Bacillus subtilis Mafic-240428 and application thereof

Abstract

The invention discloses bacillus subtilis Mafic-240428 and an application of the bacillus subtilis Mafic-240428. The bacillus subtilis Mafic-240428 (the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.33720) has good antibacterial activity on common gram-positive pathogenic bacteria, and meanwhile, the bacillus subtilis Mafic-240428 can have the same antibacterial effect on methicillin-resistant staphylococcus aureus. According to the fermentation conditions provided by the invention, the fermentation effect of the strain Mafic-240428 can be obviously improved, and the production cost is reduced; the strain Mafic-240428 does not have hemolytic activity, does not have drug resistance to common antibiotics, is high in safety and can be widely applied to animal husbandry production.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Bacillus subtilis Mafic-240428 and applications thereof. Background Art

[0002] In 1928, Fleming noticed that Penicillium could inhibit the growth of Staphylococcus aureus. Penicillin was subsequently extracted and widely used in production. However, just two years after penicillin's widespread use, scientists discovered drug-resistant Staphylococcus aureus. Agriculture and animal husbandry are the largest users of antibiotics, with approximately 100,000 to 200,000 tons of antibiotics used annually worldwide, accounting for over 70% of total global antibiotic use.

[0003] In recent years, research on the antimicrobial effects of probiotics has made significant progress. Numerous studies have shown that the use of probiotics as feed additives can reduce animal disease and antibiotic use. Probiotics possess a variety of antimicrobial mechanisms, with the production of antimicrobial substances such as bacteriocins, which directly inhibit the growth of pathogens, being a primary mechanism. Danzhou chicken, a superior local chicken breed native to Danzhou, Hainan, has developed a strong resistance to many common diseases through long-term natural selection, reducing disease risks in livestock farming. However, the probiotics in its gut have not been fully developed.

[0004] Bacillus subtilis can synthesize a variety of antimicrobial compounds, including surfactant, fengycin, and iturin, which inhibit the growth of various pathogens. This improves animals' disease resistance and reduces their reliance on antibiotics, aligning with the current trend of banning antibiotics in feed in the livestock industry. Optimizing fermentation conditions is crucial for probiotics to produce antimicrobial substances, as their yield and activity are highly dependent on the environmental parameters and nutritional conditions during the fermentation process. Optimizing fermentation conditions not only increases antimicrobial yields but also reduces production costs, increasing the feasibility of industrial production. Summary of the Invention

[0005] The purpose of the present invention is to provide Bacillus subtilis Mafic-240428 and application thereof.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a strain Mafic-240428 with antibacterial activity obtained by separation and purification from Danzhou chicken cecal chyme, which is classified and named Bacillus subtilis ( Bacillus subtilis ), the strain has been deposited in the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with deposit number CGMCC No.33720 and deposit date March 5, 2025.

[0007] In a second aspect, the present invention provides a bacterial agent containing Bacillus subtilis Mafic-240428.

[0008] In a third aspect, the present invention provides use of Bacillus subtilis Mafic-240428 in preparing an antibacterial agent.

[0009] Furthermore, the antibacterial agent has antibacterial activity against Gram-positive bacteria.

[0010] The Gram-positive bacteria include but are not limited to Staphylococcus aureus ( Staphylococcus aureus ), drug-resistant Staphylococcus aureus (such as methicillin-resistant Staphylococcus aureus).

[0011] In a fourth aspect, the present invention provides any of the following uses of Bacillus subtilis Mafic-240428: 1) Used for preparing feed additives; 2) Used for livestock and poultry breeding; 3) Used in livestock production; 4) Used for sterilization (such as Gram-positive pathogens).

[0012] In a fifth aspect, the present invention provides a feed additive or feed containing Bacillus subtilis Mafic-240428 or a bacterial agent thereof.

[0013] In a sixth aspect, the present invention provides a fermentation method for Bacillus subtilis Mafic-240428, wherein the optimal carbon source in the fermentation medium is corn flour, and the optimal addition amount is 4%; the optimal nitrogen source is peptone, and the optimal addition amount is 4%; The optimum fermentation temperature was 34℃; the initial pH of the fermentation was 6; the shaking speed was 220 rpm; and the antibacterial activity began to appear after 18 h of fermentation.

[0014] Furthermore, the optimal fermentation medium formula is: 4% corn flour, 4% peptone and 0.25% NaCl, prepared in water.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (1) The Bacillus subtilis Mafic-240428 provided by the present invention has good antibacterial activity against common Gram-positive pathogens, and can also have the same antibacterial effect against methicillin-resistant Staphylococcus aureus.

[0016] (2) The fermentation conditions provided by the present invention can significantly improve the fermentation effect of Bacillus subtilis Mafic-240428 and reduce production costs.

[0017] (3) The Bacillus subtilis Mafic-240428 provided by the present invention has no hemolytic activity and is not resistant to common antibiotics. It is highly safe and can be widely used in animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the antibacterial spectrum of Bacillus subtilis Mafic-240428 in a preferred embodiment of the present invention.

[0019] Figure 2 This is the effect of fermentation parameter optimization on antibacterial activity in a preferred embodiment of the present invention.

[0020] Figure 3 This is the effect of culture medium optimization on antibacterial activity in a preferred embodiment of the present invention.

[0021] Figure 4 The results are the acid and bile resistance evaluation results of strain Mafic-240428 in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0022] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0023] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage. However, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0024] Example 1 Screening and identification of strains Weigh approximately 1 g of Danzhou chicken cecal digesta into a sterile 50 mL centrifuge tube, add 10 mL of pre-chilled sterile saline, and pipette until smooth. Centrifuge at 100 × g for 5 minutes to remove large particles. Remove the supernatant, add 25% sterile glycerol, aliquot into sterile cryovials, and store in an ultra-low-temperature freezer. Heat the cecal bacterial suspension at 80°C for 20 minutes, perform a gradient dilution, and plate 0.1 mL onto LB plates.

[0025] Initial screening was performed based on colony morphology, selecting single colonies that were white, with rough edges, grooves, a central protrusion, a circular apex, and an overall shape resembling a volcano. Using the Bergey's Manual of Bacterial Identification and the Manual for Systematic Identification of Common Bacteria, selected single colonies were stained using a spore staining kit (Qingdao Haibo, China) and observed under a microscope to identify strains with spores.

[0026] The strains obtained from the preliminary screening were purified three times, and the bacterial genomic DNA was extracted using a bacterial DNA extraction kit (OMEGA, USA). The DNA was then sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rDNA sequencing. The sequence is shown in SEQ ID NO: 1.

[0027] Example 2 Antibacterial activity determination Bacillus subtilis Mafic-240428 and indicator bacteria (Escherichia coli K88, E. coli O157:H7, Salmonella pullorum CVCC 1791, Staphylococcus aureus S. aureus CVCC1882, S. aureus ATCC43300) were activated separately and then cultured to the logarithmic phase.

[0028] Use a sterile cotton swab to dip different indicator bacteria liquids and evenly spread them on the surface of the LB plate. Then use a hole puncher to evenly punch 6 wells on each plate. Add 200 μL of sterile LB medium to the control group wells and add 200 μL of Bacillus subtilis Mafic-240428 fermentation supernatant to the treatment group wells. Repeat 3 times for each group. Place the plate in a 37°C incubator and culture for about 12 hours. After the indicator bacteria fill the plate, observe whether an inhibition zone appears. The results show that it has good antibacterial activity against common Gram-positive pathogens, and can also have the same antibacterial effect on methicillin-resistant Staphylococcus aureus. The results are shown in Table 1 and Figure 1 .

[0029] Table 1 Antibacterial spectrum of Bacillus subtilis Mafic-240428

[0030] Example 3 Optimization of fermentation conditions Methicillin-resistant Staphylococcus aureus (S. aureus ATCC43300) was used as the indicator bacteria, and the diameter of the inhibition zone was used as the indicator. The seed solution was cultured for 10 h and the OD was adjusted. 600 To 1.0, an inoculum size of 0.2% was used. After fermentation, the fermentation supernatant was collected and the inhibition zone size of the different treatment groups was measured. Using LB medium, 220 rpm, 37°C, and 24 h as the basic fermentation conditions, single-factor optimization of fermentation parameters such as time, temperature, and initial pH was performed. Using 1% carbon source, 1% nitrogen source, and 1% NaCl as the basic fermentation medium, single-factor optimization of the type and amount of carbon source, nitrogen source, and inorganic salts in the medium was performed.

[0031] 1. Fermentation parameter optimization The optimal fermentation temperature was determined by measuring the inhibition zone size of the fermentation supernatant of Bacillus subtilis Mafic-240428 at 30°C, 32°C, 34°C, 36°C, 38°C, and 40°C after fermentation at 220 rpm and 37°C for 24 h. The optimal pH was determined by measuring the inhibition zone size of the fermentation supernatant after fermentation at 220 rpm and 37°C for 24 h. The optimal fermentation time was determined by measuring the inhibition zone size of the fermentation supernatant using the optimized fermentation medium at 220 rpm and 37°C for 12 h, 18 h, 24 h, 30 h, and 36 h. The optimal fermentation time was determined by measuring the inhibition zone size using the same method. The optimal fermentation time was determined by measuring the inhibition zone size of the fermentation supernatant using the optimized fermentation medium at 220 rpm and 37°C for 36 h.

[0032] The results are as follows Figure 2 As shown in the figure, the optimal fermentation temperature was preliminarily determined to be 34°C. The antibacterial activity of the fermentation broth would decrease when the fermentation temperature was higher than 34°C. The antibacterial activity reached the highest when the initial pH was 6. Excessive acidity or alkalinity was not conducive to the production of antibacterial substances. The antibacterial activity began to appear at 18 h of fermentation, and then slowly increased, reaching the highest at 30 h.

[0033] 2. Culture medium optimization (1) Optimization of carbon source type and concentration 1% maltose, soluble starch, corn flour, and glycerol were added as carbon sources, 1% peptone was used as a nitrogen source, and 1% NaCl was added to different treatment groups. The size of the inhibition zone was determined using the above method, with three replicates per group. The best carbon source was selected for concentration optimization, and 1%, 2%, 3%, 4%, and 5% carbon sources were added to different treatment groups, with 1% peptone as a nitrogen source and 1% NaCl added, with three replicates per group. The size of the inhibition zone was determined using the above method.

[0034] (2) Optimization of nitrogen source type and concentration Based on the type and concentration of the optimal carbon source, 1% peptone, beef meal, yeast extract powder, soybean meal, ammonium sulfate, ammonium chloride, and sodium nitrate were added as nitrogen sources to different treatment groups, along with 1% NaCl. Three replicates were performed in each group, and the inhibition zone size was measured according to the above method to determine the optimal nitrogen source. The best nitrogen source was selected for concentration optimization, and 0.5%, 1%, 2%, 4%, 6%, and 8% nitrogen sources were added to different treatment groups, along with 1% NaCl. Three replicates were performed in each group, and the inhibition zone size was measured according to the above method to determine the optimal nitrogen source concentration.

[0035] (3) Optimization of inorganic salt types Based on the optimal carbon and nitrogen source types and concentrations, as well as 1% NaCl, 1 mmol / L of ZnSO4, MnSO4, MgSO4, CaCO3, and KH2PO4 were added to different treatment groups, with three replicates per group. The inhibition zone size was measured using the above method to determine whether other inorganic salts could enhance antibacterial activity. The effect of NaCl concentration on antibacterial activity was then explored. The NaCl concentrations in the different treatment groups were adjusted to 0%, 0.25%, 0.5%, 0.75%, and 1%, and the inhibition zone size was measured using the above method to determine the optimal NaCl concentration.

[0036] The results are as follows Figure 3 As shown in the figure, the best carbon source is corn flour, and the optimal addition amount is 4%; the best nitrogen source is peptone, and the optimal addition amount is 4%; adding different types of metal ions on the basis of 1% NaCl all led to different degrees of decrease in antibacterial activity, indicating that there is no lack of trace elements in the culture medium; 0.25% is the optimal addition amount of NaCl, but compared with other influencing factors, the size of the inhibition zone of the treatment groups with different inorganic salt addition amounts is smaller, indicating that NaCl concentration is not the main influencing factor.

[0037] Example 4 Safety Evaluation 1. Antibiotic sensitivity test Testing was conducted in accordance with the CLSI M100 antimicrobial susceptibility testing standards specified by the National Committee on Antimicrobial Susceptibility Studies (NCCLS) of the United States. The results showed that Bacillus subtilis Mafic-240428 was sensitive to most common antibiotics, with only limited resistance to a few β-lactam antibiotics and lincomycin. Specific results are shown in Table 2.

[0038] Table 2 Antibiotic susceptibility of Bacillus subtilis Mafic-240428

[0039] Note: R stands for resistant, I stands for intermediately sensitive, and S stands for sensitive. The results are defined according to the CLSI M100 antimicrobial susceptibility test implementation standard.

[0040] 2. Hemolytic activity assay After activation, Bacillus subtilis Mafic-240428 was streaked onto the surface of Columbia blood agar and incubated overnight at 37°C. The colonies were observed for the presence of a transparent hemolytic zone. Staphylococcus aureus CVCC1882 was used as a positive control for incomplete hemolysis, while Bacillus thuringiensis was used as a positive control for complete hemolysis. The results are shown in Table 3. Bacillus subtilis Mafic-240428 had no hemolytic activity, while Bacillus thuringiensis had complete hemolytic activity and Staphylococcus aureus had incomplete hemolytic activity.

[0041] Table 3 Hemolytic activity of Bacillus subtilis Mafic-240428

[0042] Example 5 Evaluation of acid resistance and bile salt resistance 1. Acid resistance evaluation of strain Mafic-240428 LB medium was adjusted to pH 2, 3, 4, 5, 6, and 7, with 3 replicates per group. 2% seed solution cultured to the logarithmic phase was inoculated into each pH medium, and the OD was measured after culturing for 3 h. 600 .

[0043] 2. Evaluation of bile salt tolerance of strain Mafic-240428 0.05%, 0.3%, and 0.5% ox bile salts were added to LB medium, with 3 replicates per group. 2% seed solution cultured to the logarithmic phase was inoculated into the medium with different bile salt concentrations, and the OD was measured after 3 h of culture. 600 .

[0044] The results of the acid and bile resistance evaluation of strain Mafic-240428 are as follows Figure 4 shown.

[0045] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Bacillus subtilis ( Bacillus subtilis )Mafic-240428, the deposit number is CGMCC No.33720.

2. A bacterial agent containing the bacterium according to claim 1.

3. Use of the bacteria according to claim 1 in the preparation of antibacterial agents.

4. The use according to claim 3, characterized in that The antibacterial agent has antibacterial activity against Gram-positive bacteria; The Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

5. The use according to claim 4, characterized in that The Gram-positive bacteria are drug-resistant Staphylococcus aureus.

6. The use according to claim 5, characterized in that The drug-resistant Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

7. Any of the following uses of the bacterium according to claim 1: 1) Used for preparing feed additives; 2) Used for livestock and poultry breeding.

8. A feed additive or feed containing the bacterium or its bacterial agent according to claim 1.

9. The fermentation method of the bacteria according to claim 1, characterized in that: The best carbon source in the fermentation medium was corn flour, with an optimal addition amount of 4%; the best nitrogen source was peptone, with an optimal addition amount of 4%; The optimum fermentation temperature was 34℃; the initial pH of the fermentation was 6; the shaking speed was 220 rpm; and the antibacterial activity began to appear after 18 h of fermentation.

10. The method according to claim 9, characterized in that The optimum fermentation medium composition was: 4% corn flour, 4% peptone and 0.25% NaCl.

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