Bacillus velezensis b02 and application thereof

Abstract

The present application relates to a bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) B02 and its application, belonging to the field of microbial technology. The bacteria have been preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO.66439. The strain is isolated, purified and screened from soil samples. Tests show that the bacteriostatic active substances produced by fermentation of the strain have good bacteriostatic effect on Burkholderia cenocepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, Enterococcus faecalis, Candida albicans and Aspergillus brasiliensis. Moreover, the strain has excellent heat resistance, cold resistance, light stability, pH stability and preservative effect, belongs to a mild low-stimulation raw material, and can be used for preparing daily chemical products. Therefore, the bacillus velezensis B02 and the bacteriostatic active substances produced by the bacillus velezensis B02 both have broad application prospect and important transformation research value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus belye B02 and its applications. Background Technology

[0002] In the food, health product, and daily chemical industries, antimicrobial agents are typically added to inhibit the growth and reproduction of microorganisms and ensure product stability and safety within their shelf life. Cosmetics, due to their complex composition, often contain nutrients such as oils, proteins, polysaccharides, and various moisturizing factors, providing an extremely favorable environment for the growth of microorganisms such as bacteria, molds, and yeasts. Burkholderia cepacia is a common contaminant in cosmetics, and this strain easily develops drug resistance, posing a challenge to preservation methods in the cosmetics industry. Therefore, effectively preventing spoilage while ensuring product safety and stability is one of the core challenges in formulation development.

[0003] Currently, the industry primarily relies on chemical preservatives to achieve antibacterial effects, such as phenoxyethanol, methylisothiazolinone (MIT), methylchloroisothiazolinone (CMIT), benzyl alcohol, and potassium sorbate. Meanwhile, with increasing consumer preference for "natural" and "safe" ingredients, naturally derived antibacterial ingredients, such as certain plant essential oils or fermentation products, are also gaining attention. However, traditional chemical preservatives generally suffer from strong irritation, allergy risks, and potential toxicity. For example, phenoxyethanol has been proven to be associated with adverse skin reactions such as contact allergies, eczema, and urticaria; MIT and CMIT are controversial due to their potential cytotoxicity and neurotoxicity; and benzyl alcohol and sodium benzoate may cause skin irritation or allergic dermatitis. Furthermore, some preservatives have strong odors and affect formulation stability. Natural antibacterial agents, on the other hand, face challenges such as unstable active ingredients, narrow antibacterial spectrum, high cost, or impact on product sensory characteristics (such as color and odor), making it difficult to meet practical application needs.

[0004] Existing research has found that some microorganisms can produce stable, broad-spectrum antibacterial natural substances. These substances are highly effective at inhibiting bacteria and are safe for the skin, making them a promising alternative to traditional preservatives. Currently, commonly used fermentation-derived preservatives in the daily chemical industry include nisin, polylysine, and natamycin, but none of these have an inhibitory effect on Burkholderia cepacia. Therefore, developing highly effective, safe, and stable natural antibacterial ingredients, especially those with good antibacterial properties against Burkholderia cepacia, is of great significance for improving product quality and safety. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Bacillus belye B02 that produces high antibacterial activity. This strain was isolated and purified from soil samples collected from a pond in the Baiyun District Private Science and Technology Park in Guangzhou and has been deposited in the Guangdong Provincial Microbial Culture Collection Center with the accession number GDMCC NO.66439.

[0006] In a first aspect, the present invention provides a strain of Bacillus belyssus ( Bacillus velezensis The bacteria was deposited on May 29, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.66439, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] Secondly, the present invention provides the application of Bacillus berleis B02 as described in the first aspect in the preparation of antibacterial active substances.

[0008] Thirdly, the present invention provides a method for producing antibacterial active substances by fermentation of Bacillus belye BO2 as described in the first aspect, comprising the following steps:

[0009] S1. Inoculate Bacillus belye BO2 into liquid fermentation medium and ferment; after fermentation, centrifuge and retain the fermentation supernatant;

[0010] S2. Take the fermentation supernatant, adjust the pH to 2.0 with 6 mol / L HCl solution, let it stand overnight at 4℃, centrifuge, and collect the precipitate;

[0011] S3. Take the precipitate and methanol, extract and centrifuge at pH=7, collect the methanol extract, add methanol back to the precipitate, repeat the above extraction and centrifugation steps 3 times, mix all the methanol extracts, and adjust the pH of the extract to 7.0 with 2 mol / L NaOH solution to obtain mixture I;

[0012] S4. After concentrating the mixture I in a rotary evaporator, freeze-dry it under vacuum to obtain mixture I; add methanol to dissolve the mixture I and freeze-dry it under vacuum again to obtain the antibacterial active substance.

[0013] Preferably, in step S1, the fermentation temperature is 27-37℃, the fermentation rotation speed is 180-220 r / min, and the fermentation time is 48-60 h.

[0014] Preferably, the centrifugation in step S1 is performed at 4500-5500g and 3-6℃ for 15-25 minutes.

[0015] Preferably, the centrifugation in steps S2 and S3 is performed at 10,000-13,000 r / min at 3-6℃ for 8-12 min.

[0016] Preferably, the ratio of precipitate to methanol in step S3 is 1 g: 8 mL.

[0017] Preferably, in step S4, the mixture is concentrated by rotary evaporation to 1 / 2 to 1 / 5 of the original volume of the mixture I.

[0018] Fourthly, the present invention provides the use of the antibacterial active substance prepared by the method described in the third aspect in the preparation of daily chemical products.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The inventors of this application isolated and purified Bacillus belye B02 from soil samples collected from a pond in the Baiyun District Private Science and Technology Park in Guangzhou.

[0021] 1. The antibacterial test results show that the antibacterial active substances produced by the fermentation of Bacillus belyss isolated from soil samples in this application have good antibacterial effects against Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, Enterococcus faecalis, Candida albicans and Aspergillus brasiliensis. The diameter of the inhibition zone of the above eight tested bacteria is greater than 15 mm, and this bacterium has a better antibacterial effect than Bacillus belyss in the prior art.

[0022] 2. The results of the minimum inhibitory concentration (MIC) test showed that the antibacterial active substances prepared by fermentation of strain B02 had a minimum inhibitory concentration (MIC) of less than 25 μg / mL against Candida albicans, Aspergillus brasiliensis, and Staphylococcus aureus; a minimum inhibitory concentration (MIC) of less than 100 μg / mL against Pseudomonas aeruginosa; a minimum inhibitory concentration (MIC) of less than 50 μg / mL against Escherichia coli, Enterococcus faecalis, and Pseudomonas putida; and a minimum inhibitory concentration (MIC) of less than 12.5 μg / mL against Burkholderia cepacia. This indicates that the fermentation products of strain B02 have excellent antibacterial properties against the above-mentioned tested bacteria.

[0023] 3. The results of heat resistance, cold resistance, light resistance, and pH stability tests showed that the antibacterial active substance produced by Bacillus belyssus B02 fermentation in this application, after treatment at 4℃ (refrigerated and protected from light), 25℃ (room temperature and protected from light), 45℃ (high temperature and protected from light), and 25℃ (continuous light exposure) for 3, 7, 15, and 30 days respectively, still maintained strong antibacterial activity against Burkholderia cepacia in the heat resistance, cold resistance, and light stability groups. The diameter of the inhibition zone was greater than 16 mm in all cases. This indicates that the antibacterial active substance produced by Bacillus belyssus B02 fermentation has excellent heat resistance, cold resistance, and pH stability. Furthermore, the antibacterial active substances produced by the fermentation of Bacillus berleis B02 prepared in this application, after different pH treatments, all exhibited antibacterial effects against Burkholderia cepacia within the pH range of 1-13, with inhibition zone diameters all greater than 12 mm. This indicates that the antibacterial active substances produced by Bacillus berleis B02 isolated from soil in this application can maintain antibacterial properties within the pH range of 1-13. In summary, this demonstrates that the antibacterial active substances produced by the fermentation of strain B02 possess excellent heat resistance, cold resistance, light stability, and pH stability.

[0024] 4. The preservation test results show that, after 7 days of treatment, the viable bacterial count in each group of the test subjects prepared by this invention is no higher than 100 CFU / mL; while after 14 to 28 days of treatment, the viable bacterial count in both the bacterial and fungal groups is 0, indicating that the test subjects pass the test and have excellent preservation effects. The above test results indicate that the antibacterial active substance produced by Bacillus belyeis B02 fermentation can be used as an antibacterial and preservative raw material. When applied to the preparation of daily chemical products, it exhibits excellent preservative performance and has broad application prospects and important value for transformational research.

[0025] In summary, the antibacterial active substances produced by Bacillus belyssus B02 with accession number GDMCC NO.66439 have the advantages of being of pure natural origin, highly effective and broad-spectrum antibacterial, mild and non-irritating, and having good stability. They can be widely used in daily chemical products, skin care products, etc.

[0026] A strain of Bacillus belye B02, classified and named Bacillus velezensis It was deposited on May 29, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.66439, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0027] Figure 1 This is a colony morphology diagram of Bacillus belyssus B02 in Example 1;

[0028] Figure 2 The image shows the antibacterial effect of Bacillus berberis B02 fermentation broth on Staphylococcus aureus in Example 3.

[0029] Figure 3 The image shows the antibacterial effect of Bacillus berberis B02 fermentation broth on Candida albicans (i.e., Candida albicans) in Example 3.

[0030] Figure 4 The figure shows the pH stability test results of the antibacterial active substance prepared from Bacillus beryl B02 in Example 6. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0033] The culture medium involved in this invention:

[0034] Liquid fermentation medium: 10 g / L mannitol, 15 g / L maltose, 10 g / L dextrin, 20 g / L tryptone, 20 g / L yeast extract, 10 g / L ammonium sulfate, distilled water, natural pH.

[0035] Solid fermentation medium: 10 g / L mannitol, 15 g / L maltose, 10 g / L dextrin, 20 g / L tryptone, 20 g / L yeast extract, 10 g / L ammonium sulfate, 15 g / L agar, distilled water, natural pH.

[0036] Nutrient agar medium: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, distilled water.

[0037] Nutrient broth culture medium: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, distilled water.

[0038] All of the above culture media must undergo sterilization at 121°C for 20 minutes.

[0039] Example 1: Isolation, purification and screening of bacterial strains

[0040] Soil samples were collected from a pond near the Private Science and Technology Park in Baiyun District, Guangzhou City, Guangdong Province. 10g of the collected soil sample was weighed and added to 90mL of liquid fermentation medium. After enrichment culture at 37℃ and 200rpm in a constant temperature shaking incubator for 3 days, an appropriate amount of the enriched culture solution was taken and the bacterial strains were isolated using a 10-fold serial dilution method. 100μL of each diluted 10-fold strain was then used for further isolation. -3 10 -4 10 -5The diluted solution was spread onto nutrient agar plates and incubated at 37°C. Observations were made daily until single colonies appeared. Each single colony was transferred to a fresh nutrient agar plate, and the streak plate method was used for repeated isolation and purification. The purified strains were then numbered and recorded. The purified bacteria were inoculated into liquid fermentation medium for liquefaction. After obtaining the bacterial culture, sterile glycerol was added to prepare a 20% glycerol-containing cryopreservation solution, which was stored at -80°C.

[0041] The isolated strains were screened for antibacterial activity. The specific steps were as follows: Nutrient agar plates were prepared; 100 μL of *Burkholderia cepacia* suspension in the logarithmic growth phase was evenly spread onto the nutrient agar plates; after the plates were dried, the purified and isolated strains were inoculated into nutrient agar medium containing the test bacteria, and incubated at 36℃ for 24 h. The antibacterial activity of each isolated strain was measured, and the antagonistic strain B02 with good inhibitory effect against *Burkholderia cepacia* was screened.

[0042] Example 2: Strain Identification

[0043] Morphological characteristics: After being cultured on nutrient agar medium at 36°C for 1 day, strain B02 produced large, slightly yellow colonies with irregular edges, raised outer rings, and a rough, dry surface (as shown in the image). Figure 1 (As shown); the Gram staining reaction was positive.

[0044] Molecular biological identification: 16S rRNA identification was performed. Genomic DNA of strain B02 was extracted according to the instructions of the bacterial genomic DNA extraction kit and used as a template. PCR amplification was performed using universal primers (27F: AGAGTTTGATCMTGGCTCAG (SEQ.ID.NO.2), 1492R: TACGGYTACCTTGTTACGACTT (SEQ.ID.NO.3)) to obtain its 16S rRNA. Sequencing was subsequently performed by BGI Genomics. The sequence information (SEQ.ID.NO.1) was compared with the NCBI database for sequence alignment and homology analysis, with a sequence homology of 98.31%. Based on its morphological characteristics, strain B02 was identified as *Bacillus belye* and classified as *Bacillus*. Bacillus velezensis, It was named Bacillus belyssus B02 and preserved.

[0045] The 16S rRNA sequence of this strain is shown in SEQ ID NO.1:

[0046]

[0047] Example 3 Antibacterial test of fermentation broth

[0048] Test substances: fermentation broth ①-④;

[0049] Fermentation broth ①: Fermentation broth of Bacillus belysii B02; Bacillus belysii B02 activated and cultured for two generations was inoculated into 100 mL of liquid fermentation medium at an inoculation rate of 3% by volume, and fermented at 35℃ and 200 r / min for 48 h. After fermentation, it was centrifuged at 5000 g and 4℃ for 20 min, the bacterial cells were discarded, and the fermentation supernatant was retained. The supernatant was then ultrafiltered through a ceramic membrane with a pore size of 20 nm to obtain fermentation broth ①.

[0050] Fermentation broth ②: Fermentation broth of Bacillus belyssus SMB1 (see CN119662464A, accession number: CCTCCNO: M20242647, donated by Shandong University); the preparation method is exactly the same as that of fermentation broth ①, the only difference is the strain;

[0051] Fermentation broth ③: Fermentation broth of Bacillus belyssus DJ1 (see CN115975875B, accession number: CGMCCNo.25972, donated by the Institute of Microbiology, Heilongjiang Academy of Sciences); the preparation method is exactly the same as that of fermentation broth ①, the only difference is the strain;

[0052] Fermentation broth ④: Fermentation broth of Bacillus belyssus ST7-6 (see CN115044505B, accession number: GDMCC No: 62393, donated by Jiangnan University); the preparation method is exactly the same as that of fermentation broth ①, the only difference is the strain;

[0053] Test bacteria: Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, Enterococcus faecalis, Candida albicans, and Aspergillus brasiliensis in the logarithmic growth phase were selected.

[0054] The specific steps of the antibacterial test are as follows:

[0055] The experiment employed the double-layer agar diffusion method. 10 mL of sterile 1% water agar medium was poured into a sterile Petri dish and allowed to solidify before being placed in an Oxford cup. 200 μL of the test bacterial suspension was added to 20 mL of nutrient agar medium at approximately 45°C and mixed thoroughly. The final concentration of the test bacteria was 3.0 × 10⁻⁶. 8CFU / mL, pour well-mixed nutrient agar medium containing the test bacteria into a sterile Petri dish; after the nutrient agar medium has completely solidified, remove the Oxford cup and add 100 μL of the test substance (fermentation broth ①-④) to each sample well. The blank control group is replaced with an equal volume of sterile water. Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, and Enterococcus faecalis are incubated at 36℃ for 24 h, and Candida albicans and Aspergillus brasiliensis are incubated at 28℃ for 72 h. The diameter of the inhibition zone is measured. Each test bacteria is set up in 3 parallel experiments, and each inhibition zone is measured in 3 directions. The data are presented as average values.

[0056] The experimental results are shown in Table 1 and Figures 2-3 (The figure only shows part of the results) As shown, the inhibition zone diameters of fermentation broth ① against Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, Enterococcus faecalis, Candida albicans, and Aspergillus brasiliensis were 22.92 mm, 16.35 mm, 15.71 mm, 20.84 mm, 16.03 mm, 17.26 mm, 19.77 mm, and 21.58 mm, respectively, indicating that the fermentation product of Bacillus belyssus B02 has a good antibacterial effect.

[0057] Based on the antibacterial effects of fermentation broths ①-④ in Table 1 on Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, Enterococcus faecalis, Candida albicans, and Aspergillus brasiliensis, it can be seen that not all Bacillus bereaves species possess antibacterial activity against the above eight bacteria. The Bacillus bereaves B02 isolated in this invention can ferment to produce active substances with good antibacterial properties.

[0058] Table 1. Data on the diameter of the inhibition zone

[0059]

[0060] Note: Unit (mm), "-" indicates no obvious antibacterial activity.

[0061] Example 4 Preparation of antibacterial active substances

[0062] A method for preparing antibacterial active substances by fermentation of Bacillus belye includes the following steps:

[0063] S1. Take two generations of activated Bacillus berberis B02 and inoculate it into 1000 mL of liquid fermentation medium at a volume fraction of 3%. Ferment at 35℃ and 200 r / min for 48 h. After fermentation, centrifuge at 5000 g and 4℃ for 20 min, discard the bacterial cells, and retain the fermentation supernatant.

[0064] S2. Take 200 mL of fermentation supernatant, adjust the pH to 2.0 with 6 mol / L HCl solution, let it stand overnight at 4℃, and then centrifuge at 12000 r / min for 10 min at 4℃ to collect the precipitate.

[0065] S3. Take the precipitate and methanol (analytical grade), mix them well and extract them. The mixing ratio of the precipitate and methanol is 1g:8mL. Extract for 8h at pH=7, centrifuge at 4℃ and 12000 rmin for 10 min, collect the methanol extract, add methanol to the precipitate again and repeat the above extraction and centrifugation steps 3 times. Mix all the collected methanol extracts and adjust the pH of the extract to 7.0 with 2mol / L NaOH solution to obtain mixture I.

[0066] S4. Place the mixture I in a rotary evaporator for rotary evaporation and concentration until the volume of the original mixture I is 1 / 4. Then freeze-dry under vacuum to obtain mixture I. Add methanol to the mixture I to dissolve it and freeze-dry under vacuum again to obtain the antibacterial active substance.

[0067] Example 5: Determination of the minimum inhibitory concentration (MIC) of antibacterial active substances

[0068] Test substance: 0.1 g of the antibacterial active substance prepared in Example 4 was dissolved in 10 mL of sterile water, filtered through a 0.2 μm filter membrane to remove bacteria, and the test solution was then tested for minimum inhibitory concentration (MIC). The test solution was then diluted with nutrient broth to concentrations of 250, 200, 100, 50, 25, and 12.5 μg / mL for further testing of MIC.

[0069] Test bacteria: *Burkholderia cepacia*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Pseudomonas putida*, *Enterococcus faecalis*, *Candida albicans*, and *Aspergillus brasiliensis* in the logarithmic growth phase were selected; each test bacteria was diluted with sterile physiological saline to a concentration of 3.0 × 10⁻⁶. 8 CFU / mL concentration to be used;

[0070] Experimental group: 100 μL of test substances with concentrations of 250, 200, 100, 50, 25, and 12.5 μg / mL were added sequentially to rows 1 to 7 of a 96-well plate, followed by 100 μL of test bacteria in each well, and the mixture was gently aspirated. Blank control group: Sterile water was added to each well in row 8 of a 96-well plate according to the amount of sterile water in each concentration of test substance, and the volume was brought up to 100 μL with nutrient broth. Then, 100 μL of test bacteria was added and the mixture was gently aspirated. Each test bacteria in each group was set up in 3 replicates. Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, and Enterococcus faecalis were incubated at 36°C and 120 rpm for 24 h, while Candida albicans and Aspergillus brasiliensis were incubated at 28°C and 120 rpm for 30 h. The turbidity of each well was observed, and the results were recorded. The data are presented as average values.

[0071] As shown in Table 2, the minimum inhibitory concentration (MIC) data of the antibacterial active substance prepared by fermentation of Bacillus berleis B02 isolated from soil in this application has an MIC of less than 25 μg / mL against Candida albicans, Aspergillus brasiliensis and Staphylococcus aureus, an MIC of less than 100 μg / mL against Pseudomonas aeruginosa, an MIC of less than 50 μg / mL against Escherichia coli, Enterococcus faecalis and Pseudomonas putida, and an MIC of less than 12.5 μg / mL against Burkholderia cepacia. This indicates that the fermentation product of strain B02 has excellent antibacterial properties against the above-mentioned tested bacteria.

[0072] Table 2. Minimum Inhibitory Concentration (MIC) Data

[0073]

[0074] Note: "+" indicates turbidity, meaning it cannot inhibit the growth of the test bacteria; "-" indicates clarity, meaning it can inhibit the growth of the test bacteria.

[0075] Example 6: Heat resistance, cold resistance, light exposure, and pH stability tests of antibacterial active substances.

[0076] Test substance: 0.1 g of the antibacterial active substance prepared in Example 4 was dissolved in 10 mL of sterile water, filtered through a 0.2 μm filter membrane to remove bacteria, and the test solution was then subjected to tests for heat resistance, cold resistance, light exposure, and pH stability.

[0077] Test bacteria: Burkholderia cepacia in the logarithmic growth phase;

[0078] 1) The test samples were placed at 25℃ (room temperature, protected from light), 4℃ (refrigerated, protected from light), 45℃ (high temperature, protected from light), and 25℃ (continuous light exposure), respectively. The samples were removed on days 3, 7, 15, and 30, and the inhibition diameter was determined according to the method in Example 3. The concentration of the test bacteria was 3.0 × 10⁻⁶. 8 CFU / mL, results are shown in Table 3;

[0079] 2) Adjust the pH to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 respectively using 2 mol / L HCl and 2 mol / L NaOH, and then adjust back to neutral (pH=7) after 24 h. The concentration of the test bacteria is 3.0 × 10⁻⁶. 8 The CFU / mL inhibition diameter was determined according to the method in Example 3, and the results are shown below. Figure 4 .

[0080] The results of the heat resistance, cold resistance, and light stability tests are shown in Table 3. The antibacterial active substance prepared in Example 4 of this application was treated at 4℃ (refrigerated and protected from light), 25℃ (room temperature and protected from light), 45℃ (high temperature and protected from light), and 25℃ (uninterrupted light) for 3 days, 7 days, 15 days, and 30 days, respectively. The heat resistance, cold resistance, and light stability groups all maintained strong antibacterial activity against Burkholderia cepacia, and the diameter of the inhibition zone was greater than 16 mm. This indicates that the antibacterial active substance produced by Bacillus belyssus B02 fermentation has excellent heat resistance, cold resistance, and light stability.

[0081] pH stability test results are as follows: Figure 4 It is known that the antibacterial active substances produced by the fermentation of Bacillus belyss B02 prepared in this application have good antibacterial effects against Burkholderia cepacia in the pH range of 1-13, and the diameter of the inhibition zone is greater than 12 mm. This indicates that the antibacterial active substances produced by Bacillus belyss B02 isolated from soil in this application can maintain strong antibacterial performance in the pH range of 1-13 and have excellent pH stability.

[0082] Table 3. Test data on cold storage, light exposure, and heat resistance stability.

[0083]

[0084] Note: Diameter of the inhibition zone (mm);

[0085] Example 7: Preservative Test of Antibacterial Active Substances

[0086] Test substance: Take 2 g of the antibacterial active substance prepared in Example 4, add 8 mL of sterile water to dissolve it, filter it with a 0.2 μm filter membrane to remove bacteria, and then use the solution to be tested for preservation.

[0087] Test bacteria: Burkholderia cepacia ATCC 25416, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, and Pseudomonas putida ATCC 17485 were used for bacteria; Candida albicans ATCC 10231 was used for fungi.

[0088] The test substance was added to cosmetics as a natural preservative, and preservative tests were conducted in accordance with the well-known microbial challenge test methods of the Cosmetic, Toiletries and Fragrance Association (CTFA) and the United States Pharmacopeia. The specific test procedures are as follows:

[0089] The test substance was added to the toner for preservative efficacy testing. The amount of the test substance added was 0.15% (mass fraction). The ingredient list and manufacturing process of the toner are shown in Table 4.

[0090] Table 4. Ingredients and Manufacturing Process of Softening Toner

[0091]

[0092] The test bacterial cultures in the logarithmic growth phase were diluted with PBS buffer to a concentration of 3 × 10⁻⁶. 8 CFU / mL, appropriate amounts were added to the softening lotion. For both the bacterial and fungal groups, the final concentration of each bacterium in the softening lotion was as shown in Table 5 (0d). After thorough mixing, the bacterial group was incubated at 36℃, and the fungal group at 28℃. An equal volume of sterile water was used to replace the test substance in the blank control group. Three portions of each type of softening lotion were prepared for each group. Following the colony count test method in Chapter 5 of the 2015 edition of the Cosmetic Safety Technical Specifications, the viable bacterial count in each experimental group was measured at 7, 14, and 28 days after inoculation to determine the preservative efficacy of the cosmetic. The judgment criteria were: if the number of viable bacteria in each sample decreased to no more than 0.1% of the initial concentration on day 7 after inoculation, and then gradually decreased, with no sterile growth on day 28, then the preservative was effective and passed the test; otherwise, the preservative was ineffective and failed the test.

[0093] The results of the preservation test are shown in Table 5 (data are presented as average values). After 7 days of treatment, the viable bacterial count in each group of the test compound was no higher than 100 CFU / mL; from 14 to 28 days of treatment, the viable bacterial count in both the bacterial and fungal groups was 0, indicating that the test compound passed the test and had excellent preservation effect. These results indicate that the antibacterial active substances produced by Bacillus belyeis B02 fermentation can be used as antibacterial and preservative raw materials. When applied to the preparation of daily chemical products, they exhibit excellent preservative performance, have broad application prospects, and significant value for transformational research.

[0094] Table 5. Test data on the antiseptic efficacy of the toner

[0095]

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of Bacillus velezensis (B. velezensis) B02, characterized in that, Bacillus velezensis ) the 16S rRNA gene sequence of SEQ ID NO: 1, Its accession number is GDMCCNO.66439.

2. Use of B. velezensis B02 according to claim 1 for the preparation of a bacteriostatic active substance, characterized in that, The antibacterial active substances have antibacterial effects against Burkholderia cepacia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas putida, Enterococcus faecalis, Candida albicans, and Aspergillus brasiliensis.

3. A method for producing bacteriostatic active substances by fermentation using the Bacillus velezensis B02 according to claim 1, characterized in that, Includes the following steps: S1. Inoculate Bacillus belye BO2 into liquid fermentation medium and ferment; after fermentation, centrifuge and retain the fermentation supernatant; S2. Take the fermentation supernatant, adjust the pH to 2.0 with 6 mol / L HCl solution, let it stand overnight at 4℃, centrifuge, and collect the precipitate; S3. Take the precipitate and methanol, extract and centrifuge at pH=7, collect the methanol extract, add methanol back to the precipitate, repeat the above extraction and centrifugation steps 3 times, mix all the methanol extracts, and adjust the pH of the extract to 7.0 with 2 mol / L NaOH solution to obtain mixture I; S4. After concentrating the mixture I in a rotary evaporator, freeze-dry it under vacuum to obtain mixture I; add methanol to dissolve the mixture I and freeze-dry it under vacuum again to obtain the antibacterial active substance.

4. The method of claim 3, wherein, In step S1, the fermentation temperature is 27-37℃, the fermentation speed is 180-220 r / min, and the fermentation time is 48-60 h.

5. The method of claim 3, wherein, The centrifugation in step S1 is: 4500-5500g, 3-6℃ for 15-25min.

6. The method of claim 5, wherein, The centrifugation described in steps S2 and S3 is: 10000-13000 r / min, 3-6℃ for 8-12 min.

7. The method of claim 5, wherein, In step S3, the mixing ratio of the precipitate and methanol is 1g:8mL.

8. The method of claim 5, wherein, In step S4, the mixture is concentrated by rotary evaporation to 1 / 2 to 1 / 5 of the original volume of the mixed liquid I.

9. The use of the antibacterial active substance prepared by the method according to any one of claims 3-8 in the preparation of daily chemical products.

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