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Effect of temperature on moromi fermentation of soy sauce with intermittent aeration


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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.
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African Journal of Biotechnology Vol. 9(5), pp. 702-706, 1 February, 2010
Available online at
DOI: 10.5897/AJB09.1548
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: or 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
moromi fermentation.
Koji fermentation
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.
Moromi fermentation
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
independent trials.
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 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
transfer rate.
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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... These observations could be explained based on higher endogenous proteases and amylases activities at elevated temperature and low brine content. In particular, proteases hydrolyzed soybean proteins into peptides and amino acids, whereas amylases hydrolyzed the starch into dextrin, oligosaccharide and glucose (Chen et al., 2015;Feng et al., 2012;Van Der Sluis et al., 2001;Wu, Kan, Siow, & Palniandy, 2010). The slightly different contents of AN and RS between T4 and T1, T2, and T3 were explained by the different ratios of koji/brine applied. ...
... At the second stage, the results exhibited the similar trends in the TN contents of both T5 and T6, which increased rapidly, reaching their maximum after 2 months of fermentation, and then slightly decreased thereafter. These results were in agreement with the composition of previously examined soy sauce (Gao et al., 2010;Wu et al., 2010), contributing that the rapid increase in TN content during the initial fermentation period was attributed to the action of fungal proteases activities. Proteases released from the koji mold (Aspergillus oryzae) were active during koji and moromi fermentations and they could hydrolyze soybean and wheat proteins to form peptides and amino acids. ...
... It could be seen that 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 presence of TN and AN during soy sauce fermentation could be attributed to the activities of proteases and peptidases, which were generally low due to enzyme denaturation in high brine content and fluctuated temperature during the fermentation time, thus consequently exhibited low TN and AN contents, as well as prolong the fermentation time required (Chen et al., 2015;Feng et al., 2012;Huang & Teng, 2004;Van Der Sluis et al., 2001;Wu et al., 2010). From the above results of TN and AN, it is, however, suggested that the final contents of TN and AN are not very depend on temperature in the long-term fermentation process (≥3 months). ...
A two-stage moromi fermentation method was employed to the soy sauce making in order to shorten the overall fermentation time. At the first stage, the koji was mixed with 10% brine solution and incubated at 40 °C for 5 days. The second fermentation stage was conducted under three different treatments, including reduced temperature from 40 °C to 30 °C, outdoor without temperature control, and indoor ambience temperature up to 3 months with the brine content increased from 10% to 18%. The increases of total nitrogen (TN) and amino nitrogen (AN) contents were mainly occurred at the first stage while maintain constant during the second stage. Both TN and AN values satisfied the requirement of first-grade soy sauce of Taiwan. Unlike TN and AN, the reducing sugar and total acid contents still increased with fermentation time during the second stages. Since most of the changes of these biochemical properties occurred within 5 days’ moromi fermentation, the use of high temperature and low salt fermentation condition in the first stage was very important to rapid fermentation process of soy sauce.
... The increased protease activity of meju and soy sauce can lead to proteolysis of raw materials into small splinters of amino acids and peptides (Wu et al., 2010). The soy sauce protease activity tended to increase continuously with increasing fermentation period (Chen et al., 2015). ...
We developed a low‐salt soy sauce from soy residue and T. molitor larvae powder. The salinity of low‐salt soy sauce replaced by okara (LSO), T. molitor larvae powder (LST), or both okara and T. molitor larvae powder (LSOT) ranged between 9.87–12.47 % with a pH of 5.4–6.3. LSO exhibited lower acidity (0.58) than LST (1.27) and LSOT (1.34). LSOT showed the highest protease activity, total nitrogen, and amino nitrogen content among the soy sauces. Glutamic acid was ten times higher in LST and LSOT than in LSO. LST and LSOT had twice higher antioxidant capacities, whereas LSO had twice higher ACE inhibitory activity than low‐salt or regular high‐salt soy sauces. T. molitor larvae powder supplementation in soy sauce fermentation led to a significant increase in overall quality. Moreover, the synergistic effect of the mixture of okara and T. molitor larvae powder improved the functionality of low‐salt soy sauce.
... Soy sauce is a traditional condiment that is used in many Asian countries. It can bring an appetizing taste to a certain food [1]. At present, the annual production of soy sauce in the world is about 8 million tons. ...
Full-text available
Soy sauce is a common condiment that has a unique flavor, one that is derived from its rich amino acids and salts. It is known that excessive intake of high-sodium food will affect human health, causing a series of diseases such as hypertension and kidney disease. Therefore, removing sodium from the soy sauce and retaining the amino acids is desirable. In this study, electrodialysis (ED) was employed for the desalination of soy sauce using commercial ion exchange membranes (IEMs). The influence of the current density and initial pH on the desalination degree of the soy sauce was explored. Results showed that the optimal desalination condition for ED was reached at a current density of 5 mA/cm2 and pH of 5, with the desalination degree of 64% and the amino acid loss rate of 29.8%. Moreover, it was found that the loss rate of amino acids was related to the initial concentration and molecular structure. In addition, the amino acid adsorption by IEMs was explored. Results implied that the molecular weight and structure affect amino acid adsorption. This study illustrated that the ED process can successfully reduce the salt content of the soy sauce and retain most of the amino acids without compromising the original flavor.
... In general, the color of soy sauce is blackish brown; this color can occur during the fermentation process and during the last processing, where the heating process (pasteurization) causes browning reaction on soy sauce. Wu et al. (2010) reported that the brine color at 25°C changed into a darker color after two days of fermentation. Differences in the mold activity also affect the activity of the enzymes produced, so the components of the fermentation process are related to the release of color-forming compounds, both the natural color of the raw material and the color formed during the soy sauce processing. ...
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This study aimed at examining the effect of waru leaves (Hibiscus tiliaceus L.) as a source of molds, Apergillus oryzae, and Rhizopus oligosporus in the process of solid-state fermentation (koji making) and salinity in the process of brine fermentation to yield moromi (baceman fermentation) towards the characteristics of pigeon pea sauce. This study used a factorial randomized block design (RBD) consisting of two factors. The first factor was the inoculum treatments, which included: (1) control: the process of making koji using waru leaves (H. tiliaceus L.); (2) the process of making koji using Rhizopus oligosporus; (3) the process of making koji using Aspergillus oryzae; and (4) the process of making koji using R. oligosporus and A. oryzae. The second factor was soaking in a saline solution with concentrations of 15%, 20%, 25%, and 30% (w/v) and the fermented pigeon peas were grouped twice according to the time of processing. The results showed that the mixed inoculum treatments and 20% salt content produced the best pigeon pea sauce characteristics. The characteristics of the pigeon pea sauce were as follows: water content 80.33%, protein content 4.04%, salt content 19.41%, pH 6.2, total acid 0.22%, lactic acid bacteria 2.1 x 10 7 CFU/g, color (neutral-like), aroma (neutral-like), flavor (neutral-like), and overall acceptance (neutral-like). The pigeon pea sauce produced met the Indonesian National Standard and no aflatoxin B1 contamination was found in the products. Keywords: waru leaf, A. oryzae, R. oligosporus, pigeon pea, sauce Permana IDGM, Duniaji AS, Wisaniyasa NW, Gunam IBW (2020) The effect of inoculation of Aspergillus oryzae and Rhizopus oligosporus in molds fermentation on the characteristics of pigeon pea sauce (Cajanus cajan (L.) Millsp.). Eurasia J Biosci 14: 3527-3535.
... Besides, the decrease metabolism of microorganisms especially molds and yeasts, the enzyme activity was reduced and prolonged the fermentation time, thereby reduced protein decomposition and consequently exhibited low ANC (Wu, Kan, Siow, & Palniandy, 2010). Figure 1b shows that the ANC of WSS increased gradually as the temperature increased, and the highest value was obtained when the fermentation temperature was 44°C. ...
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Remaining walnut meal after oil extraction still contains many nutrients. However, these by‐products have not been effectively used. In this study, walnut meal and Aspergillus oryzae (3.042) were used in combination to prepare a soy sauce‐like material with high amino nitrogen content (ANC). The optimal conditions for the preparation of walnut soy sauce (ratio of brine:koji of 1.7:1 for 6 days at 45°C) were determined using response surface experiments (RSE), which showed maximum ANC of 855 mg/100 ml. The results of amino acid analysis indicated that walnut soy sauce had a similar amino acid composition compared with three commercial soy sauces. It contained all the essential amino acids and had a high content of umami amino acids such as Glu and Asp, which may give it a stronger umami taste. Moreover, the crude walnut soy sauce was extracted sequentially using ethyl acetate, n‐butanol, acetone and water, and the total phenols, total flavonoids, reducing sugars, and the peptides of different solvent extracts were measured. Results showed that the total phenolic and flavonoid contents were highest in the ethyl acetate extracts. However, water residue had the highest levels of reducing sugars and peptides. In vitro, the water residue showed the highest antioxidant capacity and angiotensin‐converting enzyme (ACE) inhibitory activity, due to more reducing sugars and peptides. These results indicated that walnut soy sauce may have significant antioxidant and ACE inhibitory activity. The findings provide a scientific basis for developing a replacement for soy sauce and broaden the beneficial application of walnut meal. The optimal conditions for the preparation of walnut soy sauce were determined. Walnut soy sauce has high umami amino acid content. All four extracts had antioxidant and ACE inhibitory activities.
... mg/L) compared to the nearest counterpart (646.78 mg/L) by imposing the carbon to nitrogen ratio strategy in Table 5. In traditional Malaysian two-step soy sauce production, koji fermentation was initially done prior the moromi fermentation (Wu et al., 2010). The current enhancement provides an alternative strategy to increase GABA compound prior moromi fermentation. ...
In submerged-liquid fermentation, seven key parameters were assessed using one-factor-at-a-time to obtain the highest GABA yield using an industrial soy sauce koji Aspergillus oryzae strain NSK (AOSNSK). AOSNSK generated maximum GABA at 30 °C (194 mg/L) and initial pH 5 (231 mg/L), thus was able to utilize sucrose (327 mg/L of GABA) for carbon source. Sucrose at 100 g/L, improved GABA production at 646 mg/L. Single nitrogen sources failed to improve GABA production, however a combination of yeast extract (YE) and glutamic acid (GA) improved GABA at 646.78 mg/L. Carbon-to-nitrogen ratio (C8:N3) produced the highest cell (24.01 g/L) and GABA at a minimal time of 216 h. The key parameters of 30 °C, initial pH 5, 100 g/L of sucrose, combination YE and GA, and C8:N3 generated the highest GABA (3278.31 mg/L) in a koji fermentation. AOSNSK promisingly showed for the development of a new GABA-rich soy sauce. (Full-text access:
... Maturity is generally proportional to concentration of soluble peptides or amino acids produced from soy protein by catalysis of microbial enzymes. The production efficiency of soluble peptides and amino acids may be dependent upon physiological activity of microorganisms and environmental conditions [31]. However, if the substrate (soybean protein) is not limited by microbial growth, production of soluble peptides and amino acids may be more influenced by microbial activity than substrate concentration. ...
This study for the first time explores the possibility of using Corynebacterium glutamicum Ⅲ (C. glutamicum) to shorten the soy sauce fermentation period without compromising product quality. C. glutamicum was added to soy sauce that had been fermented with Aspergillus oryzae for 30 days (SS30) to initiate a further 3‐day fermentation (yielding SS33). The 3‐day fermentation exerted similar impacts on SS30 as the standard fermentation for a further 60 days (yielding SS90): i.e. increased the contents of total nitrogen, total free amino acids (FAAs), umami FAAs, acids, esters, ketones, furans and sulfur‐containing compounds, whilst decreasing the contents of alcohols and phenols. This explained the similarities in taste, aroma and overall liking between the SS33 and SS90. The SS90 was the darkest among the three soy sauces. Compared with the SS90, the SS33 was less salty and smoky, more umami and kokumi, softer and more mellow, higher overall liking, with more 0.5‐1 kDa peptides and fewer peptides <0.5 kDa. Therefore, a 3‐day fermentation with C. glutamicum after an initial 30 days of a normal soy sauce fermentation represents a simple and effective way to shorten the soy sauce production time from 90 to 33 days whilst increasing greatly the umami taste and mellow sensation. This approach enhances product competitiveness via lowering manufacturing cost.
Soy sauce contains several nitrogen-containing compounds, most of which are proteins and their decomposition products during the fermentation process. These compounds include potentially harmful ammonium salts. In this study, the effects of changing the koji making and fermentation conditions of soy sauce on the ammonium salt and amino acid nitrogen content in soy sauce are researched. The results indicate that mixed microbial koji making can significantly influence the ammonium salt content in soy sauce, with the increase in the amount of microbial inoculated, the ammonium salt content exhibited an overall upward trend, and increase the total amount of free amino acids. Besides, the ammonium salt content and brine concentration was a significant negative correlation between the two. Moreover, as the temperature of koji making increases, the ammonium salt content first decreases and then increases, and the content of ammonium salt gradually decreased with the increasing fermentation temperature. This study demonstrates that changes in soy sauce brewing conditions will influence the formation of ammonium salts and amino acid nitrogen in soy sauce, providing a foundation for further optimization of the soy sauce brewing process.
This is the first report on the effect of low temperature stress applied during initial moromi fermentation on the quality and taste of soy sauce. Koji was prepared to yield initial moromi under three comparative fermentation conditions over 9 days: (1) 4 °C and 0% brine (i.e., water) (LTSF); (2) 4 °C and 16% w/w brine (LTSH); (3) 25 °C and 16% w/w brine (the control, CRTH). Greater extent of autolysis in samples was found under low temperature stress conditions (i.e., at 4 °C, a temperature much lower than the normal temperature range like 25 °C for natural microbial growth and performance). Compared to CRTH, LTSF had a two-fold increase of glutaminase activity in dregs and 65.17% increase in supernatant, and after 60 days of moromi fermentation, a 5.73% and 3.47% increase, respectively, in the contents of glutamic acid (Glu) and aspartic acid (Asp). LTSF had the highest total free amino acid content due to both the low temperature stress and absence of salt. The intensity ranking of umaminess and kokumi sensation (LTSF > LTSH > CRTH) revealed by sensory analysis followed the changing trends of their umami and sweet amino acid contents with a trend reversal in the bitter amino acid content. Low temperature (4 °C) without brine for initial moromi fermentation seemed beneficial, leading to a soy sauce product with desired taste and amino acid contents.
In order to investigate fermenting conditions and the microorganisms necessary for factory production of traditional Korean soy sauce, we manufactured soy sauce made by Bacillus species SSA3-2M1 and fused ST723-F31 with aeration (1/30 vvm, 1/3 vvm and 2/3 vvm) at 30°C for 40 days. This method was chosen to investigate the changes of dissolved oxygen, pH, cell number, flavor and the taste components during fermentation. When air was supplied (2/3 vvm) to the fermentor during fermentation, the flavor of the soy sauce and the composition of taste components (free amino acids, free sugars and organic acids) were similar to that of traditional Korean soy sauce after 22 days. The results of our experiments indicates that the mass production of traditional Korean soy sauce is possible using Bacillus species SSA3-2M1 and fused ST723-F31 given sufficient aeration.
A new method for producing fermented fish sauce was developed to improve the taste of the fish sauce. The method involved the production of a fermented sauce, seasoning liquid from salmon that was enzymatically hydrolyzed with the wheat gluten koji, lactic acid bacteria and yeast. During fermentation, the total nitrogen, lactic acid, and ethanol contents in the fish sauce increased continuously and then plateaued 3 months after fermentation had begun. We investigated the chemical composition and the sensory properties of the fish sauce prepared by this 3-month fermentation, and we compared these characteristics with those of the fish sauce from salmon similarly hydrolyzed with soy sauce koji prepared from soybean and wheat. The fish sauce produced using wheat gluten was very light-colored and had a higher content of free amino acids, especially glutamic acid. The peptides consisted mainly of Glx, Asx and Pro, compared with Asx, Glx, and Gly in the liquid from soy sauce koji. Sensory evaluation revealed that the fish sauce derived from the wheat gluten koji had an intense umami taste and a fine flavor better than that of soy sauce koji.
Soy sauce was prepared with raw materials which were subjected to extrusion pre-treatment or traditional pre-treatment. Biochemical changes during the aging of the soy sauce mash were examined. Results revealed that after a 180-day aging period, the contents of total nitrogen, amino nitrogen, free amino acids and reducing sugars, and the protein utilization rate were higher in soy sauce prepared with extruded raw material than with traditional raw material. No marked difference in pH was noted between the two types of soy sauce prepared. However, a much higher increase in the intensity of brown color was noted in soy sauce prepared with extruded substrate than that prepared with traditional substrate. ©
The effects of pH, temperature, aeration, and residence time on the continuous production of 4-ethyl-guaiacol (4-EG), which is one of the characteristic aroma components in soy sauce, by immobilized cells of the salt-tolerant yeast Candida versatilis were investigated using an airlift reactor. The optimum pH and temperature were about 4.0 and 30–33°C, respectively. The amount of 4-EG in the liquid was constant even during alterations of nitrogen/air ratio in the supplied gas. A large amount of 4-EG (over 20 ppm) was produced at a residence time from 5 to 28 h and 1–3 ppm of 4-EG, which was the optimum concentration in conventional soy souce, was produced at a shorter residence time of 0.5 h. The 4-EG production by immobilized C. versatilis cells using the airlift reactor was stable for 40 d. It was found that the immobilized cell method was effective for the production of 4-EG by C. versatilis cells.
Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25degreesC. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of a-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30degreesC. The results obtained under various incubation conditions with (deepwell) microtiter plates were validated in Erlemneyer shake-flask cultures.
Growth of lactic acid bacteria and amino acid production at an Indonesian soy sauce manufacturer, employing modern Japanese process technology, indicated that brine fermentation for one month is sufficient for industrial kecap production. Compared to traditional Indonesian soy sauce fermentation, application of modern Japanese process technology resulted in an obvious but not essential yeast fermentation.