Aloe vera extract-probiotic composite hydrogel for pet skin diseases

Abstract

The present invention belongs to the technical field of pharmaceutical preparations for pets, and particularly relates to an aloe vera extract-probiotic composite hydrogel for treating pet skin diseases. The preparation method comprises: under sterile conditions, resuscitating Lactobacillus zonitrosum ZYpet-012 (CGMCC No. 32008) in a solid slant culture medium, culturing in a liquid culture medium, and then centrifuging and washing to obtain a probiotic suspension; preparing a blank hydrogel using carbomer 940, triethanolamine, and zinc hyaluronate; mixing the aloe vera extract solution with the blank hydrogel, and then adding the probiotic suspension to produce the composite hydrogel. This invention utilizes the anti-inflammatory, antibacterial, and prebiotic properties of aloe vera extract, combined with the skin microbiome-regulating effects of Lactobacillus zonitrosum ZYpet-012, to prepare a composite hydrogel that is effective in treating pet skin diseases, offering advantages such as natural safety and precise targeting.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations for pets, and in particular relates to an aloe vera extract-probiotic composite hydrogel for pet skin diseases. Background Art

[0002] Pet skin diseases, particularly those caused by bacterial infections, are among the most common and recurring ailments in dogs and cats. Staphylococcus pseudintermedius is one of the most common pathogens of bacterial skin diseases in pets, with studies showing it accounts for a significant proportion of these cases. These infections not only cause localized skin lesions such as redness, swelling, and abscesses, but in severe cases can even lead to systemic infections, significantly increasing the difficulty of treatment and the cost of care.

[0003] Currently, pet skin care products on the market primarily target Staphylococcus aureus and Escherichia coli, lacking specific inhibition of Staphylococcus pseudintermedius and resulting in inaccurate treatment. Furthermore, traditional treatments generally rely on antibiotics or antiparasitic drugs, such as ketoconazole, florfenicol, and terbinafine hydrochloride. However, these drugs are associated with significant side effects, increased drug resistance, and environmental pollution, making them difficult to meet the needs of long-term, safe, and sustainable treatment. Summary of the Invention

[0004] In order to overcome the problems of the prior art, the present invention proposes an innovative treatment strategy: taking pet-derived probiotics as the core, combining the prebiotic properties of aloe vera extract, and using hydrogel as a three-dimensional carrier to construct a treatment system, a kind of aloe vera extract-probiotic composite hydrogel for pet skin diseases is provided. The composite hydrogel not only has good moisture retention, air permeability and biocompatibility, but also can simulate the extracellular matrix structure, provide a protective microenvironment for probiotics, improve its colonization rate and stability, and achieve sustained release and local targeting of probiotics. Through the synergy of the three, a set of pet skin disease treatment system with high efficiency, safety, low toxicity, and dual functions of specific antibacterial and healing promotion is constructed, filling the technical gap in the current market.

[0005] With modern medicine's in-depth exploration of microecological therapies, the field of skin disease treatment is undergoing a transformation from traditional drugs to bioactive preparations. During the research and development process, it was discovered that probiotics, with their safe and non-resistant biological properties, have emerged as a promising candidate for skin disease treatment. Systematic research into the pet skin microecological environment confirmed that specific probiotic strains are highly ecologically compatible with pet skin flora. By regulating microbial community structure, strengthening the skin's physical barrier, and competitively inhibiting pathogen adhesion, they demonstrate significant advantages in the targeted treatment of pet skin diseases. Furthermore, in the screening of natural plant active ingredients, aloe vera extract has been validated across multiple dimensions for its pharmacological activity, demonstrating not only anti-inflammatory, antibacterial, and tissue repair-accelerating properties, but also, due to its prebiotic properties, significantly enhancing the metabolic efficiency and antibacterial efficacy of probiotics, providing an innovative direction for the development of new pet skin disease treatments.

[0006] In view of this, the purpose of the present invention is achieved like this:

[0007] The first aspect of the present invention provides a method for preparing an aloe vera extract-probiotic composite hydrogel for treating pet skin diseases, comprising the following steps:

[0008] (1) Preparation of probiotic suspension: Under sterile conditions, freeze-dried animal combined lactobacillus ( Ligilactobacillus animalis ZYpet-012 was inoculated onto MRS solid slant medium and revived at 37°C. The cells were then transferred to MRS liquid medium and cultured overnight at 37°C in a shaker. The culture was centrifuged, the supernatant discarded, and the cells were washed with sterile PBS and resuspended to obtain a probiotic suspension.

[0009] The animal-associated Lactobacillus ZYpet-012 is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No.32008 and the deposit date: September 20, 2024;

[0010] (2) Preparation of blank hydrogel: Weigh carbomer 940 powder, add it to ultrapure water, stir thoroughly, let it stand at room temperature to fully swell, then add triethanolamine and zinc hyaluronate in sequence, stir evenly, and prepare blank hydrogel;

[0011] (3) Preparation of aloe vera extract solution: weigh aloe vera extract and dissolve it in deionized water. Stir it with a magnetic stirrer at 30-40°C until it is completely dissolved. Cool it to room temperature to obtain the aloe vera extract solution. Seal it in a volumetric flask and set aside.

[0012] (4) The aloe vera extract solution and the blank hydrogel are mixed and stirred evenly, and then the probiotic suspension prepared in step (1) is added and mixed evenly to obtain the aloe vera extract-probiotic composite hydrogel for pet skin diseases.

[0013] Furthermore, in step (1), the number of viable bacteria of animal-associated Lactobacillus ZYpet-012 in the probiotic suspension reaches 1.0×10 8 ~1.0×10 10 CFU / mL.

[0014] Furthermore, in step (2), the mass concentration of carbomer 940 powder in the blank hydrogel is 0.2% to 1.0%, the mass concentration of triethanolamine is 0.1% to 0.5%, and the mass concentration of zinc hyaluronate is 0.1% to 1.0%.

[0015] Furthermore, in step (3), the volume ratio of the aloe extract solution to the blank hydrogel is 1:15-20; and the mass concentration of the aloe extract in the aloe extract solution is 0.1%-1.0%.

[0016] A second aspect of the present invention provides an aloe vera extract-probiotic composite hydrogel for pet skin diseases, wherein the composite hydrogel is prepared by the method described in the first aspect.

[0017] The third aspect of the present invention provides an animal-associated lactobacillus ( Ligilactobacillus animalis ) ZYpet-012, deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCC No.32008, and the deposit date: September 20, 2024.

[0018] The fourth aspect of the present invention provides the use of the animal-associated Lactobacillus ZYpet-012 described in the first aspect in the preparation of a product for preventing and treating pet skin diseases.

[0019] Furthermore, the pathogenic bacteria of the pet skin disease is Staphylococcus pseudintermedius.

[0020] Furthermore, the products include pet care products and external medicines.

[0021] Furthermore, the pet washing and care products include bath liquid, hair conditioner, and pet wipes.

[0022] Culture deposit information:

[0023] Lactobacillus animalis ZYpet-012, deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC); Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; Deposit number: CGMCC No. 32008, Deposit date: September 20, 2024; Taxonomic name: Lactobacillus animalis Ligilactobacillus animalis .

[0024] The advantages and beneficial effects of the present invention are:

[0025] (1) The present invention innovatively utilizes probiotics with specific inhibitory activity against Staphylococcus pseudintermedius to achieve precise treatment of bacterial skin diseases in pets, effectively circumventing the problem of antibiotic resistance and meeting the needs of pet owners for natural, safe and green pet care products.

[0026] (2) The present invention uses aloe vera extract as a co-carrier of probiotics, innovatively exerting its prebiotic properties, producing a synergistic effect with the metabolic activity of probiotics, greatly improving the antibacterial and wound repair capabilities of probiotics, and innovatively developing a pet topical dressing with biological activity.

[0027] (3) The present invention utilizes the special three-dimensional network structure characteristics of the hydrogel material to effectively improve the long-term release ability of probiotics and aloe vera extracts on the skin surface. At the same time, it has excellent air permeability, moisturizing properties and affinity, forming an ideal wound healing microenvironment, significantly improving the treatment effect and animal comfort.

[0028] (4) The present invention is specifically designed for Staphylococcus pseudintermedius, the most common cause of pet skin diseases, filling the market gap in the specific antibacterial aspect of current pet skin disease care products. It is highly targeted and has significant therapeutic effects.

[0029] (5) The present invention uses natural biocompatible ingredients (probiotics, aloe vera extract and hydrogel carrier), which have low toxicity and side effects, high safety, and are suitable for pregnant pets and young pets. It has no obvious side effects after long-term use and meets the safety needs of pet owners for pet care products.

[0030] (6) The present invention, through the introduction of probiotics and the regulation of the hydrogel sustained-release system, constructs a cat-derived probiotic combination suitable for cat skin microecology through specific strains, which can avoid the problem of poor effect caused by microecological mismatch, achieve balanced regulation of cat skin microecology, provide a new entry point for the cure of pet skin diseases, and has high clinical application potential.

[0031] (7) The present invention innovatively proposes a new strategy for the treatment of bacterial skin diseases in pets based on a composite hydrogel of probiotics and natural aloe vera extract. This strategy not only achieves precise and efficient inhibition of Staphylococcus pseudintermedius, but also provides a natural, green, and non-resistant solution for the safe and effective treatment of pet skin diseases, and is expected to provide new ideas for the development of pet skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the accompanying drawings and examples.

[0033] Figure 1Shows the live / dead bacterial staining image (scale bar 50 μm) and bacterial survival rate of free bacteria in the probiotic hydrogel of Test Example 1 of the present invention;

[0034] Figure 2 1 is an appearance picture of different hydrogels in Test Example 2 of the present invention and a scanning electron microscope structure picture at a magnification of 300×;

[0035] Figure 3 1 is a diagram showing the relaxation time (T2) distribution results of the LF-NMR spectra of different hydrogels in Test Example 3 of the present invention;

[0036] Figure 4 4 is a graph showing the swelling rate measurement results of different hydrogels in Test Example 4 of the present invention;

[0037] Figure 5 This is a graph showing the hemolysis rate determination results of different hydrogels in Test Example 5 of the present invention;

[0038] Figure 6 This is a graph showing the antibacterial performance of different hydrogels in Test Example 6 of the present invention;

[0039] Figure 7 : This is a graph showing the results of bacterial scanning electron microscopy analysis in Test Example 7 of the present invention; A shows Staphylococcus aureus treated with CON-hydrogel, B shows Staphylococcus aureus treated with LP-hydrogel, C shows Staphylococcus aureus treated with LP+AVE-hydrogel, and D shows the normal bacterial morphology of LP.

[0040] Figure 8 This is a diagram showing the therapeutic effect of bacterial skin disease on pet cats in Test Example 8 of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In the following examples, aloe vera extract was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and carbomer 934 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0045] The data in the following examples are expressed as "mean ± standard deviation" and SPSS 22.0 software was used for one-way analysis of variance. P A value <0.05 indicates a significant difference. The graphs in the examples were created using GraphPad (GraphPad Prism, version 8.0.2, USA).

[0046] Example 1: Screening and identification of animal-associated lactobacilli

[0047] This example provides a strain screening and identification process:

[0048] (1) Isolation of strains: Skin samples of healthy cats were collected from catteries and animal rescue stations in Yancheng District, Luohe City. 0.5 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 ~10 7 100 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 hours to isolate lactic acid bacteria from the skin.

[0049] (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.

[0050] (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.

[0051] (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.

[0052] (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.

[0053]

[0054] As can be seen from Table 1, strain ZYpet-012 is a homofermentative lactic acid bacterium, with Gram staining results being positive and catalase negative.

[0055] (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-012 strain was identified as Lactobacillus animalis ( Lactobacillus plantarum ), named animal-associated Lactobacillus ZYpet-012.

[0056] ZYpet-012 strain 16S rRNA gene sequence:

[0057]

[0058] Example 2: Preparation of Aloe Vera Extract-Probiotic Composite Hydrogel for Pet Skin Diseases

[0059] This embodiment provides an aloe vera extract-probiotic composite hydrogel for pet skin diseases, and the preparation method is as follows:

[0060] (1) Preparation of probiotic suspension: Under sterile conditions, freeze-dried animal combined lactobacillus ( Ligilactobacillus animalis ZYpet-012 was inoculated onto MRS solid slant medium and allowed to recover at 37°C for 24 hours. The cells were then transferred to 100 mL of MRS liquid medium and cultured overnight at 37°C at 130 rpm. The culture was centrifuged at 3000 rpm for 5 minutes, the supernatant discarded, and the suspension was washed with sterile PBS and resuspended to obtain a probiotic suspension. The viable count of the suspension reached approximately 1.0 × 10 9 CFU / mL.

[0061] The animal-associated Lactobacillus ZYpet-012 is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No.32008 and the deposit date: September 20, 2024;

[0062] Preparation of blank hydrogel: Weigh 0.5% carbomer 940 powder, add it to ultrapure water, stir thoroughly, and let it stand at room temperature for 12 hours to fully swell. Then, add 0.25% triethanolamine and 0.5% zinc hyaluronate in sequence and stir evenly to prepare blank hydrogel (CON-hydrogel).

[0063] (3) Preparation of aloe vera extract solution: Weigh 0.6% aloe vera extract in a suitable amount of deionized water, stir magnetically at 700 rpm / min at 30-40°C for 20 min until completely dissolved, and cool to room temperature to obtain aloe vera multi-extract solution, which is then sealed in a volumetric flask for later use.

[0064] (4) The aloe extract solution and the blank hydrogel were mixed in a volume ratio of 1:19, stirred at 300 rpm for 1 h to mix evenly, and then the probiotic suspension prepared in step (1) was added and mixed to obtain the aloe extract-probiotic composite hydrogel for pet skin diseases (LP+AVE-hydrogel), which was sealed and stored at 4°C for later use.

[0065] Comparative Example 1: Preparation of probiotic carbomer composite hydrogel for pet skin diseases

[0066] This embodiment provides a probiotic composite hydrogel for treating pet skin diseases. The preparation method is similar to that of Example 2, except that no aloe extract solution is added in step (3). That is, the probiotic suspension prepared in step (1) is directly added to the blank hydrogel (CON-hydrogel), and the mixture is stirred at 300 rpm to obtain a probiotic carbomer composite hydrogel (LP-hydrogel). The hydrogel is sealed and stored at 4°C for later use.

[0067] Test Example 1: Determination of probiotic activity in hydrogel

[0068] This test example measured the probiotic activity in different hydrogels. Specifically, the LP+AVE-hydrogel, LP-hydrogel, and free probiotic IP-Free prepared in Example 2 and Comparative Example 1 were stored at 4°C for 48 hours. Live and dead bacteria were then stained with 5 μM SYTO@9 and 2 μg / mL PI, respectively. The staining was incubated at 37°C in the dark for 15 minutes, after which the staining solution was discarded and washed away with sterile saline. A 10 μL droplet of the bacterial solution was placed on a glass slide, covered with a coverslip, and imaged at 200x magnification using a laser confocal microscope (OLYMPUS FV1200). Green fluorescence indicates live bacteria, while red fluorescence indicates dead bacteria. Finally, the bacterial survival rate in the different treatment groups was calculated using fluorescence intensity using ImageJ software.

[0069] Depend on Figure 1 Live / Dead staining using laser confocal microscopy revealed that the survival of probiotics after 48 hours of storage was significantly reduced for the free probiotic LP-Free at 4°C. However, the probiotics loaded into LP-hydrogel and LP+AVE-hydrogel exhibited significantly higher survival rates, stabilizing at approximately 90% over 48 hours. These rates were significantly higher than those of free probiotics, and the probiotic activity in LP+AVE-hydrogel was higher than that in LP-hydrogel. Both fluorescence imaging and quantitative analysis demonstrated that the hydrogel matrix significantly maintained probiotic activity, and that the addition of aloe vera extract, a prebiotic, provided additional protection for probiotic survival within the hydrogel.

[0070] Test Example 2: Appearance and SEM analysis of hydrogel

[0071] In this test example, a scanning electron microscope (SEM: Sigma 30, Zeiss, Germany) was used to investigate the morphology of sample cross-sections. The LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogels prepared in Example 2, Comparative Example 1, and a blank control group were freeze-dried and then frozen and fractured with liquid nitrogen. The fracture surfaces were then gold-sprayed and transferred to a SEM sample stage for structural observation at 300x magnification. The internal morphology, including pore size and porosity, was observed.

[0072] Depend on Figure 2 It can be seen that both CON-hydrogel and LP-hydrogel hydrogels appear as transparent and uniform gels, while LP+AVE-hydrogel hydrogel has a slightly milky white appearance, which may be due to the addition of aloe vera extract. This difference in appearance indicates that the aloe vera extract has good compatibility with the hydrogel system and is successfully loaded into the interior of the gel. At the same time, SEM images show that each group of hydrogels presents a typical three-dimensional porous network structure, indicating that the addition of probiotics and aloe vera extract does not destroy the structural properties of the hydrogel. In addition, this unique internal pore structure gives the hydrogel an efficient exudate adsorption capacity, which can effectively reduce the risk of wound infection and retain a large amount of water, thereby creating an ideal moist microenvironment for wound healing. At the same time, compared with CON-hydrogel, the pore structures of the hydrogel LP-hydrogel with added probiotics and the aloe extract-probiotic hydrogel LP+AVE-hydrogel increased successively. This may be because the addition of probiotics makes the internal structure of the gel system loose and porous through the spatial effect and metabolic action of the cells themselves; and aloe extract, as a highly hydrophilic biomacromolecule, further significantly expands the internal pore structure of the gel by enhancing the hydrophilicity of the system and promoting local phase separation during the gel formation process, which is more conducive to the fixation, protection, nutrient transfer and metabolism of probiotics.

[0073] Test Example 3: Low-field NMR analysis of hydrogel flow

[0074] In this test example, a low-field nuclear magnetic resonance spectrometer (LF-NMR, Bruker Minispec mq20) was used to measure the water state inside the LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogel systems prepared in Example 2, Comparative Example 1, and the blank control group.

[0075] The specific method is as follows: an appropriate amount of sample is placed in a glass sample tube, stabilized, and placed in a nuclear magnetic resonance instrument for measurement at 32°C ± 0.5°C. Instrument parameters are set as an echo interval (TE) of 0.5 ms, 8000 echoes, and 16 repetitions. T2 relaxation time curve data of the gel sample is collected using a Carr-Purcell-Meiboom-Gill (CPMG) sequence. The obtained T2 relaxation time curve data are fitted using MultiExp Inv software to obtain the peak distribution of free and bound water states in the gel system, thereby evaluating the gel's water binding effect and water retention capacity.

[0076] like Figure 3 As shown, low-field nuclear magnetic resonance (LF-NMR) analysis of the water content of the gel system revealed that CON-hydrogel exhibited a significant free water peak at longer relaxation times (approximately 1000–10,000 ms), indicating a relatively loose internal structure and a high proportion of free water. In contrast, LP-hydrogel and LP+AVE-hydrogel hydrogels exhibited distinct bound water peaks within the shorter relaxation time range (approximately 10–100 ms), indicating that the introduction of probiotics and aloe vera extract significantly enhanced the binding of water molecules within the gel network. In particular, the T2 relaxation time peak in the LP+AVE-hydrogel group was more concentrated and stronger, demonstrating that the aloe vera extract further improved the water retention of the gel system, helping to maintain a long-term moist wound microenvironment and promoting wound healing.

[0077] Test Example 4: Determination of hydrogel swelling properties

[0078] This test example analyzed and measured the swelling properties of different hydrogels. The specific method was as follows: a certain amount of freeze-dried LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel (m0 / g) prepared in Example 2, Comparative Example 1, and a blank control group was soaked in 0.01M phosphate buffer (pH 7.4) for 2 hours. Excess surface water was then removed with filter paper to a constant weight (m1 / g). The swelling ratio was calculated as follows: Swelling ratio = (m1 - m0) / m0 × 100%.

[0079] The results of gel swelling properties are as follows Figure 4 As shown in the figure, all the gels in the treatment groups showed rapid water absorption and swelling characteristics in the initial immersion period (100 min), and then gradually stabilized. Compared with CON-hydrogel, LP-hydrogel and LP+AVE-hydrogel hydrogels showed significantly higher swelling rates ( P< 0.05), with the LP + AVE-hydrogel group showing the best swelling performance, reaching a maximum swelling ratio of nearly 1500%. This indicates that the addition of aloe vera extract significantly enhances the hydrophilicity and water retention capacity of the gel system, providing a more suitable microenvironment for probiotics and further improving their stability and survival rate in practical applications.

[0080] Test Example 5: Determination of Hemolytic Properties of Hydrogel

[0081] This test example analyzed and measured the hemolytic properties of different hydrogels. The specific method was as follows: a certain amount of sterile anticoagulated rabbit blood was diluted with physiological saline, centrifuged at 14,000 rpm for 4 minutes, and the precipitate was diluted for later use. A certain amount of sterile anticoagulated rabbit blood was diluted with physiological saline, centrifuged at 1,400 rpm for 4 minutes, and the precipitate was diluted for later use. LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogels prepared from freeze-dried Example 2, Comparative Example 1, and a blank control group were used as experimental groups. PBS served as the negative control group, and 0.2% Triton-X100 was used as the positive control group. From each group, 0.4 mL of solution was transferred to a 2 mL centrifuge tube, 0.2 mL of red blood cell solution was added, and the mixture was incubated at 37°C for 2 hours. The mixture was then centrifuged at 2,500 rpm for 4 minutes. 200 μL of the supernatant was added to a 96-well plate, and the absorbance at 545 nm was measured using a multifunctional microplate reader. The calculation formula of hemolysis rate is as follows: hemolysis rate = (ODs-ODn) / ODP-ODn×100%.

[0082] like Figure 5 As shown in the results, the hemolysis rates of CON-hydrogel, LP-hydrogel and LP+AVE-hydrogel hydrogels were all lower than 5% (<5%), which was comparable to the PBS negative control and much lower than the positive control (Triton), indicating that neither LP-hydrogel nor LP+AVE-hydrogel hydrogels had obvious hemolytic effects, had good biocompatibility and safety, and could be used as probiotic delivery carriers in the field of pet medicine.

[0083] Test Example 6: Determination of antibacterial properties of hydrogel

[0084] This test example analyzes and measures the antibacterial properties of different hydrogels. The specific method is as follows: 100 μl of LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel prepared in Example 2, Comparative Example 1, and the blank control group were mixed with 3 mL of bacterial suspension (the concentration of Staphylococcus pseudintermedius was 1×10 7CFU / m1) were mixed in different sterile centrifuge tubes. Bacteria treated with PBS were used as blank controls. After culturing in a full-temperature shaking incubator at 37°C and 220 rpm for 24 hours, 0.1 mL of bacterial culture solution was spread on an LB solid culture plate and the number of bacterial colonies in the culture plate was quantified. Colony counts were performed when the number of colonies was between 30 CFU and 300 CFU. The experiment was repeated three times and the experimental data were averaged. The results were expressed as log 10 cfu / mL.

[0085] The total colony count (CFU / mL) was calculated as follows: colony count × dilution factor × 10 (0.1 mL of dilution solution was applied to the plate).

[0086] like Figure 6 As shown in the figure, there are significant differences in the antibacterial activity of different hydrogel systems against Staphylococcus pseudintermedius. CON-hydrogel hydrogel has almost no inhibitory effect on Staphylococcus pseudintermedius, while the colony count of LP-hydrogel hydrogel group is significantly lower than that of CON-hydrogel hydrogel group ( P <0.05), reflecting the inhibitory effect of probiotics on the growth of Staphylococcus pseudintermedius; while the LP+AVE-hydrogel hydrogel with aloe vera extract showed more significant antibacterial ability, significantly reducing the number of surviving Staphylococcus pseudintermedius compared with the LP-hydrogel group ( P <0.05). These results further suggest that aloe vera extract can significantly improve the antibacterial properties of probiotic hydrogels by enhancing probiotic metabolic activity, suggesting promising applications in inhibiting pathogenic infections. This may be because probiotics themselves produce antibacterial substances (such as lactic acid, organic acids, and antimicrobial peptides) that directly inhibit the growth of Staphylococcus pseudintermedius. The addition of aloe vera extract significantly enhances probiotic metabolic activity, increasing the production of these antimicrobial metabolites (such as organic acids and lactic acid), further improving the antibacterial properties of the probiotic hydrogels.

[0087] Test Example 7: Bacterial Scanning Electron Microscope Analysis

[0088] This test example uses scanning electron microscopy to analyze the effects of different hydrogels on bacteria. The specific method is as follows: LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel prepared in Example 2, Comparative Example 1, and the blank control group are placed in MRS liquid culture, cultured at 37°C for 24 hours, and the supernatants are centrifuged and taken. 6A suspension of Staphylococcus pseudintermedius (CFU / mL) was incubated in a constant-temperature shaking incubator at 37°C and 200 rpm for 24 hours, after which the cells were collected by centrifugation. The cells were then fixed with 2.5% glutaraldehyde solution at 4°C in the dark for 12 hours. The samples were then dehydrated using a gradient of ethanol solutions containing 30%, 50%, 70%, 80%, 90%, and 95%, with each treatment lasting 15 minutes. The samples were then dehydrated twice in 100% ethanol, each for 20 minutes. The samples were then treated with a mixture of ethanol and isoamyl acetate (1:1) for 30 minutes and pure isoamyl acetate for 1 hour. Finally, after critical point drying, the cells were plated and the bacterial morphology was observed and recorded using a scanning electron microscope.

[0089] like Figure 7 Scanning electron microscopy observations of the damaging effects of gel treatment on Staphylococcus aureus revealed that the morphology of Staphylococcus aureus treated with CON-hydrogel was more regular, with a smooth, plump surface and clear, neat arrangement, indicating that the blank hydrogel itself had little significant effect on Staphylococcus aureus growth. However, the surface of Staphylococcus aureus treated with the probiotic hydrogel (LP-hydrogel) was noticeably wrinkled and deformed, and aggregation occurred. Staphylococcus aureus treated with the aloe vera extract probiotic hydrogel (LP+AVE-hydrogel) exhibited even more severe cell damage, with severe wrinkling and aggregation, significantly disrupting cell integrity. Furthermore, the probiotics in the LP-hydrogel and LP+AVE-hydrogel groups exhibited a typical, intact rod-shaped structure, showing no obvious damage or deformation, in stark contrast to the damaged morphology of Staphylococcus aureus. These results confirm that the combined action of aloe vera extract and probiotics significantly enhances the damage to the pathogen's cell structure, thereby improving overall antibacterial activity.

[0090] Test Example 8: Clinical bacterial skin disease treatment effect test for pet cats

[0091] To systematically verify the clinical efficacy and safety of the aloe vera extract-probiotic composite hydrogel for pet skin diseases (Example 2: LP+AVE-hydrogel) described in the present invention in treating bacterial skin diseases in pet cats, a control group was established and efficacy evaluation was conducted to comprehensively analyze its actual effects in alleviating skin lesion symptoms, controlling pathogens, and inhibiting recurrence.

[0092] 1. Experimental animals and grouping

[0093] Sample source: A total of 16 cats clinically diagnosed with bacterial skin diseases were collected, including different breeds, sexes, and ages (6 months to 8 years old), with a balanced distribution of cases.

[0094] Grouping method: Divide into two groups by random block method, 8 in each group:

[0095] Experimental group (n=8): topical application of the aloe extract-probiotic composite hydrogel (LP+AVE-hydrogel) of the present invention;

[0096] Control group (n=8): blank control hydrogel (CON-hydrogel) was applied topically.

[0097] Baseline balance assessment: All animals underwent health assessments including body temperature, weight, mental state, and biochemical indicators before the experiment, and photos of the lesion sites were taken for archiving to ensure that the two groups were comparable in terms of disease severity and general physical condition, with no significant differences.

[0098] 2. Experimental methods

[0099] Intervention process:

[0100] Experimental group: After cleaning the affected area, apply LP+AVE-hydrogel once every 12 hours for 14 consecutive days;

[0101] Control group: In addition to routine cleaning treatment, no antibacterial intervention was performed, and only blank gel was applied to simulate the usage experience.

[0102] Symptom assessment time point:

[0103] Day 0 (before medication), day 7 (mid-treatment), day 14 (end of treatment);

[0104] The treatment effects are divided into three categories: cure, significant effect (effective) and ineffective.

[0105] Cure: The inflammatory secretions of the affected skin are completely cleared, there is no abnormal odor, the rashes, dandruff, pustules and other pathological manifestations are completely subsided, and the skin tissue returns to normal.

[0106] Markedly effective: Inflammatory secretions were significantly reduced, odor was significantly alleviated, and symptoms such as rashes, dandruff, and pustules were significantly improved compared to before treatment, but not completely eliminated.

[0107] Ineffective: After treatment, the symptoms of the affected area have not been significantly improved, inflammatory secretions still exist, accompanied by odor, and skin lesions such as rashes, dandruff, and pustules persist or tend to worsen.

[0108]

[0109] The data in Table 2 show that in the experimental group, 6 cats were completely cured and 1 showed marked improvement, with a total effective rate of 87.5% and a cure rate of 75%. In the control group, none of the 8 cats were cured or improved, with a total effective rate of 0% and an ineffective rate of 100%. The above data clearly demonstrate that the aloe extract-probiotic composite hydrogel of the present invention has significant efficacy in the treatment of bacterial skin diseases in cats. It not only effectively alleviates the rashes, desquamation, pustules, and exudation at the lesion site, but also achieves full restoration of skin integrity and hair condition in most cases.

[0110] Figure 8 The treatment process records of typical individuals in the trial were further provided. It can be seen that on the seventh day of treatment, the inflammation of the skin lesions in the experimental group of cats was significantly reduced, and exudation was well controlled. By the 14th day, the skin in the lesion area had completely healed, hair began to regrow, and the overall appearance returned to normal.

[0111] In summary, the aloe vera extract-probiotic composite hydrogel of the present invention exhibits a rapid, safe, and low-recurrence skin repair effect in the context of non-antibiotic intervention, providing a new and effective alternative to traditional drug treatment for bacterial skin diseases in pet cats, and has extremely high potential for promotion and application.

[0112] 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 method for preparing an aloe vera extract-probiotic composite hydrogel for treating pet skin diseases, characterized in that: The pathogenic bacteria of the pet skin disease is Staphylococcus pseudintermedius, and the preparation method comprises the following steps: (1) Preparation of probiotic suspension: Under sterile conditions, freeze-dried animal combined lactobacillus ( Ligilactobacillus animalis ZYpet-012 was inoculated into MRS solid slant medium and cultured at 37°C for recovery. The cells were then transferred to MRS liquid medium and cultured overnight at 37°C in a shaker. The cultured bacteria were centrifuged, the supernatant discarded, and the cells were washed with sterile PBS and resuspended to obtain a probiotic suspension with a viable count of 1.0 × 10 9 CFU / mL; The animal-associated Lactobacillus ZYpet-012 is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No.32008 and the deposit date: September 20, 2024; (2) Preparation of blank hydrogel: Carbomer 940 powder was weighed, added to ultrapure water, stirred thoroughly, and allowed to stand at room temperature for full swelling. Triethanolamine and zinc hyaluronate were then added in sequence and stirred evenly to prepare a blank hydrogel; the mass concentration of carbomer 940 powder, triethanolamine, and zinc hyaluronate in the blank hydrogel was 0.5%, 0.25%, and 0.5%, respectively. (3) Preparation of aloe vera extract solution: weigh aloe vera extract in deionized water, stir magnetically at 30-40°C until completely dissolved, cool to room temperature to obtain aloe vera extract solution, and seal in a volumetric flask for later use; the volume ratio of aloe vera extract solution to blank hydrogel is 1:19; the mass concentration of aloe vera extract in the aloe vera extract solution is 0.6%; (4) The aloe vera extract solution and the blank hydrogel are mixed and stirred evenly, and then the probiotic suspension prepared in step (1) is added and mixed evenly to obtain the aloe vera extract-probiotic composite hydrogel for pet skin diseases.

2. An aloe vera extract-probiotic composite hydrogel for pet skin diseases, characterized in that: The composite hydrogel is prepared by the method according to claim 1.

3. Animal-associated Lactobacillus ( Ligilactobacillus animalis ) Application of ZYpet-012 in the preparation of an external-use product for preventing and treating pet skin diseases, characterized in that: The animal-associated lactobacillus ZYpet-012 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number: CGMCC No. 32008 and the deposit date: September 20, 2024. The pathogenic bacteria of the pet skin disease is Staphylococcus pseudintermedius.

4. The use according to claim 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 washing and care products include bath liquid, hair conditioner and pet wipes.

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