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Study on the Production of Fermented Soybean Sauce by Using Aspergillus

oryzae and Aspergillus flavus

Article · March 2013

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VOLUME 2 ISSUE 2
ISSN: 2230-4818

JOURNAL OF SCIENTIFIC & INNOVATIVE RESEARCH

ORIGINAL RESEARCH ARTICLE

Study on the Production of Fermented Soybean Sauce by Using Aspergillus

oryzae and Aspergillus flavus

Tin Mar Lynn *1, Kyaw Nyein Aye 2, Khin Mar Khaing 2
1. Department of Biotechnology, Mandalay Technological University, Mandalay, Myanmar
2. Department of Chemical Engineering and Biotechnology, Yangon Technological University,
Myanmar

ABSTRACT

Soy sauce has been made for centuries by traditional methods, and consumed as the source of protein and vitamins.
In this research work, the two fermenting microbes Aspergillus oryzae and Aspergillus flavus were mainly employed
to produce soya sauce. Aspergillus oryzae was isolated from moldy soybeans. Also, Aspergillus flavus strain was
received from the Department of Biotechnology, Yangon Technological University, Myanmar and recharacterized to
confirm the correct strain. Four types of soy sauce products obtained from separated fermentation of each strain
within two periods of different fermentation time (1.5 months and 3 months) and were analyzed for protein content,
fat content, reducing sugar content, and alcohol content respectively after fermentation. Analysis of commercial soy
sauce product from local market was also done as a comparative study for locally strain produced products. Finally,
amino acid composition of each product was detected by the Thin Layer Chromatographic method as a part of
research work. By comparing the physical and chemical analysis results, soy sauce produced by A. oryzae, brine
ageing time 3 months had the best quality.

Keywords: Soy sauce, Aspergillus oryzae, Aspergillus flavus, Thin Layer Chromatography, Brain ageing.

1. INTRODUCTION every time. Protein is an important type in a

Nowadays, the world’s population increases rapidly balanced diet. Soybean product has a large amount

every day. Also, food demand is increasing as of protein content, and so it was consumed as the
protein source. Soy sauce contains essential amino
Address for correspondence: acids such as Valine, Tryptophan, Lysine, and
Tin Mar Lynn*
Department of Biotechnology, Mandalay Histidine and also contains vitamins (especially
Technological University, Mandalay, Myanmar vitamin B6) and antioxidants (isoflavones).1
E-mail: light.lynn@gmail.com

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Indeed, soy sauce has been produced for centuries from the moldy soybean sauce. The strain
under natural conditions. Soy sauce is one of the characterization of receiving Aspergillus flavus
world’s oldest condiments and has been used in strain was examined. The fermenting activities on
China for more than 2,500 years. Soy sauce is a the main substrate soybean were studied on two
hydrolysis product of the soybean. Soy sauce is a conditions: 1.5 months fermentation time and 3
dark brown liquid with a pleasant aroma, used month period, respectively. Microbial safety of the
primarily as flavoring agents for meat, poultry, fish, end products was also examined in this work. The
vegetables and rice. Its high salt content of about presence of some amino acid in each produced soy
18% and distinct flavor makes it a useful adjunct sauce was also displayed by the Thin Layer
for many of the bland food products in which it is Chromatography method.
used.
The objectives of this research work are: to
The preparation of soy sauce is known to involve promote the role of soybean food consumption, to
the action of molds, yeasts, and bacteria such as improve the traditional methods of soybean food
Aspergillus oryzae, Zygosaccharomyces soya, and production and to study and investigate the quality
Lactobacillus species. Yokosuka and Sasaki2 stated improvement of soy sauce by using Aspergillus
that Aspergillus oryzae and A. soyae are used by oryzae.
manufacturer. These fungal Aspergilli species are
2. MATERIALS AND METHODS
widely found in various sources. Some species are
food grade species and others are pathogenic for 2.1. Materials
human beings. Aspergillus oryzae has been used in
2.1.1. Media and Chemicals
soybean fermentation for several years and it was
known as koji mold. Culture media, media ingredients and other
chemicals used were from “Australia Medical
Fermentation increases protein content, eliminates
Diagnostics (AMD) Co., Ltd”, “HiMedia
trypsin inhibitors, and reduces the peptide size in
Laboratories Limited, Mumbai, India”, and
soybean meals. These effects of fermentation might
Analytical grade chemicals available from local
make soy foods more useful in human diets as a
markets have also been used.
functional food and benefit livestock as a novel
feed ingredient. 2.1.2. Microorganisms

In this research study, emphasis was made on the Aspergillus oryzae were isolated from moldy
isolation and identification of Aspergillus oryzae soybeans. Aspergillus flavus were isolated from the

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chili. These two microorganisms are used for the To identify the colony morphology, A. oryzae were
production of soy sauce. Strain recharacterization incubated on Czapek`s Dox Agar, Sabouraud
of Aspergillus flavus. The flavours were also done Dextrose Agar, Potato Dextrose Agar and Malt
to confirm the correct strain. Agar. And then colonies were observed by colony
size, color, reverse position and growth pattern.
2.1.3. Raw Materials for Soy Sauce Production
The only one biochemical test for Aspergillus
12 kg soybeans, 12 kg roasted wheat flour and 5 kg
oryzae is Kojic acid test. Among the Aspergillus
salt were used for soy sauce production. They were
species, A. albus, A. candidus, A. nidulans, and A.
bought from the local market in Yangon.
oryzae produce Kojic acid.
2.2. Methods
2.2.3. Soy Sauce Production
2.2.1. Isolation of Aspergillus oryzae
An amount of 2 kg soybeans was soaked in water
The agar plate method was used to isolate for overnight. Next, the soaked soybeans were
Aspergillus colonies occurring in moldy soybeans. cooked in autoclave at 121˚C with 15 psi for 30
The moldy soybeans were placed on Czapek`s Dox minutes. After that they were drained for 3-4 hours
Agar, introduction 4 each in 9 cm Petri dishes. The in a contamination free area. And then they were
plates were incubated at room temperature for 1 to mixed with 2 kg of wheat flour. Finally,
7 days. After incubation for 48 hours, the plates Aspergillus oryzae were added to the mixture of
were observed in young fungus colonies. soybeans and wheat flour by spore count method.
The inoculum size was 2.4× 108. The incubation
2.2.2. Identification of Aspergillus oryzae
period was 5 days. The same steps were made for
Aspergillus oryzae can be identified by colony the production of soy sauce by Aspergillus flavus.
morphology and microscopic characteristics. Hypo-
After 5 days of fermentation by fungi, the mixtures
septate, reproductive organs, the structure of
were fermented by salt brine. This was called the
fruiting organs, details of spores of the fungus were
brine fermentation stage. The brine fermentation
observed and matched with the synoptically keys of
was stirred at intervals of 2 days to supply more
Thom and Church (1926), Thom and Raper (1945)
oxygen into the glass containers during brine
and Raper and Fennell (1965).3-5 Measurements of
ageing. After ageing for 1-3 months, the soy sauce
vegetative and reproductive parts of the fungus
was filtered and pasteurized at 70˚C for 30 minutes.
were also taken from the state micrometer set.
And then soy sauce was ready for consumption.

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Four types of soy sauces were produced in this incubated at 30˚C for one day and the individual
experiment for three replicates. They were soy colonies developed on the nutrient agar were
sauce produced by A. oryzae (brine ageing time 1.5 examined for microbial safety of the products.
months), soy sauce produced by A. oryzae (brine
2.2.5. Separation and Identification of Amino
ageing time 3 months), soy sauce produced by A.
Acids in Soy Sauces by One Dimensional TLC
flavus (brine ageing time 1.5 months), soy sauce
produced by A. flavus (brine ageing time 3 Preparation of solvent system
months). Each of these soy sauces was measured by
The solvent system was prepared by mixing n-
the appropriate parameters to determine the product
butanol (40 ml), acetic acid (10 ml) and water (10
qualities. One type of soy sauce from local market
ml).
was also measured by the above parameters to
compare the qualities. Preparation of standard amino acid solution

Physical Analysis Standard solutions of nine different amino acids

were prepared in 75% ethanol (v/v) with the
(1) pH measurements
addition.
(2) Viscosity measurements
Preparation of 0.2 % ninhydrin reagent
Chemical Analysis
Ninhydrin powder (0.2 gram) was dissolved in 100
(1) Protein analysis ml acetone.

(2) Fat analysis Preparation of Sample

(3) Determination of reducing sugar by Lane and 5 ml of sample was put into the separation funnel.
Eynon method 5ml of n-butanol was added into the funnel and
shaken vigorously. The layers were separated by
(4) Alcohol analysis
placing in standing position for 15 minutes. Then
(5) Salt analysis the upper layer was taken to use for TLC method.6-8

2.2.4. Microbial Safety for Soy Sauce Products 3. RESULTS

Each aliquot with 25µl of four types of soy sauce The isolation of A. oryzae was done by the agar
sample was spread on the nutrient agar before and plate method in Czapek’s Dox agar, selective agar.
after pasteurization. The plate was inverted and The rate of growth was very rapid. The microscopic

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morphology and cultural characteristics of A. The pH and viscosity of soy sauces were measured
oryzae and A. flavus was shown in Figure 1, 2, 3 and the results were described in Table 2.
and 4. The species was identified by comparing
3.4. Chemical Analysis
with the reference (Raper and Fennel, 1965).5
The protein%, fat%, reducing sugar% and
The colony morphology of A. oryzae was
alcohol% of soy sauces were measured in this
examined by plating on four different media and
experiment and the results were shown in Table 3.
the results were shown in Table 1.
The salt content of soy sauces was described in
3.1. Biochemical Characteristics
Table 4.
Red blood color was obtained by the filtrates of A.
3.5. Microbial Safety of Soy Sauce Products
oryzae and that of Aspergillus flavus. The flavors
were not changed in Kojic acid test. On account of Some colony of Bacillus species were observed in
these character the specimen was identified before pasteurized sample. But, no microorganism
tentatively as Aspergillus oryzae. was observed in after pasteurization sample.

3.2. Soy Sauce Production 3.6. Separation and Identification of Amino

Acids in Protein Hydrolysate of Soy Sauces by
Four types of soy sauces were produced in this
TLC
experiment. The yield of the finished products was
90% of the salt brine. By the comparison of Rf values of individual
standard amino acids and sample hydrolyzate
3.3. Physical Analysis
amino acids, the results were shown in Table 5.

Table 1: Colonial Morphology and Growth Characteristics of A. oryzae on Different Media

Types of Growth pattern Size of Surface colony Reverse colony

media colony color color
(inch)
Czapek’s Growth rate rapidly 2 Brownish yellow White
Dox agar
SDA More rapidly of growth 3 Dark yellow Brownish yellow
PDA Growth rate moderately, 1.5 Pale yellow Yellowish brown
(with other contaminants)
Malt Growth rate slowly, present 1 yellow Yellowish brown
Extract many contamination
agar

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Table 2: pH and Viscosity Measurements of Soy Sauces at Room Temperature

Types of pH Viscosity
Soy Sauces (Centipoises)
Sory(1.5) 5.14 1.6

Sory(3) 4.84 1.739

Sfla(1.5) 5.21 1.6

Sfla(3) 4.99 1.922

S(Wai Weng) 4.7 3.561

Table 3: Protein%, Fat%, Reducing Sugar%, Alcohol% of Soy Sauce

Types of Protein % Fat % Reducing Sugar Alcohol %

Soy Sauces %

Sory(1.5) 2.98±0.18 0.99±0.175 4.15±0.26 0.99

Sory(3) 3.88±0.29 0.048±0.007 5.3±0.42 1.00

Sfla(1.5) 2.43±0.37 0.99±0.21 2.71±0.36 0.99

Sfla(3) 3.43±0.55 0.032±0.009 3.1±0.27 0.99

S(Wai Weng) 2.05±0.0.14 0.077±0.008 9.56±0.85 0.33

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Table 4: Salt Content of Soy Sauce Products

Types of Salt content

Soy Sauces (ppm)

Sory(1.5) 140
Sory(3) 155
Sfla(1.5) 140
Sfla(3) 150
S(Wai Weng) 160

Table 5: Rf values of Individual Standard Amino Acids and Amino Acids in the Protein Hydrolyzate
of Soy Sauces

Rf values Rf values of Rf values Rf values for

No Amino Acids (standard) A. flavus of Wai Weng
products A.oryzae
product
1 Valine 0.396 0.396 0.385 0.396
2 Threonine 0.231 0.233 - -
3 Phenylalanine 0.462 - - -
4 Lysine 0.044 - 0.032 -
5 Isoleucine 0.495 - - -
6 Histidine 0.055 - 0.088 0.088
7 Leucine 0.593 - - -
8 Methionine 0.407 - - -
9 Tryptophan 0.516 0.516 0.516 -

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Figure 1: Microscopic Morphology of A. oryzae Figure 2: Microscopic Morphology of A. flavus

Figure 3: A. oryzae on Czapek`s Dox agar Figure 4: A. flavus on Czapek`s Dox Agar

4. DISCUSSION confirm the correct strain of Aspergillus oryzae.

Moreover, according to Table 1, A. oryzae grow
To obtain the pure culture of Aspergillus oryzae
rapidly in Czapek’s Dox agar and Sabouraud
was one of the main research works. Czapek’s Dox
dextrose agar. But, potato dextrose agar and malt
medium is a selective medium and were continually
extract agar gave slower growth rate. This may be
employed in subculture preparation until pure
due to other contaminating microbes which inhibit
culture was obtained. Morphological characteristics
the growth of A. oryzae. In the comparison of
such as growth rate, colony size, color and
conidiophore formation etc. were enough to
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colony size, the smallest colonial size was found in comprises only the two amino acids (Valine and
malt agar. Histidine). So, A.oryzae product had more suitable
for commercial production as a human diet. Soy
In the study of biochemical test with Kojic acid
sauce produced by A. oryzae contains more amino
test, only A. oryzae changed into red blood color. A.
acids than soy sauce produced by A. flavus because
flavus remained without any changes. From this
A.oryzae produced peptidase and proteinase than A.
point, we can distinguish the species identification
flavus.
of A. oryzae and A. flavus fungal strain.
By comparing the brine ageing time (1.5 months
According to the observed data in Table 2, the
and 3 months), better results were obtained from 3
surest we say was that the smaller the pH value, the
months. So, brine ageing time was also important
higher the viscosity in soy sauce products. But, all
for quality improvement of soy sauce.
pH values existed in the specified range of soy
sauce products (within 4.6-5.2). In viscosity 5. CONCLUSION
measurements, Sory (1.5) and Sfla (1.5) had the same
The two fermented fungal strains A. oryzae and A.
value in each product.
flavus were mainly employed throughout this study.
In the study of protein contents, Table 3 indicated Not only morphological, microscopical and
that Sory (3) had better protein content than that of biochemical test but also culture test on four
others. Moreover, its fat % and reducing sugar % different media were studied. Analysis of soy sauce
were reliable for commercial production. product was performed by determining pH,
viscosity, and protein %, fat %, reducing sugar %,
In the study of salt content described in Table 4,
alcohol %, salt content and finally amino acid
Sory (1.5) and Sfla (1.5) had the same content of 140
composition. Produced soy sauces Sory (1.5) and Sfla
ppm respectively.
(1.5) had better viscosity activities (1.6 cp) and Sory
As a part of research work, the presence of amino (3) had more protein content (3.88) than that of the
acids in soy sauce products was detected by Thin other types. In A. flavus fermented product, only
Layer Chromatographic technique. According to three amino acids (Valine, Threonine and
Table 5, only three amino acids (Valine, Threonine Tryptophan) were found. A. oryzae fermented soy
and Tryptophan) were present in A. flavus product. sauce had four amino acids composition (Valine,
Aspergillus oryzae product contributes four amino Lysine, Histidine and Tryptophan). But, in the
acids (Valine, Lysine, Histidine and Tryptophan). analysis of commercial product (Wai Weng), only
However, commercial “Wai Weng” product two amino acids (Valine and Histidine) were found.
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Analysis of amino acid composition was performed 4. Thom, C., and Church, M.B. “The Aspergilli”.
by one dimensional Thin Layer Chromatographic Copyright. The Willams and Wilkins Company.
method (TLC). In the overview study of this U.S.A, 1926.
research work, Sory (3) had better condition than
5. Raper, K.B., and Fennell, D.I. “The Genus
that of the other types.
Aspergillus”: Robert. E. Krieger Publishing
ACKNOWLEDGEMENT Company: Hunlignton. New York, 1965.

The author would like to express her deepest 6. Bui, T. T. “A Study of Vietnamese soy sauce
gratitude to Dr. Mya Mya Oo, Rector, Yangon fermentation”. M.Sc. Thesis. University of Western
Technological University and Dr. Khin Maung Sydney. Australia, 2003.
Saing for their kind help in various ways and
7. Kyaw Myo Win. “Isolation and identification of
invaluable suggestions. The author would like to
Aspergillus flavus and A. oryzae from local
express special thanks to Ma Yadanar Win Myint
sources”. M.Sc.Thesis. Department of
(Medical Biotechnology) for help in giving the
Biotechnology. YTU, Myanmar, 2003.
strain of Aspergillus flavus.
8. Okazaki, H.; Akiba, M. and Kimura, S.
REFERENCES
“Investigation on the antimutagenic substance in
1. Ebine,H. “Advances in microbiology for miso miso”. Presented in the Annual meeting of the
fermentation”. Nippon Jozo Kyokai Zasshi. Japan, Agricultural Chemistry Society of Japan, Tokyo,
1985. 1984.

2. Yokosuka, T. and Sasaki, M. Ferm-ented Protein

Foods in the Orient: Shoyu and miso in Japan.
“Microbiology of Fer-mented Foods”. Brian, J.B.,
Wood. Eds. Blackie Academic and Professional,
London, 1998.

3. Thom, C., and Raper, K.B. “A Manual of the

Aspergilli”. The Willams and Wilkins Company.
U.S.A, 1945.

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