Compositions for the prevention or treatment of fungal infections, their formulation, method of preparation and use

Abstract

The present application relates to the field of veterinary medicine, in particular to a composition for preventing or treating pet skin fungal infection and preparation method and application thereof, the composition comprises pythium oligandrum spore powder or pythium oligandrum fermentation supernatant or their combination and the combination of anti-staphylococcus aureus active component. The composition and preparation thereof can safely and effectively prevent or treat animal fungal skin infection, the curative effect is remarkable, and there is no toxic side effect and drug resistance.

Description

Technical Field

[0001] This invention relates to the field of veterinary medicine, and more specifically to a composition for the prevention or treatment of fungal skin infections in pets such as dogs and cats, as well as its formulation, preparation method, and application. Background Technology

[0002] Fungal skin diseases in pets such as dogs and cats, also known as tinea, refer to fungal infections (mainly caused by Microsporum gypseum, Microsporum canis, Trichophyton mentagrophytes, etc.) that infect the epidermis and its appendages (hair, horns, claws). It has become one of the major diseases affecting the health of dogs and cats. It is also a common zoonotic disease, affecting not only the appearance of dogs and cats but also causing itching, and in severe cases, pain.

[0003] Currently, the most commonly used antifungal drugs in the pet medical industry and pet market are chemical antifungal products, such as terbinafine, itraconazole, and chlorhexidine acetate compound preparations. These drugs have long treatment courses, are prone to recurrence, and all have certain toxic side effects, mainly manifesting as gastrointestinal discomfort, and occasionally erythema, swelling, and neurological symptoms. They are not suitable for widespread and long-term use and easily lead to drug resistance. In veterinary clinical practice, the general treatment for fungal skin infections is oral or injectable antifungal drugs, combined with external antifungal drugs to achieve therapeutic effects. However, oral or injectable antifungal drugs have certain hepatotoxic and nephrotoxic effects on animals, especially in young pets, senior pets, pets with diabetes or liver disease, and pets with weakened immune systems, where the side effects are particularly severe. However, when using the above-mentioned topical antifungal medications, pets must wear an Elizabethan collar (head cover) to prevent the animal from licking the affected area, which could cause drug toxicity and reduced efficacy. Many animals (especially cats) exhibit lethargy, loss of appetite, and resistance when wearing an Elizabethan collar. Furthermore, topical antifungal medications containing hormones or other chemical components cannot be used long-term and all carry the risk of drug resistance. In addition, puppies and senior dogs commonly suffer from mixed bacterial and fungal infections due to their low immunity. Staphylococcus aureus infections can cause dermatitis, and long-term latent skin infections can lead to immunosuppression of the skin surface, further increasing the difficulty of fungal treatment. Therefore, finding broad-spectrum, highly effective, and low-toxicity antibacterial ingredients that can simultaneously address mixed bacterial and fungal infections has become a recognized important research direction.

[0004] Currently available topical antifungal medications (chemical drugs, etc.) primarily inhibit the synthesis of the cell walls of pathogenic fungi, thereby damaging the cell walls of fungi and bacteria, causing the fungi to swell or lyse and die. In practical use, they mainly target pathogenic fungi that are currently multiplying or growing. However, when drug-resistant pathogens multiply and are passaged, continued use of the drug carries the risk of developing resistance.

[0005] Current reports indicate that when using Pythium ospore preparations, the fungal infection is often interfered with by various environmental factors, preventing the applied ospores from germinating and exerting their effects under suitable conditions. Furthermore, the disease course is dynamic, and secondary bacterial infections can occur, making ospore preparations alone insufficient. Additionally, there are no reports of successful application of Pythium ospore fermentation broth to fungal infections in animals; successful application to fungal infections in plants has only been reported. Moreover, the simple application of Pythium ospore fermentation broth cannot resolve other symptoms arising from fungal infections in animals, such as itching, dandruff, redness, and inflammatory exudation.

[0006] Patent application CN110225759A – A preparation containing the live fungal parasitic microorganism Pythium oligandrum for treating dermatophytes and yeast infections on the skin and mucous membranes, a method for determining the cell viability of Pythium oligandrum, and a method for administering the preparation – discloses a preparation containing the fungal parasitic microorganism Pythium oligandrum for treating dermatophytes and yeast infections on the skin and mucous membranes. The antibacterial active ingredient of this preparation is the live cells of Pythium oligandrum, which contain dormant oospores, zoospores of tunicae, and live multinucleate mycelia. This patent application uses dormant oospores of Pythium oligandrum as the antibacterial active ingredient and has achieved certain clinical effects in treating experimental dermatophytes in guinea pigs. However, since animal fungal skin diseases are a dynamic pathological process, in most cases they are not a single fungal infection, but may be a mixed skin disease accompanied by bacterial (such as Staphylococcus aureus) infection. Therefore, further improvements are needed in the treatment of animal fungal skin diseases.

[0007] Patent application CN102655754A – An antifungal mixture containing the fungal organism *Pythium oligandrum* – discloses an antifungal mixture containing the fungal organism *Pythium oligandrum*. The antifungal active ingredient in this mixture is live *Pythium oligandrum* cells, including zoospores and zoosporangia. Its antifungal mechanism of action involves the germination of *Pythium oligandrum* live cell oospores, which then reparasitize and kill the pathogenic fungus. Simultaneously, after parasitizing the pathogenic fungus, *Pythium oligandrum* produces various enzymes to decompose the host organism, thus achieving an antifungal effect. This patent application discloses several formulation components using live *Pythium oligandrum* cells as the antifungal active ingredient, combined with several conventional excipients. These components can be used in human and veterinary medicine, cosmetics, etc., for maintaining cleanliness, eliminating pathogenic fungi, and eliminating microorganisms on objects that come into contact with humans or animals. Because the surface environment of human and animal bodies differs fundamentally from the living environment of *Pythium oligandrum*, a soil-growing fungus, whether the live oospores of *Pythium oligandrum* in this patent application can successfully colonize the body surface of humans and animals and exert corresponding hyperparasitic effects requires further verification. Furthermore, as an obligate hyperparasitic pathogenic fungus, there are no literature reports of *Pythium oligandrum* also exhibiting hyperparasitic or anti-Staphylococcus aureus effects.

[0008] In combination with the course of fungal skin diseases in animals and the microecological environment of the skin surface, it is necessary to address potential bacterial infections of the skin in addition to treating fungal skin infections, and at the same time alleviate various symptoms caused by mixed fungal skin diseases, in order to improve the overall treatment effect of fungal skin diseases.

[0009] Existing antifungal products for dogs and cats only address the fungal infection itself, failing to consider the weakened skin immunity caused by latent Staphylococcus aureus infections from the perspective of the skin's microbial ecosystem. This makes them prone to recurrent fungal infections. There is an urgent need for a safe, effective, and stable antifungal product that is non-toxic to animals, can be used as a routine skin care product without inducing antimicrobial resistance, and is suitable for the prevention and treatment of fungal skin infections in animals. Summary of the Invention

[0010] In view of the above-mentioned technical status, the present invention provides a composition, preparation method and application thereof that can simultaneously control Staphylococcus aureus and other common pathogenic fungal infections in pets. The composition can be used for a long time without producing drug resistance, and achieves long-term and effective control of skin pathogenic fungal infections by regulating the micro-ecosystem of the pet's skin surface.

[0011] The composition for preventing or treating fungal infections according to the present invention comprises an active component of Pythium oligandrum and an active component against Staphylococcus aureus, wherein the active component of Pythium oligandrum is Pythium oligandrum oospore powder, Pythium oligandrum fermentation supernatant, or a combination thereof; wherein the active component against Staphylococcus aureus is endolysin or lysozyme.

[0012] In this invention, as one embodiment, the composition comprises a combination of Pythium oligandrum oospore powder and endosomalin or lysozyme; a combination of Pythium oligandrum fermentation supernatant and endosomalin or lysozyme; or a combination of Pythium oligandrum oospore powder and Pythium oligandrum fermentation supernatant and endosomalin or lysozyme.

[0013] In this invention, as one embodiment, the mass ratio of the oligopeptidase active component to the anti-Staphylococcus aureus active component in the composition is 2.4 to 3.4:1. As an example, it can be a mass ratio including but not limited to 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, or 3.4:1.

[0014] In this invention, as one embodiment, the Oligopyrophyll fermentation supernatant, endosomalin, or lysozyme is in the form of lyophilized powder.

[0015] In this invention, the *Pythium oligandrum* oospore powder or *Pythium oligandrum* fermentation supernatant can inhibit the germination of fungal spores and the growth of fungal hyphae in pet skin infections, and even cause them to die; the endolysin or lysozyme can kill or inhibit *Staphylococcus aureus*, solving the problem of latent bacterial infection in pet skin diseases, thereby improving the immunity of the pet's skin, and enabling the effective antifungal components of *Pythium oligandrum* to play a better antifungal role. The two play a good synergistic role in solving mixed fungal skin diseases in pets.

[0016] In this invention, as one embodiment, the composition further includes traditional Chinese medicine ingredients, which are selected from tangerine peel powder, paeonol, eugenol, or alum, or a combination of two or more of them; preferably, the traditional Chinese medicine ingredients are selected from a combination of tangerine peel powder, paeonol, eugenol, and alum.

[0017] In this invention, as one embodiment, the mass ratio of the oligopeptidase active component, the anti-Staphylococcus aureus active component, and the traditional Chinese medicine component in the composition is 2.4-3.4:1:1.

[0018] In this invention, the traditional Chinese medicine components help to resolve and alleviate various symptoms such as itching and scaling caused by fungal infections in pets, thereby accelerating the recovery of the disease course and skin ecology, and playing a further synergistic role.

[0019] The present invention also provides a formulation comprising the above-described composition, the formulation comprising, by weight percentage in the formulation,

[0020] The amount of the oligoandromeda oospore powder or oligoandromeda fermentation supernatant freeze-dried powder or oligoandromeda oospore powder or a combination thereof is 5% to 40%, preferably 10% to 25%, and most preferably 12% to 17%.

[0021] The amount of the endolysin or lysozyme or a combination thereof is 0.5% to 20%, preferably 0.5% to 10%, and most preferably 5%.

[0022] The dosage of the herbal ingredients is 0% to 20%, preferably 3% to 10%, and most preferably 5%.

[0023] In this invention, as one embodiment, the traditional Chinese medicine components, by weight percentage in the preparation, include:

[0024] The amount of orange peel powder is 0% to 5%, preferably 3%;

[0025] The amount of paeonol is 0% to 5%, preferably 0.5%;

[0026] The amount of eugenol is 0% to 5%, preferably 1%;

[0027] The amount of calcined alum is 0% to 5%, preferably 0.5%.

[0028] In this invention, as one embodiment, the 5% traditional Chinese medicine component in the preparation, by weight percentage, consists of 3% tangerine peel powder, 0.5% paeonol, 1% eugenol, and 0.5% alum.

[0029] In this invention, as one embodiment, the formulation further includes excipients selected from buffers, adsorbents, or humectants, or combinations of two or more of them; preferably, the excipients are selected from combinations of buffers, adsorbents, and humectants.

[0030] In this invention, as one embodiment, the preparation is preferably a tablet, capsule, powder, or aqueous solution.

[0031] As one embodiment of the present invention, the formulation contains ， The buffer is selected from anhydrous citric acid, sodium carbonate, or sodium bicarbonate, or a combination of two or more of them; preferably a combination of citric acid, sodium carbonate, and sodium bicarbonate; the amount of buffer is preferably 0.5% to 45% by weight in the formulation; more preferably 5% to 30% anhydrous citric acid, preferably 23%; 5% to 30% sodium bicarbonate, preferably 18%; and 0.5% to 15% sodium carbonate, preferably 2%.

[0032] In this invention, as one embodiment, the adsorbent in the formulation is selected from silica, and the amount of silica used is 0.5% to 20% by weight percentage in the formulation, preferably 8%.

[0033] In one embodiment of the present invention, the humectant in the formulation is selected from PEG6000, sorbitol, or a combination thereof; preferably a combination of PEG6000 and sorbitol; the amount of the humectant is 0.5% to 30% by weight percentage in the formulation; in one embodiment, the amount of PEG6000 is 0.5% to 20%, preferably 3%; in one embodiment, the amount of sorbitol is 5% to 30%, preferably 24%.

[0034] As one embodiment of the present invention, the present invention also provides a method for preparing Pythium oligoandrogenes oospore powder, wherein the Pythium oligoandrogenes oospore powder is obtained by the following method:

[0035] A. After the oligomale pythium strain was revived by PDA plates, it was stored in slant and kept at 2-8℃ for later use.

[0036] B. After activating the Oligocytic Pythium strain from step A in PDA medium for 3-5 days, transfer it to liquid culture medium PD for shake flask culture for 3-5 days, and then transfer the shake flask seed culture to a fermenter for large-scale culture.

[0037] C. After 7-10 days of expanded culture and spore production, collect the mycelium and oospores of Pythium oligoandrogenes, and spray dry to obtain Pythium oligoandrogenes oospore powder.

[0038] As one embodiment, this invention also provides a method for preparing freeze-dried powder of Pythium oligandrum fermentation supernatant, wherein the freeze-dried powder of Pythium oligandrum fermentation supernatant is obtained by the following method:

[0039] A. After the oligomale pythium strain was revived by PDA plates, it was stored in slant and kept at 2-8℃ for later use.

[0040] B. After activating the Oligocytic Pythium strain from step A in PDA medium for 3-5 days, transfer it to liquid culture medium PD for shake flask culture for 3-5 days, and then transfer the shake flask seed culture to a fermenter for large-scale culture.

[0041] C. After 7-10 days of expanded culture, collect the fermentation broth, centrifuge and filter to obtain the supernatant of Pythium oligandrum fermentation;

[0042] D. After the collected Pythium oligandrum fermentation supernatant is freeze-dried in a vacuum freeze dryer, nitrogen gas is purged and the freeze-dried powder is collected to obtain Pythium oligandrum fermentation supernatant freeze-dried powder.

[0043] The present invention also provides a method for preparing an agent for preventing or treating fungal infections, the method comprising: mixing lyophilized powder of Pythium oligandrum oospores and / or lyophilized supernatant of Pythium oligandrum fermentation, lyophilized powder of endosomalin or lysozyme, optional traditional Chinese medicine ingredients and excipients in proportion using a homogenizer, and then compressing the mixture into tablets under a dry and low-temperature environment.

[0044] Application of the Oligopyrophylloides antifungal compound tablets described in this invention:

[0045] For use, dissolve one tablet (3g) in 150-200ml of water for every 10kg of body weight, and apply or spray to the affected area. Use 1-2 times daily for 7-14 consecutive days.

[0046] The technical effects of this invention are as follows:

[0047] The composition of this invention contains Pythium oligandrum oospore powder or Pythium oligandrum fermentation supernatant freeze-dried powder, which, when combined with the active component against Staphylococcus aureus, has a synergistic antifungal effect. In addition, the addition of anti-inflammatory and antipruritic traditional Chinese medicine components further enhances the antifungal effect.

[0048] 1. Oospore powder or freeze-dried powder of Pythium oligomangum fermentation supernatant has a certain inhibitory effect on the hyphae and spore germination and growth of pathogenic fungi such as Microsporum gypseum, Trichophyton mentagrophytes, and Microsporum canis in dogs and cats, and can prevent or treat fungal skin infections in pets. Oospore powder or freeze-dried powder of Pythium oligomangum fermentation supernatant, lysozyme, or lysozyme can be formulated into a topical antibacterial preparation in a certain proportion. This preparation enhances the antifungal effect by regulating the skin's microecological system, achieving a synergistic effect. Oospore powder, freeze-dried powder of Pythium oligomangum fermentation supernatant, lysozyme, and lysozyme are all safe and do not induce drug resistance, and can safely and effectively prevent or treat fungal skin infections in pets.

[0049] 2. When Pythium oligandrum oospore powder was combined with anti-Staphylococcus aureus active components, namely endolysin or lysozyme, the average treatment period for pets to recover was shortened by approximately 4 days compared to the use of Pythium oligandrum oospore powder alone. When Pythium oligandrum fermentation supernatant lyophilized powder was combined with endolysin or lysozyme, the average treatment period for pets to recover was 7.2 days, which was shortened by approximately 3 days compared to the use of Pythium oligandrum fermentation supernatant lyophilized powder alone. Observation of the overall skin condition during the treatment of various cases revealed that in cases treated with the antifungal combination preparation, pathological symptoms were significantly reduced, the course of the disease was shortened, and the coat and other physical characteristics improved after recovery. This indicates that endolysin or lysozyme, after inhibiting Staphylococcus aureus, improved the skin's microecological system, enhanced skin immunity, and further promoted the efficacy of the antifungal active components of Pythium oligandrum, playing a synergistic role.

[0050] 3. The addition of anti-inflammatory and antipruritic traditional Chinese medicine ingredients to the compound preparation has an auxiliary and synergistic effect on the antifungal active ingredients of Pythium oligandrum. During the treatment process, it improved the symptoms of itching and scratching in the pets, and further enhanced the antifungal efficacy of the compound preparation. Attached Figure Description

[0051] Figure 1 Determination of the antibacterial activity of Pythium oligandrum oospore powder using the plate confrontation method;

[0052] Figure 2 The fermentation supernatant of Pythium oligandrum has an inhibitory effect on the spore germination and growth of pathogenic fungi such as Microsporum gypseum, Trichophyton mentagrophytes, and Microsporum canis. Detailed Implementation

[0053] The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the invention in any way.

[0054] Example 1: Preparation of Lyophilized Pythium oligandrum oospore powder / Lyophilized Pythium oligandrum fermentation supernatant powder and its in vitro antibacterial verification

[0055] 1. Preparation of Oospore Pythium oligosporus powder:

[0056] A. After the oligomale Pythium strain was revived via PDA plates, it was stored on slant agar and kept at 2-8℃ for later use.

[0057] B. Activate and culture the oligo-male Pythium strain from step A in PDA medium. After culturing in the dark at 28°C for 3 days, cut the mycelial blocks from the plate using the cutting method and inoculate them into liquid culture medium PD for shake-flask culture. After shaking-flask culture at 25-28°C and 190-220 rpm / min for 3-5 days, transfer the shake-flask seed culture to a large fermenter for large-scale culture.

[0058] C. After 7-8 days of expanded culture and sporulation, centrifuge to collect the mycelium and oospores of Pythium oligandii, and spray dry to obtain Pythium oligandii oospore powder.

[0059] Determination of viable spore rate in *Pythium oligoandrogenes* oospore powder: Take 1g of the prepared *Pythium oligoandrogenes* oospore powder, prepare a spore suspension, and take 1ml (1×10⁻⁶) of the suspension. 7 (Spores / g) were inoculated into PD liquid medium and cultured in shake flasks at 28°C for 12 hours. The germination rate of spores was examined by microscopic examination. Spores with a germ tube length ≥ spore diameter were considered to have germinated. The viable spore rate (≥ 85%) was calculated.

[0060] Preparation of freeze-dried powder from Pythium oligandrum fermentation supernatant:

[0061] A. After the oligomale Pythium strain was revived via PDA plates, it was stored on slant agar and kept at 2-8℃ for later use.

[0062] B. Activate and culture the Oligopyrophyll strain from step A in PDA medium. After culturing in the dark at 25-28℃ for 3 days, cut off the Oligopyrophyll mycelial blocks by the cutting method and transfer them to liquid culture medium PD for shake flask culture. After shaking flask culture at 25-28℃ and 190-220 rpm / min for 3-5 days, transfer the shake flask seed culture to a large fermenter for large-scale culture.

[0063] C. After 8 days of culture, the fermentation broth was collected, centrifuged at 10,000 rpm for 15 minutes, and filtered to obtain the supernatant of Pythium oligandrum fermentation.

[0064] D. After the collected Pythium oligandrum fermentation supernatant is freeze-dried in a vacuum freeze dryer for 48 hours, nitrogen gas is purged and the freeze-dried powder is collected to obtain Pythium oligandrum fermentation supernatant freeze-dried powder.

[0065] 2. In vitro antibacterial validation of Pythium oligandrum oospore powder and Pythium oligandrum fermentation supernatant lyophilized powder:

[0066] 1) In vitro antibacterial verification of Pythium oligandrum oospore powder

[0067] By using 1g (1×10) of Pythium ospore powder 7 (Spores / g, viable spore rate ≥85%), after being activated in agar plates, were inoculated onto plates pre-inoculated with pathogen indicator fungi (such as Microsporum gypseum). The inhibitory effect of Pythium oligosporum colonies on indicator fungi colonies was calculated using the plate confrontation method. Through the control of plates inoculated with pathogen indicator fungi alone, it was found that on the pathogen indicator fungi plates without Pythium oligosporum oospore powder, the pathogen indicator fungi grew normally, with an average colony diameter of 7.5 cm; while on the pathogen indicator fungi plates inoculated with Pythium oligosporum oospore powder, the growth of pathogen indicator fungi was inhibited, with an average colony diameter of 2.5 cm.

[0068] The pathogenic fungi that serve as indicator bacteria are: Microsporum gypseum, Trichophyton mentagrophytes, and Microsporum canis.

[0069]

[0070] (Note: X is the average colony diameter of indicator bacteria in the control group; Y is the average colony diameter of indicator bacteria after their growth is inhibited in the confrontation group.)

[0071] The antibacterial activity of *Pythium oligandrum* oospore powder was determined using the plate confrontation method. See details... Figure 1 :

[0072] in Figure 1 The left image shows the confrontation culture between Pythium oligomangiosus and the pathogenic fungus. Pythium oligomangiosus is the dominant fungus and clearly covers and parasitizes the pathogenic fungus colony, inhibiting the growth of the pathogenic fungus colony. In the right image, the growth of the pathogenic fungus is not affected in the control group.

[0073] 2) In vitro antibacterial validation of lyophilized Pythium oligandrum fermentation supernatant powder

[0074] By reconstituted 1g of lyophilized *Pythium oligandrum* fermentation supernatant powder and mixing it with a suspension of pathogenic fungal spores, the mixture was catalytically reacted at 2-8℃ for 6-24 hours. The treated suspensions were then inoculated onto PDA plates at different reaction times to observe the germination and growth of the pathogenic fungi. The results showed that *Pythium oligandrum* fermentation supernatant had a certain inhibitory effect on the germination and growth of pathogenic fungal spores. The pathogenic fungal spore suspension treated with *Pythium oligandrum* fermentation supernatant failed to germinate and grow normally, while the untreated control group of pathogenic fungal spores germinated and grew normally.

[0075] The pathogenic fungal spore suspensions used as indicator bacteria include: Microsporum gypseum, Trichophyton mentagrophytes, Microsporum canis, etc.

[0076]

[0077] (Note: X represents the average number of surviving bacteria per 1 ml of sample; Y represents the decrease in the average number of surviving bacteria per 1 ml of sample.)

[0078] The fermentation supernatant of *Pythium oligandrum* inhibits the spore germination and growth of pathogenic fungi such as *Microsporum gypseum*, *Trichophyton mentagrophytes*, and *Microsporum canis*. See details... Figure 2 :

[0079] in Figure 2 In the left image, pathogenic fungal spores were cultured after treatment with the supernatant of Pythium oligomangum fermentation, and it can be seen that the germination and growth of pathogenic fungal spores were inhibited. In the right image, pathogenic fungal spores of the control group were cultured after treatment with sterile water, and it can be seen that the germination and growth of pathogenic fungal spores were not affected, and they grew into the corresponding colony morphology normally.

[0080] Example 2: Treatment of fungal skin disease cases with Oomycetes oospore powder and treatment of fungal skin disease cases with a combination of Oomycetes oospore powder and endosomalin.

[0081] Formulation 1: Oospore powder of *Pythium oligomangum* is used as the antifungal component. After reconstitution, each ml of the suspension contains 30-50 live *Pythium oligomangum* ospores. It is formulated with excipients in a specific ratio to create an antifungal preparation comprising *Pythium oligomangum* ospore powder, anhydrous citric acid, sodium bicarbonate, silica, PEG6000, sodium carbonate, and sorbitol.

[0082]

[0083] Group A patients were given preparation 1:

[0084] This group of animals was diagnosed with fungal skin infections (such as Microsporum canis) at a veterinary hospital. The group consisted of 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs. The cats had an average weight of 2.15 kg, the dogs an average weight of 7.58 kg, and an average age of 6.3 months. None had a history of other diseases. During the clinical trial, the animals were fed a fixed amount of commercial food, with no restrictions on water intake, and received routine vaccinations. Prior to the clinical trial, each animal underwent a physical examination, including measuring weight, temperature, respiration, heart rate, appetite, and coat condition, and blood tests (complete blood count and blood glucose) were performed.

[0085] Observe the affected pet's pathological changes daily, such as rashes, dandruff, hair loss, broken hair, hairlessness, pustules, and dryness / moisture levels. Record clinical symptoms and lesions before (baseline values) and after application of Preparation 1. Examine the hair at the affected area daily with a Wood's lamp, and scrape tissue from the lesion for fungal culture and identification. Continue observation until the condition improves and then discontinue application of Preparation 1.

[0086] Formulation 2: Ozymospore powder of Pythium oligandrum is used as an antifungal component and is formulated into a compound antifungal preparation with endosomalin and excipients in a certain proportion. The final composition of the antifungal compound preparation is: Ozymospore powder of Pythium oligandrum, endosomalin, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate, and sorbitol.

[0087]

[0088] Group B patients were given preparation 2:

[0089] This group of animals was diagnosed with fungal skin infections (such as Microsporum canis or Microsporum gypseum) at a veterinary hospital. The group consisted of 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs. The cats had an average weight of 2.58 kg, the dogs an average weight of 8.25 kg, and an average age of 5.8 months. None had a history of other diseases. During the clinical trial, the animals were fed a fixed amount of commercial food with no water restrictions and received routine vaccinations. Prior to the clinical trial, each animal underwent a physical examination, including measuring weight, temperature, respiration, heart rate, appetite, and coat condition. Blood tests (complete blood count and blood glucose) were also performed.

[0090] Observe the affected pet's pathological changes daily, such as rashes, dandruff, hair loss, broken hair, hairlessness, pustules, and dryness / moisture levels. Record clinical symptoms and lesions before (baseline values) and after application of Preparation 2. Examine the affected area's fur daily with a Wood's lamp and scrape tissue for fungal culture and identification. Continue observation until the condition improves, then discontinue application of Preparation 2.

[0091] Application method: Dissolve the preparation used in the example in water, apply or spray the fully dissolved suspension to the affected area and let it air dry naturally. Apply 1-2 times a day until the condition improves and then discontinue use.

[0092] result:

[0093] Correlation of clinical status changes in group A pets after administration of preparation 1

[0094]

[0095] Correlation of clinical status changes in group B pets after administration of formulation 2

[0096]

[0097]

[0098] From the data in Example 2, we can see that:

[0099] In Group A, the average treatment period for pets treated with formulation 1 was 13.2 days. Post-treatment Wood's lamp examination and microbiological examination revealed no fungal hyphae. During treatment, the pets exhibited itching and scratching; Elizabethan collars were used to prevent scratching and to support treatment. Post-treatment, coat growth was slow. This indicates that while the single application of *Pythium oligandrum* oospore powder can achieve antifungal efficacy, it did not significantly improve clinical signs. Furthermore, the treatment course was essentially the same as that of traditional antifungal drugs or products, with no significant difference (P > 0.05).

[0100] In Group B, the average treatment period for pets treated with formulation 2 was 8.9 days. During treatment, the pets maintained good mental condition, with a significant reduction in inflammatory exudate. Occasionally, the pets scratched, and hair regrowth recovered quickly after recovery. This indicates that the combined formulation of *Pythium osmidrosis* oospore powder and endolysin significantly improved the overall condition of the pets and shortened the treatment course. This demonstrates that in the treatment of fungal skin infections, the endolysin addresses bacterial infection, improves the skin's microecological system, and enhances the skin's own immunity, thereby strengthening the antifungal efficacy of *Pythium osmidrosis* oospore powder. Therefore, the combination of *Pythium osmidrosis* oospore powder and endolysin improves the overall antifungal efficacy.

[0101] Example 3: Treatment of fungal skin disease cases with lyophilized powder of Pythium oligandrum fermentation supernatant and treatment of fungal skin disease cases with a compound preparation of lyophilized powder of Pythium oligandrum fermentation supernatant and endolysin.

[0102] Formulation 3: The lyophilized supernatant of Pythium oligandrum fermentation liquid is used as the antifungal active ingredient and is formulated with excipients in a certain proportion to form an antifungal preparation. Its composition is: lyophilized supernatant of Pythium oligandrum fermentation liquid, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate, and sorbitol.

[0103]

[0104]

[0105] Group C patients were given preparation 3:

[0106] This group of animals was diagnosed with fungal skin infections (such as Microsporum canis or Trichophyton mentagrophytes) at a veterinary hospital. The group consisted of 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs. The cats had an average weight of 3.12 kg, the dogs an average weight of 7.05 kg, and an average age of 6.4 months. None had a history of other diseases. During the clinical trial, the animals were fed a fixed amount of commercial food, with no restrictions on water intake, and received routine vaccinations. Prior to the clinical trial, each animal underwent a physical examination, including measuring weight, temperature, respiration, heart rate, appetite, and coat condition, and blood tests (complete blood count and blood glucose) were performed.

[0107] Observe the affected pet's pathological changes daily, such as rashes, dandruff, hair loss, broken hair, hairlessness, pustules, and dryness / moisture levels. Record clinical symptoms and lesions before (baseline values) and after application of preparation 3. Examine the affected area's fur daily with a Wood's lamp and scrape tissue for fungal culture and identification. Continue observation until the condition improves and discontinue application of preparation 3.

[0108] Formulation 4: The lyophilized supernatant of Pythium oligandrum fermentation liquid is used as an antifungal component and is formulated into a compound antifungal preparation with endosomalin and excipients in a certain proportion. The composition of the antifungal compound preparation is: lyophilized supernatant of Pythium oligandrum fermentation liquid, endosomalin, anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate, and sorbitol.

[0109]

[0110]

[0111] Group D patients were given preparation 4:

[0112] This group of animals consisted of 2 male cats, 3 female cats, 2 male dogs, and 3 female dogs, all diagnosed with fungal (such as Microsporum canis) skin infections at a veterinary hospital. The cats had an average weight of 2.96 kg, the dogs an average weight of 6.95 kg, and the average age was 6.4 months. None of the animals had a history of other diseases. During the clinical trial, the animals were fed a fixed amount of commercial pet food with no water restrictions and received routine vaccinations. Prior to the clinical trial, each animal underwent a physical examination, including measuring weight, temperature, respiration, heart rate, appetite, and coat condition, and blood tests (complete blood count and blood glucose) were performed.

[0113] Observe the affected pet's pathological changes daily, such as rashes, dandruff, hair loss, broken hair, hairlessness, pustules, and dryness / moisture levels. Record clinical symptoms and lesions before (baseline values) and after application of preparation 4. Examine the affected area's fur daily with a Wood's lamp and scrape tissue for fungal culture and identification. Continue observation until the condition improves and discontinue application of preparation 4.

[0114] Application method: Dissolve the preparation used in the example in water, apply or spray the fully dissolved suspension to the affected area and let it air dry naturally. Apply 1-2 times a day until the condition improves and then discontinue use.

[0115] result:

[0116] Correlation of clinical status changes in group C pets after administration of the preparation 3

[0117]

[0118]

[0119] Correlation of clinical status changes in group D pets after administration of the preparation 4 days

[0120]

[0121] As can be seen from the data in Example 3:

[0122] In Group C, the average treatment period for recovery after treatment with formulation 3 was 10.4 days. No fluorescence was observed in Wood's lamp examination after treatment. Before treatment, the pets suffered from bacterial infections such as hair loss and skin ulcers due to itching and scratching; these conditions improved after treatment, with reduced inflammatory secretions. The lyophilized supernatant of *Pythium oligandrum* fermentation supernatant alone also achieved antifungal efficacy and showed some improvement in clinical signs, with a correspondingly shorter treatment course. This indicates that the antifungal efficacy of *Pythium oligandrum* fermentation supernatant is superior to that of *Pythium oligandrum* oospore powder.

[0123] In Group D, the average treatment period for recovery after treatment with formulation 4 was 7.3 days. During treatment, inflammatory exudate at the affected area was significantly reduced, the pets experienced no discomfort, and hair regrowth recovered quickly after recovery. The combined formulation of lyophilized Oligopyroxburghii fermentation supernatant powder and endolysin significantly improved the clinical condition of the pets, reduced inflammatory secretions, and shortened the treatment course. This indicates that the combined formulation of lyophilized Oligopyroxburghii fermentation supernatant powder and endolysin greatly enhanced the antifungal effect of the lyophilized Oligopyroxburghii fermentation supernatant powder, improved the skin micro-ecosystem of the pets, and enhanced the skin's own immunity, thereby strengthening the overall therapeutic effect.

[0124] Example 4: Treatment of fungal skin disease cases using a compound preparation of lyophilized Pythium oligandrum fermentation supernatant powder, endosomalin, and traditional Chinese medicine components.

[0125] Formulation 5: The lyophilized supernatant of Pythium oligandrum fermentation liquid is used as the antifungal active ingredient. It is formulated into a compound antifungal preparation by combining it with endosomalin, traditional Chinese medicine ingredients and excipients in a certain proportion. The final composition of the antifungal compound preparation is as follows: lyophilized supernatant of Pythium oligandrum fermentation liquid, endosomalin, traditional Chinese medicine ingredients (orange peel powder, paeonol, eugenol, alum), anhydrous citric acid, sodium bicarbonate, silicon dioxide, PEG6000, sodium carbonate, and sorbitol.

[0126]

[0127]

[0128] Group E patients were given preparation 5:

[0129] This group of animals was diagnosed with fungal skin infections (such as Microsporum canis) at a veterinary hospital. The group consisted of 2 male cats, 3 female cats, 4 male dogs, and 1 female dog. The cats had an average weight of 3.25 kg, the dogs an average weight of 8.05 kg, and an average age of 8.3 months. None had a history of other diseases. During the clinical trial, the animals were fed a fixed amount of commercial food with no water restrictions and received routine vaccinations. Prior to the clinical trial, each animal underwent a physical examination, including measuring weight, temperature, respiration, heart rate, appetite, and coat condition. Blood tests (complete blood count and blood glucose) were also performed.

[0130] Observe the affected pet's pathological changes daily, such as rashes, dandruff, hair loss, broken hair, hairlessness, pustules, and dryness / moisture levels. Record clinical symptoms and lesions before (baseline values) and after application of preparation 5. Examine the affected area's hair daily with a Wood's lamp and scrape tissue for fungal culture and identification. Continue observation until the condition improves and discontinue application of preparation 5.

[0131] Application method: Dissolve the preparation used in the example in water, apply or spray the fully dissolved suspension to the affected area and let it air dry naturally. Apply 1-2 times a day until the condition improves and then discontinue use.

[0132] result:

[0133] Correlation of clinical status changes in group E pets 5 days after administration of the preparation

[0134]

[0135]

[0136] As can be seen from the data in Example 4:

[0137] In Group E, the average treatment period for recovery after administration of the formulation was 7.2 days. The pets showed significant improvement in clinical symptoms, with reduced inflammatory exudate. No significant itching or scratching was observed two days after administration. Microbiological examination of the treated area after recovery revealed negative results for both pathogenic fungi and pathogenic bacteria (Staphylococcus aureus). The skin's microecological system was improved and restored. No recurrence was observed during a three-month follow-up period. This indicates that the compound formulation is more effective than the single application of the lyophilized powder of *Pythium oligandrum* fermentation supernatant, and can be used long-term as routine care with little risk of developing drug resistance, thus improving the immunity of the pet's skin.

[0138] *Note: Evaluation criteria for clinical animal administration of the formulation in this example.

[0139] Cure: After application of the formulation in the example, fungal culture showed that the rate of negative conversion of skin fungi (Microsporum canis / Microsporum gypseum / Trichophyton mentagrophytes, etc.) was ≥95%. At the same time, inflammatory secretions disappeared significantly, and the affected area had no odor, rashes, dandruff, pustules, etc. disappeared significantly, and itching symptoms disappeared significantly.

[0140] Significant efficacy: After application of the formulation in the example, fungal culture showed a skin fungal conversion rate of ≥80%, while inflammatory secretions disappeared significantly, and the affected area had no odor, rashes, dandruff, pustules, etc., and itching symptoms disappeared significantly.

[0141] Effectiveness: After application of the formulation in the example, fungal culture showed a skin fungal conversion rate of ≥60%, while inflammatory secretions were reduced, and the affected area was free of odor, with a reduction in rashes, dandruff, pustules, etc., and a relief of itching symptoms.

[0142] Ineffective: After application of the formulation in the example, fungal culture showed a skin fungal clearance rate of <60%, while inflammatory secretions were still present, and the affected area had an odor, and there was no reduction in rashes, dandruff, pustules, etc., and the itching symptoms were still obvious.

[0143] Negative conversion rate of pathogenic fungi / bacteria to skin infections (%): Samples were collected daily from each affected pet for microbial culture and identification. Coat samples were collected from five affected areas: face, external ear, radius of the forelimb, thoracolumbar junction, and tail. The negative conversion rate of pathogenic fungi / bacteria was determined by observing the coat microbial culture results.

[0144] Clinical symptom and sign scoring: Clinical symptoms of fungal skin diseases include itching, papules, erythema, keratosis, and scaling. The severity of infection is evaluated using a scoring system, with the following evaluation criteria:

[0145] 0 = None; no obvious clinical symptoms were observed throughout the body, and the patient's condition was good.

[0146] 1 = Mild, with minor skin damage and low lesion density, and lesions appearing only in a small area;

[0147] 2 = In this case, there is damage to a small area of ​​skin all over the body, with a high degree and density of damage, or symptoms appear over a large area of ​​the body;

[0148] 3 = Severe, indicating a high degree and density of skin damage or extensive skin damage throughout the body.

[0149] Clinical symptom and sign score reduction index = (total score before treatment - total score after treatment) / total score before treatment * 100%.

[0150] The efficacy of the preparations used in the examples against fungal infections was determined as follows:

[0151] Cure: During the administration of the formulation described in the example and for 2 weeks after discontinuation, the in vitro fungi disappeared, the clinical skin symptoms caused by fungal infection (erythema, itching, hair loss, pustules, scaling, erosion and keratosis) disappeared, the animal's physical signs returned to normal, and the microbiological examination was negative.

[0152] Significant efficacy: During the administration of the formulation in the example and for 2 weeks after discontinuation, in vitro fungi basically disappeared, and the incidence of skin clinical symptoms (erythema, itching, hair loss, pustules, scaling, erosion and keratosis) caused by fungal infection was >60%, animal signs basically returned to normal, and microbiological examination was negative.

[0153] Improvement: During the administration of the formulation described in the example and for 2 weeks after discontinuation, the number of fungi in the scraping examination was between 20% and 60%, the animal's physical signs improved but did not return to normal, and the microbiological examination was positive.

[0154] Ineffective: During administration of the formulation of the example and for 2 weeks after discontinuation, the number of in vitro fungi decreased by <20% or even increased, the signs were more severe, and the microbiological examination was positive.

[0155] Overall effective rate (%) = (Number of cured animals + Number of animals showing significant improvement) / Total number of treated animals * 100%

[0156] *Note: Although the above embodiments did not involve the combination of lysozyme, based on the fact that lysozyme has the same anti-Staphylococcus aureus effect as endosomalin, it can be inferred that it will have the same efficacy when combined with the active ingredient of Pythium oligandrum.

Claims

1. A composition for the prevention or treatment of fungal infections, characterized in that, The composition comprises an active component of Pythium oligandrum and an active component against Staphylococcus aureus, wherein the active component of Pythium oligandrum is Pythium oligandrum oospore powder, Pythium oligandrum fermentation supernatant, or a combination thereof; wherein the active component against Staphylococcus aureus is an endosomalin. The fermentation supernatant of *Pythium oligandrum* and the endolysin are in the form of lyophilized powder. The lyophilized powder of the Oligomaetum fermentation supernatant was obtained by the following method: A. After the oligomale pythium strain was revived by PDA plates, it was stored in slant and kept at 2-8℃ for later use. B. After activating the Oligocytic Pythium strain from step A in PDA medium for 3-5 days, transfer it to liquid culture medium PD for shake flask culture for 3-5 days, and then transfer the shake flask seed culture to a fermenter for large-scale culture. C. After 7-10 days of expanded culture, collect the fermentation broth, centrifuge and filter to obtain the supernatant of Pythium oligandrum fermentation; D. After the collected Pythium oligandrum fermentation supernatant is freeze-dried in a vacuum freeze dryer, nitrogen gas is purged and the freeze-dried powder is collected to obtain Pythium oligandrum fermentation supernatant freeze-dried powder. The mass ratio of the oligopeptidase active component to the anti-Staphylococcus aureus active component in the composition is 2.4~3.4:

1.

2. The composition according to claim 1, characterized in that, The composition comprises a combination of Pythium oligandrum oospore powder and endosomalin; a combination of Pythium oligandrum fermentation supernatant and endosomalin; or a combination of Pythium oligandrum oospore powder and Pythium oligandrum fermentation supernatant and endosomalin.

3. The composition according to claim 1, characterized in that, The composition also includes traditional Chinese medicine ingredients selected from tangerine peel powder, paeonol, eugenol, or alum, or a combination of two or more of them.

4. The composition according to claim 3, characterized in that, The Chinese medicine ingredients are selected from a combination of tangerine peel powder, paeonol, eugenol, and alum.

5. The composition according to claim 3, characterized in that, The mass ratio of the active component of *Pythium oligandrum*, the active component against *Staphylococcus aureus*, and the traditional Chinese medicine component is 2.4~3.4:1:

1.

6. A formulation comprising the composition according to any one of claims 3 to 5, characterized in that, The formulation comprises, by weight percentage in the formulation, The amount of the oligoandromeda oospore powder or oligoandromeda fermentation supernatant lyophilized powder, or a combination thereof, is 5% to 40%. The amount of the endolysin is 0.5% to 20%; The amount of the Chinese herbal medicine ingredients is 0% to 20%.

7. A formulation comprising the method according to claim 6, characterized in that, The formulation comprises, by weight percentage in the formulation, The amount of the oligoandromeda oospore powder or oligoandromeda fermentation supernatant lyophilized powder, or a combination of both, is 10% to 25%. The amount of the endolysin is 0.5% to 10%; The amount of the Chinese herbal medicine ingredients is 3% to 10%.

8. A formulation comprising the method according to claim 7, characterized in that, The formulation comprises, by weight percentage in the formulation, The amount of the oligoandromeda oospore powder or oligoandromeda fermentation supernatant lyophilized powder, or a combination thereof, is 12% to 17%. The amount of the endosomal is 5%; The amount of the herbal ingredient is 5%.

9. The formulation according to claim 6, characterized in that, In the formulation, the traditional Chinese medicine components, by weight percentage in the formulation, include: The amount of orange peel powder is 0%~5%; The amount of paeonol is 0%~5%; The amount of eugenol is 0%~5%; and The amount of calcined alum is 0%~5%.

10. The formulation according to claim 9, characterized in that, In the formulation, the traditional Chinese medicine components, by weight percentage in the formulation, include: The amount of orange peel powder is 3%; The amount of paeonol was 0.5%; The amount of eugenol was 1%; and The amount of calcined alum is 0.5%.

11. The formulation according to claim 8, characterized in that, In the preparation, the 5% traditional Chinese medicine ingredients, by weight percentage in the preparation, consist of 3% tangerine peel powder, 0.5% paeonol, 1% eugenol and 0.5% alum.

12. The formulation according to claim 6, characterized in that, The formulation also includes excipients selected from buffers, adsorbents or humectants, or combinations of two or more of them.

13. The formulation according to claim 12, characterized in that, The excipients are selected from a combination of buffers, adsorbents and humectants.

14. The formulation according to claim 12, characterized in that, The preparation is a tablet, capsule, powder, or liquid.

15. The formulation according to claim 12, characterized in that, In the formulation, the buffer is selected from anhydrous citric acid, sodium carbonate, or sodium bicarbonate, or a combination of two or more of them.

16. The formulation according to claim 15, characterized in that, In the formulation, the buffer is selected from a combination of citric acid, sodium carbonate, and sodium bicarbonate.

17. The formulation according to claim 12, wherein the amount of buffer is 0.5 to 45% by weight in the formulation.

18. The formulation according to claim 17, wherein the amount of anhydrous citric acid is 5% to 30% by weight in the formulation; the amount of sodium bicarbonate is 5% to 30%; and the amount of sodium carbonate is 0.5% to 15%.

19. The formulation according to claim 18, wherein the amount of anhydrous citric acid is 23% by weight percentage in the formulation; the amount of sodium bicarbonate is 18%; and the amount of sodium carbonate is 2%.

20. The formulation according to claim 12, characterized in that, In the formulation, the adsorbent is selected from silica, and the amount of silica used is 0.5% to 20% by weight in the formulation.

21. The formulation according to claim 20, characterized in that, In the formulation, the amount of silica is 8% by weight.

22. The formulation according to claim 12, characterized in that, In the formulation, the humectant is selected from PEG6000, sorbitol, or a combination thereof.

23. The formulation according to claim 22, characterized in that, In the formulation, the humectant is selected from a combination of PEG6000 and sorbitol.

24. The formulation according to claim 12, characterized in that, The amount of the humectant is 0.5-30% by weight percentage in the formulation.

25. The formulation according to claim 24, characterized in that, The amount of PEG6000 used in the formulation is 0.5% to 20% by weight; the amount of sorbitol used is 5% to 30%.

26. The formulation according to claim 25, characterized in that, The amount of PEG6000 in the formulation is 3% by weight; the amount of sorbitol is 24%.

27. The composition according to claim 1, characterized in that, The oligoandromycete oospore powder was obtained by the following method: A. After the oligomale pythium strain was revived by PDA plates, it was stored in slant and kept at 2-8℃ for later use. B. After activating the Oligocytic Pythium strain from step A in PDA medium for 3-5 days, transfer it to liquid culture medium PD for shake flask culture for 3-5 days, and then transfer the shake flask seed culture to a fermenter for large-scale culture. C. After 7-10 days of expanded culture and spore production, collect the mycelium and oospores of Pythium oligoandrogenes, and spray dry to obtain Pythium oligoandrogenes oospore powder.

28. A method for preparing an agent for the prevention or treatment of fungal infections according to any one of claims 12-26, characterized in that, The method includes: mixing the lyophilized powder of Pythium oligandii oospores and / or the lyophilized powder of Pythium oligandii fermentation supernatant, the lyophilized powder of endosomalin, the traditional Chinese medicine ingredients and excipients in a proportion using a homogenizer, and then compressing the mixture into tablets under a dry and low-temperature environment.

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