KR102334456B1 - Pantoea ananatis 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 a use thereof for improving skin conditions. The present invention can be usefully used to moisturize the skin and strengthen a barrier.

Description

Pantoea ananatis strain and skin condition improving use thereof {Pantoea ananatis 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 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 squames that have undergone the final differentiation process repeat the process of constantly being removed from the skin tissue.

CN 001958785 A (2007.05.09)

One aspect is to provide a strain Pantoea ananatis ( Pantoea ananatis ) belonging to the genus Pantoea ( ( Pantoea 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 Pantoea ananatis I25 strain.

The Pantoea ananatis ( Pantoea ananatis ) I25 strain may be a strain deposited with accession number KCCM12702P.

The Pantoea ananatis ( Pantoea ananatis ) I25 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 skin moisturizing and skin barrier-related hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3), filagrin, or loricrin. The increase in markers such as HAS3, filaggrin, or loricrin 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.

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 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 lower 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 solution may be obtained by culturing the Dokdo strain in a medium (eg, R2A medium or TSA medium) at any temperature above 10°C or below 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 Pantoea ananatis ( Pantoea ananatis ) Culture medium and culture conditions for culturing the I25 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 a fraction 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 with a strain belonging to the genus Pantoea, another strain having a skin improvement effect, to show a synergistic effect. The strain belonging to the genus Pantoea may include, for example, P. agglomerans, P. dispersa, P. septica, P. eucalypit , 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 exhibited.

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 extract, various herbal medicines, drugs such as tocopherol acetate, glitylic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, 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 I25 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.
3 is a graph showing the effect of a strain according to an embodiment on the expression of loricrin.

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

Soil and seawater samples collected from Seodo, Dokdo were inoculated into liquid 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 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 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, and 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 from the isolated and cultured microbial colonies was compared with other strains registered with the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI), homology was less than 97%. Only novel species of were selected and used, and among them, a novel microorganism Pantoea ananatis with homology of 97% or less ( Pantoea ananatis ) strain (hereinafter referred to as "I25") was selected. The selected I25 strain was deposited with the Korea Microbial Conservation Center on April 27, 2020 and was given accession number KCCM12702P, and the I-25 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 I25 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'smodified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum, all of which were cultured at 37°C 5°C. % CO _{2 in an} incubator. The cultured cell line was treated with the culture solution of the I25 strain (1%, 10% (w/w)) 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, along with primers and cDNA for filaggrin and loricrin were added together with a real-time PCR machine (Step One Plus, Applied Biosystems, USA) performed real-time polymerase chain reaction. 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.

3 is a graph showing the effect of a strain according to an embodiment on the expression of loricrin.

As a result, as shown in FIGS. 1 to 3 , it was confirmed that the strain according to one embodiment significantly increased the expression levels of HAS3, filaggrin and loricrin.

Therefore, the I25 strain according to one embodiment can be usefully used for strengthening the skin barrier and moisturizing the skin.

Korea Microorganism Conservation Center (Overseas) KCCM12702P 20200427

<110> Cosmax, INC. <120> Pantoea ananatis strain and skin condition improving uses of it <130> PN132097 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1438 <212> RNA <213> Artificial Sequence <220> <223> Pantoea ananatis I25 <400> 1 gagtggcgga cgggtgagta atgtctgggg atctgcccga tagaggggga aataaccact 60 ggaaacggtg gctaataccg cataacgtcg caagaccaaa gactggggga ccttcgggcc 120 tctcactatc ggatgaaccc agatgggatt agcttcagta ggcggggtaa cggcccacct 180 aggcgacgat ccctagctgg tctgagaaag gatgaccagc cacactggaa ctgagacacg 240 gtccagactc ctacgggacc ggcagcagtg gggaatattg cacaatgggc gcaagcctga 300 tgcagccatg gcccgcgtgt atgaagaagg ccttcgggtt gtaaagtact ttcagcgggg 360 aggcgaaggc gatgtggtta ataaccgcgt cgattgacgt tacccgcaga agaatcgcac 420 cggctaactc cgtgccagca gccgcggtaa tacggagggt gcaagccagt taatcggaat 480 tactgggcgt aaagcgcacg caggcggtct gttaagtccc agatgtgaaa tccccgggct 540 taacctggga actgcatttg aaactggcag gtgcttgagt ctcgtagagg ggggtagaat 600 tccaggtgta gcggtgaaat gccggtagag atctggagga ataccggtgg cgaaggcggc 660 cccctggacg aagatcctga cgctcaggtg cgaaagcgtg gggagcaaac aggattagat 720 accctggcgt agtccacgcc gtaaacgatg tcgacttgga ggttgttcct tgaggagtgg 780 cttccggagc taacgcgtta agtcgaccgc ctggggagta cggccgcaag gttaaaactc 840 aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gatgcaacgc 900 gaagaacctt acctactctt gacatccaga gaacttagca gagatgcttt ggtgccttcg 960 ggaactctga gacaggtgct gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta 1020 agtcccgcaa cgagcgcaac ccttatcctt tgttgccagc gattcggtcg ggaactcaaa 1080 ggagactgcc ggtgataaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1140 acgagtaggg ctacacacgt gctacaatgg cgcatacaaa gagaagcgac ctcgcgagag 1200 caagcggacc tcataaagtg cgtcgtagtc cggatcggag tctgcaactc gactccgtga 1260 agtcggaatc gctagtaatc gtggatcaga atgccacggt gaatacgttc ccgggccttg 1320 tacacaccgc ccgtcacacc atgggagtgg gttgcaaaag aagtaggtag cttaaccttc 1380 gggagggcgc ttaccacttt gtgattcatg actggggtga agtcgtacaa agggtaac 1438

Claims (7)

Deposited with accession number KCCM12702P, Pantoea genus ( Pantoea sp. ) Pantoea ananatis belonging to ( Pantoea ananatis ) I25 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 composition of claim 3, wherein the cosmetic composition is for improving skin condition or for moisturizing, wherein the skin condition is skin barrier strengthening, skin wrinkles, or skin elasticity. delete Accession number: deposited as KCCM12702P, Pantoea ananatis ( Pantoea ananatis ) culturing I25; and
A method for preparing a strain culture solution comprising the steps of centrifuging the obtained cell culture solution after culturing to remove the cells and recovering the supernatant. The method according to claim 6, wherein the culture medium increases the expression of HAS3, filaggrin, or loricrin.

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