KR102273232B1 - Micrococcus lylae strain and skin condition improving uses of thereof - Google Patents

Abstract

The present invention relates to a novel microorganism, a lysate thereof, a culture solution, an extract of the culture solution, and use thereof for improving skin conditions. The present invention can be usefully used for the prevention, improvement, or treatment of skin-related conditions or inflammation-related conditions.

Description

Micrococcus lylae strain and its skin condition improving use {Micrococcus lylae strain and skin condition improving uses of thereof}

It relates to a novel microorganism, its lysate, a culture solution, an extract of the culture solution, and their use for improving skin condition.

The skin ecosystem provides various types of habitat for microorganisms, and a wide range of microorganisms live there. Human beings, the host, form a symbiotic relationship with them and are known to have many positive effects on the host. The skin constitutes various types of habitats, such as depressions and specialized crevices, and helps a wide range of microorganisms to grow. Basically, the skin forms a physical barrier and helps to defend against potential hazards and toxic substances from the outside. The skin becomes a connection point with the external environment and is also a gathering place for various microorganisms (fungi, bacteria, viruses and small larvae). In accordance with the selection of physical and chemical functions, microorganisms adapt to specialized niches and prepare habitats. In general, the skin is cold, acidic, and remains dry. Structurally, the epidermis forms the skin barrier, blocks the penetration of microorganisms and toxins, and plays an important role in maintaining moisture. The uppermost layer of the epidermis is composed of the stratum corneum. The epidermis has a so-called 'brick and mortar structure', and the skin tissue undergoes a continuous self-healing process, and the scales (squames) that have undergone the final differentiation process repeat the process of being continuously removed from the skin tissue.

Probiotics are microorganisms that have beneficial effects on the human body. Most probiotics known to date are known as lactic acid bacteria. Probiotics have been reported to produce effective efficacy through various beneficial effects on the human body, but studies on the interaction between skin flora and skin are scarce.

Accordingly, there is a need for the development of skin flora that can be usefully used for skin-related conditions.

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M Wieser et al. Emended descriptions of the genus Micrococcus, Micrococcus luteus (Cohn 1872) and Micrococcus lylae (Kloos et al. 1974). Int J Syst Evol Microbiol. 2002 Mar;52(Pt 2):629-37.

One aspect is to provide a strain of Micrococcus lylae belonging to the genus Micrococcus sp.

Another aspect is to provide a lysate of the strain, a culture solution, or an extract of the culture solution.

Another aspect is to provide a composition comprising the strain, a lysate thereof, a culture solution, and an extract of the culture solution.

One aspect provides a Micrococcus lylae strain.

The micrococcus lylae ( Micrococcus lylae ) strain may be a strain deposited with accession number KCCM12684P.

The Micrococcus lylae strain may be a strain comprising 16s rRNA of SEQ ID NO: 1.

The strain may have a skin beauty or skin condition improvement effect, for example, skin regeneration, skin moisturizing effect, or skin soothing effect.

The strain promotes skin regeneration by increasing cell migration to the skin damaged site, or increases the expression of skin moisturizing-related hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3) or aquaporin 3 (AQP3: Aquaporin 3) or inhibiting the expression of inflammatory cytokines (eg, IL-1β), or reducing the expression of itch-related factors (eg, TSLP).

Another aspect provides a lysate of the strain, a culture solution, or an extract of the culture solution.

The specific details of the strain are as described above.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and is capable of supplying nutrients so that a Micrococcus larae strain can grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain in a medium for a certain period of time, its metabolites, and extra nutrients. In addition, the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution. On the other hand, the liquid from which the cells have been removed from the 　 culture solution 　 is also called a “supernatant”, and the 　 culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the lower layer, remove the cells through filtration, It can be obtained by removing the precipitation of and taking only the liquid at the top. The "cell" of the present invention refers to the strain itself of the present invention, and includes the strain isolated and selected from a skin sample, or the strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the 　 culture solution to take the part that has sunk to the lower layer, or by removing the liquid from the upper layer after leaving it still for a certain period of time because it sinks into the lower layer of the culturing solution by gravity.

The culture medium may include the culture medium itself obtained by culturing the strain, the concentrate thereof, or the lyophilisate or the culture supernatant obtained by removing the strain from the culture medium, the concentrate or the lyophilisate thereof.

The culture medium is a medium (e.g., R2A medium, RCM medium, or TSA medium) of the micrococcus strain in a medium (eg, R2A medium, RCM medium, or TSA medium) at any temperature of more than 10 ° C or less than 40 ° C for a certain time, for example, 4 to 50 hours. may be obtained.

In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture to remove the strain.

In another embodiment, the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture solution itself, or the culture solution using centrifugation or a filter.

The culture medium and culture conditions for culturing the Micrococcus lirae strain may be appropriately selected or modified by those of ordinary skill in the art.

As used herein, the term “lysate” may refer to a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.

As used herein, the term "culture solution extract" means extraction from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and a fraction obtained by fractionating it. can

Another aspect provides the use of the strain, a lysate of the strain, a culture solution, or an extract of the culture solution. Specifically, it provides a composition comprising a Micrococcus lirae strain, a lysate thereof, a culture solution, or an extract of the culture solution.

The use of the strain may include improving skin condition, improving skin beauty, preventing, improving or treating skin diseases, preventing, improving, or treating skin inflammatory diseases.

The skin condition improvement or skin beauty improvement may be skin wound improvement, skin cell regeneration promotion, or skin moisturizing.

The "skin moisturizing" may refer to any action that maintains skin moisture or prevents moisture loss.

As used herein, the term "anti-inflammatory" may be used interchangeably with "inhibiting or improving inflammation", and may refer to any action in which an immune response is alleviated to suppress NO production.

The "skin disease" may be a disease caused by damage to the skin barrier function, skin aging, skin wounds, skin scars, or skin inflammation. The term “prevention” includes inhibiting the development of a disease. The term "treatment" includes inhibiting, alleviating, or eliminating the development of a disease.

The damage to the skin barrier function may refer to any change that appears in the skin as the function of the skin barrier is reduced or damaged. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.

The skin inflammatory disease may be any one selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, weak rash, and acne.

In another embodiment, the strain may be used together with a strain belonging to another micrococcus genus having a skin improvement effect to show a synergistic effect. The strains belonging to the genus Micrococcus are, for example, Micrococcus agilis, Micrococcus aloeverae, Micrococcus antarcticus, Micrococcus cohnii, Micrococcus endophyticus, Micrococcus flavus, Micrococcus halobius, Micrococcus kristinae, Micrococcus lactis, Micrococcus selenus rose, Micrococcus selenius roseenus, Micrococcus se. Micrococcus terreus, Micrococcus varians, Micrococcus yunnanensis, Neomicrococcus aestuarii, Neomicrococcus lactis, Halomicrococcus hydrotolerans, or Methanimicrococcus blatticola , and the like.

The composition comprises 0.001 wt% to 80 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, based on the total weight of the composition %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of a strain, a lysate thereof, a culture solution, or an extract of a culture solution thereof.

As used herein, the term "included as an active ingredient" means that the extract of the strain of the present specification, its lysate, culture, or its culture is added to the extent that it can exhibit the above-mentioned effects, drug delivery and stabilization, etc. For this purpose, it is meant to include being formulated in various forms by adding various components as sub-components.

The composition may be a cosmetic composition.

The cosmetic composition may have a cosmetic formulation of, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin adhesion type.

Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include.

In addition, the composition may be a composition for external application to the skin.

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component normally used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

In another embodiment, the composition may be a pharmaceutical composition.

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 the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection.

The composition may be a health functional food composition.

The health functional food composition may be used alone or in combination with other foods or food ingredients of the strain or its culture, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as additional components, like a conventional beverage. 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 the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition may also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages the carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.

Another aspect also provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition described above.

The subject's condition may be a condition related to skin or a condition related to inflammation.

As used herein, the terms "administering", "introducing", and "implanting" are used interchangeably and into a subject by a method or route that results in at least partial localization of a composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to one embodiment of the.

Administration may be administered by methods known in the art. Administration can be administered directly to a subject by any means, for example, by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of skin beauty improvement, for example, skin moisturizing, skin barrier strengthening, skin inflammation suppression, and skin wrinkle improvement effect.

The administration is 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to per day of the composition according to one embodiment 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site, and total daily or at intervals of 2 to 5 days. The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of humans per kg, or the above dose is converted, for example, by the volume ratio (for example, the average value) of the target animal and the organ (heart, etc.) One dose can be administered.

According to the novel Micrococcus leirae strain according to an aspect, it can be usefully used for the prevention, improvement, or treatment of skin-related conditions or inflammation-related conditions.

1 is a graph showing the effect of a strain according to an embodiment on relative wound density and cell mobility.
Figure 2 is a view showing the effect of the strain according to an embodiment on the recovery of the skin damaged area.
3 is a graph showing the effect of a strain according to an embodiment on the expression of AQP3.
4 is a graph showing the effect of a strain according to an embodiment on inflammatory cytokines.
5 is a graph showing the effect of the strain according to one embodiment on the expression of TSLP, which is a factor related to atopy and itch.

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

[Example]

Example 1. Isolation and identification of strains

A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy woman with sterile distilled water was inoculated into R2A (Reasoner's 2A) medium (Becton Dickinson, Cockeysville, MD). After inoculation, 100 colonies formed after culturing in a 28 ℃ incubator for 48 hours after inoculation were purely separated and cultured, and cultured in a 28 ℃ incubator for 48 hours. The cultured colonies were subjected to 16s rRNA gene sequence identification. The primers used in this case were designed to amplify in response only to bacteria (SEQ ID NOs: 2 and 3). PCR amplification was carried out in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 75°C for 1 minute and 30 seconds, and finally, after treatment at 72°C for 8 minutes, it was stored at 4°C. After the PCR reaction, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA). When the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies is compared with other strains registered with the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI), homology is 99% or less of a novel microorganism Micrococcus lylae (hereinafter referred to as "YM-5") was selected. The selected YM-5 strain was deposited with the Korea Microbial Conservation Center on March 27, 2020 and was given accession number KCCM12684P, and the YM-5 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

[Experimental example]

Analysis of skin regeneration activity

The effect of the YM-5 strain culture medium isolated in Example 1 on skin regeneration was analyzed.

Specifically, a human dermal fibroblast (Hs68) cell line ^{was aliquoted at 5x10 5} in a 6-well plate, and then cultured for 24 hours in an incubator at 37 ° C. 5% CO ₂ condition to obtain a 100% confluent state. made it Thereafter, using a cell scraper, a wound was randomly scraped across the center of the well, and the medium containing the YM-5 strain culture solution (1% (w/w)) and the medium without it were replaced. It was further incubated for the next 24 hours. After 24 hours, it was photographed with a fluorescence microscope to see how much the wounds randomly inflicted on the cells were recovered with and without the strain culture medium, and the results are shown in FIGS. 1 and 2 .

1 is a graph showing the rate of cell movement by recovery of cell wounds by the strain culture solution according to one embodiment (Migration rate) and the degree of cell saturation (Relative wound density) in the culture dish accordingly.

Figure 2 is a photograph showing the degree of recovery after 24 hours of cells wounded by the treatment with a strain culture solution according to an embodiment.

As shown in FIGS. 1 and 2 , when cultured in a medium containing the strain culture solution according to one embodiment, it was found that the migration of fibroblasts to the injured site was significantly increased compared to the control group.

These results mean that the YM-5 strain according to one embodiment has an effect of promoting skin regeneration.

Skin barrier strengthening and moisturizing activity analysis

The effect of the YM-5 strain culture medium isolated in Example 1 on skin barrier strengthening and skin moisturizing activity was analyzed.

Specifically, HaCaT cells, a human keratinocyte cell line, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were cultured at 37°C and 5%. It was performed in a _{CO 2 incubator.} The cultured cell line was treated with the culture solution (1% (w/w)) of the YM-5 strain and further cultured for 24 hours. As a positive control, 1 uM of retinoic acid (RA) was used. The expression level of AQP3 was measured in the same manner as in Experimental Example, except that a primer for AQP3 (aquaporin), a factor related to skin barrier strengthening and skin moisturizing, was used. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that the strain according to one embodiment significantly increased the expression level of AQP3.

These results mean that the YM-5 strain according to one embodiment can be usefully used for strengthening the skin barrier and moisturizing the skin.

Anti-inflammatory, anti-atopic and anti-itch activity assay

Whether the YM-5 strain culture isolated in Example 1 had anti-inflammatory, anti-atopic and itch inhibitory activity was analyzed.

Specifically, HaCaT cells, which are human keratinocytes, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were cultured at 37°C and 5%. It was performed in a _{CO 2 incubator.} The medium was changed every 3 to 4 days, and subculture was performed when the cells were excessively proliferated. Thereafter, the cells were aliquoted at 5x10 ⁵ /well, and after 24 hours of culture, they were washed with phosphate buffered saline solution (PBS). Cells were aliquoted into each well containing DMEM medium without FBS, and Poly I:C 10 μg/ml, and IL-4 were added at 10 ng/ml to induce inflammation in the keratinocyte line. Next, the culture solution of the strain was treated at a concentration of 1%, and further cultured for 4 hours. As a positive control, 1 μM of dexamethasone was used. The expression levels of IL-1β and TSLP were analyzed in the same manner as in Experimental Example, except that primers for IL-1β and TSLP (Thymic stromal pymphopoietin) were used. The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIGS. 4 and 5 , respectively.

4 is a graph showing the effect of a strain according to an embodiment on inflammatory cytokines.

5 is a graph showing the effect of the strain according to one embodiment on the expression of TSLP, which is a factor related to atopy and itch.

As shown in FIG. 4 , it was confirmed that the strain according to one embodiment significantly reduced the expression of IL-1β, an inflammatory cytokine, compared to the untreated control group.

In addition, as shown in FIG. 5 , it was found that the strain according to one embodiment reduced the expression of TSLP, a factor that suppresses atopy and itch, compared to the untreated control group.

These results show that the YM-5 strain according to one embodiment can be usefully used for the prevention, improvement, or treatment of skin inflammatory diseases, as well as prevention, improvement, or treatment of atopy, or prevention, treatment of itch (pruritus) Or it means that it can be usefully used for improvement.

Korea Microorganism Conservation Center (Overseas) KCCM12684P 20200327

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Claims (7)

It is characterized in that it has a 16s rRNA sequence represented by SEQ ID NO: 1, deposited with accession number KCCM12684P, belonging to the genus Micrococcus ( Micrococcus sp.) Micrococcus lylae ( Micrococcus lylae ) strain. The culture solution of the strain of claim 1. A cosmetic composition comprising the strain of claim 1, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof. The cosmetic composition according to claim 3, wherein the cosmetic composition is for skin regeneration, skin moisturizing, skin inflammation improvement, atopy improvement, or itch improvement. The cosmetic composition according to claim 3, wherein the strain has an ability to increase AQP3 expression, an ability to inhibit IL-1β expression, and an ability to inhibit TSLP expression. delete delete

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