A pet skin antibacterial repair gel based on cat-derived probiotics and aloe polysaccharides

Abstract

The application provides a pet skin antibacterial repair gel based on cat-derived probiotics and aloe polysaccharide, wherein a cat-derived probiotic with the function of resisting pathogenic bacteria of skin diseases is screened, the strain is identified as lactobacillus plantarum, the preservation number is CGMCC No. 32010, and the classification name is lactobacillus plantarum Lactobacillus plantarum The strain is applied to a pet gel product, and the pet skin antibacterial repair gel based on cat-derived probiotics and aloe polysaccharide is provided, wherein the cat-derived lactobacillus plantarum is combined with aloe polysaccharide-carbomer composite hydrogel with good biocompatibility to form an intelligent dressing system integrating antibacterial, repair and controlled release, and a new perspective based on'microecological therapy' is provided for pet skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of pet pharmaceutical preparation technology, and specifically relates to a pet skin antibacterial repair gel based on feline probiotics and aloe polysaccharides. Background Technology

[0002] With the continuous increase in the number of pet owners, pet health management has become a focus of social attention. Canine and feline fungal dermatitis, a common clinical disease in pets, suffers from high incidence, high recurrence rates, and lengthy treatment cycles, severely impacting not only the quality of life and animal welfare of pets but also imposing a heavy economic and care burden on pet owners. Statistics show that over 90% of pet fungal dermatitis infections are caused by *Microsporum canis*. This fungus causes complex symptoms, primarily circular hair loss (ringworm in cats), erythema, dandruff, accompanied by hair breakage and loss. It is highly contagious, and without timely intervention, it can easily lead to deep tissue infections or systemic secondary infections. More seriously, *Microsporum canis* has zoonotic characteristics, posing a potential public health and safety hazard and threatening the health of pet owners and their families.

[0003] Currently, clinical treatment of fungal skin diseases in pets still primarily relies on traditional methods such as antibiotics. However, the long-term and excessive use of antibiotics has led to increasingly serious drug resistance in *Microsporum canis*, and the emergence of multidrug-resistant strains has rendered conventional treatments increasingly ineffective. Furthermore, antibiotics have significant toxic side effects, potentially causing adverse reactions such as intestinal flora imbalance and liver and kidney damage, impacting the overall health of pets. In addition, the non-targeting nature of traditional treatments makes it difficult for drugs to precisely target the lesions, not only reducing treatment effectiveness but also prolonging the treatment cycle, further exacerbating drug resistance and severely hindering the sustainable development of pet dermatology treatment.

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

[0005] To address the problems existing in the prior art, the purpose of this invention is to propose a novel treatment strategy based on probiotic hydrogels, and to provide a pet skin antibacterial repair gel based on feline probiotics and aloe polysaccharides. In this invention, feline lactobacillus plantarum is combined with aloe polysaccharide-carbomer composite hydrogel with good biocompatibility to form an intelligent dressing system that integrates antibacterial, repair and controlled release.

[0006] The objective of this invention is achieved through the following technical solution: The first aspect of the present invention provides a strain of feline lactobacillus ( Lactobacillus plantarumZYpet-014, the Lactobacillus plantarum ZYpet-014, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.32010 and deposit date of September 20, 2024.

[0007] The second aspect of this invention provides the application of Lactobacillus plantarum ZYpet-014 described in the first aspect in the preparation of products for the prevention and treatment of pet skin diseases.

[0008] Furthermore, the pathogen causing the pet skin disease is Microsporum canis.

[0009] Furthermore, the products include pet grooming products and topical medications.

[0010] Furthermore, the pet grooming products include shampoo, conditioner, and pet wipes.

[0011] Furthermore, the topical medication is one of the following: liquid preparation, ointment, or gel.

[0012] The third aspect of this invention provides a pet skin antibacterial repair gel based on feline probiotics and aloe polysaccharides, wherein the raw materials of the antibacterial repair gel include: Lactobacillus plantarum ZYpet-014, aloe polysaccharides, carbomer, triethanolamine, zinc hyaluronic acid and water as described in the first aspect; The concentration of *Lactobacillus plantarum* ZYpet-014 was 1.0 × 10⁻⁶ viable cells. 8 ~1.0×10 10 CFU / mL; Based on the total amount of gel, the weight percentages of other ingredients are as follows: Aloe polysaccharides: 0.25%~0.8% Carbomer 0.2%~1.0% Triethanolamine 0.1%~0.5% Zinc hyaluronic acid 0.1%~1.0%.

[0013] The fourth aspect of this invention provides a method for preparing the pet skin antibacterial repair gel described in the third aspect, comprising the following steps: (1) Preparation of probiotic bacterial suspension: The feline Lactobacillus plantarum ZYpet-014 was inoculated onto the slant medium for recovery, cultured at 37℃ for 24h, and then transferred once. Single colonies were picked and inoculated onto MRS broth liquid medium, cultured overnight at 37℃ on a shaker, and stored at 4℃ for later use. The amplified bacterial suspension was taken, centrifuged, and the precipitate was taken and washed with sterile PBS. The washed probiotics were resuspended in PBS to obtain Lactobacillus plantarum ZYpet-014 bacterial suspension. (2) Preparation of blank hydrogel: Carbomer was weighed according to the proportion and added to ultrapure water. After stirring thoroughly, it was soaked at room temperature to allow it to fully hydrate and swell. Then, triethanolamine and zinc hyaluronic acid were added and stirred evenly to obtain blank hydrogel. (3) Preparation of aloe polysaccharide solution: Weigh aloe polysaccharide into an appropriate amount of deionized water, stir magnetically at 600 rpm / min for 15 min at 30-40℃ until completely dissolved, cool to room temperature to obtain aloe polysaccharide solution, and seal in volumetric flask for later use. (4) Mix the prepared aloe polysaccharide solution with the blank hydrogel, stir and react thoroughly, add Lactobacillus plantarum ZYpet-014 bacterial solution, stir evenly, and the pet skin antibacterial repair gel is obtained.

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

[0015] Information on strain preservation:

[0016] Lactobacillus plantarum ZYpet-014, deposited by the China General Microbiological Culture Collection Center (CGMCC); address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCCNo.32010; deposit date: September 20, 2024; classification and nomenclature: Lactobacillus plantarum Lactobacillus plantarum .

[0017] The advantages of this invention compared to the prior art are as follows:

[0018] 1. This invention screened and obtained a cat-derived probiotic strain with anti-dermatological pathogenic bacteria. This strain was identified as *Lactobacillus plantarum* through preservation and identification, with the preservation number CGMCC No. 32010 and the classification name *Lactobacillus plantarum*. Lactobacillus plantarum This provides a new perspective on pet skin diseases based on "skin microecology therapy".

[0019] 2. Traditional skin disease treatments often rely on antibiotics, but antibiotic overuse leads to drug resistance. In contrast, probiotics, by utilizing their antibacterial and immunomodulatory properties, can not only reduce pathogenic bacterial infections but also avoid drug resistance and secondary infections. Feline probiotics have better adaptability to the cat's skin surface and higher compatibility with the cat's skin microecological environment, thus offering greater specificity and effectiveness in treatment. This not only enables precise screening of functional probiotics but also further expands the application prospects of probiotics in the treatment of pet diseases.

[0020] 3. The aloe polysaccharides added to the product in this invention have been extensively studied for their excellent anti-inflammatory and healing-promoting properties. Furthermore, the active ingredients of probiotics, when combined with aloe polysaccharides possessing prebiotic properties, may synergistically enhance their antibacterial and repair functions. This type of hydrogel provides a platform for the sustained release of probiotics while utilizing 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 a dual function of "antibacterial and repair," providing an innovative application strategy for the healing of skin disease wounds.

[0021] 4. This invention uses hydrogel as a carrier for probiotics, providing ample growth space. Probiotics encapsulated within the hydrogel matrix are less affected by environmental stressors and conditions. It is characterized by its softness, good conformability, and excellent breathability, making it highly suitable for treating skin wounds and as a drug sustained-release carrier. As a carrier, the hydrogel can achieve sustained release of probiotics and aloe polysaccharides by adjusting its pore size and hydrophilicity, thereby prolonging the residence time of active ingredients on the skin surface and enhancing local therapeutic effects.

[0022] 5. This invention is specifically designed for Microsporum canis, the main pathogen causing fungal skin diseases in pets, thus improving the efficiency and effectiveness of treating fungal skin diseases in pets and demonstrating strong targeting.

[0023] 6. This invention utilizes feline probiotics as its core, combined with a low-toxicity ecological treatment platform using aloe vera polysaccharide hydrogel. Since both probiotics and aloe vera polysaccharides are natural ingredients, and a biocompatible hydrogel carrier is selected, this platform exhibits excellent biodegradability and low toxicity. It provides pets with a safer, less side-effect-prone treatment for skin diseases and wound healing. It is suitable for pregnant and young pets, ensuring their long-term health and offering high safety.

[0024] 7. The pet skin antibacterial repair gel prepared by this invention can significantly reduce the growth, adhesion 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 care products and has good application prospects. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The scanning electron microscope (SEM) images of different hydrogels in Test Example 1 at 20000× magnification; Figure 2 The rheological properties of different hydrogels in Test Example 2 are shown in the figure; where A is the shear stress, B is the apparent viscosity, and C is the dynamic viscoelastic modulus (G' and G''). Figure 3 The result of pH release rate of different hydrogels in Test Example 3 is shown in the figure; Figure 4 The results of probiotic release rate in different hydrogels in Test Example 4 are shown in the figure. Figure 5 The results of the inhibition zone determination in different hydrogels in Test Example 5 are shown in Figure A, where A is the plate inhibition zone result of different hydrogels and B is the statistical result of the inhibition zone diameter of different hydrogels. Figure 6 The results of the biofilm inhibition rate measurement of different hydrogels in Test Example 6 are shown in Figure A, where A is the absorbance value of different hydrogels measured at OD595 nm, and B is the biofilm inhibition rate of different hydrogels against pathogenic bacteria. Figure 7 The image shows the therapeutic effect of the pet skin antibacterial repair gel in Case 7 on fungal skin disease in a pet cat. Detailed Implementation

[0026] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0027] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0029] In the following examples, aloe polysaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and carbomer 934 was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0030] Example 1: Screening and Identification of Lactobacillus plantarum

[0031] This embodiment provides the process for strain screening and identification: (1) Isolation of strains: Fecal samples were collected from healthy cats at cat shelters 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 the mixture was thoroughly shaken to prepare the initial 10-fold dilution. Subsequent serial dilutions were performed to 10-fold.3 and 10 5 100 μL of different concentrations of the diluted solution were inoculated onto MRS solid culture plates and cultured at 30°C under anaerobic conditions for 48 hours to isolate intestinal lactic acid bacteria.

[0032] (2) Purification of the strain: Colonies of varying sizes and with milky white protrusions were selected from the primary culture plates for single colony selection. The three-zone streak method was used to repeatedly inoculate the MRS plates, and the plates were anaerobically cultured at 37°C for 48 hours. This process was repeated for 3 rounds to finally obtain a stable and purified target strain.

[0033] (3) Morphological identification: Gram staining technique was used to make a preliminary observation of the morphology of the strain. A small number of single colonies were evenly spread on a glass slide in a sterile water droplet and air-dried. They were then stained with crystal violet, mordanted with iodine solution, destained with 95% alcohol, and counterstained with safranin in sequence. After rinsing and air-drying, the morphological characteristics and staining reaction of the bacterial cells were observed under an optical microscope to determine their Gram properties.

[0034] (4) Glucose gas production experiment: Selected single colonies of lactic acid bacteria were 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 Durham tubes at a 1% inoculation rate and cultured statically at 30°C for 7 days. Observe whether bubbles are formed in the Durham tubes to determine whether the strain is homofermentation (aerobic) or heterofermentation (gas-producing).

[0035] (5) Physiological experiment: Add 100 μL of 3% hydrogen peroxide solution to a petri dish, place the test colony in the solution, and observe whether a bubble reaction occurs. If bubbles appear, the catalase is positive; if no bubbles are produced, the result is negative. This further helps to determine the classification characteristics of the strain.

[0036]

[0037] As shown in Table 1, strain ZYpet-014 is a homofermentative lactic acid bacterium, with a positive Gram staining result and a negative catalase result.

[0038] (6) Strain identification: The strain was anaerobically cultured on MRS plates at 30ºC for 48 h, and single colonies were picked for PCR. The primers were the universal 16S rRNA primers 27F and 1492R. The PCR reaction conditions were: 95ºC pre-denaturation for 3 min; 95ºC denaturation for 15 s, 60ºC annealing for 15 s, 72ºC extension for 90 s, 35 cycles; and 72ºC final extension for 5 min. The obtained 16S rRNA was compared with the sequences of known strains in GenBank using BLAST analysis. The ZYpet-014 strain was identified as *Lactobacillus plantarum* (Lactobacillus plantarum). Lactobacillus plantarumIt was named Lactobacillus plantarum ZYpet-014.

[0039] ZYpet-014 strain 16S rRNA gene sequence:

[0040] Example 2: Preparation of an antibacterial and repairing gel for pet skin based on feline probiotics and aloe polysaccharides

[0041] This embodiment provides a pet skin antibacterial repair gel based on feline probiotics and aloe polysaccharides, prepared by the following method: (1) Preparation of probiotic culture: Under aseptic conditions, *Lactobacillus plantarum* ZYpet-014 from lyophilized tubes was inoculated onto slant agar for resuscitation. After incubation at 37°C for 24 h, the culture was transferred once. A single colony was picked and inoculated into 100 mL of MRS broth. The culture was incubated overnight at 37°C and 130 rpm / min on a shaker, and stored at 4°C for later use. The amplified culture was centrifuged at 3000 rpm for 5 min, and the precipitate was collected and washed with sterile PBS. The washed probiotics were resuspended in PBS for later use, so that the concentration of the culture was approximately 1.0 × 10⁻⁶. 9 CFU / mL yields Lactobacillus plantarum ZYpet-014 bacterial suspension.

[0042] (2) Preparation of blank hydrogel: Carbomer 934 was weighed according to the proportion and added to ultrapure water to make its mass concentration 0.5%. After stirring magnetically at 1200 r / min for 1 h, it was soaked at room temperature for 12 h to fully hydrate and swell. Then, 0.25% triethanolamine and 0.5% zinc hyaluronic acid were added and stirred evenly to obtain blank hydrogel (CON-hydrogel).

[0043] (3) Preparation of aloe polysaccharide solution: Weigh 0.5% aloe polysaccharide into an appropriate amount of deionized water, stir magnetically at 600 rpm / min for 15 min at 30-40℃ until completely dissolved, cool to room temperature to obtain aloe polysaccharide solution, and seal in volumetric flask for later use.

[0044] (4) The prepared aloe polysaccharide solution and blank hydrogel were mixed at a volume ratio of 1:19. After mixing and stirring at 300 rpm for 1 h, Lactobacillus plantarum ZYpet-014 bacterial solution was added and stirred evenly to obtain the pet skin antibacterial repair hydrogel (LP+AVE-hydrogel). It was sealed and stored in a refrigerator at 4℃.

[0045] Comparative Example 1: Preparation of Probiotic Carbomer Composite Hydrogel

[0046] This comparative example prepared a composite hydrogel according to the method described in Example 2, the difference being that aloe polysaccharides were not added to the blank hydrogel in step (4). Specifically: (4) Add Lactobacillus plantarum ZYpet-014 bacterial solution to the blank hydrogel, stir evenly, and you will get probiotic carbomer composite hydrogel (LP-hydrogel). Seal and store in a refrigerator at 4°C.

[0047] Test Example 1: Scanning Electron Microscopy Characterization of Hydrogels

[0048] This test case investigates the internal structure 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, then freeze-dried in a vacuum freeze dryer for 48 hours. The freeze-dried hydrogels were then brittle-crystallized with liquid nitrogen, and cross-sections were sputter-coated with gold under vacuum. The structure was observed using a scanning electron microscope (SEM: Sigma30, Zeiss, Germany) with an accelerating voltage of 20kV and a magnification of 2000x to observe the internal morphology and distribution of probiotics in the hydrogels.

[0049] Depend on Figure 1 As observed by high-magnification SEM, compared with CON-hydrogel 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, maintaining their morphology intact and exhibiting a typical rod-like structure. Compared with LP-hydrogel, the LP+AVE-hydrogel hydrogel described in Example 2 showed a richer distribution of probiotics with more regular cell morphology. This indicates that the addition of aloe polysaccharides can improve the microstructure characteristics of the hydrogel, promote probiotic proliferation, and protect the integrity of bacterial cells, allowing more probiotics to successfully attach to the gel matrix. This may have a positive impact on its metabolic activity and storage stability.

[0050] Test Example 2: Determination of Rheological Properties of Hydrogels

[0051] This test case investigates the rheological properties of different hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. The specific methods are as follows: A rheometer (Anton Paar-MCR301) with a cone or plate clamp was used. The test temperature was set to 25°C, the gap to be 1.0 mm, and the shear rate to be 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 frequency sweep test was conducted with a frequency range of 0.1–100 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 system, analyzing the elastic and viscoelastic characteristics of the gel. Each sample was tested three times.

[0052] like Figure 2 As shown, all three gel systems (CON-hydrogel, LP-hydrogel, and LP+AVE-hydrogel) exhibit typical shear-thinning characteristics, meaning that shear stress and apparent viscosity gradually decrease with increasing shear rate. The shear stress and viscosity of the LP+AVE-hydrogel system are significantly lower than the other two groups, indicating that the addition of aloe polysaccharides effectively reduces the cohesive force of the gel network and increases the flexibility and spreadability of the gel. Furthermore, dynamic rheological analysis shows that the storage modulus (G') of each gel system is significantly higher than the loss modulus (G''), exhibiting obvious elastic gel characteristics. The storage modulus and loss modulus of LP+AVE-hydrogel are both lower than the other two groups, further indicating that the addition of aloe polysaccharides effectively improves the softness and deformability of the gel system, suggesting that its structure is softer and easier to spread.

[0053] The above results indicate that the LP+AVE-hydrogel prepared in Example 2 has both good flexibility and a stable gel structure, making it more suitable for clinical application in wound dressings.

[0054] Test Example 3: Determination of pH value in the hydrogel microenvironment

[0055] This test case investigates the microenvironment pH of different hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. The specific method is as follows: 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 PBS (pH 7.4) at 25°C for 72 h. The pH of the immersion solution was measured with a pH meter at specific time intervals to evaluate the pH of the microenvironment formed by the different hydrogels.

[0056] 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 to inhibit the growth of pathogens and enhance the skin's microecological balance. It is a new treatment method that promotes wound healing by inducing the microenvironment.

[0057] Results of pH changes in the hydrogel microenvironment as follows Figure 3 As shown, the CON-hydrogel group maintained a stable neutral pH (approximately 7.0) during cultivation, while the pH of both the LP-hydrogel and LP+AVE-hydrogel groups showed a gradual decreasing trend. The LP-hydrogel system exhibited a more significant pH decrease, reflecting the probiotics' strong ability to metabolize organic acids (such as short-chain fatty acids and lactic acid). The LP+AVE-hydrogel group showed a similar but more stable pH trend, suggesting that the addition of aloe polysaccharides may regulate the metabolic activity of probiotics, maintaining a stable acidic microenvironment. This acidic microenvironment is beneficial for inhibiting the growth of pathogenic microorganisms and promoting skin lesion healing.

[0058] Test Example 4: Determination of Probiotic Release Rate in Hydrogels

[0059] This test case investigates the probiotic release rate of different hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group. The specific experimental 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. At predetermined time intervals, 100 μL of the surrounding PBS was spread on MRS solid medium for colony counting. The release rate of probiotics was calculated based on the percentage of probiotics in the release medium relative to the total amount of encapsulated probiotics.

[0060] like Figure 4 As shown, both LP-hydrogel and LP+AVE-hydrogel hydrogels exhibited a continuous and stable probiotic release pattern. Compared to the LP-hydrogel group, the LP+AVE-hydrogel group showed a significantly higher probiotic release rate at all time points. PThe cumulative release rate of aloe vera polysaccharides (<0.05%) reached approximately 35% by the end of 6 hours, significantly higher than that of the LP-hydrogel group. This indicates that the addition of aloe vera polysaccharides optimized the structural characteristics of the hydrogel system, effectively improved the release efficiency of probiotics, and further demonstrated its excellent performance as a probiotic carrier material.

[0061] Test Example 5: Determination of Antibacterial Properties of Hydrogels

[0062] This test case investigates 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 causing pet dermatitis, was determined using a perforation method. In a clean bench, LB solid medium sterilized by high temperature and autoclave was cooled to 45-50℃, and a suspension of 1% *Microsporum canis* was added (10... 7 After mixing thoroughly (CFU / mL), pour the mixture into a plate (approximately 20 mL / plate) and allow it to solidify horizontally for later use. Using a sterile punch, make three holes (10 mm in diameter) in the test plate. Inject 200 μL of the LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel samples prepared in Example 2, Comparative Example 1, and the blank control group into each hole, respectively. Incubate at 37°C for 24 h. Remove the plates and determine the antibacterial ability of the hydrogels by the diameter of the inhibition zone.

[0063] Experimental Results: The antibacterial properties of each group of gels were evaluated using the inhibition zone experiment. For example... Figure 5 As shown, the CON-hydrogel hydrogel did not exhibit significant antibacterial activity, while the LP-hydrogel hydrogel formed a distinct inhibition zone (approximately 15 mm), indicating that the probiotics possess certain antibacterial activity against Microsporum canis. This may be due to the release of probiotic metabolites such as lactic acid, organic acids, or other antibacterial substances. Further addition of aloe polysaccharides significantly increased the diameter of the inhibition zone in the LP+AVE-hydrogel hydrogel (approximately 18 mm). P The result (<0.05) indicates that aloe polysaccharides can significantly enhance the antibacterial properties of probiotics. This result reveals that the combined use of aloe polysaccharides and probiotics has a synergistic antibacterial effect. Aloe polysaccharides may promote the metabolic activity and proliferation of probiotics through the prebiotic effect, enabling probiotics to secrete more antibacterial metabolites, thereby enhancing the antibacterial effect. This further expands the functional potential of probiotic preparations.

[0064] Test Example 6: Determination of the inhibition rate of hydrogels on pathogenic bacterial biofilms

[0065] This test case investigates the inhibition rate of different hydrogels prepared in Example 2, Comparative Example 1, and the blank control group on pathogenic bacterial biofilms. The specific experimental method is as follows: Using an inoculation loop, pick up the Microsporum canis inoculum from the slant of the test tube and place it in sterile water containing glass beads. Shake by hand for several minutes to disperse the spores, then filter to prepare a concentration of 10. 7 CFU / mL mixed spore suspension was added to 24-well plates. 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 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 stain the biofilm for 15 min. The staining solution was removed, and excess crystal violet was washed away. 30% acetic acid was added to the stained biofilm to dissolve the pigment. The dissolved staining solution was diluted 5 times, and the absorbance at OD595 nm was measured using a microplate reader. The inhibition rate of the biofilm was calculated.

[0066] Biofilm inhibition rate (%) = (Ax - Ay) / Ax × 100%.

[0067] In the formula, Ax: the OD595 nm value measured in the control group; Ay: the OD595 nm value measured in the experimental group.

[0068] Experimental Results: The inhibitory effects of CON-hydrogel, LP-hydrogel, and LP+AVE-hydrogel hydrogels on biofilm formation were determined by violet staining. Biofilm inhibition results are shown below. Figure 6 As shown, violet staining revealed that the empty CON-hydrogel group exhibited the strongest biofilm formation ability, while LP-hydrogel significantly reduced the formation of Microsporum canis biofilm. P <0.05%. Further addition of aloe polysaccharides enhanced the biofilm inhibition effect of the LP+AVE-hydrogel group, with an inhibition rate significantly higher than that of the LP-hydrogel group ( P <0.05). This result indicates that aloe polysaccharides have significant prebiotic properties, can enhance the metabolic function of probiotics, and effectively inhibit the formation of pathogenic biofilms, demonstrating high potential for antibacterial and anti-biofilm applications.

[0069] Test Example 7: Clinical Trial of Treatment Efficacy for Fungal Dermatitis in Pet Cats

[0070] To verify the efficacy 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, effectiveness rate, and ineffectiveness rate, the following animal experiments were conducted in this test case: 1. Experimental animals and grouping A total of 12 pet cats with clinically diagnosed fungal dermatitis were selected and randomly divided into two groups: Experimental group (n=6): Received treatment 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, animals underwent health assessments, case registration, and photographic recording of affected areas, ensuring that there were no significant differences in their physical condition.

[0071] 2. Experimental Methods Initial examination; Severity of itching: Observe whether the animal frequently scratches, bites, or rubs the affected area; Hair removal details: Record the number, distribution range, and edge clarity of localized or ring-shaped hair removal patches; Skin lesion characteristics: Assess the presence of erythema, scaling, crusting, hyperkeratosis, and annular lesions on the skin surface; Lesion exudation and crusting: Record whether there is exudate, dry crust, or thick grayish-white or yellow crust.

[0072] Intervention plan: The experimental group applied the compound 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.

[0073] Observation records: Changes in the affected area were recorded on days 0, 7, and 14 of the trial. Photos were taken for comparison and scored independently by two clinical veterinarians to ensure the objectivity of the assessment.

[0074] 3. Criteria for Evaluating Therapeutic Effect Cure: The symptoms of fungal infection, such as erythema, hair loss, scaling, and crusting, on the affected skin completely disappear; the skin color and texture return to normal, and hair regrows to cover the lesion area; there is no recurrent itching or scratching, and microscopic examination for fungi is negative.

[0075] Effective: The area of ​​skin lesions is significantly reduced, symptoms such as erythema, scaling, and hair loss are significantly relieved, and hair begins to regenerate; clinical symptoms are reduced by more than 60%, the frequency of itching in animals decreases, and the degree of fungal positivity in microscopic examination is significantly reduced.

[0076] Ineffective: The lesion area does not shrink significantly after treatment, and symptoms such as hair loss, scabbing, and itching persist or worsen; signs of fungal infection do not improve, and microscopic examination still shows a large number of spores or hyphae.

[0077]

[0078] As shown in Table 2, 5 cats in the experimental group were completely cured and 1 cat showed significant improvement, with an overall cure rate of 83.33% and an effectiveness rate of 100%. In the control group, none of the 6 cats showed improvement, with a cure rate and effectiveness rate of 0% and an ineffective rate of 100%.

[0079] Figure 7 The following is a comparison of the treatment process of two typical cats in the experimental group: On day 0, the cats exhibited obvious symptoms of fungal infection, such as scaly skin and hair loss (ringworm), especially concentrated on the face, ear margins, and head and neck. On day 7, the inflammatory response significantly decreased, the damaged skin areas scabbed over, the area around the hair became smooth, and the lesion area shrank. By day 14, the affected skin had basically returned to normal color and texture, the hair had re-covered the skin, and the clinical symptoms completely disappeared. Comparing the data and image results with the control group, it can be clearly pointed out that the cats that did not receive the skin antibacterial repair hydrogel treatment in Example 2 did not show significant improvement in their condition during the observation period, and some even experienced further hair loss and increased redness and swelling. The above results further verify the efficacy stability and wide applicability of the product of this invention in actual clinical practice. Its compound components can not only effectively inhibit the growth and adhesion of the pathogenic fungus Microsporum canis, but also achieve comprehensive control of animal skin lesions by promoting skin barrier repair and inflammation relief.

[0080] In summary, this invention, through the synergistic effect of natural plant extract aloe polysaccharides and feline probiotic components, demonstrates strong clinical value in the treatment of fungal dermatitis in pets. It is particularly suitable as a replacement for traditional antibiotics, providing a natural, gentle, and highly effective solution for the safe treatment of pet skin diseases. This product has a solid foundation for market transformation and promising prospects for application and promotion.

[0081] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A pet skin antibacterial and repairing gel based on feline probiotics and aloe polysaccharides, characterized in that, The raw materials of the antibacterial repair gel are: Lactobacillus plantarum ZYpet-014, aloe polysaccharide, carbomer, triethanolamine, zinc hyaluronic acid and water; The *Lactobacillus plantarum* ZYpet-014 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32010 and deposit date of September 20, 2024; the pathogen causing the pet skin disease is *Microsporum canis*. Based on the total amount of gel, the weight percentages of other ingredients are as follows: Aloe polysaccharides 0.5% Carbomer 0.5% Triethanolamine 0.25% Zinc hyaluronic acid 0.5%; The preparation method of the pet skin antibacterial repair gel includes the following steps: (1) Preparation of probiotic culture: Lactobacillus plantarum ZYpet-014 (feline origin) was inoculated onto slant agar for resuscitation. After incubation at 37°C for 24 h, it was transferred once. Single colonies were picked and inoculated onto MRS broth liquid medium and cultured overnight at 37°C with shaking. The culture was then stored at 4°C for later use. The amplified culture was centrifuged, and the precipitate was washed with sterile PBS. The washed probiotics were resuspended in PBS to achieve a bacterial concentration of 1.0 × 10⁻⁶. CFU / mL yields Lactobacillus plantarum ZYpet-014 bacterial suspension; (2) Preparation of blank hydrogel: Carbomer was weighed according to the proportion and added to ultrapure water. After stirring thoroughly, it was soaked at room temperature to allow it to fully hydrate and swell. Then, triethanolamine and zinc hyaluronic acid were added and stirred evenly to obtain blank hydrogel. (3) Preparation of aloe polysaccharide solution: Weigh aloe polysaccharide according to the proportion and add it to deionized water. Stir magnetically at 600 rpm / min at 30~40℃ until completely dissolved. Cool to room temperature to obtain aloe polysaccharide solution and seal it in volumetric flask for later use. (4) The prepared aloe polysaccharide solution and blank hydrogel are mixed at a volume ratio of 1:

19. After stirring and reacting fully, Lactobacillus plantarum ZYpet-014 bacterial solution is added and stirred evenly to obtain the pet skin antibacterial repair gel.

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