Bacillus amyloliquefaciens capable of degrading allergens and application of bacillus amyloliquefaciens

Abstract

The invention provides bacillus amyloliquefaciens capable of degrading allergens and application of the bacillus amyloliquefaciens. The bacillus amyloliquefaciens is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 9, 2025, the preservation number is CGMCC No.36139, and the bacillus amyloliquefaciens can be used for degrading dust mite allergens, cat allergens and dog allergens.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a Bacillus amyloliquefaciens capable of degrading allergens and application thereof. BACKGROUND

[0002] Currently, the products commonly used on the market to remove allergens mainly apply the principle of physical filtration. Although these products can effectively remove allergens after use, they do not truly decompose allergens, thus there is a risk of secondary release. In contrast, microorganisms can effectively decompose allergens and have strong sustained capacity. Therefore, products for removing allergens based on microorganisms are increasingly attracting attention. Currently, the commonly used types of microorganisms for removing allergens include Bacillus, yeast and Lactobacillus, among which Bacillus is often used as one of the components for removing allergens because it can form heat-resistant and stress-resistant spores to maintain its activity.

[0003] Bacillus amyloliquefaciens is a gram-positive, spore-forming bacterium that is widely distributed in the environment such as soil and air. This bacterium is known for its ability to secrete hydrolytic enzymes (such as amylase and protease) and inhibit pathogenic microorganisms, and has the characteristics of high temperature resistance and acid and alkali resistance. The spores formed by the bacterium can survive for a long time in harsh environments. The colony is milky white or light yellow in color and has a rough surface. The most notable feature is the secretion of various enzymes during the metabolic process, including amylase, cellulase, protease, etc., which can decompose complex organic matter into small molecular nutrients. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a Bacillus amyloliquefaciens capable of degrading allergens and application thereof.

[0005] As an aspect of the present application, the present application provides a Bacillus amyloliquefaciens capable of degrading allergens, which has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on October 09, 2025, and the deposit number is CGMCC No. 36139.

[0006] As an aspect of the present application, the present application provides an application of a Bacillus amyloliquefaciens capable of degrading allergens, which is the application of the above-mentioned Bacillus amyloliquefaciens in degrading one or several of dust mite allergen Der P1, cat allergen Fel d1 and dog allergen Can f1.

[0007] Preferably, the degradation of dust mite Der P1 allergen and / or cat Fel d1 allergen is carried out by growing and degrading with Bacillus amyloliguefaciens and NB liquid medium.

[0008] Preferably, the NB liquid medium formula is peptone 5 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, pH 7.0.

[0009] Preferably, the inoculation amount of Bacillus amyloliguefaciens is 10 4 -10 5 CFU / mL.

[0010] Compared with the prior art, the present application has the following advantages: The Bacillus amyloliguefaciens obtained by the present application can effectively degrade allergens, especially in the aspects of degrading dust mite allergen, cat allergen and dog allergen: after 24h degradation, the cat allergen Fel d1 is completely degraded, with a degradation rate of 100%; after 24h degradation, the degradation rate of dust mite allergen Der P1 is more than 98%; and after 72h degradation, the degradation rate of dog allergen Can f1 is close to 85%. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 It is a schematic diagram for collecting and single-bacterium separation of environmental microorganisms in the present application; Figure 2 It is a purification result diagram for single-bacterium separation in the present application; Figure 3 It is a result diagram of water-soluble protein degradation experiment for primary screening of proteolytic circle in the present application; Figure 4 It is a result diagram of water-soluble protein degradation experiment for secondary screening of larger proteolytic circle in the present application; Figure 5 It is a phylogenetic tree diagram of strains in the present application; Figure 6 It is a plate streaking diagram of Bacillus amyloliquefaciens of the largest proteolytic circle colony in the present application; Figure 7 It is a spore production microscope diagram of the best Bacillus amyloliquefaciens obtained in the present application; Figure 8 It is a standard curve diagram of cat allergen Fel d1 in the present application; Figure 9Standard curve for dust mite allergen; Figure 10 Standard curve for dog allergen Can f1. DETAILED DESCRIPTION

[0012] In order to have a clearer understanding of the technical features, objectives and effects of the present application, the specific embodiments of the present application will now be described.

[0013] Example 1 Strain isolation, purification, and bacterial morphology identification In the present application, the Bacillus amyloliquefaciens is isolated from an indoor environment. In the indoor environment where pets are raised, a nutrient agar plate is placed open to allow environmental microorganisms to freely settle on the plate. After 1-2 hours, it is placed in a 37°C incubator for culture, and single colonies are picked. The results are shown in Figure 1 . Single colonies are continuously picked and streaked on LB solid medium for multiple times of purification, and the results are shown in Figure 2 . The preparation method of the nutrient agar plate is as follows: 10.0 g of proteose peptone, 3.0 g of beef extract powder, 5.0 g of sodium chloride, and 15.0 g of agar powder are weighed into a 1000 ml beaker, 1000 ml of purified water is added to dissolve them, and then boiled and cooled to 60°C. The pH value is adjusted to 7.2-7.4 with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution, and then divided into 250 ml triangular flasks, sterilized at 121°C for 20 min, and then cooled and poured into plates for standby use.

[0014] The bacterial morphology characteristics of the Bacillus amyloliquefaciens strain: on the nutrient agar plate medium, the colony is milky white and opaque, with a halo on the surface and no protrusions, and the edge is irregular.

[0015] Example 2 Screening of strain proteolytic ability The typical formula of nutrient broth includes 10.0 grams of proteose peptone, 3.0 grams of beef extract powder or beef paste powder, and 5.0 grams of sodium chloride per liter of culture medium. Proteose peptone and beef extract powder provide the nitrogen source, carbon source, vitamins, and amino acids required for bacterial growth, and sodium chloride is used to maintain the osmotic pressure balance of the culture medium.

[0016] Nutrient broth medium is selected, and cultured at 37°C for 24 h with the pH value controlled at 7.2±0.2 (25°C). The bacterial solution is spotted on a nutrient agar plate containing 2% skim milk to screen bacteria with protein-dissolving rings, and the results are shown in Figure 3 . Bacteria with larger protein-dissolving rings are continuously spotted on a nutrient agar plate containing 2% skim milk, and the one with the largest protein-dissolving ring is picked as the candidate strain, and the results are shown in Figure 4 .

[0017] According to Figure 3 , Figure 4The results show that the proteolytic effect of Bacillus amyloliquefaciens is obviously different. The candidate strain is identified by molecular biology, and the 16s rRNA gene sequence is determined. The sequencing result is shown as SEQ ID NO. 1, and the sequence alignment result is shown in the phylogenetic tree of Figure 5 , which determines that the strain is Bacillus amyloliquefaciens, as shown in Figure 6 , Figure 7 The spore microscope of the strain is shown in the figure, which shows that it is a spore bacterium. The strain was preserved in China General Microbiological Culture Collection Center (CGMCC) on October 09, 2025, and the preservation number is CGMCC No. 36139.

[0018] Identification result SEQ ID NO. 1:

[0019] Strains allergen degradation of example 3 The candidate strains were prepared into overnight culture, 1ml of the overnight culture was inoculated into 100ml of NB liquid medium containing allergen, so that the final concentration of the bacteria was 10 5 CFU / mL, and then placed in a shaking incubator at 37℃ and 120rpm, and sampled at regular intervals. The NB liquid medium formula was as follows: peptone 5g / L, beef extract powder 3g / L, sodium chloride 5g / L, pH 7.0.

[0020] The ELISA method was used to determine the content of allergen, and the degradation rate was calculated. Table 1 is the degradation rate of the strain degrading cat allergen Fel d1, Figure 8 and the standard curve of cat allergen Fel d1 is shown in Table 1; Table 2 is the degradation rate of the strain degrading dust mite allergen Der P1, Figure 9 and the standard curve of dust mite allergen Der P1 is shown in Table 2; Table 3 is the degradation rate of the strain degrading dog allergen Can f1, Figure 10 and the standard curve of dog allergen Can f1 is shown in Table 3.

[0021] Table 1 Time (h) OD value Allergen content (ng / mL) Degradation rate (%) 0 0.285 2.212609388 0 4 0.264 1.730648352 21.69 8 0.257 1.574345776 28.76 12 0.255 1.530087488 30.77 24 0.103 0 100 48 0.122 0 100 72 0.101 0 100 Table 2 Time (h) OD raw data OD value Allergen content (ng / mL) Degradation rate (%) 0 0.435 0.383 10.43867732 0 4 0.381 0.329 3.690345226 64.65 8 0.34 0.288 1.484525369 85.78 24 0.257 0.205 0.145035283 98.61 48 0.173 0.121 0.003934749 99.96 72 0.199 0.147 0.014902799 99.86 Table 3 Time (h) OD value Allergen content (ng / mL) Degradation rate (%) 0 0.306 1.05210178 2 0.305 1.044114234 0.76 4 0.297 0.981460206 6.71 8 0.284 0.884335155 15.95 12 0.266 0.759295029 27.83 24 0.188 0.338450714 67.83 48 0.173 0.278881732 73.49 72 0.139 0.167559165 84.07 In the degradation experiment, the NB liquid medium was inoculated with a strain at a concentration of 10 5 CFU / mL, and the strain simultaneously grew and proliferated and degraded allergen, achieving the synergistic effect of less strain dosage and high degradation rate. According to Tables 1-3, the degradation rates of the strain degrading the three kinds of allergens at different times were different, and the cat allergen Fel d1 was completely degraded after 24h, the degradation rate of the dust mite allergen Der P1 was more than 98% after 24h, and the degradation rate of the dog allergen Can f1 was close to 85% after 72h.

[0022] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, and are not a limitation on the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A Bacillus amyloliquefaciens strain capable of degrading allergens, characterized in that: It was deposited on October 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36139.

2. The application of a Bacillus amyloliquefaciens that can degrade allergens, characterized in that: The use of Bacillus amyloliguefaciens as described in claim 1 in degrading one or more of the following: dust mite allergen Der P1, cat allergen Fel d1, and canine allergen Can f1.

3. The application of the allergen-degrading Bacillus amyloliquefaciens according to claim 2, characterized in that: The degradation of dust mite Der P1 allergen and / or cat Fel d1 allergen is carried out by growing and degrading Bacillus amyloliguefaciens as described in claim 1 and NB liquid culture medium.

4. The application of the allergen-degrading Bacillus amyloliquefaciens according to claim 4, characterized in that: The NB liquid culture medium formula is 5 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, and pH 7.

0.

5. The application of the allergen-degrading Bacillus amyloliquefaciens according to claim 4, characterized in that: The inoculation amount of Bacillus amyloliquefaciens was 10. 4 -10 5 CFU / mL.

Images