Pet hair decomposer with synergistic effect of bacillus subtilis and keratinase

Abstract

The invention provides a pet hair decomposer with synergistic effect of bacillus subtilis and keratinase, the pet hair decomposer comprises bacillus subtilis ZYpet-015 and keratinase, the preservation number of the bacillus subtilis ZYpet-015 is CGMCC (China General Microbiological Culture Collection Center) No.34790, and the preservation date is June 6, 2025; the protease activity of the bacillus subtilis is greater than or equal to 129.92 U, the survival rate of the bacillus subtilis in simulated gastric juice within 3 hours is greater than 50%, and the tolerance rate of the bacillus subtilis in intestinal juice within 4 hours is greater than 90%. According to the pet hair decomposing agent, hair keratin is synergistically degraded through protease secreted by the bacillus subtilis ZYpet-015 and keratinase, and the hair decomposing efficiency reaches 28.07% + / -2.79% and is remarkably higher than that of a single treatment group. Meanwhile, experiments prove that the pet hair decomposer can effectively destroy the hair cuticle structure, is suitable for a pet hair-melting nutritional supplement and can relieve the hair ball symptom.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, specifically to the cross-application of microbial engineering and enzyme engineering in pet health care, and particularly relates to a pet hair decomposing agent based on synergistic action of Bacillus subtilis and keratinase. BACKGROUND

[0002] Hairball is a common digestive system disease in pets such as cats and dogs, which is closely related to the daily grooming behavior of pets. Studies have shown that cats lick their fur 600-800 times a day, and about 30% of their waking time is spent grooming their fur. The barbs on the tongue surface can cause a large amount of shed hair to be ingested into the gastrointestinal tract. Under normal circumstances, about 2 / 3 of the hair can be excreted with feces, but when the pet is a long-haired breed, is in the molting season, or has skin diseases, the amount of ingested hair will significantly increase, exceeding the excretion capacity of the intestinal tract, causing hair to accumulate and harden in the stomach, causing severe problems such as vomiting, constipation, intestinal obstruction, and even organ failure, posing a significant threat to pet health.

[0003] With the vigorous development of the pet economy, the demand for pet health care continues to rise. Hair removal products have become a core consumer category in pet health care products, and hair health management has become a major concern for pet owners.

[0004] However, existing hair removal products have obvious limitations:

[0005] (1) Insufficient decomposition ability: The mainstream products on the market rely on physical means such as mineral oil and dietary fiber to promote hair excretion, only through lubricating the intestinal tract or stimulating peristalsis to promote hair excretion, and cannot decompose the core component of hair, keratin, leading to the formation of large hair balls in the intestinal tract, making it difficult to completely solve the hairball problem.

[0006] (2) Lack of nutritional improvement: Existing products focus on the "hair removal" function and do not consider the fundamental improvement of pet hair health, which cannot reduce the frequency of licking and the total amount of ingested hair, and cannot prevent hairball from the source.

[0007] (3) Safety hazards: Excessive intake of dietary fiber may damage the gastrointestinal tract of pets, causing adverse reactions such as soft stools and diarrhea, affecting the health of the digestive system of pets.

[0008] Keratinase, as a kind of enzyme that can specifically hydrolyze keratin, shows potential in hair decomposition, but its decomposition efficiency is limited when used alone, making it difficult to meet the actual application needs. Bacillus, as a kind of microorganism widely existing in nature, can secrete proteases that can participate in the process of hair decomposition, and has strong resistance to gastrointestinal environment, and can maintain activity in the intestinal tract of pets, but its single application in hair degradation technology has not been developed.

[0009] At present, there is no research on the application of pet-derived Bacillus and keratinase in targeted decomposition of pet hair. The synergistic effect of the two is expected to break through the bottleneck of existing technology, realize efficient decomposition of hair, and balance nutrition and safety. Therefore, the development of hair-removing products based on the synergistic effect of pet-derived Bacillus and keratinase has important technical innovation value and broad market application prospect. SUMMARY

[0010] In view of the problems of the existing pet hair-removing products, such as limited physical excretion effect, lack of nutrition and safety, the purpose of the present application is to provide a pet hair-decomposing agent based on the synergistic effect of Bacillus subtilis and keratinase, which can effectively decompose pet hair, fundamentally solve the hairball problem, and balance the nutritional needs of pets and improve the health of pet hair.

[0011] The purpose of the present application is achieved by the following technical solutions:

[0012] The present application provides a Bacillus subtilis that can efficiently decompose hair. The Bacillus subtilis (ZYpet-015) is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 34790, and the deposit date is June 6, 2025.

[0013] Further, the protease activity of the bacteria is ≥129.92 U, the survival rate after 3 hours of treatment in simulated gastric juice is >50%, and the tolerance rate after 4 hours of treatment in simulated intestinal juice is >90%.

[0014] The present application provides the use of the Bacillus subtilis ZYpet-015 of the first aspect in the preparation of pet hair-removing products.

[0015] The present application provides a pet hair-decomposing agent based on the synergistic effect of Bacillus subtilis and keratinase, which includes the Bacillus subtilis ZYpet-015 of the first aspect and keratinase.

[0016] Further, the concentration of the Bacillus subtilis ZYpet-015 bacteria solution in the pet hair-decomposing agent is 1x10 9 CFU / mL, and the dosage ratio of the Bacillus subtilis ZYpet-015 bacteria solution to keratinase is 1 mL:250 mg.

[0017] The present application provides a preparation method of the pet hair-decomposing agent of the first aspect, which includes the following steps:

[0018] (1) Bacillus subtilis ZYpet-015 is inoculated into NA liquid medium and cultured at 37℃ and 180rpm for 24 hours to obtain a live bacterial solution;

[0019] (2) The bacterial solution obtained in step (1) is mixed with keratinase in a certain proportion to form the pet hair decomposing agent.

[0020] The fifth aspect of the present application provides the use of the pet hair decomposing agent of the first aspect in the preparation of a pet hair removal product.

[0021] Further, the pet hair removal product includes a nutritional supplement, an oral liquid or a feed additive.

[0022] Strain preservation information:

[0023] Bacillus subtilis ZYpet-015 is preserved in the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101. The preservation number is CGMCC No. 34790, and the preservation date is June 6, 2025. The classification name is Bacillus subtilis.

[0024] The present application has the following advantages over the prior art:

[0025] 1. Efficient hair decomposition, significantly improving hair removal effect

[0026] The present application screens a strain of probiotics that can efficiently decompose hair. The strain is identified as Bacillus subtilis, with a preservation number of CGMCC No. 34790 and a classification name of Bacillus subtilis. The strain has good gastrointestinal environment tolerance, intestinal colonization ability and proteinase activity. Further, through the synergistic effect of Bacillus subtilis ZYpet-015 and keratinase, efficient degradation of pet hair is achieved, with a hair decomposition efficiency of 28.07% ± 2.79%, which is significantly higher than that of single keratinase group and single Bacillus subtilis group, with an improvement of more than 40%. The synergistic effect of the two can destroy the structure of hair scales, hydrolyze keratin peptide bonds and disulfide bonds, and fundamentally decompose hair, solving the problem of incomplete removal of hair balls by traditional physical promotion.

[0027] 2. Excellent gastrointestinal environment adaptability, ensuring in vivo activity

[0028] The Bacillus subtilis ZYpet-015 has a survival rate of more than 50% in simulated gastric juice (pH 2.0) for 3 hours and a tolerance rate of more than 90% in simulated intestinal juice for 4 hours, which is much higher than the industry qualified standard (gastric juice survival rate > 15%, intestinal juice tolerance rate > 50%), and can smoothly pass through the gastrointestinal tract of pets and colonize to ensure continuous decomposition in the intestinal tract; and after the keratinase and the Bacillus subtilis ZYpet-015 are synergized, the defect that the single enzyme preparation is easily inactivated by digestive juice is further avoided, and the in-vivo action stability is enhanced.

[0029] 3. High safety and no side effect risk

[0030] The Bacillus subtilis ZYpet-015 is isolated from pet feces and is a pet intestinal tract source microorganism, and has no exogenous pathogenicity; through drug sensitivity experiment verification, the strain is highly sensitive to common antibiotics such as ceftriaxone and ciprofloxacin, and has no drug resistance transmission risk. No chemical synthetic components are added in the preparation production process, and the problem of gastrointestinal injury such as soft stool and diarrhea caused by excessive dietary fiber in traditional dehairing products is avoided, which meets the development trend of "natural and healthy" pet food.

[0031] 4. Nutritional improvement and reduction of hairball formation from the source

[0032] The present application can improve pet hair health by adding nutrients such as fish oil, reduce hair loss and total amount of ingestion, and reduce the risk of hairball from the source; at the same time, the Bacillus subtilis ZYpet-015 as probiotics can assist in regulating the balance of pet intestinal flora and improve the overall health level, realizing the dual effect of "decomposing existing hairball + preventing new hairball formation".

[0033] 5. Wide application scenarios and broad market prospects

[0034] The pet hair decomposing agent can be used as a dehairing nutrient supplement alone, or can be made into various forms such as oral liquid and feed additive, which is suitable for different pets (cats, dogs, etc.) and use scenarios (daily care, prevention during hair replacement season, etc.). BRIEF DESCRIPTION OF DRAWINGS

[0035] The present application will be further described below in combination with the drawings and examples:

[0036] Figure 1 The figure shows the bacteriostatic circle of bacillus in skim milk plate;

[0037] Figure 2 The figure shows the survival rate of bacillus-77 (Bacillus subtilis ZYpet-015) and bacillus-90 in artificial simulated gastric and intestinal juice;

[0038] Figure 3The scanning electron microscope contrast chart of hair decomposition is shown for blank group, keratinase group, strain, and combined group.

[0039] Figure 4 The hair decomposition efficiency histogram is shown. DETAILED DESCRIPTION

[0040] The technical solutions of the present application will be further described below in combination with examples and drawings, and the advantages and characteristics of the present application will be more apparent with the description. However, it should be understood that the examples are only exemplary and do not limit the scope of the present application.

[0041] It should be noted that, unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application relates.

[0042] In the following examples, keratinase was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0043] Example 1: Isolation, activation and screening of Bacillus subtilis

[0044] Strain screening was performed on the bacterial flora isolated from cat feces, and the specific steps were as follows:

[0045] Isolation and activation of strains: 105 strains of Bacillus (such as Bacillus subtilis, B. licheniformis, etc.) glycerol bacterial liquid isolated from cat feces were obtained from the strain bank, and after thawing and dissolving, streak inoculation was performed on LB plates, and incubation was performed at 37°C for 24 hours. Transfer to a new LB solid culture medium plate and purify culture for 3 times to obtain 105 candidate Bacillus strains. From the cultured plate, single colonies were selected and inoculated in LB liquid medium, and the inoculated liquid medium was placed in a 37°C, 180r / min shaker for shaking culture to obtain seed liquid (OD600≈1.0).

[0046] The medium formula is as follows: LB solid culture medium (per liter): tryptone 10g, yeast extract 5g, NaCl 10g, agar 15g, pH 7.0±0.2, 121℃ sterilization for 15 minutes. LB liquid medium: without agar, the rest is the same as above.

[0047] With protease activity as an indicator, 105 candidate Bacillus strains after activation were screened:

[0048] (1) Preliminary screening (bacteriostatic circle experiment): Prepare skim milk solid medium (formula: skim milk powder 10 g, peptone 5 g, beef extract 3 g, agar 15 g, add water to 1 L, pH 7.2±0.2, sterilize at 115°C for 15 minutes). According to the formula, weigh the skim milk powder, water, agar and other ingredients, mix and dissolve, sterilize and pour flat. The seed liquid cultured in step (1) is inoculated on the skim milk flat plate, and the inoculated flat plate is placed in a constant temperature incubator, the formation of the bacteriostatic circle is observed, the experimental results are recorded by taking pictures, and the diameter of the bacteriostatic circle around each colony is measured with a ruler and the data is recorded.

[0049] Inoculate the candidate strain into the solid medium containing skim milk, and measure the diameter of the bacteriostatic circle after 48 hours of culture at 37°C. Select 54 strains with a bacteriostatic circle diameter ≥20 mm Figure 1 ).

[0050] (2) Rescreening (enzyme activity determination):

[0051] Reagent formula: Substrate solution: 0.5% (w / v) casein (Sigma) dissolved in 50 mM phosphate buffer (pH 7.5). Stop solution: 0.4 M trichloroacetic acid (TCA).

[0052] Take the 54 strains obtained by preliminary screening, inoculate them in NA liquid medium (formula: peptone 10 g, beef extract 3 g, sodium chloride 5 g, add water to 1 L, pH 7.0±0.2, sterilize at 121°C for 20 minutes), and cultivate at 37°C, 180 r / min for 24 hours. Obtain the bacterial liquid.

[0053] Determine the keratinase activity by spectrophotometry: Centrifuge the bacterial liquid (8000 r / min, 10 minutes), take 1 ml of supernatant, add 2.0 ml of 0.05 mol / L Tris / HCL buffer (pH 7.5), then add 10 mg of soluble keratin, and react in a constant temperature water bath at 37°C. Take out and shake vigorously at regular intervals, add 2.0 ml of 10% TCA to terminate the reaction after 1 hour, centrifuge at 10000 g and 4°C for 15 min, and take out the supernatant for colorimetric determination at 280 nm. The treatment with TCA before reaction is used as a control.

[0054] Enzyme activity definition: A 280 value increases by 0.1, which is 1 U, and the average value is taken for 3 times. The results show that the keratinase activity of No. 77 strain is 126.19 U, which is significantly higher than that of other strains.

[0055] The strain No. 77 is identified as Bacillus subtilis. The strain was preserved in China General Microbiological Culture Collection Center on June 6, 2025, with the preservation number of CGMCC No. 34790, and was named as Bacillus subtilis ZYpet-015.

[0056] Example 2: Property detection of Bacillus subtilis

[0057] 1. Simulation of artificial gastrointestinal fluid experiment

[0058] Preparation of artificial gastrointestinal fluid:

[0059] Artificial gastric juice: take dilute hydrochloric acid 16.4 mL, dilute with water to 1000 mL, adjust pH to 2.0, add pepsin (1 g / L), filter sterilization; artificial intestinal fluid: take potassium dihydrogen phosphate 6.8 g, dissolve in water to 500 mL, adjust pH to 8.0 with 0.1 mol / L sodium hydroxide, add trypsin (1 g / L), filter sterilization.

[0060] Tolerance experiment:

[0061] Take 1 mL of liquid culture of Bacillus subtilis-77 (Bacillus subtilis ZYpet-015) and Bacillus subtilis-90 strain respectively, add 3 mL of artificial gastric juice, 37℃, 150 r / min shaking culture for 3 hours (simulate gastric environment), take sample dilution and spread on solid culture medium, 37℃ culture for 48 hours to count viable bacteria number (CFU / mL) (Table 1), and further calculate the gastric juice tolerance rate of 60.41% and 56.56% respectively. Figure 2

[0062] Take 1 mL of the above gastric juice treated bacterial solution, add 3 mL of artificial intestinal fluid, 37℃, 150 r / min shaking culture for 4 hours (simulate intestinal environment), count viable bacteria number (CFU / mL) by the same method (Table 1), and further calculate the intestinal fluid tolerance rate of 91.04% and 84.11% respectively, which is significantly higher than the qualified standard (gastric juice 4 hours survival rate > 15%, intestinal fluid tolerance rate > 50%) specified in the Journal of Nutrition. Figure 2

[0063] Table 1: Comparison of gastric juice and intestinal fluid tolerance rates of different Bacillus subtilis strains

[0064]

[0065] 2. Safety verification (drug sensitivity experiment)

[0066] ​​The sensitivity of the strain ZYpet-015 to 10 common antibiotics was determined according to the method of the Clinical and Laboratory Standards Institute (CLSI), including gentamicin (GEN), ciprofloxacin (CIP), ceftriaxone (CTR), erythromycin (E), ampicillin (AMP), tetracycline (TET), sulfamethoxazole-trimethoprim (SXT), chloramphenicol (C), lincomycin (MY) and penicillin (PEN).

[0067] ZYpet-015 liquid was added to LB solid medium cooled to about 45°C according to a 5% inoculation amount, and antibiotic sensitivity sheets were placed on the surface, with the strain cultured on agar plates without antibiotics as a control. After 24 h of culture at 37°C, the diameter of the inhibition zone was measured using a vernier caliper, and the sensitivity of the strain to the 10 antibiotics was determined according to the diameter.

[0068] The antibiotic-containing sensitivity paper sheets (including ceftriaxone, ciprofloxacin, chloramphenicol, erythromycin, etc.) were attached, and the culture was carried out at 37°C for 24 h. The diameters of the inhibition zones were measured: 49.75±1.84 mm for ceftriaxone, 48.3±2.98 mm for ciprofloxacin, and 43.45±0.7 mm for chloramphenicol, all showing high sensitivity and no drug resistance, meeting the safety requirements for pet food raw materials.

[0069] Table 2: ZYpet-015 antibacterial circle diameter results table of drug sensitivity test against different antibiotics

[0070]

[0071] Example 3: Pet hair decomposing agent

[0072] The present example provides a pet hair decomposing agent in which Bacillus subtilis and keratinase synergistically act, comprising Bacillus subtilis ZYpet-015 and keratinase. The ratio of the use amounts of Bacillus subtilis ZYpet-015 and keratinase in the pet hair decomposing agent is 1×10 9 CFU / mL: 250 mg.

[0073] The preparation method of the pet hair decomposing agent comprises the following steps:

[0074] (1) Bacillus subtilis ZYpet-015 was inoculated into NA liquid medium and cultured at 37℃ and 180rpm for 24 hours to obtain live bacterial culture.

[0075] (2) The bacterial solution obtained in step (1) is mixed with keratinase in a certain proportion to form the pet hair decomposing agent.

[0076] Test example: In vitro decomposition experiment of pet hair

[0077] To investigate the ability of Bacillus subtilis ZYpet-015, keratinase, and the combination of the two to decompose pet hair, the following in vitro pet hair decomposition experiment was conducted in this test case:

[0078] Hair pretreatment: Take healthy adult cat hair (1-2cm in length), wash with 1% SDS, rinse 3 times with distilled water, and dry at 60℃ to constant weight.

[0079] The experimental groups are as follows:

[0080] (1) Blank group: hair + sterile water;

[0081] (2) Bacillus subtilis group: hair + No. 77 bacterial solution (10 9 (CFU / mL);

[0082] (3) Keratinase group: hair + keratinase (250mg);

[0083] (4) Keratinase + Bacillus subtilis combined group: hair + No. 77 bacterial solution (10 9 CFU / mL) + keratinase (250mg).

[0084] Each group was cultured in a shaker at 37℃ and 150 rpm for 7 days, with 1 mL of nutrient solution (containing 10 g / L glucose and 5 g / L peptone) added daily. After the culture was completed, the hair was removed, dried at 60℃ to constant weight, and the hair decomposition was observed and the data were recorded.

[0085] The surface structure of hair was observed by scanning electron microscopy, the mechanism of action of bacteria and enzymes on hair decomposition was analyzed, and the decomposition efficiency was calculated: Decomposition efficiency = (initial mass - remaining mass) / initial mass × 100%.

[0086] Experimental results:

[0087] Analysis of pet hair electromicroscopy results: such as Figure 3The results show that the hair surface structure of the blank group is complete, and the scales are tight and undamaged. The scales of the keratinase group are partially peeled off and broken, exposing the cortex layer, which confirms the specific hydrolysis of enzymes on keratin. The scales of the Bacillus subtilis group are partially raised, showing microbial erosion marks. The scale structure of the combined treatment group of keratinase and Bacillus subtilis shows disintegration and loose scale structure, confirming that the synergistic effect of enzymatic hydrolysis and microbial fermentation can efficiently decompose hair.

[0088] As shown in Figure 4 The decomposition efficiency of the blank group is 3.42%, which is speculated to be due to slight physical breakage caused by shaking and temperature; the decomposition rate of the keratinase solution is 21.24%, and the single keratinase can hydrolyze peptide bonds, but it is difficult to break the scale layer and disulfide bonds of hair; the experimental results of the Bacillus subtilis group are 14.47%, and the secreted keratinase and auxiliary protease and metabolic products (organic acids) can assist in softening the hair. The decomposition rate of the combined group of keratinase and Bacillus subtilis is 28.07%±2.79%, which is about 32.1% higher than that of the keratinase group and about 94.0% higher than that of the Bacillus subtilis group, and more than 40% higher than that of the single treatment group, further confirming the significance of the synergistic effect. This high efficiency may be due to the connection of the two in different dimensions: the pretreatment of the hair by the metabolic products of the bacteria (such as the softening of the scale layer) provides a more accessible substrate surface for the subsequent targeted hydrolysis of keratinase, and the immediate hydrolysis of keratinase can reduce the physical barrier of undecomposed hair to bacterial proliferation, forming a dynamic and positive cycle.

[0089] In summary, the pet hair decomposing agent provided by the present application can effectively decompose pet hair and relieve pet hair ball syndrome by screening and culturing Bacillus subtilis ZYpet-015 with high protease activity and combining it with keratinase.

[0090] Finally, it should be noted that the above is only used to illustrate the technical solutions of the present application and is not limiting. Although the present application has been described in detail with reference to the preferred arrangement, 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 spirit and scope of the technical solutions of the present application.

Claims

1. A Bacillus subtilis which can efficiently decompose hair, characterized by, The Bacillus subtilis strain was named ZYpet-015. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34790 and deposit date of June 6, 2025.

2. The Bacillus subtilis according to claim 1, characterized in that, The protease activity of this bacterium is ≥129.92U, and its survival rate after treatment in simulated gastric fluid for 3 hours is >50%, while its tolerance rate after treatment in simulated intestinal fluid for 4 hours is >90%.

3. The application of Bacillus subtilis ZYpet-015 as described in claim 1 or 2 in the preparation of pet fur products.

4. A pet hair decomposing agent with the synergistic effect of Bacillus subtilis and keratinase, characterized in that, The pet hair decomposing agent comprises Bacillus subtilis ZYpet-015 as described in claim 1 or 2 and keratinase.

5. The pet hair decomposing agent according to claim 4, characterized in that, The concentration of the Bacillus subtilis ZYpet-015 bacterial solution in the pet hair decomposing agent is 1×10 9 CFU / mL, and the dosage ratio of the Bacillus subtilis ZYpet-015 bacterial solution to the keratinase is 1 mL: 250 mg.

6. The method for preparing the pet hair decomposing agent as described in claim 4 or 5, characterized in that, The preparation method includes the following steps: (1) Bacillus subtilis ZYpet-015 was inoculated into NA liquid medium and cultured at 37℃ and 180rpm for 24 hours to obtain live bacterial culture. (2) The bacterial solution obtained in step (1) is mixed with keratinase in a certain proportion to form the pet hair decomposing agent.

7. The use of the pet hair decomposing agent as described in claim 4 or 5 in the preparation of pet hair-removing products.

8. The application according to claim 3 or 7, characterized in that, The pet hair removal products include nutritional supplements, oral liquids, or feed additives.

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