WO2024196194A1 - Novel enterococcus faecalis hl1 strain and use thereof - Google Patents

Abstract

The present invention relates to a novel (Enterococcus faecalis HL1 strain and a use thereof. The novel (Enterococcus faecalis HL1 strain, isolated and identified from pediatric feces, or a culture thereof, reduces serum IgE levels and IL-4 (interleukin-4) expression in an atopic dermatitis animal model, thereby alleviating immune responses and symptoms of atopic dermatitis. Therefore, the novel Enterococcus faecalis HL1 strain or a culture thereof can be advantageously used in the treatment of atopic dermatitis and is expected to help prevent the occurrence of allergic march by controlling atopic dermatitis.

Description

Novel Enterococcus faecalis HL1 strain and uses thereof

The present invention relates to a novel Enterococcus faecalis HL1 strain and uses thereof.

This invention claims the benefit of priority from Republic of Korea Patent Application No. 10-2023-0038200, filed on March 23, 2023, the entire contents of which are incorporated herein by reference.

Atopic dermatitis (AD) is an inflammatory disease accompanied by itching, dry skin, and eczema, and the exact cause has not yet been identified. The causes of atopic dermatitis known to date are known to be largely genetic causes inherited from parents, environmental causes due to urbanization and industrialization, and immunological causes. Atopic dermatitis, which was previously commonly called jaundice and often occurred in infancy and childhood, has recently become a disease that is not cured even in adulthood, causing many people to suffer mentally and physically.

Most patients with atopic dermatitis are characterized by increased levels of IgE in their blood, and macrophages activated in this inflammatory response are characterized by the release of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6.

Currently used general atopic dermatitis symptom relievers include moisturizers, antihistamines that reduce itching, and topical steroids that have therapeutic effects through anti-inflammation, vasoconstriction, and immunosuppressive effects. However, in severe cases, steroid-based drugs such as glucocorticoids and cyclosporine can cause diabetes and high blood pressure, and cause serious side effects such as Cushing's syndrome, eye disease (cataracts, glaucoma), and kidney and liver toxicity. Therefore, there is a growing need for new treatments without side effects.

Meanwhile, lactic acid bacteria are widely distributed in the digestive tract, oral cavity, feces, various fermented foods, and soil of humans and mammals, and are closely related to human life. Lactic acid bacteria that represent probiotics include Lactobacillus and Bifidobacterium , and these probiotics have various functional effects such as improving intestinal flora, improving lactose intolerance, preventing blood lipids and heart disease, anticancer effects, and preventing hypertension, and are receiving attention as therapeutic substances that can replace existing compound-based treatments.

The present inventors have made extensive research efforts to find probiotics that can change the intestinal microbiome and to apply them to the treatment of atopic dermatitis. As a result, they have confirmed that a new strain of Enterococcus genus, Enterococcus faecalis HL1, isolated and identified from pediatric feces, can alleviate atopic dermatitis symptoms and immune responses to a level similar to that of a normal group, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide an Enterococcus faecalis HL1 strain deposited under the deposit number KCTC 15339BP.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

Another object of the present invention is to provide a cosmetic composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

Another object of the present invention is to provide a skin external preparation for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

To achieve the above purpose, the present invention provides Enterococcus faecalis HL1 strain deposited under the deposit number KCTC 15339BP.

In one embodiment of the present invention, the strain may include a DNA sequence encoding the 16S rRNA sequence of sequence number 1, but is not limited thereto.

In another embodiment of the present invention, the strain may have an effect of preventing, treating, or improving atopic dermatitis, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

In addition, the present invention provides a food composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

In addition, the present invention provides a skin external preparation for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

In one embodiment of the present invention, the Enterococcus faecalis HL1 strain or a culture thereof may satisfy one or more of the following characteristics, but is not limited thereto:

(a) Inhibits IL-4 (interleukin-4) expression;

(b) decrease the amount of IgE in serum;

(c) reducing transepidermal water loss of the skin;

(d) reducing the epidermal thickness within the skin tissue; and

(e) Reduces inflammatory cell infiltration into skin tissue.

In another embodiment of the present invention, the pharmaceutical composition may be for oral administration, but is not limited thereto.

In another embodiment of the present invention, the food composition may be, but is not limited to, a health functional food composition.

In another embodiment of the present invention, the cosmetic composition may be in the form of, but is not limited to, a serum, a toner, a paste, a patch, a gel, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a cream, a massage cream, a nourishing cream, a mist, a moisture cream, a hand cream, a hand lotion, a foundation, an essence, a nourishing essence, a pack, a soap, an oil, a foundation, a makeup base, a wax, a spray, a cleansing foam, a cleansing lotion, a cleansing cream, a cleansing oil, a cleansing balm, a body lotion, or a body cleanser.

In addition, the present invention provides a method for preventing or treating atopic dermatitis, comprising a step of administering a composition comprising the Enterococcus faecalis HL1 strain according to the present invention or a culture solution thereof to a subject in need thereof.

In addition, the present invention provides a use of a composition comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention for preventing or treating atopic dermatitis.

In addition, the present invention provides a use of the Enterococcus faecalis HL1 strain according to the present invention or a culture solution thereof for the manufacture of a therapeutic agent for atopic dermatitis.

The present invention relates to a novel Enterococcus faecalis HL1 strain and a use thereof. The novel Enterococcus faecalis HL1 strain isolated and identified from pediatric feces or a culture thereof reduces the serum IgE concentration and IL-4 (interleukin-4) expression in an atopic dermatitis animal model, thereby alleviating the immune response and atopic dermatitis symptoms. Therefore, the novel Enterococcus faecalis HL1 strain or a culture thereof can be usefully utilized not only for the treatment of atopic dermatitis, but also is expected to help prevent the occurrence of allergic march by controlling atopic dermatitis.

[Correction under Rule 91 13.08.2024]
[Correction under Rule 91 24.05.2024]
Figure 1 is a diagram showing a phylogenic tree of Enterococcus faecalis .

Figure 2 is a diagram showing an experimental protocol to confirm the effect of Enterococcus faecalis HL1 strain on atopic dermatitis.

Figure 3 is a drawing showing the results of confirming changes in skin lesions in an atopic dermatitis animal model administered Enterococcus faecalis HL1 strain. Scale bar = 200 μm.

Figure 4 is a drawing showing the results of confirming the epidermal thickness and inflammatory cell infiltration in skin tissue by administration of Enterococcus faecalis HL1 strain in an animal model of atopic dermatitis through hematoxylin and eosin (H&E) staining.

Figure 5 is a diagram showing the results of confirming transepidermal water loss (A) and atopic dermatitis clinical score (B) in an atopic dermatitis animal model administered Enterococcus faecalis HL1 strain.

Figure 6 is a diagram showing the results of confirming the concentration of IgE in serum (A) and the expression of IL-4 in skin tissue (B) in an atopic dermatitis animal model administered Enterococcus faecalis HL1 strain. con: control, AD: atopic dermatitis.

The present inventors have made extensive research efforts to find probiotics that can change the intestinal microbiome and to apply them to the treatment of atopic dermatitis. As a result, they have confirmed that a new strain of Enterococcus genus, Enterococcus faecalis HL1, isolated and identified from pediatric feces, can alleviate atopic dermatitis symptoms and immune responses to a level similar to that of a normal group, thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides Enterococcus faecalis HL1 strain deposited under the deposit number KCTC 15339BP.

The present inventors compared and analyzed the 16S rRNA sequences of the strain isolated from the pediatric feces, and confirmed that the 16S rRNA of the strain was encoded by the base sequence of sequence number 1, and confirmed that it was a novel strain with 99.56% similarity to the existing strain ATCC 19433, and named it Enterococcus faecalis HL1, and deposited it with the Korea Research Institute of Bioscience and Biotechnology on March 7, 2023 (Accession number: KCTC 15339BP).

In one embodiment of the present invention, the Enterococcus faecalis HL1 strain isolated and identified from pediatric stool was confirmed to be a novel strain with 99.56% similarity to the existing ATCC 19433 (see Example 1).

In another embodiment of the present invention, when Enterococcus faecalis HL1 strain was administered to an atopic dermatitis animal model, it was confirmed that skin lesions were alleviated, the thickness of the skin tissue epidermis and the degree of inflammatory cell infiltration into the dermis layer were reduced, and the transepidermal water loss and atopic dermatitis clinical scores, which are clinical indicators of atopic dermatitis, were reduced (see Example 2).

In another embodiment of the present invention, when Enterococcus faecalis HL1 strain was administered to an animal model of atopic dermatitis, it was confirmed that the serum IgE level was reduced and the expression of IL-4, a Th2-related cytokine, was suppressed (see Example 3).

In the present invention, the strain may include a DNA sequence encoding the 16S rRNA sequence of sequence number 1, but is not limited thereto.

In the present invention, the strain may have an effect of preventing, treating, or improving atopic dermatitis, but is not limited thereto.

In the present invention, "atopic dermatitis" or "atopic dermatitis" is a skin disease that exhibits a chronic inflammatory skin condition with severe itching, which usually appears in infancy and childhood and continues into adulthood or even throughout life. It has a prevalence rate of 10-20% in children and 1-3% in adults, and the prevalence rate has been increasing in adults recently.

Extrinsic dermatitis, which accounts for 70-80% of the above atopic dermatitis, is caused by an IgE-related immune mechanism, and in this case, there are many reports that a delayed immune response due to T cell abnormality is involved. In the area where atopic dermatitis occurs, the infiltration of immune-related cells such as macrophages, Th lymphocytes, and mast cells increases significantly. In the case of atopic dermatitis patients, the concentration of blood IgE is high because the number of Th2 cells increases and the Th2 cytokines such as IL-4 and IL-13 secreted by these cells stimulate IgE secretion through B lymphocyte stimulation. IL-4 and IL-13 play an important role, especially in the case of early atopic dermatitis (Donald Y.M. Leung et al., J Clin Invest. 2004, 113, 651-657).

In the present invention, the term "allergic march" refers to cases where newborns born with atopic predisposition grow up to suffer from allergic asthma or rhinitis. This is a phenomenon in which allergic diseases appear simultaneously or with a time difference due to changes in the immune system, and atopic dermatitis, asthma, rhinitis, etc. appear in sequence.

In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an active ingredient.

In the present invention, the Enterococcus faecalis HL1 strain or a culture solution thereof may satisfy one or more of the following characteristics, but is not limited thereto:

(a) Inhibits IL-4 (interleukin-4) expression;

(b) decrease the amount of IgE in serum;

(c) reducing transepidermal water loss of the skin;

(d) reducing the epidermal thickness within the skin tissue; and

(e) Reduces inflammatory cell infiltration into skin tissue.

In this specification, "IgE (immunoglobulin E)" refers to immunoglobulin E, which can cause allergic diseases such as allergic asthma, urticaria, and atopic dermatitis by binding to surface receptors of mast cells or basophils in the blood and initiating an IgE-mediated allergic reaction.

In this specification, "IL-4 (interleukin 4)" refers to a Th2 cytokine secreted from activated mast cells, which can cause an allergic reaction by promoting the expression of pro-inflammatory signal molecules.

According to one embodiment of the present invention, the Enterococcus faecalis HL1 strain or a culture thereof can prevent, improve, or treat symptoms of atopic dermatitis by significantly reducing the level of IgE in serum or IL-4 in skin lesions, thereby reducing allergic reactions or inflammatory reactions.

In general, atopic dermatitis is characterized by a breakdown of the skin barrier, poor moisturizing, and loss of moisture. According to one embodiment of the present invention, the Enterococcus faecalis HL1 strain or a culture solution thereof can prevent, improve, or treat symptoms of atopic dermatitis by reducing the amount of transepidermal moisture loss of the skin.

According to one embodiment of the present invention, the Enterococcus faecalis HL1 strain or the culture solution thereof reduces the epidermal thickness in skin tissue and reduces the infiltration of inflammatory cells in skin tissue. Accordingly, the Enterococcus faecalis HL1 strain or the culture solution thereof can prevent, improve or treat symptoms of atopic dermatitis by inhibiting or improving epidermal hyperproliferation, which is a skin symptom commonly observed in atopic dermatitis, and inhibiting or improving the inflammatory state.

In this specification, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the Enterococcus faecalis HL1 strain can grow and survive in vitro. In addition, the culture medium may mean a culture medium obtained by culturing the strain and removing the bacterial cells from the bacterial cell culture. Meanwhile, the liquid from the culture medium from which the bacterial cells have been removed is also called a "supernatant", and may be obtained by allowing the culture medium to stand still for a certain period of time and taking only the liquid in the upper layer excluding the portion that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the lower layer and taking only the liquid at the upper layer. The above "bacteria" refers to the Enterococcus faecalis HL1 strain of the present invention itself, and includes a strain itself isolated and selected from pediatric feces, etc., or a strain isolated from a culture medium by culturing the strain. The bacteria can be obtained by centrifuging the culture medium and taking the portion that has settled to the lower layer, or can be obtained by allowing the bacteria to settle to the lower layer of the culture medium by gravity for a certain period of time and then removing the upper liquid. The culture medium can include the culture medium itself obtained by culturing the strain, a concentrate thereof, or a lyophilized product, or a culture supernatant obtained by removing the strain from the culture medium, a concentrate thereof, or a lyophilized product.

In the present invention, the "pharmaceutical composition" means a composition manufactured for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated and used in the form of external preparations, suppositories, and sterile injection solutions.

The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipses, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigants, ointments, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

Additives for tablets, powders, granules, capsules, pills and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethyl cellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinyl pyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

Additives that can be used in the liquid formulation according to the present invention include water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc.

The syrup according to the present invention may use a solution of white sugar, other sugars or sweeteners, and may also use a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity increasing agent, etc., as needed.

Purified water may be used in the emulsion according to the present invention, and an emulsifier, a preservative, a stabilizer, a fragrance, etc. may be used as needed.

The suspension according to the present invention may use suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, and the like. Surfactants, preservatives, stabilizers, colorants, and fragrances may also be used as needed.

The injection according to the present invention includes: solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, nonvolatile oils such as sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethyl acetamide, butazolidine, propylene glycol, Tween, nitrile acid amide, hexamine, and dimethyl acetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO ₃ ), carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cisplatinum, sodium alginate, Tween 80, and aluminum monostearate.

The suppository according to the present invention comprises cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranette wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, and for example, it can be administered by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc. According to one embodiment of the present invention, the pharmaceutical composition comprising the Enterococcus faecalis HL1 strain or a culture thereof can be administered orally, but is not limited thereto.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

In addition, the present invention provides a method for preventing or treating atopic dermatitis, comprising a step of administering a composition comprising the Enterococcus faecalis HL1 strain according to the present invention or a culture solution thereof to a subject in need thereof.

In addition, the present invention provides a use of a composition comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention for preventing or treating atopic dermatitis.

In addition, the present invention provides a use of the Enterococcus faecalis HL1 strain according to the present invention or a culture solution thereof for the manufacture of a therapeutic agent for atopic dermatitis.

In the present invention, the term "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

In the present invention, “administration” means providing a composition of the present invention to a subject by any appropriate method.

In the present invention, “prevention” means any act of inhibiting or delaying the onset of a target disease, “treatment” means any act of improving or beneficially changing a target disease and its resulting metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any act of reducing a parameter related to a target disease, for example, the degree of symptoms, by administering a composition according to the present invention.

In addition, the present invention provides a food composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

In the present invention, the food composition may be a health functional food composition, but is not limited thereto, and the health functional food composition may be used simultaneously with or separately from a drug for treatment before or after the onset stage of the disease in order to prevent or improve atopic dermatitis.

When the Enterococcus faecalis HL1 strain of the present invention or the culture solution thereof is used as a food additive, the Enterococcus faecalis HL1 strain or the culture solution thereof can be added as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the effective ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverage, the Enterococcus faecalis HL1 strain of the present invention or the culture solution thereof can be added in an amount of 15 wt% or less, or 10 wt% or less, with respect to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the effective ingredient can also be used in an amount greater than the above range.

There are no special restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In this specification, the term "health functional food" is the same as food for special health use (FoSHU), and means a food with high medical and therapeutic effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. The food can be manufactured in various forms such as tablets, capsules, powder, granules, liquid, and pills in order to obtain a useful effect in preventing or improving atopic dermatitis.

The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time by using food as a raw material, and can be highly portable.

In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

The formulation of the cosmetic composition according to the present invention may be a serum, toner, paste, patch, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, cream, massage cream, nutritional cream, mist, moisture cream, hand cream, hand lotion, foundation, essence, nutritional essence, pack, soap, oil, foundation, makeup base, wax, spray, cleansing foam, cleansing lotion, cleansing cream, cleansing oil, cleansing balm, body lotion, or body cleanser formulation.

The cosmetic composition of the present invention may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, and sphingolipids.

Any water-soluble vitamin that can be mixed in cosmetics may be used, but examples thereof include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinamide, folic acid, vitamin C, vitamin H, etc. Their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbate-2-phosphate, magnesium ascorbate-2-phosphate, etc.) are also included in the water-soluble vitamins that can be used in the present invention. Water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from a microbial culture, enzymatic method, or chemical synthesis method.

Any useful vitamin that can be mixed in cosmetics may be used, but examples thereof include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), and their derivatives (ascorbic acid palmitate, ascorbic acid stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinate, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ether, etc.). The useful vitamins can be obtained by conventional methods such as microbial transformation, purification from a microbial culture, enzymatic or chemical synthesis, etc.

As for the high molecular weight peptide, any polymer that can be mixed in cosmetics may be used, and examples thereof include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. The high molecular weight peptide can be purified and obtained by conventional methods such as purification from a culture solution of microorganisms, enzymatic methods, or chemical synthesis methods, or can be purified and used from natural products such as the dermis of pigs or cows, and silkworm fibers.

Any polymeric polysaccharide that can be mixed into cosmetics may be used, but examples thereof include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or its salts (sodium salt, etc.). For example, chondroitin sulfate or its salts can usually be purified from mammals or fish and used.

Anything that can be mixed into cosmetics can be used as a sphingolipid, but examples include ceramide, phytosphingosine, and sphingoglycolipids. Sphingolipids can be purified by conventional methods from mammals, fish, shellfish, yeast, or plants, or can be obtained by chemical synthesis.

In addition to the above essential ingredients, the cosmetic composition of the present invention may contain other ingredients commonly contained in cosmetics as needed.

In addition, other compounding ingredients that may be added include oil components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, circulation promoters, cooling agents, antiperspirants, purified water, etc.

Examples of maintenance ingredients include ester-based fats, hydrocarbon-based fats, silicone-based fats, fluorine-based fats, animal fats, and plant fats.

As ester-based fats and oils, tri-2-ethylhexanoate glyceryl, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentanoate, tri(caprylic, capric)glyceryl, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, tetra-2-ethylhexanoate pentaellistolate, cetyl caprylate, decyl laurate, Hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isostearyl laurate, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isostearate isopropyl, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, di(capryl, capric acid) propylene glycol, propylene glycol dicaprylate, neopentyl glycol dicaprate, neopentyl glycol dioctanoate, glyceryl tricaprylate, Triundecyl glyceryl, triisopalmitate glyceryl, triisostearate glyceryl, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleate ester, polyglycerol isostearate ester, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malate, hydroxystearic acid Examples thereof include esters such as 2-ethylhexyl, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesterol stearate, cholesterol isostearate, cholesterol hydroxystearate, cholesterol oleate, dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate, and isostearyl 12-stearoylhydroxystearate.

Examples of hydrocarbon-based fats include squalene, liquid paraffin, alpha-olefin oligomers, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybdenum, microcrystalline wax, and vaseline.

Examples of silicone-based oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane/methyl stearoxysiloxane copolymer, alkyl-modified silicone oil, and amino-modified silicone oil.

Examples of fluorinated oils include perfluoropolyether.

Examples of animal or plant fats include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil, cottonseed oil, coconut oil, kukui nut oil, wheat germ oil, rice germ oil, shea butter, colostrum oil, marc damia nut oil, meadowsweet oil, egg yolk oil, beef tallow, horse oil, mink oil, orange rapeseed oil, jojoba oil, candelilla wax, carnauba wax, liquid lanolin, and hydrogenated castor oil.

Examples of moisturizers include water-soluble low-molecular-weight moisturizers, fat-soluble molecular-weight moisturizers, water-soluble polymers, and fat-soluble polymers.

Examples of water-soluble low-molecular-weight moisturizers include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or higher), polypropylene glycol (polymerization degree n = 2 or higher), polyglycerin B (polymerization degree n = 2 or higher), lactic acid, and lactate salts.

Examples of fat-soluble low-molecular-weight moisturizers include cholesterol and cholesterol esters.

Examples of water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water-soluble chitin, chitosan, dextrin, etc.

Examples of lipid-soluble polymers include polyvinylpyrrolidone-eicosene copolymer, polyvinylpyrrolidone-hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of emollients include long-chain acylglutamic acid cholesteryl ester, hydroxystearate cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

Surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Nonionic surfactants include self-emulsifying monostearate glycerin, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POEㆍPOP (polyoxyethyleneㆍpolyoxypropylene) copolymer, POEㆍPOP alkyl ether, polyether-modified silicone, lauric acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc.

Examples of anionic surfactants include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkyl naphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkyl amide sulfates, alkyl phosphates, POE alkyl phosphates, alkyl amide phosphates, alkyloyl alkyl taurine salts, N-acyl amino acid salts, POE alkyl ether carboxylates, alkyl sulfosuccinates, sodium alkyl sulfoacetate, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphate esters.

Examples of cationic surfactants include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzylammonium chloride, behenyl trimethyl ammonium bromide, benzalkonium chloride, diethyl amino ethyl amide stearate, dimethyl amino propyl amide stearate, and quaternary ammonium salts of lanolin derivatives.

Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amidesulfobetaine type, phosphobetaine type, aminocarboxylic acid salt type, imidazoline derivative type, and amideamine type.

Organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, and complexes thereof; Examples thereof include organic pigments such as polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene-styrene copolymer, silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of inorganic pigments and organic pigments.

Examples of the organic powder include metal soaps such as calcium stearate; alkyl phosphate metal salts such as sodium zinc cetylphosphate, zinc laurylate, and calcium laurylate; acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroyl glycine calcium; amide sulfonic acid polyvalent metal salts such as N-lauroyl-calcium taurine, and N-palmitoyl-calcium taurine; N-acyl basic amino acids such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoyl lysine, N-alpha-paritoyl olnithine, N-alpha-lauroyl arginine, and N-alpha-hydrogenated beef tallow fatty acid acylarginine; N-acyl polypeptides such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride, etc.

As ultraviolet absorbers, para-aminobenzoic acid, para-aminobenzoic acid ethyl, para-aminobenzoic acid amyl, para-aminobenzoic acid octyl, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzyl cinnamic acid, para-methoxycinnamic acid-2-ethoxyethyl, para-methoxycinnamic acid octyl, dipara-methoxycinnamic acid mono-2-ethylhexaneglyceryl, para-methoxycinnamic acid isopropyl, diisopropyl-diisopropyl cinnamic acid ester mixture, urocanic acid, ethyl urocanic acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzophenone, tetrahydroxybenzophenone, Examples include 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy)-1,3,5-triazine, and 2-(2-hydroxy-5-methylphenyl)benzotriazole.

Examples of disinfectants include hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zincpyrithione, benzalkonium chloride, photosensitive agent No. 301, sodium mononitroguaiachol, and undecylenic acid.

Antioxidants include butylated hydroxyanisole, propyl gallic acid, and ellisorbic acid.

pH adjusters include citric acid, sodium citrate, malic acid, sodium malate, formaldehyde, sodium formaldehyde, succinic acid, sodium succinate, sodium hydroxide, and sodium hydrogen phosphate.

As for alcohol, we can mention higher alcohols such as cetyl alcohol.

In addition, the mixing components that may be added are not limited to these, and any of the above components may be mixed within a range that does not impair the purpose and effect of the present invention, but may be mixed at a weight percentage of 0.01-5% or a weight percentage of 0.01-3% with respect to the total weight.

When the formulation of the present invention is a lotion, paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether may be additionally included.

When the formulation of the present invention is a solution or emulsion, a solvent, a solvating agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as carrier components.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

In addition, the present invention provides a skin external preparation for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain or a culture solution thereof according to the present invention as an effective ingredient.

The above skin external preparation may be any one selected from the group consisting of ointments, creams, lotions, solutions, dressings, patches, blisters, tapes, mists, external powders, and sprays, but is not limited thereto.

In this specification, “active ingredient” means an ingredient that exhibits the intended activity alone or can exhibit the intended activity together with a carrier or the like that is inactive on its own.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Experimental method]

1. Strain isolation and sequencing

Enterococcus faecalis ( E.faecalis ) HL1 strain was isolated and identified from pediatric stool using MRS medium (BD-Difco) and WizardGenomic DNA of the strain was extracted using a Genomic DNA Purification Kit (Promega, USA). 4 μg of genomic DNA was used for library construction, and whole-genome sequencing was performed using the PacBio Sequel IIe (Pacific Biosciences) sequencing platform. The 16S rRNA sequence was targeted to the V1-V9 region using the primers 9F (SEQ ID NO: 2): 5'-GAGTTTGATCCTGGCTCAG-3' and 1542R (SEQ ID NO: 3): 5'-AGAAAGGAGGTGATCCAGCC-3', and strain identification was performed using the EzBioCloud Database.

2. Animal model

All procedures for mouse animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul Asan Medical Center and were performed in accordance with the guidelines and regulations of the Ministry of Food and Drug Safety (MFDS) of the Republic of Korea. To determine the effect of Enterococcus faecalis HL1 on atopic dermatitis, an antigen-induced atopic dermatitis animal model was created, as shown in Fig. 2 . Specifically, female BALB/c mice (4 weeks old, n = 5/group) were purchased from Orient Bio (Seongnam, Korea). To induce an imbalance in the intestinal flora, mice were administered antibiotics (1 g/L ampicillin (Sigma Aldrich, St Louis, MO), 500 mg/L vancomycin hydrochloride (Sigma Aldrich, St Louis, MO), 1 g/L neomycin (Sigma Aldrich, St Louis, MO), and 1 g/L metronidazole (Fresenius, Germany)) in the drinking water for 1 week. One week after the end of antibiotic administration, an atopic dermatitis mouse model was created through antigen induction. Atopic dermatitis was induced through skin sensitization with ovalbumin (OVA; grade V; Sigma, St Louis, MO). There were 1-week skin sensitization period and 2-week rest period, and a total of 2 sensitization periods were performed.

3. Administration of bacterial strains

Enterococcus faecalis HL1 cultured in MRS medium (BD-Difco) was cultured overnight at 37°C under anaerobic conditions. Then, the bacteria at an optical density (OD) of 600 = 1 were washed once with sterile phosphate-buffered saline (PBS, pH 7.4) and resuspended in 200 μl sterile PBS. Enterococcus faecalis HL1 or PBS was orally administered daily from 1 week of antibiotic treatment until the end of the atopic dermatitis experiment.

4. Evaluation of transepidermal water loss, clinical scoring of skin lesions, and histopathological analysis

Transepidermal water loss (TEWL) was measured using a vapometer (SWL-3; Delfin Technologies Ltd., Kuopio, Finland) before the start of the experiment and after each sensitization.

Clinical scores for skin lesions in mice were scored using a 0-3 scoring system for erythema, keratinization, and edema as follows: 0 (asymptomatic), 1 (mild), 2 (moderate), and 3 (severe).

Changes in epidermal thickness and the degree of inflammatory cell infiltration within the dermal layer were confirmed through histopathological analysis using hematoxylin and eosin (H&E) staining.

5. Check serum IgE and cytokine levels in skin tissue

Serum total IgE concentrations were measured using a mouse Immunoglobulin E (IgE) enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA, USA). To characterize the expression of the Th2-related cytokine interleukin (IL)-4 in mice, RNA was extracted from mouse skin tissues, and cDNA was synthesized using a WizScript cDNA synthesis Kit (Wizbiosolutions, Korea). qPCR was measured using the TaqMan method on an ABI 7900 system (Applied Biosystems, Piscataway, NJ). Each signal was normalized to the level of GAPDH in the same sample.

[Example]

Example 1. Novel strain Enterococcus faecalis ( Enterococcus faecalis ) Confirmation of HL1

Enterococcus faecalis, one of the probiotics, was isolated from pediatric feces, and the isolated strain was confirmed to contain a DNA sequence encoding the 16S rRNA sequence of sequence number 1. In addition, as a result of comparative analysis of the isolated strain with the 16S rRNA sequences of the EzBioCloud Database, it was confirmed to be a novel strain with 99.56% similarity to the existing ATCC 19433 strain, as shown in Fig. 1.

Example 2. Enterococcus faecalis ( Enterococcus faecalis ) Confirmation of skin changes in animal models of atopic dermatitis caused by HL1

To confirm the effect of Enterococcus faecalis HL1 on atopic dermatitis, skin lesion changes, transepidermal water loss (TEWL), atopic dermatitis clinical scores, and skin tissue changes (H&E) caused by Enterococcus faecalis HL1 were confirmed in an atopic dermatitis-induced animal model.

As a result of observing changes in skin lesions, as shown in Fig. 3, it was confirmed that skin lesions were alleviated in the Enterococcus faecalis HL1 administration group compared to the atopic dermatitis group.

In addition, as a result of confirming skin tissue changes through H&E staining, as shown in Fig. 4, it was confirmed that the thickness of the skin tissue epidermis and the degree of inflammatory cell infiltration into the dermis layer were reduced in the Enterococcus faecalis HL1 administration group compared to the atopic dermatitis group.

In addition, as a result of checking the transepidermal water loss and atopic dermatitis clinical score, as shown in Fig. 5, it was confirmed that the TEWL and clinical score tended to decrease in the Enterococcus faecalis HL1 administration group compared to the atopic dermatitis group.

From the above results, it was found that the novel strain Enterococcus faecalis HL1 can effectively alleviate atopic dermatitis symptoms.

Example 3. Enterococcus faecalis on systemic immune allergic response and cytokine expression Enterococcus faecalis ) Confirming the effectiveness of HL1

To determine the improving effect of Enterococcus faecalis HL1 on serum IgE levels and the expression of Th2-related cytokines in the skin, Enterococcus faecalis HL1 was orally administered to an atopic dermatitis-induced animal model, and then compared with the atopic dermatitis group.

As a result, as shown in Fig. 6, it was confirmed that the total serum IgE level was relatively reduced in the Enterococcus faecalis HL1 administration group compared to the atopic dermatitis group.

In addition, when measuring the expression of IL-4, a Th2-related cytokine in the skin, it was confirmed that IL-4 expression was suppressed in the Enterococcus faecalis HL1 administration group compared to the atopic dermatitis group.

From the above results, it was confirmed that administration of Enterococcus faecalis HL1 suppressed systemic allergic and Th2-related immune responses.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The present invention relates to a novel Enterococcus faecalis HL1 strain and a use thereof. The novel Enterococcus faecalis HL1 strain isolated and identified from pediatric feces or a culture thereof reduces the serum IgE concentration and IL-4 (interleukin-4) expression in an atopic dermatitis animal model, thereby alleviating the immune response and atopic dermatitis symptoms. Therefore, the novel Enterococcus faecalis HL1 strain or a culture thereof can be usefully utilized not only for the treatment of atopic dermatitis but also for the prevention of allergic march by controlling atopic dermatitis, and thus has industrial applicability.

<Acceptance number>

Name of depositor: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC 15339BP

Date of acceptance: 20230307

Claims (13)

Enterococcus faecalis HL1 strain deposited under the accession number KCTC 15339BP. In the first paragraph, A strain characterized in that the strain comprises a DNA sequence encoding the 16S rRNA sequence of sequence number 1. In the first paragraph, The above strain is characterized by having an effect of preventing, treating, or improving atopic dermatitis. A pharmaceutical composition for preventing or treating atopic dermatitis, comprising the Enterococcus faecalis HL1 strain of claim 1 or a culture solution thereof as an active ingredient. In paragraph 4, A pharmaceutical composition characterized in that the Enterococcus faecalis HL1 strain or a culture solution thereof satisfies at least one of the following characteristics: (a) Inhibits IL-4 (interleukin-4) expression; (b) decrease the amount of IgE in serum; (c) reducing transepidermal water loss of the skin; (d) reducing the epidermal thickness within the skin tissue; and (e) Reduces inflammatory cell infiltration into skin tissue. In paragraph 4, A pharmaceutical composition, characterized in that the pharmaceutical composition is for oral administration. A food composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain of claim 1 or a culture solution thereof as an effective ingredient. A cosmetic composition for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain of claim 1 or a culture solution thereof as an active ingredient. In Article 8, The cosmetic composition is characterized in that the cosmetic composition is in the form of a serum, a toner, a paste, a patch, a gel, a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a cream, a massage cream, a nourishing cream, a mist, a moisture cream, a hand cream, a hand lotion, a foundation, an essence, a nourishing essence, a pack, a soap, an oil, a foundation, a makeup base, a wax, a spray, a cleansing foam, a cleansing lotion, a cleansing cream, a cleansing oil, a cleansing balm, a body lotion, or a body cleanser. A skin external preparation for preventing or improving atopic dermatitis, comprising the Enterococcus faecalis HL1 strain of Article 1 or a culture solution thereof as an active ingredient. A method for preventing or treating atopic dermatitis, comprising a step of administering a composition containing the Enterococcus faecalis HL1 strain of claim 1 or a culture solution thereof to a subject in need thereof. Use of a composition containing the Enterococcus faecalis HL1 strain of claim 1 or a culture solution thereof for the prevention or treatment of atopic dermatitis. Use of Enterococcus faecalis HL1 strain of Article 1 or a culture solution thereof for the manufacture of a therapeutic agent for atopic dermatitis.

Images