Microbial cleaning agent for removing allergens of pets in indoor environment, deodorizing and inhibiting bacteria and application of microbial cleaning agent

Abstract

The invention discloses a microbial cleaning agent for removing pet allergens in an indoor environment, deodorizing and inhibiting bacteria and application, the microbial cleaning agent comprises bacillus subtilis YHX001J, bacillus amyloliquefaciens YHX002J, bacillus velezensis YHX003J and bacillus velezensis YHX-DH37, and the microbial cleaning agent has the following effects: (1) the pet allergens are degraded; comprise cat allergen, dog allergen, rabbit allergen and mouse allergen; (2) peculiar smell of pets is removed, especially the degradation rate of ammonia gas in the environment is 95.9%, and the degradation rate of hydrogen sulfide is 98.5%; and (3) inhibiting canine microspore bacteria which are pathogenic bacteria of canine moss, wherein the bacteriostasis rate reaches 97.14%. The microbial detergent consists of natural probiotics, does not contain a chemical protein denaturant or a chemical disinfectant, is safe and non-irritant, is friendly to allergic people, and is suitable for indoor places such as pet keeping families, pet hospitals and pet shops.

Description

Technical Field

[0001] This invention belongs to the field of indoor environmental purification technology, specifically relating to the preparation and application of a microbial cleaning agent that removes pet allergens and deodorizes and inhibits bacteria in the indoor environment. Background Technology

[0002] With social development and accelerated urbanization, the number of pet owners is increasing year by year. Pets not only enrich people's lives but also serve as important emotional anchors, becoming an indispensable part of the family. Reports show that the total number of pets in China reached 180 million in 2024, and is expected to grow significantly in the next decade. Indoor pet ownership in cities can have negative impacts on the home environment, such as excessive levels of allergens, harmful microorganisms, and harmful gases. The salivary glands, sebaceous glands, and anal glands of pets such as cats, dogs, and rabbits produce large amounts of allergenic proteins. The activities of pets carry these allergenic proteins to various corners of the indoor environment. When the accumulation of allergenic proteins in the environment exceeds a certain threshold, people will experience allergic reactions, such as itchy and swollen skin, conjunctivitis, coughing, difficulty breathing, and even life-threatening variant asthma. Public data shows that the probability of people being allergic to cats, dogs, and other pets is about 10% to 20%. Based on this, it is estimated that approximately 20 to 40 million people in China are suffering from pet allergies. Pet excrement and secretions, when present in indoor environments for extended periods, produce foul-smelling gases such as ammonia and hydrogen sulfide. They also easily breed mites and harmful microorganisms, impacting human health. *Microsporum canis* is the primary pathogen causing dermatophytes in cats and dogs (especially cats), and can also be transmitted to humans, causing various types of ringworm. It typically manifests as round or irregular patches of hair loss (most commonly on the face, ears, and limbs), with lesions possibly presenting as erythema, scaling, crusting, purulent discharge, and itching. After an animal becomes infected, its fur easily carries *Microsporum canis* spores, which are continuously spread into the environment, thus infecting humans. Therefore, the market urgently needs safe and effective products that remove indoor environmental allergens, deodorize, and inhibit bacteria.

[0003] Currently, most common allergen removal products on the market are a combination of traditional disinfectant ingredients such as benzalkonium chloride, hypochlorous acid, quaternary ammonium salts, and nano-silver with plant-based antibacterial ingredients. While these reduce the irritation of chemical components to the human body to some extent, they cannot completely eliminate the harm caused by these chemicals. It is worth noting that allergy sufferers often have varying degrees of damage to their skin and respiratory systems, making them more susceptible to irritation and exacerbation of allergy symptoms from chemical disinfectants. Studies have reported that respondents who used household cleaning and disinfection products daily had a 2-3 times increased risk of asthma attacks. These household cleaning products included both traditional chemical disinfectants and plant-based disinfectants. While extensive use of these products can reduce the abundance of allergens in the environment in the short term, it is detrimental to the recovery of patients' health.

[0004] Bacillus has a long history of safe use as a probiotic in humans, with no adverse events reported. It is frequently used as a food supplement to rebalance the gut microbiota, as an additive in fermented foods, as a supplement in animal husbandry, and as an enhancer and bactericide in agriculture. Among them, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus coagulans have been recognized by the EFSA (European Food Safety Authority) as safe strains with no pathogenic risk to humans.

[0005] In recent years, Bacillus probiotics have been used as indoor environmental cleaners or air purifiers. By competing with harmful microorganisms for ecological niches and nutrients, they reduce the growth of harmful bacteria, fungi, and other harmful microorganisms, helping to restore a more balanced indoor microbial environment. Bacillus probiotics can germinate and multiply on hard surfaces and secrete various antimicrobial peptides, organic acids, chitinases, glucanases, proteases, and other metabolites, thereby decomposing allergenic particles, organic matter, and inorganic compounds in the environment. Compared to traditional chemical cleaners, probiotic cleaning methods have many advantages, including no secondary pollution to the environment, no irritation to the human body, long-lasting effectiveness, and more thorough cleaning. However, the effect and scope of a single strain are limited; therefore, a combination of several strains is usually used to achieve a more comprehensive cleaning effect. Summary of the Invention

[0006] This invention aims to solve problems such as allergies in humans, unpleasant odors in the environment, and dermatophyte infections in pets caused by indoor pet ownership. It provides an environmental cleaner containing beneficial Bacillus subtilis. After these beneficial microorganisms are released into the indoor environment, they can produce proteases to reduce the abundance of protein allergens in the environment, alleviate allergy symptoms, and degrade odor molecules in the environment through a series of cellular activities. They also have the effect of inhibiting the pathogenic fungus Microsporum canis, which causes dermatophytes in pets.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Microbial cleaning agents include Bacillus subtilis YHX001J with accession number CCTCC NO: M2022266, Bacillus amyloliquefaciens YHX002J with accession number CCTCC NO: M2022265, Bacillus belycei with accession number CCTCC NO: M2023973, and Bacillus belycei with accession number CCTCC NO: M 20232409; Preferably, the viable cell ratio of Bacillus subtilis YHX001J, Bacillus amyloliquefaciens YHX002J, Bacillus belyssus YHX003J, and Bacillus belyssus YHX-DH37 is 1:1:1:1; Preferably, it also contains the inorganic salt NaCl.

[0008] The above-mentioned microbial cleaning agent is used to degrade pet allergens, including cat allergens, dog allergens, rabbit allergens, and mouse allergens.

[0009] The aforementioned microbial cleaners are used to remove pet odor gases, including ammonia and hydrogen sulfide.

[0010] The above-mentioned microbial cleaning agent is used in the preparation of a preparation that inhibits the pathogenic bacterium Microsporum canis, which causes tinea in cats and dogs. Detailed Implementation

[0011] The method of the present invention will be described in detail below with reference to specific embodiments. These embodiments are implemented under the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0012] Biological material description: Bacillus subtilis YHX001J has the accession number CCTCC NO: M2022266, Bacillus amyloliquefaciens YHX002J has the accession number CCTCC NO: M2022265, Bacillus velezensis YHX003J has the accession number CCTCC NO: M2023973, and Bacillus velezensis YHX-DH37 has the accession number CCTCC NO: M20232409.

[0013] Example 1: Preparation method of microbial cleaning agent This embodiment provides a method for preparing a microbial cleaning agent, which is used to obtain a Bacillus bacterial solution with specific functions and then dilute and compound it into a cleaning agent product for indoor use. The specific steps are as follows: 1. Preparation of concentrated bacterial cultures of Bacillus subtilis YHX001J, Bacillus amyloliquefaciens YHX002J, Bacillus belyceta var. ... (1) Activation of strains: The strains were activated by streaking on nutrient agar medium at 37°C for 24 h. (2) Preparation of seed culture: The activated single colony was inoculated into the nutrient broth medium and cultured at 37°C with shaking for 24-48 h. Samples were taken for identification such as staining to prevent contamination. (3) Fermentation: A 60 L stainless steel fermenter and its fermentation medium were used. The inoculum was 3%. Fermentation was carried out at 37℃ for 24 h. The spore rate was observed under a microscope. Fermentation was stopped when the spore rate reached more than 90%. The composition of the fermentation medium was as follows: starch 2%, peptone 3%, yeast extract 1%, corn steep liquor 2%. Aeration was carried out at 30℃ and 200 rpm at 0.85 vvm. (4) Membrane filtration concentration: After fermentation, the fermentation broth is transferred to the material tank of the membrane filtration (membrane pore diameter is 200 nm) equipment, the cells are membrane filtered and concentrated to obtain concentrated liquid and spores are counted.

[0014] 2. Dilution and filling of concentrated bacterial solution Based on the spore count results of the membrane filtration concentrate and the final filling concentration (4×10⁻⁶), 7 After determining the appropriate dilution ratio for each strain (CFU / mL), an appropriate amount of concentrated solution was taken according to the bacterial concentration ratio of 1:1:1:1 and diluted with sterile purified water. NaCl (final concentration 0.5 mg / L) was then added, and the solution was dispensed into small bottles using a small filling machine to obtain the microbial cleaner.

[0015] Example 2: Repeated skin and eye irritation tests of microbial cleaning agents 1. Skin allergy test According to Part II (2.3.6) of the "Disinfection Technical Specifications" (Ministry of Health, 2002 edition), a skin allergy test was conducted on the microbial cleaning agent. Forty-four healthy adult white guinea pigs were selected, including 22 males and 22 females, with a weight range of 200-300g.

[0016] (1) Remove hair from the left side of the back of the experimental animal 24 hours before the experiment, with a hair removal area of ​​3 cm × 3 cm; (2) Take 0.5 mL of the inducing test substance (microbial cleaner prepared in Example 1) and apply it directly to the hair removal area of ​​the left side of the test animal (2 cm × 2 cm). Cover it with two layers of gauze and one layer of cellophane or similar material, and then seal and fix it with non-irritating tape for 6 h. Remove the residual test substance with warm water. Repeat the same method on the same hair removal area on the 7th and 14th days. (3) On the 14th day after the last induction, take 0.5 mL of the stimulation test substance (microbial cleaner prepared in Example 1) and apply it directly to the hairless area of ​​2 cm × 2 cm on the right side of the test animal (hair was removed 24 h before the test). Then cover it with two layers of gauze and one layer of cellophane, and then seal and fix it with non-irritating tape for 6 h. After that, remove the residual test substance with warm water. (4) Observe skin reactions 24 h and 48 h after stimulation and score them; (5) The negative control group was only given the test substance stimulation treatment: 0.5 mL of stimulation test substance was applied directly to the hair removal area of ​​2 cm × 2 cm on the right side of the test animal (hair was removed 24 h before the test), then covered with two layers of gauze and one layer of cellophane, and then sealed and fixed with non-irritating tape for 6 h. The residual test substance was then removed with warm water.

[0017] Experimental results: The skin reaction score of the test substance was 0, and the sensitization intensity grade was no skin allergic reaction, which meets the standard requirements in Part II (2.3.13) of the "Disinfection Technical Specifications" (Ministry of Health, 2002 edition) (In the skin allergy test, if the disinfectant has only a very mild sensitizing effect on the skin, it can pass).

[0018]

[0019] 2. Acute eye irritation test According to Part II (2.3.4) of the "Disinfection Technical Specifications" (Ministry of Health, 2002 edition), an acute eye irritation test was conducted on the prepared microbial cleaning agent. Three ordinary-grade female New Zealand rabbits with a weight range of 2.0-2.2 kg were selected.

[0020] (1) 24 hours before the experiment, routine examination of both eyes of the experimental animals was performed. During the experiment, 0.1 mL of microbial cleaning agent was instilled into the conjunctival sac, and the eyes were passively closed for 4 seconds to prevent loss of the test sample.

[0021] (2) Rinse with physiological saline after 30 seconds. Use physiological saline as a normal control on the other side.

[0022] (3) Eye examinations should be performed at 1 h, 24 h, 48 h, 72 h, 7 d, 14 d, and 21 d after the end of the test. If no irritation response occurs within 72 h, or if the eye irritation response completely recovers on the 7th or 14th day, the test can be terminated early. Changes in the cornea, iris, and conjunctiva should be examined using a 2% sodium fluorescein solution during observation. The score of the eye irritation response should be recorded according to the eye damage scoring criteria at each examination.

[0023] (4) Evaluation of experimental results: The acute irritation response of the rabbit's cornea, iris, and conjunctiva was scored, and the average scores for corneal damage, iris damage, conjunctival hyperemia, and conjunctival edema were calculated for each animal at three different observation times (24 h, 48 h, and 72 h). The average scores and recovery time of corneal, iris, and conjunctival hyperemia and edema were used for evaluation, and the intensity of the test substance's stimulation to the eye was determined according to the eye irritation response grading standard.

[0024]

[0025] Note: The average score is the sum of the scores for each animal at 24 h, 48 h, and 72 h, divided by the number of observations (3).

[0026] Experimental results: Under the conditions of this experiment, the test substance showed no irritation in the New Zealand acute eye irritation test.

[0027] Example 3: Degradation effect of microbial cleaning agents on common indoor pet allergens The feline allergens Fel d 1 and Fel d 4, the canine allergen Can f 1, the rabbit allergen Ory c 1, and the mouse allergen Mus m1 protein and detection kits were all purchased from Indoor Biotech.

[0028] 1. Inoculate the prepared microbial cleaner or single bacterial culture into 100 mL of YPD liquid culture medium and incubate overnight with shaking, approximately 18-24 h.

[0029] 2. For the experimental group, 90 μL of the bacterial culture that had been cultured overnight was transferred to a sterile test tube. The allergen stock solution was appropriately diluted, and 10 μL was transferred to a sterile test tube and mixed with the bacterial culture. The mixture was then allowed to stand at room temperature for 5 days.

[0030] 3. In the control group, 90 μL of sterile water was added to 10 μL of diluted allergen solution and allowed to react for the corresponding time.

[0031] 4. The negative control group was inoculated with the same batch of culture medium as the test medium or sterile water and cultured to observe for sterile growth.

[0032] 5. After 5 days of treatment, the concentration of allergens in the solution was measured using a double-antibody sandwich ELISA method. The experiment was repeated 3 times.

[0033] 6. Result Calculation: Allergen Removal Rate In the formula, T oi The concentration (ng / mL) of the allergen solution in the control group after 5 days of treatment is represented by T. i This indicates the concentration (ng / mL) of the allergen solution in the test group after 5 days of treatment.

[0034] Table 1. Effect of microbial cleaning agents on the removal of feline Fel d4 allergens.

[0035]

[0036] Table 2. Degradation effect of microbial cleaning agents on other pet allergens

[0037]

[0038] As can be seen from Tables 1 and 2, the compounded microbial cleaner has a good removal effect on a variety of pet allergens.

[0039] Example 4: The degradation effect of microbial cleaning agents on pet allergens in cat-owning households 1. For cat-owning households, use a vacuum cleaner to collect environmental dust from areas such as sofas, floors, carpets, and beds before use. Remove large particles and collect only 1g of dust. Collect dust three times.

[0040] 2. Fill the prepared microbial cleaning agent into small spray bottles and spray at 50 m... 2 Use the microbial cleaner three times a day in the designated area, with each application being 3 mL. Continue spraying for two months.

[0041] 3. Every ten days, use a vacuum cleaner to collect environmental dust from areas such as sofas, floors, carpets, and beds. Remove large particles and collect only 1 g of dust. Collect dust three times.

[0042] 4. Detect the allergen concentration in dust samples before and after use. The detection method is based on the kit instructions. The kit was purchased from Indoor Biotechnologies.

[0043] Table 3. Effects of microbial cleaning agents on the degradation of allergens in the indoor environment of cat-owning households.

[0044]

[0045] As can be seen from Table 3, the use of microbial cleaners has a good degradation effect on allergens in the indoor environment of cat-owning households.

[0046] Example 5: Application of microbial cleaning agents for deodorization According to the requirements of OB / T 2761-2006 "Test Method for Purification Effect of Indoor Air Purification Products", the specific test steps are as follows: Pour 100 mL of microbial cleaning agent into the ultrasonic atomizer, and place the atomizer in a 15 m... 3 The experiment was conducted in long-range spray mode inside the test chamber for 24 hours.

[0047] Table 4. Effects of microbial cleaning agents and single-strain bacterial solutions on the removal of ammonia, a component of odor in the air.

[0048]

[0049] Table 5. Effects of microbial cleaning agents and single-strain bacterial solutions on the removal of hydrogen sulfide, an odor component in the air.

[0050]

[0051] As can be seen from Tables 4 and 5, the compounded microbial cleaner has a good removal effect on ammonia and hydrogen sulfide, the odor components in the air of pet-owning households.

[0052] Example 6: Inhibitory effect of microbial cleaning agents on Microsporum canis Pathogenic experimental strain: Microsporum canis ATCC 10214

[0053] 1. Preparation of Sabouraud dextrose liquid medium: Weigh 10 g of peptone and 40 g of glucose into a beaker, add 1000 mL of purified water to dissolve them, boil, cool to 60℃, adjust the pH to 5.4-5.6, dispense into 500 mL Erlenmeyer flasks, 89 mL per flask, stopper with a breathable stopper, autoclave at 115℃ for 30 min, and refrigerate for later use.

[0054] 2. Preparation of potato glucose culture medium: Weigh out 300 g of potato, 20 g of glucose, and 20 g of agar powder respectively; peel and cut the potato into pieces, add 1000 mL of distilled water, boil for 10-20 min, filter with gauze, and add distilled water to 1000 mL; then add glucose and agar powder to dissolve, boil, cool to 60℃, dispense into 250 mL Erlenmeyer flasks, and autoclave at 115℃ for 30 min.

[0055] 3. Antibacterial test procedure: Under aseptic conditions, use an inoculation loop to pick up one loopful of freeze-dried Microsporum canis powder, add it to Sabouraud dextrose liquid medium and mix well. Incubate at 28°C for 24 h. Use an inoculation loop to streak the cultured bacterial suspension onto potato dextrose agar plates and incubate at 28°C for 24 h. Use an inoculation loop to pick up a single colony, add it to Sabouraud dextrose liquid medium and mix well. Incubate at 28°C for 24 h.

[0056] 4. Determine the bacterial count of the bacterial suspension using the plate count method: After appropriate dilution with physiological saline, select 2-3 dilutions. Pipette 1 mL of *Microsporum canis* suspension into sterile bacterial culture dishes, inoculating three dishes for each sample. Pour in potato dextrose agar cooled to 40-45℃, mixing thoroughly in a figure-eight motion. After the agar has solidified, invert the dishes and incubate at 28℃ for 48 hours. Count the colonies afterward. Store the bacterial suspension at 4℃ until the bacterial count results are available. The bacterial suspension concentration should be 10⁻⁶. 6 -10 7 If the CFU / mL result is higher than this range, dilute with physiological saline; if the result is lower than this range, concentrate it by centrifugation.

[0057] 5. In the experimental group, 1 mL of *Microsporum canis* suspension was inoculated into 89 mL of Sabouraud dextrose liquid medium, mixed thoroughly, and then 10 mL of a microbial cleaner / *Bacillus subtilis* YHX001J / *Bacillus amyloliquefaciens* YHX002J / *Bacillus vesiliflorus* YHX003J / *Bacillus vesiliflorus* YHX-DH37 suspension was added (to control the bacterial concentration at 10). 6 -10 7 (CFU / mL), and another 1 mL of Microsporum canis suspension was inoculated into 89 mL of Sabouraud dextrose liquid medium, and 10 mL of sterile water was added as a blank control group. Mix well.

[0058] 6. Place the two mixed cultures at 30℃ for static incubation. After 48 h, measure the number of experimental strains in the two mixed cultures after co-culturing.

[0059] 7. Calculation formula: In the formula, X represents the inhibition rate (%), A represents the average number of colonies in the control group after co-culture, and B represents the average number of colonies in the experimental group after co-culture.

[0060] Table 6. Antibacterial effect of microbial cleaning agents on Microsporum canis

[0061]

[0062] As can be seen from Table 6, the microbial cleaning agent prepared by compounding Bacillus subtilis YHX001J, Bacillus amyloliquefaciens YHX002J, Bacillus belyssus YHX003J, and Bacillus belyssus YHX-DH37 has a good antibacterial effect against the pathogenic microsporum canis.

Claims

1. A microbial cleaning agent, characterized in that, This includes Bacillus subtilis YHX001J with accession number CCTCC NO: M2022266, Bacillus amyloliquefaciens YHX002J with accession number CCTCC NO: M2022265, Bacillus belycei with accession number CCTCC NO: M2023973, and Bacillus belycei with accession number CCTCC NO: M 20232409.

2. The microbial cleaning agent according to claim 1, characterized in that, The viable cell ratio of Bacillus subtilis YHX001J, Bacillus amyloliquefaciens YHX002J, Bacillus belyssus YHX003J, and Bacillus belyssus YHX-DH37 was 1:1:1:

1.

3. The microbial cleaning agent according to claim 2, characterized in that, It also contains the inorganic salt NaCl.

4. The use of the microbial cleaner according to any one of claims 1 to 3 in degrading pet allergens.

5. The application according to claim 4, characterized in that, The allergens include cat allergens, dog allergens, rabbit allergens, and mouse allergens.

6. The application of the microbial cleaner of claim 1 in removing pet odor gases.

7. The application according to claim 6, characterized in that, The pet odor gases include ammonia and hydrogen sulfide.

8. The use of the microbial cleaner according to claim 1 in the preparation of a preparation that inhibits the pathogenic bacterium Microsporum canis of cats and dogs causing tinea.

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