J. Trop. Biodiv. Biotech., Vol.

3 (2018), 8—17

Journal of Tropical Biodiversity and Biotechnology

journal homepage: http://jtbb.or.id

The production of corn kernel miso based on rice-koji fermented by

Aspergillus oryzae and Rhizopus oligosporus
Diah Ratnaningrum1*, Thelma Agustina Budiwati1, Tri Darsini2, Panji Cahya Mawarda1
1
Research Unit for Clean Technology, Indonesian Institute of Sciences, Jalan Cisitu/Sangkuriang, Bandung, 40135, Indonesia
2
Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sebelas Maret, Jl. Ir. Sutami No. 36A,
Jebres, Surakarta, 57126, Indonesia
*Corresponding author: [email protected]

ARTICLE INFO ABSTRACT

Article history: The suitability of corn kernel as raw material to produce miso fermented by rice-koji containing
Received 05/10/2017 Aspergillus oryzae and Rhizopus oligosporus has been investigated. The optimization was
Received in revised form 08/03/2018
conducted on two important factors in miso production namely mold composition in rice-koji
Accepted 10/03/2018
and salt concentration. The mold composition was prepared by inoculating the spores of 2%
Keywords: A. oryzae, 2% R. oligosporus, and 2% the mixture of both in a ratio of 1:1, 2:1, and 1:2 (v/v) into
Aspergillus oryzae
different rice media. The mold composition was optimized to produce rice-koji with high
corn kernel
miso α-amylase and protease activity. Different NaCl concentrations of 10%, 15%, and 20% were
rice-koji subjected to optimization process and added to each mixture after five days of fermentation.
Rhizopus oligosporus The salt concentration was also optimized to produce corn kernel miso with high glucose and
DOI: 10.22146/jtbb.28765 high dissolved protein concentration. The result showed that rice-koji containing A. oryzae and
© 2018 JTBB R. oligosporus in the ratio of 1:1 had the highest α-amylase and protease activity of 0.42 U/mL
and 0.45 U/mL respectively. In addition, the presence of 10% NaCl in corn kernel miso fermented
by A. oryzae and R. oligosporus in the ratio of 1:1 exhibited the highest glucose and dissolved
protein concentration of 0.64 mg/mL and 8.80 mg/mL respectively. The optimized corn kernel
miso by A. oryzae and R. oligosporus in the ratio of 1:1 with 10% NaCl was subjected to nutrient
content analysis and compared to the result before the corn kernel was fermented. The nutrient
content analysis showed nutrient enhancement after corn kernel was fermented and
transformed into a miso. Glucose, dissolved protein, and fat content increased 6.74, 1.34, 7.63
times respectively. This study concludes corn kernel could be utilized to produce a novel corn
kernel miso for dietary diversification and for improving nutritional and health status.

1. Introduction content characteristic. This food is usually made from koji, a

Miso (fermented soybean paste) is a traditional solid state fermented grain by Aspergilus oryzae served as
fermented food originally from Japan and has been used starter culture in addition to soybean, salt, and water
worldwide as a basic ingredient to cook soups as well as (Bourdichon et al., 2012).
seasoning for side dishes (Kusumoto and Rai, 2017). Miso has As a starter culture, koji has several types which are
several health benefits due to its bioactive compounds such determined by the substrates (soybean, barley, or rice) in
as peptides and isoflavones released during fermentation. which the koji molds grow. The most common type of koji
This food is capable of reducing cancer cell proliferation and used for miso production is rice-koji fermented by A. oryzae
possesses anti-diabetic, anti-hypertensive as well as which accounts 80% of miso production in Japan (Machida et
antioxidant properties (Hirota et al., 2000; Kwon et al., 2010; al., 2008). However, Aspergillus sojae, Aspergillus tamarii,
Zaid and El Shenawy, 2010; Shimizu et al., 2015; Kusumoto Aspergillus awamori, Aspergillus saitoi, Aspergillus kawachi,
and Rai, 2017). Miso has sweet, salty, and umami taste, and Rhizopus oligosporus have also been used in Japanese
presents in a paste-like half-solid food form with high protein food industries for the production of koji (Esaki et al., 1997;

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Lin et al., 2006). The molds in koji produce hydrolytic done to know the amount of glucose, dissolved protein, and
enzymes for instance proteases and amylases for degrading fat content in the optimized final product. The result of
biological polymers such as protein and carbohydrate. The nutrient content analysis was compared with the analysis
monomer produced from various enzymatic reactions conducted before the corn kernel was fermented to know
happened in the fermentation process gave sweet and whether the nutrients increased or decreased after
umami tastes of miso. These enzyme activities are optimized fermentation. This study would give an opportunity to
and controlled to yield optimum starter cultures for introduce a novel corn kernel miso for dietary diversification
fermentation by manufacturers (Marui et al., 2013). to improve nutritional and health status.
Beside koji, the type of raw material is important to
diversify the production of different miso types. In addition to 2. Materials and Methods
soybean, there are a lot of potential grains that could be 2.1 Microorganisms and Chemicals
utilized to produce miso. The use of other grains as raw Aspergillus oryzae and Rhizopus oligosphorus were
material substitute for producing miso could support dietary obtained from Research Unit for Chemistry, Indonesian
diversity to overcome nutrition and health status issues Institute of Science. Corn kernel and rice were purchased
(Fanzo et al., 2013). Dietary diversity directly influences diet from local store. (Hayden, 1923). Casein and Trichloroacetic
quality by guarantying adequate intake of essential nutrients acid (TCA) were purchased from Merck (Darmstadt, Germany)
and important non-nutrient factors. Research conducted by and used for determination of protease activity using Kunitz
Kennedy et al., (2007); Moursi et al., (2008); and Rah et al., method (Kunitz, 1950). The analytical grade of Lowry reagent
(2010) demonstrated dietary diversity has strong association was purchased from Merck (Darmstadt, Germany) and used
with nutritional status, particularly micronutrient density of for determination of dissolved protein concentration using
the diet. Therefore, substituting soybean with another type of Lowry method (Lowry, 1951). The analytical grade of Nelson
grain could offer another type of miso with different nutrient reagents and arsenomolybdate were purchased from Merck
composition and inherent health benefits. (Darmstadt, Germany) and used for determination of glucose
One of the most potential grains to substitute raw concentration using Nelson-Somogyi method (Nelson, 1944).
material of miso is corn kernel. The worldwide production of
corn is currently higher than soybean. In June 2017, the 2.2. Methods
production of corn was 1031.86 million metric tons whereas 2.2.1. Spores Suspension Preparation
the production of soybean was 344.67 million metric tons A. oryzae and R. oligosphorus were inoculated
(USDA, 2017). This higher availability renders corn more separately into different PDA and incubated at 30oC for seven
reliable for dietary diversification. Besides, the nutrient days. The spores of each mould were diluted in 0.09% NaCl,
composition of corn kernel is suitable for miso fermentation and the optical density of each pure culture was measured at
which consists of 68.40% carbohydrates, 3.62% protein, 600 nm using a spectrophotometer. The optical density was
1.42% oil (USDA, 2016). This data was based on the analysis set to 0.7 to obtain a cell concentration around 107 CFU/mL.
of corn kernel for the commodity yellow dent corn on a dry The total cell number was confirmed by plating them into
matter basis. Starch and protein contained by corn kernel can different PDA. The colony number of each medium was
be biochemically converted into essential amino acids, simple observed and counted after 24 h of incubation at 30 oC. One
carbohydrates, and a wide diversity of phenolic compounds colony formed is assumed coming from one spore.
that improves flavors, aromas, and textures of fermented
food products (Villeger et al., 2017). 2.2.2. Koji fermentation Process
Hence, the purpose of this study was to obtain the Rice was cleansed, washed, and mixed with distilled
composition of A. oryzae and R. oligosporus culture ratio, as water in a ratio of 1:1 (m/v). This mixture was sterilised at
well as NaCl concentration which can improve the nutritional 121oC for 15 minutes and cooled down to room temperature.
status of miso. The fermentation optimization results were The spores of 2% A. oryzae, 2% R. oligosphorus, and 2% the
evaluated with enzyme activity at the time of fermentation mixture of both in a ratio of 1:1, 2:1, and 1:2 (v/v) were
and nutritional status of miso. The optimization of salt inoculated into different rice media. Each medium was
concentration was also conducted and evaluated by incubated at 30oC for 48 h and dried inside the oven at 50 oC
measuring the concentration of glucose and dissolved protein for 24 h. Each dried culture was then subjected to a grinding
on each fermented corn kernel. Nutrient content analysis was process to obtain rice-koji in the form of powder.

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2.2.3. Corn Kernel Fermentation phosphate buffer pH 7.6 to casein solution and incubated for
Corn kernel was weight to give a mass of 100 g. Corn 20 min at 35oC. To deactivate the enzyme, 1.5 mL of 5% TCA
kernel was then cleaned, washed, sliced into pieces, soaked, was added to the mixture and incubated at room
and sterilised at 121oC for 15 minutes. After it was cooled temperature for 30 min. The blank solution was prepared by
down to room temperature, corn kernel was mixed with adding 1 mL of distilled water to casein solution and was
different rice-koji composition mentioned above. Each rice- treated the same as sample solution. Each solution was
koji added to corn kernel was 5% (m/m). Fermentation was centrifuged at 2500 rpm for 20 min in the temperature of 4 oC.
o
done at 30 C for five days inside an incubator. During five Each supernatant was collected and subjected to absorbance
days-fermentation, the sampling process was conducted. measurement at 280 nm using spectrophotometer. One unit
of protease activity was defined as the amount of enzyme
2.2.4. Enzymatic Activity Analysis that releases 1 µg of amino acid as tyrosine for 20 min at 35 oC
Fermented corn kernel of 10 g was sampled every day and expressed as units per mL supernatant.
and diluted to 100 mL of phosphate buffer. The suspension
was shaken at 120 rpm for 90 min and centrifuged at 2500 2.2.7. Optimization of Salt Concentration and Chemical
o
rpm in the temperature of 4 C for 20 min. The supernatant Analysis
was collected and subjected to α-amylase and protease Different salt concentrations of 10%, 15%, and 20%
enzymatic activity analysis. (m/m) were added to each fermented corn kernel after five
days of fermentation. The mixtures were subjected to further
2.2.5. α-Amylase Assay fermentation for eight weeks more. After eight weeks of
The solution containing seven mL of 1% soluble starch fermentation, fermented corn kernel of 10 g was sampled
o
in 0.1 M phosphate buffer (pH 7.0) was incubated at 40 C for and diluted to 100 mL of phosphate buffer. The suspension
5 min. The sample solution was prepared by adding 1 mL of was shaken at 120 rpm for 90 min and centrifuged at 2500
the supernatant as crude enzyme extract to the soluble rpm in the temperature of 4oC for 20 min. The supernatant
o
starch solution and incubated for 30 min at 40 C. was collected and subjected to glucose concentration analysis
Deactivation of the enzymes was done by adding 1.5 mL of using Nelson-Somogyi method (Somogyi, 1952) and dissolved
0.67 N sulfuric acid and 0.5 mL of 10% sodium tungstate to protein concentration analysis using Lowry method (Lowry et
the mixture. The blank solution was prepared by adding 1 mL al., 1951).
of distilled water to starch solution and was treated the same
as sample solution. Each solution was centrifuged at 2500 2.2.8. Nutrient content analysis
o
rpm for 20 min in the temperature of 4 C. Each supernatant The nutrient content analysis was done before the
was collected and subjected to reducing sugar analysis using corn kernel was fermented and in the final product of corn
Folin Wu method (Folin and Wu, 1919). Folin-Wu method was kernel miso that has been optimized. This was conducted to
based on the ability of reducing sugars to reduce alkaline compare the nutrient before and after fermentation process.
copper tartrate. The product of this reaction was used to The analysis includes glucose concentration analysis using
oxidise phosphomolybdic acid to yield a blue compound Nelson-Somogyi method (Somogyi, 1952), dissolved protein
measured using spectrophotometer at 420 nm. The result concentration analysis using Lowry method (Lowry et al.,
was compared with enzymatic activity of standard α-amylase 1951), and fat content analysis using soxhlet extraction
obtained through the same method. One unit of α-amylase method (Manirakiza et al., 2001).
activity was defined as the amount of enzyme that releases 1
µg of reducing sugar as glucose for 30 min at 40oC and 3. Results and Discussions
expressed as units per mL supernatant. 3.1. Physical properties of koji and Miso
Koji made from rice that has been steamed and
2.2.6. Protease Assay overgrown with mould spores (yeast tempeh) and then
The protease assay was performed based on the incubated for three days. During koji fermentation, various
method proposed by Kunitz (1971). The solution containing enzymes (proteases, amylases and lipases) will be produced
o
0.5 mL of 1% casein was incubated at 35 C for 20 min. The by moulds and have an important role in the process of
sample solution was prepared by adding 0.25 mL of the overhauling raw material macromolecules into simpler
supernatant as crude enzyme extract and 0.25 mL of molecules (Wahyuhapsari et al., 2013; Andarti et al., 2015).

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Figure 1. The enzymatic activity of α-amilase in rice-koji containing different mold composition.

The second stage of miso production is fermentation with a entirely competitive. This binding site is large enough to
mixture of salt, peanuts and koji to form a miso flavour. Miso accommodate two molecules of maltose. Hence, when
is shaped like peanut butter, or coloured peanut butter with a maltose concentration is high and a second molecule may
creamy yellowish, light brown, dark brown to blackish with bind at the active site, the affinity of this binding step is
texture is like peanut butter with salty tasted. Miso produces approximately 6.5-fold weaker.
a savoury and salty taste unique and commonly used as a The highest α-amylase activity was shown by rice-koji
spice on a variety of Japanese cuisine for example used as a containing A. oryzae and R. oligosporus in the ratio of 1:1.
sauce, soup sauce and soaked spices for pickles. This mixture gave α-amylase activity of 0.423 U/mL (Figure 1).
This result is similar to the study conducted by Takefuji et al.
3.2. The Activity of α-Amylase Enzyme (2016) who showed rice-koji containing A. oryzae and R.
Regarding α-amylase activity, the result showed each oryzae in the ratio of 1:1 had higher α-amylase activity
mold composition had different activity to hydrolyze starch compared to the other ratios. Figure 1 also showed A. oryzae
from corn kernel (Figure 1). All samples had similar profiles produced α-amylase enzyme more than R. oligosporus. This
where the activity increased from the first day until the can be attributed to the fact that the growth of Rhizopus
fourth day of fermentation and tend to diminish on the fifth species in steamed polished rice culture is delayed due to
day. This was possibly caused by decreasing nutrient in the inability of the microbes to utilize heat-denatured proteins
media and the stationary phase regulation leading to the contained in rice (Tanaka et al., 1982). Rhizopus strains also
reduction of molds’ metabolism rate. This restrains the lack carboxypeptidase activity which in turn makes them
formation of α-amylase enzyme as it has been thought to be incapable of breaking down heat-denatured proteins. In
a way for microorganisms to save energy (Coronado et al., addition, the study conducted by Harayama et al. (1989) also
2000). Moreover, the excessive amount of maltose as one of reported the α-amylase activity of R. oryzae IFO 5418 and R.
α-amylase hydrolysates is able to hedge the production of α- oligosporus NRRL 2710 was extremely weak in rice and
amylase enzyme. The presence of hydrolysates in a certain soybean-koji. Hence, the enzyme activity of koji containing A.
concentration could inhibit the activity of hydrolase enzyme oryzae was generally higher than the ones containing R.
itself through product inhibition mechanism (Qian et al., oligosporus with a higher ratio. The result showed during the
1995). This result was confirmed by Frederick et al. (2012) first few days of fermentation, the mixed culture exhibited
who found increasing maltose concentration could enhance lower α-amylase activity. This was probably because the
the dissociation constant for starch binding with the enzyme. growth of each mold in the mixed culture was still in the lag
The binding analysis shows the kinetic action of maltose is phase for adaptation process leading to less α-amylase

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enzyme production. Moreover, in the mixed culture, there The optimization study of mold composition showed
was competition between both molds to get nutrient from rice-koji containing A. oryzae and R. oligosporus in the ratio of
the media. Hence, A. oryzae and R. oligosporus in the ratio of 1:1 exhibited higher α-amylase and protease activities
2:1 and 1:2 exhibited lesser α-amylase activity compared to compared to the others. This result suggested controlling the
balance ratio of 1:1. spore numbers of A. oryzae and R. oligosporus could control
the production of α-amylase and protease enzymes.
3.3. The Activity of Protease Enzyme Furthermore, the data shown above also imply that
Other than α-amylase activity, protease activity of coculturing A. oryzae and R. oligosporus could raise the
each mold composition in rice-koji was also investigated. The production of α-amylase and protease enzymes. In solid-state
result showed all mold composition reached its maximum fermentation, these molds colonized corn kernel as the media
activity on the fourth day of fermentation (Figure 2). This through two types of penetrative hyphae. The long ones, also
finding conforms to other studies suggesting that highest called scouting hyphae are used to help the mold explore the
protease activity of R. olisgosporus and A. oryzae was usually composition of the medium and seek new colonization sites
obtained at 48 h of incubation and after (de Castro et al., across the medium. These hyphae are formed during the lag
2014; Han et al., 2003). The protease activity of R. oligosporus and exponential phase where there is still a significant
was higher than A. oryzae, however, the highest protease amount of O2 in the medium. Meanwhile, the short hyphae,
activity was shown by rice-koji containing A. oryzae and R. also called vegetative hyphae are used to anchor the colony
oligosporus in the ratio of 1:1. This mixture gave protease to the solid medium preventing the removal of the entire
activity of 0.455 U/mL (Figure 2). The same result was also colony due to physical disturbances like wind and agitation of
shown by Starzyńska-Janiszewska et al. (2015) who observed the fermentation bed. These hyphae are used to improve
the improvement of protein hydrolysis when the dose of A. substrate hydrolysis by secreting large quantities of hydrolytic
oryzae spores was equal and lower than that of R. oligosporus enzymes into the medium (Sugai-Guérios et al., 2016).
in inoculum. Yigzaw et al. (2004) also discovered the
improvement of amino acid profile when teff and grass-pea 3.4. The Optimization of Salt Concentration
were fermented by A. oryzae and R. oligosporus. This was Salt is one of main components to produce miso which
probably caused by the ability of A. oryzae to eliminate most also needs to be optimized. Different concentrations of salt
of protease inhibitors in soya bean increasing the degree of were added to the mixture in order to know which salt
proteolysis when it is mixed with R. oligosporus during the concentration produced higher concentration of glucose and
fermentation (Hong et al., 2004). protein as indicators for better fermentation result. The study
conducted by Ito et al. (2017) revealed an interesting fact

Figure 2. The protease activity in rice-koji containing different mold composition

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that the salt contained in miso did not increase blood possessed by R. oligosporus probably requires a higher
pressure and was not associated with hypertension in concentration of chloride ions to be active. Hence, the
humans. However, the reason remains unclear and the study production of glucose as one of the hydrolysis products was
still needs to be conducted further. The result showed when reduced when 10% of NaCl was applied in corn kernel miso
10% of NaCl was added to corn kernel, the one fermented by fermented by R. oligosporus. However, the glucose
A. oryzae: R. oligosporus in the ratio of 1:1 produced the concentration decreased when 20% of NaCl was added. High
highest glucose concentration of 0.64 mg/mL compared to concentration of salt could also inhibit the growth of the
the others (see Figure 3). This mold composition had the molds especially if both molds are not halophile microbes.
highest α-amylase activity as described above leading to the Therefore, when 20% NaCl was added to the mixture, the
highest glucose concentration. However, the concentration of production of α-amylase in each mold composition was
glucose in corn kernel miso fermented by those molds limited leading to lower production of glucose. This finding
decreased as salt concentration increased. The result also was in line with some scientific reports (Almansouri et al.,
showed the optimum salt concentration for each mold 2001; Dutta et al., 2006; Fentahun and Kumari, 2017).
composition was different. When corn kernel was fermented In addition to glucose, the protein concentration of
by R. oligosporus the sample reached its maximum glucose each corn kernel miso in different salt concentration was also
concentration in the presence of 15% NaCl. A similar result measured. The result showed corn kernel miso fermented by
was shown when A. oryzae and R. oligosporus in the ratio of A. oryzae: R. oligosporus in the ratio of 1:1 produced the
1:2 and 2:1 was used to ferment corn kernel. The maximum highest dissolved protein concentration of 8.8 mg/mL when
glucose concentration was also reached when 15% of NaCl 10% of NaCl was added to the mixture (Figure 4). This was
was added to the mixture. because according to the previous result, this composition
The result also showed all corn kernel miso had the had the highest protease activity. Moreover, all mold
lowest concentration of glucose in the presence of 20% NaCl compositions in this study reached maximum dissolved
except for R. oligosporus which had the lowest glucose protein concentration in the presence of 10% NaCl. The more
concentration when 10% NaCl was added to the mixture. In NaCl added to the mixture, the lower the protein
the case of R. oligosporus, this was probably caused by the concentration detected due to salting out and protease
low concentration of chloride ion in the 10% of NaCl which is denaturation (Su et al., 2005). High salt concentrations also
one of the allosteric activators for amylase enzyme (Kumar inhibit the growth of many microorganisms, which in turn
and Khare, 2015; Mohapatra et al., 1998; Obo and Ajele, greatly decreases the likelihood of the protease enzymes
1997). Chloride ions could bind and interact with catalytic production. These are responsible for protein hydrolysis to
residues and ultimately turn on -amylase activity (Maurus et form amino acids. Therefore, the result showed when salt
al., 2008; Williams et al., 2006). The amylase enzyme concentration increased to 15% and 20%, dissolved protein in

Figure 3. The effect of various salt concentration with ratio comparison of A oryzae dan R. oligosporus on glucose
concentration in corn kernel Miso

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Figure 4. The Interaction between various of salt concentration and composition A. oryzae and R. oligosporus on
protein concentration in corn kernel Miso

corn kernel miso decreased. This finding was similar to some process. The fat content of corn kernel miso also increased
reported articles saying proteolytic activity decreased as salt 7.63 times from 1.94% to 14.80% due to lipase activity
concentration increased (Dodia et al., 2006; Gardini et al., produced by R. oligosporus and A. oryzae which aids the
2001; Patel et al., 2005; Kurniawan et al 2008). Based on the hydrolysis of triglyceride to form fatty acids and glycerol. Fat
optimization results above, in the presence of 10% NaCl, corn content is an important factor to determine the aroma of
kernel miso fermented by A. oryzae and R. oligosporus in the miso since fatty acids are prone to oxidation which in turn
ratio of 1:1 produced highest glucose and dissolved protein form volatile compounds for specific aromas (Peinado et al.,
concentration. This mold composition has also been proven 2016).
to reduce 80% of the neurotoxin β-N-oxalyl-α,β-
diaminopropionic acid (β-ODAP) in fermented grass pea Table 1. Nutritional content Analysis of corn kernel Miso
(Yigzaw et al., 2004). fermented by A. oryzae: R. oligosporus in the ratio of 1:1
Samples
Indicator Corn kernel before Optimized corn
3.5. Nutrient Content Analysis
fermentation kernel Miso
Once mold composition and salt concentration have Dissolved protein
2.34 ± 0.07 16.03 ± 0.01
been optimized, the chosen corn kernel miso fermented by A. (mg/mL)
oryzae and R. oligosporus in the ratio of 1:1 with 10% NaCl Glucose (mg/mL) 0.36 ± 0.01 0.50 ± 0.04
Fat Content (%) 1.94 ± 0.02 14.85 ± 0.02
was subjected to nutrient content analysis and compared to
corn kernel before it was fermented. There were three
measured indicators including dissolved protein 4. Conclusion
concentration, glucose concentration, and fat content. This The corn kernel employed in this study was suitable to
was done to determine whether the nutrient content be used as a raw material for miso production. The
increased or decreased after fermentation. The result showed combination between A. oryzae and R. oligosporus in the
that all measured indicators increased after fermentation. ratio of 1:1 inside the rice-koji could yield highest α-amylase
The concentration of dissolved protein in corn kernel and protease activity of 0.42 U/mL and 0.45 U/mL
increased 6.74 times after fermentation from 2.38 mg/mL to respectively. The addition of 10% NaCl influenced better
16.03 mg/mL (Table 1). Dissolved protein of corn kernel miso fermentation results as the glucose and dissolved protein
was concentrated due to water loss in the drying process and concentrations were the highest measured in the miso
increased due to protease activity produced by A. oryzae and fermented by the optimised rice-koji. The glucose and
R. oligosporus. Meanwhile the glucose concentration dissolved protein concentration measured in the optimised
increased 1.34 times from 0.37 mg/mL to 0.49 mg/mL due to product was 0.64 mg/mL and 8.80 mg/mL respectively. These
amylase activity by those molds during the fermentation data imply increased concentration of glucose, protein, α-

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amylase activity, and protease activity could be obtained by Dutta, T. K., Jana, M., PAHARI, P. R., & Bhattacharya, T., 2006,
coculturing mould composition and controlling salt The effect of temperature, pH, and salt on amylase in
concentration. The nutrient content enhanced as shown by Heliodiaptomus viduus (Gurney) (Crustacea: Copepoda:
glucose, dissolved protein, and fat content in corn kernel Calanoida), Turkish Journal of Zoology 30 (2), 187-195.
miso was higher than corn kernel before it was fermented. To Esaki, H., Onozaki, H., Kawakishi, S., & Osawa, T., 1997,
the best of our knowledge, this is the first report of miso Antioxidant activity and isolation from soybeans
production from corn kernel. Further research should be fermented with Aspergillus spp, Journal of Agricultural
focused on the organoleptic study and bioactivity studies of and Food Chemistry 45(6), 2020-2024.
the corn kernel miso for the exploration of health benefits. Fanzo, J., Hunter, D., Borelli, T., & Mattei, F., 2013,
Diversifying food and diets using agricultural
Acknowledgement biodiversity to improve nutrition and health. New York:
This work was supported by Indonesian Institute of Biodiversity International.
Sciences. The authors would like to thank to Chelsie Kennedy Fentahun, M., & Kumari, P. V., 2017, Isolation and screening
from UK Defense Science and Technology Laboratory and Tim of amylase producing thermophilic spore forming
O’Brien from NYC Office of the Chief Medical Examiner for Bacilli from starch rich soil and characterization of
their useful comments and suggestions from a linguistic point their amylase activity, African Journal of Microbiology
of view. Research 11(21), 851-859.
Folin, O., & Wu, H., 1919, A system of blood analysis, J Biol
References Chem. 38(1), 81-110.
Gardini, F., Martuscelli, M., Caruso, M.C., Galgano, F.,
Almansouri, M., Kinet, J. M., & Lutts, S., 2001, Effect of salt Crudele, M.A., Favati, F., Guerzoni, M.E. & Suzzi, G.,
and osmotic stresses on germination in durum wheat 2001, Effects of pH, temperature and NaCl
(Triticum durum Desf.), Plant and Soil 231 (2), 243-254. concentration on the growth kinetics, proteolytic
Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J.C., Gerds, activity and biogenic amine production of
M.L., Hammes, W.P., Harnett, J., Huys, G., Laulund, S., Enterococcus faecalis, International Journal of Food
Ouwehand, A., & Powell, I.B., 2012, Food Microbiology 64(1), 105-117.
fermentations: microorganisms with technological Han, B. Z., Ma, Y., Rombouts, F. M., & Nout, M. R., 2003,
beneficial use, International Journal of Food Effects of temperature and relative humidity on
Microbiology 154 (3), 87-97. growth and enzyme production by Actinomucor
Coronado, M. J., Vargas, C., Hofemeister, J., Ventosa, A., & elegans and Rhizopus oligosporus during sufupehtze
Nieto, J. J., 2000, Production and biochemical preparation, Food Chemistry 81(1), 27-34.
characterization of an α-amylase from the moderate Hirota, A., Taki, S., Kawaii, S., Yano, M., & Abe, N., 2000, 1, 1-
halophile Halomonas meridian, FEMS Microbiology Diphenyl-2-picrylhydrazyl radical-scavenging
Letters 183(1), 67-71. compounds from soybean Miso and antiproliferative
de Castro, R. J. S., & Sato H. H., 2014, Production and activity of isoflavones from soybean Miso toward the
biochemical characterization of protease from cancer cell lines, Bioscience, Biotechnology, and
Aspergillus oryzae: an evaluation of the physical– Biochemistry 64 (5), 1038-1040.
chemical parameters using agroindustrial wastes as Hong, K. J., Lee, C. H., & Kim, S. W., 2004, Aspergillus oryzae
supports, Biocatalysis and Agricultural Biotechnology 3 GB-107 fermentation improves nutritional quality of
(3), 20-25. food soybeans and feed soybean meals, Journal of
Dodia, Mital S., Joshi, Rupal H., Patel, Rajesh K., Singh, & Medicinal Food 7(4), 430-435.
Satya P., 2006, Characterization and stability of Kennedy, G. L., Pedro, M. R., Seghieri, C., Nantel, G., &
extracellular alkaline proteases from halophilic and Brouwer, I., 2007, Dietary diversity score is a useful
alkaliphilic bacteria isolated from saline habitat of indicator of micronutrient intake in non-breast-feeding
coastal Gujarat, India. Brazilian Journal of Filipino children, The Journal of Nutrition 137(2), 472-
Microbiology 37(3), 276-282. 477.

15
J. Trop. Biodiv. Biotech., Vol. 3 (2018), 8—17

Kumar, S., & Khare, S. K., 2015, Chloride activated halophilic α Moursi, M. M., Arimond, M., Dewey, K. G., Trèche, S., Ruel,
-amylase from Marinobacter sp EMB8: production M. T., & Delpeuch, F., 2008, Dietary diversity is a good
optimization and nanoimmobilization for efficient predictor of the micronutrient density of the diet of 6-
starch hydrolysis, Enzyme Research 2015. to 23-month-old children in Madagascar, The Journal
Kunitz, M. T., 1947, Crystalline soybean trypsin inhibitor, The of Nutrition 138(12), 2448-2453.
Journal of General Physiology 30(4), 291-310. Oboh, G., & Ajele, J. O., 1997, Effects of some metallic
Kusumoto, K. I., and Rai, A. K., 2017, ‘Miso, the Traditional chlorides on the activity of ß-amylase from sweet
Fermented Soybean Paste of Japan’ in Ray, R.C. and potatoes, Nigerian, Journal Biochemistry Molecular
Montet, D., (Eds.), Fermented Foods, Part II: Biology 12, 73-75.
Technological Interventions, Florida: CRC Press. Patel, R., Dodia, M., Singh, & S. P., 2005, Extracellular alkaline
Kwon, D. Y., Daily, J. W., Kim, H. J., & Park, S., 2010, protease from a newly isolated haloalkaliphilic Bacillus
Antidiabetic effects of fermented soybean products on sp.: Production and optimization, Process Biochemistry
type 2 diabetes, Nutrition Research 30(1), 1-13. 40(11), 3569-3575.
Lin, C. H., Wei, Y. T., & Chou, C. C, 2006, Enhanced Peinado, I., Miles, W., & Koutsidis, G., 2016, Odour
antioxidative activity of soybean koji prepared with characteristics of seafood flavour formulations
various filamentous fungi, Food Microbiology 23(7), produced with fish by-products incorporating EPA,
628-633. DHA and fish oil, Food Chemistry 212, 612-619.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J., Qian, M., Haser, R., & Payan, F., 1995, Carbohydrate binding
1951, Protein measurement with the Folin phenol sites in a pancreatic α‐amylase‐substrate complex,
reagent, Journal of Biological Chemistry 193(1), 265- derived from X‐ray structure analysis at 2.1 Å
275. resolution. Protein Science 4(4), 747-755.
Machida, M., Yamada, O., & Gomi, K., 2008, Genomics of Rah, J.H., Akhter, N., Semba, R.D., De Pee, S., Bloem, M.W.,
Aspergillus oryzae: learning from the history of Koji Campbell, A.A., Moench-Pfanner, R., Sun, K., Badham,
mold and exploration of its future, DNA Research 15 J., & Kraemer, K., 2010, Low dietary diversity is a
(4), 173-183. predictor of child stunting in rural
Manirakiza, P., Covaci, A., & Schepens, P., 2001, Comparative Bangladesh, European Journal of Clinical Nutrition 64
study on total lipid determination using Soxhlet, Roese (12), 1393-1398.
-Gottlieb, Bligh and Dyer, and modified Bligh and Dyer Shimizu, N., Du, D. D., Sakuyama, H., Ito, Y., Sonoda, M.,
extraction methods, Journal of Food Composition and Kawakubo, K., & Uehara, Y., 2015, Continuous
Analysis 14(1), 93-100. Subcutaneous Administration of Miso Extracts
Marui, J., Tada, S., Fukuoka, M., Wagu, Y., Shiraishi, Y., Attenuates Salt-Induced Hypertension in Dahl Salt-
Kitamoto, N., Sugimoto, T., Hattori, R., Suzuki, & S., Sensitive Rats, Food and Nutrition Sciences 6(8), 693.
Kusumoto, K.I. (2013). Reduction of the degradation Somogyi, M, 1952, Determination of reducing sugars by
activity of umami-enhancing purinic ribonucleotide Nelson–Somogyi method. Journal Biological Chemistry
supplement in Miso by the targeted suppression of 200, 245.
acid phosphatases in the Aspergillus oryzae starter Starzyńska-Janiszewska, A., Stodolak, B., & Wikiera, A., 2015,
culture, International Journal of Food Microbiology 166 Proteolysis in tempeh-type products obtained with
(2), 238-243. Rhizopus and Aspergillus strains from grass pea
Maurus, R., Begum, A., Williams, L. K., Fredriksen, J. R., Zhang, (Lathyrus sativus) seeds, Acta Scientiarum Polonorum
R., Withers, S. G., & Brayer, G. D., 2008, Alternative Technologia Alimentaria 14(2), 125-132.
catalytic anions differentially modulate human α- Su, N. W., Wang, M. L., Kwok, K. F., & Lee, M. H., 2005, Effects
amylase activity and specificity, Biochemistry 47(11), of temperature and sodium chloride concentration on
3332-3344. the activities of proteases and amylases in soy sauce
Mohapatra, B. R., Banerjee, U. C., & Bapuji, M., 1998, koji, Journal of Agricultural and Food Chemistry 53(5),
Characterization of a fungal amylase from Mucor sp. 1521-1525.
associated with the marine sponge Spirastrella
sp, Journal of Biotechnology 60(1), 113-117.

16
 J. Trop. Biodiv. Biotech., Vol. 3 (2018), 8—17

Sugai-Guérios, M. H., Balmant, W., Krieger, N., Junior, A. F., & Villeger, R., Cachon, R., & Urdaci, C.M., 2017, ‘Fermented
Mitchell, D. A., 2016, Colonization of solid particles by Foods, Microbiology, biochemistry, and
Rhizopus oligosporus and Aspergillus oryzae in solid- Biotechnology’, in: RC. Ray and D. Montet, (Eds.),
state fermentation involves two types of penetrative Fermented Foods, Part II: Technological Interventions,
hyphae: A model-based study on how these hyphae pp 1-20, CRC Press, Florida
grow, Biochemical Engineering Journal 114, 173-182. Watson, S. A., 2003, ‘Description, development, structure and
Takefuji, H., Ninomiya, J., & Morita, H., 2016, ‘Improving the composition of the corn kernel’ in: J. White, and L.
Yield of Glucoamylase and [alpha]-amylase in Solid- Johnason, (Eds.), Corn: Chemistry and Technology, pp
state Co-culture’, Hong Kong Chemical, Biological & 69−101, American Association of Cereal Chemists, St.
Environmental Engineering Society: 3rd International Paul.
Conference on Chemical and Food Engineering, MATEC Williams, D. N., Ehrman, S. H., & Holoman, T. R. P., 2006,
62 Proceeding, Tokyo, Japan, April 8-9, 2016, pp. 1-6 Evaluation of the microbial growth response to
Tanaka, T., Okazaki, N., & Kitani, M., 1982, Growth of mold on inorganic nanoparticles, Journal of
uncooked grains: Comparison of growth and enzyme Nanobiotechnology 4(1), 3.
production between Aspergillus oryzae and Rhizopus Yigzaw, Y., Gorton, L., Solomon, T., & Akalu, G., 2004,
sp, Journal of the Brewing Society of Japan 77(11), 831- Fermentation of seeds of Teff (Eragrostis teff), grass-
835. pea (Lathyrus sativus), and their mixtures: aspects of
United States Department of Agriculture (USDA) & Food nutrition and food safety, Journal of Agricultural and
Agricultural Service (FAS), 2017, ‘World Agricultural Food Chemistry 52(5) 1163-1169.
Production’, in FAS Website, viewed 17 August 2017, Yu, K. W., Lee, S. E., Choi, H. S., Suh, H. J., Ra, K. S., Choi, J. W.,
from https://apps.fas.usda.gov/psdonline/circulars/ & Hwang, J. H, 2012, Optimization for rice koji
production.pdf preparation using aspergillus oryzae CJCM-4 isolated
United States Department of Agriculture (USDA), 2016, from a korean traditional meju, Food Science and
‘National Nutrient Database for Standard Reference’ Biotechnology 21(1), 129-135.
in US Department of Agriculture, Agriculture Research Zaid, A. A. & El-Shenawy, N. S, 2010, Effect of Miso (A
Service Website Release 28 Website, viewed 12 March soybean fermented food) on some human cell lines;
2018, from https://ndb.nal.usda.gov/ndb/foods/show/ HEPG2, MCF7 and HCT116, Journal of American
Science 12, 6.

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