Course ...

Literature review 1:
PROBIOTIC CHARACTERISTICS AND THE ABILITY TO DISSOLUTE
THROT OF THE Bacterial Strain Bacillus subtilis NATTO

Student name: Le Thi Thu Trang

Student code: 22125327

Due date: Saturday, March 23, 2024

Word Count: 3009 (excluding References and Cover Pages)

Table of contents

List of tables................................................................... .............................

List of images

I. Introduce
II. Overview of Natto
1. What is natto?
2. Types of natto
3. Nutritional value and benefits of Natto
4. Natto processing process
III. Overview of Bacillus subtilis Natto bacteria
1. Bacillus subtilis Natto classification system
2. Discovery history
3. Distribution
4. Characteristic
a. Morphological characteristics
b. Physiological characteristics
c. Biochemical characteristics
5. Benefits of bacteria
6. The process of research and isolation of Bacillus subtilis natto
7. Research situation at home and abroad
8. The influence of external factors related to the research process
IV. Experiments serve to study the characteristics of Bacillus subtilis Natto
1. Standard bag Huyen bacterial edema Bacillus subtilis natto
 2. Investigation of resistance to low pH, artificial gastric juice and bile salts
3. Determine cell density by colony counting method
4. Surveying the ability to fight against pathogenic bacteria
5. Survey of antibiotic resistance
6. Investigation of the ability to dissolve thrombus
V. Discuss results
1. Resistance to low pH environments
2. Resistance to artificial gastric juice of Bacillus subtilis Natto strain
3. Resistance to bile salt environment of Bacillus subtilis Natto strain
4. Kha power resistance bacteria belong to Bacillus subtilis Natto strain
5. Kha power resistance resistance born belong to Bacillus subtilis N atto strain
6. Kha power dissolve blood block belong to Bacillus subtilis strain N atto

VI. Conclude
References

I. Introduction
Stroke not only ranks highest in adult disability but is also the third-greatest cause of death, after cancer
and heart disease ( Feigin et al., 2009). Globally, 15 million people suffer from strokes each year; this
includes 5 million deaths and an additional 5 million people permanently disabled. These statistics have
a significant negative impact on families, society, and developing nations like Vietnam ( Feigin et al.,
2014). Natto is a traditional Japanese dish fermented with Bacillus subtilis Natto bacteria in warm
conditions (40 ℃ ) for 15 – 24 hours. It was first identified by Sumi Hiroyuki in 1980. Strong
fibrinolytic action of nattokinase allows it to both act directly on fibrin and dissolve it simultaneously,
enhancing the ability of other enzymes in the body to break up blood clots. According to a study, B.
subtilis natto strains possess adhesion, which is an attribute of advantageous gut bacteria (Le and
Nguyen, 2016). The pH value of the human stomach usually fluctuates between 1 and 3. Some studies
have investigated the survival ability of bacterial strains used for human probiotics in artificial gastric
fluid conditions with pH values from 1 to 3 or from 2 to 2.5, bile salt concentrations from 0.3% to 0.5%
( Cenci et al., 2006, Sangtiago et al., 2008), pepsin concentration from 0.2% to 0.3% ( Agung et al.,
2015). Therefore, some surveys on the characteristics of Bacillus subtilis Natto bacteria: resistance to
low pH, artificial gastric juice; resistance to pathogenic bacteria, antibiotic resistance; thrombolysis will
be discussed below.

I. Overview of Natto
1. What is natto?
Natto are soft-boiled soybeans that are fermented with Bacillus subtilis Natto bacteria in warm conditions
(40℃) for 15 - 24 hours. Fermented beans that meet the requirements are brown in color, highly sticky,
have a viscous surface, and especially have a very strong and intense scent.
2. Types of Natto
- Itohiki Natto
 - Tera Natto
3. Nutritional value and benefits of Natto
3.1. Nutritional value of Natto
Table 1.1. Main nutritional ingredients of Natto

Ingredient Weight (g)

Total fat 19
Cholesterol 0
Nattokinase twelfth
Total carbohydrates 25
The protein thirty first

Figure 1.1. Nutritional composition of Natto

3.2. Benefits of Natto
- Prevent cancer
- Prevent osteoporosis
- Prevent heart attack, stroke and physical weakness
- Antibacterial effect
- Improves digestion and prevents intestinal disorders
- Helps brain development

4. Overview of the traditional production process of Japanese Natto fermented soybean products
To make Natto, people choose small soybeans to make the fermentation process easier. Soybeans are
washed and soaked in water for about 12 - 24 hours. Then the soybeans are steamed until soft for about
6 hours. Finally, the soybeans are mixed with Natto yeast solution. And from now on, people will have
to be very careful to avoid harmful bacteria from entering. The mixture of soybeans and yeast is
incubated at 40oC for more than 1 day, then it is cooled and placed in the refrigerator for about a week
so that Natto produces viscous fibers. In the past, people stored steamed soybeans in straw baskets.
 Figure 1.6. Natto wrapped in straw in the traditional method

II. Overview of Bacillus subtilis natto bacteria.

1. Bacillus subtilis Natto classification system

Gender Bacteria
Branch Firmicute
Class Bacilli
Set Bacillales
Surname Bacillaceae
Alike Bacillus
Species Subtilis
Subspecie Bacillus subtilis Natto
s

2. Discovery history
B. subtilis Natto was discovered by Dr. Shin Sawamura in 1906 and named Bacillus Natto Sawamura.
When it was first discovered, B. subtilis Natto was considered a new species of microorganism.
Currently, based on the results of B. subtilis chromosomal DNA analysis by Seki in 1975 and Tamang
in 2002, classification keys have classified B. subtilis Natto as a strain of the B. subtilis species but
distinct from other strains. Thanks to its ability to ferment, it creates natto products.
3. Distribution
B. subtilis Natto is abundant in straw, hay... and grows well in beans, especially soybeans.
4. Function of B. subtilis Natto bacteria
- Enhance beneficial bacteria
- Inhibits harmful intestinal bacteria
- Production of enzymes that decompose proteins and starch
- Building a B vitamin system
5. Characteristics of B. subtilis Natto:
a. Morphological characteristics
B. subtilis Natto is an aerobic bacillus that stains purple on Gram stain, with rod-shaped endospores
measuring less than 1µm. Bacterial cells stand alone or are joined together in chains.

subtilis Natto bacterial cell (100x magnification)

(Source: Dinh Thu Huong (2013))

b) Physiological characteristics
B. subtilis Natto is an obligate aerobic organism that requires oxygen for growth and can grow at oxygen
concentrations greater than 3%. B. subtilis Natto grows in a wide temperature range (30 – 50℃), the
optimum is 37℃. At 55℃, bacterial growth stops and vegetative cell lysis occurs.
c. Biochemical characteristics
B. subtilis Natto is a heterotrophic organism, capable of using many carbohydrate sources. Nitrogen
sources necessary for bacterial growth are proteins and amino acids. B. subtilis Natto can easily use
glutamic acid, arginine, aspartic acid... but cannot use threonine, tryptophan, phenylalanine and
methionine.
Table 1.1. Results of biochemical reactions of B. subtilis Natto strain

Biochemical reaction Result

Catalase activity +
Born indole -
MR +
VP +
Use citrate +
Nitrate removal +
Melt the gelatin +
Starch breakdown +
Arabinose +
Xylose +
Saccharose +
Mannitol +
Glucose +
Lactose -
 Maltose +
(according to Holt, 1992)
4. Research situation at home and abroad:
a. Research situation abroad
In 1998, Hidehiro Miyamura and his colleagues studied the fermentation of Bacillus subtilis on soybean
residue under appropriate conditions of temperature 37℃, humidity 80%, and 24 hours.
b. researches in our country
In 2012, Le Thi Bich Phuong and her colleagues successfully isolated two strains of Bacillus sp.7.2 and
Bacillus sp.NP3 that synthesize nattokinase enzyme in steamed soybean culture on Petri dishes and trays.

II. Experiments serve to study the characteristics of Bacillus subtilis Natto

1. Standard bag Huyen Bacterial edema B. subtilis natto
Culture B. subtilis Natto bacteria in 10 mL of NB medium (10 g/L meat extract; 10 g/L peptone; 5 g/L
NaCl) shaking at 150 rpm, 37℃ for 24 hours. Centrifuge the biomass at 5,000 rpm, 15 minutes, 4 ℃. B.
subtilis natto biomass suspension was obtained in 10 mL of PBS buffer (8.0 g NaCl; 0.2 g KCl; 1.44 g
Na2HPO4; 0.24 g KH2PO4, pH 7.4).

2. Investigation of resistance to low pH, artificial gastric juice and bile salts
-For low pH conditions: Pipet 2 mL of the suspension into test tubes containing 2 mL of PBS solution at
pH 2; pH 3 and pH 7. Incubate at 37℃ (each pH value repeated 3 times). After 0, 1, 2 and 3 hours of
incubation, 0.1 mL of bacterial solution was checked for bacterial population on nutrient agar (NA)
medium using the colony counting method.
-For artificial gastric fluid conditions: perform as above but the test tubes have conditions of pH 2.5 and
contain pepsin concentrations of 0%; 0.2% and 0.3% (Agung et al., 2015). For bile salt conditions:
perform as above but the test tubes have a bile salt concentration of 0%; 0.3% and 0.6% (Quach Duc
Tin et al., 2013).

3. Determine cell density by colony counting method

Samples were taken from the times before and after incubation above, extracted 100 µL and diluted 10 to
10 -7 . Spread 100 µL of the last three dilutions on NA medium, incubate at 37 ℃. After 18 hours, count the
number of colonies growing on the plate and calculate the cell density according to the formula N =
(nD)/V.

4. Surveying the ability to fight against pathogenic bacteria

B. subtilis Natto is resistant to 4 pathogenic strains Escherichia coli ATCC 8739, Salmonella typhimurium
ATCC 14028, Staphylococcus aureus ATCC 25923, Pseudomonas agurinosa ATCC 25853. B. subtilis natto
is cultured in NB medium for 24 hours, centrifuged to get the solution, discard biomass. Pathogenic
bacteria were also cultured in liquid NB overnight, centrifuged and mixed with physiological saline so that
the bacterial solution had a turbidity of 0.5 Mc Farland. Spread 100 µL of this bacterial solution on the
surface of the NA medium plate. Punch jelly holes with a diameter of 5 mm. Put into each agar hole 50 µL
of B. subtilis natto bacterial culture solution , repeat 3 times with each type of pathogenic bacteria. Each
agar plate has a control well containing 50 µL of physiological saline. Leave for 30 minutes for the
solution to absorb into the agar and incubate at 37 ℃. Measure the diameter of the sterile ring after 24
hours.

5. Survey antibiotic resistance

The disk diffusion method was used to evaluate antibiotic resistance. B. subtilis Natto was grown in NB
medium, shaking at 150 rpm, 37℃ for 24 hours. Collect biomass and mix with physiological saline to
achieve turbidity of 0.5 Mc Farland. Use a sterile cotton swab to soak in the bacterial solution and spread
evenly onto the surface of the MHA agar plate (Mueller Hinton Agar - 300 g/L beef extract, 17.5 g/L
casein, 1.5 g/L starch, 17 g/L agar pH 7.3), let the agar surface dry for 10 minutes. The antibiotic plate was
placed on the agar surface and incubated at 37 ℃ for 24 hours. Sterile zone diameter was compared with
CLSI, 2012 standards to conclude antibiotic susceptibility.

5. Investigation of the ability to dissolve thrombus

Culture of B. subtilis Natto bacteria in lip school NB at 37 ℃ , shaking at 150 rpm. After 24 hours , 36
hours and 48 hours raise , separate Center 5,000 rpm for 15 minutes , quit born block , collect bacterial
epidemic . Weigh 2 g of blood pig fresh enter tube test , add 2 mL of solution bacterial extraction collect be
above enter tube test , sample opposite to proof positive Add 2 mL of nattokinase enzyme solution love
products ( Hau Giang Pharmaceutical Company , Vietnam . Hoa one pellets nattokinase love Products
Have active Calculate 670 FU/ tablet into 2 mL of water salt born reason ), sample opposite to proof minus
Add 2 mL of water store enter coincide . Incubate at 37 ℃ , after 2 hours and 4 hours , weigh again block
quantity block blood still again and count Billion rule dissolve blood block . Each alms experience repeat
again father times (Le Thi Bich Phuong and et al ., 2012 ).

III. Discuss results

1. Resistance to low pH environments

subtilis natto strain has the ability to survive in low pH conditions, the cell density that survives after 3
hours of incubation at low pH values remains at 106 CFU/mL. The pH value 7 is suitable for bacteria to
grow, the cell density after 3 hours of incubation is 8.35±0.06 log CFU/mL, corresponding to 98.24%
compared to the initial density of 8. ,50±0.09 log CFU/mL. At pH value 3, the cell survival rate after 1
hour was up to 98.16%, corresponding to a density of 8.35±0.04 log CFU/mL. After 3 hours of incubation,
the survival rate decreased to 81.11% corresponding to a bacterial density of 6.90±0.05 log CFU/mL.

At a pH value of 2, this is considered a harsh pH condition, and is a selective factor for acid-resistant
bacteria that can survive in the stomach. When microorganisms fall into an environment with low pH,
membrane channel proteins will be damaged, water and nutrients entering and leaving the membrane are
not regulated, the osmotic pressure of the membrane changes, and intracellular enzymes are inhibited. , the
lower the pH, the faster the cell membrane will break down, leading to stagnation of metabolism, which in
turn leads to cell death (Agung et al., 2015). Therefore, the density of B. subtilis Natto bacteria decreased
significantly after incubation in an environment with pH 2. After 2 hours and 3 hours of incubation, the
density of viable bacteria decreased by 90.98% and 79.11, respectively. %. In research on B. subtilis B20.1
strain, it was shown that their ability to survive at pH 3 after 3 hours was over 90% (Ho Thi Truong Thy et
al., 2015).

Table 1: Secret degree international cell B. subtilis Natto in other pH solutions together after time
incubation period
 pH 7 pH 3 pH 2

Incubat Density (log Ratio (%) Density (log Ratio (%) Density Ratio
ion CFU/mL) CFU/mL) (log (%)
time CFU/mL)
(hours)

0 8.50±0.09 100.00 8.50±0.09 100.00 8.50±0.08 100.00 2. Resistance to

artificial
first 8.46±0.02 99.49 8.35±0.04 98.16 8.25±0.03 97.02 gastric juice
of Bacillus
2 8.43±0.02 99.10 7.91±0.04 92.98 7.74±0.05 90.98 subtilis Natto
strain
3 8.35±0.06 98.24 6.90±0.05 81.11 6.73±0.04 79.11
Strain B. subtilis Natto has the ability to survive over 80% in an environment containing artificial gastric
juice with pepsin concentrations of 0%, 0.2% and 0.3%, respectively (Table 2). However, the survival rate
decreases over time. In the pH 7 environment without pepsin, the initial bacterial cell density was
8.05±0.01 log CFU/mL, the bacterial density after 3 hours of incubation had no significant difference,
maintained at level 8.07±0.01 log CFU/mL. In the pH 2.5 environment and 0.2% pepsin concentration, the
bacterial density after 1 hour of incubation decreased to 7.75±0.04 log CFU/mL, equivalent to 96.31%.
After 2 hours and 3 hours of incubation, the number of viable bacterial cells continued to decrease,
remaining at 7.13±0.04 log CFU/mL (88.61%) and 6.75±0, respectively. 06 log CFU/mL (83.93%). In
medium with 0.3% pepsin, the number of surviving cells was lower than in medium with 0.2% pepsin.
Specifically, after 1 hour of incubation, the cell density reached 7.35±0.04 log CFU/mL (91.14%). After 2
hours and 3 hours of incubation, the remaining cell density was 7.04±0.02 log CFU/mL (87.49%) and
6.71±0.02 log CFU/mL (83.31), respectively. %). While in the study of Huynh and Nguyen (2016) showed
that under artificial gastric fluid conditions with pepsin concentration of 0.3% and pH 2, the density of B.
subtilis Natto bacteria decreased from 8.06±0, 11 log CFU/mL down to 4.81±0.14 log CFU/mL after 2
hours of incubation.

Table 2. Density of Bacillus subtilis Natto cells in artificial gastric fluid after incubation

0% Pepsin 0.2% Pepsin 0.3% Pepsin

Incubation Density (log Ratio (%) Density (log Ratio (%) Density (log Ratio
time (hours) CFU/mL) CFU/mL) CFU/mL) (%)

0 8.05±0.01 100.00 8.05±0.02 100.00 8.05±0.01 100.00

first 8.02±0.02 99.63 7.75±0.04 96.31 7.35±0.04 91.14

2 8.04±0.02 99.83 7.13±0.04 88.61 7.04±0.02 87.49

 3 8.07±0.01 100.29 6.75±0.06 83.93 6.71±0.02 83.31

i. Resistance to bile salt environment of B. subtilis Natto strain

Results from Table 3 show that the cell density in the solution containing 0.3% bile salts after 1 hour
decreased from 8.13±0.07 to 7.99±0.12 log CFU/mL, after 2 hours to 7. 96±0.11 log CFU/mL and after 3
hours it was 7.87±0.08 log CFU/mL, corresponding to 96.80% of the initial density. At the same time, the
presence of bile salts reduces the surface tension of lipid molecules on the cell membrane. With the impact
of intestinal motility, lipid particles will burst, the transmembrane lipoprotein structure is destroyed and the
cell cells will be destroyed (Verónica and Josep, 2017). Therefore, bile salt conditions in the intestine are
often a criterion for evaluating the probiotic properties of a microbial strain. In a solution containing 0.6%
bile salts, the viability of the B. subtilis Natto strain gradually decreased over time. The initial density of
8.11±0.12 log CFU/mL decreased to 7.77±0.11 log CFU/mL, equivalent to 95.57% after 1 hour of
incubation. After 2 hours of incubation, the cell density decreased slightly to 95.20% and decreased
significantly after 3 hours of incubation, remaining 6.95±0.10 log CFU/mL, corresponding to 85.49%
compared to First value.

subtilis Natto cell density at different bile salt concentrations

0% bile salts 0.3% bile salts 0.6% bile salts

Incu Density (log Ratio (%) Density (log Ratio (%) Density (log Ratio (%)
bati CFU/mL) CFU/mL) CFU/mL)
on
time
(hou
rs)

0 8.13 ± 0.05 100 8.13 ± 0.07 100 8.11 ± 0.12 99.75

first 8.10 ± 0.13 99.63 7.99 ± 0.12 98.28 7.77 ± 0.11 95.57

2 8.12 ± 0.03 99.88 7.96 ± 0.11 97.91 7.74 ± 0.04 95.20

3 8.15 ± 0.02 100.25 7.87 ± 0.08 96.80 6.95 ± 0.10 85.49

ii. Kha power resistance bacteria belong to Bacillus subtilis Natto strain
The antibacterial circle diameter of B. subtilis Natto against bacteria Escherichia coli ATCC 8739,
Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, Psedomonus aegurinosa
ATCC 25853 are 8.00±0.76, respectively; 9.00±0.40; 10.00±0.58 and 8.00±0.29. This result shows that
B. subtilis Natto bacteria has the ability to produce compounds that are resistant to pathogenic bacteria.

iii. Antibiotic resistance of Bacillus subtilis Natto strain

Results of antibiotic resistance survey
 Figure 2: Antibiotic paper plate on Bacillus subtilis Natto culture medium plate

B. subtilis Natto is resistant to 1/9 antibiotics surveyed, namely clindamycine of the lincosamide group
(this group has the effect of inhibiting protein biosynthesis) and sensitive to 8/9 antibiotics surveyed,
namely streptomycin of the amino glycosides group. , ciprofloxacin belongs to the quinolone group,
chloramphenicol belongs to the phenicol group, erythromycin belongs to the macrolines group,
tetracycline belongs to the tetracycline group (these 5 groups have the effect of inhibiting protein
biosynthesis); amoxicillin belongs to the β -lactam group, vancomycin belongs to the glycopeptide group
and cefotaxime belongs to the cephalosporin group (these 3 groups inhibit bacterial cell wall synthesis).
This shows that the investigated bacterial strain is safe, does not show resistance to many antibiotics, and
avoids the risk of spreading resistance genes to other bacteria.

Table 4: Diameter of the sterile zone of Bacillus subtilis Natto with antibiotics

Type of antibiotic Diameter Antibiotic sensitivity Result

of
antibacteri Resistance Intermediate I Susceptible
al ring R (Intermediate)
(Resistant)

Amoxicillin 28.67±1.53 ≤ 13 14 – 17 ≥18 S

Streptomycin 17.00±1.73 ≤11 12 – 14 ≥ 15 S

Ciprofloxacin 26.00±2.65 ≤15 16 – 20 ≥21 S

Chloramphenicol 29.00±2.00 ≤12 13 – 17 ≥18 S

Erythromycin 28.67±0.58 ≤13 14 – 22 ≥23 S

Tetracycline 30.33±2.52 ≤11 12 – 14 ≥15 S

Cefotaxime 37.33±3.21 ≤22 23 – 25 ≥26 S

Vancomycin 20.33±3.21 ≤14 15 – 16 ≥17 S

Clindamycin 13.33±2.52 ≤15 16 – 18 ≥19 S

iv. Kha power dissolve blood block belong to Bacillus subtilis strain N atto
A characteristic feature of the B. subtilis Natto strain is its ability to dissolve blood clots. The survey
results showed that the hemolytic ability of the bacterial solution containing Nattokinase produced after
24, 36 and 48 hours of culture was different (Table 5).
Table 5: Thrombodissolving ability of Bacillus subtilis Natto bacterial solution

Sample 2 hours incubation 4 hours incubation

Reduced blood Ratio (%) Reduced blood Ratio (%)

volume (g) volume (g)

Nattokinase 670 1.46±0.26 73.0 1.52±0.17 76.0

FU

Extract after 24 0.28±0.10 14.0 0.43±0.10 21.5

hours of culture

Extract after 36 0.40±0.04 20.0 0.49±0.03 24.5

hours of culture

Extract after 48 0.49±0.05 24.5 0.50±0.05 25.0

hours of culture

Distilled water 0.01±0.01 0.5 0.02±0.01 1.0

The longer the bacteria were cultured during the survey, the more enzymes they produced, as shown by the
clot-dissolving ability of the bacterial extracts. Bacterial fluid after 24 hours of incubation reduces the
initial blood volume by 24.5% after 4 hours of incubation. The culture fluid after 36 hours had a thrombus
dissolution rate after 2 hours of incubation of 20% and after 4 hours it increased to 24.5%, corresponding
to the hemolysis mass of 0.40±0.04 g and 0.49± 0.03 g. The culture fluid after 48 hours has a higher
hemolytic ability than the culture fluid after 36 hours, the hemolysis rate is 24.5% after 2 hours of
incubation. Thus, the time for bacteria to produce many enzymes is about 36 to 48 hours. Le Thi Bich
Phuong and colleagues. (2012) concluded that 40 hours is the appropriate time for Bacillus sp.7.2 and
Bacillus NP3 to produce the highest enzyme in soybean seed medium. Nattokinase produced in the above
study has an activity equivalent to 470 FU/g. It is noteworthy that, when diluting the content of
commercial nattokinase enzyme tablets to 111.7 FU, the hemolytic ability of this enzyme solution is
similar to that of bacterial solution after culturing for 36 hours. This result shows that the investigated
bacterial strain is capable of producing thrombolytic nattokinase but does not have high activity. Therefore,
culture conditions need to be investigated to help bacteria produce more nattokinase, such as culturing at
45oC, pH 5.0 will produce higher amounts of enzyme (Kawther et al., 2015).
3. Conclude

subtilis Natto bacteria were surveyed to have the ability to survive in harsh conditions such as low pH
environment, artificial gastric fluid, bile salt fluid, and are resistant to pathogenic bacteria, resistant to
antibiotics and ability to dissolve blood clots. With an initial density of 108 CFU/mL, after 3 hours of
incubation in environments simulating artificial stomach conditions, the bacterial density still remains
above 106 CFU/mL, this density ensures Ensuring bacteria exert beneficial effects on the body. In
addition, the surveyed bacteria were resistant to 4 strains of common pathogenic bacteria and were
resistant to only 1/9 of the surveyed antibiotics. In particular, the bacterial strain when grown in culture
medium can produce nattokinase to dissolve blood clots. The above characteristics show that the B. subtilis
natto strain has the potential to produce probiotics or as a supplement for functional foods, contributing to
reducing stroke, thrombotic stroke and protecting health. community health.

References
(Nguyet et al. , 2017)
(Uy, 2017)
(Luan et al. , 2022)
(Thanh et al. , 2020)

Luan, MT, Thuong, HTL, & Anh, NTM (2022). STUDY ON ISOLATION AND BIOLOGY OF THE BACTERIAL
SPECIES BACILLUS SUBTILIS VARNATTO. Hong Duc University Science Magazine (62), 102-109.
Nguyet, NTM, Hanh, TTM, & Cuong, BV (2017). Building and selection of optimal mathematical model for
natto fermentation for nattokinase synthesis by using bacillus subtilis natto. Journal of Science and
Technology-Da Nang University , 66-70.
Thanh, D. TN, Ngoan, PTT, & Lai, PNK (2020). Probiotic properties and thrombolytic ability of Bacillus
subtilis natto strain. Can Tho University Science Magazine , 56 (1), 104-110.
Uy, V. D. (2017). Survey of biological characteristics of Bacillus bacteria and their ability to produce
enzymes, Amylase, and protease.