High-density fermentation method of bacillus subtilis
By using Bacillus subtilis SKC8-ob and its NAC addition in the fermentation system, the problem of high cost in high-density fermentation was solved, achieving a high-efficiency and low-cost fermentation process, increasing the number of viable bacteria and spore rate, and enhancing the biocontrol effect.
Patent Information
- Application Number
- CN202611569869.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-10-12
- Publication Date
- 2026-11-06
AI Technical Summary
The high-density fermentation of Bacillus subtilis in existing technologies is costly and has a long fermentation cycle, resulting in low market acceptance and restricting its promotion and application in the agricultural field.
A strain of Bacillus subtilis SKC8-ob was used, and N-acetylcysteine (NAC) was added to the fermentation system. By utilizing its special tolerance properties, the physiological activity of the bacteria was improved, the logarithmic growth cycle was extended, the number of viable bacteria and the spore rate in the fermentation broth were increased, and the post-fermentation treatment process was simplified.
It significantly improves fermentation efficiency, reduces production costs, simplifies post-fermentation processing, reduces equipment and operating costs, increases viable cell count and spore rate, and enhances biological control effects.
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Figure CN123042871A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a high-density fermentation method for a strain of Bacillus subtilis. Background Technology
[0002] Bacillus subtilis is widely distributed in nature. This bacterium exhibits diverse physiological and biochemical characteristics, has low nutritional requirements, is easily isolated and purified, grows rapidly without polluting the environment, and is harmless to humans and animals. Bacillus subtilis also possesses strong broad-spectrum antibacterial activity and can produce dormant spores with environmental resistance in its later growth stages. Therefore, Bacillus subtilis is an ideal biocontrol microorganism.
[0003] Bacillus subtilis demonstrates remarkable performance in agriculture, primarily in three areas: biological control, growth promotion, and soil improvement. In biological control, Bacillus subtilis inhibits the growth of various pathogenic fungi, activates the plant's own defense system, and enhances crop resistance to diseases. Regarding growth promotion, Bacillus subtilis secretes substances similar to plant growth hormones (such as indoleacetic acid) and converts nutrients such as phosphorus and potassium, which are not easily absorbed by the soil, into soluble forms for plant utilization. In improving soil properties, Bacillus subtilis accelerates the decomposition of organic matter in the soil, improves soil aggregate structure, alleviates soil compaction, thereby increasing fertilizer utilization and creating a better growth environment for roots.
[0004] Although Bacillus subtilis preparations have shown significant advantages in the agricultural sector, market acceptance remains low, with product price being a major factor restricting their widespread application. Bacillus fertilizers are generally more expensive than chemical fertilizers. This is primarily due to the lower high-density fermentation cell count of Bacillus subtilis, resulting in higher production and fermentation costs, a longer fermentation cycle, and ultimately, higher unit production costs.
[0005] Therefore, if a low-cost, high-density fermentation method for preparing Bacillus subtilis can be provided, it will be of great significance to promoting the high-quality development of green agriculture. Summary of the Invention
[0006] The purpose of this invention is to provide a high-density fermentation method for a strain of Bacillus subtilis.
[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a strain of Bacillus subtilis (… Bacillus subtilis SKC8-ob was deposited on February 3, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.67800, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0008] Accordingly, a bacterial preparation was prepared using the Bacillus subtilis SKC8-ob.
[0009] Correspondingly, biopesticides, plant growth promoters, and soil conditioners are prepared using the Bacillus subtilis SKC8-ob or the bacterial preparation.
[0010] Accordingly, a method for fermenting the Bacillus subtilis SKC8-ob involves adding N-acetyl-L-cysteine (NAC) to the fermentation system. The NAC content in the fermentation system is 10 mg / L to 50 mg / L; more preferably, it is 10 mg / L to 30 mg / L; even more preferably, it is 20 mg / L to 30 mg / L.
[0011] Accordingly, the Bacillus subtilis SKC8-ob bacteria preparation was prepared using the method described above.
[0012] The present invention has the following beneficial effects:
[0013] As a precursor for the synthesis of Bacillithiol (BSH, Bacillus thiol), NAC can enhance the endogenous antioxidant level of Bacillus subtilis, fundamentally strengthening the cell's own "anti-aging" ability. At the same time, NAC can directly remove reactive oxygen species in the fermentation broth environment, creating a low-oxidative-stress external environment for cells, reducing the damage of oxidative stress to cells, and enabling bacteria to maintain high-speed growth and division for a longer period of time.
[0014] However, in practice, NAC often does not promote bacterial growth and may even inhibit it. This is because: (1) The -SH in NAC can damage the integrity of the bacterial cell wall / membrane, steal disulfide bonds from proteins, leading to enzyme inactivation, structural protein denaturation, and inhibiting DNA replication and transcription. (2) It causes redox imbalance: Bacteria need a moderate redox potential (ORP) to respire / metabolize. NAC can cause the electron transport chain (ETC) to be paralyzed, ATP synthesis to be blocked, and growth to stop. (3) Osmotic stress: High concentrations of NAC can also cause excessively high osmotic pressure in the environment, which in turn leads to bacterial dehydration, shrinkage, and growth inhibition. (4) It inhibits quorum sensing (QS): NAC can quench QS signaling molecules, making it impossible for bacteria to coordinate growth, virulence, and biofilm, thus inhibiting overall growth. (5) It causes metabolic burden: After being ingested by bacteria, NAC requires a large amount of energy for detoxification / metabolism, which in turn crowds out growth resources. Based on the above limitations, NAC has not yet been applied in practice to directly promote bacterial growth.
[0015] This invention provides a novel Bacillus subtilis strain SKC8-ob that exhibits exceptional tolerance to NAC. Based on Bacillus subtilis SKC8-ob and its unique properties, this invention specifically adds NAC during the cultivation of Bacillus subtilis SKC8-ob. This helps the bacteria resist various environmental stresses such as oxidation, acid, and heat, improves the physiological activity of the bacteria, and extends the logarithmic growth cycle of the bacteria. This effectively increases the viable cell count and spore rate in high-density Bacillus subtilis fermentation broth, significantly improving fermentation efficiency and reducing production costs.
[0016] Increased spore count in fermentation broth simplifies post-fermentation processing. Direct spraying can be used to produce microbial powder (after fermentation broth is shut down, it is not centrifuged and concentrated; instead, spray powder additives are added directly and then the mixture is sprayed into a spray drying tower for drying). This eliminates the traditional centrifugal concentration step, reduces the manpower required for centrifugal equipment and processes, avoids the generation of supernatant wastewater from traditional centrifugal processes, and reduces the wastewater treatment load and operating costs. Attached Figure Description
[0017] Figure 1 The colony morphology of Bacillus subtilis SKC8-ob on LB agar plates;
[0018] Figure 2 This is a plate confrontation inhibition test of Bacillus subtilis SKC8-ob against different plant pathogens; where A is the pathogen of apple rot; B is the pathogen of cucumber black spot; C is the pathogen of wheat scab; D is the pathogen of rice sheath blight; E is the pathogen of solanum wilt; and F is the pathogen of stem base rot.
[0019] Figure 3 These are the results of tolerance tests on Bacillus subtilis SKC8-ob, SKC1, and SKC2 to different concentrations of NAC; where A represents the OD of the fermentation broth. 600 Test results; B represents the result of effective viable bacteria count detection;
[0020] Figure 4 Bacillus subtilis SKC8-ob was cultured in a 50L tank and measured using OD. 600 Growth curve plotted from net increment;
[0021] Figure 5 These are the microscopic examination results of Bacillus subtilis SKC8-ob at the end of fermentation in a 50L tank; where A is the microscopic examination result of the control group at the end of fermentation; and B is the microscopic examination result of the experimental group at the end of fermentation. Detailed Implementation
[0022] This invention provides a novel *Bacillus subtilis* SKC8-ob strain, deposited on February 3, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC NO. 67800, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The 16S rDNA sequence of the *Bacillus subtilis* SKC8-ob is shown in SEQ ID NO: 1.
[0023] This invention also provides a method for high-density fermentation of the Bacillus subtilis SKC8-ob, specifically comprising adding the antioxidant NAC to the culture medium. Preferably, the concentration of NAC in the culture system is 10–50 mg / L, more preferably 10–30 mg / L, and even more preferably 20–30 mg / L.
[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values obtained after at least three repetitions, and each repetition yields valid data.
[0025] Example 1: Screening, Identification and Preservation of Strains
[0026] 1. Screening
[0027] Soil from the experimental field in Shuangliu District, Chengdu City, Sichuan Province was taken and serially diluted. After the diluted solution was heated in an 80℃ water bath for 20 minutes, it was spread onto LB medium and incubated at 37℃ for 24 hours. Single colonies of suitable size were selected as strains for preliminary screening.
[0028] The initially screened strains were streaked on LB medium containing 200 mg / L NAC and incubated at 37°C for 24 hours. The colony growth was observed, and the largest colony was selected. After purification, a single colony strain SKC8-ob with some tolerance to NAC was obtained.
[0029] 2. Identification
[0030] (1) Physiological and biochemical characteristics: The colony morphology of strain SKC8-ob is shown in the figure below. Figure 1As shown. After incubation on LB medium at 37°C for 1 day, the colonies were off-white, 5–8 mm in diameter, with irregular edges and wrinkled surfaces. Strain SKC8-ob was Gram-positive, with rod-shaped cells 2–3 μm long and 0.7–0.8 μm wide. The SKC8-ob strain can survive in a temperature range of 15–50°C, with an optimal temperature range of 33–38°C; it can survive in a pH range of 5.0–9.0, with an optimal pH range of 6.5–7.5.
[0031] The carbon sources available to strain SKC8-ob include glucose, sucrose, fructose, galactose, and corn starch; the nitrogen sources available include beef extract, peptone, yeast powder, soybean meal, ammonium sulfate, and sodium nitrate.
[0032] (2) Molecular identification: Genomic DNA of strain SKC8-ob was extracted using the Shanghai Sangon Biotech Bacterial Genome Extraction Kit. The 16S rRNA of SKC8-ob was amplified using the universal primer pair 27F-1492R for bacterial 16S rRNA. The amplified target fragment was sequenced using an ABI 3730 sequencer. The 16S rRNA gene sequence of strain SKC8-ob is shown in SEQ ID NO:1.
[0033] Sequence alignment analysis using the NCBI database showed that strain SKC8-ob had 100% similarity to Bacillus subtilis (Sequence ID: NC_000964.3) in the database.
[0034] Based on comprehensive physiological and biochemical characteristics and molecular identification results, strain SKC8-ob was identified as Bacillus subtilis. It was deposited on February 3, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO.67800, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The 16S rDNA sequence of Bacillus subtilis SKC8-ob is shown in SEQ ID NO: 1.
[0035] Example 2: Biocontrol and antibacterial effects of Bacillus subtilis SKC8-ob
[0036] To verify the biocontrol and antibacterial effects of Bacillus subtilis SKC8-ob, a plate confrontation inhibition test was conducted using six common plant pathogens: apple rot pathogen, cucumber black spot pathogen, wheat scab pathogen, rice sheath blight pathogen, solanum falcatum pathogen, and stem rot pathogen. The specific experimental procedures are as follows:
[0037] (1) Preparation of pathogen spore culture: The above pathogens were inoculated into PDA liquid culture medium and cultured at 28℃ and 180rpm for 5 days to obtain the spore culture medium of each pathogen.
[0038] (2) Preparation of SKC8-ob bacterial culture: SKC8-ob bacteria were added to LB medium and cultured at 37℃ and 180 rpm for 24 hours to obtain SKC8-ob bacterial culture.
[0039] (3) Plate confrontation test: Using a sterile pipette, 5 μL of each pathogen spore suspension was inoculated into the center of a new PDA plate, and 5 μL of SKC8-ob bacterial suspension was inoculated at equal distances of 3 cm on both sides of the pathogen. The plate inoculated only with the pathogen served as a control. Each treatment was repeated 3 times and incubated at 28℃ for 7 days. The diameter of the inhibition zone was observed and recorded, and the inhibition rate was calculated according to the following formula:
[0040] Antibacterial rate = ×100%.
[0041] The antibacterial effects of Bacillus subtilis SKC8-ob against common plant pathogens are shown in Table 1. *Data in Table 1 are mean ± standard deviation.
[0042] Table 1: Antifungal effects of SKC8-ob against common plant pathogens
[0043]
[0044] The antibacterial effect of the plate confrontation is as follows Figure 2 As shown in Table 1, there are obvious inhibition zones between Bacillus subtilis SKC8-ob and plant pathogens, indicating that Bacillus subtilis SKC8-ob has a good inhibitory effect on common plant pathogens. The inhibition rate of Bacillus subtilis SKC8-ob against common plant pathogens is shown in Table 1. SKC8-ob has an inhibition rate of more than 60% against common plant pathogens, and the inhibition effect is obvious. Among them, the inhibition effect against cucumber black spot pathogen is the best, with an inhibition rate of (78.43±1.35)%.
[0045] Example 3: Demonstration of Bacillus subtilis SKC8-ob's tolerance to NAC
[0046] Bacillus subtilis SKC8-ob and other Bacillus subtilis strains SKC1 and SKC2 preserved in the inventor's laboratory were selected to determine the tolerance of different strains to NAC. The specific experiments are as follows:
[0047] (1) Activation of bacterial strains: SKC8-ob, SKC1 and SKC2 bacterial strains were streaked onto LB agar plates and cultured at 37°C for 18 hours. Single colonies of appropriate size were picked, streaked onto LB agar slants, and cultured at 35°C for 24 hours to obtain activated bacterial strains.
[0048] (2) Preparation of seed culture in shake flask: 200 mL of LB seed culture medium was added to each of the 500 mL Erlenmeyer flasks. The activated strains SKC8-ob, SKC1 and SKC2 in step (1) were inoculated into the seed culture medium in the shake flasks using an inoculation loop. The culture conditions were: temperature 37℃, shaker speed 180 r / min and culture time 20 h.
[0049] (3) Preparation of shake flask fermentation medium: Prepare shake flask fermentation medium with the following formula: corn starch 40 g / L, soybean meal 20 g / L, corn steep liquor powder 5 g / L, yeast powder 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, manganese sulfate 0.2 g / L, calcium chloride 1 g / L, pH=7.6, sterilize at 121℃ for 20 min. After sterilization, filter NAC through a sterile filter membrane in a sterile room and add it to the Erlenmeyer flask to make the NAC concentrations 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 500 mg / L, respectively.
[0050] (4) Inoculation and culture: The SKC8-ob, SKC1, and SKC2 shake flask seeds cultured in step (2) were inoculated into NAC fermentation medium of different concentrations prepared in step (3) in an aseptic room. After culturing at 37°C and 240 r / min for 24 h, the fermentation broth was diluted 200 times and the OD was measured. 600 Samples were simultaneously taken to a sterile room for viable bacteria counting.
[0051] The results are as follows Figure 3 As shown, Figure 3 A is OD 600 , Figure 3 B represents the effective viable cell count. Results showed that after 24 hours of culture, the OD of the SKC8-ob fermentation broth... 600 The OD of the SKC8-ob fermentation broth initially increased with increasing NAC concentration. At an NAC concentration of 30 mg / L, the OD... 600 The maximum value of 178.9 was reached, and the effective viable bacteria count was 2.84 × 10⁻⁶. 10 CFU / mL, followed by OD 600 The concentration of NAC decreases rapidly with increasing concentration, and when the NAC concentration reaches 500 mg / L, the bacteria are completely inhibited and cease to grow.
[0052] The overall growth trends of SKC1 and SKC2 strains were similar: at low concentrations of NAC, their growth was not significantly affected; as the concentration of NAC increased, their inhibitory effect on bacterial growth was significantly enhanced; when the concentrations of NAC reached 200 mg / L and 50 mg / L, respectively, the growth of SKC1 and SKC2 strains was completely inhibited.
[0053] The results showed that low concentrations of NAC had a weak effect on the growth of *Bacillus subtilis*, while at a concentration of 30 mg / L, NAC significantly inhibited the growth of *Bacillus subtilis*. However, for *Bacillus subtilis* SKC8-ob of this invention, NAC at concentrations of 50 mg / L and below significantly promoted its growth.
[0054] Example 4: NAC promotes high-density fermentation of Bacillus subtilis SKC8-ob
[0055] 1. 50L tank test
[0056] (1) Activation of bacterial strain: Bacillus subtilis SKC8-ob was streaked onto LB agar plates and cultured at 35°C for 24 h. Then, a single colony of suitable size was streaked onto LB agar slants and cultured at 35°C for 24 h to obtain the activated bacterial strain.
[0057] (2) Preparation of seed culture in shake flask: 500 mL of LB seed culture medium was added to a 1000 mL Erlenmeyer flask. The activated strains in step (1) were inoculated into the seed culture medium in the shake flask using an inoculation loop. The culture conditions were: temperature 35℃, shaker speed 180 r / min, and culture time 18 h.
[0058] (3) Preparation of fermentation medium in a 50L tank: Fermentation medium for strain SKC8-ob was prepared and divided into a control group and an experimental group. The experimental group consisted of the following: 50g / L corn starch, 25g / L soybean meal, 5g / L corn steep liquor powder, 5g / L yeast powder, 1.5g / L dipotassium hydrogen phosphate, 0.5g / L magnesium sulfate, 0.2g / L manganese sulfate, 1g / L calcium chloride, 10mg / L NAC, pH=7.6. All components except NAC were sterilized at 121℃ for 20min. After sterilization and inoculation, the fermentation volume in the fermenter was 30L. NAC was sterilized separately by filtration, filtered through a 0.22μm sterile membrane in a sterile room into a sterilized Erlenmeyer flask. In the control group, no NAC was added, but an equal amount of sterile water was added, and the rest were the same.
[0059] (4) 50L tank inoculation and fermentation: The seed liquid cultured in step (2), the fermentation medium prepared in step (3) and the sterilized NAC were poured into a 50L fermenter by fire ring inoculation (the control group used sterile water of the same amount as NAC instead of NAC). The aeration rate was 1.2VVM (36L / min), the rotation speed was linked to the dissolved oxygen control, the dissolved oxygen control range was 30-70%, the fermentation temperature was 35℃, and the tank pressure was constant at 0.05MPa.
[0060] (5) Sample testing: Starting from 0h after inoculation, samples were taken from the experimental group and the control group every 1h, diluted 200 times, and their OD was measured. 600 The growth curves of SKC8-ob under two culture media were plotted, and the cell morphology was observed under a microscope. Fermentation was stopped when the spore germination rate in the microscopic field of view was greater than 80% and the spore degermination rate was greater than 50%. Samples were taken in a sterile room, diluted and spread to calculate the number of viable bacteria and spores in the fermentation broth, and the spore rate was calculated.
[0061] OD of Bacillus subtilis SKC8-ob experimental group and control group 600 The net incremental growth curve (excluding the background OD value of the 0h fermentation broth) is shown in the figure. Figure 4 As shown, the OD of the experimental group 600 The OD level was significantly increased compared to the control group, which had an OD level of 24 hours after fermentation. 600 The OD value reached its maximum of 169.1 after 28 hours of fermentation in the experimental group. 600 The maximum value was reached at 219.7, which was 29.9% higher than that of the control group. The control group reached the fermentation endpoint after 24 hours (at which point the spore germination rate was highest under microscopic examination), with a spore germination rate of 88.4% and a spore shedding rate of 55.9%. At this point, the viable cell count in the fermentation broth was 2.82 × 10⁻⁶. 10 CFU / mL, spore count 2.61 × 10⁻⁶ 10 The CFU / mL concentration was 92.3%; the experimental group reached the fermentation endpoint after 28 hours (at which point the spore germination rate was highest under microscopic examination), with a spore germination rate of 97.4% and a spore shedding rate of 86.5% under microscopic examination. At this point, the viable cell count in the fermentation broth was 3.87 × 10⁻⁶. 10 CFU / mL, spore count 3.77 × 10⁻⁶ 10 CFU / mL, spore count 97.3%. Microscopic examination results of fermentation endpoint in the experimental and control groups are as follows: Figure 5 As shown; Figure 5 A represents the microscopic examination results of the control group (24h). Figure 5B shows the microscopic examination results of the experimental group (28h). The results show that: at the end of fermentation, the spore synchronicity of the control group was low, with a germination rate of 88.4%, and some spores in the field of view germinated and underwent secondary growth; the spore synchronicity of the experimental group was high, with a germination rate of 97.4%, and there were basically no vegetative cells in the field of view. This shows that the addition of NAC can prolong the logarithmic growth cycle of SKC8-ob, while improving the synchronicity of spore germination, increasing the germination rate and spore shedding rate.
[0062] 2.5T tank pilot test
[0063] The strain activation and seed liquid preparation were the same as those tested in the 50L tank.
[0064] (1) Preparation of fermentation broth for primary seed tank in 500L tank: Prepare primary seed tank culture medium in 500L tank with a liquid volume of 300L. The formula of primary seed tank culture medium is as follows: corn starch 15g / L, soybean meal 10g / L, yeast powder 4g / L, corn steep liquor powder 4g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, silicone oil defoamer 1g / L, pH=7.5, sterilize at 121℃ for 30min. Then, inoculate the seed liquid into the 500L seed tank through a fire ring with an inoculation amount of 1%, fermentation temperature of 37℃, fermentation tank rotation speed of 150r / min, aeration rate of 1.0VVM, tank pressure of 0.04Mpa, and culture for 8 hours to obtain primary seed tank fermentation broth.
[0065] (2) High-density fermentation in a 5T tank: A high-density fermentation medium was prepared in a 5T fermenter at a rate of 3T. The medium formula was as follows: corn starch 60g / L, soybean meal 35g / L, corn steep liquor powder 10g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 2g / L, manganese sulfate 0.2g / L, calcium chloride 1.5g / L, silicone oil defoamer 1g / L, pH 7.5, sterilized at 121℃ for 30min. After sterilization of the 5T fermenter, 45g of filtered sterilized NAC was added to the tank through a fire ring to make the NAC concentration in the 5T tank 15mg / L (experimental group). Then, all the fermentation liquid from the primary seed tank prepared in step (1) was transferred to the 5T fermenter through the transfer pipeline. The inoculum amount was 10%, the fermentation temperature was 37℃, the fermenter rotation speed was 180r / min, the aeration rate was 1.5VVM, and the tank pressure was 0.04Mpa. A control group was set up with all other conditions being the same except that an equal amount of sterile water was used instead of NAC.
[0066] (3) Sample testing and results: After 10 hours of fermentation, samples were taken every hour for microscopic examination and plating to observe the cell growth. The fermentation endpoint was determined based on the microscopic examination. After 27 hours of fermentation, microscopic observation showed that the fermentation endpoint of the experimental group had been reached. At this point, the budding rate was 98.3% and the debudding rate was 89.1%. Fermentation was stopped, and samples were taken in a sterile room for dilution and plating to calculate the viable cell count, which was 4.28 × 10⁻⁶. 10CFU / mL, spore count 4.13 × 10⁻⁶ 10 CFU / mL, spore count 96.5%.
[0067] After 22 hours of fermentation, microscopic examination of the control group revealed that fermentation had reached its endpoint. At this point, the budding rate of the control group was 92.6%, and the debudding rate was 63.4%. Fermentation was then stopped, and samples were taken in a sterile room, diluted, and plated. The viable cell count at this point was calculated to be 3.03 × 10⁻⁶. 10 CFU / mL, spore count 2.82 × 10⁻⁶ 10 CFU / mL, spore rate 93.1%.
[0068] 3. Scale-up of 50T tank production
[0069] The strain activation and seed liquid preparation were the same as in the 50L tank test; the fermentation broth in the 500L primary seed tank was the same as in the 5T tank pilot test.
[0070] (1) Secondary seed culture medium in a 5T fermenter: Secondary seed culture medium was prepared in a 5T fermenter at a rate of 2.5T. The culture medium formula was as follows: corn starch 20g / L, soybean meal 15g / L, yeast powder 5g / L, corn steep liquor powder 4g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 1g / L, silicone oil defoamer 0.5g / L, pH 7.6, sterilized at 121℃ for 30min. After sterilization of the 5T fermenter, the fermentation liquid from the 500L primary seed tank was transferred to the 5T fermenter through the transfer pipeline. The inoculum size was 8%, the fermentation temperature was 37℃, the fermenter rotation speed was 150r / min, the aeration rate was 1.0VVM, the tank pressure was 0.04Mpa, and the culture was carried out for 5h to obtain the secondary seed tank fermentation liquid.
[0071] (2) High-density fermentation in a 50T tank: A high-density fermentation medium was prepared in a 50T fermenter at a rate of 33T. The medium formula was as follows: corn starch 70g / L, soybean meal 40g / L, corn steep liquor powder 5g / L, yeast powder 2g / L, dipotassium hydrogen phosphate 1.5g / L, magnesium sulfate 2g / L, manganese sulfate 0.4g / L, calcium chloride 0.3g / L, silicone oil defoamer 1.5g / L, pH 7.6, sterilized at 121℃ for 30min. After sterilization of the 50T fermenter, 600g of filtered and sterilized NAC was added to the 5T seed tank through a fire ring to make the NAC concentration in the 50T tank 20mg / L (experimental group). Then, all the fermentation liquid from the secondary seed tank was transferred to the 50T fermenter through the transfer pipeline. The inoculum size was 8.1%, the fermentation temperature was 37℃, the fermenter rotation speed was 150r / min, the aeration rate was 1.5VVM, and the tank pressure was 0.06Mpa. A control group was set up with all other conditions being the same except that an equal amount of sterile water was used instead of NAC.
[0072] (3) Sample testing and results: After 10 hours of fermentation, samples were taken every hour for microscopic examination and spread to observe the cell growth. The fermentation endpoint was determined based on the microscopic examination. After 25 hours of fermentation, microscopic examination showed that the fermentation endpoint of the experimental group had been reached. At this time, the budding rate of the cells was 95.7%, and the debudding rate was 84.5%. Fermentation was stopped, and samples were taken in a sterile room for dilution and spread to calculate the viable cell count, which was 4.42 × 10⁻⁶. 10 CFU / mL, spore count 4.35 × 10⁻⁶ 10 CFU / mL, spore rate 98.4%.
[0073] The control group reached the fermentation endpoint after 22 hours. Microscopic examination showed a budding rate of 93.6% and a debudding rate of 68.6%. Fermentation was then stopped, and samples were taken in a sterile room, diluted, and spread to calculate the viable cell count, which was 3.49 × 10⁻⁶. 10 CFU / mL, spore count 3.27 × 10⁻⁶ 10 CFU / mL, spore rate 93.7%.
[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for fermenting Bacillus subtilis, characterized in that: The Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis SKC8-ob was deposited on February 3, 2026, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.67800, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The method includes adding N-acetylcysteine to the fermentation system; the content of N-acetylcysteine in the fermentation system is 10 mg / L to 50 mg / L.
2. The method according to claim 1, characterized in that: In the fermentation system, the content of N-acetylcysteine is 10 mg / L to 30 mg / L.
3. The method according to claim 1, characterized in that: The content of N-acetylcysteine in the fermentation system is 20 mg / L to 30 mg / L.