Aloe extract-probiotic composite hydrogel for skin diseases of pets

Abstract

The invention belongs to the technical field of medical preparations for pets, and particularly relates to an aloe extract-probiotic composite hydrogel for pet dermatosis, a preparation method comprises the following steps: under an aseptic condition, animal combined lactobacillus ZYpet-012 with the preservation number of CGMCC No.32008 is subjected to solid slant culture medium recovery and liquid culture, and then the animal combined lactobacillus ZYpet-012 with the preservation number of CGMCC No.32008 is subjected to freeze-drying to obtain the aloe extract-probiotic composite hydrogel for pet dermatosis. Centrifugally washing to obtain a probiotic suspension; the preparation method comprises the following steps: preparing blank hydrogel from carbomer 940, triethanolamine, zinc hyaluronate and the like; and mixing the aloe extract solution with the blank hydrogel, and then adding the probiotic suspension to prepare the composite hydrogel. According to the present invention, by using the anti-inflammatory, antibacterial and prebiotic characteristics of the aloe extract and combining the skin micro-ecology adjusting effect of the animal and the lactobacillus ZYpet-012, the prepared composite hydrogel can effectively treat the skin diseases of pets, and has advantages of nature, safety, accurate targeting and the like.

Description

Technical Field

[0001] 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 pet skin diseases. Background Art

[0002] Pet skin diseases, especially those caused by bacterial infections, are the most common and recurrent stubborn diseases in the clinics of dogs and cats. Among them, Staphylococcus pseudintermedius is one of the main pathogenic bacteria of pet bacterial skin diseases. Relevant research shows that it accounts for a very high proportion in pet bacterial skin diseases. Such infections can not only cause local skin lesions such as swelling and abscesses, but may even lead to systemic infections in severe cases, significantly increasing the treatment difficulty and nursing cost.

[0003] Currently, most pet skin disease care products on the market target mainly at Staphylococcus aureus, Escherichia coli, etc., lacking the specific inhibitory function against Staphylococcus pseudintermedius, and the treatment accuracy is insufficient. At the same time, traditional treatment methods generally rely on antibiotics or anti-parasitic drugs such as ketoconazole, florfenicol, terbinafine hydrochloride, etc., but these drugs have problems such as large toxic and side effects, strong drug resistance, and environmental pollution, and it is difficult to meet the long-term, safe and sustainable treatment needs. 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, providing an aloe vera extract-probiotic composite hydrogel for pet skin diseases. 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 their colonization rate and stability, and achieve the continuous slow release and local targeting effect of probiotics. Through the synergy of the three, a set of efficient, safe, low-toxic pet skin disease treatment system with specific antibacterial and wound healing dual functions is constructed, filling the technical gap in the current market.

[0005] With the in - depth exploration of microecological therapy in modern medicine, the field of dermatological treatment for pets is undergoing an innovation from traditional drugs to bioactive preparations. During the research and development process, it has been found that probiotics, with their biological characteristics of safety and no drug resistance, have emerged in the research of pet dermatological treatment. Through systematic research on the microecological environment of pet skin, it is confirmed that specific probiotic strains have a high degree of ecological adaptability with pet skin flora, and through mechanisms such as regulating the microbial community structure, strengthening the physical skin barrier, and competitively inhibiting the adhesion of pathogenic bacteria, they show significant advantages in targeted treatment of pet skin diseases. At the same time, in the screening of natural plant active ingredients, aloe vera extract has been verified by multi - dimensional efficacy. It not only has pharmacological activities such as anti - inflammation, antibacterial, and accelerating tissue repair, but also, due to its prebiotic properties, can significantly improve the metabolic efficiency and antibacterial efficacy of probiotics, providing an innovative direction for the development of new pet dermatological treatment programs.

[0006] In view of this, the purpose of the present invention is achieved as follows: The first aspect of the present invention provides a preparation method of an aloe vera extract - probiotic composite hydrogel for pet skin diseases, comprising the following steps: (1) Preparation of probiotic suspension: Under aseptic conditions, inoculate freeze - dried Lactobacillus animalis Ligilactobacillus animalis ) ZYpet - 012 on an MRS solid slant medium and culture it at 37 °C for resuscitation; then transfer it to an MRS liquid medium and culture it overnight on a shaker at 37 °C; centrifuge the cultured bacterial liquid, discard the supernatant, wash it with sterile PBS and resuspend it to obtain a probiotic suspension; The Lactobacillus animalis ZYpet - 012 is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number: CGMCC No. 32008, and the preservation date: September 20, 2024; (2) Preparation of blank hydrogel: Weigh carbomer 940 powder, add it to ultrapure water, stir well, let it stand at room temperature for sufficient swelling, and then add triethanolamine and zinc hyaluronate in sequence, stir evenly to prepare a blank hydrogel; (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 an aloe vera extract solution, and encapsulate it in a volumetric flask for standby; (4) Mix the above - mentioned aloe vera extract solution with the blank hydrogel, stir evenly, and then add the probiotic suspension prepared in step (1) and mix well to obtain the aloe vera extract - probiotic composite hydrogel for pet skin diseases.

[0007] Further, in step (1), in the probiotic suspension, the viable count of Lactobacillus animalis ZYpet - 012 reaches 1.0×10 8 ~1.0×10 10CFU / mL.

[0008] Further, in step (2), the mass concentration of carbomer 940 powder in the blank hydrogel is 0.2% - 1.0%, the mass concentration of triethanolamine is 0.1% - 0.5%, and the mass concentration of zinc hyaluronate is 0.1% - 1.0%.

[0009] Further, in step (3), the volume ratio of the aloe extract solution to the blank hydrogel is 1:15 - 20; the mass concentration of aloe extract in the aloe extract solution is 0.1% - 1.0%.

[0010] The second aspect of the present invention provides an aloe extract - probiotic composite hydrogel for pet skin diseases, and the composite hydrogel is prepared by the method described in the first aspect.

[0011] The third aspect of the present invention provides a Lactobacillus animalis ( Ligilactobacillus animalis ) ZYpet - 012, which is deposited in the General Microbiology Center of the China Center for Type Culture Collection, with the deposit number: CGMCC No. 32008, and the deposit date: September 20, 2024.

[0012] The fourth aspect of the present invention provides the application of the Lactobacillus animalis ZYpet - 012 described in the first aspect in the preparation of products for preventing and treating pet skin diseases.

[0013] Further, the pathogenic bacterium of the pet skin disease is Staphylococcus pseudintermedius.

[0014] Further, the products include pet washing and care products and topical medications.

[0015] Further, the pet washing and care products include bath lotion, hair conditioner, and pet wipes. Strain preservation information:

[0016] Lactobacillus animalis ZYpet - 012, deposit institution: General Microbiology Center of the China Center for Type Culture Collection (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 . The advantages and beneficial effects of the present invention are:

[0017] (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 avoiding the problem of antibiotic resistance and meeting the needs of pet owners for natural, safe and green pet care products.

[0018] (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.

[0019] (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.

[0020] (4) The present invention is specifically designed for Staphylococcus pseudointermedius, 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 has strong targeting and significant efficacy.

[0021] (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. There are no obvious side effects after long-term use, which meets the safety requirements of pet owners for pet care products.

[0022] (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, and provide a new entry point for the cure of pet skin diseases, with high clinical application potential.

[0023] (7) The present invention innovatively proposes a new strategy for the treatment of bacterial skin diseases in pets with a composite hydrogel of probiotics and natural aloe vera extract as the core. 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

[0024] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0025] Figure 1 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 are shown; Figure 2 are the appearance diagrams of different hydrogels and the scanning electron microscope structure diagrams at a magnification of 300× in Test Example 2 of the present invention; Figure 3 is the distribution result diagram of the relaxation time (Relaxation time, T2) of the LF-NMR spectra of different hydrogels in Test Example 3 of the present invention; Figure 4 is the determination result diagram of the swelling ratio of different hydrogels in Test Example 4 of the present invention; Figure 5 is the determination result diagram of the hemolysis ratio of different hydrogels in Test Example 5 of the present invention; Figure 6 is the determination result diagram of the antibacterial performance of different hydrogels in Test Example 6 of the present invention; Figure 7 is the determination result diagram of the bacteria scanning electron microscope in Test Example 7 of the present invention; wherein, A is Staphylococcus aureus after treatment with CON-hydrogel, B is Staphylococcus aureus after treatment with LP-hydrogel, C is Staphylococcus aureus after treatment with LP+AVE-hydrogel, and D is the normal cell morphology of LP; Figure 8 is the treatment effect diagram of bacterial skin diseases of pet cats in Test Example 8 of the present invention. Detailed implementation manners

[0026] The examples are given to better illustrate the present invention, but the content of the present invention is not limited to the given examples only. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners according to the above invention content still fall within the protection scope of the present invention.

[0027] In the ranges disclosed herein, the endpoints and any values 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, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0028] 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 an exemplary manner, and are not used to limit the present invention.

[0029] In the following examples, aloe vera extract was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and carbomer 934 was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0030] The data in the following examples are expressed as "mean ± standard deviation" and one-way analysis of variance was performed using SPSS 22.0 software. P <0.05 indicates a significant difference. The graphs in the examples were created using GraphPad (GraphPad Prism, version 8.0.2, USA). Example 1: Screening and identification of animal-associated lactobacilli

[0031] This example provides a strain screening and identification process: (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 the 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 of different concentrations were taken and inoculated on MRS solid culture medium plates, and cultured under anaerobic conditions at 30°C for 48 hours to isolate lactic acid bacteria from the skin.

[0032] (2) Purification of strains: Select colonies of different sizes and milky white protrusions from the primary culture plate for single colony selection. Repeatedly inoculate on the MRS plate using the three-zone streak method, culture anaerobically at 37°C for 48 hours, and perform three rounds of operation to finally obtain a stable and purified target strain.

[0033] (3) Morphological identification: Gram staining technique is used to make a preliminary observation of the strain morphology. A small amount of single colony is evenly smeared on a glass slide in a drop of sterile water and air-dried. It 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 its Gram properties.

[0034] (4) Glucose gas production experiment: The selected lactic acid bacteria colony was inoculated into MRS liquid culture medium and cultured at 30°C for 24 hours. Then, the bacterial liquid was inoculated into MRS liquid culture medium containing inverted Duroc tubes at a 1% inoculum and cultured at 30°C for 7 days. Observe whether bubbles are formed in the Duroc tubes to determine whether the strain is homotypic (no gas) or heterotypic (gas-producing) type.

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

[0036]

[0037] As can be seen from Table 1, strain ZYpet-012 is a homofermentative lactic acid bacterium, with a positive result in Gram staining and negative catalase.

[0038] (6) Strain identification: The strain was anaerobically cultured on MRS plates at 30 °C for 48 h, and single colonies were picked for PCR. The primers were the 16S rRNA universal primers 27F and 1492R. The PCR reaction conditions were: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 90 s, for 35 cycles; final extension at 72 °C for 5 min. The obtained 16S rRNA was subjected to BLAST analysis with the sequences of known strains in the GenBank website. Strain ZYpet-012 was identified as Lactobacillus plantarum Lactobacillus animalis, and named Lactobacillus animalis ZYpet-012.

[0039] 16S rRNA gene sequence of strain ZYpet-012: Example 2: Preparation of Aloe Extract-Probiotic Composite Hydrogel for Pet Skin Diseases

[0040] This example provides an aloe extract-probiotic composite hydrogel for pet skin diseases, and the preparation method is as follows: (1) Preparation of probiotic suspension: Under sterile conditions, freeze-dried Lactobacillus animalis Ligilactobacillus animalis ) ZYpet-012 was inoculated on MRS solid slant medium and cultured at 37 °C for 24 h for resuscitation; then transferred to 100 mL of MRS liquid medium and cultured overnight on a shaker at 37 °C and 130 rpm; the cultured bacterial liquid was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and after washing with sterile PBS, it was resuspended to obtain a probiotic suspension. Among them, the viable count of the bacterial liquid reached about 1.0×10 9 CFU / mL.

[0041] The Lactobacillus animalis ZYpet-012 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number was: CGMCC No. 32008, and the deposit date was: September 20, 2024; Preparation of blank hydrogel: Weigh 0.5% of Carbomer 940 powder, add it to ultrapure water, stir well, let it stand at room temperature for 12 h for sufficient swelling, then add 0.25% of triethanolamine and 0.5% of zinc hyaluronate in sequence, stir evenly to prepare a blank hydrogel (CON-hydrogel).

[0042] (3) Preparation of aloe extract solution: Weigh 0.6% of aloe extract in proportion in an appropriate amount of deionized water, stir magnetically at 700 rpm / min at 30 - 40 °C for 20 min until completely dissolved, cool to room temperature to obtain an aloe multi-extract solution, and seal it in a volumetric flask for standby.

[0043] (4) Mix the aloe extract solution with the above blank hydrogel according to a volume ratio of 1:19, stir evenly at 300 rpm for 1 h, and then add the probiotic suspension prepared in step (1) and mix well to obtain the aloe extract-probiotic composite hydrogel (LP+AVE-hydrogel) for pet skin diseases, and store it sealed at 4 °C for standby. Comparative Example 1: Preparation of Probiotic Carbomer Composite Hydrogel for Pet Skin Diseases

[0044] This example provides a probiotic composite hydrogel for pet skin diseases. The preparation method refers to Example 2, with the difference that the aloe extract solution is not added in step (3). That is, the probiotic suspension prepared in step (1) is directly added to the blank hydrogel (CON-hydrogel), and after stirring evenly at 300 rpm, the probiotic carbomer composite hydrogel (LP-hydrogel) is obtained. After sealing, it is stored at 4 °C for later use. Test Example 1: Determination and analysis of probiotic activity in hydrogel

[0045] In this test example, the probiotic activities in different hydrogels were determined. The specific method was as follows: 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. Then, the live / dead bacteria were stained with 5 μM SYTO@9 and 2 μg / mL PI respectively. After staining in the dark at 37 °C for 15 min, the staining solution was discarded, and the staining solution was washed off with sterile normal saline. 10 μL of the bacterial solution was dropped on a glass slide, covered with a coverslip, and photographed at 200 times magnification on a laser confocal microscope (OLYMPUS FV1200). Among them, the green fluorescence represented live bacteria, and the red fluorescence represented dead bacteria. Finally, the bacterial survival rates of different treatment groups were calculated by the fluorescence intensity using ImageJ software.

[0046] It can be seen from Figure 1 the observation of the survival of probiotics after 48 hours of storage by laser confocal microscopy Live / Dead staining that the bacterial survival rate of the free probiotic LP-Free decreased significantly after being stored at 4 °C for 48 hours, while the probiotics loaded in the LP-hydrogel and LP+AVE-hydrogel showed significantly higher survival rates, which were stable at about 90% within 48 hours, both significantly higher than the free probiotics, and the probiotic activity in LP+AVE-hydrogel was higher than that in LP-hydrogel. Both the fluorescence images and quantitative analysis showed that the hydrogel matrix had a significant effect on maintaining the probiotic activity, and the addition of aloe extract as a prebiotic could produce an additional protective effect on the survival of probiotics in the hydrogel. Test Example 2: Appearance and scanning electron microscopy analysis of hydrogel

[0047] In this test example, a scanning electron microscope SEM (SEM: Sigma30, Zeiss, Germany) was used to conduct a morphological study on the cross-section of the sample. The LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogels prepared in Example 2, Comparative Example 1, and the blank control group were freeze-dried, then cryogenically crushed with liquid nitrogen, the fracture surface was sputter-coated with gold, transferred to the scanning electron microscope sample stage, and magnified 300 times for structural observation to observe the internal morphological structures such as the pore size and porosity of the hydrogels.

[0048] It can be seen that Figure 2 both the CON-hydrogel and LP-hydrogel hydrogels showed a transparent and homogeneous gel state, while the LP+AVE-hydrogel hydrogel presented a slightly milky white appearance, which might be due to the addition of aloe extract. This difference in appearance indicates that the aloe extract has good compatibility with the hydrogel system and has been successfully loaded into the gel interior. At the same time, the SEM images showed that each group of hydrogels presented a typical three-dimensional porous network structure, indicating that the addition of probiotics and aloe extract did not destroy the structural characteristics of the hydrogels, and this unique internal pore structure enabled the hydrogels to have high liquid absorption capacity, effectively reducing the risk of wound infection and retaining a large amount of moisture, thus creating an ideal moist microenvironment for wound healing. At the same time, compared with the CON-hydrogel, the pore structures of the probiotic-added hydrogel LP-hydrogel and the aloe extract-probiotic hydrogel LP+AVE-hydrogel increased in sequence. This might be because the addition of probiotics made the internal structure of the gel system become loose and porous through the spatial effect and metabolic action of the cells themselves; while aloe extract, as a highly hydrophilic biological macromolecule, further significantly enlarged the internal pore structure of the gel by enhancing the hydrophilicity of the system and promoting the local phase separation during the gel formation process, which was more conducive to probiotic immobilization, protection, nutrient transport, and probiotic metabolism. Test Example 3: Low-field nuclear magnetic resonance analysis of hydrogels

[0049] In this test example, a low-field nuclear magnetic resonance instrument (LF-NMR, Bruker Minispec mq20) was used to measure the moisture 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.

[0050] The specific method is as follows: Take an appropriate amount of the sample and place it in a glass sample tube. After stabilization, place it in a nuclear magnetic resonance instrument and measure it at 32°C ± 0.5°C. The instrument parameters are set as the echo spacing TE of 0.5 ms, the number of echoes of 8000, and the number of repeated scans of 16 times. The T2 relaxation time curve data of the gel sample is collected through the Carr-Purcell-Meiboom-Gill (CPMG) sequence. The obtained T2 relaxation data is fitted using the MultiExp Inv software to obtain the peak distribution of the free water and bound water states of water in the gel system, so as to evaluate the binding effect of the gel on water and the water retention ability.

[0051] As Figure 3 shown, the water state of the gel system was analyzed by low-field nuclear magnetic resonance (LF-NMR). A significant free water peak appeared in the CON-hydrogel at a relatively long relaxation time (about 1000 - 10000 ms), indicating that the internal structure of the gel was relatively loose and the proportion of free water was high. While obvious bound water peaks appeared in the LP-hydrogel and LP+AVE-hydrogel within a relatively short relaxation time range (about 10 - 100 ms), indicating that the introduction of probiotics and aloe vera extract significantly enhanced the binding effect of the gel network on water molecules. In particular, the T2 relaxation time peaks of the LP+AVE-hydrogel group were more concentrated and stronger, reflecting that the aloe vera extract further improved the water retention performance of the gel system, contributed to maintaining the moist microenvironment of the wound for a long time, and was more conducive to promoting the wound healing process. Test Example 4: Determination of the swelling performance of hydrogels

[0052] In this test example, the swelling performance of different hydrogels was analyzed and determined. The specific method is as follows: Take a certain amount of LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel prepared in Example 2, Comparative Example 1, and the blank control group after freeze-drying (recorded as m0 / g), soak them in a phosphate buffer solution with a concentration of 0.01M and a pH value of 7.4 for 2 h, and then use filter paper to absorb the excess water on the surface until a constant weight (recorded as m1 / g). The calculation formula for the swelling ratio is as follows: Swelling ratio = (m1 - m0) / m0 × 100%.

[0053] The results of the gel swelling performance are as Figure 4 shown. All treatment group gels showed rapid water absorption and swelling characteristics in the initial stage of soaking (100 min), and then gradually tended to be stable. Compared with CON-hydrogel, the LP-hydrogel and LP+AVE-hydrogel showed significantly higher swelling ratios ( P< 0.05), and the swelling performance of the LP+AVE-hydrogel group was the best, with the highest swelling rate approaching 1500%. This indicates that the addition of aloe extract significantly enhances the hydrophilicity and water retention capacity of the gel system, provides a more suitable microenvironment for probiotics, and further improves the stability and survival rate of probiotics in practical applications. Test Example 5: Determination of Hemolytic Properties of Hydrogels

[0054] In this test example, the hemolytic properties of different hydrogels were analyzed and determined. The specific method was as follows: A certain amount of sterile anticoagulated rabbit blood was diluted with physiological saline and centrifuged at 14000 r / min for 4 min, and the precipitate was taken and diluted for standby. A certain amount of sterile anticoagulated rabbit blood was diluted with physiological saline and centrifuged at 1400 r / min for 4 min, and the precipitate was taken and diluted for standby. The LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogels prepared in Freeze-dried Example 2, Comparative Example 1, and the blank control group were used as the experimental groups, PBS was used as the negative control group, and Triton-X100 with a volume fraction of 0.2% was used as the positive control group. 0.4 mL of the solution was taken from each group and placed in a 2 mL centrifuge tube, 0.2 mL of the erythrocyte solution was added, and the mixture was incubated at 37 °C for 2 h, then centrifuged at 2500 for 4 min. 200 μL of the supernatant was taken and added to a 96-well plate, and the absorbance at 545 nm was measured using a multifunctional microplate reader. The calculation formula for the hemolysis rate is as follows: Hemolysis rate = (ODs - ODn) / (ODP - ODn) × 100%.

[0055] As Figure 5 shown, the hemolysis rates of the CON-hydrogel, LP-hydrogel, and LP+AVE-hydrogel hydrogels were all lower than 5% (<5%), comparable to the PBS negative control, and much lower than the positive control (Triton), indicating that the LP-hydrogel and LP+AVE-hydrogel hydrogels do not have obvious hemolytic effects, have good biocompatibility and safety, and can be used as probiotic delivery carriers in the field of pet medicine. Test Example 6: Determination of Antibacterial Properties of Hydrogels

[0056] In this test example, the antibacterial properties of different hydrogels were analyzed and determined. The specific method was as follows: 100 μL of the LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel hydrogels prepared in Example 2, Comparative Example 1, and the blank control group were respectively mixed with 3 mL of the bacterial suspension (the concentration of Staphylococcus pseudintermedius was 1×10 7CFU / m1) were mixed in different sterile centrifuge tubes. The PBS-treated bacteria served as the blank control. After culturing in a constant temperature shaking incubator at 37 °C and 220 rpm for 24 h, 0.1 mL of the bacterial culture solution was taken and spread on an LB solid culture dish, and the number of bacterial colonies in the culture dish was quantified. Colonies with a count between 30 CFU and 300 CFU were selected for colony counting. This experiment was repeated three times, and the experimental data were averaged. The results were expressed as log 10 cfu / mL.

[0057] Calculation method for total number of colonies (CFU / mL): number of colonies × dilution factor × 10 (0.1 mL of diluted solution spread on the plate).

[0058] As Figure 6 shown, there were significant differences in the antibacterial activities of different hydrogel systems against Staphylococcus pseudintermedius. The CON-hydrogel had almost no inhibitory effect on Staphylococcus pseudintermedius, while the number of colonies in the LP-hydrogel group was significantly lower than that in the CON-hydrogel group ( P <0.05), indicating the inhibitory effect of probiotics on the growth of Staphylococcus pseudintermedius; while the LP+AVE-hydrogel containing aloe extract showed a more significant antibacterial ability, significantly reducing the survival number of Staphylococcus pseudintermedius compared with the LP-hydrogel group ( P <0.05). Further, this result suggests that aloe extract can significantly improve the antibacterial performance of probiotic hydrogels by enhancing the metabolic activity of probiotics and has good application prospects in inhibiting pathogen infections. This may be because probiotics themselves can produce antibacterial active substances (such as lactic acid, organic acids, antibacterial peptides), which have a direct inhibitory effect on the growth of Staphylococcus pseudintermedius, and the addition of aloe extract significantly enhances the metabolic activity of probiotics, increasing the production of these antibacterial metabolites (such as organic acids, lactic acid, etc.), thus further improving the antibacterial performance of probiotic hydrogels. Test Example 7: Bacterial Scanning Electron Microscopy Analysis

[0059] In this test example, the effects of different hydrogels on bacteria were analyzed by scanning electron microscopy. The specific method was as follows: The LP+AVE-hydrogel, LP-hydrogel, and CON-hydrogel prepared in Example 2, Comparative Example 1, and the blank control group were placed in MRS liquid culture and cultured at 37 °C for 24 h. The supernatant was taken by centrifugation respectively. The supernatant was mixed with 10 6Mix with Staphylococcus pseudintermedius suspension at CFU / mL, culture in a constant temperature shaking incubator at 37°C and 200 rpm for 24 hours, and then centrifuge to collect the bacteria. Then fix with 2.5% glutaraldehyde solution in the dark at 4°C for 12 hours, dehydrate the sample step by step with gradient concentration solutions containing 30%, 50%, 70%, 80%, 90% and 95% ethanol, treat each concentration for 15 min, perform two dehydration treatments in 100% ethanol, each for 20 minutes. Then treat the sample with a mixture of ethanol and isoamyl acetate (1:1) for 30 min and pure isoamyl acetate for 1 h. Finally, coat the film after critical point drying, and observe and record the morphology of the bacteria using a scanning electron microscope.

[0060] As Figure 7 shown, the damage effect of gel treatment on Staphylococcus bacteria was observed by scanning electron microscopy. After treatment with CON-hydrogel, the morphology of Staphylococcus bacteria was relatively regular, the surface was smooth and plump, and the arrangement was clear and neat, indicating that the blank hydrogel itself had almost no obvious effect on the growth of Staphylococcus. However, the surface of Staphylococcus treated with probiotic hydrogel (LP-hydrogel) was significantly shrunk and deformed, and aggregation occurred. In the group treated with aloe vera extract probiotic hydrogel (LP+AVE-hydrogel), the Staphylococcus showed more serious bacterial damage, with severe surface shrinkage and aggregation into clusters, and the integrity of the bacteria was significantly damaged. At the same time, the probiotics in the LP-hydrogel and LP+AVE-hydrogel groups showed a typical complete rod-shaped structure, without obvious damage or deformation, forming a sharp contrast with the damaged morphology of Staphylococcus. These results confirmed that the combined action of aloe vera extract and probiotics could significantly enhance the damage effect on the cell structure of pathogenic bacteria, thereby improving the overall antibacterial activity. Test Example 8: Treatment Effect Test of Clinical Bacterial Skin Diseases in Pet Cats

[0061] In order to systematically verify the clinical efficacy and safety of the aloe vera extract-probiotic composite hydrogel (Example 2: LP+AVE-hydrogel) for treating pet skin diseases of the present invention in the treatment of bacterial skin diseases in pet cats. By setting up a control group and carrying out an evaluation of the efficacy level, comprehensively analyze its actual effects on the relief of skin disease symptoms, the control of pathogenic bacteria and the inhibition of recurrence.

[0062] 1. Experimental Animals and Grouping Sample Source: A total of 16 cats clinically diagnosed with bacterial skin diseases were collected, including different breeds, genders and ages (6 months to 8 years), and the case distribution was balanced.

[0063] Grouping Method: Divide into two groups according to the random block method, with 8 in each group: Experimental group (n = 8): Topically apply the aloe vera extract-probiotic composite hydrogel (LP+AVE-hydrogel) of the present invention; Control group (n = 8): Topically use a blank control hydrogel (CON-hydrogel).

[0064] Baseline balance assessment: All animals were subjected to health assessments such as body temperature, body weight, mental state, and biochemical indices before the experiment, and the lesion sites were photographed and archived to ensure the comparability of the two groups in terms of disease severity and general physical condition, with no significant differences.

[0065] 2. Experimental methods Intervention process: Experimental group: After cleaning the affected area, apply LP+AVE-hydrogel topically once every 12 hours for 14 consecutive days; Control group: Except for routine cleaning, no antibacterial intervention was performed, and only the blank gel was applied to simulate the usage experience.

[0066] Symptom assessment time points: Day 0 (before medication), Day 7 (mid-treatment), Day 14 (treatment endpoint); The treatment effects are divided into three categories: cured, markedly effective (effective), and ineffective.

[0067] Cured: The inflammatory secretions of the skin at the affected area are completely cleared, there is no abnormal odor, and the lesions such as red rashes, dandruff, and pustules have completely subsided, and the skin tissue has returned to normal.

[0068] Markedly effective: The inflammatory secretions are significantly reduced, the odor is significantly alleviated, and the symptoms such as red rashes, dandruff, and pustules are significantly improved compared with before treatment, but not completely eliminated.

[0069] Ineffective: The symptoms at the affected area after treatment have not improved significantly, the inflammatory secretions still exist, accompanied by an odor, and the skin lesions such as red rashes, dandruff, and pustules persist or show a tendency to worsen.

[0070]

[0071] According to the data in Table 2: In the experimental group, 6 cats were completely cured and 1 was markedly effective, 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 ineffectiveness rate of 100%. The above data clearly show that the aloe vera extract-probiotic composite hydrogel of the present invention has a significant effect in the treatment of feline bacterial dermatosis, and can not only effectively relieve the red rashes, desquamation, pustules, and exudation at the lesion site, but also achieve a comprehensive recovery of skin integrity and hair condition in most cases.

[0072] Figure 8The treatment process records of typical individuals in the experiment are further provided. It can be seen that in the experimental group of cats, on the 7th day of treatment, the inflammation of the skin lesions was significantly reduced, and the exudation was well controlled; by the 14th day, the skin in the lesion area had completely healed, hair began to regenerate, and the overall appearance returned to normal.

[0073] Based on the above results, the aloe extract-probiotic composite hydrogel described in the present invention exhibits rapid, safe, and low-recurrence skin repair effects under the background of non-antibiotic intervention, providing an effective and alternative new solution to traditional drug treatment for bacterial skin diseases in pet cats, and having extremely high potential for popularization and application.

[0074] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the preferred arrangement, those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A preparation method of an aloe extract-probiotic composite hydrogel for pet skin diseases, characterized in that, The preparation method includes the following steps: (1) Preparation of probiotic bacterial suspension: Under aseptic conditions, freeze-dried and preserved Lactobacillus animalis Ligilactobacillus animalis ) ZYpet-012 was inoculated onto an MRS solid slant medium and cultured at 37 °C for resuscitation; subsequently, it was transferred to an MRS liquid medium and cultured overnight on a shaker at 37 °C; the cultured bacterial solution was centrifuged, the supernatant was discarded, and after washing with sterile PBS, it was resuspended to obtain a probiotic bacterial suspension; The Ligilactobacillus animalis ZYpet-012 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC No. 32008, and the deposit date: September 20, 2024; (2) Preparation of the blank hydrogel: Weigh carbomer 940 powder, add it to ultrapure water, stir well, let it stand at room temperature to fully swell, then sequentially add triethanolamine and zinc hyaluronate, and stir evenly to obtain the blank hydrogel; (3) Preparation of the aloe extract solution: Weigh the aloe extract in deionized water, stir magnetically at 30 - 40 °C until completely dissolved, cool to room temperature to obtain the aloe extract solution, and seal it in a volumetric flask for standby; (4) Mix the above aloe extract solution with the blank hydrogel, stir evenly, and then add the probiotic suspension prepared in step (1) and mix well to obtain the aloe extract - probiotic composite hydrogel for treating pet skin diseases.

2. The preparation method according to claim 1, wherein In step (1), in the probiotic bacterial suspension, the viable count of Lactobacillus animalis subsp. lactis ZYpet-012 reaches 1.0×10 8 ~1.0×10 10 CFU / mL.

3. The preparation method according to claim 1, wherein In step (2), the mass concentration of carbomer 940 powder in the blank hydrogel is 0.2% - 1.0%, the mass concentration of triethanolamine is 0.1% - 0.5%, and the mass concentration of zinc hyaluronate is 0.1% - 1.0%.

4. The preparation method according to claim 1, wherein In step (3), the volume ratio of the aloe extract solution to the blank hydrogel is 1:15 - 20; the mass concentration of the aloe extract in the aloe extract solution is 0.1% - 1.0%.

5. An aloe vera extract-probiotic composite hydrogel for pet skin diseases, characterized in that, The composite hydrogel is prepared by the method according to any one of claims 1 - 4.

6. A Lactobacillus animalis ( Ligilactobacillus animalis ), ZYpet-012, characterized in that Deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC No. 32008, and the deposit date: September 20, 2024.

7. Use of the Ligilactobacillus animalis ZYpet-012 according to claim 1 in the preparation of products for preventing and treating pet skin diseases.

8. The application according to claim 7, characterized in that, The pathogenic bacterium of the pet skin disease is Staphylococcus pseudintermedius.

9. The application according to claim 7 or 8, characterized in that, The products include pet washing and care products and topical medications.

10. The application according to claim 9, characterized in that, The pet washing and care products include shower gels, hair conditioners, and pet wet wipes.

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