Preparation method and application of bacillus subtilis antibacterial peptide

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method and application of a bacillus subtilis antibacterial peptide. By accurately controlling the inoculation density culture in the logarithmic phase, the biomass density of the thalli can be increased by 2-3 times, and the fermentation period is shortened; by optimizing the composition of a fermentation culture medium, fermentation conditions and a downstream treatment method, the yield and activity of the bacillus subtilis antibacterial peptide can be remarkably improved, the broad-spectrum bacteriostasis is enhanced, the drug resistance is remarkable, and the bacillus subtilis antibacterial peptide has the tendency of selective intelligent bacteriostasis due to low concentration, namely, the bacillus subtilis antibacterial peptide has no obvious inhibition effect on skin beneficial bacteria staphylococcus epidermidis; and the traditional Chinese medicine composition has a relatively obvious inhibition effect on staphylococcus aureus, propionibacterium acnes and other strains. Moreover, only water, inorganic salt and food-grade raw materials are used in the whole process, no organic solvent is left, and compared with a traditional ethanol / acetone extraction method, the method better meets the FDA / EFSA standard.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for preparing and applying Bacillus subtilis antimicrobial peptides. Background Technology

[0002] Bacillus subtilis ( Bacillus subtilis Bacillus subtilis is a Gram-positive bacterium with a strong metabolic capacity, capable of secreting various bioactive substances, including antimicrobial peptides and lipopeptides. These metabolites play a role in inhibiting the growth of other microorganisms in the competition for survival. During fermentation, Bacillus subtilis regulates its metabolic pathways, converting nutrients such as carbon and nitrogen sources into various metabolites. When fermentation conditions (such as pH, temperature, and aeration) are suitable, Bacillus subtilis synthesizes precursors of antimicrobial peptides via ribosomes, and then, through a series of modification processes (such as dehydration and cyclization), generates mature antimicrobial peptides. These antimicrobial peptides are secreted extracellularly through the cell membrane, thus accumulating in the fermentation broth. Antimicrobial peptides inhibit the growth of pathogens by disrupting the integrity of the cell membrane. For example, antimicrobial peptides produced by Bacillus subtilis fermentation can increase cell membrane permeability, leading to the leakage of intracellular potassium ions and proteins. Furthermore, antimicrobial peptides can also inhibit the protein synthesis of pathogens, thereby further inhibiting their growth. The molecular weight is typically between 1 and 5 kDa, with good water solubility. The antibacterial mechanism primarily involves disrupting the cell membrane integrity of pathogenic microorganisms, making it less likely to induce drug resistance. It remains stable at high temperatures (above 100°C) and over a wide pH range. It can be completely degraded by the body without residual toxicity. However, existing preparation methods, due to improper procedures, generally result in poor antibacterial effects and a tendency to induce drug resistance in the obtained Bacillus subtilis antimicrobial peptides. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing Bacillus subtilis antimicrobial peptides, thereby enhancing the selective antimicrobial activity and resistance to drug resistance of Bacillus subtilis antimicrobial peptides.

[0004] This invention provides a method for preparing Bacillus subtilis antimicrobial peptides, comprising the following steps: Bacillus subtilis was activated and cultured to the logarithmic growth phase to obtain seed culture; The seed culture was inoculated into a fermentation medium and fermented to obtain a fermentation broth; The fermentation broth was subjected to solid-liquid separation, and the supernatant was collected; The supernatant was subjected to ultrafiltration using an ultrafiltration membrane, and the filtrate was collected; the filtrate contained the Bacillus subtilis antimicrobial peptide; The inoculation amount of the seed solution is 1~3% v / v; The fermentation medium comprises 15-25 g / L soybean meal, 3-10 g / L corn flour, 0.3 g / L inorganic salt and the rest water. The fermentation culture has a temperature of 32-42℃, a time of 10-24 h, a pH of 5.0-7.0 and a rotating speed of 150-400 rpm.

[0005] Preferably, the activated culture has a temperature of 30-40℃ and a time of 15-48 h.

[0006] Preferably, the inorganic salt comprises sodium chloride.

[0007] Preferably, the ultrafiltration membrane has a molecular weight cut-off of 1000-2000 Da.

[0008] Preferably, the Bacillus subtilis is Bacillus subtilis gs-11061, and the preservation number is CGMCC No. 13932.

[0009] Preferably, after obtaining the filtrate, the filtrate is concentrated to obtain a concentrated solution, and the concentrated solution is vacuum freeze-dried to obtain the powder-like Bacillus subtilis antimicrobial peptide.

[0010] Preferably, the concentration has a vacuum degree of -0.15 Pa and a temperature of 45℃. The vacuum freeze-drying comprises the following steps: under a vacuum degree of 0.24 MPa, the temperature of the concentrated solution is increased from -40℃ to 0℃ within 5 h to obtain a first treated sample. Under a vacuum degree of 0.24 MPa, the temperature of the first treated sample is increased from 0℃ to 5℃ within 12 h to obtain a second treated sample. Under a vacuum degree of 0.24 MPa, the temperature of the second treated sample is increased from 5℃ to 10℃ within 10 h to obtain a third treated sample. Under a vacuum degree of 0.24 MPa, the temperature of the third treated sample is increased from 10℃ to 20℃ within 3 h to obtain the powder-like Bacillus subtilis antimicrobial peptide.

[0011] The present application provides the Bacillus subtilis antimicrobial peptide obtained by the preparation method.

[0012] The present application provides the use of the Bacillus subtilis antimicrobial peptide in the preparation of an antibacterial product and / or an anti-inflammatory product.

[0013] Preferably, the antibacterial product comprises a product inhibiting one or more pathogenic bacteria of Staphylococcus aureus, Escherichia coli and Propionibacterium acnes.

[0014] Beneficial effects: The application provides a preparation method of bacillus subtilis antibacterial peptide, and comprises the following steps: culturing bacillus subtilis to logarithmic growth phase to obtain seed liquid; inoculating the seed liquid into fermentation medium to obtain fermentation liquid; performing solid-liquid separation on the fermentation liquid to collect supernatant; performing ultrafiltration on the supernatant by using an ultrafiltration membrane to collect filtrate; the filtrate contains the bacillus subtilis antibacterial peptide; the inoculation amount of the seed liquid is 1-3% v / v; the fermentation medium comprises 15-25 g / L soybean meal, 3-10 g / L corn flour, 0.3 g / L inorganic salt and the rest water; the fermentation culture temperature is 32-42 DEG C, the time is 10-24 h, the pH is 5.0-7.0, and the rotating speed is 150-400 rpm. The present application can improve the biomass density of the bacterial body by 2-3 times and shorten the fermentation period by precisely controlling the inoculation density in the logarithmic growth phase. The yield and activity of the bacillus subtilis antibacterial peptide can be significantly improved by optimizing the composition of the fermentation medium, the fermentation conditions and the downstream processing method, the broad-spectrum antibacterial effect is enhanced, the drug resistance is significantly reduced, and the selective intelligent antibacterial trend is achieved at a low concentration, that is, the bacillus subtilis antibacterial peptide has no obvious inhibitory effect on the skin beneficial bacteria staphylococcus epidermidis, but has obvious inhibitory effect on staphylococcus aureus and propionibacterium acnes. Moreover, only water, inorganic salt and food-grade raw materials are used in the whole process of the present application, and there is no organic solvent residue, so the present application is more in line with the FDA / EFSA standard compared with the traditional ethanol / acetone extraction method. The preparation method of the present application is simple in process, easy to operate, low in cost, and suitable for industrial production, and has obvious practicability and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0016] Figure 1 It is the real object diagram of the bacillus subtilis antibacterial peptide powder of Example 1; Figure 2 It is the antibacterial circle real object diagram of the bacillus subtilis antibacterial peptide powder of Example 1 on escherichia coli; Figure 3 It is the antibacterial circle real object diagram of the bacillus subtilis antibacterial peptide powder of Example 1 on staphylococcus epidermidis; Figure 4 It is the antibacterial circle real object diagram of the bacillus subtilis antibacterial peptide powder of Example 1 on staphylococcus aureus; Figure 5 It is the antibacterial circle real object diagram of the bacillus subtilis antibacterial peptide powder of Example 1 on propionibacterium acnes; Figure 6 It is the antibacterial circle real object diagram of the bacillus subtilis antibacterial peptide powder of the negative control (0.9% sodium chloride solution) on propionibacterium acnes. DETAILED DESCRIPTION

[0017] The application provides a preparation method of bacillus subtilis antibacterial peptide, comprising the following steps: Activating and culturing the bacillus subtilis to logarithmic growth phase to obtain a seed liquid; Inoculating the seed liquid into a fermentation medium to perform fermentation culture to obtain a fermentation liquid; Performing solid-liquid separation on the fermentation liquid to collect supernatant; Performing ultrafiltration on the supernatant by using an ultrafiltration membrane to collect a filtrate; the filtrate contains the bacillus subtilis antibacterial peptide; The inoculation amount of the seed liquid is 1-3% v / v; The fermentation medium comprises 15-25 g / L soybean meal, 3-10 g / L corn flour, 0.3 g / L inorganic salt and the rest water; The fermentation culture is performed at a temperature of 32-42 ℃ for 10-24 h, at a pH of 5.0-7.0 and at a rotating speed of 150-400 rpm.

[0018] The bacillus subtilis is activated and cultured until the bacillus subtilis enters logarithmic growth phase to obtain a seed liquid.

[0019] As an embodiment, the temperature of the activation culture is 30-40 ℃; as another embodiment, the temperature of the activation culture is 32-38 ℃; as another embodiment, the temperature of the activation culture is 35-37 ℃. As an embodiment, the time of the activation culture is 15-48 h; as another embodiment, the time of the activation culture is 20-40 h; as another embodiment, the time of the activation culture is 30-35 h.

[0020] After obtaining the seed liquid, the seed liquid is inoculated into a fermentation medium to perform fermentation culture to obtain a fermentation liquid.

[0021] The inoculation amount of the seed liquid is 1-3% v / v. As an embodiment, the inoculation amount of the seed liquid is 1.5-2% v / v.

[0022] The fermentation medium comprises 15-25 g / L soybean meal, 3-10 g / L corn flour, 0.3 g / L inorganic salt, and the balance of water. As an embodiment, the fermentation medium comprises 20 g / L soybean meal, 5 g / L corn flour, 0.3 g / L inorganic salt, and the balance of water. As an embodiment, the inorganic salt comprises sodium chloride. The accurate proportioning of the novel cheap substrate composite carbon and nitrogen source is the key to improving the yield of antibacterial peptides. In the present application, the combination of soybean meal and corn flour is selected, corn flour is used as the carbon source, and soybean meal is used as the nitrogen source. The complementary soybean meal and corn flour provide complete amino acid spectrum and growth factors. The soybean meal-corn flour composite substrate is used to replace expensive peptone (the cost is only 1 / 5-1 / 3 of the LB medium), and complex additives are avoided.

[0023] The fermentation culture temperature is 32-42℃. As an embodiment, the fermentation culture temperature is 37-40℃. The fermentation culture time is 10-24 h. As an embodiment, the fermentation culture time is 16-20 h. The fermentation culture pH is 5.0-7.0. As an embodiment, the fermentation culture pH is 5.5-6.5; as another embodiment, the fermentation culture pH is 6.0. The fermentation culture rotation speed is 150-400 rpm. As an embodiment, the fermentation culture rotation speed is 200-300 rpm; as another embodiment, the fermentation culture rotation speed is 240 rpm. The stirring speed (rotation speed) affects the oxygen transfer efficiency. The present application limits the rotation speed to provide appropriate oxygen supply, significantly promotes the growth of Bacillus subtilis and the synthesis of antibacterial peptides, and avoids the deviation of metabolic pathways caused by excessive dissolved oxygen and the limitation of bacterial growth caused by low dissolved oxygen.

[0024] After obtaining the fermentation broth, the present application performs solid-liquid separation on the fermentation broth to collect the supernatant. The present application does not have strict requirements for the solid-liquid separation method, and a conventional method in the art can be used, such as centrifugation.

[0025] After obtaining the supernatant, the present application uses an ultrafiltration membrane to perform ultrafiltration on the supernatant to collect the filtrate; the filtrate contains the Bacillus subtilis antibacterial peptide.

[0026] As an embodiment, the ultrafiltration membrane has a molecular weight cut-off of 1000-2000 Da. As an embodiment, the present application uses an ultrafiltration membrane to perform overnight ultrafiltration on the supernatant. As an embodiment, the ultrafiltration is performed at room temperature, which avoids the loss of activity of heat-sensitive antibacterial peptides and is easy to scale up to continuous industrial production.

[0027] As an embodiment, the Bacillus subtilis in the present application is Bacillus subtilis gs-11061, and the preservation number is CGMCC No. 13932. The Bacillus subtilis gs-11061 in the present application has certain particularity in single nucleotide polymorphism (SNP) and the sequence of specific genes, and is a natural strain for high expression of protein peptides. The Bacillus subtilis gs-11061 can use bean peel (the leftover of defatted soybean) as raw material, reduce production cost, and realize the recycling of the leftover of defatted soybean. Compared with other Bacillus subtilis, the peptide obtained after fermentation has a significant bacteriostatic effect, and has a selective and intelligent bacteriostatic trend, that is, the peptide has no obvious inhibitory effect on Staphylococcus epidermidis which is a skin beneficial bacterium, but has obvious inhibitory effect on Staphylococcus aureus and Propionibacterium acnes. The source of the Bacillus subtilis gs-11061 in the present application is not strictly required, and the Bacillus subtilis gs-11061 can be purchased regularly.

[0028] As an embodiment, after obtaining the filtrate, the present application concentrates the filtrate to obtain a concentrated solution, and then the concentrated solution is subjected to vacuum freeze-drying to obtain a powder-like Bacillus subtilis antibacterial peptide. As an embodiment, the vacuum degree of the concentration in the present application is -0.15 Pa, and the temperature is 45°C. As an embodiment, the vacuum freeze-drying in the present application comprises the following steps: under the condition of a vacuum degree of 0.24 MPa, the temperature of the concentrated solution is increased from -40°C to 0°C within 5h to obtain a first treated sample; under the condition of a vacuum degree of 0.24 MPa, the temperature of the first treated sample is increased from 0°C to 5°C within 12h to obtain a second treated sample; under the condition of a vacuum degree of 0.24 MPa, the temperature of the second treated sample is increased from 5°C to 10°C within 10h to obtain a third treated sample; under the condition of a vacuum degree of 0.24 MPa, the temperature of the third treated sample is increased from 10°C to 20°C within 3h to obtain a powder-like Bacillus subtilis antibacterial peptide. The present application concentrates and dries the filtrate, which is convenient for subsequent storage and transportation.

[0029] The present application provides a Bacillus subtilis antibacterial peptide obtained by the preparation method of the above technical solution.

[0030] The present application provides an application of the Bacillus subtilis antibacterial peptide of the above technical solution in the preparation of an antibacterial product and / or an anti-inflammatory product. As an embodiment, the antibacterial product in the present application comprises a product for inhibiting one or more pathogenic bacteria of Staphylococcus aureus, Escherichia coli and Propionibacterium acnes. As an embodiment, the antibacterial product in the present application comprises a product having a skin bacteriostatic effect.

[0031] In order to further illustrate the present application, a preparation method and application of a bacillus subtilis antibacterial peptide provided by the present application are described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0032] Example 1 A preparation method of a bacillus subtilis antibacterial peptide, characterized by comprising the following steps: 1. Strain activation: inoculate bacillus subtilis gs-11061 (purchased from Nanjing University of Technology, preservation number CGMCC No. 13932) into LB culture medium, and culture at 30-40℃ for 15-48h, measure the OD value of the fermentation liquor every 2h until the strain enters the logarithmic growth phase, and obtain a seed liquor. 600

[0033] 2. Inoculate the seed liquor obtained in step 1 into a fermentation culture medium (soybean peptone 5g / L, corn meal 5g / L, sodium chloride 0.3g / L and the rest water) at an inoculation amount of 1% (v / v), adjust the pH to 5.5, and ferment at 37℃ and 240rpm for 16h to obtain a fermentation culture liquor, then centrifuge and collect the supernatant.

[0034] 3. Concentrate the supernatant obtained in step 2 by using an ultrafiltration membrane with a molecular weight cut-off of 1000-2000Da overnight to obtain a concentrated liquor.

[0035] 4. Concentrate the concentrated liquor obtained in step 3 to 1 / 3 of the original volume at-0.15Pa and 45℃, and then remove the water in the antibacterial peptide solution by using a vacuum freeze-drying method, and desolvate at 20-30℃ for 6h to obtain bacillus subtilis antibacterial peptide powder (BAP-1); wherein the freezing temperature of the vacuum freeze-drying is-40℃, the vacuum degree is 0.24 MPa, and the temperature is raised according to the program: -40℃→0℃ / 5h, 0℃→5℃ / 12h, 5℃→10℃ / 10h, 10℃→20℃ / 3h. Figure 1

[0036] Examples 2-8 The bacillus subtilis antibacterial peptide is prepared according to the steps of Example 1, and the differences between Examples 2-8 and Example 1 are that the inoculation amount (A), fermentation temperature (B), fermentation time (C), fermentation pH (D) and fermentation rotation speed (E) in step 2 are different from those of Example 1, which are listed in Table 1.

[0037] Table 1 Related parameters in the preparation process of Examples 1-8

[0038] Comparative Examples 1-6 ​​The bacillus subtilis antibacterial peptide was prepared according to the steps of Example 1, and the differences between Examples 2-8 and Example 1 are that the inoculation amount (A), fermentation temperature (B), fermentation time (C), fermentation pH (D), fermentation rotation speed (E), concentration of soybean meal in the fermentation medium (F), concentration of corn meal in the fermentation medium (G), concentration method (H) in step 3 and drying method (I) in step 4 are different from those of Example 1, and the specific parameters are listed in Table 2.

[0039] Table 2: Related parameters in the preparation process of Examples 1 and Comparative Examples 1-6

[0040] The salting-out step is as follows: slowly add ammonium sulfate to the supernatant to 10% saturation at 25°C, stand at 4°C for 2h, centrifuge at 8500r / min for 10min to remove impurities; then add ammonium sulfate to the supernatant to 60% saturation, stand at 4°C for 4h, centrifuge at 12000r / min for 20min to collect the precipitate, dissolve with 0.02mol / L Tris-HCl to obtain the concentrated solution; The acid precipitation step is as follows: adjust the pH of the supernatant to 2.0 with 0.1mol / L dilute hydrochloric acid, mix well and stand for 24h to form a flocculent precipitate, which is collected by filtration or centrifugation; after redissolving with a small amount of deionized water, the crude antibacterial peptide solution is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 1000-2000 Da overnight, and the filtrate is discarded.

[0041] The inlet air temperature for spray drying is 180-230°C, and the outlet air temperature is 80-100°C, or the inlet air temperature is 130-160°C and the exhaust air temperature is 65-75°C.

[0042] Test Example 1 Drug resistance test (safety test) Test strain: Staphylococcus aureus TSA medium: weigh 40.0g of TSA medium dry powder, heat and stir to dissolve in 1000ml of distilled water, distribute into triangular flasks, autoclave at 121°C for 15min, pour into sterile plates and cool for standby.

[0043] Drug stock solution: the bacillus subtilis antibacterial peptide powders obtained from Examples 1-8 and Comparative Examples 1-6 were used as samples, and 12.8mg / ml stock solutions were prepared for standby.

[0044] 1. Primary minimum inhibitory concentration (MIC) test 1) Staphylococcus aureus USA300 was inoculated on the medium and incubated at 37°C for 1 day to grow single colonies.

[0045] 2) Using a sterile inoculation loop, pick a mature monoclonal strain and inoculate it into 5 mL of liquid TSB medium. Incubate at 37°C with shaking until OD (October Expiratory Time) reaches 100°C. 600 It is approximately 0.6.

[0046] 3) Inoculate the bacterial solution into the liquid culture medium at a ratio of 1‰ (v / v) to obtain the bacterial culture medium; take 1 mL of the bacterial culture medium and add the test sample (final concentration 128 μg / ml) to prepare the drug-containing bacterial solution.

[0047] 4) Initial drug addition to the experimental group: Add 200 μl of drug-containing bacterial culture to the initial well of the 96-well plate, and add 100 μl of bacterial culture medium to the remaining wells of the 96-well plate. Then, add 100 μl of liquid from the initial well to the bacterial culture in the second well, mix, and then add 100 μl of liquid from the second well to the third well, and so on up to the eleventh well. Discard the 100 μl of liquid from the eleventh well, and incubate at 37°C for 1 day.

[0048] 5) Remove the 96-well plate, observe the bacterial cell precipitation with the naked eye, and determine the minimum inhibitory concentration (MIC). The results are shown in Tables 3 and 4.

[0049] 2. 10th generation induction 1) Add the sample to the TSA medium in a certain proportion to make the sample concentration in the liquid medium 1 / 2 MIC, that is, 0.125 μg / ml of drug-containing medium.

[0050] 2) Streak Staphylococcus aureus USA300 on TSA agar plates and incubate at 37°C until visible bacterial plaques are visible.

[0051] 3) Pick a single colony and inoculate it into 5 ml of TSA liquid medium. Incubate at 37°C and 200 rpm until OD reaches 100%. 600 It is 0.6.

[0052] 4) Inoculate OD at a ratio of 1‰ (v / v) 600 USA300 with an OD600 of 0.6 was cultured in 1 ml of drug-containing medium at 37°C and 200 rpm until the OD600 reached 0.6 again.

[0053] 5) Repeat step 4) until the drug induction is completed 10 times.

[0054] 3. Minimum inhibitory concentration (MIC) test after induction Following the procedure in step 1, the MIC of Staphylococcus aureus USA300 after 10 generations of induction in step 2 was detected, and the results are shown in Tables 3 and 4.

[0055] Table 3. MIC (μg / mL) of different Bacillus subtilis antimicrobial peptide samples against Staphylococcus aureus.

[0056] Note: Compared with Example 1,** P <0.01.

[0057] Table 4 MIC of different Bacillus subtilis antibacterial peptide samples against Staphylococcus aureus (μg / mL)

[0058] Note: Compared with Example 1,*** indicates P <0.001.

[0059] According to Table 3 and Table 4, it can be seen that compared with each example, in the primary MIC test: the MIC value of Bacillus peptide in Example 1 against Staphylococcus aureus is 0.25 μg / ml; after 10 generations of induction: the MIC value of Bacillus peptide in Example 1 against induced Staphylococcus aureus is 0.25 μg / ml; it shows that Staphylococcus aureus does not enhance its drug resistance after being induced for 10 generations by the Bacillus subtilis antibacterial peptide provided by the application. The rest of the examples and comparative examples also do not show obvious drug resistance. Compared with each MIC value, the MIC value of Example 1 is the lowest, indicating that it has stronger bacteriostatic property and can achieve significant bacteriostatic effect at a lower concentration.

[0060] Test Example 2 Endotoxin test (safety test) 1. Bacillus subtilis antibacterial peptide powders obtained from Examples 1-8 and Comparative Examples 1-6 were respectively taken as samples, and 0.5% sample aqueous dilutions were prepared.

[0061] 2. Sample dilution 40 times: 10 μl of 0.5% sample aqueous dilution was taken and added to a sample tube, and 390 μl of water was added and mixed well.

[0062] 3. Preparation of endotoxin standard: (1) Take 4 sterile ep tubes and label them E10, E1, E0.5, and E0.25, respectively. (2) Break open one endotoxin test water, add 900 μl to E1, 500 μl to E0.5, and 500 μl to E0.25. (3) Break open one endotoxin standard, add 1 ml of endotoxin test water, mix well after blowing, and transfer to E10 tube, vortex to mix thoroughly. (4) E1: Take 100 μl of E10 and add to E1 tube to obtain 1 ml of E1, mix well by blowing. (5) E0.5: Take 500 μl of E1 and add to E0.5 tube to obtain 1 ml of E0.5, mix well by blowing. (6) E0.25: Take 500 μl E0.5 into the E0.25 tube, get 1 ml E0.25, mix well, and wait for use.

[0063] 4. Take out 4 tachypleus amebocyte lysate (Zhanjiang Anduosi Biology), marked as positive (+), negative (-), sample 1, and sample 2. Add 200 μl of endotoxin test water to the negative (-) tube, add 100 μl of endotoxin test water and 100 μl of E0.25 to the positive (+) tube, add 100 μl of endotoxin test water and 100 μl of 40-fold diluted sample to the sample tube, mix well, seal with sealing film, and incubate at 37°C for 60±2 min. 5. Results: Take out the reaction tube, slowly invert 180°, if a milky white gel is formed in the tube, and the gel does not deform and does not slide off the tube wall, it is positive; if no gel is formed or the formed gel is not firm, deformed and slides off the tube wall, it is negative. The results are shown in Table 5.

[0064] Table 5 Endotoxin test results of different Bacillus subtilis antibacterial peptide samples

[0065] As can be seen from Table 5, the examples and comparative examples are all negative, indicating that the Bacillus subtilis antibacterial peptide provided by the present application has no endotoxin.

[0066] Test Example 3 Bacteriostatic test 1. Test sample: The Bacillus subtilis antibacterial peptide powders obtained in Examples 1-8 and Comparative Examples 1-6 were respectively configured into 0.05% dilution solution (m / v) using TSA liquid medium to obtain the test sample.

[0067] 2. Test strain type: Staphylococcus aureus, Staphylococcus epidermidis (skin beneficial bacteria), Escherichia coli and Propionibacterium acnes.

[0068] 3. Reference standard: WS / T 650-2019, Antibacterial and Bacteriostasis Efficacy Evaluation Method.

[0069] 4. Test method: The test strain 24h fresh slant culture was washed with PBS and diluted to 5.0×10 5 ~5.0×10 6CFU / mL standby. Preparation of bacteriostatic tablets: directly make the sample to be tested into a 5 mm diameter, thickness not more than 4 mm disc (block), every 4 pieces as a group. The negative control sample is made of the same material without bacteriostatic ingredients, and the size of the disc (block) is the same as the test group. Use a sterile cotton swab to dip the test bacteria suspension with a concentration of 5.0 x 10 5 ~5.0 x 10 6 CFU / mL test bacteria suspension, evenly smear 3 times on the surface of the appropriate medium plate. Each time, the plate should be rotated by 60°, and finally the cotton swab is smeared around the edge of the plate. Cover the plate and dry at room temperature for 5 min. Place 1 bacterized plate for each test, 4 test sample pieces, 1 negative control sample piece, a total of 5 pieces on each plate. Use a sterile forceps to place the sample pieces on the plate surface, the negative control sample piece is placed at the center of the plate, and the test sample pieces are placed around the plate. After placing, use a sterile forceps to press the sample pieces tightly against the plate surface. The center of each sample piece is more than 25 mm apart, and the edge of the plate is more than 15 mm apart. Cover the plate and incubate at 36℃±1℃ for 16h~18h, then observe the results. The test is repeated 3 times. Measure the diameter of the bacteriostatic ring (including the sample piece) with a vernier caliper and record it. When measuring the bacteriostatic ring, choose the uniform and completely sterile growth bacteriostatic ring, and measure the diameter with the outer edge of the bacteriostatic ring as the boundary. The results are shown in Figures 2-6 and Table 6.

[0070] Table 6 Bacteriostatic ring size (mm) of different Bacillus subtilis antibacterial peptides on test strains

[0071] Note: the diameter of the bacteriostatic ring is >7.8 mm, which is judged to have bacteriostatic effect; the diameter of the bacteriostatic ring is ≤7.8 mm, which is judged to have no bacteriostatic effect; compared with Example 1, P <0.01, P <0.01, P <0.005.

[0072] According to Figures 2-6 and Table 6, the diameter of the bacteriostatic ring of Example 1 is 41.27 mm ( Figure 4 ) at a concentration of 0.05% (m / v) on Staphylococcus aureus, has no bacteriostatic effect on Staphylococcus epidermidis, and the diameter is still 7.8 mm ( Figure 3 ), which is consistent with the negative control ( Figure 6 ), the bacteriostatic diameter on Escherichia coli is 15.28 mm ( Figure 2 ), and the bacteriostatic diameter on Propionibacterium acnes is 28.76 mm ( Figure 5). The rest of the examples have obvious bacteriostatic effect, but the bacteriostatic diameter is less than that of Example 1. In the comparative examples, each sample has different degrees of bacteriostatic effect on S. aureus, E. coli and P. acnes, but on S. epidermidis, comparative examples 2, 3 and 5 show a certain bacteriostatic effect, which may be related to the over-high pH value in comparative example 2, the over-long fermentation time in comparative example 3 and the over-high fermentation temperature in comparative example 5, resulting in bacteriostatic components that inhibit the growth of S. epidermidis.

[0073] Test Example 4 Anti-inflammatory test (ELISA detection) 1. Experimental reagent: IL-1β, IL-6, TNF-α, IL-10, TGF-β inflammatory factor kit.

[0074] 2. Cells: Hacat human immortalized epidermal cell line.

[0075] 3. Sample to be tested: The Bacillus subtilis antibacterial peptide powders obtained from Examples 1-8 and Comparative Examples 1-6 are respectively configured into 0.01% aqueous solution (m / v) to obtain the sample to be tested.

[0076] 4. Operation method: (1) Cell culture and treatment. Cell culture: HaCaT cells were cultured at an appropriate density (1 x 10 5(1) Cell culture: Cells were seeded in culture dishes or 96-well plates at a density of 1 x 104cells / well and cultured in RPMI-1640 or DMEM medium containing 10% fetal bovine serum (FBS). (2) Stimulation: Cells were treated with inflammatory factors (100 μg / mL of each inflammatory factor IL-1β, IL-6, TNF-α, IL-10, and TGF-β) for 24 h. Then, 0.3 mL of the sample to be tested was added to the stimulation model for further incubation for 24 h. (3) Collection of supernatant: After treatment, the cells were washed with PBS, the culture medium was removed, and the cell supernatant was collected. After centrifugation to remove cell debris, the supernatant was stored in a -80°C refrigerator for testing. (4) Preparation of ELISA kit: According to the instructions of the ELISA kit used, prepare the standard, detection antibody, peptide-labeled secondary antibody, and other reagents. Standard curve preparation: Set up standard wells on the ELISA plate, dilute the standard according to the instructions, and add it to the wells. Each concentration sets 3 replicate wells. At the same time, set up blank wells and sample wells. (5) Sample treatment: Dilute the stored cell supernatant according to the kit requirements, and add it to the sample wells of the ELISA plate, about 100 μL per well. (6) Incubation and washing: Incubate according to the kit instructions. Usually, first incubate the detection antibody, then incubate the peptide-labeled secondary antibody. After each incubation, wash the plate with washing solution to remove unbound components. Color development and reading: After adding the color developing solution, incubate at room temperature in the dark. After the color changes, add the stop solution to terminate the reaction. Use the peptide marker instrument to read the absorbance value at 450 nm wavelength. (7) Data analysis: Calculate the concentration of inflammatory factors in the sample according to the standard curve, and express it in pg / mL or ng / mL. Calculate the change rate of inflammatory factors compared with the stimulation model, and express it in %. The results are shown in Tables 7 and 8.

[0077] Table 7 Change rate of inflammatory factors (%) under Bacillus subtilis antibacterial peptides of Examples 1-8

[0078] Note: Compared with Example 1, * P <0.05,** P <0.01.

[0079] Table 8 Change rate of inflammatory factors (%) under Bacillus subtilis antibacterial peptides of Comparative Examples 1-6

[0080] Note: Compared with Example 1, *** indicates P <0.001.

[0081] As can be seen from Table 7 and Table 8, Example 1 has inhibitory effect on inflammatory factors IL-1β, IL-6 and TNF-α, and has promoting effect on anti-inflammatory factors IL-10 and TGF-β, thereby playing an overall inhibitory effect on inflammation. In the comparative examples, Comparative Examples 2, 3 and 5 have certain promoting effect on IL-6 and TNF-α, and also have certain promoting effect on anti-inflammatory factor IL-10, indicating that side product inflammatory reactions occur on the three inflammatory factors, which is probably caused by imbalance due to improper fermentation conditions.

[0082] As can be seen from the above, the bacillus subtilis antibacterial peptide prepared by the preparation method has low-concentration selective intelligent bacteriostatic trend, enhanced broad-spectrum bacteriostasis and significant resistance to drug resistance.

[0083] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A method for preparing an antibacterial peptide of Bacillus subtilis, characterized by, The method comprises the following steps: activating culture of Bacillus subtilis to logarithmic growth phase to obtain seed liquid; inoculating the seed liquid into fermentation medium to carry out fermentation culture to obtain fermentation liquid; carrying out solid-liquid separation on the fermentation liquid to collect supernatant; carrying out ultrafiltration on the supernatant using an ultrafiltration membrane to collect filtrate; the filtrate contains the Bacillus subtilis antibacterial peptide; the inoculation amount of the seed liquid is 1-3% v / v; the fermentation medium comprises 15-25 g / L soybean meal, 3-10 g / L corn meal, 0.3 g / L inorganic salt and the rest water; the fermentation culture is carried out at a temperature of 32-42 ℃ for 10-24 h, at a pH of 5.0-7.0 and at a rotating speed of 150-400 rpm.

2. The production method according to claim 1, characterized by, the activation culture is carried out at a temperature of 30-40 ℃ for 15-48 h.

3. The production method according to claim 1, characterized by, the inorganic salt comprises sodium chloride.

4. The method of claim 1, wherein, the ultrafiltration membrane has a molecular weight cut-off of 1000-2000 Da.

5. The preparation method according to claim 1, characterized in that, the Bacillus subtilis is Bacillus subtilis gs-11061, and the preservation number is CGMCC No. 13932.

6. The method of any one of claims 1 to 5, wherein the method further comprises the step of: after obtaining the filtrate, the filtrate is concentrated to obtain concentrated liquid, and the concentrated liquid is subjected to vacuum freeze-drying to obtain the powder-like Bacillus subtilis antibacterial peptide.

7. The preparation method according to claim 6, characterized in that, the concentration vacuum degree is -0.15 Pa, and the temperature is 45 ℃; the vacuum freeze-drying comprises the following steps: under a vacuum degree of 0.24 MPa, the temperature of the concentrated liquid is increased from -40 ℃ to 0 ℃ within 5 h to obtain a first treated sample; under a vacuum degree of 0.24 MPa, the temperature of the first treated sample is increased from 0 ℃ to 5 ℃ within 12 h to obtain a second treated sample; under a vacuum degree of 0.24 MPa, the temperature of the second treated sample is increased from 5 ℃ to 10 ℃ within 10 h to obtain a third treated sample; under a vacuum degree of 0.24 MPa, the temperature of the third treated sample is increased from 10 ℃ to 20 ℃ within 3 h to obtain the powder-like Bacillus subtilis antibacterial peptide.

8. The Bacillus subtilis antibacterial peptide obtained by the preparation method in any one of claims 1-7.

9. The use of the Bacillus subtilis antibacterial peptide in claim 8 in the preparation of antibacterial products and / or anti-inflammatory products.

10. Use according to claim 9, characterized in that, The antibacterial products comprise products for inhibiting one or more pathogenic bacteria of Staphylococcus aureus, Escherichia coli and Propionibacterium acnes.

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