Paenibacillus polymyxa B01 and application thereof

Abstract

This invention relates to a polymyxin Bacillus strain ( Paenibacillus polymyxa This invention relates to B01 and its applications, belonging to the field of microbial technology. The *Bacillus polymyxa* B01 strain disclosed in this invention has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.66594. This strain was isolated, purified, and screened from soil samples. Experiments show that this strain has excellent pH and high-temperature tolerance, and the antibacterial active substances produced by its fermentation have good antibacterial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Pseudomonas putida*, *Staphylococcus aureus*, *Candida albicans*, *Aspergillus brasiliensis*, *Propionibacterium acnes*, and *Malassezia*. Furthermore, the antibacterial active substances exhibit excellent heat resistance, cold resistance, light stability, pH stability, and preservative effects. It is a mild and low-irritant raw material that can be used to prepare daily chemical products and pharmaceuticals, possessing broad application prospects and significant value for transformational research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Paenibacillus polymyxa B01 and application thereof. BACKGROUND

[0002] With the acceleration of modern life pace and the increase of social pressure, people's skin health is facing more and more challenges, such as dryness, aging, acne, dull skin and other problems are becoming increasingly common, which promotes the rapid development of the functional cosmetic market. In order to ensure the stability and safety of the product in complex environment, bacteriostatic ingredients are usually added in the formula to prevent microbial contamination. The ideal bacteriostatic agent should not only have good antibacterial ability, but also meet the multiple requirements of green environmental protection, no irritation to human body, no damage to the formula system, etc., which puts forward higher standards for the design of preservative system.

[0003] At present, many traditional chemical preservatives in practical applications have obvious defects. For example, phenoxyethanol may cause contact dermatitis, the safety of methylisothiazolinone components is questioned due to potential neurotoxicity and sensitization risk, and benzoic acid, sorbic acid and the like are harsh in application conditions such as pH, and may cause skin allergy. The above problems prompt the industry to turn its attention to natural source antibacterial substances. Among them, plant source bacteriostatic agent active ingredients are favored due to their "mildness", "degradability" and other advantages, but they are generally prone to oxidation inactivation, poor water solubility, strong odor or affect product color, etc., which limits their application.

[0004] Existing research shows that part of microorganisms can produce natural substances with broad-spectrum antibacterial activity and stable properties in the metabolic process. Such substances not only have excellent bacteriostatic effect, but also are safe and non-irritating to the skin, and have the potential to replace or partially replace traditional preservatives. Therefore, developing a natural bacteriostatic ingredient of microbial origin with high efficiency, broad spectrum, safety and good stability has become an important direction to improve the safety and functionality of products, which can reduce the dependence on chemical preservatives and provide better solutions for daily chemical products, skin care products, eye products (health care eye drops) and the like. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provide a Paenibacillus polymyxa B01 producing high bacteriostatic activity substances. The strain is isolated and purified from soil samples collected from the pond beside the civilian science and technology park in Baiyun District of Guangzhou, and has been preserved in Guangdong Microbial Culture Collection Center with the preservation number of GDMCC NO.66594.

[0006] In a first aspect, the present application provides a Paenibacillus polymyxa (B01) strain. Paenibacillus polymyxa), which was preserved in Guangdong Microbial Culture Collection Center on June 26, 2025, with a preservation number of GDMCC NO. 66594 and a preservation address of No. 59 Building, 5th Floor, Guangzhou Institute, 100 Middle Liujie Road, Guangzhou.

[0007] In a second aspect, the present application provides an application of the Paenibacillus polymyxa B01 in the first aspect in the preparation of bacteriostatic active substances.

[0008] In a third aspect, the present application provides a preparation method of the Paenibacillus polymyxa B01 in the first aspect for fermenting and producing bacteriostatic active substances, comprising the following steps:

[0009] S1, inoculating the Paenibacillus polymyxa B01 into a liquid fermentation medium for fermentation;

[0010] S2, collecting the fermentation broth after the fermentation is completed, filtering the fermentation broth through a 0.22 μm ceramic membrane filter to obtain a supernatant, adding ammonium sulfate solid to the supernatant until saturation, and collecting the precipitate by centrifugation;

[0011] S3, redissolving and dialyzing the precipitate prepared in step S2, purifying and concentrating the dialysate to obtain the bacteriostatic active substance.

[0012] Preferably, the fermentation temperature in step S1 is 32-38℃, the culture rotation speed is 180-220 rpm, and the culture time is 2-3 d.

[0013] Preferably, the specific steps in step S3 include: dialyzing with a dialysis membrane after redissolving with ultrapure water; collecting the supernatant after dialysis is completed, freeze-drying and concentrating to obtain a freeze-dried powder, preparing a sample solution with a mass concentration of 3% from the freeze-dried powder with ultrapure water, purifying through a Sephadex G-50 column, and using ultrapure water as an eluent, collecting one tube every 5 mL, and vacuum concentrating the eluent capable of making coomassie brilliant blue discolor after elution is completed to obtain the bacteriostatic active substance.

[0014] Further preferably, the sample volume for Sephadex G-50 column purification is 10 mL, and the elution flow rate is 0.8-1.2 mL / min.

[0015] Further preferably, the molecular weight of the dialysis membrane is 500 Da.

[0016] Further preferably, the dialysis membrane dialysis time is 2-3 d.

[0017] In a fourth aspect, the present application provides a product comprising the Paenibacillus polymyxa B01 in the first aspect.

[0018] In a fifth aspect, the present application provides an application of the bacteriostatic active substance prepared by the method in the third aspect in the preparation of daily chemical products and medicines.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] The present application inventors isolated and purified Paenibacillus polymyxa B01 from a soil sample collected from a pond in Guangzhou Baiyun District Private Science and Technology Park.

[0021] 1. The results of pH and temperature tolerance tests show that the pH tolerance of Paenibacillus polymyxa B01 is pH 5-11, and the OD value (absorbance value) of the bacteria is the highest at pH=10; the temperature tolerance of Paenibacillus polymyxa B01 is 30℃-38℃, and the OD value (absorbance value) of the bacteria is the highest at a temperature of 36℃; as can be seen from the above, the Paenibacillus polymyxa B01 isolated by the application has excellent pH tolerance and high temperature tolerance.

[0022] 2. The results of the bacteriostatic test show that the bacteriostatic active substances produced by the Paenibacillus polymyxa isolated from the soil sample have good bacteriostatic effect on Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia, and the bacteriostatic circle diameters of the above eight indicator bacteria are all greater than 17mm.

[0023] 3. The results of heat resistance, cold resistance, light and pH stability tests show that after the bacteriostatic active substances produced by the Paenibacillus polymyxa B01 of the application are 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 5 months, the minimum bacteriostatic concentrations of the heat-resistant, cold-resistant, light-stable group to Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida are still all below 25mg / L, the minimum bacteriostatic concentrations to Staphylococcus aureus are all below 50mg / L, the minimum bacteriostatic concentrations to Candida albicans are all below 62.5mg / L, the minimum bacteriostatic concentrations to Aspergillus brasiliensis are all below 100mg / L, and the minimum bacteriostatic concentrations to Propionibacterium acnes and Malassezia are all below 12.5mg / L; and after the bacteriostatic active substances produced by the Paenibacillus polymyxa B01 of the application are treated at different pH, the bacteriostatic circle diameters of the bacteriostatic active substances to Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia in the pH=1-11 range are not significantly different from the data in Example 5, and the bacteriostatic circle diameters are all greater than 17mm, indicating that the bacteriostatic active substances produced by the Paenibacillus polymyxa B01 isolated from the soil of the application can maintain very strong bacteriostatic performance in the pH=1-11 range; as can be seen from the above, the bacteriostatic active substances produced by the strain B01 have excellent heat resistance, cold resistance, light stability and pH stability.

[0024] 4. The irritation test results show that the antibacterial active substance prepared in this application is a mild and low-irritant raw material, which has broad prospects in skin care products.

[0025] 5. The preservation test results showed that the viable bacterial counts in both the bacterial and fungal groups decreased at day 0; at day 7, the viable bacterial counts in each group were no higher than 100 CFU / mL; and from day 14 to 28, the viable bacterial counts in both the bacterial and fungal groups were 0, indicating that the test substance passed the test and had excellent preservative effects. This suggests that the antibacterial active substances produced by the fermentation of Bacillus polymyxa B01 can be used as antibacterial and preservative raw materials, exhibiting excellent preservative performance when applied to the preparation of daily chemical products, and possessing broad application prospects and significant value for transformational research.

[0026] In summary, the Bacillus polymyxa B01 with accession number GDMCC NO.66594 not only has excellent pH and temperature tolerance, but also produces antibacterial active substances with advantages such as pure natural source, high efficiency and broad spectrum antibacterial effect, mild and non-irritating properties, and good stability. It can be widely used in daily chemical products, skin care products, and eye products (health care eye drops).

[0027] Preservation of biological materials

[0028] A strain of *Bacillus polymyxa* B01, classified and named Paenibacillus polymyxa It was deposited on June 26, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.66594, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0029] Figure 1 The image shows the inhibitory effect of Bacillus polymyxa B01 on Aspergillus brasiliensis in Example 1.

[0030] Figure 2 This is a colony morphology diagram of Bacillus polymyxa B01;

[0031] Figure 3 To investigate the effect of different pH values ​​on the growth of Bacillus polymyxa B01 and Bacillus polymyxa SP1, the absorbance values ​​(OD) of the bacterial solutions at different pH values ​​were analyzed. 600nm );

[0032] Figure 4 To investigate the effect of different temperatures on the growth of Bacillus polymyxa B01 and Bacillus polymyxa SP1, the absorbance values ​​(OD) of the bacterial solutions at different temperatures were analyzed. 600nm );

[0033] Figure 5SDS-PAGE electrophoresis and glycoprotein staining of the bacteriostatic substance ① of Bacillus polymyxa B01 in Example 4, wherein band 1 is the SDS-PAGE electrophoresis chart, and band 2 is the glycoprotein staining chart. DETAILED DESCRIPTION

[0034] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples.

[0035] Other materials, reagents, etc. used in the examples can be obtained from commercial channels unless otherwise specified.

[0036] Culture medium involved in the present application:

[0037] Liquid fermentation medium: 20 g / L glucose, 10 g / L peptone, 5 g / L beef infusion powder, 5 g / L sodium chloride, distilled water, pH natural.

[0038] Solid culture medium: 20 g / L glucose, 10 g / L peptone, 5 g / L beef infusion powder, 5 g / L sodium chloride, 15 g / L agar, distilled water, pH natural.

[0039] Nutrient agar medium: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, distilled water.

[0040] Nutrient broth medium: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, distilled water.

[0041] The above culture media all need to go through a sterilization step, and the sterilization condition is 121℃ for 20 min.

[0042] Example 1: Isolation, purification and screening of strains

[0043] A soil sample was collected from a pond beside a private science and technology park in Baiyun District, Guangzhou, Guangdong Province. 10 g of the collected soil sample was weighed, 90 mL of liquid fermentation medium was added, and after 3 days of enrichment culture in a 37℃ constant temperature shaking incubator at 200 rpm, a suitable amount of the enrichment culture was used to isolate strains by 10-fold gradient dilution method. 100 μL of the dilution solution with dilution multiples of 10 -3 , 10 -4 , and 10 -5 were respectively streaked on nutrient agar medium plates, and incubated at 37℃. Observations were made every day until single colonies were grown. Each single colony was picked and transferred to a new solid fermentation medium plate, and repeated isolation and purification were performed by plate streaking method to obtain purified strains and record the numbers. Each strain was inoculated into liquid fermentation medium for liquid culture, and after obtaining the bacterial solution, a sterile glycerol was added to prepare a strain cryopreservation solution containing 20% glycerol, which was stored in a -80℃ refrigerator.

[0044] The isolated strains were screened for antibacterial activity. The specific steps were as follows: a nutrient agar plate was prepared, 100 μL of the Aspergillus brasiliensis in the logarithmic growth phase was uniformly coated on the nutrient agar plate, the plate surface was dried, each strain obtained by purification and separation was inoculated into the nutrient agar medium containing the indicator bacteria, and incubated at 36°C for 24 h. The antibacterial activity of each isolated strain was determined. The preliminary screening experiment (results shown in Figure 1 ) showed that strain 1A had obvious antibacterial activity against Aspergillus brasiliensis, so the strain was identified and subsequent verification experiments were performed.

[0045] Example 2: Strain identification

[0046] Morphological characteristics: after strain 1A was incubated at 36°C for 1 day, the colony was round, the surface was milky white, the surface was moist and sticky (as shown in Figure 2 ); microscopic examination showed that the bacterial body was short rod-shaped, the spore was oval, and the flagellum was peritrichous; the gram staining reaction was positive.

[0047] Molecular biology method identification: 16S rRNA identification was performed. The genomic DNA of strain 1A was extracted according to the operation instruction of the bacterial genomic DNA extraction kit, and used as a template for PCR amplification of 16S rRNA using universal primers (27F: AGAGTTTGATCMTGGCTCAG (SEQ. ID. NO. 2), 1492R: TACGGYTACCTTGTTACGACTT (SEQ. ID. NO. 3)). The sequence information (SEQ. ID. NO. 1) obtained by sequencing was subjected to sequence alignment and homology analysis in the NCBI database, and the sequence homology was 98.68%; and combined with the morphological characteristics, it was determined that strain 1A was Paenibacillus polymyxa, and was classified and named as Paenibacillus polymyxa, It was renamed as Paenibacillus polymyxa B01 and preserved.

[0048] The 16S rRNA sequence of the strain is shown as SEQ ID NO. 1:

[0049]

[0050] Example 3 pH tolerance and temperature tolerance test of the strains

[0051] The pH tolerance and temperature tolerance of Paenibacillus polymyxa B01 and Paenibacillus polymyxa SP1 with the preservation number of GDMCC No: 61605 (donated by South China Agricultural University) were determined respectively, and the specific steps were as follows:

[0052] The activated culture liquid of the two strains of Paenibacillus polymyxa was taken respectively, and inoculated into the nutrient broth medium with different pH values (3, 5, 7, 9, 10, 11, 12) at a volume fraction of 3%, and cultured at 30°C and 180 r / min for 24 h. Then the activated culture liquid of the two strains of Paenibacillus polymyxa was taken respectively, and inoculated into the nutrient broth medium at a volume fraction of 3%, and placed in different temperatures (28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C) at 200 r / min for 24 h. The growth of the tested strains was observed, and the OD value (absorbance value) of the culture liquid was measured by an enzyme marker. If the strain could grow normally, it indicated that the strain could tolerate the pH or temperature; otherwise, it indicated that the strain could not tolerate the pH or temperature. 600nm

[0053] The pH tolerance results are shown in Figure 3 The OD value (absorbance value) of the two strains showed a trend of first increasing and then decreasing under different pH values, and the over-acid or over-alkali would inhibit the growth and reproduction of the strains. Among them, the growth of Paenibacillus polymyxa B01 was obviously inhibited when the pH was less than 5 or greater than 10, the OD value (absorbance value) of the strain was the highest at pH 10, and the cell density of Paenibacillus polymyxa B01 in the pH range of 5-11 was significantly higher than that of Paenibacillus polymyxa SP1, indicating that Paenibacillus polymyxa B01 had excellent pH tolerance and its amplification activity was better than that of Paenibacillus polymyxa SP1.

[0054] The temperature tolerance results are shown in Figure 4 When the temperature was not more than 30°C, it had basically no effect on the growth of the two strains. When the temperature was greater than 30°C, the cell density of Paenibacillus polymyxa SP1 gradually decreased. When the temperature was greater than 36°C, the OD value (absorbance value) of Paenibacillus polymyxa B01 gradually decreased, and the OD value (absorbance value) of the strain was the highest at 36°C, and the cell density of Paenibacillus polymyxa B01 in the temperature range of 30°C-38°C was significantly higher than that of Paenibacillus polymyxa SP1, indicating that Paenibacillus polymyxa B01 had excellent high-temperature tolerance and was better than Paenibacillus polymyxa SP1.

[0055] Example 4 Preparation, purification and identification of bacteriostatic active substances

[0056] ​1) The preparation method of the bacteriostatic active substance ①, specifically comprising the following steps:

[0057] S1, inoculate the activated Paenibacillus polymyxa B01 bacterial solution into a liquid fermentation medium, and ferment at 36°C and 200 rpm for 2 days;

[0058] S2, after the fermentation is completed, the fermentation broth is filtered through a 0.22 μm ceramic membrane filter to obtain a fermentation supernatant; slowly add ammonium sulfate solid powder to the fermentation supernatant to saturation in an ice bath environment, and continuously stir while adding; after adding, continue to gently stir on ice for 1 hour to complete the reaction, and collect the precipitate after centrifugation at 15000 g for 20 min;

[0059] S3, take 2g of the precipitate, redissolve it with 200mL of ultrapure water, and then dialyze it for 2 days using a 500Da dialysis membrane; after dialysis, freeze-dry the clear liquid to obtain a freeze-dried powder; prepare a sample solution with a mass concentration of 3% from the freeze-dried powder using ultrapure water, purify it through a dextran gel column G-50, use ultrapure water as the eluent, load 10mL of the sample, and elute at a flow rate of 1mL / min, collecting one tube every 5mL; after elution, vacuum concentrate the eluent that can change the color of coomassie brilliant blue to obtain the bacteriostatic active substance ①.

[0060] 2) Prepare an SDS-PAGE gel using a Tris-Tricine-SDS-PAGE gel preparation kit (Solabio), use Solabio ultra-low molecular weight protein Marker (3.3-20.1kDa) as the protein Marker, and perform protein property identification on the bacteriostatic active substance ①: prepare 4%-10%-20% interlayer glue according to the kit instructions, prepare a protein sample with a concentration of 1mg / ml, mix it with 2x loading buffer, load 5μL per well, and run the gel at 30v for 1 hour, then adjust the voltage to 100v until the electrophoresis is completed; after electrophoresis, dye the gel with coomassie brilliant blue rapid staining solution (Yaenzyme Biological) for 30min, and decolorize with pure water to obtain the protein sample SDS-PAGE gel result.

[0061] In addition, the PAGE gel glycoprotein staining kit (Beijing Baolaibo Technology Co., Ltd.) is used to stain the gel after electrophoresis of the bacteriostatic active substance ①:

[0062] (1) Take out the SDS-PAGE gel after electrophoresis, add it to a container containing 100mL of 50% methanol solution, soak completely for 30 minutes, and then pour out the methanol solution;

[0063] (2) Wash the gel block with 100mL of 3% acetic acid solution twice, each time for 10 minutes with gentle shaking;

[0064] (3) Transfer the PAGE gel slab into a vessel containing 25 mL of the oxidizing reagent and gently shake for 15 minutes;

[0065] (4) Wash the gel slab with 100 mL of 3% acetic acid solution for 3 times, each time gently shaking for 5 minutes;

[0066] (5) Transfer the gel slab into a vessel containing 25 mL of the glycoprotein staining reagent and gently shake for 15 minutes;

[0067] (6) Transfer the gel slab into a vessel containing 25 mL of the reducing reagent and gently shake for 5 minutes;

[0068] (7) Wash the gel slab with 3% acetic acid solution and then with ultrapure water until color development, the glycoprotein will appear as a magenta band, and the gel slab is preserved in 3% acetic acid solution.

[0069] From the SDS-PAGE gel results and PAGE gel glycoprotein staining of Figure 5 It can be seen that the bacteriostatic active substance ① in the present application is a bacteriostatic active glycoprotein, and the electrophoresis band is diffused due to glycosylation, and according to the electrophoresis results, the molecular weight of the bacteriostatic active glycoprotein of the B01 strain is about 6-7 kDa.

[0070] Bacteriostatic active substance ②: Compared with the preparation steps of the bacteriostatic active substance ①, the only difference is that the Paenibacillus polymyxa S3 with the accession number CCTCC NO: M 2022950 (donated by Hunan Normal University) is used instead of the Paenibacillus polymyxa B01 in the present application to prepare the bacteriostatic active substance through fermentation, purification and other steps, and other steps and parameters are consistent with those of the bacteriostatic active substance ①.

[0071] Bacteriostatic active substance ③: Compared with the preparation steps of the bacteriostatic active substance ①, the only difference is that the Paenibacillus polymyxa SAMM2-1G with the accession number CCTCC NO: M20221521 (donated by Jiangsu San Yi Biological Engineering Co., Ltd.) is used instead of the Paenibacillus polymyxa B01 in the present application to prepare the bacteriostatic active substance through fermentation, purification and other steps, and other steps and parameters are consistent with those of the bacteriostatic active substance ①.

[0072] Example 5 Bacteriostatic test of bacteriostatic active substance

[0073] Test substance: 0.05 g of each of the bacteriostatic active substances ①-③ prepared in Example 4 was added into 5 mL of sterile water and mixed uniformly, and then filtered with a 0.2 μm filter membrane to obtain a test solution for bacteriostatic experiment.

[0074] Indicator bacteria: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia were selected in the logarithmic growth phase;

[0075] The specific steps of the bacteriostatic test are as follows:

[0076] The test adopts a double-layer agar diffusion method, 10 mL of sterilized 1% water agar medium is poured into a sterile flat dish, and after it is solidified, an Oxford cup is placed; 200 μL of an indicator bacteria solution is added to 20 mL of nutrient agar medium at about 50°C, and mixed, wherein the final concentration of the indicator bacteria is 3.0 x 10 6 CFU / mL, the mixed nutrient agar medium containing the indicator bacteria is poured into a sterile flat dish; after the nutrient agar medium is completely solidified, the Oxford cup is taken out, and 200 μL of the test substance is added to the sample hole, and the blank control group is replaced with an equal amount of sterile water instead of the test substance; the escherichia coli, pseudomonas aeruginosa, malodor pseudomonas, staphylococcus aureus and malassezia are cultured in a 36°C, 120 rpm shaker for 24 hours, the propionibacterium acnes is cultured in an anaerobic bag at 30°C, 120 rpm for 24 hours, and the aspergillus brasiliensis and candida albicans are cultured in a 28°C, 120 rpm shaker for 72 hours, and the diameter of the bacteriostatic circle is measured; wherein three parallel experiments are set for each indicator bacteria, and three directions are measured for each bacteriostatic circle, and the data is presented as an average value.

[0077] The test results are shown in Table 1: the diameters of the bacteriostatic circles of the bacteriostatic active substance ① on escherichia coli, pseudomonas aeruginosa, malodor pseudomonas, staphylococcus aureus, candida albicans, aspergillus brasiliensis, propionibacterium acnes and malassezia are 21.14 mm, 18.56 mm, 19.39 mm, 25.94 mm, 27.05 mm, 28.23 mm, 21.37 mm and 24.81 mm respectively, indicating that the fermentation product of bacillus polymyxa B01 has good bacteriostatic effect.

[0078] According to the bacteriostatic effect of bacteriostatic active substances ①-③ on escherichia coli, pseudomonas aeruginosa, malodor pseudomonas, staphylococcus aureus, candida albicans, aspergillus brasiliensis, propionibacterium acnes and malassezia in Table 1, it can be known that not all bacillus polymyxa has bacteriostatic effect on the above-mentioned eight kinds of bacteria, and the bacillus polymyxa B01 separated and obtained in the present application can ferment to produce active substances with good bacteriostatic performance.

[0079] Table 1 Diameter of bacteriostatic circle data

[0080] Indicator bacteria Antibacterial active substance 1 Antibacterial active substance 2 Antibacterial active substance 3 Escherichia coli 21.14 11.25 16.64 Pseudomonas aeruginosa 18.56 13.09 - Pseudomonas putida 19.39 16.14 12.75 Staphylococcus aureus 25.94 10.38 18.71 Candida albicans 27.05 - - Aspergillus brasiliensis 28.23 - - Propionibacterium acnes 21.37 14.13 - Malassezia 24.81 - -

[0081] Note: "-" indicates no bacteriostatic effect, and the diameter of the bacteriostatic circle (mm).

[0082] Example 6 Heat resistance, cold resistance, light resistance and pH stability test of bacteriostatic active substance

[0083] Test substance: 0.2 g of the bacteriostatic active substance 1 prepared in Example 4 was respectively added into 20 mL of sterile water, mixed uniformly, and filtered to remove bacteria using a 0.2 μm filter membrane to obtain a test solution for heat resistance, cold resistance, light resistance, and pH stability tests;

[0084] Indicator bacteria: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes, and Malassezia were selected in the logarithmic growth phase;

[0085] 1) The test substance was respectively placed at 25°C (normal temperature and light avoidance), 4°C (cold storage and light avoidance), 45°C (high temperature and light avoidance), and 25°C (continuous light) for 5 months, and then the test substance was taken out and tested according to the minimum bacteriostatic concentration (nutrient broth dilution method) in 2.1.8.4.2 of the Disinfection Technical Specification, and the final concentration of the indicator bacteria was 2.0×10 7 CFU / mL. The minimum bacteriostatic concentration of the test substance treated under different conditions on different indicator bacteria was detected;

[0086] 2) The pH was adjusted to 1, 3, 5, 7, 9, 11, and 12 using 2 mol / L HCl and 2 mol / L NaOH, and then placed for 24 h, and then adjusted back to neutral (pH=7). The final concentration of the indicator bacteria was 2.0×10 7 CFU / mL. The bacteriostatic test was performed according to the method in Example 5, and the results are shown in Table 3.

[0087] The heat resistance, cold resistance, and light resistance test results are shown in Table 2. After the bacteriostatic active substance 1 prepared in Example 4 was treated at 4°C (cold storage and light avoidance), 25°C (normal temperature and light avoidance), 45°C (high temperature and light avoidance), and 25°C (continuous light) for 5 months, the minimum bacteriostatic concentration of the heat resistance, cold resistance, and light resistance groups on Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida was all less than 25 mg / L, the minimum bacteriostatic concentration on Staphylococcus aureus was all less than 50 mg / L, the minimum bacteriostatic concentration on Candida albicans was all less than 62.5 mg / L, the minimum bacteriostatic concentration on Aspergillus brasiliensis was all less than 100 mg / L, and the minimum bacteriostatic concentration on Propionibacterium acnes and Malassezia was all less than 12.5 mg / L, indicating that the bacteriostatic active substance produced by Bacillus polymyxa B01 fermentation has excellent heat resistance, cold resistance, and light resistance.

[0088] The pH stability test results are shown in Table 3. The bacteriostatic active substances produced by the fermentation of Paenibacillus polymyxa B01 prepared in the application have no significant difference in the inhibition zone diameters of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia against the data in Example 5 in the pH range of 1-11, and the inhibition zone diameters are all greater than 17 mm, indicating that the bacteriostatic active substances produced by the Paenibacillus polymyxa B01 isolated from the soil in the application can maintain very strong bacteriostatic performance in the pH range of 1-11, and have excellent pH stability.

[0089] Table 2. Cold storage, light, heat resistance stability test data

[0090] Minimum inhibitory concentration mg / L Control Cold-resistant Heat-resistant Light Escherichia coli 62.5 62.5 62.5 62.5 Pseudomonas aeruginosa 100 100 100 100 Pseudomonas putida 100 100 100 100 Staphylococcus aureus 25 25 25 25 Candida albicans 12.5 12.5 12.5 12.5 Aspergillus brasiliensis 12.5 12.5 12.5 12.5 Propionibacterium acnes 62.5 62.5 62.5 62.5 Malassezia 25 25 25 25

[0091] Table 3. pH stability data

[0092] Indicator bacteria / pH 1 3 5 7 9 11 12 Escherichia coli 21.08 20.53 20.82 22.04 20.73 20.19 13.32 Pseudomonas aeruginosa 18.77 19.05 19.24 18.19 18.35 17.96 10.95 Pseudomonas putida 19.46 18.84 20.07 20.10 19.09 19.18 15.92 Staphylococcus aureus 25.32 25.99 26.15 26.09 26.34 25.12 16.83 Candida albicans 26.14 27.64 28.21 27.73 27.11 26.83 11.44 Aspergillus brasiliensis 27.66 27.92 27.34 28.01 26.93 26.31 15.60 Propionibacterium acnes 21.11 21.98 21.14 21.53 21.87 20.34 14.51 Malassezia 24.32 24.12 24.17 24.50 23.01 23.13 12.63

[0093] Note: "-" indicates no bacteriostatic effect, and the inhibition zone diameter (mm).

[0094] Example 7. Irritancy test of bacteriostatic active substances

[0095] Test substance: 0.5% test solution was prepared by adding sterile water to the bacteriostatic active substance ① prepared in Example 4, mixing uniformly, and filtering sterilization with a 0.2 μm filter membrane;

[0096] In this example, the irritancy intensity of the test substance was tested according to the chicken embryo chorioallantoic membrane test in SN / T 2329 Cosmetics Eye Irritation and Corrosion, and the corneal cell test method in SN / T 3084.2 Part 2: Eye Irritation Test for Imported and Exported Cosmetics.

[0097] As shown by the test results in Tables 5-6, the bacteriostatic active substance ① prepared in the application is a mild and low-irritation raw material, which has a broad prospect in skin care product applications.

[0098] Table 4. Evaluation criteria for chicken embryo chorioallantoic membrane test-reaction time scoring method

[0099]

[0100] Table 5. Test results of test substance chicken embryo chorioallantoic membrane test

[0101] Irritation test Test eggs Number of valid eggs NC value Irritation grade 0.5% test substance in water 6 6 IS = 0.8 No irritation Positive control (0.1 mol / L NaOH) 3 3 IS = 13.59 Strongly irritating / corrosive Negative control (0.9% NaCl) 6 6 IS = 0 No irritation

[0102] Table 6. Test results of test substance corneal cell test

[0103]

[0104] Example 8 Antiseptic test

[0105] Test substance: 1 g of the bacteriostatic active substance 1 prepared in Example 4 was added to 10 mL of sterile water, mixed uniformly, and filtered to sterilize using a 0.2 μm filter membrane to obtain a test solution for the antiseptic test;

[0106] Indicator bacteria: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Pseudomonas putida ATCC 17485; and fungi Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 were used.

[0107] The test substance was added as a natural preservative to a cosmetic product, and an antiseptic test was performed according to the microbial challenge test method of the well-known Cosmetic, Toiletry, and Fragrance Association (CTFA) and the United States Pharmacopoeia, and the specific test procedure was as follows:

[0108] The test substance was added to the serum for antiseptic efficacy testing, and the amount of the test substance added was 0.2% (mass fraction), and the composition table and production process of the serum are shown in Table 7:

[0109] Table 7 Composition table and production process of serum

[0110]

[0111] The indicator bacteria in the logarithmic growth phase were diluted to 4 x 10 9 CFU / mL with PBS buffer, and an appropriate amount of each was added to the serum, so that the final concentration of each bacterium in the serum was 3.0 x 10 7 CFU / mL, and the final concentration of each fungus in the serum was 2.0 x 10 6 CFU / mL, and the bacteria group was placed in a 36°C incubator for culture, and the fungus group was placed in a 28°C incubator for culture; the blank control group was replaced with an equal amount of sterile water instead of the test substance, and each group had three serum samples for each indicator bacteria; according to the colony count test method in Chapter 5 of the Microbiological Test Method in the Cosmetic Safety Technical Specification 2015 edition, the viable cell count was determined at 0d, 7d, 14d, and 28d after inoculation to determine the antiseptic efficacy of the cosmetic product; the judgment criteria are as follows: when the viable cell count of each sample after inoculation is reduced to not more than 0.1% of the initial concentration on the 7th day, and then gradually reduced, and no bacteria grow on the 28th day; then the preservative is effective and passes the test; otherwise, the preservative is ineffective and fails the test.

[0112] The results of the preservative test are shown in Table 8 (data are presented as average values). The number of viable bacteria in the bacteria group and the number of viable fungi in the fungi group showed a downward trend at 0 days of action; at 7 days of action, the number of viable bacteria in each group was not higher than 100 CFU / mL; at 14 to 28 days of action, the number of viable bacteria in the bacteria group and the number of viable fungi in the fungi group were both 0, indicating that the tested material passed the test and had excellent preservative effect. It is shown that the bacteriostatic active substance produced by fermentation of Paenibacillus polymyxa B01 can be used as a bacteriostatic preservative raw material, and when applied to the preparation of daily chemical products, it has excellent preservative performance, has a broad application prospect and important transformation research value.

[0113] Table 8. Data of preservative efficacy test of serum

[0114]

[0115] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A strain of Polymyxin Bacillus ( Paenibacillus polymyxa B01, characterized in that, The preservation number is GDMCC NO. 66594.

2. The use of Bacillus polymyxa B01 according to claim 1 for the production of a bacteriostatic active substance, characterized in that The bacteriostatic active substance has bacteriostatic effects on Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia.

3. A method for producing a bacteriostatic active substance using the Bacillus polymyxa B01 according to claim 1, characterized by, The method comprises the following steps: S1, inoculating Paenibacillus polymyxa B01 into a liquid fermentation medium for fermentation; S2, collecting the fermentation broth after fermentation, filtering the fermentation broth through a 0.22 μm ceramic membrane filter to obtain supernatant, adding ammonium sulfate solid to the supernatant until saturation, and collecting the precipitate by centrifugation; S3, redissolving and dialyzing the precipitate prepared in step S2, purifying and concentrating the dialysate to obtain the bacteriostatic active substance.

4. The method of claim 3, wherein, The fermentation temperature in step S1 is 32-38℃, the culture rotation speed is 180-220 rpm, and the culture time is 2-3 d.

5. The method of claim 3, wherein, The specific steps in step S3 include: redissolving with ultrapure water and dialyzing with a dialysis membrane; after dialysis, collecting the supernatant, freeze-drying and concentrating it into a freeze-dried powder, preparing a sample solution with a mass concentration of 3% from the freeze-dried powder with ultrapure water, purifying it through a Sephadex G-50 column, using ultrapure water as the eluent, collecting one tube every 5 mL, vacuum concentrating the eluent that can make coomassie brilliant blue change color after elution to obtain the bacteriostatic active substance.

6. The method of claim 5, wherein, The elution flow rate during Sephadex G-50 purification is 0.8-1.2 mL / min.

7. The method of claim 5, wherein, The molecular weight of the dialysis membrane is 500 Da.

8. The method of claim 5, wherein, The dialysis time of the dialysis membrane is 2-3 d.

9. A product characterized by, The bacteriostatic active substance has bacteriostatic effects on Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia.

10. The use of the bacteriostatic active substance obtained by the process according to any one of claims 3 to 8 for the preparation of a daily hygiene product, a pharmaceutical product, characterized in that, The bacteriostatic active substance has bacteriostatic effects on Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis, Propionibacterium acnes and Malassezia.

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