processes

Article
Optimization of Sporulation Conditions for Bacillus subtilis
BSNK-5
Zhiliang Tian, Lizhen Hou, Miao Hu, Yaxin Gao, Danfeng Li, Bei Fan, Fengzhong Wang * and Shuying Li *

Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, No. 2 Yuan Ming Yuan West
Road, Beijing 100193, China; tianzhiliang0520@[Link] (Z.T.); houlizhen2020@[Link] (L.H.);
humiao7890@[Link] (M.H.); gaoyx1997@[Link] (Y.G.); ldf970920@[Link] (D.L.); fanbei@[Link] (B.F.)
* Correspondence: wangfengzhong@[Link] (F.W.); lishuying@[Link] (S.L.);
Tel.: +86-010-62815977 (F.W.); +86-010-62810295 (S.L.)

Abstract: Bacillus subtilis spores have important biological applications; however, high spore-cell
densities and sporulation efficiencies in fermentation is poorly reported. This study systematically
analyzed the spore densities and formation efficiency of B. subtilis BSNK-5 in different culture
substrates. A response surface regression equation was established based on the results of single
factor and Box–Behnken experimental designs. The optimal medium formulation, as predicted from
the equation, consisted of soluble starch at 3 g·L−1 , soybean flour at 12 g·L−1 , and MgSO4 at 5 g·L−1 .
The spore yield reached 2.43 × 109 CFU·mL−1 , and the sporulation rate was 83.3%, which was nearly
three times higher than before optimization using an optimized medium at 36 ◦ C and 200 rpm for
60 h.

Keywords: Bacillus subtilis; fermentation medium; sporulation rate; optimization

Citation: Tian, Z.; Hou, L.; Hu, M.; 1. Introduction

Gao, Y.; Li, D.; Fan, B.; Wang, F.; Li, S. Bacillus subtilis is an aerobic Gram-positive probiotic which is tolerated by humans
Optimization of Sporulation
and animals. It can form endospores that have a thick cell wall, contains no built-in
Conditions for Bacillus subtilis
toxins, and is extremely resistant to all manner of harsh treatments [1]. When nutrients are
BSNK-5. Processes 2022, 10, 1133.
limited, B. subtilis stops growing and begins sporulation, releasing mature spores through
[Link]
asymmetric cell division, endocytosis, and mother cell lysis. When a dormant spore
Academic Editors: Tiane Finimundy, encounters the right environmental stimulus, it initiates the germination process, and if
Taofiq Oludemi and Filipa S. Reis sufficient nutrients are present, the spore germinates into a vegetative bacterium [2]. In
recent years, research into the application of Bacillus in various aspects of life has increased
Received: 11 May 2022
Accepted: 30 May 2022
all over the world. This probiotic can be used to replace antibiotics in agriculture and
Published: 6 June 2022
aquaculture or to produce new food and dietary supplements in human products [3].
As a probiotic preparation, B. subtilis is widely used in industry and is the most
Publisher’s Note: MDPI stays neutral studied spore-forming bacterium [4]. B. subtilis can be used as a dietary supplement to
with regard to jurisdictional claims in
maintain intestinal microecological balance. Through colonizing and consuming oxygen
published maps and institutional affil-
in the intestinal environment, it can promote the growth of anaerobic probiotics (e.g.,
iations.
lactic acid bacteria, bifidobacteria) and prevent the reproduction of some harmful aerobic
bacteria (e.g., salmonella), resulting in the ability to both prevent gastrointestinal diseases
and treat uremia [5]. In poultry farming, B. subtilis can be directly added to feed to replace
Copyright: © 2022 by the authors.
antibiotics, improve the digestibility of nutrients, promote body growth, enhance immunity,
Licensee MDPI, Basel, Switzerland. and provide enzymes needed by a variety of animals [6]. In addition, with the help of
This article is an open access article fermentation technology, B. subtilis can be used to make foods, among which natto is
distributed under the terms and the most prominent. Microbial metabolism can improve the nutritional composition of
conditions of the Creative Commons soybean, and enrich the bioactive ingredients of functional peptides, aglycone isoflavones,
Attribution (CC BY) license (https:// nattokinase, vitamin K, and polyglutamic acid, playing an important role in the prevention
[Link]/licenses/by/ of cardiovascular disease and osteoporosis [7,8].
4.0/).

Processes 2022, 10, 1133. [Link] [Link]

Processes 2022, 10, 1133 2 of 10

The aforementioned studies have shown that B. subtilis and its metabolites can affect
growth, digestion, and immune performance. The concentration of B. subtilis is the main
factor in determining its prebiotic effect. In the production process, complex processing
technology and a harsh processing environment will reduce the survival rate of strains.
The formation of spores can make the bacteria more resistant to extreme conditions, such
as pH, heat, cold, and pressure and improve the survival rate of strains under different
production processes, as well as improving the colonization rate in the stomach. The higher
the spore content, the more suitable it is for industrial processing. During fermentation, the
content of spores is also an important index to evaluate the fermentation effect [9]. Although
there have been many studies on improving the spore yield of Bacillus by changing the
medium components and culture conditions, different strains require their own specific
culture medium components [10]. For example, for Bacillus cereus, the presence of glutamate
in the medium favors its growth, but this compound inhibits the growth of other species of
Bacillus [11]. Therefore, it is of great significance to study the spore-forming characteristics of
B. subtilis BSNK-5, a high-yield nattokinase strain isolated and identified by our laboratory.
In order to make better use of the spores of BSNK-5, this study optimized the medium
composition using response surface methodology to enhance the sporulation efficiencies of
BSNK-5. Response surface analysis is a collection of mathematical and statistical techniques
widely used in the food industry [12]. It uses multiple quadratic regression equations to
find functional relationships between factors and response values, evaluate the relation-
ship between independent variables and predicted values of dependent variables, and
analyze and solve multivariate problems [13]. The use of response surface analysis in
the optimization of Bacillus culture medium components can reduce development costs,
optimize experimental conditions, and improve production efficiency [14]. Therefore, it is
an effective method to solve the optimization of multiple components.

2. Materials and Methods

2.1. Strain Culture
2.1.1. Microorganism and Culture Mediums
The BSNK-5 strain was screened from fermented soybean products and identified by
the 16S rDNA gene in our laboratory [15]. The bacteria were stored in Luria-Bertani (LB)
broth containing 20% v/v glycerol at −80 ◦ C and activated in an LB solid plate for 12 h at
37 ◦ C before any experimental use.
The LB broth medium was composed of yeast extract (5 g·L−1 ), tryptone (10 g·L−1 ),
and NaCl (10 g·L−1 ). The agar (0.1 g·L−1 ) was added to LB liquid medium to form the
LB solid medium. The optimized medium was changed based on the composition of
carbon source (yeast extract), nitrogen source (tryptone), and inorganic salt (NaCl) of the
LB medium.

2.1.2. Culture Conditions

A single colony of activated BSNK-5 in the LB solid plate was first inoculated into
100 mL of LB broth medium and grown at 37 ◦ C and 200 rpm for 12 h. An amount of 1 mL
of LB broth medium was transferred to 100 mL optimized media and grown at 37 ◦ C and
200 rpm for 60 h.

2.2. Measurement of Total Cell Density and Spore Density

The total cell density and the spore density were calculated according to the plate
count method. Serial tenfold dilutions of the cell suspensions of BSNK-5 were prepared.
Then, 0.1 mL of the 10−6 and 10−7 dilutions were spread in an LB solid plate. These plates
were incubated at 37 ◦ C for 12 h. Viable cell concentration was calculated and expressed as
colony-forming units per milliliter (CFU·mL−1 ). The cell suspensions were heated at 80 ◦ C
for 20 min to harvest the spores. The spores were counted using the same method. The
spore rate was calculated as the percentage of the spore count to the total number of viable
cells of BSNK-5.
Processes 2022, 10, 1133 3 of 10

2.3. Single Factor Test in Culture Medium

2.3.1. Carbon Source Screening
Based on the LB medium, different carbon sources (yeast extract, glucose, sucrose,
soluble starch or lactose) with a concentration of 5 g·L−1 were used to replace the yeast
extract of the LB medium. The optimal carbon source was determined according to the
amount of spore formation. Then, based on the best carbon source, five concentrations of 1,
3, 5, 7 and 9 g·L−1 were set to determine the optimal concentration. The total cell density,
spore cell density and sporulation efficiency were calculated after being cultured for 60 h at
37 ◦ C and 200 rpm.

2.3.2. Nitrogen Source Screening

Based on the LB medium, different nitrogen sources (tryptone, soybean flour, beef
extract, (NH4 )2 SO4 or urea) with a concentration of 10 g·L−1 were used to replace the
tryptone of the LB medium. The optimal nitrogen source was determined according to the
amount of spore formation. Then, based on the best nitrogen source, five concentrations
of 6, 8, 10, 12 and 14 g·L−1 were set to determine the optimal concentration. The total cell
density, spore cell density and sporulation efficiency were calculated after being cultured
for 60 h at 37 ◦ C and 200 rpm.

2.3.3. Inorganic Salts Screening

Based on the LB medium, using different inorganic salts (NaCl, MgSO4 , K2 HPO4 ,
CaCO3 or KCl) with a concentration of 5 g·L−1 , the optimal inorganic salt was determined
according to the amount of spore formation. For the best inorganic salt, five concentrations
of 1, 3, 5, 7, and 9 g·L− 1 were set to determine the optimal concentration. The total cell
density, spore cell density and sporulation efficiency were measured after being cultured
for 60 h at 37 ◦ C and 200 rpm.

2.4. Optimize Culture Medium by Response Surface Methodology

The Box–Behnken design (BBD) of RSM demonstrates a mathematical model of the
interaction between independent variables in medium composition. Important variables
affecting sporulation in BSNK-5 were optimized using a BBD.
According to the results of the single factor experiments, the optimal combination
of the best carbon source, nitrogen source and inorganic salt was further examined by
using the zero-level center point of the response surface. High (+1) and low (−1) levels
are one actual concentration higher or lower, respectively. Design Expert [Link] software
was used to design response surface experiments and perform the regression and graphic
analysis. Variance (ANOVA) and LSD multiple comparison tests were performed on the
obtained data.

2.5. Statistical Analysis

All treatments were performed in triplicate, and data are expressed as mean ± SD.
The data of the total cell and spore cell density (CFU·mL−1 ) were transformed using
a logarithmic scale (Log10). Graphs were generated using GraphPad Prism 8 software
(GraphPad Software Inc., La Jolla, CA, USA). Statistical comparisons were made by a
two-tailed Student’s t-test using IBM SPSS software (Version 22, New York, NY, USA).
Differences were regarded as statistically significant for p < 0.05, p < 0.01 and p < 0.001. The
combined graphs were generated using Adobe Photoshop CS5 and Adobe Illustrator CS5
(Adobe Systems Inc., San Jose, CA, USA).

3. Results
3.1. Screening the Best Carbon Source and Its Optimum Concentration
Among the five carbon sources (yeast extract, glucose, sucrose, soluble starch and
lactose) with a concentration of 5 g·L−1 , the total cell density and spore density of soluble
starch were the highest, followed by those of yeast extract, lactose, glucose and sucrose.
Processes 2022, 10, 1133 4 of 10

Processes 2022, 10, x FOR PEER REVIEW 5 of 12

However, in terms of sporulation efficiency, soluble starch, glucose and lactose were
the highest, followed by lactose and yeast extract (Figure 1a).

Figure1.1. Screening
Figure ofdifferent
Screening of differentcarbon
carbon sources:
sources: (a,b),
(a,b), nitrogen
nitrogen sources
sources (c,d), (c,d), and inorganic
and inorganic salts
salts (e,f)
(e,f) in medium.
in medium.
Processes 2022, 10, 1133 5 of 10

Based on the above experimental results, soluble starch was selected as the best carbon
source for inducing BSNK-5 to produce spores, and its optimal concentration was further
studied. When different concentrations (1, 3, 5, 7 and 9 g·L−1 ) of soluble starch were
used as the only carbon source, the total cell density, spore cell density and sporulation
efficiency showed a trend of first increasing and then decreasing with the increase in
soluble starch concentration. The peak appeared at a concentration of 3 g·L−1 of soluble
starch, in which the total cell density, spore cell density, and sporulation efficiency were
4.8 × 108 CFU·mL−1 , 4.2 × 108 CFU·mL−1 , and 86.8%, respectively (Figure 1b). Therefore,
soluble starch with a concentration of 3 g·L−1 was selected as the best carbon source and
the best concentration.

3.2. Screening the Best Nitrogen Source and Its Optimum Concentration
As shown in Figure 1c, among the five nitrogen sources (tryptone, soybean flour, beef
extract, (NH4 )2 SO4 and urea) with a concentration of 10 g·L−1 , on the whole, the total cell
density, spore density and sporulation efficiency of the organic nitrogen sources (tryptone,
soybean flour, and beef extract) were higher than those of the inorganic nitrogen sources
((NH4 )2 SO4 and urea). The total cell density, spore density, and sporulation efficiency of
soybean flour were the highest in organic nitrogen, followed by those of tryptone and
beef extract.
Then, different concentrations (6, 8, 10, 12, and 14 g·L−1 ) of soybean flour were used
as the only nitrogen source to analyze the effect of BSNK-5 on spore formation. The total
cell density, spore cell density and sporulation efficiency showed a trend of first increasing
and then decreasing with the increase in soybean flour concentration. The maximum value
appeared at the concentration of 12 g·L−1 , at which the total cell density, spore cell density
and sporulation efficiency were 1.2 × 109 CFU·mL−1 , 9.8 × 108 CFU·mL−1 , and 82.6%,
respectively (Figure 1d). Therefore, soybean flour with a concentration of 12 g·L−1 was
selected as the best nitrogen source and the optimal concentration.

3.3. Screening the Best Inorganic Salts and Their Optimum Concentrations
As shown in Figure 1e, based on five inorganic salts (NaCl, KCl, CaCO3 , MgSO4 ,
or K2 HPO4 ) with a concentration of 5 g·L−1 , the total cell density, spore cell density and
sporulation efficiency were analyzed. The formation of the spores had no significant
difference (p > 0.05) among NaCl, KCl and CaCO3 . The number of spores of MgSO4 or
K2 HPO4 were higher than that of NaCl, CaCO3 or KCl. When MgSO4 was used as the
only inorganic salt, the total cell density, spore density and sporulation efficiency were
the highest.
When different concentrations (1, 3, 5, 7, and 9 g·L−1 ) of MgSO4 were used as the only
inorganic salt, the total cell density, spore cell density and sporulation efficiency showed a
trend of first increasing and then decreasing with the increase in MgSO4 concentration. The
maximum value appeared at a concentration of 5 g·L−1 , in which the total cell density, spore
cell density and sporulation efficiency were 8.7 × 108 CFU·mL−1 , 7.3 × 108 CFU·mL−1 ,
and 84.3% (Figure 1f). Therefore, MgSO4 was selected as the best inorganic salt, and 5 g·L−1
was chosen as the optimal concentration.

3.4. Optimizing Combination of Medium Composition by RSM

According to the single factor test, the best components and optimal concentrations
of the spore-forming medium for BSNK-5 were determined as soluble starch at 3 g·L−1 ,
soybean flour at 12 g·L−1 , and MgSO4 at 5 g·L−1 . The BBD of RSM was performed to
further investigate the correlation between the spore density and the three variables (A:
soluble starch, B: soybean flour, C: MgSO4 ) of three levels (soluble starch: 2, 3 or 4 g·L−1 ;
soybean flour: 11, 12 or 13 g·L−1 ; MgSO4 : 4, 5 or 6 g·L−1 ). Response surface analysis was
carried out with soluble starch at 3 g·L−1 , soybean flour at 12 g·L−1 , and MgSO4 at 5 g·L−1
as the center points. The levels of the respective variables are shown in Table 1.
 x FOR PEER REVIEW
Processes 2022, 10, 1133 76of
of 12
10

Table 1. Experimental
Table 1. Experimental factors
factors with
with three
three levels.
levels.

Levels ·L−−11 )
(g·L
Levels (g
Symbol
Symbol Variables
Variables
−11
− 00 11
A
A Soluble Starch
Soluble Starch 22 33 44
B Soybean Flour
Soybean Flour 11 12 13
C
C MgSO
MgSO44 44 55 66

According
According to to the
the BBD,
BBD, aa total
total of
of 1515 experiments
experiments were were performed,
performed, and and their
their results
results are
are
shown
shown in Figure 2 and Table 2. Figure 2a shows the effects of soluble starch, soybean flour
in Figure 2 and Table 2. Figure 2a shows the effects of soluble starch, soybean flour
and their concentrations
and their concentrations on on the
the spore
spore density
density when
when the the addition
additionof ofMgSO was55gg·L
MgSO44 was ·L−−11..
The minimum value value ofofspore
sporedensity
densitywas was1.33
1.33×× 9
1010CFU·mL
9
CFU·mLwhen
−1 − 1 the soluble
when starch
the soluble was
starch
was ·L−soybean
2 g·L2−1 gand 1 and soybean
flour was flour13was
g·L−113 g·L−1the
. When concentrations
. When of solubleof
the concentrations starch
solubleincreased
starch
−1 to 4 g·L−
from 2 g·Lfrom
increased 2 g·L −11, to
and ·L−1 , andflour
4 gsoybean increased
soybean from 11 g·L
flour increased 11 g·L−1−1to
−1 to 13 g·L
from , the · L−1 ,
13 gspore
density
the spore in density
the fermentation broth showed
in the fermentation a trend
broth showed of first increasing
a trend of firstand then decreasing.
increasing and then
When soluble starch was at 3 g·L −1 and soybean − 1
flour at
decreasing. When soluble starch was at 3 g·L and soybean flour at 12 g·L , the 12 g·L −
−1, the spore density 1 reached
spore
the maximum.
density reachedThe thesurfaces
maximum. in Figure 2b, c reflect
The surfaces the similar
in Figure interactions
2b, c reflect between
the similar soluble
interactions
starch
between and MgSOstarch
soluble 4, and and
soybean
MgSO flour and
4 , and MgSO4flour
soybean , respectively.
and MgSO4 , respectively.

Figure 2. Response
Response surface
surfaceinteraction
interactioninfluence
influencesurface
surface diagram.
diagram. Effects
Effects of of soybean
soybean flour
flour andand sol-
soluble
uble starch on spore formation (a); Effects of MgSO
starch on spore formation (a); Effects of MgSO4 and soluble starch on spore formation (b); EffectsEf-
4 and soluble starch on spore formation (b); of
fects
MgSO of MgSO 4 and soybean on spore formation (c).
and soybean on spore formation (c).
4

The results of ANOVA for the response surface quadratic model are shown in Table 3.
The multivariate correlation coefficient of the quadratic model was 0.9146, indicating
that only 8.54% of the variance could not be explained using this model. The regression
model p = 0.0319 indicated that the model was significant. The lack of fit item p-value
is 0.3930, indicating that the lack of fit was not significant, and the model had no lack
of fit phenomenon. The linear and square effects of the model were significant, and the
interaction effect was not significant.
After regression fitting, the quadratic polynomial equation was obtained: Y = 2.34
+ 0.0087A + 0.025B + 0.0063C + 0.17AB − 0.0425AC − 0.025BC − 0.4412A2 − 0.2787B2
− 0.5863C2 . According to the quadratic regression equation, the spore density reached
a maximum value of 2.34 × 109 CFU·mL−1 when the soluble starch concentration was
3 g·L−1 , soybean flour was 12 g·L−1 , and MgSO4 was 5 g·L−1 . To confirm the predicted
results, repeated experiments were performed at these concentrations. The results obtained
from three repetitions of the experiments, are shown in Table 4. The spore density was
Processes 2022, 10, 1133 7 of 10

2.43 × 109 CFU·mL−1 which was close to the expected value of 2.34 × 109 CFU·mL−1 . The
good fit of the two values confirmed the validity of the model.

Table 2. Central composite design of actual factors and responses based on actual values.

Coded Value Spore Density

Std
A B C (×109 CFU·mL− 1 )

1 1 −1 0 1.57
2 0 −1 −1 1.4
3 1 0 −1 1.24
4 0 1 −1 1.7
5 0 0 0 2.53
6 0 1 1 1.5
7 −1 0 1 1.47
8 0 0 0 2.21
9 0 0 0 2.28
10 −1 −1 0 1.82
11 1 1 0 1.76
12 1 0 1 1.33
13 0 −1 1 1.30
14 −1 1 0 1.33
15 −1 0 −1 1.21

Table 3. Analysis of variance (ANOVA) for response surface quadratic model.

Source Adj SS df Adj MS F-Value p-Value

Model 2.14 9 0.2378 5.95 0.0319 *
A-Soluble Starch 0.0006 1 0.0006 0.0153 0.9063
B-Soybean Flour 0.0050 1 0.0050 0.1250 0.7380
C-MgSO4 0.0003 1 0.0003 0.0078 0.9330
AB 0.1156 1 0.1156 2.89 0.1498
AC 0.0072 1 0.0072 0.1807 0.6885 *
BC 0.0025 1 0.0025 0.0625 0.8125 *
A2 0.7189 1 0.7189 17.98 0.0082 *
B2 0.2869 1 0.2869 7.18 0.0439 *
C2 1.27 1 1.27 31.74 0.0024 *
Residual 0.1999 5 0.0400
Lack of Fit 0.1433 3 0.0478 1.69 0.3930
Pure Error 0.0566 2 0.0283
Cor Total 2.34 14
* Coefficients of variation (CV): 12.17%, Coefficient of determination (R2): 0.9146, Adjusted R2: 0.7608, Adequate
precision: 6.6455.

Table 4. Numerical optimization and validation of pretreatment based on the regression models.

Parameters Coded Factor Levels Actual Factor Levels (g·L−1 )

Soluble Starch 0 3
Soybean Flour 0 12
MgSO4 0 5
Response Predicted Actual
Spore cell density
2.34 2.43
(×109 CFU·ml−1 )

4. Discussion
Spores are a naturally occurring form of B. subtilis. The formation of spores can keep
bacteria highly active in extreme environments and industrial production processes, which
can be used in the production and application of probiotics. It has been confirmed that
the composition of the culture medium is a key factor affecting the spore formation of
Processes 2022, 10, 1133 8 of 10

B. subtilis [16]. BSNK-5 is a lab-bred strain with a high nattokinase yield and excellent
application prospects [17]. In this research, the influence of the sporulation of a carbon
source, nitrogen source and inorganic salt on BSNK-5 were studied for the first time.
The results indicated that the best components of the spore medium for BSNK-5 were
soluble starch, soybean flour, and MgSO4 . With the concentration of soluble starch at
3 g·L−1 , soybean flour at 12 g·L−1 , and MgSO4 at 5 g·L−1 , the maximum spore density of
2.43 × 109 CFU·mL−1 was obtained.
In this study, single carbon sources (glucose, sucrose and lactose) and compound
carbon sources (yeast extract and soluble starch) were used to screen for the best carbon
source for the spore production of BSNK-5. In general, the total colony density and spore
density of compound carbon sources were higher than those of single carbon sources.
Soluble starch showed the best advantage in promoting spore formation. This may be
related to the differences in basic composition and absorption and utilization efficiency of
the two kinds of carbon sources [18]. A single carbon source is composed of one component
and can be absorbed directly, while a compound carbon source is composed of several
components and needs to be degraded before absorption and utilization [19]. It can be
seen that proper nutrient supply is favorable for spore formation. Studies have shown
that glucose has a negative effect on spore formation [20], and excess glucose inhibits
sporulation by repressing the transcription of the spo0A gene. This gene is responsible
for encoding the Spo0A protein, a response regulator activated by phosphorylation in
response to several internal and external stimuli, and is the master regulator of entry into
sporulation [21,22].
Inorganic nitrogen sources ((NH4 )2 SO4 and urea) and organic nitrogen sources (tryp-
tone, soybean flour and beef extract) were used to screen for the best nitrogen source for
the spore production of BSNK-5. Nitrogen sources also showed the same trend as those
of carbon sources in this study. The results of nitrogen source optimization showed that
the total cell density and spore density of BSNK-5 were relatively low when (NH4 )2 SO4
and urea were used as nitrogen sources, which indicated that inorganic nitrogen was the
only nitrogen source that was not conducive to the growth and spore formation of BSNK-5.
When soybean flour was used as the sole carbon source, the total colony density and
spore density were highest, probably because soybean flour contains limited and complex
nutrient components, which are more difficult to digest and absorb than those of the other
nitrogen sources, and this resulted in BSNK-5 forming more spores under starvation stress.
Numerous studies have confirmed that inorganic salts can influence spore formation.
Adding inorganic salts to the medium, such as high concentrations of Na+ , Mg2+ , K+ and
Ca2+ , affected the formation of spores [23]. The addition of NaCl significantly increased
the total number of bacteria, but had little effect on the number of spores, and CaCO3
played a role in buffering pH and affecting spore formation [24]. In our experiment, Na+ ,
Mg2+ , K+ and Ca2+ all promoted the total cell density and spore density, and the addition
of MgSO4 had the most obvious improvement in the total density of colonies and spores.
This may be related to the fact that Mg2+ can stabilize the ribosome complex, which can
change the sensitivity of bacteria to antibiotics [25].
The response surface analysis is clearly accurate and widely used. At present, it is
commonly used to optimize medium composition and fermentation conditions to improve
various enzymes, exopolysaccharides, amino acids and other metabolites produced by
B. subtilis [26,27]. In this experiment, the center points and factor levels of the response
surface were determined by the single factor experiment, and then the response surface
analysis chart was obtained through BBD analysis. This can directly reflect the influence
of various factors on the response value, so as to find the optimal process parameters and
the interaction between them [28]. The greater the curvature of the convex graph, the
more significant the interaction between the two factors [29]. The response surface analysis
graphs in this experiment were all convex graphs, indicating that the interaction between
the soluble starch, soybean flour and MgSO4 were significant.
Processes 2022, 10, 1133 9 of 10

Spores are not an indispensable part of bacterial life history; they are simply a stress-
resistant dormant body formed by spore-producing bacteria during the growth process.
The formation of spores is affected by many factors. In addition to the characteristics of
the bacteria and the composition of the medium studied in this paper, some fermentation
process parameters, such as temperature, pH value, and ventilation, etc., also need to
be focused on [30]. At the same time, the effects and mechanisms of different media
components on the spore formation of BSNK-5 still need to be further explored.

5. Conclusions
High cell density and sporulation efficiency are critical for Bacillus-based products.
In this study, the BSNK-5 sporulation medium was optimized using the single factor test
and response surface methodology. Based on the single factor test, the best components
of the spore-forming medium for BSNK-5 were determined as soluble starch, soybean
flour, and MgSO4 . After composite analysis by response surface, the optimized component
concentrations were 3 g·L−1 of soluble starch, 12 g·L−1 of soybean flour, and 5 g·L−1 of
MgSO4 . With this combination, the maximum spore density of 2.43 × 109 CFU·mL−1 for
BSNK-5 was obtained, which was nearly three times higher than before. Through this study,
the production of bacterial spores was not only improved, but the amount of nutrients
required for fermentation was also determined, laying a theoretical foundation for the
application of BSNK-5 in industrial production.

Author Contributions: Conceptualization, F.W. and S.L.; Writing—original draft preparation, Z.T.;
Writing—review and editing, supervision, Z.T., D.L., M.H., Y.G. and S.L.; Investigation, Y.G., D.L.,
Z.T. and L.H.; Validation, Y.G., S.L. and Z.T.; Funding acquisition, F.W., B.F. and S.L.; Project adminis-
tration, F.W., B.F. and S.L.; Data curation, Z.T., Y.G. and D.L.; Formal analysis, L.H. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by China Agriculture Research System (CARS-04), Central
Public-interest Scientific Institution Basal Research Fund (S2021JBKY-02) and Central Public-interest
Scientific Institution Basal Research Incremental Fund (Y2020PT10).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors acknowledge the funds from the Knowledge Innovation Program
Funding of the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences.
Conflicts of Interest: The authors declare no conflict of interest.

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