Patent Description:
The present disclosure relates to a novel microorganism of the family Sporichthyaceae and use thereof.

The skin ecosystem provides diverse habitats for microorganisms, and a wide range of microorganisms occupy the skin ecosystem. It is known that these microorganisms have a symbiotic relationship with their human hosts and have many positive effects on the hosts. The skin forms a variety of habitats, such as invaginations, specified niches, etc., which help a wide range of microorganisms grow. The primary role of the skin is to form a physical barrier and to offer protection against a potential threat from the external environment and toxic substances. The skin is an interface with the external environment and is colonized by a diverse collection of microorganisms (fungi, bacteria, viruses, and small larvae). The microorganisms that are adapted to the niche they inhabit are selected according to selection of physical and chemical functions. In general, the skin is cool, acidic, and maintained in a dry state. Structurally, the epidermis plays a role in forming a physical barrier and resisting penetration by microorganisms and toxins while retaining moisture. The outermost layer of the epidermis is composed of the stratum corneum. The epidermis forms a so-called "brick and mortar" structure, the skin tissue undergoes continuous self-renewal, and squames are constantly shed from the skin surface at the final stage of differentiation.

Probiotics, collectively refers to microorganisms which have beneficial effects on the human body, and are microorganisms that offer benefit to our bodies. Most probiotics known to date are lactic acid bacteria. Probiotics have been reported to show efficacy through various beneficial effects on the human body, but there are few studies on a correlation between skin flora and skin.

The skin barrier consists of dead keratinocytes and intercellular lipids. The skin barrier is a skin protection layer that protects the skin against external stimuli and prevents evaporation of water through the skin, and plays a key role in maintaining skin health. That is, the skin barrier prevents excessive loss of water from the body and entry of harmful substances such as chemicals or microorganisms. Keratinocyte envelopes constituting the surface of dead keratinocytes play an important role in the stability of intercellular lipids. Keratinocytes undergo differentiation to form skin barriers through cornification. Functions of the skin barrier may be destroyed with aging or by external factors, and damage of the skin barrier may cause water loss of the skin and wrinkles. <NPL>, reviews the beneficial effects of probiotic bacteria on the skin. It describes the treatment of skin conditions with Vitreoscilla filiformis, Enterococcus faecalis or Lactobacillus plantarum and discusses the main mechanisms involved in health benefits of probiotics to the skin.

Accordingly, the present inventors examined what changes occur in the skin according to changes of skin flora, and moreover, they studied whether the skin environment may be potentially improved by inducing changes of skin flora. As a result, a novel microorganism of the family Sporichthyaceae was isolated and identified from the skin of a healthy adult, and it was found that the novel microorganism or a culture thereof may induce changes in a skin microbial community so as to improve the skin environment, and may be used in skin-related conditions, thereby completing the present invention.

An aspect provides a novel microorganism of the genus Epidermidibacterium belonging to the family Sporichthyaceae belonging to the family Sporichthyaceae, wherein the Epidermidibacterium keratini is deposited under the Accession No. KCCM 11843P, and having the following mycological characteristics: <NUM>) rod-shaped, (<NUM>) non-motile, (<NUM>) Gram-positive, and (<NUM>) non-spore-forming.

Another aspect provides a culture comprising the microorganism.

Still another aspect provides a cosmetic composition including the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism.

Still another aspect provides a microorganism of the genus Epidermidibacterium or the culture comprising the microorganism for use in preventing, improving, or treating skin conditions of a subject.

Still another aspect provides a pharmaceutical composition comprising the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism.

Still another aspect provides the composition comprising the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism for use in preventing, improving, or treating skin diseases or inflammatory diseases of a subject.

Still another aspect provides a health functional food composition comprising the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism.

An aspect provides a novel microorganism of the genus Epidermidibacterium belonging to the family Sporichthyaceae.

The microorganism of the genus Epidermidibacterium (Epidermidibacterium keratini sp. ) belongs to the family Sporichthyaceae, wherein the Epidermidibacterium keratini is deposited under the Accession No. KCCM 11843P and has the following mycological characteristics:.

There are obvious distinctions in phylogenetic and chemotaxonomic characteristics between the microorganism of the genus Epidermidibacterium and other strains of different genera, and thus the microorganism was identified as a novel microorganism of the genus Epidermidibacterium.

The microorganism of the genus Epidermidibacterium may include Epidermidibacterium keratini.

The Epidermidibacterium keratini may have any one or more of the following mycological characteristics:.

The microorganism of the genus Epidermidibacterium or species Epidermidibacterium keratini (Epidermidibacterium keratini sp. ), which belongs to the family Sporichthyaceae, wherein the Epidermidibacterium keratini is deposited under the Accession No. KCCM 11843P, may have any one or more of the following morphological characteristics:.

The microorganism of the genus Epidermidibacterium or species Epidermidibacterium keratini may have any one or more of the following cultural and physiological characteristics:.

The microorganism of the genus Epidermidibacterium or species Epidermidibacterium keratini (Epidermidibacterium keratini sp. ), which belongs to the family Sporichthyaceae, wherein the Epidermidibacterium keratini is deposited under the Accession No. KCCM 11843P, may have any one or more of the following biochemical characteristics:.

The Epidermidibacterium keratini may include a <NUM> rRNA gene having a nucleotide sequence of SEQ ID NO: <NUM>.

The Epidermidibacterium keratini is deposited under Accession NO. KCCM 11843P.

Further, another aspect provides a culture comprising the microorganism of the genus Epidermidibacterium or species Epidermidibacterium keratini.

As used herein, the term "culture" refers to a culture obtained by culturing the microorganism, a concentrate thereof, or a freeze-dried product thereof, or a culture supernatant obtained by removing the microorganism from the culture, a concentrate thereof, or a freeze-dried product thereof, and "culture supernatant", "conditioned culture", and "conditioned medium" may be used interchangeably therewith.

The culture may be obtained by culturing the microorganism of the genus Epidermidibacterium or species Epidermidibacterium keratini in R2A medium at any temperature of higher than <NUM> or lower than <NUM> for a predetermined time, for example, <NUM> hours to <NUM> hours.

In a specific embodiment, the culture supernatant of the microorganism may be obtained by removing the microorganism from the culture of the microorganism by centrifugation or filtration.

In another specific embodiment, a concentrate may be obtained by concentrating the culture itself or a supernatant which is obtained by centrifuging the culture or filtering the culture by using a filter.

A medium and culture conditions for culturing the microorganism of the genus Epidermidibacterium may be appropriately selected or modified by those skilled in the art.

Still another aspect provides a pharmaceutical or a cosmetic composition, including the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism.

The microorganism of the genus Epidermidibacterium and the culture thereof are the same as described above.

In a specific embodiment, the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism may increase expression of filaggrin, claudin, αSMase, CerS3, transglutaminase-<NUM>, and HAS, and decrease expression of inflammation-related factors and pruritus-related factors. Therefore, another aspect relates to the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism for use in preventing, improving or treating skin-related conditions or inflammation-related conditions.

Examples of the skin-related conditions may include skin aging, wounds, dermatitis, atopic dermatitis, pruritus, eczematous dermatosis, dry eczema, erythema, urticaria, psoriasis, drug rash, papulosquamous disease, insect and parasite-mediated diseases, superficial dermatomycosis, bacterial infectious diseases, viral diseases, sexually transmitted diseases, autoimmune bullous diseases, connective tissue disease, dyschromatosis, xeroderma pigmentosum, acne, etc..

Examples of the inflammation-related conditions may include dermatitis, allergy, atopy, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder arthritis, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases.

The composition may include <NUM>% by weight to <NUM>% by weight, for example, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, <NUM>% by weight to <NUM>% by weight, or <NUM>% by weight to <NUM>% by weight of the microorganism or the culture comprising the microorganism with respect to the total weight of the composition.

Hereinafter, use of the microorganism of the genus Epidermidibacterium according to compositions will be described in detail.

In one embodiment, the composition is a cosmetic composition.

In a specific embodiment, the cosmetic composition may enhance a skin barrier; or prevent, improve, or inhibit aging.

As used herein, the term "skin aging" refers to both tangible and intangible changes that appear on the skin with aging, and for example, decreased epidermal thickness, reduction in the number of dermal cells or blood vessels, reduced ability to repair DNA, decreased cell turnover, delayed wound healing, reduced skin barrier functions, reduced water retention in the epidermis, decreased sweat and sebum secretion, decreased vitamin D production, decreased physical damage defense, decreased chemical removal ability, decreased immune responsiveness, decreased sensory function, decreased temperature control, etc. The microorganism of the genus Epidermidibacterium or the culture comprising the mircoorganism may be used to improve skin aging caused by exogenous or endogenous factors. The exogenous factors refer to many external factors, for example, ultraviolet rays (light), and the endogenous factors, also called chronological factors, refer to factors mainly occurring over time. That is, the skin aging specifically includes not only early aging caused by external stimuli such as UV, air pollution, cigarette smoke, chemicals, etc., but also natural aging occurring due to a reduction in skin cell proliferation with aging. The skin aging is a concept including all of wrinkles, loss of elasticity, saggy skin, dryness, etc. Further, wrinkles include wrinkles caused by changes in the components constituting the skin tissue by stimulation of internal/external factors.

Therefore, since the cosmetic composition may have the above effects, it may be used for skin improvement, for example, skin moisturizing, skin aging prevention, or skin barrier reinforcement.

The cosmetic composition may have, for example, a cosmetic formulation of a softening toner, a nutrient toner, a massage cream, a nutritional cream, an essence, a pack, a gel, an ampoule, or a type of skin adhesive.

The cosmetic composition may include components commonly used in cosmetic compositions, for example, common additives and carriers such as a stabilizer, a solubilizer, vitamins, a pigment, and a fragrance, in addition to the composition as an active ingredient.

Further, the cosmetic composition may be a composition for external use.

In the present disclosure, the composition for external use may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The composition for external use may be appropriately blended with components used in compositions for external use, such as common cosmetics or medicines, for example, an aqueous component, an oily component, a powder component, alcohols, a moisturizer, a thickener, a UV absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance, a pigment, a variety of skin nutrients, or a combination thereof as needed. The composition for external use may be appropriately blended with a sequestering agent such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc., caffeine, tannine, verapamil, a licorice extract, glabridin, a hot water extract of fruits of Calin, various herbal medicines, tocopheryl acetate, glycyrrhizic acid, tranexamic acid and a derivative or salt thereof, vitamin C, magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin, Kojic acid, sugars such as glucose, fructose, treharose, etc..

In another specific embodiment, the composition is a pharmaceutical composition. In another specific embodiment, the composition is for use in preventing or treating skin diseases or inflammatory diseases.

The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and a lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrating agent, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxy cellulose, or a combination thereof. The disintegrating agent may be calcium carboxymethylcellulose, sodium starch glycolate, anhydrous dibasic calcium phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as a preparation for oral or parenteral administration. The preparation for oral administration may be granules, a powder, a liquid, a tablet, a capsule, a dry syrup, or a combination thereof. The preparation for parenteral administration may be an injectable formulation.

Another aspect of the present invention relates to a health functional food composition comprising the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism.

In the health functional food composition, the microorganism of the genus Epidermidibacterium or the culture comprising the microorganism may be used alone or in combination with other foods or food ingredients, according to a general method. A mixing ratio of the active ingredient may be determined according to the purpose of use (prevention, health, or treatment). In general, to produce foods or beverages, the composition of the present disclosure may be added in an amount of <NUM> parts by weight or less with respect to a raw material. There is no particular limitation in kinds of the health functional food. Of the kinds of the health functional food, a beverage composition may include various flavors or natural carbohydrates as an additional ingredient, like common beverages. The natural carbohydrates include 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, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The health food composition may include nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerins, alcohols, sparkling components for carbonated beverages, or a combination thereof. The health functional food composition may also include natural fruit juice, fruit juice beverages, fruit flesh for the preparation of vegetable beverages, or a combination thereof.

As mentioned above, the present invention provides the microorganism of the genus Epidermidibacterium, the culture thereof, or the composition including the same for use in a method of preventing, improving, or treating a condition of a subject, , wherein the condition is a skin-related condition or an inflammation-related condition.

The condition of a subject may be a skin-related condition or an inflammation-related condition.

The composition is the same as described above. The subject may be mammals, for example, humans, cattle, horses, pigs, dogs, sheep, goats, or cats.

The novel microorganism of the family Sporichthyaceae, according to an aspect, has phylogenetic and chemotaxonomic characteristics distinct from those of other microorganisms of different genera of the same or different family, and thus the microorganism of the family Sporichthyaceae or a culture comprising the microorganism may be effectively used for preventing, improving, or treating skin-related conditions or inflammation-related conditions.

Hereinafter, the present disclosure will be described in more detail with reference to Examples.

A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy woman with sterile distilled water was seeded in a Reasoner's 2A (R2A) medium (Becton Dickinson, Cockeysville, MD). After seeding, the sample was incubated for <NUM> hours in an incubator at <NUM>, and <NUM> colonies were isolated and cultured, followed by re-incubation for <NUM> hours in an incubator at <NUM>. The completely cultured colonies were identified by <NUM> rRNA gene sequencing. Primers (SEQ ID NOS: <NUM> and <NUM>) used at this time were designed to react and amplify only in bacteria. PCR amplification was performed for <NUM> cycles of at <NUM> for <NUM>, at <NUM> for <NUM>, and at <NUM> for <NUM> and <NUM> sec, and lastly, at <NUM> for <NUM>. A product was stored at <NUM>. DNA sequences of species isolated and cultured after PCR reaction were determined by using an ABI-3730XL (ABI, USA). Nucleotide sequences of <NUM> rRNA regions determined in the isolated and cultured colonies of the microorganisms were compared with those of other strains registered in the BLAST program provided through the National Center for Biotechnology Information (NCBI)'s homepage. Only novel species with homology of <NUM>% or less were selected and used, and of them, a novel microorganism with homology of <NUM>% or less (hereinafter, referred to as "EPI-<NUM>") was selected. EPI-<NUM> has a <NUM> rRNA sequence of SEQ ID NO: <NUM> (complementary DNA).

<NUM> rRNA homology analysis was calculated using an ExTaxon-e server (Yoon S. , (<NUM>)). Sequence data were aligned using a software package BioEdit (Hall T. (<NUM>)), and phylogenetic analysis was performed using MEGA version <NUM> (Tamura, K et al. Phylogenetic trees were constructed using Neighbor-joining (Sitouand Nei (<NUM>)), maximum-likelihood, and maixmum-parsimony algorithms, and stability of the phylogenetic trees was evaluated by bootstrap analysis (<NUM> replications) (Felsenstein <NUM>).

As a result, EPI-<NUM> was found to have <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>% homology with Modestobacter lapidis MON <NUM>, Sporichthya polymorpha DSM <NUM>, Modestobacter marinus 42H12-<NUM>, Modestobacter roseus KLBMP1279, and Modestobacter versicolor CP153-<NUM>, respectively. As shown in <FIG>, results of a constructed phylogenetic tree showed that EPI-<NUM> is a novel microorganism belonging to the family Sporichthyaceae, which has not yet been reported.

Morphological characteristics of EPI-<NUM> were analyzed as follows.

First, cell morphology of EPI-<NUM> was observed under an Olympus microscope (GX71) at magnification of <NUM> X after culturing cells in R2A medium at <NUM> for <NUM> days. Gliding motility was investigated by subjecting a hanging-drop technique to fresh EPI-<NUM> cells in R2A medium.

For scanning electron microscopy (SEM), a sample was pretreated as follows. EPI-<NUM> was seeded on R2A (Becton Dickinson, Cockeysville, MD) solid medium and incubated for <NUM> days, and then a membrane filter was attached to formed colonies.

The membrane filter having colonies attached thereto was separated and fixed in a <NUM>% glutaraldehyde solution for <NUM> hours, and then washed with PBS for <NUM> minutes twice.

Thereafter, the membrane filter was treated with a <NUM>% osmium tetroxide solution for <NUM> hour, and dehydrated with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>% ethanol. The dehydrated sample was treated with isoamylacetate to remove ethanol therefrom. The pre-treated sample was naturally dried, and sputtered with gold by using a sputter coater (SC502, Polaron). Thereafter, the sample was photographed using SEM (Hitachi S4300N, Hitachi, Japan) at magnification of <NUM> X and 100000X, and results are shown in <FIG>.

To distinguish between gram-positive and gram-negative bacteria, experiments were performed as follows.

In detail, one colony of EPI-<NUM> cultured well on the R2A solid medium was collected and fixed on a slide glass. The slide glass was completely dried, and then stained with crystal violet for <NUM> to <NUM> minutes, and washed with running water and dried again.

The dried slide was stained with iodine-potassium iodide for <NUM> minute. Thereafter, the slide was destained with ethyl alcohol, and washed with running water, followed by drying. To determine whether the sample was Gram-positive or -negative, secondary staining was performed using a red staining dye such as Safranin for <NUM> to <NUM> minutes, and the slide was washed with water and observed under a microscope (Olympus microscope, <NUM> GX71).

As a result, EPI-<NUM> was found to have the following morphological characteristics:.

To investigate culture conditions of EPI-<NUM>, an optimum culture temperature was determined by setting an optimum growth temperature from <NUM> to <NUM> (<NUM> intervals), and an optimum salt concentration was determined by adding NaCl of <NUM>%(w/v) to <NUM>%(w/v) (<NUM>% interval) to a medium and then culturing at <NUM> for <NUM> days.

pH was determined by using the following buffer system; sodium acetate/acetic acid(pH<<NUM>), Tris/HCl (pH <NUM> to pH <NUM>), and glycine/sodium hydroxide (pH><NUM>).

Further, catalase activity was determined by analyzing bubble generation in <NUM>%(v/v) H<NUM>O<NUM>, and oxidase activity was determined by using <NUM>% (w/v) tetramethyl phenylenediamine.

Further, in order to test growth media, nutrient agar (NA), yeast extract malt extract agar (ISP <NUM>), and trypticase soy agar medium (BD, USA) were used for culturing at <NUM> for <NUM> days.

Further, BioLog GP2, BioLog Gen III, API 20NE, and API ZYM (biomeriuex, France) were used to investigate substance utilization of EPI-<NUM>. A method of use was performed according to the manual provided by the manufacturer. When a positive reaction occurs, a violet color develops. After examination with the naked eye, the degree of growth was accurately determined using a microplate spectrophotometer. An API ZYM test was analyzed after incubation at <NUM> for <NUM> hours, and another API test was analyzed after incubation at <NUM> for at least <NUM> hours. Results of a comparison with other species are shown in the following Table <NUM>.

Anaerobic growth was examined in serum bottles by adding sodium thioglycollate (<NUM>/l) to R2A medium, and replacing the upper air layer with nitrogen gas.

Tests for degradability of DNA, casein, starch, tween <NUM>, and carboxymethyl cellulose were performed after incubation at <NUM> for <NUM> days.

As a result, EPI-<NUM> was found to have the following cultural and physiological characteristics:.

A DNA G+C content of EPI-<NUM> was analyzed by HPLC. In detail, experimental methods and conditions for analyzing a cellular fatty acid composition, a G+C composition, and quinone are as follows.

First, the cellular fatty acid composition of the isolated microorganism was analyzed according to Miller's method. About <NUM> of cultured cells were transferred to a tube, and <NUM> of a solution prepared by adding <NUM>% NaOH to <NUM>% methanol was added thereto, and the tube was heated to <NUM> for <NUM> minutes and cooled at room temperature. <NUM> of methanolic-HCl (a mixture of <NUM> of <NUM> N HCl and <NUM> of methanol) was added to the tube, which was then heated at <NUM> for <NUM> minutes, and then rapidly cooled. <NUM> of hexane/methyl-tert-butylether (<NUM>:<NUM>, v/v) was added thereto, and mixed well for <NUM> minutes. After being left at room temperature, when the reaction solution was separated into <NUM> layers, a lower layer was removed, and <NUM> of dilute NaOH (<NUM> of NaOH/<NUM> of D. W) was added and mixed well for <NUM> minutes and left at room temperature. About <NUM>/<NUM> of a supernatant was transferred to a screw-capped sample vial (<NUM> X <NUM>, Agilent technologies), and capped, and then used as a sample. The sample was subjected to saponification, methylation, and extraction according to a standard protocol of Sherlock MIS Software. Fatty acids were analyzed by using an Agilent Technologies <NUM> Gas Chromatograph, an A30 m X <NUM> X <NUM> methyl siloxane column (HP-<NUM>), and the TSBA40 database (MIDI, Version <NUM>) of the MIS package was used to identify the fatty acids.

All experiments were repeated at least three times, and the results are shown in the following Table <NUM>.

Further, G+C mol% was analyzed as follows. A DNA solution extracted from the microorganism was transferred to a microtube, and centrifuged at <NUM> rpm for <NUM> minutes.

Thereafter, <NUM>µl of a supernatant was collected and transferred to a new tube, and DNA denaturation was performed at <NUM> for <NUM> minutes, and then cooled. <NUM>µl (<NUM>/mL) of P1 nuclease was added thereto, and allowed to react at <NUM> for <NUM> hours. Alkaline phosphatase (<NUM>µl of buffer, and <NUM>µl of phosphatase) was added thereto, followed by incubation at <NUM> overnight. After centrifugation at <NUM> rpm for <NUM> minutes, analysis was performed under the following conditions:.

Polar lipid analysis was performed after culturing the microorganism at <NUM> for <NUM> hours in R2A medium. First, polar lipids were extracted from the microorganism according to a method described in<NPL>). Thereafter, polar lipids were identified by two-dimensional TLC (<NPL>).

As a result, EPI-<NUM> was found to have the following biochemical characteristics:.

Further, its characteristics were compared with those of other microorganisms, and summarized in the following Table <NUM>.

As shown in Table <NUM>, EPI-<NUM> is different from the closest species in terms of major biochemical characteristics, and these chemotaxonomic results and the phylogenetic results of Example <NUM>. demonstrate that EPI-<NUM> is a new species.

Taken together, it was demonstrated that EPI-<NUM> isolated from human epidermal keratinocytes is a microorganism of a novel genus belonging to the family Sporichthyaceae, and designated as Epidermidibacterium gen. according to the phylogenetic nomenclature; Epidermidibacterium [E. epidermis, -idis skin; L. bacterium, (a small rod or staff); N. (Epidermidibacterium a small rod from the skin)].

Further, as confirmed in Example <NUM>, Epidermidibacterium gen. has the following characteristics:.

Subsequently, the above results demonstrated that the Epidermidibacterium gen. strain isolated from human epidermal keratinocytes is one species, which was designated as Epidermidibacterium keratini sp. nov according to the phylogenetic nomenclature;.

Epidermidibacterium keratini (ke. keratini pertaining to keratin).

Further, as confirmed in Example <NUM>, Epidermidibacterium keratini has the following characteristics in addition to the characteristics of the Epidermidibacterium gen.

The present inventors deposited the Epidermidibacterium keratini strain belonging to the genus Epidermidibacterium at the Korean Culture Center of Microorganisms (KCCM) in the Korea Research Institute of Bioscience and Biotechnology on June <NUM>, <NUM> with Accession NO: KCCM 11843P.

In order to measure the effect of EPI-<NUM> on changes of skin flora, changes of the skin microbiome of a subject was analyzed.

In detail, EPI-<NUM> culture was applied to the skin of the subject at <NUM>-hr intervals, and skin samples were collected for a total of <NUM> hours and analyzed. Samples were collected before treatment of the culture (Epidermis A) and at <NUM> hours after treatment of the culture (Epidermis B). The samples were collected using sterile water and a scraper, and all procedures were performed on a clean bench. The samples collected from the skin were suspended in <NUM>% NaCl, and supernatants were centrifuged (<NUM>,<NUM> rpm/m) to collect cells therefrom. The cells were washed once with sterile physiological saline. Thereafter, DNA was extracted using a FastDNA SPIN KIT (MP Biomedical, France). For amplification of <NUM> ribosomal DNA genes of the extracted DNA, GC clamp (SEQ ID NO: <NUM>)-attached 341F (SEQ ID NO: <NUM>) and 518R (SEQ ID NO: <NUM>) were used. For a PCR reaction, <NUM> dNTP, <NUM> units of Taq polymerase, and <NUM>µl of <NUM> Mg<NUM>+-containing Takara Perfect Premix (Takara, Japan) were mixed with <NUM>µl of a DNA template (<NUM>µg/mL), and <NUM>µl each of <NUM> forward primer and <NUM> reverse primer. Sterile water was added to the mixture to a total volume of <NUM>µl. The PCR reaction was performed in a C1000-Dual (Bio-Rad, USA) by touchdown PCR in which temperature was decreased from <NUM> to <NUM> every <NUM> cycles, and the PCR mixture was finally treated at <NUM> for <NUM> minutes, and stored at <NUM>.

Thereafter, the resulting PCR product was used in denaturing gradient gel electrophoresis (DGGE) analysis. In the DGGE analysis, a D-code system (Bio-rad, USA) was used. A concentration of a polyacrylamide gel used was <NUM>%, and <NUM>% polyacrylamide bis-solution (<NUM>:<NUM>, <NUM>%C) (Bio-Rad, USA) was used to prepare a gel with a vertical concentration gradient of <NUM>-<NUM>%. At this time, <NUM> urea and <NUM>% (w/v) formamide (Sigma, USA) were used as a denaturant. The D-code system was filled with about <NUM> of TAE buffer (<NUM> Tris, <NUM> acetic acid, <NUM> EDTA, pH <NUM>), and equal amounts of 2X loading dye (<NUM>% bromophenol blue, <NUM>% xylene cyanol, <NUM>% glycerol) and the PCR product were mixed and electrophoresed at <NUM> V for <NUM> minutes. The electrophoresed polyacrylamide gel was stained with a Redsafe (Intron, Korea) solution for <NUM> minutes, and specific bands which appeared were analyzed. Results are shown in <FIG>. Further, images of DGGE bands were quantified and analyzed using GelCompar2 (Bionumeric, Belgium) to generate a histogram. For sequencing analysis, the DGGE gel bands were sent to Macrogen Inc. , and results are shown in <FIG>.

As shown in <FIG> and <FIG>, a decreased number of bands and strong expression of particular bands were observed in Epidermis B (after treatment of the culture), as compared to Epidermis A (before treatment of the culture).

The increased expression of bands indicates an increase in population. Most of the DGGE bands with increased intensity indicated microorganisms of the genus staphylococcus, which belong to skin flora symbiotic with the skin and effective in strengthening the skin barrier and moisturizing the skin.

These results suggest that EPI-<NUM> influences strengthening of the skin barrier and moisturizing of the skin by increasing the microorganisms.

In order to analyze skin wound healing, skin barrier strengthening, and moisturizing activities, expression levels of filaggrin, hyaluronic acid synthase3 (HAS3), claudin <NUM>, claudin <NUM>, αSMase, aquaporin <NUM> (AQP3) transglutaminase <NUM>, and CerS3, which are skin barrier strengthening and moisturizing activity-related factors, in a human keratinocyte cell line (HaCaT) were examined, according to EPI-<NUM> treatment, by RT-PCR.

In detail, HaCaT cells, which are human keratinocytes, were cultured in <NUM>% fetal bovine serum-containing DMEM (Dulbecco'smodified Eagle's Medium, Gibco <NUM>-<NUM>), and culturing was carried out in an incubator at <NUM> and <NUM>% CO<NUM>. The cultured cell line was treated with <NUM> retinol as a positive control group. As another positive control group, <NUM> of the cell culture was treated with cultures of Bifidobacterium animalis (UB) and Lactobacillus acidophilus (AB) at a concentration of <NUM>%. Further, EPI-<NUM> cultures were treated at a concentration of <NUM>%, <NUM>%, or <NUM>%.

Thereafter, HaCaT cells were further incubated for <NUM> hours, and recovered, and <NUM> of trizol (RNAiso, DAKARA, Japan) was added thereto to isolate RNA. Further, for RT-PCR, RNA was quantified by using a Nanodrop <NUM> (Thermo, USA), and reacted at <NUM> for <NUM> minutes and at <NUM> for <NUM> minutes to synthesize cDNA (Reverse Transcriptase Mix, ELPIS biotech, Korea). RT-PCR was performed by using Step One Plus (Applied Biosystems, USA), and SYBR Green supermix (Applied Biosystems, USA) was added together with primers and cDNA. Polymerase was activated at <NUM> for <NUM> minutes and then polymerization was performed at <NUM> for <NUM> seconds, at <NUM> for <NUM> minute, and at <NUM> for <NUM> minute, for <NUM> cycles.

Primers of SEQ ID NOS: <NUM> and <NUM> were used for filaggrin, primers of SEQ ID NOS: <NUM> and <NUM> for HAS3, primers of SEQ ID NOS: <NUM> and <NUM> for claudin <NUM>, primers of SEQ ID NOS: <NUM> and <NUM> for claudin <NUM>, primers of SEQ ID NOS: <NUM> and <NUM> for αSMase, primers of SEQ ID NOS: <NUM> and <NUM> for AQP3, primers of SEQ ID NOS: <NUM> and <NUM> for transglutaminase <NUM>, primers of SEQ ID NOS: <NUM> and <NUM> for CerS3, and primers of SEQ ID NOS: <NUM> and <NUM> for beta-actin.

<FIG> is a graph showing a comparison of the effect of EPI-<NUM> on filaggrin expression in human keratinocytes and a positive control group.

<FIG> is a graph showing a comparison of the effect of EPI-<NUM> on claudin <NUM> expression in human keratinocytes and a positive control group.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on claudin <NUM> expression in human keratinocytes.

<FIG> is a graph showing a comparison of the effect of EPI-<NUM> on CerS3 expression in human keratinocytes and a positive control group.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on CerS3 expression in human keratinocytes.

<FIG> is a graph showing a comparison of the effect of EPI-<NUM> on αSMase expression in human keratinocytes and a positive control group.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on αSMase expression in human keratinocytes.

<FIG> is a graph showing a comparison of the effect of EPI-<NUM> on HAS expression in human keratinocytes and a positive control group.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on HAS expression in human keratinocytes.

<FIG> is a graph showing a comparison of the effect of EPI-<NUM> on transglutaminase <NUM> expression in human keratinocytes and a positive control group.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on aquaporin <NUM> expression in human keratinocytes.

As shown in <FIG>, EPI-<NUM> was found to significantly increase expression of filaggrin which is a keratinocyte differentiation marker and a precursor protein of a natural moisturizing factor responsible for moisturizing the skin surface.

As shown in <FIG>, <FIG>, and <FIG>, EPI-<NUM> was found to significantly increase expression of claudin <NUM> and <NUM>, which are tight junction structures responsible for protecting the internal organs from the external environment and maintaining homeostasis of the human body.

As shown in <FIG> and <FIG>, EPI-<NUM> was found to significantly increase expression of CerS3 and aSMase, which are enzymes essential for ceramide synthesis.

As shown in <FIG> and <FIG>, EPI-<NUM> was found to significantly increase expression of HAS3, which is responsible for synthesis of hyaluronic acid which is an extracellular matrix component maintaining skin moisture, and showed higher efficacy than the positive control, retinol.

As shown in <FIG>, EPI-<NUM> was found to significantly increase expression of transglutaminase <NUM>, which plays a critical role in forming cornified envelopes.

As shown in <FIG>, EPI-<NUM> was found to significantly increase expression of AQP3, which plays a role in providing water for the human skin, functions as a water channel to facilitate cell migration, and functions as a glycerol transporter to regulate keratinocyte proliferation and differentiation, thereby treating wounds.

Taken together, it can be seen that EPI-<NUM> enhances the flexibility and firmness of the skin, improves the skin's water retention ability so as to exhibit a skin moisturizing effect, strengthens the skin barrier function by more effectively controlling intercellular adhesion and migration of water-containing solutes and water through the periplasmic space, and increases proliferation, differentiation, and migration of keratinocytes to exhibit a significant effect on wound healing.

In order to analyze anti-inflammatory activity of EPI-<NUM>, HaCaT cells were treated with Staphylococcus aureus (S. aureus), which is an inflammation inducer, and S. epidermidis and EPI-<NUM> were co-cultured with human HaCaT cells, and then mRNA expression levels of inflammatory factors were assessed.

In detail, HaCaT cells were cultured and seeded on a <NUM>-well plate. <NUM> hours later, a micromembrane well was placed on the top of a cell single layer, and then living cells (SA: S. aureus, SA_SE: S. aureus + S. epidermidis, SA_EPI-<NUM>: S. aureus + EPI-<NUM>) were seeded at a cell density of <NUM> × <NUM><NUM>.

The cells were further co-cultured for <NUM> hours, and then the cells and medium were removed. RT-PCR was performed in the same manner as in Example <NUM>. , except that a combination of SEQ ID NOS: <NUM> and <NUM>, a combination of SEQ ID NOS: <NUM> and <NUM>, a combination of SEQ ID NOS: <NUM> and <NUM>, and a combination of SEQ ID NOS: <NUM> and <NUM> were used as primers for TNF-α, IL-1a, TSLP, and DDIT3, respectively. Results are shown in <FIG>.

<FIG> is a graph showing effects of EPI-<NUM> on inflammatory factors.

As shown in <FIG>, EPI-<NUM> was found to significantly inhibit expression of TNF-α, IL-1a, TSLP, and DDIT3, which are skin inflammatory factors. This result suggests that EPI-<NUM> and the culture thereof may be used for skin inflammation-related diseases.

In order to analyze antipruritic activity of EPI-<NUM>, mRNA expression levels of TSLP (Thymic stromal lymphopoietin) and TARC (Thymus and activation-regulated chemokine), which are pruritic factors, were assessed. In detail, HaCaT cells were treated with polyinosinic:polycytidylic acid (Poly I:C), which is a synthetic ribonucleic acid that induces interferon production, and then cultured for <NUM> hours to stimulate HaCaT cells. Aged cells by stimulation were treated with the EPI-<NUM> culture at a concentration of <NUM>%, <NUM>%, or <NUM>%, and further cultured for <NUM> hours. RT-PCR was performed in the same manner as in Example <NUM>. , except that a combination of SEQ ID NOS: <NUM> and <NUM> was used as TARC primers.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on TSLP expression in human keratinocytes.

<FIG> is a graph showing the effects of different doses of EPI-<NUM> on TARC expression in human keratinocytes.

As shown in <FIG>, EPI-<NUM> was found to significantly decrease expression of TSLP and TARC, which are pruritic factors. This result suggests that EPI-<NUM> and the culture thereof may be used for skin inflammation-related diseases and pruritus.

In order to examine cytotoxicity of EPI-<NUM>, an MTT assay was performed.

In detail, HaCaT cells were prepared in the same manner as in Example <NUM>. , and dispensed in a <NUM>-well plate at a density of <NUM> × <NUM><NUM> cells/well. <NUM> hours later, a FBS-free medium was replaced. An EPI-<NUM> culture was diluted with the FBS-free medium at concentrations of <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%, and used to treat the cells. After incubation for <NUM> hours, the medium was removed, and <NUM>µg/mL of MTT (Sigma-Aldrich, St. Louis, MO) reagent was added to crystallize the cells, followed by further incubation for <NUM> hours. After removing the MTT reagent, crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO). Absorbance at <NUM> was measured, and compared with a non-treatment group to measure cell viability. Results are shown in <FIG>.

<FIG> is a graph showing the effect of EPI-<NUM> on cell viability.

Claim 1:
A microorganism of the species Epidermidibacterium keratini (Epidermidibacterium keratini sp.) belonging to the family Sporichthyaceae, wherein the Epidermidibacterium keratini is deposited under the Accession No. KCCM 11843P, and having the following mycological characteristics:
(<NUM>) rod-shaped;
(<NUM>) non-motile;
(<NUM>) Gram-positive; and
(<NUM>) non-spore-forming.