Preparation method and application of bacillus oily extract

Abstract

The invention discloses a preparation method and application of a bacillus oily extract. The method comprises the following steps: S1, culturing bacillus by a fed-batch fermentation method, performing multiplication culture, and freeze-drying into powder; and S2, grinding the powder at a low temperature, breaking walls by using low-frequency ultrasonic waves and high-energy microwaves, extracting by using simmondsia chinensis seed oil and oat kernel oil as extract liquor, forming a nano-scale micro-micelle structure after extraction, and filtering the micro-micelle by using a micropore to obtain the bacillus oily extract. The bacillus oily extract prepared by the invention has the characteristics of improving antibacterial peptide expression and inhibiting biological membranes formed by malassezia and staphylococcus aureus so as to improve folliculitis.

Description

Technical Field

[0001] The present invention relates to a bacillus extract, in particular to a preparation method and application of a bacillus oily extract. Background Art

[0002] Folliculitis is a common skin disease characterized by an inflammatory reaction around the hair follicles. This disease not only affects appearance but can also lead to serious complications such as abscesses and scarring. The current main treatments for folliculitis include topical and systemic treatments. Topical treatments typically include antibiotic ointments, steroid preparations, and antifungal drugs, but topical treatments are only effective for mild and moderate folliculitis and cannot treat severe folliculitis. Systemic treatments generally use oral antibiotics, especially for folliculitis caused by bacterial infection. Although it can treat severe folliculitis, the widespread use of antibiotics has also brought about the problem of drug resistance.

[0003] Common bacteria causing folliculitis include Malassezia and Staphylococcus aureus. Biofilms are complex microbial communities formed on solid surfaces, enhancing their resilience and pathogenicity. In the scalp environment, harmful bacteria such as Malassezia and Staphylococcus aureus enhance their colonization and drug resistance through biofilm formation, leading to the persistence and intractability of folliculitis.

[0004] Prior art Chinese patent number CN115491327A discloses a Paenibacillus polymyxa strain for inhibiting Malassezia and its applications, specifically disclosing that Paenibacillus polymyxa can inhibit Malassezia folliculitis. However, the Paenibacillus polymyxa strain described in the patent is typically applied in the form of a fermentation broth or lyophilized powder, and only discloses its inhibitory effect on Malassezia folliculitis. There are no public reports on the ability of Bacillus oil extracts to inhibit Malassezia and Staphylococcus aureus biofilms. Summary of the Invention

[0005] The present invention aims to provide a method for preparing an oily extract of Bacillus and its application. The oily extract of Bacillus prepared by the present invention has the characteristics of enhancing the expression of antimicrobial peptides and inhibiting the biofilm formation of Malassezia and Staphylococcus aureus, thereby improving folliculitis.

[0006] The technical solution of the present invention is a method for preparing a Bacillus oily extract, comprising the following steps: S1. Bacillus is first cultured by a fed-batch fermentation method, then amplified and then freeze-dried into a powder; S2. The powder is cryogenically ground and broken by low-frequency ultrasound and high-energy microwaves, and then extracted with jojoba seed oil and oat kernel oil as extractants. After extraction, a nano-scale micelle structure is formed, and the micelles are filtered through micropores to obtain a Bacillus oily extract.

[0007] In the aforementioned method for preparing the Bacillus oily extract, in step S1, the Bacillus is an anaerobic Bacillus.

[0008] In the aforementioned method for preparing the Bacillus oily extract, in step S1, the culture conditions of the fed-batch fermentation method are: temperature = 45°C, pH = 7.5, and pO2>35% under aerobic conditions.

[0009] In the aforementioned method for preparing the Bacillus oily extract, in step S2, the low temperature is -20°C to -80°C, and the cell wall breaking time is 10 to 20 minutes.

[0010] In the aforementioned method for preparing the Bacillus oily extract, in step S2, during extraction, the mass fraction of jojoba seed oil is 77-81 parts, the mass fraction of oat kernel oil is 12-16 parts, and the mass fraction of Bacillus powder is 5-9 parts.

[0011] A Bacillus oily extract is prepared according to the above method.

[0012] In the aforementioned Bacillus oily extract, the Bacillus oily extract contains ≥40 ppm of Iso 15:0 fatty acid.

[0013] The present invention also discloses the use of the above-mentioned Bacillus oily extract in the preparation of a shampoo and conditioner product for enhancing the expression of antimicrobial peptides, improving scalp microecology, and inhibiting folliculitis.

[0014] The invention also discloses the use of the Bacillus oily extract in preparing a cleaning product for inhibiting biofilm formation.

[0015] In the aforementioned application, the biofilm is a biofilm formed by Malassezia and / or Staphylococcus aureus.

[0016] A preparation for improving folliculitis contains the above-mentioned Bacillus oily extract, wherein the concentration of the Bacillus oily extract in the preparation is 10-3000 ppm.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The Bacillus oily extract prepared by the present invention can upregulate the expression of hBD-2 endogenous antimicrobial peptide and hBD-2 mRNA in human immortalized keratinocytes, inhibit the biofilm formed by Malassezia and Staphylococcus aureus, thereby improving the scalp condition and microecological environment, reducing the probability of folliculitis, and inhibiting folliculitis. The effect of increasing the expression of hBD-2 is better than that of Bacillus freeze-dried powder.

[0018] Therefore, the Bacillus oily extract prepared by the present invention has the characteristics of enhancing the expression of antimicrobial peptides, inhibiting the biofilm formed by Malassezia and Staphylococcus aureus, and thus improving folliculitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the cytotoxicity of Bacillus Kamchatka oil extract on keratinocytes.

[0020] Figure 2 Schematic diagram of hBD-2 immunofluorescence staining results of keratinocytes in different groups (20×).

[0021] Figure 3 This is a bar graph showing the relative quantitative expression of hBD-2 immunofluorescence in keratinocytes in different groups.

[0022] Figure 4 This is a schematic diagram of the results of mRNA detection of keratinocytes by the oily extract of Bacillus Kamchatka.

[0023] Figure 5 Schematic diagram of the staining results of Staphylococcus aureus biofilm in different groups (10×).

[0024] Figure 6 This is a bar chart showing the inhibitory effects of different groups on Staphylococcus aureus biofilm.

[0025] Figure 7 Schematic diagram of the staining results of Malassezia biofilm in different groups (10×).

[0026] Figure 8 This is a bar chart showing the inhibitory effects of different groups on Malassezia biofilm.

[0027] Figure 9 This is a scanning electron microscopy image of Staphylococcus aureus biofilm.

[0028] Figure 10 This is a scanning electron microscopy image of Malassezia biofilm. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0030] Example 1: Bacillus sp. Kamchatka Anoxybacilluskamchatkensis ), the American Type Culture Collection's accession number is ATCC BAA-549, and it is a polar thermophilic bacterium.

[0031] The preparation method of the Bacillus oily extract comprises the following steps: S1. Bacillus kamchatka was cultured by a fed-batch fermentation method at a temperature of 45°C, a pH of 7.5, and an aerobic pO2 > 35%, and the culture was expanded on a large scale and freeze-dried into a powder. S2. Grind the powder at -50°C and break the powder wall with low-frequency ultrasound of 20-30 kHz and high-energy microwave (10 kW to 30 kW per kilogram of the mixture) for 15 minutes, then extract with jojoba seed oil and oat kernel oil as extracting liquids, wherein the mass percentage of jojoba seed oil is 80%, the mass percentage of oat kernel oil is 15%, and the mass percentage of Kamchatka Bacillus powder is 5%. After extraction, a 10-nanometer micromicelle structure is formed, and the extract is filtered through a micropore to obtain a Kamchatka Bacillus oily extract containing ≥40 ppm of Iso 15:0 fatty acids.

[0032] Comparative Example 1: The Bacillus used is Anoxybacillus kamchatkensis, which has the American Type Culture Collection accession number of ATCC BAA-549 and is a polar thermophilic bacterium.

[0033] The preparation method of Bacillus lyophilized powder comprises the following steps: culturing Kamchatka Peninsula Bacillus by a batch fed-batch fermentation method under the following culture conditions: temperature of 45°C, pH=7.5, pO2>35% under aerobic conditions, large-scale amplification culture, and lyophilizing into powder to prepare Kamchatka Peninsula Bacillus lyophilized powder.

[0034] Experimental example: 1. CCK-8 cytotoxicity test 1.1 Instruments and Equipment Biological safety cabinet, low-speed centrifuge, cell counter, constant temperature water bath, inverted microscope, carbon dioxide incubator, liquid nitrogen storage tank, and microplate reader.

[0035] 1.2 Reagents and Materials Reagents: DMEM medium, fetal bovine serum, double antibody, trypsin, PBS, Cell Counting Kit-8 reagent, trypan blue, DMSO.

[0036] DMEM complete medium: 10% fetal bovine serum, 1% double antibody; (89mL DMEM + 10mL FBS + 1mL ps).

[0037] Cell material: human immortalized keratinocytes.

[0038] 1.3 Operation process Pretreatment of test substance: Use a 0.22 μm filter to sterilize and filter the sample solution before pretreatment, and then use culture medium to prepare the sample to the required test concentration according to the table.

[0039] Cell digestion: Cells reaching the logarithmic growth phase are plated in a 96-well plate for assay. When the keratinocytes in the cell culture flask are over 50-70% confluent, remove the culture medium and wash twice with 5-10 mL of sterile PBS. Gently shake to ensure no foam is formed, and then remove the wash solution. Add trypsin / EDTA solution to the flask (1 mL for a T25 cm² flask, 2 mL for a T75 cm² flask). After 30 seconds, aspirate the solution and incubate at room temperature for 3-7 minutes, monitoring the cells periodically. When over 50% of the cells are floating, gently tap the flask to detach the cells. When most of the cells are detached, add 2 times the volume of trypsin solution in room temperature complete medium containing FBS and gently pipette the cells. Transfer the cell suspension to a centrifuge tube and centrifuge at approximately 220 x g for 5 minutes. Remove the supernatant.

[0040] Cell counting and plating: Gently pipette the complete culture medium to resuspend the cell pellet to form a uniformly distributed single-cell suspension. Use a cell counter to count the cell concentration and calculate the number of viable cells and cell survival rate. After cell counting, use a multichannel pipette to plate the cells so that the culture medium volume is 100 μL / well and the number of viable cells is 1~2×10 4 cell / well.

[0041] Sample addition: 24 hours after plating, when the cell confluence rate is 40-60%, add the test substance at various concentrations to achieve the test concentration. Set up parallel wells (n ≥ 3). Test groups are set as: blank group and sample group (Example 1). Specific groupings are shown in Table 1.

[0042] Table 1. Grouping table 1.4 Experimental Results Cytotoxicity results are shown in Figure 1 and as shown in Table 2.

[0043] Table 2. Cytotoxicity of Bacillus Kamchatka oily extract on keratinocytes 2. Immunofluorescence staining to evaluate the effects of antimicrobial peptides on keratinocytes 2.1 Principle Defensin 2 (hBD-2) is a key regulatory molecule in the immune system, possessing direct bactericidal properties. It is a major antimicrobial peptide widely distributed in animals. Upregulating hBD-2 gene expression in immortalized human keratinocytes promotes defensin 2 synthesis, thereby exerting an immunomodulatory effect.

[0044] 2.2 Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, fluorescence microscope.

[0045] 2.3 Reagents and materials Cell material: human immortalized keratinocytes Reagents: hBD-2 rabbit antibody, DAPI staining solution, paraformaldehyde fixative, PBS, immunostaining blocking solution, immunostaining permeabilization solution.

[0046] 2.4 Operation process Cell plating: After counting the cells, dilute to the required concentration and add the cell suspension to the cell culture dish so that the culture medium volume is 1 mL / well and the number of viable cells is 5×10 4 cell / well.

[0047] Sample splitting: 24 hours after plating, when the cell fusion rate was 40-60%, different concentrations of the test substance were added to achieve the test concentration, and three parallel test wells were set up. The test groups were set as follows: blank group and sample group (Comparative Example 1 and Example 1). The specific grouping is shown in Table 3.

[0048] Table 3. Grouping Liquid Addition: 24 hours after sample addition, aspirate the culture medium and wash three times with PBS. Add fixative and fix at 37°C for 30 minutes. Aspirate the fixative and wash three times with wash solution, each for 3-5 minutes. Add immunostaining permeabilization solution and permeabilize at room temperature for 10 minutes. Aspirate the permeabilization solution and wash three times with wash solution, each for 3-5 minutes. Add immunostaining blocking solution and block at room temperature for 60 minutes. The amount of immunostaining blocking solution should be sufficient to cover the sample. For samples in a 24-well plate, 0.5 mL of immunostaining blocking solution is typically added. Aspirate the immunostaining blocking solution and add hBD-2 rabbit monoclonal antibody. Incubate at 4°C in the dark overnight. The amount of hBD-2 rabbit monoclonal antibody should be sufficient to cover the sample. Carefully aspirate the hBD-2 rabbit monoclonal antibody into an appropriate container. Wash three times with wash solution, each for 5-10 minutes. Add anti-rabbit secondary antibody and incubate at 37°C for 1 hour. The amount of anti-rabbit secondary antibody should be sufficient to cover the sample. Carefully aspirate the anti-rabbit secondary antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each. Add DAPI, a nuclear stain, and stain for 10 minutes at room temperature. Aspirate the nuclear stain and wash three times with washing buffer for 3-5 minutes each. Observe under a fluorescence microscope. hBD-2 staining will produce green fluorescence, while nuclei will produce blue fluorescence.

[0049] 2.5 Judgment Basis If the expression level of hBD-2 in human immortalized keratinocytes under the action of the sample group is significantly higher than that of the blank group, the sample is judged to be able to effectively upregulate the expression of hBD-2 in human immortalized keratinocytes. If the expression level of hBD-2 in human keratinocytes under the action of Example 1 is significantly higher than that of Comparative Example 1, Example 1 is judged to be superior to Comparative Example 1 in upregulating the expression of hBD-2 in human immortalized keratinocytes.

[0050] 2.6 Test results The test results are shown in Figure 2 and Figure 3 As shown, it can be seen that the Kamchatka Bacillus oily extract of Example 1 of the present invention can improve the expression and synthesis of hBD-2 in human immortalized keratinocytes at a concentration of 80 ppm to 1000 ppm, and the improvement effect is better than that of the Kamchatka Bacillus lyophilized powder of Comparative Example 1 at the same concentration.

[0051] 3. qPCR method to evaluate the effect of antimicrobial peptides on keratinocytes 3.1 Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, microplate reader, water bath, ultra-micro spectrophotometer, real-time fluorescence PCR instrument.

[0052] 3.2 Reagents and materials Reagents: BASIC H-DMEM, 4.5 g / liter glucose; BASIC L-DMEM, 1.5 g / liter glucose; FBS; 0.25% Trypsin-EDTA; FastPure Cell / Tissue Total RNA Isolation Kit; One Step RT-qPCR Kit; chloroform; anhydrous ethanol; Cell Counting Kit-8 reagents; primers.

[0053] Cell material: human immortalized keratinocytes.

[0054] 3.3 Operation process Pretreatment of test substance: Use a 0.22 μm filter to sterilize and filter the sample solution before pretreatment, and then use culture medium to prepare the sample to the required test concentration according to the table.

[0055] Digestion of cells: cells that have reached the logarithmic growth phase were seeded in 96-well plates for detection. When the keratinocytes in the cell culture flask were more than 50-70% confluent, the culture medium was removed and the cells were washed twice with 5-10 mL of sterile PBS. The cells were gently shaken to ensure that no obvious foam was generated and the washing solution was removed. Trypsin / EDTA solution was added to the culture flask (T25 cm 2 Use 1mL culture flask, T75cm 2 Use a 2 mL culture flask), aspirate after 30 seconds, and incubate at room temperature for 3-7 minutes. Observe the cells periodically. When more than 50% of the cells are floating, gently tap the flask to detach them. When the majority of cells are detached, add 2 times the volume of trypsin solution in room temperature complete medium containing FBS and gently pipette the cells. Transfer the cell suspension to a centrifuge tube; centrifuge at approximately 220 x g for 5 minutes, and remove the supernatant.

[0056] Cell counting and plating: Gently pipette the complete culture medium to resuspend the cell pellet to form a uniformly distributed single cell suspension. Use a cell counter to count the cell concentration and calculate the number of viable cells and cell survival rate. After cell counting, use a multichannel pipette to plate the cells so that the culture medium volume is 100 μL / well and the number of viable cells is 1-2×10 4 cell / well.

[0057] Cell plating: After counting the cells, dilute to the required concentration and add the cell suspension to the cell culture dish so that the culture medium volume is 4 mL / well and the number of viable cells is 5 × 10 5 cell / well.

[0058] Sample addition: 24 hours after plating, when the cell confluence rate is 40-60%, add the test substance at various concentrations to achieve the test concentration. Set up parallel wells (n ≥ 3). Test groups are set as: blank group and sample group (Example 1). Specific groupings are shown in Table 4.

[0059] Table 4. Grouping Extract mRNA: 48 hours after sample addition, remove the waste liquid, collect the cells, and extract mRNA using a cell / tissue mRNA extraction kit.

[0060] RNA quality testing: RNA purity testing: Use an ultra-micro spectrophotometer to test RNA purity, and the detection standard OD (260 / 280) = 1.8-2.1.

[0061] Primer design: Use professional primer synthesis software to synthesize specific primers for the gene to be detected (hBD-2).

[0062] RT-PCR detection of target gene expression: Add 10 µL of 2× One-Step SYBR MasterMix, 0.65 µL of RT Enzyme Mix, specific primers, and template RNA in the appropriate proportions. Make up to volume with water, mix thoroughly, and use for testing. The real-time fluorescence quantitative RNA instrument setup conditions are shown in Table 5.

[0063] Table 5. Real-time fluorescence quantitative RNA instrument setup reaction program After the reaction is completed, the amplification curve and melting curve of Real Time PCR are confirmed, and a standard curve is prepared.

[0064] 3.4 Data Statistics The average Ct value of three replicates for each cDNA sample and each gene was used as the amplification result, and the amplification amount of the GAPDH gene was used as the internal reference gene to calculate the relative gene expression.

[0065] Relative gene expression: 2 －△△CT ; Where: ① CT treated - CT internal reference (treated) = ΔCT treated; ② CT control - CT internal reference (control) = ΔCT control; ③ ΔCT treatment - ΔCT control = ΔΔCT; GraphPad Prism 8.0 statistical analysis software was used to perform one-way analysis of variance between groups. p <0.05 was considered statistically significant.

[0066] 3.5 Judgment Basis If the expression level of hBD-2 mRNA in the human immortalized keratinocytes in the sample group is significantly higher than that in the blank group, it is determined that the sample can effectively upregulate the expression of hBD-2 mRNA in the human immortalized keratinocytes.

[0067] 3.6 Test results The results of hBD-2 mRNA expression are shown in Figure 4 and as shown in Table 6.

[0068] Table 6. hBD-2 mRNA detection results Compared with the blank group:* p <0.05,** p <0.01.

[0069] According to the above test results, compared with the blank group, the mRNA expression level of Example 1 at concentrations of 100 ppm (0.01%) and 3000 ppm (0.3%) was significantly increased (p < 0.05), indicating that the oily extract of Bacillus Kamchatka has the effect of increasing the mRNA expression level of hBD-2.

[0070] 4. Biofilm inhibition test of Malassezia and Staphylococcus aureus by fluorescence staining 4.1 Principle Malassezia and Staphylococcus aureus are common skin bacteria that often form biofilms on the skin. By observing the effects of samples on biofilms formed by Malassezia / Staphylococcus aureus, we can determine their effects on these biofilms. This dual fluorescence staining method, using the green fluorescent DNA dye DMAO and the red fluorescent dye propidium iodide (PI), fluoresces green when live bacteria are present, while dead bacteria exhibit both green and red fluorescence. Fluorescence detection systems, such as fluorescence microscopes, can be used for detection.

[0071] 4.2 Instruments and Equipment Microbalance, clean bench, biochemical incubator, fluorescence microscope.

[0072] 4.3 Reagents and materials Malassezia, Staphylococcus aureus.

[0073] 4.4 Operation process Test substance pretreatment and test grouping: Use a 0.22 μm filter to sterilize and filter the sample solution before pretreatment, and then use PBS to prepare the sample to the required test concentration according to Table 7.

[0074] Table 7. Grouping Preparation of Malassezia / Staphylococcus aureus biofilm: Sterile 1 cm × 1 cm coverslips were placed in 6-well plates, and 1 mL of Malassezia / Staphylococcus aureus (≈105 cfu, resuspended in sterile PBS or saline) was added to each well. The cells were cultured overnight to form a mature Malassezia / Staphylococcus aureus biofilm.

[0075] Sample treatment: Wash with PBS three times to remove planktonic bacteria. Add 2 mL of sample to each well of the experimental group. Set the groups as shown in Table 6. Continue to incubate for 30 minutes and aspirate the waste liquid.

[0076] Preparation: Coverslips were fixed in 6-well plates with 4% paraformaldehyde for 2 h.

[0077] Observe the effects of samples on biofilms using a fluorescence microscope: Wash three times with sterile PBS. Take the staining reagents DMAO (1000×) and PI (1000×) from the bacterial live / death staining kit and dilute them to 100× using assay buffer. For example, add 10µl of DMAO (1000×) and 10µl of PI (1000×) to 80µl of assay buffer and mix thoroughly to make 100µl of bacterial live / death staining working solution (100×). Add 500µl of 1× staining solution to each well and mix thoroughly. Incubate at 37°C in the dark for 45 minutes. After incubation, observe the staining under a fluorescence microscope. DMAO fluoresces green with an E / E ratio of 503 / 530nm; PI fluoresces red with an E / E ratio of 535 / 617nm. Observe bacterial colonies under a fluorescence microscope, showing both green and red fluorescence.

[0078] 4.5 Judgment Basis If the fluorescence area of ​​the biofilm in the sample group is significantly lower than that in the blank group, it means that the sample has an inhibitory effect on the biofilm formation of Staphylococcus aureus / Malassezia.

[0079] 4.6 Test results The test results are shown in Figure 5-Figure 8 , as shown in Tables 8 and 9.

[0080] Table 8. Staphylococcus aureus biofilm inhibition results P Value vs BC: *, <0.05; **, <0.01.

[0081] Table 9 Malassezia biofilm inhibition results PValue vs BC: *, <0.05; **, <0.01.

[0082] According to the above test results, when the test concentrations were 10 ppm and 1000 ppm, the inhibition rates of the Kamchatka Bacillus oily extract against Staphylococcus aureus biofilm were 32.95% and 46.68% (biofilm area), and the inhibition rates against Malassezia biofilm were 31.64% and 74.82% (biofilm area), respectively.

[0083] This shows that Example 1 can significantly inhibit the biofilm formed by Staphylococcus aureus and Malassezia at the test concentrations of 10 ppm and 1000 ppm.

[0084] 5. Electron microscopy observation of biofilm inhibition test against Malassezia and Staphylococcus aureus 5.1 Instruments and Equipment Microbalance, clean bench, biochemical incubator, scanning electron microscope.

[0085] 5.2 Reagents and materials Malassezia, Staphylococcus aureus.

[0086] 5.3 Operation process Pretreatment of test substances and test grouping: Use a 0.22 μm filter to sterilize and filter the sample solution before pretreatment, and then use culture medium to prepare the sample to the required test concentration according to Table 10.

[0087] Table 10. Grouping situation Preparation of Malassezia / Staphylococcus aureus biofilm: Sterile 1 cm × 1 cm coverslips were placed in 6-well plates, and Malassezia / Staphylococcus aureus (≈10 5 cfu, resuspended in sterile PBS or saline) 1 mL, Staphylococcus aureus was cultured overnight, and Malassezia was cultured for three days until a mature Malassezia / Staphylococcus aureus biofilm was formed.

[0088] Sample treatment: Wash three times with PBS to remove planktonic bacteria. Add 2 mL of sample to each well of the experimental group. Set the groups as shown in Table 10. Continue to incubate for 30 minutes and aspirate the waste liquid.

[0089] Preparation: Coverslips were fixed in 6-well plates with 3% glutaraldehyde for 2 h.

[0090] Scanning electron microscopy was used to observe the effects of the samples on the biofilm: the samples were washed with PBS three times for 4 hours, each time for 15 minutes; the PBS was removed, 1% osmium tetroxide was added and fixed on a rotator for 1.5 hours, and then rinsed with distilled water four times for 10 minutes each time; after dehydration with gradient ethanol, the samples were first placed in a critical point dryer to dry, and then the samples were placed in a vacuum coating machine for gold spraying and then observed under a scanning electron microscope.

[0091] 5.4 Judgment Basis The biofilms formed by Malassezia and Staphylococcus aureus after the samples were treated were observed by scanning electron microscopy to determine the effects of the samples on the biofilms of Malassezia and Staphylococcus aureus.

[0092] 5.5 Test results The test results are shown in Figure 9 and Figure 10 ,according to Figure 9 and Figure 10 It can be seen that the number of colonies on the biofilm formed by Malassezia and Staphylococcus aureus after being treated with the oily extract of Bacillus Kamchatka in Example 1 was significantly reduced compared with the BC group, indicating that the oily extract of Bacillus Kamchatka has an inhibitory effect on the biofilm of Malassezia and Staphylococcus aureus.

[0093] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Those skilled in the art may modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein; and all these modifications and replacements should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a Bacillus oily extract, characterized in that: The following steps are involved: S1. Bacillus is first cultured by a fed-batch fermentation method, then amplified and freeze-dried into a powder; S2. The powder is cryogenically ground and broken by low-frequency ultrasound and high-energy microwaves, and then extracted with jojoba seed oil and oat kernel oil as extractants. After extraction, a nano-scale micelle structure is formed, and the micelles are filtered through micropores to obtain a Bacillus oily extract.

2. The method for preparing the Bacillus oily extract according to claim 1, wherein: In step S1, the culture conditions of the fed-batch fermentation method are: temperature = 45°C, pH = 7.5, and pO2>35% under aerobic conditions.

3. The method for preparing the Bacillus oily extract according to claim 1, wherein: In step S2, the low temperature is -20°C to -80°C, and the cell wall breaking time is 10 to 20 minutes.

4. The method for preparing the Bacillus oily extract according to claim 1, wherein: In step S2, during extraction, the mass fraction of jojoba seed oil is 77-81 parts, the mass fraction of oat kernel oil is 12-16 parts, and the mass fraction of Bacillus powder is 5-9 parts.

5. A Bacillus oily extract prepared according to the method according to any one of claims 1 to 4.

6. The Bacillus oily extract according to claim 5, characterized in that: The oily extract of Bacillus contains ≥40 ppm of Iso 15:0 fatty acids.

7. Use of the Bacillus oily extract according to any one of claims 5-6 in the preparation of a shampoo and conditioner for enhancing the expression of antimicrobial peptides, improving scalp microecology, and inhibiting folliculitis.

8. Use of the Bacillus oily extract according to any one of claims 5 to 6 in the preparation of a cleaning product that inhibits biofilm formation.

9. The use according to claim 8, characterized in that: The biofilm is a biofilm formed by Malassezia and / or Staphylococcus aureus.

10. A preparation for improving folliculitis, comprising the Bacillus oily extract according to any one of claims 5 to 6, characterized in that: The concentration of the Bacillus oil extract in the preparation is 10~3000ppm.

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