Study on the Production of Fermented Soybean Sauce by Using Aspergillus oryzae and Aspergillus flavus

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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.
Tin Mar Lynn et. al.
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March-April 2013 | Vol 2 |Issue 2 Journal of Scientific & Innovative Research
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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
Nowadays, the world’s population increases rapidly
every day. Also, food demand is increasing as
every time. Protein is an important type in a
balanced diet. Soybean product has a large amount
of protein content, and so it was consumed as the
protein source. Soy sauce contains essential amino
acids such as Valine, Tryptophan, Lysine, and
Histidine and also contains vitamins (especially
vitamin B6) and antioxidants (isoflavones).
1
Address for correspondence:
Tin Mar Lynn*
Department of Biotechnology, Mandalay
Technological University, Mandalay, Myanmar
E-mail: light.lynn@gmail.com
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Indeed, soy sauce has been produced for centuries
under natural conditions. Soy sauce is one of the
world’s oldest condiments and has been used in
China for more than 2,500 years. Soy sauce is a
hydrolysis product of the soybean. Soy sauce is a
dark brown liquid with a pleasant aroma, used
primarily as flavoring agents for meat, poultry, fish,
vegetables and rice. Its high salt content of about
18% and distinct flavor makes it a useful adjunct
for many of the bland food products in which it is
used.
The preparation of soy sauce is known to involve
the action of molds, yeasts, and bacteria such as
Aspergillus oryzae, Zygosaccharomyces soya, and
Lactobacillus species. Yokosuka and Sasaki
2
stated
that Aspergillus oryzae and A. soyae are used by
manufacturer. These fungal Aspergilli species are
widely found in various sources. Some species are
food grade species and others are pathogenic for
human beings. Aspergillus oryzae has been used in
soybean fermentation for several years and it was
known as koji mold.
Fermentation increases protein content, eliminates
trypsin inhibitors, and reduces the peptide size in
soybean meals. These effects of fermentation might
make soy foods more useful in human diets as a
functional food and benefit livestock as a novel
feed ingredient.
In this research study, emphasis was made on the
isolation and identification of Aspergillus oryzae
from the moldy soybean sauce. The strain
characterization of receiving Aspergillus flavus
strain was examined. The fermenting activities on
the main substrate soybean were studied on two
conditions: 1.5 months fermentation time and 3
month period, respectively. Microbial safety of the
end products was also examined in this work. The
presence of some amino acid in each produced soy
sauce was also displayed by the Thin Layer
Chromatography method.
The objectives of this research work are: to
promote the role of soybean food consumption, to
improve the traditional methods of soybean food
production and to study and investigate the quality
improvement of soy sauce by using Aspergillus
oryzae.
2. MATERIALS AND METHODS
2.1. Materials
2.1.1. Media and Chemicals
Culture media, media ingredients and other
chemicals used were from “Australia Medical
Diagnostics (AMD) Co., Ltd”, “HiMedia
Laboratories Limited, Mumbai, India”, and
Analytical grade chemicals available from local
markets have also been used.
2.1.2. Microorganisms
Aspergillus oryzae were isolated from moldy
soybeans. Aspergillus flavus were isolated from the
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chili. These two microorganisms are used for the
production of soy sauce. Strain recharacterization
of Aspergillus flavus. The flavours were also done
to confirm the correct strain.
2.1.3. Raw Materials for Soy Sauce Production
12 kg soybeans, 12 kg roasted wheat flour and 5 kg
salt were used for soy sauce production. They were
bought from the local market in Yangon.
2.2. Methods
2.2.1. Isolation of Aspergillus oryzae
The agar plate method was used to isolate
Aspergillus colonies occurring in moldy soybeans.
The moldy soybeans were placed on Czapek`s Dox
Agar, introduction 4 each in 9 cm Petri dishes. The
plates were incubated at room temperature for 1 to
7 days. After incubation for 48 hours, the plates
were observed in young fungus colonies.
2.2.2. Identification of Aspergillus oryzae
Aspergillus oryzae can be identified by colony
morphology and microscopic characteristics. Hypo-
septate, reproductive organs, the structure of
fruiting organs, details of spores of the fungus were
observed and matched with the synoptically keys of
Thom and Church (1926), Thom and Raper (1945)
and Raper and Fennell (1965).
3-5
Measurements of
vegetative and reproductive parts of the fungus
were also taken from the state micrometer set.
To identify the colony morphology, A. oryzae were
incubated on Czapek`s Dox Agar, Sabouraud
Dextrose Agar, Potato Dextrose Agar and Malt
Agar. And then colonies were observed by colony
size, color, reverse position and growth pattern.
The only one biochemical test for Aspergillus
oryzae is Kojic acid test. Among the Aspergillus
species, A. albus, A. candidus, A. nidulans, and A.
oryzae produce Kojic acid.
2.2.3. Soy Sauce Production
An amount of 2 kg soybeans was soaked in water
for overnight. Next, the soaked soybeans were
cooked in autoclave at 121˚C with 15 psi for 30
minutes. After that they were drained for 3-4 hours
in a contamination free area. And then they were
mixed with 2 kg of wheat flour. Finally,
Aspergillus oryzae were added to the mixture of
soybeans and wheat flour by spore count method.
The inoculum size was 2.4× 10
8
. The incubation
period was 5 days. The same steps were made for
the production of soy sauce by Aspergillus flavus.
After 5 days of fermentation by fungi, the mixtures
were fermented by salt brine. This was called the
brine fermentation stage. The brine fermentation
was stirred at intervals of 2 days to supply more
oxygen into the glass containers during brine
ageing. After ageing for 1-3 months, the soy sauce
was filtered and pasteurized at 70˚C for 30 minutes.
And then soy sauce was ready for consumption.
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Four types of soy sauces were produced in this
experiment for three replicates. They were soy
sauce produced by A. oryzae (brine ageing time 1.5
months), soy sauce produced by A. oryzae (brine
ageing time 3 months), soy sauce produced by A.
flavus (brine ageing time 1.5 months), soy sauce
produced by A. flavus (brine ageing time 3
months). Each of these soy sauces was measured by
the appropriate parameters to determine the product
qualities. One type of soy sauce from local market
was also measured by the above parameters to
compare the qualities.
Physical Analysis
(1) pH measurements
(2) Viscosity measurements
Chemical Analysis
(1) Protein analysis
(2) Fat analysis
(3) Determination of reducing sugar by Lane and
Eynon method
(4) Alcohol analysis
(5) Salt analysis
2.2.4. Microbial Safety for Soy Sauce Products
Each aliquot with 25µl of four types of soy sauce
sample was spread on the nutrient agar before and
after pasteurization. The plate was inverted and
incubated at 30˚C for one day and the individual
colonies developed on the nutrient agar were
examined for microbial safety of the products.
2.2.5. Separation and Identification of Amino
Acids in Soy Sauces by One Dimensional TLC
Preparation of solvent system
The solvent system was prepared by mixing n-
butanol (40 ml), acetic acid (10 ml) and water (10
ml).
Preparation of standard amino acid solution
Standard solutions of nine different amino acids
were prepared in 75% ethanol (v/v) with the
addition.
Preparation of 0.2 % ninhydrin reagent
Ninhydrin powder (0.2 gram) was dissolved in 100
ml acetone.
Preparation of Sample
5 ml of sample was put into the separation funnel.
5ml of n-butanol was added into the funnel and
shaken vigorously. The layers were separated by
placing in standing position for 15 minutes. Then
the upper layer was taken to use for TLC method.
6-8
3. RESULTS
The isolation of A. oryzae was done by the agar
plate method in Czapek’s Dox agar, selective agar.
The rate of growth was very rapid. The microscopic
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morphology and cultural characteristics of A.
oryzae and A. flavus was shown in Figure 1, 2, 3
and 4. The species was identified by comparing
with the reference (Raper and Fennel, 1965).
5
The colony morphology of A. oryzae was
examined by plating on four different media and
the results were shown in Table 1.
3.1. Biochemical Characteristics
Red blood color was obtained by the filtrates of A.
oryzae and that of Aspergillus flavus. The flavors
were not changed in Kojic acid test. On account of
these character the specimen was identified
tentatively as Aspergillus oryzae.
3.2. Soy Sauce Production
Four types of soy sauces were produced in this
experiment. The yield of the finished products was
90% of the salt brine.
3.3. Physical Analysis
The pH and viscosity of soy sauces were measured
and the results were described in Table 2.
3.4. Chemical Analysis
The protein%, fat%, reducing sugar% and
alcohol% of soy sauces were measured in this
experiment and the results were shown in Table 3.
The salt content of soy sauces was described in
Table 4.
3.5. Microbial Safety of Soy Sauce Products
Some colony of Bacillus species were observed in
before pasteurized sample. But, no microorganism
was observed in after pasteurization sample.
3.6. Separation and Identification of Amino
Acids in Protein Hydrolysate of Soy Sauces by
TLC
By the comparison of R
f
values of individual
standard amino acids and sample hydrolyzate
amino acids, the results were shown in Table 5.
Table 1: Colonial Morphology and Growth Characteristics of A. oryzae on Different Media
Types of
media
Growth pattern Size of
colony
(inch)
Surface colony
color
Reverse colony
color
Czapek’s
Dox agar
Growth rate rapidly 2 Brownish yellow White
SDA More rapidly of growth 3 Dark yellow Brownish yellow
PDA Growth rate moderately,
(with other contaminants)
1.5 Pale yellow Yellowish brown
Malt
Extract
agar
Growth rate slowly, present
many contamination
1 yellow Yellowish brown
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Table 2: pH and Viscosity Measurements of Soy Sauces at Room Temperature
Types of
Soy Sauces
pH Viscosity
(Centipoises)
S
ory(1.5)
5.14 1.6
S
ory(3)
4.84 1.739
S
fla(1.5)
5.21 1.6
S
fla(3)
4.99 1.922
S
(Wai Weng)
4.7 3.561
Table 3: Protein%, Fat%, Reducing Sugar%, Alcohol% of Soy Sauce
Types of
Soy Sauces
Protein % Fat % Reducing Sugar
%
Alcohol %
S
ory(1.5)
2.98±0.18 0.99±0.175 4.15±0.26 0.99
S
ory(3)
3.88±0.29 0.048±0.007 5.3±0.42 1.00
S
fla(1.5)
2.43±0.37 0.99±0.21 2.71±0.36 0.99
S
fla(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
Soy Sauces
Salt content
(ppm)
S
ory(1.5)
140
S
ory(3)
155
S
fla(1.5)
140
S
fla(3)
150
S
(Wai Weng)
160
Table 5: R
f
values of Individual Standard Amino Acids and Amino Acids in the Protein Hydrolyzate
of Soy Sauces
No
Amino Acids
R
f
values
(standard)
R
f
values of
A. flavus
products
R
f
values
of
A.oryzae
product
R
f
values for
Wai Weng
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
To obtain the pure culture of Aspergillus oryzae
was one of the main research works. Czapek’s Dox
medium is a selective medium and were continually
employed in subculture preparation until pure
culture was obtained. Morphological characteristics
such as growth rate, colony size, color and
conidiophore formation etc. were enough to
confirm the correct strain of Aspergillus oryzae.
Moreover, according to Table 1, A. oryzae grow
rapidly in Czapek’s Dox agar and Sabouraud
dextrose agar. But, potato dextrose agar and malt
extract agar gave slower growth rate. This may be
due to other contaminating microbes which inhibit
the growth of A. oryzae. In the comparison of
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colony size, the smallest colonial size was found in
malt agar.
In the study of biochemical test with Kojic acid
test, only A. oryzae changed into red blood color. A.
flavus remained without any changes. From this
point, we can distinguish the species identification
of A. oryzae and A. flavus fungal strain.
According to the observed data in Table 2, the
surest we say was that the smaller the pH value, the
higher the viscosity in soy sauce products. But, all
pH values existed in the specified range of soy
sauce products (within 4.6-5.2). In viscosity
measurements, S
ory
(1.5) and S
fla
(1.5) had the same
value in each product.
In the study of protein contents, Table 3 indicated
that S
ory
(3) had better protein content than that of
others. Moreover, its fat % and reducing sugar %
were reliable for commercial production.
In the study of salt content described in Table 4,
S
ory
(1.5) and S
fla
(1.5) had the same content of 140
ppm respectively.
As a part of research work, the presence of amino
acids in soy sauce products was detected by Thin
Layer Chromatographic technique. According to
Table 5, only three amino acids (Valine, Threonine
and Tryptophan) were present in A. flavus product.
Aspergillus oryzae product contributes four amino
acids (Valine, Lysine, Histidine and Tryptophan).
However, commercial “Wai Weng” product
comprises only the two amino acids (Valine and
Histidine). So, A.oryzae product had more suitable
for commercial production as a human diet. Soy
sauce produced by A. oryzae contains more amino
acids than soy sauce produced by A. flavus because
A.oryzae produced peptidase and proteinase than A.
flavus.
By comparing the brine ageing time (1.5 months
and 3 months), better results were obtained from 3
months. So, brine ageing time was also important
for quality improvement of soy sauce.
5. CONCLUSION
The two fermented fungal strains A. oryzae and A.
flavus were mainly employed throughout this study.
Not only morphological, microscopical and
biochemical test but also culture test on four
different media were studied. Analysis of soy sauce
product was performed by determining pH,
viscosity, and protein %, fat %, reducing sugar %,
alcohol %, salt content and finally amino acid
composition. Produced soy sauces S
ory
(1.5) and S
fla
(1.5) had better viscosity activities (1.6 cp) and S
ory
(3) had more protein content (3.88) than that of the
other types. In A. flavus fermented product, only
three amino acids (Valine, Threonine and
Tryptophan) were found. A. oryzae fermented soy
sauce had four amino acids composition (Valine,
Lysine, Histidine and Tryptophan). But, in the
analysis of commercial product (Wai Weng), only
two amino acids (Valine and Histidine) were found.
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Analysis of amino acid composition was performed
by one dimensional Thin Layer Chromatographic
method (TLC). In the overview study of this
research work, S
ory
(3) had better condition than
that of the other types.
ACKNOWLEDGEMENT
The author would like to express her deepest
gratitude to Dr. Mya Mya Oo, Rector, Yangon
Technological University and Dr. Khin Maung
Saing for their kind help in various ways and
invaluable suggestions. The author would like to
express special thanks to Ma Yadanar Win Myint
(Medical Biotechnology) for help in giving the
strain of Aspergillus flavus.
REFERENCES
1. Ebine,H. “Advances in microbiology for miso
fermentation”. Nippon Jozo Kyokai Zasshi. Japan,
1985.
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.
4. Thom, C., and Church, M.B. “The Aspergilli”.
Copyright. The Willams and Wilkins Company.
U.S.A, 1926.
5. Raper, K.B., and Fennell, D.I. “The Genus
Aspergillus”: Robert. E. Krieger Publishing
Company: Hunlignton. New York, 1965.
6. Bui, T. T. “A Study of Vietnamese soy sauce
fermentation”. M.Sc. Thesis. University of Western
Sydney. Australia, 2003.
7. Kyaw Myo Win. “Isolation and identification of
Aspergillus flavus and A. oryzae from local
sources”. M.Sc.Thesis. Department of
Biotechnology. YTU, Myanmar, 2003.
8. Okazaki, H.; Akiba, M. and Kimura, S.
“Investigation on the antimutagenic substance in
miso”. Presented in the Annual meeting of the
Agricultural Chemistry Society of Japan, Tokyo,
1984.

Supplementary resources

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  • A Study of Vietnamese soy sauce fermentation
    • T Bui
    Bui, T. T. " A Study of Vietnamese soy sauce fermentation ". M.Sc. Thesis. University of Western Sydney. Australia, 2003.
    • M Sc
    M.Sc.Thesis. Department of Biotechnology. YTU, Myanmar, 2003.
  • Isolation and identification of Aspergillus flavus and A. oryzae from local sources
    • Win Kyaw Myo
    Kyaw Myo Win. " Isolation and identification of Aspergillus flavus and A. oryzae from local sources ".
  • Investigation on the antimutagenic substance in miso
    • H Okazaki
    • M Akiba
    • S Kimura
    Okazaki, H.; Akiba, M. and Kimura, S. " Investigation on the antimutagenic substance in miso ". Presented in the Annual meeting of the Agricultural Chemistry Society of Japan, Tokyo, 1984.
  • Chapter
    From ancient times, nations all over the world have inherited their own alcoholic beverages, which are prepared principally by converting the sugars involved in raw materials into alcohol by the action of yeasts. At the same time, vinegars have been made from almost all of these alcoholic beverages, converting alcohol into acetic acid by the action of acetic acid bacteria. Thus, fruit wines have been made from sweet fruits such as grapes, apples, oranges, and so on. However, in the preparation of wines from a starchy raw material such as wheat, barley, rice, or corn, the raw material must be degraded into sugars, mainly glucose, in order to be fermented by yeasts. It must be noted that there is a major difference between the saccharification process of Western countries and that of the Orient. The amylolytic enzymes used for the saccharification in Western countries have been derived from malt, while in the Orient, Aspergillus or Rhizopus moulds have been utilized as the source of amylolytic enzymes. Accordingly, beer is prepared by first saccharifying the starch of barley by the use of malt, while in the preparation of alcoholic beverages from rice or wheat in the Orient, Aspergillus or Rhizopus moulds are cultured on part of these raw materials to produce amylolytic enzymes. These mould-cultured materials are called ‘koji’ in Japan, and this koji is mixed with the other remaining parts of rice or wheat and water to make mash, which is then concurrently subjected to enzymatic saccharification, lactic fermentation, and yeast fermentation.