International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 2, No. 4, July 2012

Production of Enzyme and Growth of Aspergillus oryzae S.
on Soybean Koji
Chuenjit Chancharoonpong, Pao-Chuan Hsieh, and Shyang-Chwen Sheu
production [5].
In traditional fermentation, solid-state fermentation (SSF)
is suitable for fungi growth because of its low moisture
content and permitting of the penetration of fungi
mycelium through the solid substrates. Fungal mycelium
can penetrate into the solid substrate as 4 layers of mycelium
penetration. The first layer is areal hyphae, followed byaerobic wet hyphae and anaerobic wet hyphae, the last layer
is penetrative hyphae [6]. Low humidity in a solid-state
fermentation makes microorganisms more capable ofproducing certain enzymes and metabolites which usually
will not be produced in a submerge fermentation [6], [7].
As same as traditional soy sauce production, protease andamylase are also used to hydrolyze substrate in fish sauce
production. During fermentation, enzymatic digestion may
range from 6 months for small fish to 18 months for larger
fish. The liquid removed from the fermenting fish contains anabundance of peptides, amino acids, ammonia, volatile fatty
acids, and other nutrients are considered the highest quality
product [8], [9].
However, this process takes up to one to two years forfermentation. Accordingly, there are many acceleration
techniques, such as acid or alkaline hydrolysis, enzyme
degradation, and bacterial inoculation [10], [11]. The
acceleration of fish sauce fermentation will be advantageous
if the fermentation period can be reduced. In this study, weinvestigated the production of certain enzymes in kojicontaining mixture of soybean and wheat bran which havebeen used in soy sauce production and the potential to be
applied for accelerating fish sauce production. Moreover, thegrowth of A. oryzae S. during koji fermentation was also
investigated.

Abstract—Soybean koji is an important ingredient fortraditional fermented food in South-East Asia and East Asia. It
provides large amount of enzyme from koji mold, Aspergillus
oryzae S., to digest nutrients in substrates. This study aimed at
production of certain enzymes in soybean koji and potential tobe applied for accelerating fish sauce fermentation. Koji
containing 60% soybean was used as substrate to investigate the
enzyme production by A. oryzae S. The growth of this mold wasenumerated by potato dextrose agar. The mycelium
development of A. oryzae S. was observed by scanning electron
microscope. During koji production, pH of soybean koji
increased from 6.32 to 6.07. It was caused by extracellular
proteins production. The highest neutral protease, alkaline
protease and amylase activities were 84.38, 41.35 and 200 unit/gof dry weight, respectively. Moreover, growing of enzyme
activities on soybean koji correlated with the growth of thismold. Electron micrograph showed that spores of A. oryzae S.
were formed after 48 h of cultivation period. Additionally, thehighest enzymes activities were also shown in this stage.
Index Terms—Amylase, Aspergillus oryzae S., protease,soybean koji

I. INTRODUCTION
Koji is cooked wheat and/or soybean that has been
inoculated with a fermentation culture or koji mold. The firststep in making fermented foods, such as soy sauce, miso,
mirin, sake, and other fermented foods is creating the koji.
During the koji making, the koji mold (Aspergillus oryzae S.)
produces a variety of amylases and proteases to break downcarbohydrates and proteins in wheat and soybean [1]-[3].
A. oryzae S. is a filamentous fungus, which has an ability
to secrete large amounts of hydrolytic enzymes. It is widelyused in the manufacture of traditional fermented soy sauce inAsia. The extracellular proteins in soybean koji inoculatedwith A. oryzae S. contain different protein profiles including
neutral and alkaline protease, amylase, glutaminase, andmetallopeptidase [4]. Moreover, A. oryzae S. is genomicallywell characterized and considered to be a safe organism forproduction of food enzymes because it lacks expressedsequence tags for the genes responsible for aflatoxin

II. MATERIALS AND METHODS
A. Koji Preparation
Sixty percent of soybean was soaked in water for 6-8 hthen autoclaved at 121C for 40 min. Forty percent of wheatbran was roasted to dark brown and broken. The 32% of total
wheat bran was ground to powder and mixed with culture
before spreading to raw material to ensure mixing through ofraw material. Spores of A. oryzae S. NPUST-FS-206-A1(0.1%) were inoculated into raw material and incubated at
30C for 72h. Sheet cloth was sprayed with water andcovered on fermented matter to protect moisture evaporation
during cultivation. Koji was agitated every 12 h to released
heat.

Manuscript received April 26, 2012; revised May 29, 2012.
Chuenjit Chancharoonpong is with the Department of Tropical
Agriculture and International Cooperation, National Pingtung University of
Science and Technology, Pingtung 91201, Taiwan and Kasetsart University,
Bangkok 10900, Thailand (e-mail: csncjc@ku.ac.th).
P. C. Hsieh and S. C. Sheu are with the Department of Food Science,
National Pingtung University of Science and Technology, Pingtung 91201,
Taiwan (e-mail: pchsieh@mail.npust.edu, ssheu@mail.npust.edu.tw).

DOI: 10.7763/IJBBB.2012.V2.106

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E. Scanning Electron Microscope
Samples were fixed by 3% glutaraldehyde in 0.1 M
phosphate buffer (pH 7.0), dried by a critical point dryer(HCP-2, Hitachi, and Japan) and mounted on stub holders.
The samples were then coated with gold in a coater (E-1010,
Hitachi, Japan) and investigated on a scanning electron
microscope (Model S-3000N, Hitachi, Japan).

B. Enzyme Extraction
The fermented matter was mixed with 0.1 M phosphatebuffer (pH 6.9) (1:2 w/v) using a shaking incubator (150 rpm,30C, 30 min). The supernatant was collected as crude
enzyme extract by centrifugation at 10,000 xg, 4C for 15
min.
C. Analytical Methods
The moisture content of a fermented sample wasdetermined by infrared moisture determination balance(FD-720, Kett Electric Laboratory, Tokyo, Japan). The
fermented matter was mixed with deionized water (1:2 w/v)
by blender and the pH value was measured by pH meter(DKK-TOA, HM-25G, Japan).
Casein solution (0.65%) in 0.05 M phosphate buffer (pH6.9) and 0.05 M carbonate buffer (pH 10) were used as a
substrate to analyze neutral and alkaline protease activity,
respectively. One mL of crude enzyme extract and 5 mL ofcasein were mixed and incubated at 30C for 10 min. The
reaction was arrested by adding 5 mL of trichloroacetic acid
(TCA) and incubated for 30 min. After centrifugation at
10,000 xg for 10 min at 4C, the supernatant was collected.
The supernatant (2 mL) was reacted with 5 mL of 500 mM
Na2CO3 and 1 mL of five-fold diluted Folin-Ciocalteau
reagent for 10 min. Absorbance was read against a blank at
660 nm. One unit of enzyme activity was defined as theamount of enzyme that liberated 1 µg of tyrosine per minute
under assay conditions [12].
Amylase activity was measured with 1.0% soluble starch
in 0.1 M sodium acetate buffer (pH 5.0) as a substrate. One
mL of crude enzyme extract and 1 mL of soluble starch weremixed and incubated at 30C for 10 min. After incubation,
one mL of 3,5-dinitrosalicylic acid was added, reacted in aboiling water bath for 15 min, cooled to room temperature
and added with 9 mL of deionized water. The amylase
activity was analyzed spectrophotometrically at 540 nm. One
unit of enzyme activity was defined as the amount of enzymethat released 1 mM of reducing sugar as maltose per minute
under the assay condition [13]. The enzyme activity wasreported per gram of dry koji used in the initial extraction. All
the samples were analyzed in triplicate.

F. Statistical Analysis
Data was analyzed using statistical analysis system (SPSS,
version 13) for correlation analysis between microbial
growth and enzymes production.

Fig. 2. Change of neutral protease (●), alkaline protease (), andamylase activity (▲) during soybean koji production

Fig. 3. Change of total viable count (□), and mold (○) during soybean koji
Production

III. RESULTS AND DISCUSSIONS
A. Physical Properties of Koji
As shown in Fig. 1, the initial pH of koji was 6.32, then the
pH decreased to 6.12 after 24 h of cultivation. However, it
increased to 6.97 at the end of cultivation period. Similar
results have also been reported for A. oryzae in soy sauce kojicontaining 75% soybean koji that the increasing of pH of
fermented matter was due to the microbial metabolic
activities especially various extracellular proteins production[4].
Moisture in substrates brings a suitable water activity andswells substrates for mold growth [14]. The results showedthat the initial moisture content of koji was 40% thenincreased to 45% at 12 h cultivation. However, it decreased
to 22% after 72 h (Fig. 1). However, the high moisture
content at the beginning of fermentation period resulted indecreasing the porosity of substrates and reduction of heattransfer. Consequently, low enzymes activities at the
beginning of cultivation period (Fig. 2) might be caused by
the increasing of temperature (data not shown) [15].

Fig. 1. Change of pH () and moisture content (▲) during koji
production

D. Growth of Microorganism.
At different time intervals, koji samples were taken andblended with 0.1% peptone water for 20 sec. The growth of
microorganism was enumerated by pour plate techniqueusing plate count agar for total viable count and potato
dextrose agar for growth of mold.

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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 2, No. 4, July 2012

B. Enzyme Production
The changes of neutral protease, alkaline protease andamylase production during soybean koji production were
investigated (Fig. 2). Neutral protease activities on soybean
koji increased rapidly after 12 h of cultivation. At 48 h offermentation, soybean koji showed the highest neutral
protease activity at 84.38 unit/g of dry weight. However,reason of decreasing of neutral protease activity after 60 h
could be the limitation of nutrients and variation of pH (Fig.
1) [12]. On the contrary, alkaline protease activity was
detected only a very small amount during the first 24 h. Afterthat time, the activity was slightly increased; however it was
lower than those of neutral protease. The high activity ofalkaline protease was shown after 48 h of cultivation period.
It might be cause by increasing of pH of koji (Fig. 1). The
highest alkaline protease activity was shown at the end ofcultivation period at 41.35 unit/g of dry weight. Amylase
activity was rapidly increased after 24 h of cultivation period.
However, amylase activity remained for the last 24 h ofcultivation. Finally, it showed the highest activity for 200
unit/g of dry weight, at 72 h of cultivation period.
Additionally, the results indicated that increasing of pH after
48 h of cultivation period (Fig. 1) was due to the productionof various enzymes from A. oryzae S. These similar results
were explained that production of enzymes required aminoacids by digesting proteins in substrate. A. oryzae can use apool of nitrogen sources in soybean to produce extracellular
proteins. Soybean contains high content of nitrogen to be agood substrate to produce proteolytic enzymes. Moreover,the intensity of proteins was deduced by the intensity of timedependence by digesting nutrients from substrates [4], [12],[13].
C. Growth of A. oryzae S. During Koji Production
The growth of microorganism on soybean koji wasenumerated by plate count technique. The initial cultures onsoybean koji were 6.0 and 6.5 Log CFU/g of koji for moldand total count, respectively. It was shown that there were
few of bacteria contaminated from substrates at the beginningof cultivation. The growth of culture increased continuously
during soybean koji production then reached to 10 Log
CFU/g of koji at the end of cultivation period. Moreover, theresults indicated that the most of survival cells on soybean
koji were mold. The number of total viable count and molds
were also similar (Fig. 3).

correlated between microbial growth and enzyme
production.
Moreover, the growth of A. oryzae S. during koji
production was also observed by a scanning electron
microscope. As shown in Fig. 4, A. oryzae S. grewcontinuously during soybean koji production. There was no
spore forming of mold at the beginning of cultivation.
However, spore forming of mold was observed after 48 h.
The growth of mold also showed the contrasting relation withthe moisture content (Fig. 1). The decrease of moisture
content in koji is due to the utilization of water in substrate bymold to generate mycelia [14].
(a)

(b)

(c)

(d)

TABLE I: THE CORRELATION ANALYSIS BETWEEN MICROBIAL GROWTH
ANDNEUTRAL PROTEASE, ALKALINE PROTEASE, AND AMYLASEPRODUCTION
EnzymeCorrelation coefficients

Fig. 4. Scanning electron micrographs of mycelium propagation on soybean

Neutral protease
0.827*
Alkaline protease0.899**
Amylase0.925*** Correlation is significant at the 0.05 level (2-tailed).
** Correlation is significant at the 0.01 level (2-tailed).

koji fermentation, at 0 h (a), 24 h (b), 48 h (c) and 72 h (d)Furthermore, the increasing of enzyme activity was also
related to the growth of A. oryzae S. (Fig. 2). Additionally,
the results showed that neither high content of enzyme
activity nor spore forming of mold was observed at the
beginning of cultivation (24 h). However, both of spores
formation and the highest enzyme activities were found at
48 h of cultivation. It indicated that protease production was
followed by the development of mycelium. There was similar

The correlations between microbial growth and enzyme
production were analyzed (Table I). For neutral protease,correlation coefficient was 0.827 (P<0.05). Alkaline protease
and amylase activity showed high correlation coefficients togrowth. There were 0.899 and 0.925, respectively (P<0.01).
The results indicated that it was signiﬁcant positive230

International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 2, No. 4, July 2012
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results reported that growth of mold and enzyme production
was correlated. A. niger showed the correlation between
development of conidia and protease production during thefermentation period [16]. Moreover, our results indicated thathigh activities of various enzymes were also related tosporulation of mold (Fig. 2 and 4). The reason was reported
that the asexual cycle or spore forming is related to thesecondary metabolite production, such as enzyme andorganic acid [3].

IV. CONCLUSION
The results indicated that production of enzymes from A.
oryzae S. was related to the physical properties of soybean
koji during cultivation. Moreover, the growth of fungi
showed the high correlation with enzyme production.
Furthermore, development of mycelium was also related tothe increasing of enzyme production. Particularly, both thespore forming stage of A. oryzae S. and the highest enzyme
production were at 48 h of cultivation period. It cansummarize that fermented soybean koji at 48 h might be used
as an activated enzyme source to accelerate fish sauce
fermentation.
ACKNOWLEDGMENT
We would like to thank Dr. Tsair-Bor Yen from
Department of Tropical Agriculture and International
Cooperation, National Pingtung University of Science andTechnology for availing the electron microscope facility.
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