A strain of Bacillus velezine with the efficacy of degrading allergens and inhibiting Gram-negative bacteria and fungi and its application

Abstract

The invention discloses a strain of Bacillus Velezii with the efficacy of degrading allergens and inhibiting Gram-negative bacteria and fungi and application thereof. The deposit number of the Bacillus Velezii is CCTCC NO: M 20232409. The strain has significant inhibitory effects on common Gram-negative pathogenic bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and common pathogenic fungi Aspergillus niger, Candida albicans and Aspergillus flavus. Meanwhile, the strain has strong degradation effects on common indoor protein allergens such as cat allergen Fel d 1, dog allergen Can f 1, mite allergen Der f 1, pollen allergen Bet v 1 and the like. Therefore, the Bacillus Velezii can be used to prepare microbial cleaners for home environments and public environments.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Bacillus Velezii and application of the strain in degrading common indoor allergens and inhibiting indoor Gram-negative pathogens and fungi. Background Art

[0002] In recent years, with the deepening of scientific research, people have gradually realized that the indoor environment is closely related to human health, and indoor purification has gradually become an important topic of concern. There may be a variety of harmful substances in the indoor environment, such as gaseous pollutants such as formaldehyde, benzene, ammonia, etc., microbial pollutants such as Escherichia coli, Aspergillus niger, H1N1 virus, etc., inhalable particulate matter pet allergens, mite allergens, pollen allergens, etc. These will affect people's health and even cause various respiratory diseases and allergic reactions.

[0003] The number of pet owners around the world continues to rise, and pets have become an indispensable part of family life. Keeping pets in cities will also cause many pollution problems to the indoor environment, such as excessive allergens and harmful microorganisms. Public data shows that about 10% to 20% of the world's population are allergic to cats and dogs. The salivary glands, sebaceous glands, and anal glands of pets such as cats and dogs produce a large amount of allergenic proteins. The activities of pets will carry these allergenic proteins to every corner of the indoor environment. When the allergenic proteins in the environment accumulate above the threshold, people will have allergic reactions, such as itchy and red skin, coughing and difficulty breathing, and even variant asthma that endangers life. The thick fur of pets is also prone to breeding mites and harmful microorganisms, which can easily affect human health during contact with their owners.

[0004] Common pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, etc. are all Gram-negative bacteria. However, there is currently a lack of safe and effective drugs for the treatment of Gram-negative bacterial infections in clinical practice, which is related to the special bacterial structure of Gram-negative bacteria. The cell membrane of Gram-negative bacteria consists of an inner membrane, periplasm, and outer membrane. The inner membrane is composed of a symmetrical phospholipid bilayer, the periplasm contains a thin layer of peptidoglycan, and the outer membrane is a highly asymmetric lipid bilayer, which has unique permeability barrier properties, allowing them to effectively block the entry of antibiotics and other drugs while allowing nutrients to enter. This is also one of the reasons why the development of Gram-negative antibiotics is more difficult. Gram-negative pathogens are widely present in nature, including soil, water, animals and the human body. These Gram-negative pathogens pose a certain threat to public health and personal health, so it is of great significance to prevent, inhibit and disinfect these pathogens.

[0005] The main ingredients of common allergen removal or antibacterial products on the market are mostly chemically synthesized protein denaturants and antibacterial agents. These chemical ingredients can solve environmental pollution problems in a short period of time, but they are easy to volatilize and become ineffective, and require multiple and large-scale use to maintain good results. In contrast, microbial cleaning products use the natural characteristics of microorganisms to degrade and utilize allergens in the environment, and inhibit the growth of harmful bacteria in the environment through the antibacterial metabolites produced. They are naturally safe; the active microbial ingredients they contain can grow and reproduce in the environment, and can continuously degrade allergens and inhibit bacteria in the environment, achieving a long-term purification effect. In microbial cleaning products, commonly used microorganisms include Bacillus, yeast, and Lactobacillus. Among them, Bacillus can form heat-resistant and stress-resistant spores to help maintain its activity, and is often used as one of the components of microbial cleaning products. Bacillus Velez has the advantages of a broad antibacterial spectrum, rapid growth, easy isolation and cultivation, strong stress resistance and high biosafety. It can secrete and produce a variety of biologically active substances, including proteases, lipopeptide antibiotics and polyketide antibiotics. Due to its antibacterial ability and protease production ability, Bacillus Velez has been widely used in agriculture, environmental protection, and animal husbandry production. However, there have been no reports in China of using it as an indoor microbial cleaner for allergen control. Summary of the invention

[0006] In order to solve the problems of allergies, harmful bacteria and mold pollution caused by keeping pets indoors, the present invention provides a strain of Bacillus Velezii that has a strong inhibitory effect on common Gram-negative pathogens and fungal pathogens. The strain can also effectively degrade common indoor allergens and can be used as a microbial cleaner for home environments and public environments.

[0007] The Bacillus velezensis YHX-DH37 described in the present invention is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20232409, a deposit address of Wuhan University, Wuhan, China, and a deposit date of December 1, 2023.

[0008] Application of Bacillus velez YHX-DH37 in degrading allergens: the allergens include cat allergen Fel d 1, dog allergen Can f 1, mite allergen Der f 1, and pollen allergen Bet v 1.

[0009] Application of Bacillus Velez YHX-DH37 in the preparation of products for inhibiting Gram-negative bacteria: the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

[0010] Application of Bacillus Velez YHX-DH37 in the preparation of fungus inhibition products: the fungi include Aspergillus niger, Aspergillus flavus, and Candida albicans. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The hydrolysis effect of Bacillus on milk protein.

[0012] Figure 2 This is the colony picture of Bacillus velez YHX-DH37.

[0013] Figure 3 This is a microscopic examination image of Bacillus Velez YHX-DH37. DETAILED DESCRIPTION

[0014] Example 1 Isolation and purification of Bacillus

[0015] 1. Collect soil, water or other samples that may contain Bacillus from the original forest, and ensure that the samples are taken from different areas with a wide range of sources, different environmental conditions and good soil fertility.

[0016] 2. Soil collection: Use a sampling shovel to remove the loose soil about 5 cm above the surface, take 10-50 g of soil sample from 5-25 cm, put it into a sampling bag and seal it; Water sample collection: Before sampling, the sampling bottles, water sample storage bottles, filters and other instruments must be sterilized and disinfected, and water layers with a depth of 20-30 cm should be selected for sampling in flowing rivers and lakes.

[0017] 3. Pre-treat the sample appropriately and remove impurities through solid-liquid separation, filtration, etc.

[0018] 4. Prepare nutrient agar solid culture medium: 0.5% yeast extract powder, 1% peptone, 1% sodium chloride, and 2% agar powder.

[0019] 5. Weigh 10g of the collected sample and pour it into a sterilized triangular flask containing glass beads and deionized water, and shake the flask for 5 minutes. Dilute the sample according to the conventional microbial dilution method, heat treat the diluted sample at 80℃ for 10 minutes, spread it on nutrient agar solid culture medium, and invert and culture it at 37℃ overnight.

[0020] 6. Observe the morphology of the grown colonies, pick the bacterial moss to observe the morphology of the spores under a microscope, and take photos for record. Select single colonies with different morphologies to streak and transfer to new plates to purify the strains, and preserve the strains.

[0021] Bacillus is a common protease-producing bacterium. After the strain that can produce extracellular protease grows on the verification plate, an obvious proteolysis circle can be formed around its colony. The ratio of the hydrolysis circle to the colony diameter is often used as a preliminary screening basis for judging the protease production ability of the strain. Inoculate Bacillus on the protein plate for cultivation and observe the hydrolysis circle on the protein plate. By calculating the ratio of the hydrolysis circle diameter (H) and the colony diameter (C), colonies with strong protease production ability can be screened. The experimental operation is as follows:

[0022] 1. Prepare LB medium (1L): peptone 10g, beef extract 5g, sodium chloride 5g. Protease screening plate (1L): peptone 10g, beef extract 5g, sodium chloride 5g, skim milk 15g, agar 20g.

[0023] 2. Prepare Bacillus carrier: 5mm diameter qualitative filter paper, sterilize it with pressure steam, dry it at 120℃, and store it for later use.

[0024] 3. Inoculate the isolated Bacillus strain into LB medium and culture overnight.

[0025] 4. Take a sterile and dry filter paper, add 20 μL of Bacillus suspension to each piece, then place the filter paper flat in a clean sterile dish, open the lid and place it in an incubator (37°C) to dry, or place it at room temperature to dry naturally for later use.

[0026] 5. Preparation of negative control samples: Take sterile dry filter paper pieces, add 20 μL of sterile distilled water to each piece, dry and set aside.

[0027] 6. Placement of Bacillus samples: Place one infected plate for each test, and place four test samples and one negative control sample on each protease screening plate, for a total of five samples. Use sterile tweezers to take the sample and place it on the surface of the plate. The distance between the centers of each sample should be more than 25 mm, and the distance from the edge of the plate should be more than 15 mm. After placing, use sterile tweezers to gently press the sample to make it close to the surface of the plate. Cover the plate, place it in a 37°C incubator, and culture it for 16h~18h.

[0028] 7. Observation results: Use a vernier caliper to measure the diameter of the hydrolysis circle (including the patch) and record it. Repeat the test 3 times. When measuring the hydrolysis circle, the diameter should be measured with the outer edge of the hydrolysis circle as the boundary.

[0029] 8. Judgment of enzyme production ability: If the diameter of the hydrolysis zone (H) minus the diameter of the colony (C) is greater than 2mm, it is judged that protease is produced. If the diameter of the hydrolysis zone minus the diameter of the colony is less than 2mm, it is judged that there is no protease. If there are hydrolysis zones in 3 repeated tests, it is judged that there is enzyme production ability. There should be no hydrolysis ring in the negative control group, otherwise the test is invalid.

[0030] Experiments on the screened Bacillus samples showed that Bacillus YHX-DH37 can effectively produce protease and may have the potential to widely degrade allergic proteins.

[0031] Example 2 Identification of Bacillus YHX-DH37 strain

[0032] Morphological identification of bacteria: observe the spore morphology, cell shape and other morphological characteristics, and compare them with the known reference standard of Bacillus Velezii. The results showed that on the nutrient agar plate medium, the colonies were light yellow and opaque, with a rough surface, wrinkles, and irregular edges ( Figure 2 ), through observation and testing under a microscope, it was found that the bacteria were thin rod-shaped, Gram-positive bacteria, the spores were oval, with blunt ends, the spore capsules were not swollen, and the meso- to sub-terminal ( Figure 3 ). The morphological characteristics of the strain were highly similar to those of Bacillus Velezii, further verifying that it might be Bacillus Velezii.

[0033] Molecular biological identification of the strain: In order to further confirm the identification results, molecular biological identification methods were used. The total DNA of the strain was extracted as a template, and the 16s rRNA and rpo B genes were amplified by PCR and sequenced. The 16srRNA gene sequence of Bacillus YHX-DH37 is shown in SEQ ID NO.1, and the rpo B gene sequence is shown in SEQ ID NO.2. The sequencing results were compared, and the strain YHX-DH37 was determined to be Bacillus velezensis. It was submitted to the China Center for Type Culture Collection for preservation on December 1, 2023, with a preservation number of CCTCC NO: M 20232409, and the classification name was Bacillus velezensis YHX-DH37.

[0034] Example 3 Degradation of common indoor allergens by Bacillus velez YHX-DH37

[0035] Cat allergen Fel d 1, dog allergen Can f 1, mite allergen Der f 1, pollen allergen Betv1 ELISA test kits and natural purified allergen proteins were purchased from Indoor Biotech.

[0036] 1. Activate the Bacillus velezensis YHX-DH37 stored in the glycerol tube by streaking, select a single colony and inoculate it into 100 mL YPD liquid medium and culture it with shaking overnight for about 18 to 24 hours.

[0037] 2. For the test group, take 90 μL of YHX-DH37 bacterial solution cultured overnight into a sterile test tube, dilute the allergen stock solution appropriately, take 10 μL into a sterile test tube and mix with the bacterial solution, and leave it at room temperature for 5 days.

[0038] 3. For the control group, take 90 μL of sterile water and add 10 μL of diluted allergen solution, and let it act for the corresponding time.

[0039] 4. For the negative control group, the culture medium is inoculated with the same batch of culture medium or sterile water as used in the experiment to observe whether there is bacterial growth.

[0040] 5. After 5 days of exposure, the allergen concentration in the solution was tested using the double antibody sandwich ELISA method. The test was repeated 3 times.

[0041] 6. Result calculation: allergen removal rate Where T oi represents the concentration of allergen solution in the control group after 5 days of action (ng / mL), T i It indicates the concentration of allergen solution in the test group after 5 days of action (ng / mL).

[0042] Table 1 Allergen degradation effect

[0043]

[0044] As can be seen from Table 1, Bacillus Velezii YHX-DH37 has a significant degradation effect on common allergens in indoor environments.

[0045] Example 4 Inhibitory Effect of Bacillus Velezii YHX-DH37 on Gram-negative Pathogenic Bacteria

[0046] Prepare Gram-negative pathogenic bacteria experimental strains: Escherichia coli MG1655, Klebsiella pneumoniae ATCC4352, Pseudomonas aeruginosa ATCC27853, and Stenotrophomonas maltophilia ATCC17666.

[0047] 1. Prepare nutrient agar medium: weigh 3 g beef extract, 10 g peptone, 5 g sodium chloride, and 15 g agar powder respectively and place them in a beaker, add 1000 mL purified water to dissolve them, boil, cool to 60°C, adjust the pH value to 7.2-7.4, dispense into 250 mL Erlenmeyer flasks, autoclave at 121°C for 30 min, and refrigerate for later use.

[0048] 2. Prepare nutrient broth medium: weigh 3 g beef extract, 10 g peptone, and 5 g sodium chloride respectively, place them in a beaker, add 1000 mL purified water to dissolve them, boil, cool to 60°C, adjust the pH value to 7.2-7.4, and dispense into 500 mL conical flasks, 89 mL per bottle, plug with a breathable stopper, autoclave at 121°C for 30 min, and refrigerate for later use.

[0049] 3. Crystal violet neutral red bile agar (VRBA): Weigh 7g of peptone, 3g of yeast extract, 10g of lactose, 5g of sodium chloride, 1.5g of bile salt, 0.03g of neutral red, 0.002g of crystal violet, and 15g of agar powder respectively and place them in a 1000mL beaker, add 1000mL of purified water to dissolve them, adjust the pH value to 7.3-7.5 with 1mol / L sodium hydroxide or 1mol / L hydrochloric acid, boil for 2min, melt the culture medium and keep the temperature at 45℃-50℃ to pour on the plate. Prepare temporarily before use, no more than 3 hours, and use it for plate colony counting in Gram-negative pathogenic bacteria inhibition experiment.

[0050] 4. Operation steps of antibacterial test: Under sterile operation, use an inoculation loop to pick up a loop of freeze-dried bacterial powder of Gram-negative pathogenic bacteria experimental strain, add it to the nutrient broth culture medium and mix it evenly, and place it at 37°C and shake it at 220rpm for 24 hours; use an inoculation loop to streak the cultured bacterial suspension on the nutrient agar culture plate, and place it in a 37°C biochemical incubator for 24 hours; use an inoculation loop to pick up a single colony, add it to the nutrient broth culture medium and mix it evenly, and place it at 37°C and shake it at 220rpm for 24 hours. Use the plate count method to determine the number of bacteria in the bacterial suspension: after appropriate dilution with physiological saline, select 2-3 dilutions and spread them on the nutrient agar culture plate, place it in a 37°C biochemical incubator for 24 hours, and count the colonies. The bacterial suspension should be stored in a 4°C refrigerator for use until the bacterial count results come out. The concentration of the bacterial suspension should be 10 6 -10 7 CFU / mL. If the test result is higher than this range, it is diluted with physiological saline; if the test result is lower than this range, it is concentrated by centrifugation. In the experimental group, 1 mL of Gram-negative pathogenic bacteria suspension was inoculated into 89 mL of nutrient broth medium and mixed evenly, and then 10 mL of Bacillus Velezii YHX-DH37 bacterial solution (bacterial solution concentration was 10 6 -10 7 CFU / mL); 1 mL of Gram-negative pathogenic bacteria experimental strain suspension was inoculated into 89 mL of nutrient broth medium, and 10 mL of sterile water was added as a blank control group, and mixed evenly. The two groups of mixed cultures were diluted and coated on crystal violet neutral red bile salt agar plates, placed at 37°C for static culture, and the number of Gram-negative pathogenic bacteria experimental bacteria in the two groups of mixed cultures after co-culture was determined after 24 hours.

[0051] Calculation formula: Wherein: X represents the inhibition rate (%), A represents the average colony count of the control group after co-culture, and B represents the average colony count of the experimental group after co-culture.

[0052] Table 2 Antibacterial effect of Bacillus velez YHX-DH37 on Gram-negative pathogens

[0053]

[0054] As can be seen from Table 2, Bacillus Velezii YHX-DH37 has a significant inhibitory effect on Gram-negative pathogenic bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

[0055] Example 5 Inhibitory Effect of Bacillus Velez YHX-DH37 on Pathogenic Fungi

[0056] Pathogenic fungal experimental strains: Aspergillus niger ATCC16404, Aspergillus flavus ATCC16883, Candida albicans ATCC10231.

[0057] 1. Prepare Sabouraud glucose liquid medium: weigh 10 g of peptone and 40 g of glucose respectively and put them into a beaker, add 1000 mL of purified water to dissolve them, boil, cool to 60°C, adjust the pH value to 5.4-5.6, and dispense into 500 mL conical flasks, 89 mL per bottle, plug with a breathable stopper, autoclave at 115°C for 30 min, and refrigerate for later use.

[0058] 2. Prepare potato glucose culture medium: Weigh 300g potato, 20g glucose, and 20g agar powder respectively, peel and cut the potato into pieces, add 1000mL distilled water, and boil for 10-20min. Filter with gauze, add distilled water to 1000mL. Add glucose and agar powder to dissolve, boil, cool to 60℃, dispense into 250mL Erlenmeyer flasks, and sterilize at 115℃ for 30min.

[0059] 3. Antibacterial test operation steps: Under sterile operation, use an inoculation loop to pick up a loop of freeze-dried bacterial powder of the experimental pathogenic fungus strain, add it to Sabouraud glucose liquid culture medium and mix evenly, and place it at 28°C for 24 hours; use an inoculation loop to streak the cultured bacterial suspension on a potato glucose medium plate, place it in a 28°C biochemical incubator for 24 hours; use an inoculation loop to pick up a single colony, add it to Sabouraud glucose liquid culture medium and mix evenly, and place it at 28°C for 24 hours.

[0060] 4. Determine the number of bacteria in the bacterial suspension using the plate count method. After appropriate dilution with physiological saline, select 2-3 dilutions. Pipette 1 mL of the pathogenic fungal experimental strain suspension and place it in a sterile bacterial culture dish. Inoculate three sterile bacterial culture dishes with each sample. Pour the potato glucose culture medium cooled to 40-45°C and mix evenly in an 8-shaped pattern. After the culture medium solidifies, turn the bacterial culture dish upside down and culture it in a 28°C incubator. Count the colonies after 48 hours. Store the bacterial suspension in a 4°C refrigerator for later use and use it after the bacterial count results come out. The concentration of the bacterial suspension should be 10 6 -10 7 CFU / mL. If the result is higher than this range, dilute it with normal saline; if the result is lower than this range, concentrate it by centrifugation.

[0061] 5. In the experimental group, 1 mL of pathogenic fungal suspension was inoculated into 89 mL of Sabouraud glucose liquid medium, mixed evenly, and then 10 mL of Bacillus Velezii YHX-DH37 bacterial solution (bacterial solution concentration was 10 6 -10 7 CFU / mL); another 1 mL of pathogenic fungus suspension was inoculated into 89 mL of Sabouraud glucose liquid medium, and 10 mL of sterile water was added as a blank control group, and mixed evenly.

[0062] 6. Place the two groups of mixed cultures at 30°C for static culture. After 48 hours, determine the number of pathogenic fungal experimental strains in the two groups of mixed cultures after co-culture.

[0063] 7. Calculation formula: Wherein X represents the inhibition rate (%), A represents the average colony count of the control group after co-culture, and B represents the average colony count of the experimental group after co-culture.

[0064] Table 3 Antibacterial effect of Bacillus velez YHX-DH37 on fungal pathogens

[0065]

[0066] As can be seen from Table 3, Bacillus Velezii YHX-DH37 has a significant inhibitory effect on common pathogenic fungi Aspergillus niger, Candida albicans and Aspergillus flavus.

[0067] Implementation 6 Preparation method of Bacillus velez YHX-DH37 bacterial liquid preparation

[0068] A preparation method of a liquid preparation containing Bacillus Velez YHX-DH37:

[0069] 1. Activation of strains: Use nutrient agar medium to streak and activate the preserved Bacillus Velez subtilis YHX-DH37 at 37°C for 24 hours.

[0070] 2. Preparation of seed solution: Inoculate the activated single colony into nutrient broth medium, place it at 37°C and shake for 24-48 hours, take samples for staining and other identification to prevent contamination;

[0071] 3. Fermentation: A 60L stainless steel fermenter and a fermentation medium were used with an inoculation amount of 3%. The culture was fermented at 37°C for 24 h. The spore rate was observed under a microscope. When the spore rate reached more than 90%, the fermentation was stopped. The composition of the fermentation medium was as follows: 2% starch, 3% peptone, 1% yeast extract powder, and 2% corn steep liquor. The culture medium was aerated at 30°C and 200 rpm with a ventilation rate of 0.85 vvm.

[0072] 4. Membrane filtration and concentration: After the fermentation is completed, the fermentation liquid is transferred to the material barrel of the membrane filtration equipment (the pore diameter of the filter membrane is 200nm), the bacteria are filtered and concentrated to obtain the concentrate and the spore count is performed;

[0073] 5. Dilution and filling: According to the spore count results of the membrane filtration concentrate and the final filling concentration (1×10 7 CFU / mL), after determining the appropriate dilution multiple, take an appropriate amount of the concentrate, add sterile purified water for dilution and use a small filling machine to pack into small bottles.

[0074] In summary, Bacillus Velezii YHX-DH37 has a strong inhibitory effect on common pathogenic microorganisms, especially Gram-negative pathogens and fungal pathogens, and has a good degradation effect on common indoor allergens (cat allergens, dog allergens, mite allergens, pollen allergens). Therefore, this strain preparation has great application potential in the preparation of microbial cleaning products for home and public environments.

Claims

1. Bacillus Velez Bacillus velezensis ) YHX-DH37, characterized in that, The strain is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20232409.

2. Use of the Bacillus Velezii YHX-DH37 according to claim 1 in degrading allergens.

3. The use according to claim 2, characterized in that: The allergens include cat allergen Fel d 1, dog allergen Can f 1, mite allergen Der f 1, and pollen allergen Bet v 1.

4. Use of the Bacillus Velez subtilis YHX-DH37 according to claim 1 in the preparation of a product for inhibiting Gram-negative bacteria.

5. The use according to claim 4, characterized in that: The Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

6. Use of the Bacillus Velez subtilis YHX-DH37 according to claim 1 in the preparation of antifungal products.

7. The use according to claim 6, characterized in that: The fungi include Aspergillus niger, Aspergillus flavus and Candida albicans.

8. A microbial cleaning preparation, characterized in that: Contains the Bacillus Velez YHX-DH37 according to claim 1.

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