KR20230168063A - Neisseria subflava strain and skin condition improving uses of thereof - Google Patents

Abstract

It relates to novel microorganisms, their lysates, culture media, extracts of culture media, and their use for improving skin condition.

Description

Neisseria subflava strain and skin condition improving uses of it}

It relates to novel microorganisms, their lysates, culture media, extracts of culture media, and their use for improving skin condition.

The skin ecosystem provides various types of habitats for microorganisms, and a wide range of microorganisms live there. Humans, the host, have a symbiotic relationship with them and are known to have many positive effects on the host. The skin constitutes various types of habitats, including indentations and specialized crevices, and helps a wide range of microorganisms to grow. Basically, the skin forms a physical membrane and helps defend against potential hazards and toxic substances from the outside. The skin serves as a point of connection 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. Typically, the skin remains cool, acidic, and dry. 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 (squames) that have completed the differentiation process constantly repeat the process of being shed from the skin tissue.

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.

Accordingly, there is a need to develop skin flora that can be useful for skin-related conditions.

One aspect is to provide a Subflava strain belonging to the genus Neisseria sp.

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

Another aspect is to provide a composition comprising the strain, its lysate, culture medium, and extract of the culture medium.

One aspect provides a Subflava strain.

The Subflava strain may be a strain belonging to the Neisseria sp.

The Subflava strain may be a strain deposited under deposit number KCCM13173P.

The Subflava strain may be a strain containing 16s rRNA of SEQ ID NO: 1. In one embodiment, the strain has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity with SEQ ID NO. It may contain 16S rRNA having .

The strain may have skin beauty improvement effects, for example, skin aging inhibition, skin whitening, skin barrier strengthening, skin wrinkle inhibition, skin moisturizing, skin regeneration, skin soothing, or skin wound recovery effect. Additionally, the strain may have the effect of preventing, treating, or improving skin inflammatory diseases.

The strain may increase the expression of filaggrin, which is involved in adhesion between hyaluronic acid and keratin and binding to membrane proteins of keratinocytes, or ABCA12 (ATP binding cassette subfamily A member 12), which is involved in lipid lamellar body transport.

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

The strain is as described above.

As used herein, the term “culture medium” may be used interchangeably with “culture supernatant,” “conditioned culture medium,” or “conditioned medium,” and can be used to provide nutrients for subflava strains 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 in the medium 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 may include the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating the strain, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product.

The culture medium is obtained by culturing the subflava strain in a medium (e.g., R2A medium, TSA medium, or TSB medium) at any temperature above 10°C or below 40°C for a certain period of time, for example, 4 to 50 hours. It may have happened.

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.

Culture media and culture conditions for culturing the subflava strain 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 a subflava 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, preventing, improving or treating skin inflammatory 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. Pigmentation refers to a condition in which the amount of pigment in the body is abnormal or there is an abnormality in the area where the pigment appears, for example, blemishes and freckles. 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 whitening” may mean not only brightening the skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots or freckles caused by ultraviolet rays, hormones, or genetics.

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 term “anti-inflammatory” may be used interchangeably with “inhibiting or improving inflammation,” and may refer to any action that alleviates the immune response and suppresses NO production.

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.

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

In another embodiment, the strain may be used together with another strain belonging to the genus Neisseria, another strain that has a skin-improving effect, to show a synergistic effect. Strains belonging to the Neisseria genus include, for example, Neisseria flava .

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.

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, and is used for various purposes such as drug delivery and stabilization. It means adding ingredients as sub-ingredients and formulating them into various forms.

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 calin. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytylitinic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately mixed.

Additionally, 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.

Additionally, 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.

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

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 per day, 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. 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, 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.

In one aspect, the novel subflava strain has effects that may be useful for preventing, improving, or treating skin-related conditions.

Figure 1 is a result showing the effect of a strain on the expression of filaggrin according to one embodiment.
Figure 2 is a result showing the effect of a strain on the expression of ABCA12 according to one embodiment.

This will be described in more detail through examples below. However, these examples are intended to illustrate 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

Neisseria subflava strains were isolated and identified from the skin of healthy women. Specifically, the cheeks, tip of the nose, and alar border of a healthy woman were swabbed two to three times with a sterile cotton swab soaked in PBS (phosphate buffer saline). A cotton swab was placed in a tube containing PBS and vortexed for 10 seconds. 10-fold, 100-fold, and 1,000-fold dilutions of the sample were prepared, and 100 μL each was added to 0.1 After culturing at ℃, 100 colonies formed were isolated and cultured in pure form, and re-cultured in an incubator at 28℃ for 48 hours. Colonies whose culture was completed were identified by 16s rRNA gene sequence. The primers used at this time were designed to amplify by reacting only with 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). When the nucleotide 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, the homology was 97.78%. A new microorganism Neisseria subflava strain (hereinafter referred to as “CM5”) was selected. The selected CM5 strain was deposited at the Korea Microbial Conservation Center on May 4, 2022 and given the deposit number KCCM13173P, and the CM5 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Example 2. Preparation of strain culture medium

A culture medium of Neisseria subflava CM5 strain was prepared. Specifically, the CM5 strain isolated in Example 1 was cultured in TSB medium, the cells were removed through centrifugation, and the supernatant was recovered and filtered to prepare a Neisseria subflava CM5 strain culture medium.

[Experimental example]

Strengthening skin barrier and confirming skin moisturizing activity

The effect of Neisseria subflava CM5 strain culture on skin barrier strengthening and moisturizing was analyzed.

Specifically, HaCaT cells, a human keratinocyte cell line, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum, and all cultures were performed at 37°C. It was performed in a % CO2 incubator. The cultured cell line was treated with the culture medium (1% (w/w)) of Example 2 and further cultured for 24 hours. As a negative control group, an untreated group of strain culture was used. Afterwards, RNA was isolated from the cells of each sample using Trizol (RNA iso, DAKARA, Japan), and 16 μL of RNA was quantified using nanodrop at 260 nm, and then treated at 70°C for 5 minutes. Afterwards, it was added to the PCR premix, vortexed gently, treated at 42°C for 55 minutes, 70°C for 15 minutes, and stored at 4°C to synthesize cDNA (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, Cyber Green (SYBR Green supermix, Applied Biosystems, USA) was added to the target genes FLG and ABCA12 genes along with primers and cDNA, and then run on a real-time PCR machine (Step One Plus, Applied Biosystems). , USA), real-time polymerase chain reaction (95°C 3 min, {95°C 30 s, 49 to 53°C 30 s, 72°C 1 min} 40 cycles) was performed.

Figure 1 is a result showing the effect of a strain on the expression of filaggrin according to one embodiment.

Figure 2 is a result showing the effect of a strain on the expression of ABCA12 according to one embodiment.

As a result, as shown in Figures 1 and 2, it was confirmed that the Neisseria subflava CM5 strain significantly increased the expression of filaggrin and ABCA12.

Therefore, the strain according to one embodiment can be usefully used to prevent or improve skin aging, including strengthening the skin barrier.

Korea Center for Microbial Conservation (KCCM) KCCM13173P 20220504

SEQUENCE LISTING <110> Cosmax, INC. <120> Neisseria subflava strain and skin condition improving uses of of that <130> PN144363 <160> 3 <170> PatentIn version 3.2 <210> 1 <211> 1389 <212> DNA <213> Artificial <220> <223> Neisseria subflava 16s rRNA <400> 1 ccctacctac ttctggtatc ccccactccc atggtgtgac gggcggtgtg tacaagaccc 60 gggaacgtat tcaccgcagt atgctgacct gcgattacta gcgattccga cttcatgcac 120 tcgagttgca gagtgcaatc cggactacga tcggttttgt gagattggct ccacctcgcg 180 gcttggctac cctctgtacc gaccattgta tgacgtgtga agccctggtc ataagggcca 240 tgaggacttg acgtcatccc caccttcctc cggcttgtca ccggcagtct cattagagtg 300 cccaactaaa tgatggcaac taatgacaag ggttgcgctc gttgcgggac ttaacccaac 360 atctcacgac acgagctgac gacagccatg cagcacctgt gttacggctc ccgaaggcac 420 tcctccgtct ctggaggatt ccgtacatgt caagaccagg taaggttctt cgcgttgcat 480 cgaattaatc cacatcatcc accgcttgtg cgggtccccg tcaattcctt tgagttttaa 540 tcttgcgacc gtactcccca ggcggtcaat ttcacgcgtt agctacgcta ctaagcaatc 600 aagttgccca acagctaatt gacatcgttt agggcgtgga ctaccagggt atctaatcct 660 gtttgctacc cacgctttcg agcatgaacg tcagtgttat cccagggaggc tgccttcgcc 720 atcggtattc ctccacatct ctacgcattt cactgctaca cgtggaattc tacctccctc 780 tgacacactc tagtcaccca gttcagaacg cagttcccag gttgagcccg gggatttcac 840 atcctgctta agtaaccgtc tgcgctcgct ttacgcccag taattccgat taacgctcgc 900 accctacgta ttaccgcggc tgctggcacg tagttagccg gtgcttattc ttcaggtacc 960 gtcatcagac aggggtatta accctatcct tttcttccct gacaaaagtc ctttacaacc 1020 cgaaggcctt cttcagacac gcggcatggc tggatcaggc ttgcgcccat tgtccaaaat 1080 tccccactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggcggatca 1140 tcctctcaga cccgctactg atcgtcgcct tggtaggcct ttaccccacc aactagctaa 1200 tcagatatcg gccgctcgaa taacgcaagg cccgaaggtc ccctgctttc ctcctcagag 1260 aatatgcggt attagctaat ctttcgatta gttatccccc attactcggt acgttccgat 1320 atattactca cccgttcgcc actcgccacc caagaagcaa gcttctctgt gctgccgtcc 1380 gactgcatg 1389 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> Forward primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial <220> <223> Reverse primer <400> 3 ggttaccttg ttacgactt 19

Claims (7)

Subflava strain belonging to the Neisseria genus ( Neisseria . sp). The strain according to claim 1, wherein the strain has been deposited under deposit number KCCM13173P. The method according to claim 1, wherein the strain contains 16s rRNA having more than 97% sequence identity with SEQ ID NO: 1. The lysate or culture medium of the strain of claim 1. A cosmetic composition comprising the strain of claim 1, its lysate, culture medium, extract of the culture medium, or a mixture thereof. The cosmetic composition according to claim 5, which is for improving skin condition. The cosmetic composition according to claim 6, wherein the improvement of skin condition includes inhibiting skin aging, strengthening the skin barrier, inhibiting skin wrinkles, moisturizing the skin, or regenerating the skin.