WO2024071736A1

WO2024071736A1 - Pharmaceutical composition for prevention or treatment of allergic diseases, containing dead cells of streptococcus pyogenes or spea protein

Info

Publication number: WO2024071736A1
Application number: PCT/KR2023/013583
Authority: WO
Inventors: 김정진
Original assignee: (주)콤비메드
Priority date: 2022-09-28
Filing date: 2023-09-11
Publication date: 2024-04-04
Also published as: KR102507470B1

Abstract

An aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of allergic diseases, containing dead cells of Streptococcus pyogenes or Streptococcal pyrogenic Exotoxin A (SpeA) protein. The dead cells of Streptococcus Pyogenes or SpeA protein according to an aspect exhibits the excellent effect in patients with allergic diseases of increasing body temperature, palliating symptoms of dermatitis and itching, reducing the thickness of epithelial tissues, and lowering the expression levels of IgE, IFN-γ, and IL-4 proteins in serum , and thus can be advantageously utilized for the prevention, alleviation, or treatment of allergic diseases.

Description

Pharmaceutical composition for preventing or treating allergic diseases containing dead cells of Streptococcus pyogenes or SPEA protein

It relates to a pharmaceutical composition for the prevention or treatment of allergic diseases containing dead cells of Streptococcus pyogenes or SpeA (Streptococcal pyrogenic Exotoxin A) protein.

It is known that atopic dermatitis is caused by excessive TH2 (T helper type 2) immune response. Accordingly, in modern standard medicine, steroid-based treatment with an immunosuppressive concept is applied as the main treatment. However, the side effects and recurrence rate of using steroid-type immunosuppressants are considerable.

Recently, the incidence of atopic dermatitis has been widespread both domestically and internationally, from children to adults, and steroid-based atopic dermatitis inhibitors are being used as treatments, but they do not provide a fundamental solution to atopic dermatitis, but only provide temporary relief. There is a side effect of developing resistance.

Accordingly, duplimab, an IL-4 (main antibody in allergic reactions among TH2 family cytokines) antibody treatment, was recently developed and is showing therapeutic effects in seriously ill patients. However, this also temporarily suppresses itching and continues to be administered. Due to limitations in the treatment required, there is a need to develop a treatment for atopic dermatitis with a low recurrence rate through fundamental improvement of the skin immune response.

One aspect is to provide a pharmaceutical composition for preventing or treating allergic diseases containing dead cells of Streptococcus Pyogenes.

Another aspect is to provide a health functional food for preventing or improving allergic diseases containing dead cells of Streptococcus Pyogenes.

Another aspect is to provide a cosmetic composition for preventing or improving allergic diseases containing dead cells of Streptococcus Pyogenes.

Another aspect is to provide a pharmaceutical composition for preventing or treating allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.

Another aspect is to provide a health functional food for preventing or improving allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.

Another aspect is to provide a cosmetic composition for preventing or improving allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.

Another aspect includes culturing a Streptococcus Pyogenes strain; and

To provide a method for producing dead cells of Streptococcus Pyogenes, including the step of applying heat and pressure to the cultured strain.

Another aspect is to provide a method for preventing or treating allergic diseases comprising administering dead cells of Streptococcus Pyogenes to an individual in need thereof.

Another aspect is to provide the use of dead cells of Streptococcus Pyogenes for the manufacture of a medicament for the prevention or treatment of allergic diseases.

Another aspect is to provide a method for preventing or treating allergic diseases comprising administering SpeA (Streptococcal pyrogenic Exotoxin A) protein to an individual in need thereof.

Another aspect is to provide the use of SpeA (Streptococcal pyrogenic Exotoxin A) protein for the manufacture of a medicament for the prevention or treatment of allergic diseases.

One aspect provides a pharmaceutical composition for preventing or treating allergic diseases containing dead cells of Streptococcus Pyogenes.

The term " Streptococcus Pyogenes" refers to a bacterium belonging to the Streptococcus genus, and when the human body is infected with the Streptococcus pyogenes, the Streptococcus pyogenes causes purulent inflammation and fever on the skin ( TH1 immune response). In one aspect, the Streptococcus pyogenes may be a Streptococcus Pyogenes strain deposited under the accession number KCTC3984.

The term "dead cells" means a product that kills live cells or suppresses their activity by methods such as heat or drying after culturing live cells under certain conditions, or isolates effective ingredients from them, and the dead cells are It does not cause disease in the body and only stimulates the immune system to induce immunity. Additionally, the dead cells may contain cytoplasm, cell wall, antibacterial active substances such as bacteriocin, polysaccharide, organic acid, etc.

In one aspect, the dead cells of Streptococcus Pyogenes are all of the culture, culture medium, supernatant, concentrate, concentrate, dried matter, dilution or dilution of Streptococcus Pyogenes. It can be included.

The term "allergic disease" refers to when a certain type of substance (antigen or allergen (causing agent of allergy)) enters an individual, an antibody is created against it, and then an antigen of the same substance enters the body again. It refers to a disease caused by an allergy, which is an antigen-antibody reaction.

In one aspect, the allergic disease may be one or more selected from the group consisting of atopic dermatitis, rhinitis, eczema, asthma, conjunctivitis, bronchiectasis, enteritis, hay fever, and urticaria, and specifically, atopic dermatitis, eczema, and asthma. , rhinitis, and urticaria, and more specifically, it may be atopic dermatitis.

The term “prevention” may refer to any action that suppresses or delays the onset of an allergic disease in an individual by administering a pharmaceutical composition according to one aspect.

The term “treatment” may refer to any action in which the symptoms of an individual's allergic disease are improved or beneficially changed by administration of a pharmaceutical composition according to one aspect.

The term “administration” refers to introducing a predetermined substance into an individual by an appropriate method, and “individual” refers to all living organisms such as rats, mice, and livestock, including humans, that may have allergic diseases. As a specific example, it may be a mammal, including humans.

In one aspect, when the pharmaceutical composition is an injection, 10 ^-10 to 0.5 mg/kg, 10 ^-8 to 0.5 mg/kg, 10 ^-6 to 0.5 mg/kg, 10 ^-5 to 0.5 mg/kg, 10 ^-4 to 0.5 mg/kg, 0.001 to 0.5 mg/kg, 0.001 to 0.4 mg/kg, 0.001 to 0.3 mg/kg, 0.05 to 0.5 mg/kg, 0.05 to 0.4 mg/kg, 0.05 to 0.3 mg/kg , may be administered at 0.01 to 0.5 mg/kg, 0.01 to 0.4 mg/kg, or 0.01 to 0.3 mg/kg.

In one aspect, when the pharmaceutical composition is an injection, if the pharmaceutical composition is administered in an amount exceeding 0.5 mg/kg, toxicity to normal cells of the administered subject may be induced.

In one aspect, when the pharmaceutical composition is an external preparation, 10 ^-10 to 0.5 mg/kg, 10 ^-8 to 0.5 mg/kg, 10 ^-6 to 0.5 mg/kg, 10 ^-5 to 0.5 mg/kg, 10 ^-4 to 0.5 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.5 mg/kg, 0.01 to 0.045 mg/kg, 0.01 to 0.04 mg/kg, 0.015 to 0.5 mg/kg, 0.015 to 0.045 mg/kg , may be applied at 0.015 to 0.04 mg/kg, 0.02 to 0.05 mg/kg, 0.02 to 0.045 mg/kg, or 0.02 to 0.04 mg/kg.

In one aspect, when the pharmaceutical composition is for external use, if the pharmaceutical composition is applied in an amount exceeding 0.5 mg/kg, toxicity to normal cells of the subject may be induced.

In one aspect, the pharmaceutical composition may reduce itching in the subject to which it is administered.

In one example, mice were observed for 10 minutes to determine whether administration of dead cells affects itching symptoms in an atopic mouse model. As a result, the number of scratches was significantly higher in the negative control group (PBS) than in the normal control group (NC). It was confirmed that when dead cells were administered, the number of scratches was significantly reduced to a significant level compared to the positive control group, DEXA group, at all concentrations (see Example 2.(1)3).

In one aspect, the pharmaceutical composition may reduce the thickness of the epithelial tissue of the administration target.

In one example, in order to histopathologically analyze the skin in an atopic mouse model, the skin tissue on the back of the mouse was fixed, stained with H&E, observed under a microscope, and the thickness of the epithelial tissue was measured. As a result, the negative control group was compared to the normal control group (NC). (PBS), the thickness of the epithelial tissue significantly increased, and it was confirmed that the thickness of the epithelial tissue significantly decreased in the group administered dead cells to a level similar to that of the DEXA group, which was a positive control (Example 2. (Example 2) See 1)4)).

Additionally, the pharmaceutical composition may contain the active ingredient alone, or may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients, or diluents.

Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. They may form complexes or associate with delivery vehicles and may be used in the art as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids. It can be transported in vivo using known delivery systems.

When the pharmaceutical composition is formulated, it may be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants. You can. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may contain at least one excipient, such as starch, calcium carbonate, or sucrose. ) or it can be prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used, and when manufacturing in the form of eye drops, known diluents or excipients can be used. there is. For example, when the pharmaceutical composition is used as an injection, the pharmaceutical composition can be mixed with D-PBS (Dulbecco's phosphate-buffered saline), and when the pharmaceutical composition is used as an external agent, the pharmaceutical composition can be mixed with D-PBS (Dulbecco's phosphate-buffered saline). The composition can be used in combination with a hyaluronic acid moisturizer.

In one aspect, the pharmaceutical composition may further include a pharmaceutical composition for preventing or treating allergic diseases other than dead cells of Streptococcus Pyogenes.

The pharmaceutical composition may be provided by mixing with a pharmaceutical composition for the prevention or treatment of other allergic diseases, and the pharmaceutical composition for the prevention or treatment of other allergic diseases may be used to prevent or treat allergic diseases known in the art. It may be a pharmaceutical composition for treatment or a newly developed pharmaceutical composition for preventing or treating allergic diseases.

When the pharmaceutical composition further contains a pharmaceutical composition for the prevention or treatment of other allergic diseases or is provided in combination with a pharmaceutical composition for the prevention or treatment of other allergic diseases, the maximum effect is achieved with the minimum amount without side effects. It is important to mix the amount that can be obtained, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition is not mixed with a pharmaceutical composition for the prevention or treatment of allergic diseases other than the dead cells of Streptococcus Pyogenes, and can be administered in parallel, simultaneously, separately, or sequentially. It can be administered, and can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition may be administered orally or parenterally. When administered parenterally, it may be administered externally to the skin or intraperitoneally, intrarectally, subcutaneously, intravenously, intramuscularly, intraarterially, intramedullary, or cardiacally. It may be administered by intradermal injection, intrathecal injection, transdermal injection, intranasal injection, intraenteral injection, local injection, sublingual injection, or intrathoracic injection. Specifically, the pharmaceutical composition is administered by external injection on the skin or by subcutaneous injection. It can be administered.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the drug's effect. It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The pharmaceutical composition may be administered once a day, or may be administered several times. For example, it may be administered every other day, or it may be administered once a week.

The pharmaceutical composition according to one aspect has an excellent effect of increasing the body temperature of patients with allergic diseases, improving dermatitis symptoms and itching, and reducing the thickness of epithelial tissue, and can be usefully used in the prevention or treatment of allergic diseases. You can.

Another aspect provides a health functional food for preventing or improving allergic diseases containing dead cells of Streptococcus Pyogenes.

The terms “ Streptococcus Pyogenes”, “dead cells”, “allergic disease”, “prevention”, etc. may be within the above-mentioned range.

The term “improvement” may refer to any action that results in at least a reduction in the severity of a parameter associated with the condition being treated, such as symptoms. At this time, the health functional food can be used simultaneously or separately with a drug for treatment before or after the onset of the disease in order to prevent or improve allergic disease.

In the health functional food, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). In general, when manufacturing a food or beverage, the health functional food may be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

The health functional food may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of carriers, diluents, excipients, and additives. Foods to which compounds according to one aspect can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

Specific examples of the carriers, excipients, diluents and additives include lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose. , a group consisting of polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. It may be at least one selected from.

In addition to containing the effective ingredients, the health functional food may contain other ingredients as essential ingredients without any particular restrictions. For example, like regular beverages, it may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. You can. The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, health functional foods according to one aspect include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.

In one aspect, the health functional food may further include a health functional food for preventing or improving allergic diseases other than the dead cells of Streptococcus Pyogenes.

The health functional food may be provided by mixing with health functional food for preventing or improving other allergic diseases, and the health functional food for preventing or improving other allergic diseases is known in the art for preventing or improving allergic diseases. It may be a health functional food for improvement or a newly developed health functional food for preventing or improving allergic diseases.

If the health functional food further includes health functional foods for preventing or improving other allergic diseases, it is important to mix the amount to obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art. You can.

Health functional foods according to one aspect are effective in increasing the body temperature of patients with allergic diseases, improving dermatitis symptoms and itching, and reducing the thickness of epithelial tissue, so they can be useful in preventing or improving allergic diseases. You can.

Another aspect provides a cosmetic composition for preventing or improving allergic diseases containing dead cells of Streptococcus Pyogenes.

The terms “ Streptococcus Pyogenes”, “dead cells”, “allergic disease”, “prevention”, “improvement”, etc. may be within the above-mentioned range.

The cosmetic composition can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil. , can be formulated as powder foundation, emulsion foundation, wax foundation, spray, etc. More specifically, lotion such as softening lotion or nourishing lotion, emulsion such as facial lotion, body lotion, etc., cream such as nutritional cream, moisture cream, eye cream, essence, cosmetic ointment, spray, gel, pack, sunscreen, makeup It can be manufactured in formulations such as foundation such as base, liquid type, solid type or spray type, powder, makeup remover such as cleansing cream, cleansing lotion, cleansing oil, cleansing foam, soap, body wash, etc. In this case, the cosmetic composition includes surfactants, emulsifiers, soapy acid, solvents, colorants, preservatives, antioxidants, anti-foaming agents, antibacterial agents, anti-redeposition agents, enzymes, plant or mineral oils, fats, fluorescent substances, fungicides, hydrotropes-inducing substances, It may contain excipients including moisturizers, fragrances, preservatives, proteins, silicones, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, waxes, etc.

In the present invention, the cosmetic composition includes ingredients commonly added to cosmetic compositions, such as purified water, surfactants, moisturizers, lower alcohols, chelating agents, disinfectants, preservatives, antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. Phosphorus auxiliaries, etc. may be additionally included, and these may be used alone or in a mixture of two or more types. Additionally, all ingredients included in the cosmetic composition do not exceed the regulations set by each country. Specifically, the cosmetic composition may contain the individual ingredients mentioned within a range that does not exceed the maximum usage amount specified in the “Cosmetic Safety and Technical Code” established by the Chinese government.

In addition, the cosmetic composition contains ingredients commonly used in external skin preparations such as cosmetics and medicines, such as water-based ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, and surfactants. , fragrance, colorant, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The cosmetic composition also contains metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glablidin, and calin. hot water extract of fruits, various herbal medicines, drugs such as tocopherol acetate, glythylitic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose Sugars such as , trehalose, etc. can also be appropriately mixed.

The cosmetic composition according to one aspect has an excellent effect of increasing the body temperature of allergic disease patients, improving dermatitis symptoms and itching, and reducing the thickness of epithelial tissue, and can be usefully used in preventing or improving allergic diseases. there is.

Another aspect provides a pharmaceutical composition for preventing or treating allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.

The terms “allergic disease,” “prevention,” “treatment,” “pharmaceutical composition,” etc. may be within the above-mentioned range.

The term "SpeA (Streptococcal pyrogenic Exotoxin A)" refers to a superantigen (exotoxin) produced by Streptococcus Pyogenes, and is a cause of the Streptococcal toxic shock syndrome. It is known to play an important role in pathogenesis. In one aspect, the SpeA (Streptococcal pyrogenic Exotoxin A) may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. Additionally, in one aspect, the SpeA (Streptococcal pyrogenic Exotoxin A) may be derived from dead cells of Streptococcus Pyogenes or Streptococcus Pyogenes .

In one example, mice were observed for 10 minutes to determine whether administration of SpeA affects itching symptoms in an atopic mouse model. As a result, the number of scratches was significantly increased in the negative control group (PBS) compared to the normal control group (NC). It was confirmed that when SpeA was administered, the number of scratches was significantly reduced to a significant level compared to the positive control group, DEXA group, at all concentrations (see Example 2.(2)3).

In one aspect, the pharmaceutical composition may reduce the expression level of one or more proteins selected from the group consisting of IgE, IFN-γ, and IL-4 in the serum of the administered subject, and specifically, in the serum of the administered subject. It may reduce the expression level of one or more proteins selected from the group consisting of IgE, IFN-γ, or IL-4, and more specifically, the expression level of IgE, IFN-γ, and IL-4 proteins in the serum of the administration subject. It may be to reduce .

In one example, changes in serum IgE, IFN-γ, and IL-4 were measured to confirm changes in IgE and cytokines in the blood of an atopic mouse model. As a result, serum IgE was significantly increased in the negative control group compared to the normal control group. It was confirmed that there was a significant decrease in both the positive control group (DEXA) and the SpeA administration group. In addition, serum IFN-γ and IL-4 were confirmed to be significantly increased in the negative control group compared to the normal control group, and significantly decreased in both the positive control group (DEXA) and the SpeA administered group (Example 2.(2) 4) see).

In one example, in order to histopathologically analyze the skin in an atopic mouse model, the skin tissue on the back of the mouse was fixed, stained with H&E, observed under a microscope, and the thickness of the epithelial tissue was measured. As a result, the negative control (NC) was compared to the normal control (NC). It was confirmed that the thickness of the epithelial tissue significantly increased in PBS), and that the thickness of the epithelial tissue significantly decreased in the SpeA-administered group to a level similar to that of the positive control group, DEXA group (Example 2.(2) 5) see).

The pharmaceutical composition according to one aspect has the effect of increasing body temperature in patients with allergic diseases, improving dermatitis symptoms and itching, and reducing the thickness of epithelial tissue and the expression levels of IgE, IFN-γ and IL-4 proteins in serum. Since it is excellent, it can be usefully used in the prevention or treatment of allergic diseases.

Another aspect provides a health functional food for preventing or improving allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.

The terms “SpeA (Streptococcal pyrogenic Exotoxin A) protein”, “allergic disease”, “prevention”, “improvement”, “health functional food”, etc. may be within the above-mentioned range.

Health functional foods according to one aspect have the effect of increasing body temperature in patients with allergic diseases, improving dermatitis symptoms and itching, and reducing the thickness of epithelial tissue and the expression levels of IgE, IFN-γ and IL-4 proteins in serum. Since it is excellent, it can be usefully used to prevent or improve allergic diseases.

Another aspect provides a cosmetic composition for preventing or improving allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.

The terms “SpeA (Streptococcal pyrogenic Exotoxin A) protein”, “allergic disease”, “prevention”, “improvement”, “cosmetic composition”, etc. may be within the above-mentioned range.

A cosmetic composition according to one aspect has the effect of increasing the body temperature of patients with allergic diseases, improving dermatitis symptoms and itching, and reducing the thickness of epithelial tissue and the expression levels of IgE, IFN-γ and IL-4 proteins in serum. Because it is excellent, it can be useful in preventing or improving allergic diseases.

Another aspect includes culturing a Streptococcus Pyogenes strain; and

A method for producing dead cells of Streptococcus Pyogenes is provided, including the step of applying heat to the cultured strain.

The " Streptococcus Pyogenes", "dead cells", etc. may be within the above-mentioned range.

In one aspect, the step of applying heat is performed at 80 to 110°C, 80 to 105°C, 80 to 100°C, 85 to 110°C, 85 to 105°C, 85 to 100°C, 90 to 110°C, 90 to 105°C. Alternatively, it may be performed at a temperature of 90 to 100°C.

In addition, the step of applying heat is 20 to 40 minutes, 20 to 36 minutes, 20 to 32 minutes, 24 to 40 minutes, 24 to 36 minutes, 24 to 32 minutes, 28 to 40 minutes, 28 to 36 minutes, or 28 to 36 minutes. It may be performed for 32 minutes.

In one aspect, when the step of applying heat is performed at a temperature of less than 80° C., sterilization or sterilization is not effective, and Streptococcus Pyogenes strains may not be inactivated or killed, 110 When performed at a temperature exceeding ℃, even the effective ingredients for the prevention or treatment of allergic diseases within the Streptococcus Pyogenes strain or its dead cells may be denatured.

In one aspect, the step of applying the heat may be applying pressure along with the heat.

In one aspect, the applied pressure is 10 to 20 psi, 10 to 18 psi, 10 to 16 psi, 12 to 20 psi, 12 to 18 psi, 12 to 16 psi, 14 to 20 psi, 14 to 18 psi, or It may be 14 to 16 psi.

In addition, when the pressure is applied below 10 psi, sterilization or sterilization is not effective, and Streptococcus Pyogenes strains may not be inactivated or killed, and when the pressure is applied above 20 psi, Streptococcus pyogenes strains may not be inactivated or killed. Even the effective ingredients for preventing or treating allergic diseases within Streptococcus Pyogenes strains or their dead cells may be denatured.

In one aspect, the method may further include the step of crushing the cell wall after the step of applying heat.

If the method further includes the step of crushing the cell wall after the step of applying heat, effective ingredients for the prevention or treatment of allergic diseases can be easily obtained from the Streptococcus Pyogenes strain. .

The active ingredient enhances immune activity and may be, for example, dead cells of Streptococcus Pyogenes or SpeA protein.

In one aspect, the step of pulverizing the cell wall may be performed through one or more pulverization methods selected from the group consisting of centrifugation, ultrasonic treatment, compression, bead crushing, homogenizer, and lysozyme, specifically. , may be performed through one or more pulverization methods selected from the group consisting of centrifugation, sonication, and homogenizer, and more specifically, may be performed through sonication.

In one aspect, the method may further include centrifuging the cell wall after pulverizing it to obtain a supernatant.

If the method further includes the step of obtaining a supernatant by centrifugation after pulverizing the cell wall, the active ingredient for the prevention or treatment of allergic diseases can be easily obtained from the Streptococcus Pyogenes strain. can do.

When using the method for producing dead cells of Streptococcus Pyogenes according to one aspect, dead cells of Streptococcus Pyogenes , which are useful for preventing or treating allergic diseases, are quickly produced in high yield. can do.

Another aspect provides a method for preventing or treating allergic diseases, comprising administering dead cells of Streptococcus Pyogenes to an individual in need thereof.

The terms “ Streptococcus Pyogenes”, “dead cells”, “individual”, “administration”, “allergic disease”, “prevention”, “treatment”, etc. may be within the above-described scope.

Another aspect provides the use of dead cells of Streptococcus Pyogenes for the manufacture of a medicament for the prevention or treatment of allergic diseases.

The terms “allergic disease,” “prevention,” “treatment,” “ Streptococcus Pyogenes,” “dead cells,” etc. may be within the above-mentioned range.

Another aspect provides a method for preventing or treating allergic diseases, comprising administering SpeA (Streptococcal pyrogenic Exotoxin A) protein to a subject in need thereof.

The terms “SpeA (Streptococcal pyrogenic Exotoxin A) protein”, “individual”, “administration”, “allergic disease”, “prevention”, “treatment”, etc. may be within the above-mentioned scope.

Another aspect provides the use of Streptococcal pyrogenic Exotoxin A (SpeA) protein for the manufacture of a medicament for the prevention or treatment of allergic diseases.

The terms “allergic disease,” “prevention,” “treatment,” “SpeA (Streptococcal pyrogenic Exotoxin A) protein,” etc. may be within the above-mentioned range.

According to one aspect, the dead cells of Streptococcus Pyogenes or SpeA (Streptococcal pyrogenic Exotoxin A) protein increases body temperature in patients with allergic diseases, improves dermatitis symptoms and itching, and increases the thickness of epithelial tissue and serum. Since it has an excellent effect in reducing the expression levels of IgE, IFN-γ, and IL-4 proteins, it can be useful in preventing, improving, or treating allergic diseases.

Figure 1 is a diagram showing the number of times a test substance is administered after dermatitis is induced in an atopic mouse model by treating it with DNCB.

Figure 2 is a diagram showing the results of confirming changes in dermatitis symptoms due to administration and application of dead cells in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: * <0.05).

Figure 3 is a diagram showing the results of confirming changes in itching symptoms due to administration of dead cells in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: *** <0.001).

Figure 4 is a diagram showing the results of confirming changes in skin epithelial tissue thickness due to administration of dead cells in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: ** <0.01, * ** <0.001).

Figure 5 is a diagram showing the results of confirming changes in dermatitis symptoms due to SpeA administration in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: ** <0.01, *** <0.001) ).

Figure 6 is a diagram showing the results of confirming changes in itching symptoms due to SpeA administration in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: *** <0.001).

Figure 7 is a diagram showing the results of confirming changes in serum IgE and cytokines due to SpeA administration in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: ** <0.01, ** *<0.001).

Figure 8 is a diagram showing the results of confirming changes in skin epithelial tissue thickness by SpeA administration in an atopic mouse model (when compared to the NC group: ### <0.001, when compared to the PBS group: *** <0.001).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

실시예Example

1. 실험 재료 및 방법1. Experimental materials and methods

(1) 시험계의 선택 및 사육 환경(1) Selection of test system and rearing environment

Mice are small and easy to handle, making them convenient for animal testing. NC/Nga mice were selected because they are animals optimized for inducing atopy, as diseases similar to atopic dermatitis naturally occur at the age of about 6 to 7 weeks under conventional conditions. 68 NC/Nga mice were raised in an SPF environment, specifically, under environmental conditions of a temperature of 22 ± 1°C, relative humidity of 55 ± 15%, and lighting for 12 hours (lights on at 7 a.m. - lights off at 7 p.m.). bred.

NC/Nga mice were reared in breeding boxes measuring W 200 Individual identification was done by marking the tail using a permanent marker. Solid feed for laboratory animals was freely consumed, and water was freely consumed using a water bottle. This test was approved by the Animal Ethics Committee of Kyung Hee University based on the Animal Protection Act (Enacted on May 31, 1991, Law No. 4379, partially revised on February 29, 2008, Law No. 8852) (Serial Number: 22-051).

(2) 피하주사용 시험물질의 제조(2) Manufacture of test substances for subcutaneous injection

Streptococcus Pyogenes (Accession number: KCTC3984) strain BD 211768, which can be easily obtained in the art, was inoculated at 1% in 10 ml of Trypic Soy Broth (TSB) medium and cultured at 37°C for 24 hours. The cultured strain was cultured again in an amount of 100 ml for 24 hours, sterilized in an autoclave at 95°C for 30 minutes, and centrifuged at a speed of 4,500 rpm. At this time, the obtained bacteria were diluted in physiological saline and stored. The volume was prepared to contain ⁵ It was centrifuged at a speed of , and the supernatant was used. In addition, glass beads were added in 1/3 of the amount of the supernatant, vortexed for 30 minutes, centrifuged at 12,000 rpm, and the supernatant was used, and 0.28 mg was added to D-PBS (Dulbecco's phosphate-buffered saline). A solution preparation diluted to a concentration of /kg, 0.028 mg/kg or 0.0028 mg/kg and filtered through a 0.45 or 0.2 ㎛ filter was used as a dead cell of Streptococcus Pyogenes for subcutaneous injection among the test substances. .

In addition, SpeA (Streptococcal pyrogenic exotoxin A) (SEQ ID NO: 1: QQDPDPSQLHRSSLVKNLQNIYFLYEGDPVTHENVKSVDQLLSHDLIYNVSGPNYDKLKTELKNQEMATLFKDKNVDIYGVEYYHLCYLCENAERSACIYGGVTNHEGNHLEIPKKIVVKVSIDGIQSLSFDIETNKKMVTAQELDYKVRKYLTDNKQLY TNGPSKYETGYIKFIPKNKESFWFDFFPEPEFTQSKYLMIYKDNETLDSNTSQIEVYLTTK) is dissolved in D-PBS at 1 mg/ml and then diluted in D-PBS to a concentration of 1.0 mg/kg or 2.5 mg/kg for subcutaneous injection. The material used was SpeA (Streptococcal pyrogenic exotoxin A) purchased from CUSABIO (cat # CSB-YP350473SMR).

(3) 외용제용 시험물질의 제조(3) Manufacture of test substances for external use

In order to use dead cells of Streptococcus Pyogenes as a test substance for external use, the solution preparation filtered through a 0.45 or 0.2 ㎛ filter prepared by the preparation method in Example 1. (2) above was 5 100 ml of ⁹ cells/ml was evenly mixed with 900 ml of hyaluronic acid moisturizer, and at this time, the concentration of Streptococcus Pyogenes ^dead cells was prepared to be 5

(4) 피하주사용 시험물질의 투여량 및 시험군의 구성(4) Dosage of test substances for subcutaneous injection and composition of test group

Tables 1 and 2 below show the dosages of test substances and test groups.

군army		투여물질administered substance	투여용량 (mg/kg)Dosage (mg/kg)	투여경로Route of administration	동물 수number of animals	투여 횟수Number of doses
정상군Jeongsang-gun	VehicleVehicle	PBSPBS		s.cs.c.	88	주2회2 times a week
아토피 유발군Atopic dermatitis group	Negative con.Negative con.	PBSPBS		s.cs.c.	88	주2회2 times a week
	Positive con.Positive con.	DEXADEXA	1010	s.cs.c.	88	주2회2 times a week
	시험물질1 (H)Test substance 1 (H)	사균dead bacteria	0.280.28	s.cs.c.	88	주2회2 times a week
	시험물질1 (M)Test substance 1 (M)	사균dead bacteria	0.0280.028	s.cs.c.	88	주2회2 times a week
	시험물질1 (L)Test substance 1 (L)	사균dead bacteria	0.00280.0028	s.cs.c.	88	주2회2 times a week
	시험물질1 (외용제)Test substance 1 (external preparation)	사균dead bacteria	0.0280.028	피부도포 Skin application	88	주2회2 times a week

군army		투여물질administered substance	투여용량 (mg/kg)Dosage (mg/kg)	투여경로Route of administration	동물 수number of animals	투여 횟수Number of doses
정상군Jeongsang-gun	VehicleVehicle	PBSPBS		s.cs.c.	44	주2회2 times a week
아토피 유발군Atopic dermatitis group	Negative con.Negative con.	PBSPBS		s.cs.c.	44	주2회2 times a week
	Positive con.Positive con.	DEXADEXA	1010	s.cs.c.	44	주2회2 times a week
	시험물질2 (L)Test substance 2 (L)	사균dead bacteria	1.01.0	s.cs.c.	44	주2회2 times a week
	시험물질2 (H)Test substance 2 (H)	사균dead bacteria	2.52.5	s.cs.c.	44	주2회2 times a week

(5) 시험물질의 투여 및 도포(5) Administration and application of test substances

The back of the NC/Nga mouse was disinfected with a 70% alcohol cotton pad and then administered subcutaneously using an insulin syringe. The above administration method was selected to evaluate the efficacy of the test substance as an injectable drug when administered subcutaneously. Specifically, NC/Nga mice were treated with DNCB to induce dermatitis, and then the test substance was administered twice a week until the end of the test, and the volume of excipient was 100 ㎕. More specifically, the back of 6- to 7-week-old NC/Nga mice (SLC. Japan) was hair removed and left for 24 hours to allow microwounds on the skin to heal. Prepare a reaction solution of 1% DNCB (Sigma, USA) in a 4:1 mixture of acetone and olive oil, and apply 200 ㎕ of the mixture to the back area twice a week for 2 weeks. It was applied during. Afterwards, 200 ㎕ of a 1% DNCB solution was applied to the back once a week to induce dermatitis for 3 weeks, and 100 ㎕ of a PBS solution containing the test substance was injected subcutaneously twice a week, and changes in symptoms were observed ( Figure 1).

In addition, 100 ul of an external preparation prepared using dead cells of Streptococcus Pyogenes was applied to the skin twice a week.

(6) 주사제용 시험물질 투여 후 피부 병변 평가(6) Evaluation of skin lesions after administration of test substances for injections

The severity of DNCB-induced atopic dermatitis-like skin lesions was determined using previously established methods (Choi, Yoon Jung, et al. “Therapeutic effects and immunomodulation of suanbo mineral water therapy in a murine model of atopic dermatitis.” Annals of dermatology 25.4 (2013): 462-470) was clinically evaluated. Skin lesions on the back were scored according to four symptom criteria: erythema/hemorrhage, edema, abrasion/erosion, and dryness/scaling. The dermatitis score is defined as the sum of individual scores graded as follows: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; and 3, severe symptoms. In addition, the degree of scratching (scratching due to itching) was measured by number of times and compared between groups.

(7) 주사제용 시험물질 투여 후 혈액 내 IgE 및 사이토카인 발현량 측정(7) Measurement of IgE and cytokine expression in the blood after administration of test substances for injections

Blood samples from NC/Nga mice administered the test substance were centrifuged at 5,000 g for 20 minutes to collect serum, and changes in expression were measured using a mouse IgE ELISA Kit (BD Biosciences, San Diego, CA, USA). did. A 96-well plate was coated O/N at 4°C with anti-mouse IgE mAB. After washing with 0.05% PBS-tween 20 solution, blocking was performed with 10% FBS at room temperature for 1 hour. 100 μl of serum sample diluted 1:700 was incubated for 2 hours at room temperature. After washing with 0.05% PBS-tween 20 solution, the cells were incubated with secondary peroxidase-labeled biotinylated anti-rat IgE mAb for 1 hour. The enzymatic reaction was initiated by adding TMB substrate solution (BD Biosciences) for 30 min, and the reaction was stopped by adding 50 μl of stop solution. Absorbance was measured at 450 nm using a micro plate reader (SOFT max PRO, version 3.1. Molecular Devices, Sunnyvale, CA, USA), and the lower limit of detection for IgE ELISA was 1.5 ng/ml.

(8)(8) 주사제용For injection 시험물질 투여 후 조직학적 분석Histological analysis after test substance administration

For histopathological examination, the dorsal skin of each mouse was collected and fixed with 4% neutral buffered formalin for 24 hours at 4°C. The tissue was dehydrated, embedded in paraffin, and cut to a thickness of 4 μm using a rotary microtome. Sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich), and images were acquired using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and imaged using Image Pro-Plus 5.1 software (Media Quantification was performed using Cybemetics Inc., Silver Spring, MD, USA).

(9) 통계 분석(9) Statistical analysis

Data are expressed as mean ± SEM. Significance difference analysis for all test results was performed by analyzing the average value using Graphpad Prism software (Version 5, CA, USA), and in case of multiple comparisons, the p -value was calculated according to one-way Anova, Tukey HSD test, and unpaired T test. If it was less than 0.05, it was judged to indicate a statistically significant difference. All experiments were performed blindly and repeated independently under identical conditions.

2. 실험 결과2. Experimental results

(1) 아토피 마우스 모델(NC/Nga 마우스)에서 스트렙토코커스 피오게네스((1) Streptococcus pyogenes ( Streptococcus PyogenesStreptococcus Pyogenes )) 사균체 투여에 의한 아토피 증상 완화Relief of atopy symptoms by administration of dead cells

1) 아토피 마우스 모델에서 사균체 투여에 의한 체온 변화 확인1) Confirmation of body temperature changes due to administration of dead cells in an atopic mouse model

In an atopic mouse model (NC/Nga mouse), the body temperature of the mouse was measured using a rectal thermometer before and after administration of dead cells, compared to the normal control (NC), negative control (PBS), and positive control (DEXA). It was confirmed that body temperature increased significantly in both groups.

2) 아토피 마우스 모델에서 사균체 투여 및 도포에 의한 피부염 증상의 변화 확인2) Confirmation of changes in dermatitis symptoms due to administration and application of dead cells in an atopic mouse model

In an atopic mouse model (NC/Nga mouse), the dermatitis score was significantly increased in the negative control group (PBS) treated with DNCB compared to the normal control group (NC), and the dermatitis score was significantly increased in the negative control group (PBS) treated with dead cells and topical preparations using dead cells were administered and applied, respectively. As a result, it was confirmed that the dermatitis score was significantly reduced to a level similar to that of DEXA, the positive control group (Figure 2).

3)3) 아토피 마우스 모델에서 사균체 투여에 의한 가려움 증상의 변화 확인Confirmation of changes in itching symptoms due to administration of dead cells in an atopic mouse model

To determine whether the administration of dead cells affected itching symptoms in an atopic mouse model (NC/Nga mouse), the mice were observed for 10 minutes and the number of scratches (scratching due to itching) was measured. As a result, the number of scratches significantly increased in the negative control group (PBS) compared to the normal control group (NC), and when dead cells were administered, the number of scratches was significantly lower than that in the positive control group, DEXA group, at all concentrations. A decrease was confirmed (Figure 3).

4)4) 아토피 마우스 모델에서 사균체 투여에 의한 피부 조직학적 변화 확인Confirmation of skin histological changes caused by administration of dead cells in an atopic mouse model

To histopathologically analyze the skin in an atopic mouse model (NC/Nga mouse), skin tissue from the back of the mouse was fixed, stained with H&E, observed under a microscope, and the thickness of the epithelial tissue was measured. As a result, the thickness of the epithelial tissue was significantly increased in the negative control group (PBS) compared to the normal control group (NC), and the thickness of the epithelial tissue was significantly increased in the group administered dead cells to a level similar to that in the DEXA group, the positive control group. It was confirmed that it decreased (Figure 4).

(2) 아토피 마우스 모델(NC/Nga 마우스)에서 SpeA(Streptococcal pyrogenic Exotoxin A) 투여에 의한 아토피 증상 완화(2) Alleviation of atopic symptoms by administration of SpeA (Streptococcal pyrogenic Exotoxin A) in an atopic mouse model (NC/Nga mouse)

1)One) 아토피 마우스 모델에서 SpeA 투여에 의한 체온 변화 확인Confirmation of body temperature change due to SpeA administration in atopic mouse model

In an atopic mouse model (NC/Nga mouse), the body temperature of the mouse was measured using a rectal thermometer before and after SpeA administration. As a result, body temperature was significantly increased in the group administered SpeA compared to the normal control, negative control, and positive control groups. Confirmed.

2)2) 아토피 마우스 모델에서 SpeA 투여에 의한 피부염 증상 확인Dermatitis symptoms confirmed by SpeA administration in atopic mouse model

In an atopic mouse model (NC/Nga mouse), the dermatitis score was significantly increased in the negative control group (PBS) compared to the normal control group (NC), and when SpeA was administered, the dermatitis score was significantly increased in the positive control group, DEXA, and the SpeA group. It was confirmed that was decreased (Figure 5).

3)3) 아토피 마우스 모델에서 SpeA 투여에 의한 가려움 증상 확인Confirmation of itching symptoms caused by SpeA administration in atopic mouse model

To determine whether administration of SpeA affects itching symptoms in an atopic mouse model (NC/Nga mouse), the mice were observed for 10 minutes and the number of scratches (scratching due to itching) was measured. As a result, the number of scratches significantly increased in the negative control group (PBS) compared to the normal control group (NC), and when SpeA was administered, the number of scratches significantly decreased at all concentrations to a level significantly different from the positive control group, DEXA group. This was confirmed (Figure 6).

4)4) 아토피 마우스 모델에서 SpeA 투여에 의한 혈청 IgE 및 사이토카인 변화 확인Confirmation of changes in serum IgE and cytokines due to SpeA administration in atopic mouse model

To check changes in IgE and cytokines in the blood of an atopic mouse model (NC/Nga mouse), blood was collected at the end of the experiment to obtain serum. Changes in serum IgE, IFN-γ, and IL-4 were measured using an ELISA kit. Serum IgE significantly increased in the negative control group compared to the normal control group, and significantly decreased in both the positive control (DEXA) and SpeA administered groups. In addition, serum IFN-γ and IL-4 were confirmed to be significantly increased in the negative control group compared to the normal control group, and significantly decreased in both the positive control (DEXA) and SpeA administered groups (Figure 7).

5)5) 아토피 마우스 모델에서 SpeA 투여에 의한 피부 조직학적 변화 확인Confirmation of skin histological changes caused by SpeA administration in an atopic mouse model

To histopathologically analyze the skin in an atopic mouse model (NC/Nga mouse), skin tissue from the back of the mouse was fixed, stained with H&E, observed under a microscope, and the thickness of the epithelial tissue was measured. As a result, the thickness of the epithelial tissue significantly increased in the negative control group (PBS) compared to the normal control group (NC), and the thickness of the epithelial tissue significantly increased in the SpeA-administered group to a similar level as the positive control group, DEXA group. A decrease was confirmed (Figure 8).

3. 소결3. Sintering

In general, atopic disease is caused by Th2 and IgE reacting to environmental and food antigens, but in healthy individuals, atopy development is suppressed by the activity of regulatory T cells that prevent natural T helper cell precursors from differentiating into the Th2 phenotype. . In vivo, IgE is produced by the interaction of antigen-specific B lymphocytes and Th2 lymphocytes, IgE antibodies are closely related to the interaction of T/B cells and the cytokine IL-4, and the presence of specific Th2 lymphocytes It is also related to

Therefore, through experiments in the embodiments of the present invention,

test substances

1 and 2 of the present invention show an effect that acts by a similar mechanism not only on atopic dermatitis but also on IgE- or regulatory T cell-mediated allergic diseases such as asthma and rhinitis. You can.

Claims

A pharmaceutical composition for preventing or treating allergic diseases containing dead cells of Streptococcus Pyogenes.
The composition according to claim 1, wherein the pharmaceutical composition reduces itching in the subject to which it is administered.
The composition according to claim 1, wherein the pharmaceutical composition reduces the thickness of the epithelial tissue of the administration target.
The composition according to claim 1, wherein the allergic disease is at least one selected from the group consisting of atopic dermatitis, rhinitis, eczema, asthma, conjunctivitis, bronchiectasis, enteritis, hay fever, and urticaria.
A health functional food for preventing or improving allergic diseases containing dead cells of Streptococcus Pyogenes.
A cosmetic composition for preventing or improving allergic diseases containing dead cells of Streptococcus Pyogenes.
A pharmaceutical composition for preventing or treating allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.
The composition according to claim 7, wherein the pharmaceutical composition reduces itching in the subject to which it is administered.
The composition according to claim 7, wherein the pharmaceutical composition reduces the expression level of one or more proteins selected from the group consisting of IgE, IFN-γ, and IL-4 in the serum of the administered subject.
The composition according to claim 7, wherein the pharmaceutical composition reduces the thickness of the epithelial tissue of the administration target.
A health functional food for preventing or improving allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.
A cosmetic composition for preventing or improving allergic diseases containing SpeA (Streptococcal pyrogenic Exotoxin A) protein.
Culturing a Streptococcus Pyogenes strain; and

A method of producing dead cells of Streptococcus Pyogenes, comprising the step of applying heat to the cultured strain.
The method of claim 13, wherein the method further comprises the step of pulverizing the cell wall after the step of applying heat.
The method of claim 14, wherein the step of pulverizing the cell wall is performed through one or more pulverizing methods selected from the group consisting of centrifugation, sonication, compression, bead crushing, homogenizer, and lysozyme.
The method of claim 14, wherein the method further includes the step of centrifuging the cell wall after pulverizing it to obtain a supernatant.