KR102368736B1 - Microbacterium oleivorans strain and skin condition improving uses of thereof - Google Patents

Abstract

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

Description

Microbacterium oleivorans strain and skin condition improving uses thereof {Microbacterium oleivorans 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 to prepare habitats. In general, the skin is cold, acidic, and remains dry. Structurally, the epidermis forms a 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 squames that have undergone the final differentiation process repeat the process of constantly being removed from the skin tissue.

JP 2020-508357 A

One aspect is to provide a microbacterium oleivorans ( Microbacterium oleivorans ) strain belonging to the genus Microbacterium ( Microbacterium 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 Microbacterium oleivorans I-46 strain.

The Microbacterium oleivorans I-46 strain may be a strain deposited with accession number KCCM12703P.

The Microbacterium oleivorans I-46 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 aging suppression, skin whitening, skin barrier strengthening, skin wrinkle suppression, or skin moisturizing effect.

The strain may be to increase the expression of hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3) or filagrin related to skin moisturizing and skin barrier. The increase in markers such as HAS3 or filaggrin means that filaggrin, a precursor of NMF, and intercellular lipids were normalized in keratinocytes through the formation of a normal stratum corneum. Therefore, to prevent skin aging and maintain skin health from external environmental changes such as atmospheric drying, ultraviolet rays and various pollutants, to strengthen the skin barrier to prevent moisture loss in the skin tissue while promoting the maintenance of moisture in the skin tissue It may be to form a stratum corneum.

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

Specific details of the strain are the same as described above.

As used herein, the term "culture medium" can be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and is a medium capable of supplying nutrients so that Dokdo strains can grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain for a certain period, its metabolites, extra nutrients, and the like. 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 medium 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 bottom layer, remove the cells through filtration, or 　 centrifuge the culture solution to the lower part 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 medium and taking 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 to the lower layer of the 　 culture solution by gravity.

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

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

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 Microbacterium oleivorans I-46 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 crushing 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 fractions obtained by fractionation thereof. 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 Dokdo strain, a lysate thereof, a culture medium, or an extract of the culture medium.

Uses of the strain may include improving skin condition, improving skin beauty, preventing, improving or treating skin diseases.

The skin condition improvement or skin beauty improvement may be skin aging inhibition or improvement, anti-wrinkle, skin whitening, skin barrier strengthening, or skin moisturizing.

As used herein, the term "skin aging" refers to the tangible and intangible changes that appear on the skin with age, for example, a phenomenon in which the thickness of the epidermis becomes thin, the number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound healing, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and temperature regulation.

The strain or its culture medium may be for improving skin aging caused by extrinsic factors or intrinsic factors. The extrinsic factor refers to various external factors, such as ultraviolet light (light), and the intrinsic factor is also referred to as a chronological factor and refers to a factor mainly caused by the passage of time. That is, the skin aging is specifically a natural aging phenomenon that occurs as the proliferation of skin cells decreases due to aging, as well as premature aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemical substances, etc. It is a concept that includes all of wrinkles, elasticity reduction, skin sagging and dryness. In addition, wrinkles include those that cause wrinkles by changing the components constituting the skin tissue by stimulation by changes in internal and external factors.

The aging may be photoaging. The term “photoaging” is a phenomenon induced by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cleavage of matrix proteins, and abnormal cross-linking, and repetition of these mechanisms leads to apparent skin aging.

As used herein, the term "wrinkle" refers to a state in which elasticity of the skin is lost and loosened, and for example, the skin may be folded. The "skin wrinkle prevention or improvement" may refer to any action of preventing or improving wrinkles by inhibiting the expression of factors related to wrinkles, or increasing the total amount of collagen.

The "skin whitening" may mean not only to brighten skin tone by inhibiting the synthesis of melanin pigment, but also to improve skin hyperpigmentation such as spots or freckles caused by ultraviolet rays, hormones, or heredity. The "pigmentation" refers to a condition in which the amount of pigment in the body is abnormal or there is an abnormality in the place where the pigment appears, and may be, for example, spots and freckles.

The "skin barrier strengthening" may refer to any action that enhances the function of the skin barrier that is located at the outermost part of the skin and prevents loss of moisture and nutrients.

The "skin moisturizing" may refer to any action of maintaining skin moisture or preventing moisture loss.

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.

In another embodiment, the strain may be used together with a strain belonging to another microbacterium genus having a skin improvement effect to show a synergistic effect. The strain belonging to the genus Microbacterium may include, for example, M.pramxydans, M. aurum , M. foliorum, M. hominis, M. flavescens , 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 the strain, a lysate thereof, a culture medium, or an extract of the culture medium thereof. At this time, when the content of the extract of the strain, its lysate, the culture medium, or its culture medium is less than the above range, the skin condition improvement effect, for example, skin aging inhibition, skin whitening, skin barrier strengthening, skin wrinkle inhibition, or skin moisturizing There is a problem that the effect is not sufficiently exerted.

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, transdermally delivered patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually 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-blocking 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 extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and 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 an additional component 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 taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages 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 may be administered directly to a subject by any means, for example, 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 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 reaction 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 a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.

According to the novel I-46 strain according to an aspect, it can be usefully used for skin moisturizing and barrier strengthening.

1 is a graph showing the effect of a strain according to an embodiment on the expression of HAS3.
2 is a graph showing the effect of a strain according to an embodiment on the expression of filaggrin.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Isolation and identification of strains

Soil and seawater samples collected from Seodo, Dokdo were inoculated into liquid medium or solid medium (Becton Dickinson, Cockeysville, MD) containing TSB (Tryptic soy broth). After inoculation, 100 colonies formed after culturing in a 28 ℃ incubator for 48 hours after inoculation were separated and cultured in pure water, and cultured in a 28 ℃ incubator for 48 hours. The cultured colonies were subjected to 16s rRNA gene sequence identification. The primers used at this time were designed to amplify in response only to bacteria. 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 less than 97% Only novel species of were selected and used, and among them, a novel microorganism Microbacterium oleivarans with less than 97% homology (hereinafter referred to as "I-46") was selected. The selected I-46 strain was deposited with the Korea Microbial Conservation Center on April 27, 2020 and was given accession number KCCM12709P, and the I-46 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

[Experimental example]

Skin barrier strengthening and skin moisturizing activity analysis

The effect of the I-46 strain culture medium isolated in Example 1 on skin barrier strengthening and skin moisturizing activity was analyzed.

Specifically, HaCaT cells, a human keratinocyte, were cultured in DMEM medium (Dulbecco'smodified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum, and all cultures were cultured at 37°C CO ₂ was performed in an incubator. The cultured cell line was treated with the culture medium (1%, 10% (w/w)) of the I-46 strain and further cultured for 24 hours.

As a positive control, 1 μM of retionic acid was used. After that, RNA was isolated from the cells of each sample using trizol (RNA iso, DAKARA, Japan), and RNA was quantified at 260 nm with nanodrops, and cDNA was synthesized in an amplifier using 2 μg of RNA each. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, the target gene, HAS3 (hyaluronic acid synthase), a factor related to skin barrier strengthening and moisturizing, and primers and cDNA for filaggrin were added together with a real-time PCR machine (Step One Plus, Real-time polymerase chain reaction was performed in Applied Biosystems, USA). The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in FIG. 1 .

1 is a graph showing the effect of a strain according to an embodiment on the expression of HAS3.

2 is a graph showing the effect of a strain according to an embodiment on the expression of filaggrin.

As a result, as shown in FIGS. 1 and 2 , it was confirmed that the strain according to one embodiment significantly increased the expression levels of HAS3 and filaggrin.

Therefore, the I-46 strain according to one embodiment can be usefully used for strengthening the skin barrier and moisturizing the skin.

Korea Microorganism Conservation Center (Overseas) KCCM12703P 20200427

<110> Cosmax, INC. <120> Microbacterium oleivorans strain and skin condition improving uses of <130> PN141371KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1397 <212> RNA <213> Artificial Sequence <220> <223> Microbacterium oleivorans I46 <400> 1 accatgcagt cgaacggtga gcagagcttg ctctgtggat cagtggcgaa cgggtgagta 60 acacgtgagc aatctgcccc tgactctggg ataagcgctg gaaacggcgt ctaataccgg 120 atacgagctg cgaaggcatc ttcagcagtt ggaaagaatt tcggtcaggg atgagctcgc 180 ggcctatcag cttgttggtg aggtaatggc tcaccaaggc gtcgacgggt agccggcctg 240 agagggtgac cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 300 tggggaatat tgcacaatgg gcgcaagcct gatgcagcaa cgccgcgtga gggatgacgg 360 ccttcgggtt gtaaacctct tttagcaggg aagaagcgaa agtgacggta cctgcagaaa 420 aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcaa gcgttatccg 480 gaattattgg gcgtaaagag ctcgtaggcg gtctgtcgcg tctgctgtga aatcccgagg 540 ctcaacctcg ggtctgcagt gggtacgggc agactagagt gcggtagggg agattggaat 600 tcctggtgta gcggtggaat gcgcagatat caggaggaac accgatggcg aaggcagatc 660 tctgggccgt aactgacgct gaggagcgaa agggtgggga gcaaacaggc tagataccc 720 tggtagtcca ccccgtaaac gttgggaact agttgtgggg tccattccac ggattccgtg 780 acgcagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct aaaactcaaa 840 ggaattgacg gggacccgca caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa 900 gaaccttacc aaggcttgac atatacgaga acgggctaga aatagtcaac tctttggaca 960 ctcgtaaaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1020 ccgcaacgag cgcaaccctc gttctatgtt gccagcacgt aatggtggga actcatggga 1080 tactgccggg gtcaactcgg aggaaggtgg gccgatgacg tcaaatcatc atgcccctta 1140 tgtcttgggc ttcacgcatg ctacaatggc cggtacaaag ggctgcaata ccgtgaggtg 1200 gagcgaatcc caacaaaagc cggtcccagt tcggattgag gtctgcaact cgacctcatg 1260 aagtcggagt cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgctggt 1320 cttgtacaca ccgcccgtca agtcatgaaa gtcggtaaca cctgaagccg gtggcccaac 1380 ccttgtggag ggagccg 1397

Claims (5)

As a cosmetic composition comprising a microbacterium oleivorans strain, a lysate thereof, a culture solution, an extract or a mixture thereof, or a mixture thereof belonging to the genus Microbacterium deposited with accession number KCCM12703P, HAS3 , or a cosmetic composition that increases the expression of filaggrin. The cosmetic composition of claim 1, wherein the cosmetic composition is for skin barrier strengthening, skin wrinkles, skin elasticity, or moisturizing. delete Microbacterium oleivorans belonging to the genus Microbacterium deposited with accession number KCCM12703P, a lysate thereof, a culture medium, an extract of the culture medium, or a mixture thereof. A composition for external application for skin for wrinkles, skin elasticity, or moisturizing, wherein the composition for external application for skin increases the expression of HAS3 or filaggrin. Microbacterium oleivorans belonging to the genus Microbacterium deposited with accession number KCCM12703P, a lysate thereof, a culture medium, an extract of the culture medium, or a mixture thereof. A health functional food composition for wrinkles, skin elasticity or moisturizing.

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