Pet skin antibacterial repair gel based on cat-source probiotics and aloe polysaccharide

Abstract

The invention provides pet skin antibacterial repair gel based on cat probiotics and aloe polysaccharide, a strain of cat probiotics with a function of resisting pathogenic bacteria of skin diseases is obtained through screening, the strain is preserved and identified as lactobacillus plantarum, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.32010, and the strain is classified and named as lactobacillus plantarum. The bacterial strain is applied to a pet gel product, the pet skin antibacterial repairing gel based on cat-source probiotics and aloe polysaccharide is provided, cat-source lactobacillus plantarum is combined with aloe polysaccharide-carbomer composite hydrogel with good biocompatibility, an intelligent dressing system integrating antibacterial, repairing and controlled release is formed, and the antibacterial repairing gel is applied to the pet skin. And a new perspective based on micro-ecological therapy is provided for the skin diseases of the pets.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations for pets, and in particular relates to an antibacterial repair gel for pet skin based on cat-derived probiotics and aloe polysaccharides. Background Art

[0002] With the continued growth in the number of pets, pet health management has become a focus of social attention. Fungal skin diseases in dogs and cats are common clinical diseases among pets. Due to their high incidence, high recurrence rates, and lengthy treatment cycles, they not only severely impact the quality of life and welfare of pets, but also place a heavy financial and care burden on pet owners. Statistics show that over 90% of fungal skin infections in pets are caused by Microsporum canis. The symptoms caused by this bacterium are complex, primarily manifested by circular hair loss (ringworms), erythema, dandruff, and hair breakage and loss. It is highly contagious and, without timely intervention, can easily lead to deep tissue infection or systemic secondary infection. More seriously, Microsporum canis is zoonotic, posing a potential public health and safety risk and threatening the health of pet owners and their family members.

[0003] Currently, the clinical treatment of fungal skin diseases in pets still primarily relies on traditional methods such as antibiotics. However, the long-term and extensive use of antibiotics has led to an increasingly serious problem of drug resistance in Microsporum canis, and the emergence of multidrug-resistant strains has gradually rendered conventional treatment options ineffective. Furthermore, antibiotics have significant drug toxicity and side effects, which can cause adverse reactions such as intestinal dysbiosis and liver and kidney damage in pets, affecting their overall health. Furthermore, the non-targeted nature of traditional treatments makes it difficult for drugs to precisely target lesions, which not only reduces treatment efficacy but also prolongs the treatment cycle, further exacerbating the problem of drug resistance and severely restricting the sustainable development of pet skin disease treatment.

[0004] Therefore, the development of new, safe, efficient and targeted pet skin antibacterial repair products has become the key to solving the difficulties in treating pet fungal skin diseases. Summary of the Invention

[0005] In response to the problems existing in the prior art, the purpose of the present invention is to propose a new treatment strategy based on probiotic hydrogel, and provide an antibacterial and repair gel for pet skin based on cat-derived probiotics and aloe polysaccharides. In particular, cat-derived plant lactobacillus is combined with an aloe polysaccharide-carbomer composite hydrogel with good biocompatibility to form an intelligent dressing system that integrates antibacterial, repair, and controlled release.

[0006] The purpose of the present invention is achieved through the following technical solutions: The first aspect of the present invention provides a cat-derived Lactobacillus plantarum ( Lactobacillus plantarum) ZYpet-014, the Lactobacillus plantarum ZYpet-014 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCC No.32010, and the deposit date: September 20, 2024.

[0007] The second aspect of the present invention provides the use of the Lactobacillus plantarum ZYpet-014 described in the first aspect in the preparation of a product for preventing and treating pet skin diseases.

[0008] Furthermore, the pathogenic bacteria of the pet skin disease is Microsporum canis.

[0009] Furthermore, the products include pet care products and external medicines.

[0010] Furthermore, the pet washing and care products include bath liquid, hair conditioner, and pet wipes.

[0011] Furthermore, the external medicine is one of a liquid preparation, an ointment, and a gel.

[0012] The third aspect of the present invention provides an antibacterial repair gel for pet skin based on cat-derived probiotics and aloe polysaccharides, wherein the raw materials of the antibacterial repair gel include: Lactobacillus plantarum ZYpet-014 as described in the first aspect, aloe polysaccharides, carbomer, triethanolamine, zinc hyaluronate and water; Among them, the concentration of Lactobacillus plantarum ZYpet-014 was 1.0×10 8 ~1.0×10 10 CFU / mL; Based on the total amount of gel, the weight percentages of other raw materials are as follows: Aloe polysaccharide 0.25%~0.8% Carbomer 0.2%~1.0% Triethanolamine 0.1%~0.5% Zinc hyaluronate 0.1%~1.0%.

[0013] The fourth aspect of the present invention provides a method for preparing the pet skin antibacterial repair gel according to the third aspect, comprising the following steps: (1) Preparation of probiotic liquid: inoculate cat-derived Lactobacillus plantarum ZYpet-014 onto a slant culture medium for recovery, culture at 37°C for 24 hours, transfer once, pick a single colony and inoculate into MRS broth liquid culture medium, culture overnight on a shaker at 37°C, and store at 4°C for later use; take the amplified bacterial liquid, centrifuge it, take the precipitate, wash it with sterile PBS, and resuspend the washed probiotics in PBS to obtain Lactobacillus plantarum ZYpet-014 liquid; (2) Preparation of blank hydrogel: Carbomer was weighed in proportion and added to ultrapure water. After thorough stirring, the mixture was immersed in water at room temperature to allow it to fully hydrate and swell. Triethanolamine and zinc hyaluronate were then added and stirred to obtain a blank hydrogel. (3) Preparation of aloe polysaccharide solution: weigh aloe polysaccharide in appropriate proportion and add it to an appropriate amount of deionized water. Stir the solution with a magnetic stirrer at 600 rpm / min at 30-40°C for 15 min until it is completely dissolved. Cool the solution to room temperature to obtain the aloe polysaccharide solution. Seal the solution in a volumetric flask for later use. (4) The prepared aloe polysaccharide solution and the blank hydrogel were mixed, stirred for sufficient reaction, and then Lactobacillus plantarum ZYpet-014 bacterial solution was added and stirred evenly to obtain the pet skin antibacterial repair gel.

[0014] Furthermore, in step (4), the volume ratio of the aloe polysaccharide solution to the blank hydrogel is 1:15-20. Culture deposit information:

[0015] Lactobacillus plantarum ZYpet-014, deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC); Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; Deposit Number: CGMCC No. 32010, Deposit Date: September 20, 2024; Taxonomic Name: Lactobacillus plantarum Lactobacillus plantarum . The beneficial effects of the present invention compared to the prior art are:

[0016] 1. The present invention screened and obtained a cat-derived probiotic strain with the function of resisting skin disease pathogens. The strain was identified as Lactobacillus plantarum, with a preservation number of CGMCC No.32010 and a classification name of Lactobacillus plantarum. Lactobacillus plantarum , providing a new perspective based on "skin microecological therapy" for pet skin diseases.

[0017] 2. Traditional skin disease treatments rely heavily on antibiotics, and the overuse of these antibiotics can lead to drug resistance. In contrast, probiotics, by leveraging their antibacterial and immunomodulatory properties, not only reduce pathogenic bacterial infections but also prevent drug resistance and secondary infections. Feline probiotics are better adapted to cat skin surfaces and are more compatible with the cat's skin microecological environment, making them more specific and effective in treatment. This not only enables the precise screening of functional probiotics, but also further expands the application prospects of probiotics in the treatment of pet diseases.

[0018] 3. The aloe polysaccharides added to the product of this invention have been extensively studied for their excellent anti-inflammatory and healing properties. The active ingredients of probiotics, when combined with the prebiotic aloe polysaccharides, may synergistically enhance their antibacterial and repair functions. This hydrogel not only provides a platform for the sustained release of probiotics, but also leverages the healing properties of aloe polysaccharides to create a more favorable healing environment for the affected area. This design combines the advantages of probiotics and hydrogel materials, achieving dual functions of "antibacterial and repair," providing an innovative application strategy for wound healing in skin diseases.

[0019] 4. The present invention uses hydrogel as a carrier for probiotics, providing ample space for their growth. Encapsulated within the hydrogel matrix, the probiotics are less susceptible to environmental stressors and conditions. The hydrogel matrix is ​​soft, conformable, and breathable, making it ideal for treating skin wounds and as a sustained-release drug carrier. By adjusting properties such as pore size and hydrophilicity, the hydrogel can achieve sustained release of probiotics and aloe polysaccharides, thereby extending the residence time of the active ingredients on the skin surface and enhancing local therapeutic efficacy.

[0020] 5. The present invention is specially designed for Microsporum canis, the main pathogenic bacteria of pet fungal skin diseases, which improves the efficiency and effect of solving pet fungal skin diseases and is highly targeted.

[0021] 6. This invention utilizes a low-toxicity eco-therapeutic platform based on cat-derived probiotics, combined with aloe polysaccharide hydrogel. Because both probiotics and aloe polysaccharides are natural ingredients, and the biocompatible hydrogel carrier is used, this platform exhibits excellent biodegradability and low toxicity, providing a safer, low-side effect treatment for skin diseases and wound healing in pets. It is suitable for both pregnant and young pets, ensuring their long-term health and safety.

[0022] 7. The pet skin antibacterial repair gel prepared by the present invention can significantly reduce the growth ability, adhesion ability and bactericidal ability of Microsporum canis. It not only provides a safe and effective solution for pet skin diseases, but also avoids the risk of pet owners contracting skin diseases. At the same time, it meets the market demand for healthy and natural pet skin disease care products and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below with reference to the accompanying drawings and examples: Figure 1 The scanning electron microscope structure of different hydrogels in Test Example 1 at a magnification of 20,000×; Figure 2 The graph shows the rheological properties of different hydrogels in Test Example 2; A is shear stress, B is apparent viscosity, and C is dynamic viscoelastic modulus (G' and G''); Figure 3 This is the pH release rate result of different hydrogels in Test Example 3; Figure 4 This is the result diagram of the probiotic release rate in different hydrogels in Test Example 4; Figure 5 Figure 5 is a graph showing the results of the inhibition zone determination in different hydrogels in Test Example 5, where A is a graph showing the results of the flat plate inhibition zone of different hydrogels, and B is a graph showing the statistical results of the inhibition zone diameters of different hydrogels; Figure 6 Figure 6 shows the results of the biofilm inhibition rate determination of different hydrogels in Test Example 6, where A shows the absorbance values ​​of different hydrogels measured at OD595 nm, and B shows the results of the biofilm inhibition rate of different hydrogels against pathogenic bacteria; Figure 7 This is a diagram showing the therapeutic effect of Test Example 7 Pet Skin Antibacterial Repair Gel on fungal skin diseases in pet cats. DETAILED DESCRIPTION

[0024] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0025] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0026] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not intended to limit the present invention.

[0027] In the following examples, aloe polysaccharide was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and carbomer 934 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Example 1: Screening and identification of Lactobacillus plantarum

[0028] This example provides a strain screening and identification process: (1) Isolation of strains: Fecal samples of healthy cats were collected from catteries and animal rescue stations in Yancheng District, Luohe City. 1 g of sample was weighed and placed in a 50 mL centrifuge tube. 9 mL of sterile water was added and thoroughly shaken to mix. This was used as the initial 10-fold dilution. Subsequently, gradient dilutions were performed to 10 3 and 105 100 μL of the dilutions at different concentrations were taken and inoculated onto MRS solid culture medium plates, which were then cultured at 30°C under anaerobic conditions for 48 h to isolate intestinal lactic acid bacteria.

[0029] (2) Purification of strains: From the primary culture plate, select colonies of varying sizes and milky white protrusions for single colony selection. Repeatedly inoculate on MRS plates using the three-zone streak method and culture anaerobically at 37°C for 48 hours for three consecutive rounds to obtain a stable and purified target strain.

[0030] (3) Morphological identification: Gram staining is used to perform a preliminary observation of the strain morphology. A small number of single colonies are evenly smeared on a glass slide in a drop of sterile water and air-dried. The slide is then stained with crystal violet, mordanted with iodine solution, decolorized with 95% alcohol, and counterstained with safranin. After rinsing and air-drying, the morphological characteristics and staining reaction of the bacteria are observed under an optical microscope to determine their Gram properties.

[0031] (4) Glucose gas production test: A single colony of selected lactic acid bacteria was inoculated into MRS liquid medium and cultured at 30°C for 24 hours. Subsequently, the bacterial solution was inoculated into MRS liquid medium containing inverted Dulbecco's tubules at a 1% inoculum and cultured at 30°C for 7 days. The formation of bubbles in the Dulbecco's tubules was observed to determine whether the strain was homofermentative (no gas) or heterofermentative (gas-producing) type.

[0032] (5) Physiological experiment: Add 100 μL of 3% hydrogen peroxide solution to a culture dish, place the test colony in the solution, and observe whether bubbles are produced. If bubbles appear, it is catalase positive; if no bubbles are produced, it is negative, which further assists in determining the taxonomic characteristics of the strain.

[0033]

[0034] As can be seen from Table 1, strain ZYpet-014 is a homofermentative lactic acid bacterium, with Gram staining results being positive and catalase negative.

[0035] (6) Strain identification: The strain was cultured anaerobically at 30°C on an MRS plate for 48 h, and a single colony was picked for PCR. The primers were 16S rRNA universal primers 27F and 1492R. The PCR reaction conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 90 s, 35 cycles; 72°C final extension for 5 min. The obtained 16S rRNA was subjected to BLAST analysis with the sequences of known strains in the GenBank website. The ZYpet-014 strain was identified as Lactobacillus plantarum ( Lactobacillus plantarum ), named Lactobacillus plantarum ZYpet-014.

[0036] ZYpet-014 strain 16S rRNA gene sequence: Example 2: Preparation of pet skin antibacterial repair gel based on cat-derived probiotics and aloe polysaccharides

[0037] This embodiment provides a pet skin antibacterial repair gel based on cat-derived probiotics and aloe polysaccharides, and the preparation method is as follows: (1) Preparation of probiotic liquid: Under aseptic conditions, inoculate the feline plantarum Lactobacillus ZYpet-014 stored in a freeze-dried tube onto a slant culture medium for recovery. After culturing at 37°C for 24 h, transfer once and pick a single colony to inoculate into 100 mL of MRS broth liquid culture medium. Incubate overnight at 37°C and 130 rpm / min in a shaking incubator and store at 4°C for later use. Take the amplified bacterial liquid, centrifuge at 3000 rpm for 5 min, take the precipitate, wash with sterile PBS, and resuspend the washed probiotics in PBS for later use to make the bacterial liquid concentration approximately 1.0 × 10 9 CFU / mL, and the bacterial liquid of Lactobacillus plantarum ZYpet-014 was obtained.

[0038] (2) Preparation of blank hydrogel: Carbomer 934 was weighed in proportion and added to ultrapure water to a mass concentration of 0.5%. After magnetic stirring at a speed of 1200 r / min for 1 hour, it was soaked at room temperature for 12 hours to allow it to fully hydrate and swell. Then, 0.25% triethanolamine and 0.5% zinc hyaluronate were added and stirred evenly to obtain blank hydrogel (CON-hydrogel).

[0039] (3) Preparation of aloe polysaccharide solution: Weigh 0.5% aloe polysaccharide in a suitable amount of deionized water, stir magnetically at 600 rpm / min at 30-40°C for 15 min until completely dissolved, cool to room temperature to obtain aloe polysaccharide solution, and seal in a volumetric flask for later use.

[0040] (4) The prepared aloe polysaccharide solution and blank hydrogel were mixed in a volume ratio of 1:19, stirred at 300 rpm for 1 h, and then Lactobacillus plantarum ZYpet-014 bacterial solution was added and stirred evenly to obtain the pet skin antibacterial repair hydrogel (LP+AVE-hydrogel), which was sealed and stored in a refrigerator at 4°C. Comparative Example 1: Preparation of probiotic carbomer composite hydrogel

[0041] This comparative example prepared a composite hydrogel by referring to the method described in Example 2, except that no aloe polysaccharide was added to the blank hydrogel in step (4). Specifically: (4) Add Lactobacillus plantarum ZYpet-014 bacterial solution to the blank hydrogel and stir evenly to obtain the probiotic carbomer composite hydrogel (LP-hydrogel), which is then sealed and stored in a refrigerator at 4°C. Test Example 1: Scanning Electron Microscopy Characterization of Hydrogel

[0042] This test example explores the internal structures of different hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. The specific method is as follows: The LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group were pre-frozen at -80°C overnight and then freeze-dried for 48 h using a vacuum freeze dryer. The freeze-dried hydrogels were then fractured with liquid nitrogen, and cross-sections were subjected to gold spraying under vacuum. Structural observation was performed using a scanning electron microscope (SEM: Sigma30, Zeiss, Germany) with an accelerating voltage of 20 kV and a magnification of 2000 times to observe the internal morphology of the hydrogels and the distribution of probiotics.

[0043] Depend on Figure 1 High-magnification SEM observations revealed that, compared with CON-hydrogel, probiotics were effectively embedded within the gel network or attached to the surface of the gel matrix in both LP-hydrogel and LP+AVE-hydrogel hydrogels, with the probiotic morphology remaining intact, exhibiting a typical rod-shaped structure. Compared with LP-hydrogel, the probiotics in the LP+AVE-hydrogel hydrogel described in Example 2 were more abundantly distributed and had a regular bacterial morphology. This suggests that the addition of aloe polysaccharides can improve the microstructural properties of the hydrogel, aiding probiotic proliferation and protecting bacterial cell integrity, allowing more probiotics to successfully attach to the gel matrix, which may have a positive impact on their metabolic activity and storage stability. Test Example 2: Determination of rheological properties of hydrogel

[0044] This test example explores the rheological properties of different hydrogel systems prepared in Example 2, Comparative Example 1, and a blank control group. The specific method is as follows: A rheometer (Anton Paar-MCR301) equipped with a cone-plate or plate fixture was used. The test temperature was set at 25°C, the gap was 1.0 mm, and the shear rate range was 0.1-200 s⁻¹. Shear stress and apparent viscosity were measured at different shear rates to analyze the shear-thinning behavior and rheological properties of the LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. Furthermore, a dynamic frequency sweep test was performed with a frequency range of 0.1100 rad / s and a fixed strain amplitude of 1% (within the linear viscoelastic region) to measure the storage modulus (G') and loss modulus (G'') of the gel systems and analyze the elastic and viscoelastic properties of the gels. Each sample was tested three times.

[0045] like Figure 2 As shown in the figure, the three gel systems (CON-hydrogel, LP-hydrogel and LP+AVE-hydrogel) all exhibited typical shear-thinning properties, that is, the shear stress and apparent viscosity gradually decreased with the increase of shear rate. The shear stress and viscosity of the LP+AVE-hydrogel system were significantly lower than those of the other two groups, indicating that the addition of aloe polysaccharides effectively reduced the cohesion of the gel network and increased the flexibility and spreadability of the gel. In addition, dynamic rheological analysis showed that the storage modulus (G') of each gel system was significantly higher than the loss modulus (G''), showing obvious elastic gel characteristics. The storage modulus and loss modulus of LP+AVE-hydrogel were lower than those of the other two groups, further indicating that the addition of aloe polysaccharides effectively improved the softness and deformability of the gel system, indicating that its structure was softer and easier to apply.

[0046] The above results show that the LP+AVE-hydrogel prepared in Example 2 has both good flexibility and stable gel structure, and is more suitable for clinical application of wound dressing. Test Example 3: Determination of pH value of hydrogel microenvironment

[0047] This test example explores the microenvironment pH of different hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. The specific method is: 1.0 g of LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel samples prepared in Example 2, Comparative Example 1, and the blank control group were immersed in 4 mL of 25°C PBS (pH 7.4) for 72 h. The pH of the immersion solution was measured at specific time intervals using a pH meter to assess the pH of the microenvironment formed by the different hydrogels.

[0048] pH value is an important parameter for wound dressings. The pH value of normal skin is slightly acidic, ranging from 4 to 6, while the pH value of infected wounds is alkaline. The acidic microenvironment of the probiotic gel system helps inhibit the growth of pathogens and enhance the balance of skin microecology. It is a new treatment method that promotes wound healing by inducing the microenvironment.

[0049] The results of pH changes in the hydrogel microenvironment are as follows Figure 3 As shown, the CON-hydrogel group maintained a stable neutral pH (approximately 7.0) during incubation, while the pH in both the LP-hydrogel and LP+AVE-hydrogel groups showed a gradual decrease. The pH drop was particularly significant in the LP-hydrogel system, reflecting the probiotic's strong ability to metabolize organic acids (such as short-chain fatty acids and lactic acid production). The LP+AVE-hydrogel group exhibited a similar but more stable pH change trend, suggesting that the addition of aloe polysaccharides may regulate probiotic metabolic activity and maintain a stable acidic microenvironment. This acidic microenvironment is beneficial for inhibiting the growth of pathogens and promoting skin wound healing. Test Example 4: Determination of probiotic release rate in hydrogel

[0050] This test example investigates the probiotic release rates of different hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. The specific test method is as follows: The LP+AVE-hydrogel and LP-hydrogel hydrogels prepared in Example 2 and Comparative Example 1 were immersed in 10 mL of PBS and placed in an incubator at 37°C. Within a predetermined time interval, 100 μL of the surrounding PBS was applied to MRS solid culture medium for colony counting. The release rate of the probiotics was calculated based on the percentage of the amount of probiotics in the release medium to the total amount of encapsulated probiotics.

[0051] like Figure 4 As shown, both LP-hydrogel and LP+AVE-hydrogel hydrogels showed a sustained and stable probiotic release pattern. Compared with the LP-hydrogel group, the probiotic release rate of the LP+AVE-hydrogel group at each time point was significantly increased ( P<0.05), with a cumulative release rate of approximately 35% by the end of 6 hours, significantly higher than that of the LP-hydrogel group. This indicates that the addition of aloe polysaccharides optimizes the structural properties of the hydrogel system, effectively improving the release efficiency of probiotics and further demonstrating its excellent performance as a probiotic carrier material. Test Example 5: Determination of antibacterial properties of hydrogel

[0052] This test example explores the antibacterial properties of different hydrogels prepared in Example 2, Comparative Example 1, and the blank control group. The specific test method is as follows: The antibacterial activity of different hydrogels against Microsporum canis, a major pathogen of pet skin diseases, was determined by the punch method. In a clean bench, LB solid culture medium sterilized by high temperature and high pressure was cooled to 45-50°C and a 1% suspension of Microsporum canis (10 7 CFU / mL), mix thoroughly, and pour onto a plate (approximately 20 mL / plate). Set aside horizontally for solidification before use. Use a sterile hole puncher to punch three holes (10 mm in diameter) on the test plate. Inject 200 μL of each of the LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel samples prepared in Example 2, Comparative Example 1, and the blank control into the circular holes. Incubate in an incubator at 37°C for 24 h. Remove the plate and determine the antibacterial activity of the hydrogels by measuring the diameter of the inhibition zone.

[0053] Test results: The antibacterial properties of each group of gels were evaluated by the inhibition zone test. Figure 5 As shown in the figure, CON-hydrogel hydrogel did not show obvious antibacterial activity, while LP-hydrogel hydrogel formed a clear inhibition zone (about 15 mm), indicating that probiotics have certain antibacterial activity against Microsporum canis, which may be due to the release of probiotic metabolites such as lactic acid, organic acids or other antibacterial substances. After further addition of aloe polysaccharide, the diameter of the inhibition zone of LP+AVE-hydrogel hydrogel increased significantly (about 18 mm, P <0.05), indicating that aloe polysaccharides significantly enhance the antibacterial properties of probiotics. This result reveals that the combination of aloe polysaccharides and probiotics has a synergistic antibacterial effect. Aloe polysaccharides may promote the metabolic activity and proliferation of probiotics through a prebiotic effect, enabling probiotics to secrete more antibacterial metabolites, thereby enhancing antibacterial effects. This further expands the functional potential of probiotic preparations. Test Example 6: Determination of the inhibition rate of hydrogel on pathogenic bacteria biofilm

[0054] This test example explores the inhibition rate of pathogenic bacteria biofilm by different hydrogels prepared in Example 2, Comparative Example 1 and the blank control group. The specific test method is as follows: Use an inoculating loop to pick up the canis microsporum from the slant of the test tube and put it in sterile water with glass beads. Shake it by hand for a few minutes to disperse the spores. After filtering, make a 10 7 CFU / mL mixed spore suspension, 0.5 mL of LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel samples prepared in Example 2, Comparative Example 1, and the blank control group were mixed with 2 mL of bacterial suspension and added to a 24-well plate and incubated at 37°C for 24 h. The culture supernatant was discarded, and the biofilm was washed 2-3 times with sterile PBS. After drying in a 37°C oven for 10 min, 0.1% crystal violet was added to the biofilm and stained for 15 min. The stain was removed and the excess crystal violet was washed away. 30% acetic acid was added to the stained biofilm to dissolve the pigment. The dissolved stain was diluted 5-fold, and its absorbance at OD595 nm was measured using a microplate reader, and the biofilm inhibition rate was calculated.

[0055] Biofilm inhibition rate (%) = (Ax-Ay) / Ax×100%.

[0056] Wherein, Ax: the value measured at OD595 nm of the control group; Ay: the value measured at OD595 nm of the experimental group.

[0057] Experimental results: The inhibition effect of CON-hydrogel, LP-hydrogel and LP+AVE-hydrogel on biofilm formation was determined by crystal violet staining method: The biofilm inhibition results are as follows: Figure 6 As shown in Figure 2, crystal violet staining showed that the empty CON-hydrogel group had the strongest biofilm formation ability, while LP-hydrogel significantly reduced the formation of Microsporum canis biofilm ( P <0.05). After further addition of aloe polysaccharide, the biofilm inhibition effect of the LP+AVE-hydrogel hydrogel group was further enhanced, and the inhibition rate was significantly higher than that of the LP-hydrogel group ( P <0.05). This result shows that aloe polysaccharides have obvious prebiotic properties, can enhance the metabolic function of probiotics, and effectively inhibit the formation of pathogenic biofilms, reflecting a high antibacterial and anti-biofilm application potential. Test Example 7: Clinical Fungal Skin Disease Treatment Effect Test for Pet Cats

[0058] To verify the effectiveness and safety of the pet skin antibacterial repair gel described in Example 2 in treating fungal skin diseases in pet cats in actual clinical practice, and to evaluate its clinical cure rate, efficacy, and inefficiency, the following animal experiments were conducted in this test example: 1. Experimental animals and grouping A total of 12 pet cats with clinically confirmed fungal skin diseases were selected and randomly divided into two groups: Experimental group (n=6): treated with the pet skin antibacterial repair gel (LP+AVE-hydrogel) of Example 2; Control group (n=6): received blank hydrogel (CON-hydrogel) intervention, serving as the blank control group; Before the experiment, the animals completed health assessment, case registration, and photographic records of the affected parts, and ensured that there was no significant difference in body condition.

[0059] 2. Experimental methods Initial diagnosis examination; Itch severity: Observe whether the animal frequently scratches, bites, or rubs the affected area; Hair loss condition: record the number, distribution range and edge clarity of local or annular hair loss patches; Skin lesion characteristics: assess the presence of erythema, scaling, crusting, hyperkeratosis, and annular lesions on the skin surface; Lesion exudation and crusting: record the presence of exudate, dry crust, grayish white or yellow thick crust.

[0060] Intervention plan: The experimental group applied the composite hydrogel topically every 12 hours for 14 consecutive days; the control group did not receive any drug treatment, only daily cleaning and care, and avoided other intervention factors.

[0061] Observation records: Changes in the affected area were recorded on days 0, 7, and 14 of the experiment, photographed and compared, and scored independently by two clinical veterinarians to ensure the objectivity of the evaluation.

[0062] 3. Criteria for determining efficacy Cure: Symptoms of fungal infection such as erythema, hair loss, scaling, and scabs on the affected skin completely disappear; skin color and texture return to normal, and hair regrows to cover the affected area; there is no recurrent itching or scratching behavior, and microscopic examination is negative for fungi.

[0063] Effective: The scope of skin lesions was significantly reduced, symptoms such as erythema, scaling, and hair loss were significantly alleviated, and hair began to regenerate; clinical symptoms were alleviated by more than 60%, the frequency of animal itching decreased, and the degree of fungal positivity in microscopic examination was significantly reduced.

[0064] Ineffective: The lesion area does not shrink significantly after treatment, and symptoms such as hair loss, scabs, and itching persist or worsen; there is no improvement in the signs of fungal infection, and microscopic examination still shows a large number of spores or hyphae structures.

[0065]

[0066] From the data in Table 2, it can be seen that: 5 cats in the experimental group were completely cured, 1 showed significant improvement, the overall cure rate was 83.33%, and the effective rate was 100%; none of the 6 cats in the control group showed improvement, the cure rate and effective rate were both 0%, and the ineffective rate was 100%.

[0067] Figure 7 The following chart compares the treatment progress of two typical cats in the experimental groups: On day 0, the cats displayed obvious symptoms of a fungal infection, including skin scaling and hair loss, particularly on the face, ear margins, and head and neck. By day 7, the inflammatory response had significantly subsided, with scabs forming on the damaged skin areas, the hair surrounding the area returning to a smooth surface, and the lesion area shrinking. By day 14, the affected skin had essentially returned to its normal color and texture, hair had re-covered, and the clinical symptoms had completely disappeared. Comparison of the control group data and image results clearly indicates that the cats not treated with the antibacterial skin repair hydrogel in Example 2 showed no significant improvement during the observation period, and some even experienced further hair loss and increased redness and swelling. These results further demonstrate the stability of the therapeutic efficacy and broad applicability of the present invention in actual clinical practice. Its composite ingredients not only effectively inhibit the growth and adhesion of the pathogenic bacteria Microsporum canis, but also promote skin barrier repair and alleviate inflammation, achieving comprehensive control of animal skin lesions.

[0068] In summary, the present invention, through the synergistic effect of aloe polysaccharides, a natural plant extract, and cat-derived probiotics, demonstrates strong clinical utility in the treatment of fungal skin diseases in pets. It is particularly suitable as an alternative to traditional antibiotics, providing a natural, gentle, and highly effective solution for the safe treatment of pet skin diseases. This product has a promising market potential and promising prospects for application and promotion.

[0069] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and is not limiting. Although the present invention is described in detail with reference to the preferred arrangement scheme, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of cat-derived Lactobacillus plantarum ( Lactobacillus plantarum ) ZYpet-014, characterized in that The Lactobacillus plantarum ZYpet-014 is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 32010 and a deposit date of September 20, 2024.

2. Use of Lactobacillus plantarum ZYpet-014 as claimed in claim 1 in preparing products for preventing and treating pet skin diseases.

3. The use according to claim 2, characterized in that The pathogenic bacteria of the pet skin disease is Microsporum canis.

4. The use according to claim 2 or 3, characterized in that The products include pet care products and topical medications.

5. The use according to claim 4, characterized in that The pet cleaning and care products include bath liquid, hair conditioner, and pet wipes.

6. The use according to claim 4, characterized in that The external medicine is one of a liquid preparation, an ointment, and a gel.

7. A pet skin antibacterial repair gel based on cat-derived probiotics and aloe polysaccharides, characterized in that: The raw materials of the antibacterial repair gel include: Lactobacillus plantarum ZYpet-014 as claimed in claim 1, aloe polysaccharide, carbomer, triethanolamine, zinc hyaluronate and water; Among them, the concentration of Lactobacillus plantarum ZYpet-014 was 1.0×10 8 ~1.0×10 10 CFU / mL; Based on the total amount of gel, the weight percentages of other raw materials are as follows: Aloe polysaccharide 0.25%~0.8% Carbomer 0.2%~1.0% Triethanolamine 0.1%~0.5% Zinc hyaluronate 0.1%~1.0%.

8. A method for preparing the pet skin antibacterial repair gel according to claim 7, characterized in that: The preparation method comprises the following steps: (1) Preparation of probiotic liquid: inoculate cat-derived Lactobacillus plantarum ZYpet-014 onto a slant culture medium for recovery, culture at 37°C for 24 hours, transfer once, pick a single colony and inoculate into MRS broth liquid culture medium, culture overnight on a shaker at 37°C, and store at 4°C for later use; take the amplified bacterial liquid, centrifuge it, take the precipitate, wash it with sterile PBS, and resuspend the washed probiotics in PBS to obtain Lactobacillus plantarum ZYpet-014 liquid; (2) Preparation of blank hydrogel: Carbomer was weighed in proportion and added to ultrapure water. After thorough stirring, the mixture was immersed in water at room temperature to allow it to fully hydrate and swell. Triethanolamine and zinc hyaluronate were then added and stirred to obtain a blank hydrogel. (3) Preparation of aloe polysaccharide solution: weigh aloe polysaccharide in proportion and add it to deionized water. Stir magnetically at 600 rpm / min at 30-40°C until completely dissolved. Cool to room temperature to obtain aloe polysaccharide solution, which is then sealed in a volumetric flask for later use. (4) The prepared aloe polysaccharide solution and the blank hydrogel were mixed, stirred for sufficient reaction, and then Lactobacillus plantarum ZYpet-014 bacterial solution was added and stirred evenly to obtain the pet skin antibacterial repair gel.

9. The preparation method according to claim 8, characterized in that In step (4), the volume ratio of the aloe polysaccharide solution to the blank hydrogel is 1:15-20.

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