrolyzed nutrients will be utilized by the yeast and
bacteria in moromi stage.
In Malaysia, the moromi fermentation process is usually
carried out in closed tanks that are kept under the sun for
3 - 4 months. In this process, there are three important
types of microorganisms, which play crucial roles for a
good soy sauce production. The simpler sugars from koji
fermentation are mainly metabolized into lactic acid and
acetic acid by Pediococcus halophilus (Iwasaki et al.,
1993). The natural presence of yeast in the environment,
namely Zygosaccharomyces rouxii and Candida species,
will convert the remaining sugars to ethanol and a
number of minor flavor compounds, respectively (Sasaki
and Nunomura, 2003). During moromi fermentation, etha-
nol is being processed by Z. rouxii under aerobic and
anaerobic condition (Hamada et al., 1989). Ethanol
concentration is directly proportional to the increasing
numbers in yeast cells during the moromi fermentation
(Röling et al., 1996). On the other hand, Candida species
are important for the development of aroma in soy sauce
by producing phenolic compounds such as 4-ethyl-
guaiacol (Hamada et al., 1990). For a good quality of soy
sauce, it should contain 1.0 - 1.65% total nitrogen (w/v),
2.0 - 2.5% ethanol and 17 - 19% sodium chloride (w/v)
with 45% of the total nitrogen being simple peptides and
another 45%, amino acids (Luh, 1995).
Fermentation temperature is an important factor in
defining the aging and quality of the soy sauce. In normal
production of soy sauce, the fermentation temperature is
usually dependent on the surrounding temperature.
However, it was suggested that for better aging of soy
sauce mash, the temperature for the soy sauce produc-
tion were maintained at 15°C during the first month of
fermentation and then the fermentation temperature was
(gradually) raised to 30°C (Chou and Ling, 1998; Iwasaki
et al., 1993). Later, Jansen et al. (2003) found that the
production of fusel alcohols (which are important flavor
compounds in the soy sauce) by Z. rouxii was also
dependant on the fermentation temperature.
Aeration could be considered as one of the factors that
would also influence the soy sauce production. Earlier,
Beatrice Foods Co. (1972) reported that intermittent
aeration could actually accelerate the maturity of soy
sauce. Hamada et al. (1989) found that the soy sauce
fermented by supplying air (0.02 vvm) had a higher con-
tent of aroma components. Later, Hamada et al. (1990)
proposed the supply of air to fermentation broth was
necessary for long time production of 4-ethylguaiacol in
soy sauce fermentation. When lower aeration was
supplied to the fermentation broth, the changes and pro-
duction of flavor in soy sauce was very slow, accom-
panying by unripe flavor (Kim et al., 1996).
In the present study, the moromi fermentation was
conducted in bioreactor with a maximum working volume
of 6 L. The bioreactor system was equipped with aeration
system and temperature sensor. The main aim of this
study was to investigate the effect of temperature in the
range of 25 to 45oC on the batch fermentation of soy
sauce with intermittent aeration for 10 min at interval of
three days. The quality of the soy sauce would be mea-
sured based upon the pH variations, ethanol concentrations
Wu et al. 703
and total nitrogen contents in raw soy sauce during
MATERIALS AND METHODS
For k oji production, soy beans were first s oaked in water for 10 h.
Then, the s oy beans were cooked by autoclave for 20 min at
121°C. The cooked soy beans were cooled t o room temperature.
The cooked soy beans were thoroughly mixed with wheat flour at a
ratio of 3:1. Then, A. oryzae in powder form was inoculated at 0.1%
of the cooked soybeans and spread evenly onto the mixture of
beans and flour. They were placed on the perforated trays (30 x 24
x 2.5 c m). These trays were k ept in a koji ferment er, which was
equipped with a fan blower that sent wet air to the fermenter (Indoh
et al., 2006). The koji was incubated for 72 h at temperature of
30°C throughout the experiment.
The matured k oji were equally divided and transferred separately to
the bioreactor. For each bioreactor, the matured koji was mixed with
a brine solution (20% w/v) at a ratio of 1:3. Temperature for each
bioreactor was maintained differently, namely at 25, 35 and 45°C,
while t he control was kept separately from the bioreactors under
ambient temperature (26 - 30°C). The temperatures in bioreactors
(except the control) were maintained constantly throughout the
experiment by jacketed vessel.
Also, intermittent aeration f or 10 min at interval of three days was
applied during the fermentation process. Aeration is r equired by the
yeast in s oy sauce production, in which case the yeasts are strict
aerobes and need oxygen for their growth. W hen aeration is
applied, in the early stage, it will retard the growth of salt tolerant
lactobacilli and wild yeasts (O’Toole, 1997; Sasaki and Nunomura,
No agitation was introduced in this study because the organo-
leptic properties of s oy beans in moromi stage were preferred to be
maintained as a whole beans until the end of the experiment for
reducing the problem in filtration of raw soy sauce.
Sampling and analytical methods
About 20 mL of s ample was collected after the aeration t ook place.
The sample was filtered through a muslin cloth and then under vacuum
through a Whatman No. 4 filter paper (20-25 µm). The filtrate,
regarded as raw soy sauce, was kept at 4°C for further analysis.
The pH of the s oy sauce samples could be directly measured by
using pH meter (HI 251, Hanna Instrument) (Ishigami et al., 1965).
The total nitrogen contents in the soy sauce samples were
analyzed by using Kj edhal Distillation Method while the ethanol
contents were determined by using Gas Chromatography (GC-
2010, Shimadzu). All data were reported as the mean of three
RESULTS AND DISCUSSION
The fermentation of soy sauce production was carried out
for 36 days. Moromi fermentation was carried out in three
different fermentation temperatures, namely 25, 35 and
704 Afr. J. Biotechnol.
Figure 1. The color of the brine at (a) 25, (b) 35, (c) 45°C and ( d) room temperature (as c ontrol) after one day of moromi
Figure 2. Time courses of pH variations in moromi fermentation.
45°C, while the control was kept under ambient tempe-
rature (26-30°C). During this period, the temperature was
maintained throughout the process by the jacketed vessel
in bioreactor. Aeration was supplied to each bioreactor
for 10 min at interval of 3 days, while the aeration was
controlled by valve manually.
Initially, the colors of the brine for all the investigated
temperatures were green. As the fermentation period
increases, the brownish color was formed and the color
became darker as the aging of the mashes continued.
After one day fermentation, the color of the brine appea-
red to be the darkest at 45°C while the color of the brine
at 25°C was still in greenish (Figure 1). However, the
color of the brine at 25°C changed into a darker color
after two days of fermentation.
Figure 2 shows the pH variations during the brine
fermentation. The pHs of the soy sauce at the fermen-
tation temperature of 25 and 35°C were lower than the
pH of the soy sauce at 45°C and room temperature. In
general, the acidity of the raw soy sauce increased as the
fermentation period continued. In the early stage of
moromi fermentation, the pH of the soy sauce was about
7.0. As the fermentation period continued, the pH of the
soy sauce was reduced to about 4.88 after a month of
fermentation. This might be due to the production of lactic
acid by Tetragenococccus halophilus. Lactic acid was
produced through the microbial digestion of the starch
that eventually could reduce the pH of the soy sauce
(Yong and Wood, 1976). After the pH has dropped below
5.0, T. halophilus was unable to grow and an alcoholic
fermentation by Z. rouxii would begin (Sluis et al., 2001).
The decline of pH during fermentation might also be
attributed to autolysis of microbial cells, accumulation of
free fatty acid, amino acids and peptides containing
carbolylic side chains as a result of hydrolysis of
materials in soy sauce (Kim and Lee, 2008).
Wu et al. 705
Figure 3. Time courses of ethanol concentrations (% w/v) in moromi fermentation.
Figure 4. Time courses of total nitrogen contents (% w/v) in moromi fermentation.
Z. rouxii synthesizes ethanol from the sugars that are
present during moromi fermentation (Röling, 1995).
According to the halal specification in Malaysia, the
ethanol content in any halal food must be lower than 2%.
Figure 3 shows that the ethanol contents in all trials were
below 2%, in which case the lowest content of ethanol
was found at the fermentation temperature of 45°C.
Seeing that the ethanol concentration is directly propor-
tional to the increasing numbers in yeast cells during the
moromi fermentation (Röling et al., 1996), it is not
surprising to find that the lowest content of ethanol was
found at 45°C in this experiment because the optimum
growth condition of Z. rouxii is around 25 - 27.5°C in brine
solution (Hamada et al., 1989; Sasaki and Nunomura,
Total nitrogen content is an important parameter for
evaluating the soy sauce quality (Chou and Ling, 1998).
The changes in total nitrogen contents in raw soy sauces
at different fermentation temperatures are shown in
Figure 4. This study shows that the total nitrogen con-
tents for all soy sauces at different fermentation tempe-
ratures were greater than 0.7% (w/v). The total nitrogen
contents in all trials were rapidly increasing in the begin-
ning of the fermentation process but their contents were
maintained near the end of the experiments. During the
fermentation process, the increase of total nitrogen
content in the liquid phase might be due to hydrolysis of
soy beans. Also, this study shows that the total nitrogen
706 Afr. J. Biotechnol.
content in the soy sauce was not greatly influenced by
the fermentation temperature (Figure 4). Similar result
was also observed by Kim and Lee (2008), in which case
they found that the crude protein content in soy sauce
was 2.83% at 4°C and only 3.36% (0.53% difference in
crude protein content) at 20oC after 360 days of moromi
Aeration was introduced in this study because it was
considered as one of the important factors in the pro-
duction of soy sauce. This is because the yeasts gene-
rally would not be able to survive during the brine
fermentation because of the low availability of oxygen,
which is caused by the low aeration rate and poor
solubility of oxygen in the brine solution (Sluis et al.,
2001). According to Hamada et al. (1989), a supply of air
was necessary for vigorous fermentation by Z. rouxii as
the degree of fermentation was affected by the oxygen
In general, this study indicates that the temperature used
in the moromi fermentation imposed significant effects on
soy sauce aging and quality. At the highest fermentation
temperature of 45°C, the color of the fermentation brine
appeared to be the darkest after a certain period of
maturation. Also, the lowest content of ethanol in the soy
sauce was found at the fermentation temperature of
45°C. On the other hand, the overall pHs of the soy
sauce at the fermentation temperature of 25 and 35°C
were lower than the pH of the soy sauce at 45°C and
room temperature. However, the study found out that the
total nitrogen content in the soy sauce was not signifi-
cantly influenced by the fermentation temperature. Further
investigation is required to reconfirm this observation.
The authors would like to thank the Malaysian Ministry of
Science, Technology and Innovation (MOSTI) for suppor-
ting this research work under eScienceFund 02-02-10-
SF0013. We are grateful to Malaysian Agricultural Research
and Development Institute (MARDI) for sponsoring A.
oryzae and Dr. Yong Wee Ooi for helpful discussions.
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