Probiotic liquid for removing allergen
By creating and optimizing probiotic bacterial flora and preparing allergen removal solution, the problem of difficulty in effectively degrading and inhibiting allergens in the existing technology has been solved, effective degradation and inhibition of various allergens has been achieved, and health and quality of life have been improved.
Patent Information
- Application Number
- CN202411743723.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-30
- Publication Date
- 2025-06-17
AI Technical Summary
The prior art is difficult to effectively degrade allergens such as plant pollen, pets, dust mites, insect excrement and mold spores, resulting in the widespread existence of allergic problems and affecting health and property.
An optimized group of probiotic bacteria was created, including Bacillus amyloligosaccharide, Bacillus subtilis and Bacillus Siam. Allergen removal solution was prepared by adjusting the proportion of each bacteria in the bacterial population (5:3:2), and utilizing its ability to degrade and inhibit allergens.
This probiotic solution can effectively degrade and inhibit allergens such as plant pollen, pets, dust mites, insect excrement and mold spores, significantly reduce the concentration of allergens, and improve the health status and quality of life of allergic patients.
Smart Images
Figure CN120158385A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the fields of biotechnology and fermentation engineering. By utilizing the symbiotic, mutualistic, and antagonistic effects of probiotic communities, a group of probiotic communities has been created, and the proportion of each bacterium in the probiotic communities has been tested and optimized. The functions of effectively degrading plant pollen allergens, effectively degrading pet allergens, effectively degrading dust mite allergens, effectively degrading insect excrement allergens, and effectively inhibiting mold spore allergens have been achieved. Technical Background
[0002] Plant pollen can cause allergies in people of all ages, with allergy cases reported in infants as young as 4 months old and the elderly over 60 years old. According to statistics, the number of people with pollen allergies in China reaches 200 million. The number of pet owners in China is increasing and has approached 100 million. With the continuous increase in the pet-raising rate, the number of pet allergy patients is also showing an increasing trend. Especially in big cities, due to the relatively high density of pets, the distribution of pet allergens in the environment is more extensive, leading to an increasing number of people being allergic to pet allergens. The urban permanent population in China has exceeded 900 million, with over 40 million people suffering from asthma and over 200 million people suffering from rhinitis. Moreover, the permanent population in first- and second-tier cities is more concentrated, and the attention to the problems of dust mite allergens, insect excrement allergens, and mold spore allergens in the living environment is increasing day by day. In short, the allergies caused by the above-mentioned various allergens affect physical health and cause property losses. The probiotic liquid for removing allergens has safety, no pollution, and wide applicability. Summary of the Invention
[0003] The present invention has created a group of probiotic communities, which are composed of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus siamensis. Bacillus amyloliquefaciens (YR003, deposit number: CGMCC NO.32698), Latin name Bacillus amyloliquefaciens, deposit date: November 18, 2024. Bacillus subtilis (YR002, deposit number: CGMCC NO.32697), Latin name Bacillus subtilis, deposit date: November 18, 2024. Bacillus siamensis (YR001, deposit number: CGMCC NO.32696), Latin name Bacillus siamensis, deposit date: November 14, 2024. These three strains are deposited in the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, contact phone number: 010 - 64807596.
[0004] The present invention optimizes the proportion of each bacterial content in the probiotic group. In the bacterial group, the proportion of Bacillus amyloliquefaciens (YR003, preservation number: CGMCC NO.32698), Bacillus subtilis (YR002, preservation number: CGMCC NO.32697), and Bacillus siamensis (YR001, preservation number: CGMCC NO.32696) is 5:3:2.
[0005] The present invention can effectively degrade plant pollen allergens, effectively degrade pet allergens, effectively degrade dust mite allergens, effectively degrade insect excrement allergens, and effectively inhibit mold spore allergens.
[0006] The present invention is safe, pollution-free, and has wide applicability during use. Detailed implementation method
[0007] Preparation of liquid LB medium: Tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L). Add tryptone, yeast extract, and sodium chloride to 950 ml of deionized water, and stir until completely dissolved. Adjust the pH to 7.0 with 5 mol / L NaOH. Make up the volume to 1 L with deionized water. Autoclave at 121 °C for 20 minutes.
[0008] Preparation of solid LB medium: Tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L), agar powder 15 g / L. Add tryptone, yeast extract, and sodium chloride to 950 ml of deionized water, and stir until completely dissolved. Adjust the pH to 7.0 with 5 mol / L NaOH. Make up the volume to 1 L with deionized water. Autoclave at 121 °C for 20 minutes.
[0009] Preparation of spores of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus siamensis: Inoculate the preserved Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus siamensis into the liquid LB medium in different reactors respectively, and culture at 200 r / min and 33 - 35 °C for 24 hours. Centrifuge and wash the fermentation broth to prepare a bacterial solution of 7x10 6 CFU / ml. For the prepared bacterial solution, mix the Bacillus amyloliquefaciens solution, Bacillus subtilis solution, and Bacillus siamensis solution in a volume ratio of 5:3:2 to prepare an allergen-removing bacterial solution.
[0010] Counting of spores: Take 1 ml of the bacterial solution fermented in the above liquid LB medium for 24 hours, and place it in a water bath at 80 °C for 10 minutes. Prepare 6 10-fold gradient dilution solutions from the treated bacterial solution. Dissolve 1 ml of the bacterial solution in 9 ml of sterile water to dilute it 10 times. Take 3 gradient dilution solutions, coat the plates with solid LB medium, and culture them in an inverted position at 36 °C for 48 hours. Calculate the spore content.
[0011] Detection of the degradation ability of the allergen - removing bacterial liquid on pollen allergens: Collect 1 g of poplar, willow, phoenix tree, and cypress pollen respectively, and divide them into two groups, with 0.5 g in each group. Spray 3 ml of the allergen - removing bacterial liquid on the experimental group and 3 ml of sterile water on the control group. Place them at room temperature for 2 hours. Rinse the two groups of pollen repeatedly with a filter membrane to remove the bacterial liquid, and then dry - treat the pollen. Extract the total protein using a plant protein extraction kit purchased from Bangfei Biology. Measure the protein concentration by the Bradford method. The results are as Figure 1 .
[0012] Detection of the degradation ability of the allergen - removing bacterial liquid on pet allergens: The cat allergen proteins Fel d1 and Fel d4, and the dog allergen proteins Can f1, Can f2, Can f4, and Can f6 are respectively prepared into 3 ml of 2.5 μg / ml solutions. The proteins used are purchased from Beijing Sino Biological Inc. 3 ml of sterile water. Sterilize an A4 white paper by ultraviolet light irradiation for 1 hour. In the experimental group, evenly spray the allergen protein solution on the sterile A4 paper; in the control group, evenly spray 3 ml of sterile water on the sterile A4 paper. Place them in a closed environment at 37 °C for 2 hours. Crush and soak the A4 paper pieces of the experimental group and the control group in 3 ml of PBS solvent for 2 hours. Centrifuge and take the supernatant. Detect the protein concentration using ELISA reagents respectively. The ELISA reagents used are purchased from Indoor Biotechnology. The results are as Figure 2 .
[0013] Detection of the degradation ability of the allergen - removing bacterial liquid on dust mite allergens: The dust mite allergen proteins Der f1, Der f2, Der p1, and Der p2 are prepared into 3 ml of 2.5 μg / ml solutions. The proteins used are purchased from Beijing Sino Biological Inc. 3 ml of sterile water. Sterilize an A4 white paper by ultraviolet light irradiation for 1 hour. In the experimental group, evenly spray the allergen protein solution on the sterile A4 paper; in the control group, evenly spray 3 ml of sterile water on the sterile A4 paper. Place them in a closed environment at 37 °C for 2 hours. Crush and soak the A4 paper pieces of the experimental group and the control group in 3 ml of PBS solvent for 2 hours. Centrifuge and take the supernatant. Detect the protein concentration using ELISA reagents respectively. The ELISA reagents used are purchased from Indoor Biotechnology. The results are as Figure 3 .
[0014] Detection of the ability of the allergen-removing bacterial liquid to degrade insect excrement allergens: 3 ml of a 2.5 μg / ml solution was prepared from cockroach allergen proteins Bla g1 and Blag7. The proteins used were purchased from Beijing Protein Innovation Co., Ltd. 3 ml of sterile water. An A4 white paper was sterilized by ultraviolet light irradiation for 1 hour. In the experimental group, the allergen protein solution was evenly sprayed on the sterile A4 paper; in the control group, 3 ml of sterile water was evenly sprayed on the sterile A4 paper. It was placed in a closed environment at 37 °C for 2 hours. The A4 paper pieces of the experimental group and the control group were crushed and soaked in 3 ml of PBS solvent for 2 hours. Centrifuged to obtain the supernatant. The protein concentration was detected using ELISA reagents respectively. The ELISA reagents used were purchased from Indoor Biotechnology. The results are as Figure 4 。
[0015] Detection of the ability of the allergen-removing bacterial liquid to inhibit mold allergens: Preparation of potato agar medium (200 g of peeled potato, 20 g of glucose, 15 g of agar, 1000 ml of distilled water. The cut small pieces of potato were put into 1000 ml of water and boiled for 30 minutes. Filtered with 4 layers of gauze to remove the residue. Agar was added to the filtrate and heated with stirring until completely dissolved. The volume was made up to 1000 ml with water. Measured with a pH meter. The medium was dispensed into clean glass test tubes or Erlenmeyer flasks. Sterilized in a high-pressure steam sterilizer for 30 minutes). Preparation of potato medium (200 g of peeled potato, 20 g of glucose, 1000 ml of distilled water. The cut small pieces of potato were put into 1000 ml of water and boiled for 30 minutes. Filtered with 4 layers of gauze to remove the residue. Agar was added to the filtrate and heated with stirring until completely dissolved. The volume was made up to 1000 ml with water. Measured with a pH meter. The medium was dispensed into clean glass test tubes or Erlenmeyer flasks. Sterilized in a high-pressure steam sterilizer for 30 minutes). 3 ml of the allergen-removing bacterial liquid, 7.0x10 6 cfu / ml; 3 ml of mold liquid, 7.0x10 6 cfu / ml. In the experimental group, 3 ml each of the allergen-removing bacterial liquid and the mold liquid were inoculated into the potato medium and cultured at 30 °C for 72 hours; in the control group, 3 ml of the mold liquid was inoculated into the potato medium and cultured at 30 °C for 72 hours. 1 ml of the cultured bacterial liquid of the experimental group and the control group was taken and added to a test tube containing 9 ml of sterile normal saline to prepare a 1:10 test sample, and 7 dilution gradients were made in this way. 100 μl each of the experimental group and the control group at 10 -6 、10 -7 Three dilution gradients were spread on the potato agar medium, and three parallel samples were made for each type and cultured upside down at 30 °C for 24 hours, and the number of probiotic colonies and the number of mold colonies were recorded. The results are as Figure 5 。
[0016] Inhalation toxicity test of the probiotic solution for removing allergens: Select 10 healthy mice with a body weight of 17 - 24 grams and raise them in separate cages in the same indoor environment. There are 5 mice in the experimental group. Place an automatic spray of 160 ml of the probiotic solution for removing allergens in each cage. The spray frequency is 5 times per minute, and the single spray volume is 0.15 ml per time. Continuously spray for 12 days. There are 5 mice in the control group without the automatic spray of the probiotic solution for removing allergens. Record the average body weight and the number of deaths of the mice on the 0th, 3rd, 6th, 9th, and 12th days respectively. The results are as Figure 6 。 Description of the drawings Figure 1 It is a detection chart of the degradation ability of the allergen - removing bacterial solution to pollen allergens. Figure 2 It is a detection chart of the degradation ability of the allergen - removing bacterial solution to pet allergens. Figure 3 It is a detection chart of the degradation ability of the allergen - removing bacterial solution to dust mite allergens. Figure 4 It is a detection chart of the degradation ability of the allergen - removing bacterial solution to insect excrement allergens. Figure 5 It is a detection chart of the inhibitory ability of the allergen - removing bacterial solution to mold allergens. Figure 6 It is a detection chart of the inhalation toxicity of the probiotic solution for removing allergens. Figure 7 It is a Gram - staining picture of Bacillus amyloliquefaciens (magnification 10×40). Figure 8 It is a Gram - staining picture of Bacillus subtilis (magnification 10×40). Figure 9 It is a Gram - staining picture of Bacillus siamensis (magnification 10×40).
Claims
1. A probiotic liquid for removing allergens, characterized in that: The bacterial flora is composed of Bacillus amyloliquefaciens (YR003, with a preservation number of CGMCC NO.32698), Bacillus subtilis (YR002, with a preservation number of CGMCC NO.32697), and Bacillus siamese (YR001, with a preservation number of CGMCC NO.32696).
2. A probiotic liquid for removing allergens, characterized in that: The ratio of Bacillus amyloliquefaciens (YR003, preservation number CGMCC NO.32698), Bacillus subtilis (YR002, preservation number CGMCC NO.32697), and Bacillus siamensis (YR001, preservation number CGMCC NO.32696) in the bacterial community is 5:3:
2.
3. Use of the allergen-eliminating probiotic liquid according to claims 1 and 2 in degrading pollen allergens, wherein the pollen comprises poplar pollen, willow pollen, sycamore pollen, and cypress pollen.
4. Use of the allergen-removing probiotic liquid according to claims 1 and 2 in degrading pet allergens, wherein the pet allergens include cat allergens and dog allergens.
5. Use of the allergen-eliminating probiotic liquid according to claims 1 and 2 in degrading dust mite allergens, wherein the dust mite allergens include house dust mite allergens and dust mite allergens.
6. Use of the allergen-removing probiotic liquid according to claims 1 and 2 in degrading insect excrement allergens, wherein the insect excrement allergens include cockroach allergens.
7. Use of the allergen-eliminating probiotic liquid according to claims 1 and 2 in inhibiting mold allergens, wherein the mold allergens include Penicillium allergens, Black mold allergens, Cladosporium allergens, and Aspergillus allergens.