WO2023239024A1 - Novel iamiaceae family microorganism and uses thereof - Google Patents

Abstract

The present invention relates to a microorganism belonging to the Iamiaceae family, which has phylogenetically and chemotaxonomically different characteristics compared to strains from the same or different genus. In addition, the strain or a culture solution thereof may be effectively used for preventing, improving, or treating skin-related conditions.

Description

New Aiaemiaceae microorganisms and their uses

It is about microorganisms of the Aemiaceae family.

The skin is a point of contact with the external environment and is also a collection site for various microorganisms (fungi, bacteria, viruses, and small larvae). Depending on the selection of physical and chemical functions, microorganisms adapt to specialized niches and establish habitats.

As a general characteristic of skin, the surface of the skin is cold, shows slightly acidic properties, and is maintained in a dry state. Structurally, the epidermis forms the skin barrier and plays an important role in blocking the penetration of microorganisms and toxins and maintaining moisture. The uppermost layer of the epidermis is composed of the stratum corneum. The epidermis has a form called a 'brick and mortar structure'. The skin tissue goes through a continuous self-recovery process, and the scales that have completed the differentiation process constantly repeat the process of being shed from the skin tissue. It has been revealed that microorganisms influence these skin changes.

Probiotics are a general term for microorganisms that have beneficial effects on the human body. They refer to microorganisms that benefit our bodies. Most probiotics known to date are known as lactic acid bacteria. Probiotics have been reported to produce effective effects through various beneficial effects on the human body, but research on the relationship between skin flora and skin is insufficient.

The skin barrier is composed of dead keratinocytes and intercellular lipids, and plays a key role in skin health as a skin protective film that protects the skin from external stimuli and prevents moisture from evaporating from the skin. In other words, it prevents excessive release of moisture from the body and prevents harmful substances such as chemicals or microorganisms from entering our body. The keratinocyte envelope, which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. Keratinocytes differentiate and create a skin barrier through the keratinization process. The skin barrier function can be destroyed by aging or external factors, and damage to the skin barrier can reduce skin moisture and cause wrinkles.

Accordingly, the present inventors observed changes in the skin due to changes in skin flora and further studied whether changes in skin flora could lead to potential improvement in the skin environment. Iamiaceae strains were isolated and identified from and confirmed to be useful for skin-related conditions, and the present invention was completed.

One aspect is to provide a new Dermatobacter hominis strain belonging to the Iamiaceae family.

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

Another aspect provides a composition comprising a Dermatobacter hominis strain, its lysate, a culture, an extract of the culture, or a mixture thereof.

One aspect provides a novel Dermatobacter hominis strain belonging to the Iamiaceae family.

Strains belonging to the Iamiaceae family may have one or more of the following mycological properties;

(1) Rod-Shaped;

(2) Non-motile;

(3) Gram positive; and

(4) Non-spore production (non-motile).

Strains belonging to the Iamiaceae family may have one or more of the following mycological properties;

(1) Rod-Shaped;

(2) Non-motile;

(3) Gram positive;

(4) non-spore producing (non-motile);

(5) DNA G+C content is 72.2 mol%;

(6) MK-9(H8) (tetrahydrogenated menaquinone with nine units) is the isoprenoid quinone detected;

(7) The main polar lipids are diphosphatidylglycerol (DPG), three unidentified phosphoglycolipid (PGL), unidentified aminophosphoglycolipid (APGL), and unidentified aminophospholipid ( at least one selected from the group consisting of unidentified aminophospholipid (APL); and

(8) Catalase positive.

The phylogenetic characteristics of the strain belonging to the Iamiaceae family show a clear difference from other genera to which other strains belong, and the chemical taxonomic characteristics also show a clear difference, resulting in the new Dermatobacter . I sympathized with him in my heart.

The Dermatobacter strain may include Dermatobacter hominis .

The Dermatobacter hominis may have one or more of the following mycological properties;

(1) The length of the cell is 0.8~1.4 ㎛ and the diameter is 0.4~0.7 ㎛;

(2) Colony shape is oval and white;

(3) Grows on R2A (Reasoner's 2A) agar, TSA (Tryptic Soy Agar), NA (Nutrient Agar), and LBA (Lysogeny Broth Agar);

(4) Grows at 18°C to 37°C in R2A medium (optimum 25°C), but does not grow at temperatures beyond this;

(5) Grows at pH 6.0 to pH 8.0 and at a salinity (NaCl) of 0 to 1% (optimum pH: 7.0%, optimal NaCl concentration: 0%);

(6) Nitrate is not reduced to nitrite;

(7) Casein, starch, DNA, cellulose, and Tween 80 do not decompose;

(8) has the properties listed in Tables 1 to 3;

(9) N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, α-D-glucose, D-mannose, D-fructose, D-galactose ), dextrin, D-fucose, inosine, lactamide, maltotriose, D-psicose, D-ribose, α- Ketovaleric Acid, D-fructose-6-PO4, L-serine, pectin, pyruvic acid, adenosine, thymidine, 2'-deoxy adenosine, adenosine-5'-monophosphate, p-hydroxy-phenylacetic acid, methyl pyruvate (methyl published patent 10-2018-0001459 - 5 - pyruvate), D -Lactic acid methyl ester, L-lactic acid, citric acid, α-keto-glutaric acid, D-malic acid, L-malic acid, bromo-succinic acid, Any selected from the group consisting of Tween 40, Tween 80, α-hydroxy-butyric acid, β-hydroxy-D,L-butyric acid, α-keto-butyric acid, propionic acid and acetic acid activity. one or more assimilations positive;

(10) D-maltose, D-cellobiose, gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, α- D-lactose, D-melibiose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-sorbitol, D-mannitol, D-arabitol, myo-inositol, glycerol, D-glucose-6-PO4, D-aspartic acid ( aspartic acid, D-serine, gelatin, glycyl-L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid ), L-histidine, L-pyroglutamic acid, D-galacturonic acid, L-galactonic acid lactone, D-gluconic acid, D-glu Glucuronic acid, glucuronamide, mucic acid, quinic acid, D-saccharic acid, γ-amino-butyric acid, formic acid (formic acid), a-cyclodextrin, β-cyclodextrin, glycogen, inulin, mannan, N-acetyl-D-glucosamine, N-acetyl-β-D- Mannosamine, amygdalin, L-arabinose, arbutin, L-fucose, D-galactose, D-galacturonic acid, D -Gluconic acid, lactulose, D-melezitose, α-methyl-D-galactoside, β-methyl-D-galactoside, 3-methyl Glucose, α-methyl-D-glucoside, β-methyl-D-glucoside, α-methyl-D-mannoside, palatinose, L-rhamnose, salicin, sedoheptul sedoheptulosan, D-tagatose, D-trehalose, turanose, xylitol, D-xylose, acetic acid, γ- Hydroxybutyric acid, p-hydroxy-phenylacetic acid, D-lactic acid methyl ester, D-malic acid, L-malic acid, pyruvatic acid methyl ester, mono-methyl succinic acid Ester (succinic acid mono-methyl ester), propionic acid, succinamic acid, N-acetyl-L-glutamic acid, L-alaninamine, D-alanine , L-alanyl-glycerin, glycylyl-L-glutamic acid, L-pyroglutamic acid, putrescine, 2,3-butanediol, uridine, thymidine -5'-monophosphate, uridine-5'-monophosphate, D-fructose-6-phosphate, α-D-glucose-1-phosphate, D-glucose-6-phosphate, and D-L-α-glycerol. A voice for one or more assimilates selected from the group consisting of phosphate;

(11) Esterase (C4), esterase lipase (C8), leucine arylamidase, crystalline arylamidase, acid phosphatase, and naphthol-AS -BI-phosphohydrolase activity. Any one or more activities selected from the group consisting of;

(12) Lipase (C14), trynsin, α-galactosidase, β-glucuronidase, β-glucosidase, α-glucosidase, N-acetyl-β- At least one activity selected from the group consisting of glucosaminidase, α-mannosidase, and α-fucosidase activities is absent (absent);

(13) The main fatty acids are C _16:0 , C _18:0, C _18:1 ω9c or C _20:1 ω9c; and

(14) Catalase positive.

The Dermatobacter hominis may include 16s rRNA containing the base sequence represented by SEQ ID NO: 1. Additionally, the Dermatobacter hominis may have been deposited under the deposit number KACC 22415.

Another aspect provides lysate, culture fluid, or extract of the culture fluid of the strain.

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 refers to a medium that can supply nutrients to enable the strain to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time. Additionally, the culture medium may refer to a culture medium obtained by removing the bacterial cells from the bacterial culture medium obtained by culturing the strain. On the other hand, the liquid from which the bacteria have been removed from the culture medium is also called "supernatant", and the culture medium is left alone for a certain period of time and only the liquid in the upper layer excluding the part that has settled in the lower layer is taken, the bacteria are removed through filtration, or the culture medium is centrifuged and the lower layer is removed. It can be obtained by removing the precipitation and taking only the upper liquid. The "bacteria" refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a skin sample, etc., or a strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the part that has sunk to the lower layer. Alternatively, since they sink to the lower layer of the culture medium by gravity, they can be obtained by leaving them still for a certain period of time and then removing the upper liquid.

The culture medium is the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating a strain belonging to the Iamiaceae family, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product. may include.

The culture medium may be obtained by culturing the strain in a medium (e.g., 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 medium to remove the strain.

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

The culture medium and culture conditions for cultivating the KERA-3 strain belonging to the Iamiaceae family can be appropriately selected or modified by those skilled in the art.

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

As used herein, the term "culture extract" refers to an extract from the culture medium or its concentrate, and may include extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, crude or purified products thereof, and fractions thereof. You can.

Another aspect provides the use of the strain, a lysate of the strain, a culture medium, or an extract of the culture medium. In detail, a composition comprising the above strain, its lysate, culture medium, or extract of the culture medium is provided.

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

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

As used herein, the term "skin aging" refers to both tangible and intangible changes that occur in the skin as one ages, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound recovery, 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 used to improve skin aging caused by exogenous factors or endogenous factors. The exogenous factors refer to various external factors, such as ultraviolet rays (light), and the endogenous factors are also referred to as chronological factors and mainly refer to factors that occur due to the passage of time. In other words, the skin aging is not only a premature aging phenomenon induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging, and dryness. In addition, wrinkles include stimulation caused by changes in internal and external factors that change the components that make up skin tissue, causing wrinkles.

The aging may be photoaging. The term “photoaging” is a phenomenon caused by external environmental factors, the most representative factor being ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cutting of matrix proteins, and abnormal cross-linking, and repetition of this mechanism causes skin aging that is evident in appearance.

As used herein, the term “wrinkle” refers to a state in which the skin loses its elasticity and becomes loose, for example, the skin may be folded. The term “prevention or improvement of skin wrinkles” may refer to any action that prevents or improves wrinkles by suppressing the expression of factors related to wrinkles, or increases the total amount of collagen.

The “skin barrier strengthening” may refer to any action that improves the function of the skin barrier, which is located on the outermost layer of the skin and prevents moisture and nutrition loss.

The term “skin moisturizing” may refer to any action that maintains skin moisture or prevents moisture loss.

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

The damage to the skin barrier function may mean any change that appears in the skin due to decreased or damaged skin barrier function. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, etc.

In another embodiment, the strain may be used together with another strain belonging to Iamiaceae that has a skin-improving effect to exhibit a synergistic effect. Strains belonging to the Aiaemia family include, for example, Desertimonas Genus (e.g., Desertimonas flava ) , Ilumatobacter Genera (e.g., Ilumatobacter coccineus ), Ilumatobacter fluminis ( Ilumatobacter fluminis ), Ilumatobacter nonamiensis ( Ilumatobacter nonamiensis ), etc. may be included.

The composition is 0.001% to 80% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% to 20% by weight, based on the total weight of the composition. %, 0.01% to 10% by weight, 0.01% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 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 It may include % to 10% by weight, or 0.1% to 5% by weight of the strain, its lysate, culture medium, or extract of the culture medium. At this time, if the content of the strain, its lysate, culture, or extract of the culture is less than the above range, the effect of improving skin condition, for example, inhibiting skin aging, whitening skin, strengthening the skin barrier, inhibiting skin wrinkles, or moisturizing the skin There is a problem that the effect is not sufficiently effective.

As used herein, the term "included as an active ingredient" means that the strain of the present specification, its lysate, culture medium, or extract of the culture medium is added to the extent that it can exhibit the effects mentioned above, drug delivery, stabilization, etc. This means that it is formulated into various forms by adding various ingredients as sub-ingredients.

The composition may be a cosmetic composition.

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

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

Additionally, the composition may be a composition for external skin application.

In this specification, the external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and various herbal medicines. , drugs such as tocopherol acetate, glythylitic acid, tranexamic acid and its derivatives or salts, and sugars such as vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, and trehalose. It can be mixed easily.

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

The pharmaceutical composition may additionally 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, disintegrant, binder, lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, 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 syrup, or combinations thereof. Parenteral dosage forms may be injectable.

The composition may be a health functional food composition.

The health functional food composition can be used alone or with other foods or food ingredients, such as the strain or its culture medium, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw materials. There are no particular restrictions on the types of health functional foods. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary 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 can be used. The health food composition also contains nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. It may contain the carbonating agent used, or a combination thereof. The health functional food composition may also contain pulp for the production of natural fruit juice, fruit juice beverage, vegetable beverage, or a combination thereof.

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

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

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

Administration can be done by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration may be administered 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 inhibition, and skin wrinkle improvement effects.

The administration of the composition according to one embodiment 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 mg per day. 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. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The frequency of administration can be once a day or two or more times within the range of clinically acceptable side effects, and can be administered at one or two or more locations, daily or at intervals of 2 to 5 days. The number of days of administration can be from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg is the same as for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and human. One dose can be administered.

According to one aspect, the new Aemiaceae family strain has phylogenetically and chemically distinct characteristics compared to strains of other or other genera of the same family. Additionally, the strain or its culture medium can be usefully used to prevent, improve, or treat skin-related conditions.

Figure 1 is a diagram showing the systematic classification results of KERA-3.

Figure 2 shows the results of confirming the morphological characteristics of KERA-3.

Figure 3 shows the results showing the effect of KERA-3 strain on the expression of filaggrin in human keratinocytes.

Figure 4 shows the results showing the effect of KERA-3 strain on the expression of HAS-3 in human keratinocytes.

Figure 5 shows the results showing the effect of KERA-3 strain on the expression of ABCA12 in human keratinocytes.

Figure 6 shows the results showing the effect of KERA-3 strain on the expression of ELOVL4 in human keratinocytes.

Figure 7 shows the results showing the effect of KER-3 strain on the expression of CERS3 in human keratinocytes.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Isolation of strains

A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy adult with sterile distilled water was inoculated into R2A (Reasoner's 2A) medium (Becton Dickinson, Cockeysville, MD) diluted 1/10. After inoculation, the colonies were cultured in an incubator at 30°C for 240 hours, and then 100 colonies formed were pure, isolated and cultured, and re-cultured in an incubator at 30°C for 240 hours. The 16s rRNA gene sequence was identified for the colonies whose culture was completed. The primers used at this time were designed to react and amplify only bacteria (SEQ ID NOs. 2 and 3). PCR amplification was performed in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, and 75°C for 1 minute and 30 seconds, and was finally treated at 72°C for 8 minutes and stored at 4°C. After the PCR reaction was completed, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA). The base sequence of the 16s rRNA region determined among the isolated and cultured microbial colonies was compared and analyzed with other strains registered with the BLAST program provided on the US National Center for Biotechnology Information (NCBI) website. Only novel species with 97% or less homology were selected and used, and among them, novel microorganisms with 94% or less homology (hereinafter referred to as “KERA-3”) were selected. KERA-3 has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Example 2. Confirmation of taxonomic and mycological properties of new microorganisms

2-1. Taxonomic properties

Homology analysis for the 16s rRNA gene was calculated using the Ezbiocloud server. Sequence data and phylogenetic analysis were performed using MEGA-X. Phylogenetic trees were drawn using neighbor-joining analysis (Sitou and Nei (1987)), maximum-likelihood and maixmum-parsimony algorithms, and the stability of the phylogenetic trees was assessed using bootstrap analysis (1000 replications) (Felsenstein 1985). evaluated.

As a result, KERA-3 was Actinomarinicola tropica SCSIO 58843, Aquihabitans daechungensis CH22-21, Iamia majanohamensis F12, Illumatobacter It was confirmed that it had 90.94%, 90.39%, 89.56%, and 89.20% homology with Ilumatobacter fluminis YM22-133, respectively.

Figure 1 is a diagram showing the systematic classification results of KERA-3.

As shown in Figure 1, KERA-3 was confirmed to be a new genus belonging to the Iamiaceae family that has never been reported so far.

2-2. mycological properties

2-2-1. Morphological properties

The morphological characteristics of KERA-3 were analyzed as follows.

First, the cell morphology of KERA-3 was cultured in R2A at 25°C for 5 days and then observed at 1000x magnification using a microscope (Olympus microscope, GX71).

The motility of the strain was evaluated by inoculating fresh cultured KERA-3 cells into 0.3% agar slant medium.

To observe SEM (Scanning Electron Microscopy), the sample was pretreated as follows. KERA-3 was inoculated on R2A (Becton Dickinson, Cockeysville, MD) solid medium, and a membrane filter was attached to the colonies formed after culturing for 5 days. The membrane filter to which the colony was attached was separated and fixed by treatment in 2.5% glutaraldehyde solution for 2 hours, and then washed by soaking in PBS for 5 minutes each. Afterwards, it was treated with 2% osmium tetroxide solution for 1 hour and dehydrated using 40, 50, 60, 70, 80, 90, and 100% ethanol. To remove ethanol remaining in the sample after completion of dehydration treatment, isoamylacetate was used to remove it. After completing pretreatment, the sample was dried naturally and coated with gold using a sputter coater (SC502, Polaron). After this, it was photographed with a SEM (Hitachi S4300N, Hitachi, Japan) at 10000x magnification, and the results are shown in Figure 2.

To determine Gram positive and negative, an experiment was conducted in the following manner. Specifically, one KERA-3 colony cultured well using R2A solid medium was collected and fixed to a glass slide. After completely drying the slide glass, it was stained with crystal violet for 1 to 2 minutes, washed in running water, and dried again. The dried slide was again stained with iodine-potassium iodide for 1 minute. Afterwards, the slide was decolorized using ethyl alcohol, washed with running water, and dried. To determine Gram positive or negative, secondary staining was performed with a red dye such as safranin for 1 to 3 minutes, washed in water, and observed under a microscope (Olympus microscope, GX71).

As a result, it was confirmed that KERA-3 had the following morphological characteristics.

(1) rod-shaped;

(2) non-motile;

(3) no flagella;

(4) Gram positive;

(5) non-spore forming;

(6) The length of the cell is 0.8~1.4 ㎛ and the diameter is 0.4~0.7 ㎛.

(7) The colony shape is oval and white.

(8) Catalase positive.

2-2-2. Culture and physiological characteristics

To confirm the culture conditions of KERA-3, the optimal growth temperature was set from 4℃ to 50℃ to confirm the optimal culture temperature, and the optimal salt concentration was determined by adding 0% to 10% (w/v) of NaCl to the medium at 25℃. It was confirmed by culturing for 10 days. pH was checked using the following buffer system; Sodium acetate/acetic acid (pH<6), Tris/HCl (pH 6 to 9), and glycine/sodium hydroxide (pH>9).

In addition, to confirm the growth medium, R2A (Reasoner's 2A) agar, TSA (Tryptic Soy Agar), NA (Nutrient Agar), and LBA (Lysogeny Broth Agar) were used and cultured at 25°C for 10 days.

Additionally, to confirm the material utilization ability of KERA-3, BioLog GP2, BioLog Gen III, API 20NE, and API ZYM (biomeriuex, France) were used. The method of use was carried out according to the manual provided by the manufacturer. In the case of determination, when a positive reaction occurred, a purple color occurred, and after visual inspection, the degree of growth was accurately determined using a microplate spectrophotometer. The API ZYM test was read after 4 hours of incubation at 25°C, and the other API tests were read after at least 48 hours of incubation at 25°C, and the results are shown in Table 1 below.

API-ZYM test control - Alkaline phosphatase - Esterase (C4) + Esterase Lipase (C8) + Lipase (C14) - Leucine arylamidase + Valine arylamidase - Cystine arylamidase - Trypsin - a-chymotrypsin - Acid phosphatase - Naphtol-AS-BI-phosphohydrolase + α-galactosidase - β-galactosidase - β-glucuronidase - α-glucosidase - β-glucosidase - N -acetyl -β-glucosaminidase - a - mannosidase - a-fucosidase - Use of carbon source Glucose, + Sucrose, + Pyruvate, + Soluble starch, + Xylose, - galactose, - Mannose, + Mannitol, - Lactose, + Fructose +

+: positive, -: negative

In addition, to confirm the nutrient source, minimal salt medium (proteose peotone 0.05%, Casamino acids 0.05%, Dipotassium phosphate, Magnesium sulfate) and carbon source (Glucose, Sucrose, Pyruvate, Soluble starch, Xylose, Galactose, Mannose, Mannitol, Lactose, Fructose) were used. ) was separately added to 0.02% (w/w) to confirm the carbon usability.

Aerobic growth was confirmed by culturing under aerobic culture conditions and anaerobic culture conditions (using an anaerobic pack).

Tests for the degradability of DNA, casein, starch, Tween 80, and carboxymethylcellulose were evaluated after incubation at 25°C for 7 days.

As a result, it was confirmed that EPI-7 had the following culture and physiological characteristics.

(1) Aerobic:

(2) Heterotrophy:

(3) Characteristics in Table 1;

(4) Grows on R2A (Reasoner's 2A) agar, TSA (Tryptic Soy Agar), NA (Nutrient Agar), and LBA (Lysogeny Broth Agar);

(5) The optimal growth medium is R2A medium, which grows at 18°C to 37°C (optimum 25°C), but does not grow at temperatures outside this;

(6) Grow at pH 6.0 to pH 8.0 and at a salinity (NaCl) of 0-1% (optimal pH: 7.0%, optimal NaCl concentration: 0%);

(7) Nitrate is not reduced to nitrite; and

(8) Casein, starch, DNA, carboxymethylcellulose, and Tween 80 do not decompose.

2-2-3. biochemical properties

The DNA G+C content of KERA-3 was confirmed by whole genome sequencing using Illumina Miseq equipment. In addition, the Cellular Fatty Acid composition, G+C composition, and Quinone analysis test methods and conditions are as follows.

First, to confirm the DNA G+C content of the isolated strain, a large amount of KERA-3 cells were obtained, and then DNA was extracted using the QIAamp PowerFecal Pro DNA Kit according to the user manual. The extracted DNA was used to create a DNA library suitable for Miseq, a genome analysis equipment, and full-length genome analysis was performed using this. The data that completed the analysis were assembled into a single genome using HGAP 3 version 2.3, and Best-placed reference protein set in the NCBI prokaryotic genome annotation pipeline (PGAP) pipeline; A full-length genome was created using the GeneMarkS-2+ method.

The cellular fatty acid composition of the isolated strains was determined according to Miller's method. After transferring about 40 mg of cultured cells to a tube, 1 ml of a solution containing 15% NaOH in 50% methanol was added, heated at 100°C for 30 minutes, and cooled to room temperature. 2 ml of methanolic-HCl (a mixed solution of 325 ml of 6.0N HCl and 275 ml of methanol) was added thereto, heated at 80°C for 10 minutes, and then rapidly cooled. Afterwards, 1.25 ml of hexane/methyl-tert-butylether (1:1, v/v) was added and mixed well for 10 minutes. After standing at room temperature, when the reaction solution was separated into two layers, only the lower liquid was removed, 3 mL of dilute NaOH (10.8 g NaOH/900 mL D.W) was added, mixed well for 10 minutes, and left at room temperature. 2% of the supernatant was added. About 3 was transferred to a screw-capped sample vial (12 Samples were saponified, methylated and extracted according to standard protocols in Sherlock MIS Software. Fatty acids were analyzed using Agilent technologies 6890 Gas chromatography and A30m This was confirmed using (MIDI, Version 4.5). All experiments were performed at least three times, and the results are shown in Table 2 below.

Fatty Acids(%) Saturated fatty acids C _10:0 N.D. C _12:0 0.47 C _14:0 1.17 C _16:0 46.82 C _17:0 3.75 C _18:0 6.81 C _19:0 1.40 C _20:0 3.21 Unsaturated fatty acids C _15:1 ω6c N.D. C _16:1 ω5c 2.72 C _17:1 ω6c N.D. C _17:1 ω8c N.D. C _18:1 ω5c N.D. C _18:1 ω9c 12.62 C _20:1 ω9c 6.24 Branched-chain fatty acid C _16:0 iso 4.22 C _16:1 iso H N.D. C _18:0 iso 1.17 C _18:1 iso H N.D. C _17:1 anteiso ω9c 0.55 C _17:0 10-methyl N.D. C _18:1 ω7c 11-methyl N.D. Hydroxy fatty acids C _16:0 3OH 1.43 Cyclo C _19:0 cyclo ω8c N.D. Other C _12:1 at 11-12 0.55 C _13:1 at 12-13 3.96 Summed features 2;C _14:0 3OH/C _16:1 iso I 2.13 3; C _16:1 ω7c/C _16:1 ω6c 0.76 8; C _18:1 ω7c/C _18:1 ω6c Total 99.98

Quinone was analyzed as follows. After the strain was cultured in R2A medium at 25°C for 96 hours, only the bacterial cells were collected and freeze-dried. The freeze-dried sample was extracted under the following conditions. Chloroform:methanol (2:1) solution was added and shaken for 3 to 4 hours. Cells were removed by filtering using filter paper (Whatman No. 2), and the filtered solution was concentrated. Afterwards, it was dissolved in 100㎕ of chloroform:methanol (8.5:1.5), centrifuged at 14000rpm for 5 minutes, and the supernatant was collected and subjected to HPLC analysis. HPLC conditions were as follows;

HPLC: YOUNG LIN YL9100 (YL9111 Binary pump)

Detector: YOUNG LIN YL9120 UV/Vis Detector

Chromatograph data system: YOUNG LIN Autochro-3000

Analysis column: Waters Spherisorb 5um ODS2 4.6mmx150mm column (column temp. 40℃)

Analysis solvent: Methanol: Isopropyl ether (4:1)

Flow rate: 1.0 mL/min

Detection wavelength: 254 mm.

Polar lipid analysis was performed after culturing the strain in R2A medium at 25°C for 96 hours. First, polar lipids from the strains were purified using Minnikin D.E. Extraction was performed according to the method described in et al (1984) (J Microbiol Methods 2, 233-241). Afterwards, polar lipids were identified using two-dimensional TLC (Komakata K et al., 1987, Methods Microbiol 19, 161-206).

As a result, it was confirmed that KERA-3 has the following biochemical characteristics;

(1) DNA G+C content is 72.2 mol%;

(2) MK-9(H8) (tetrahydrogenated menaquinone with nine units) is the isoprenoid quinone detected;

(3) The main polar lipids are diphosphatidylglycerol (DPG), three unidentified phosphoglycolipids (PGL), unidentified aminophosphoglycolipid (APGL), and unidentified aminophospholipids ( at least one selected from the group consisting of unidentified aminophospholipid (APL); and

(4) Fatty acid composition in Table 2.

In addition, the characteristics were finally compared and summarized with other species and are shown in Table 3 below.

Characteristic One 2 3 4 5 6 7 Cell morphology Rod Rod Cocobacilli Short rod Rod Rod Short rod Cell length (μm) 0.8~1.4 1.3~1.6 1.1 0.7~1.0 1.2-1.7 1.0-3.0 1.0-2.0 Cell with (μm) 0.4~0.7 0.4~0.5 0.7 0.3~0.5 0.3~0.5 0.5 0.3~0.6 Growth Condition Temperature (°C) 18~37
26~31 15~37
10~37
10~45
35~43 10~45 Optimum 25 28 30 30 35 35 NaCl tolerance (w/v) 0~1.0 0~5.0 0.5 0~1.0 0~12.5 N.D. 0~5.0 pH range 6.0~8.0 7.0~11.0 6.0~7.0 6.0~8.0 6.0~9.0 1.4~2.0 6.0~9.0 Carbon utilization Glucose + + N.D. N.D. - N.D. Maltose N.D. N.D. N.D. + - - - Ribose + - N.D. + - - N.D. Enzyme activity Nitrate reduction - N.D. + - + N.D. + DNA G+C contents (%) 73.2 68.0 70.2 71.8 74.4 54.9 72.5 Chemotaxonomic characteristics Major menaquinones MK-9 (H ₈ ) MK-9 (H ₈ ) MK-9 (H ₆ )
MK-9 (H ₈ )
MK-9 (H ₄ ) MK-9 (H ₆ ) MK-9 (H ₆ ) MK-9 (H ₁₀ ) MK-9 (H ₈ ) Diaminopimelic acid (DAP) meso -DAP LL -DAP LL -DAP meso -DAP meso -DAP meso -DAP DD -DAP Main polar lipids DPG, APL APGL, PGL N.D. DPG DPG, PI, PIM N.D. N.D. DPG, PI, PC, PIM Major fatty acids C16:0, C18:1w9c ISO-C16:0(17.9),
Iso-C17:1ω 9c(14.7);
ISO-C17:0(11.3) C16: 0, ,
iso-C16:0 16:1ω5c, 16:0,
17:1ω8c,18:1ω9c C17:0, C17:1w8c iso-C16:0,
iso-C14:0,
iso-C15:0,
ai-C15: 0,C17: 1 ω 6c. 17:1ω8c, 17:0

1: KERA-3

2: Ilumatobacter fluminis (YM22-133T)

3: Desertimonas flava (SYSU D60003)

4: Aquihabitans daechungensis (KCTC19849T)

5: Iamia majanohamensis (KCTC19879T)

6: Ferrimicrobium acidiphilum (T23T)

7: Actinomarinicola tropica (KCTC49408T)

* -: Negative, +: Positive, ND: Not Determined

* DPG: diphosphatidylglycerol, PI: phosphatidylinositol, PC: phospatidylcholine, PIM: phosphatidylinositolmannoside, unidentified phospholipids, PGL: unidentified unidentified phosphoglycolipid, APGL: unidentified aminophosphoglycolipid, APL: unidentified aminophospholipid

As shown in Table 3, it was confirmed that KERA-3 has the same characteristics as those in Table 3 above, and its main biochemical characteristics are different from the relative strain, and the combination of these chemical classification results is the result of the phylogenetic classification of the strain. Together, it proves that KERA-3 is a new species.

As a result of the above results, it was confirmed that KERA-3 isolated from human epidermal keratinocyte is a new genus of microorganism belonging to the Iamiaceae family, and it was named Dermatobacter according to the systematic nomenclature. The genus was named Hominis . In addition, the Dermatobacter Hominis strain belonging to the Iemia family was deposited in the National Institute of Agricultural Science's Microbial Bank (Korean Agricultural Culture Collection, KACC) on February 15, 2022, with deposit number KACC 22415. was granted.

Example 3. Activity of strains

In order to analyze the skin barrier strengthening and moisturizing activity of the KERA-3 strain identified in Example 2, filaggrin, a factor related to skin barrier strengthening and moisturizing activity following treatment of KERA-3 in human keratinocyte cell line (HaCaT) ), HAS3 (hyaluronic acid synthase 3), ABCA12 (ATP-binding cassette sub-family A member 12), ELOVL4 (Elongation of very long chain fatty acids protein 4), and CERS3 (Ceramide synthase 3) expression levels were confirmed by RT-PCR. did.

Specifically, HaCaT cells, a human keratinocyte cell line, and human-derived Epidermal Keratinocytes were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum. All cultures were performed in a 37°C CO ₂ incubator. As a positive control, the cultured cell lines were treated with retinol at a dose of 1mM. Additionally, KERA-3 cultures were treated at concentrations of 1.0% and 10.0%. Afterwards, HaCaT cells were cultured for an additional 24 hours, then recovered, and RNA was isolated by adding 1 ml of Trizol (RNA iso, DAKARA, Japan). Additionally, for RT-PCR, RNA was quantified using Nanodrop 2000 (Thermo, USA), and cDNA was synthesized by reacting at 42°C for 55 minutes and 70°C for 15 minutes (Reverse Transcriptase Mix, ELPIS biotech, Korea) ). RT-PCR was performed using Step One Plus (Applied Biosystems, USA), and Cybergreen (SYBR Green supermix, Applied Biosystems, USA) was added along with primers and cDNA to activate the polymerase at 94°C for 5 minutes. Polymerization was performed in 40 cycles of 95°C for 30 seconds, 54°C for 1 minute, and 72°C for 1 minute.

Figure 3 shows the results showing the effect of KERA-3 strain on the expression of filaggrin in human keratinocytes.

Figure 4 shows the results showing the effect of KERA-3 strain on the expression of HAS-3 in human keratinocytes.

Figure 5 shows the results showing the effect of KERA-3 strain on the expression of ABCA12 in human keratinocytes.

Figure 6 shows the results showing the effect of KERA-3 strain on the expression of ELOVL4 in human keratinocytes.

Figure 7 shows the results showing the effect of KER-3 strain on the expression of CERS3 in human keratinocytes.

As a result, as shown in Figures 3 to 7, KERA-3 culture medium (Example 1) compared to the untreated control and KERA-3 culture medium (Comparative Example 1) of filaggrin, HAS3, ABCA12, ELOVL4 and CERS3 It was confirmed that expression was significantly high.

Therefore, the novel strain belonging to the Aemia family according to one embodiment can be used to improve skin conditions such as inhibiting or improving skin aging, anti-wrinkle, strengthening the skin barrier, or moisturizing the skin.

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

<Accession number>

Name of depository institution: Korea Agricultural and Biotechnology Research Institute

Accession number: KACC22415

Trust date: 20220215

Claims (12)

A new Dermatobacter Hominis strain belonging to the Iamiaceae family and having the following mycological properties; (1) Rod-Shaped; (2) Non-motile; (3) Gram positive; and (4) Non-spore production (non-motile). The method according to claim 1, wherein the strain further has any one of the following mycological properties; (1) DNA G+C content is 72.2 mol%; (2) MK-9(H8) (tetrahydrogenated menaquinone with nine units) is the isoprenoid quinone detected; and (3) The main polar lipids are diphosphatidylglycerol (DPG), three unidentified phosphoglycolipids (PGL), unidentified aminophosphoglycolipid (APGL), and unidentified aminophospholipids ( It is one or more selected from the group consisting of unidentified aminophospholipid (APL). The method according to claim 1, wherein the strain is a strain having any one or more of the following mycological properties; (1) The length of the cell is 0.8~1.4 ㎛ and the diameter is 0.4~0.7 ㎛; (2) Colony shape is oval and white; (3) Grows on R2A (Reasoner's 2A) agar, TSA (Tryptic Soy Agar), NA (Nutrient Agar), and LBA (Lysogeny Broth Agar); (4) Grows at 18°C to 37°C in R2A medium (optimum 25°C), but does not grow at temperatures beyond this; (5) Grows at pH 6.0 to pH 8.0 and at a salinity (NaCl) of 0 to 1% (optimum pH: 7.0%, optimal NaCl concentration: 0%); (6) Nitrate is not reduced to nitrite; (7) Catalase positive; (8) Casein, starch, DNA, cellulose, and Tween 80 do not decompose; and (9) It has the characteristics shown in Tables 1 to 3. The method according to claim 1, wherein the strain is Dermatobacter genus Hominis ( Hominis ). The method according to claim 4, wherein the Dermatobacter hominis ( Dermatobacter Hominis ) strain has a 16s RNA sequence including the base sequence represented by SEQ ID NO: 1. The method according to claim 4, wherein the Dermatobacter hominis ( Dermatobacter Hominis ) is a strain deposited under the deposit number KACC22415. A lysate, culture medium, or extract of the culture medium of the strain of claim 1. The method according to claim 7, wherein the culture medium is the culture medium itself obtained by culturing the strain, or a concentrate or freeze-dried product thereof, or a culture supernatant obtained by removing the strain from the culture medium, an extract thereof, or a freeze-dried product. A cosmetic composition comprising a Dermatobacter Hominis strain belonging to the Iamiaceae family, its lysate, culture medium, extract of the culture medium, or a mixture thereof. The cosmetic composition according to claim 9, which is used for strengthening the skin barrier, preventing or improving skin aging, moisturizing the skin, or improving skin elasticity. Dermatobacter Hominis ( Dermatobacter Hominis ) strain belonging to the Iamiaceae family, its lysate, culture, extract of the culture, or a mixture thereof for external application to the skin for the prevention, improvement or treatment of skin conditions Composition. A health functional food composition comprising a Dermatobacter Hominis strain belonging to the Iamiaceae family, its lysate, culture medium, extract of the culture medium, or a mixture thereof.

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