KR102021557B1 - Novel lactic acid bacteria and composition and healthfunctional food comprising the same - Google Patents

Abstract

The present invention relates to Enterococcus faecium L-15 KCTC13498BP and a composition comprising the same. The strain exhibits various activities such as regeneration and growth promoting effect of skin stem cells, maintenance capacity enhancement of skin stem cells, fat differentiation promotion of skin stem cells, and cartilage differentiation promotion of stem cells. Thus, the composition can be preferably used as a pharmaceutical composition, cosmetic composition, health functional food and the like.

Description

Novel Lactic Acid Bacteria and COMPOSITION AND HEALTHFUNCTIONAL FOOD COMPRISING THE SAME

The present invention relates to a novel lactic acid bacterium, a composition comprising the same and a dietary supplement.

Lactobacillus is one of the most beneficial microorganisms available to human beings and has a long history of direct and indirect relationships with human life. Lactic acid bacteria are widely distributed not only in food but also in the natural world such as the oral cavity, digestive tract and soil of mammals, and are beneficial strains for human life that play important roles related to food preservation and sensory characteristics.

The name lactic acid bacteria is conceptual and is not a taxonomy of bacteria. A bacterium that produces about 50% more lactic acid as a metabolite for sugars consumed is called lactic acid bacteria. Unlike E. coli, it does not produce rot products. Therefore, the bacterium that produces a large amount of lactic acid from sugars and has a beneficial effect such as preventing corruption without generating substances harmful to the human body in the intestines of humans and animals is conceptually referred to as lactic acid bacteria. Conventional concepts of lactic acid bacteria are Gram-positive cocci or bacilli, do not form catalase negative (anaerobic fermentation), spores, and are generally non-motile.

In the gastrointestinal tract of animals including humans, living organisms that improve the host's intestinal microbial environment and have beneficial effects on the health of the host are collectively called probiotics. In order to be effective as a probiotic, when ingested orally, most of them must reach the small intestine and adhere to the intestinal surface. Therefore, the resistance to acid, bile and intestinal epithelial cells should be excellent. Lactic acid bacteria are used as probiotics because they play a role in breaking down fiber and complex proteins into important nutrients while living in the digestive system of the human body. Lactobacillus has been reported to show the effects of maintaining normal intestinal flora, improving intestinal flora, antidiabetic and antihyperlipidemic effects, inhibiting carcinogenesis, inhibiting colitis, and nonspecific activity of the host's immune system.

As various physiological activities of these lactic acid bacteria are known, researches to develop lactic acid bacteria strains which are safe for humans and excellent in function and apply them as pharmaceuticals or functional foods are being actively conducted. For example, Korean Laid-Open Patent Publication No. 10-2009-0116051 discloses Lactobacillus brevis HY7401, which has the effect of treating and preventing colitis.

Korean Patent Publication No. 10-2009-0116051

An object of the present invention is to provide a novel lactic acid bacteria.

An object of the present invention is to provide a composition, health functional food utilizing various activities, functionalities, etc. of the novel lactic acid bacteria.

Enterococcus faecium L-15 KCTC13498BP strain.

2. according to the above 1, the strain is capable of promoting the regeneration or growth of skin stem cells; Or a strain having a differentiation promoting ability of stem cells into chondrocytes.

3. The composition for skin improvement or regeneration comprising the above strain 1 or its culture.

4. Composition for inducing differentiation into chondrocytes from stem cells comprising the above strain 1 or its culture.

5. The composition of 4 above, wherein the stem cells are pulp stem cells.

6. Cosmetic composition comprising the strain of 1 or its culture.

Enterococcus faecium L-15 (Enterococcus faecium L-15) KCTC13498BP strain of the present invention is the effect of promoting the regeneration and growth of skin stem cells, improving the maintenance capacity of skin stem cells, promote the differentiation of skin stem cells, cartilage differentiation of stem cells Promoting effect. Thus, the strain, its culture or extract thereof may be utilized as a pharmaceutical composition, cosmetic composition, health functional food and the like.

1 to 8 show the effects of enterococcus faecium L-15 (Enterococcus faecium L-15) KCTC13498BP on the regeneration and growth of skin stem cells, and the improvement of the maintenance ability of skin stem cells.
9 to 12 show the cartilage differentiation promoting effect of Enterococcus faecium L-15 KCTC13498BP.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel lactic acid bacterium Enterococcus faecium L-15 KCTC13498BP.

The strain was deposited with the Korea Biotechnology Research Institute Bioresource Center in 2018.3.15, accession number KCTC13498BP.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13498BP

Deposit Date: 20180315

The strain or its culture has several excellent activities. For example, the effect of promoting the regeneration and growth of skin stem cells, improving the maintenance ability of skin stem cells, promote the differentiation of skin stem cells, the effect of promoting the differentiation of cartilage of stem cells. Thus, the strain, its culture or extract thereof may be utilized as a pharmaceutical composition, cosmetic composition, health functional food and the like.

The culture may include the strain, or the strain may be an isolated culture.

The present invention also relates to a composition comprising Enterococcus faecium L-15 KCTC13498BP or a culture thereof.

The composition may be a composition for skin improvement or regeneration.

Enterococcus faecium L-15 KCTC13498BP, or its culture, has an effect of promoting the proliferation of skin stem cells and shows a stem cell maintenance effect. As a result, the skin may be regenerated and improved.

In the present specification, the "stem cell" is a cell capable of cell division by itself and having the ability to differentiate into a wide variety of specific cell types. The type of such stem cells is not particularly limited, and in one embodiment, the stem cells may be skin stem cells. The term “skin stem cell” refers to stem cells capable of differentiating into cells forming the skin (epidermis, dermis and subcutaneous fat layer). The skin-forming cells include keratinocytes, melanocytes and fibroblasts (mainly responsible for the biosynthesis of collagen and elastin) present in the epidermis.

The type of skin stem cells is not particularly limited. Skin stem cells used in the present invention can be used regardless of where it comes from. For example, dermal stem cells can be obtained from known dermal stem cell sources, such as the hair follicles or the basal layer of the epidermis, and the animal being harvested can be a mammal. In one embodiment, the mammal may include, but is not limited to, humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cattle, sheep, antelopes, dogs, or cats. Preferably the mammal may be a human. Such methods for obtaining skin stem cells from skin stem cell sources are well known in the art.

Skin stem cells refer to all stem cells derived from the skin, and specifically, may be a skin-derived precursor cell, but is not limited thereto.

In the present invention, the "stem cell activity promoting effect" refers to a stem cell proliferation promoting effect, and / or the specific indicator molecules of stem cells and the effect of maintaining the stem cell properties of self-dividing properties.

In the present invention, "skin regeneration effect" refers to the recovery of skin tissue against damage caused by external and internal causes of skin as the activity of skin stem cells is promoted. In this case, the damage caused by the external causes may include ultraviolet rays, external pollutants, wounds or traumas, and the damage caused by the internal causes may include stress. Preferably, the skin regeneration is associated with skin stem cells that play an important role in maintaining skin health and physiological and biochemical homeostasis. Enterococcus faecium L-15 KCTC13498BP, its culture or its extract can significantly enhance skin regeneration effect by promoting the proliferation of skin stem cells and maintaining the skin stem cell properties.

In addition, the composition may further contain one or more active ingredients exhibiting the same or similar functions. For example, it may include known skin regeneration. Inclusion of additional skin regeneration components may further enhance the skin regeneration effects of the compositions of the present invention. When the ingredient is added, skin safety, ease of formulation, and stability of the active ingredients may be considered. In one embodiment of the invention, the composition is a skin regeneration component known in the art, retinoic acid, TGF, protein from animal placenta, betulinic acid and chlorella extract, as antioxidant ingredients known in the art, tocopherol, selenium , Vitamin C and phenolic compounds, as whitening ingredients known in the art, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, vitamin-C (L- Ascorbic acid) and derivatives thereof and various plant extracts may further comprise one or two or more components selected from the group consisting of. Additional components may be included in 0.0001% to 10% by weight relative to the total composition weight, the content range is skin safety, the Enterococcus faecium L-15 KCTC13498BP, its culture or its It may be adjusted according to requirements such as the ease in formulating the extract.

The composition may be a composition for inducing chondrocyte differentiation.

Enterococcus faecium L-15 KCTC13498BP, its culture or its extract can promote differentiation from stem cells to chondrocytes. Thus, it may act to promote cartilage cell differentiation, cartilage regeneration, etc., and may exhibit drug efficacy and health function against cartilage damage, cartilage insufficiency, or a disease caused by it.

The stem cells may be, for example, embryonic stem cells, adult stem cells or induced pluripotent stem cells, adult stem cells may be mesenchymal stem cells, more specifically, dental pulp stem cells However, it is not limited thereto.

The composition may be a pharmaceutical composition for the prevention or treatment of arthritis.

Arthritis can be for example degenerative arthritis or rheumatoid arthritis.

Arthritis requires regeneration of damaged cartilage for treatment, Enterococcus faecium L-15 KCTC13498BP, or its culture, promotes proliferation of skin progenitor cells, increases pluripotency related marker expression, In addition, it is possible to promote cartilage regeneration by increasing the expression of chondrocyte forming markers in mesenchymal stem cells (dimension stem cells), and may exhibit excellent efficacy against arthritis.

The limitation of the existing arthritis drugs is that it is difficult to develop a therapeutic agent that specifically affects arthritis due to the wide variety of autoantigens involved in the immune response, and the existing arthritis drugs are focused on suppressing only the inflammatory response. Although there are disadvantages of long-term side effects as a substance, Enterococcus pasium L-15 is a safe lactic acid bacterium that controls immune responses without side effects and confirms that cartilage formation markers are elevated to prevent and treat arthritis. Can be.

In addition, the composition may be a composition for promoting hair growth.

Enterococcus faecium L-15 KCTC13498BP, or its culture, has the ability to stimulate the formation of globular cells of SKP, skin precursor cells in which neuronal cells, skin stem cells, and hair follicle stem cells are present in combination. Promotes the production of hair follicle cells. Thus, hair growth promoting ability can be exhibited.

In addition, the composition may be a composition for preventing or treating atopic dermatitis.

In atopic dermatitis, the basement membrane is defective and stem cells are known to be reduced. Defects in the basement membrane cause changes in the environment in which stem cells survive, reducing the number of stem cells, which is associated with slow skin regeneration and chronic recurrence in patients with atopic dermatitis. However, Enterococcus faecium L-15 KCTC13498BP, or its culture, has the ability to promote the formation of progenitor cells of the skin, thereby relieving atopic dermatitis and promoting skin regeneration to prevent chronic progression. have.

The composition can be used for the manufacture of a pharmaceutical formulation.

The pharmaceutical formulation may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules.

In addition, the composition of the present invention may further comprise a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers may contain various components such as buffers, sterile water for injection, general saline or phosphate buffered saline, sucrose, histidine, salts, polysorbates and the like.

The composition of the present invention may be administered orally or parenterally, and may be administered in the form of general pharmaceutical preparations, for example, in various dosage forms, orally and parenterally, in the case of clinical administration. , Diluents or excipients such as binders, wetting agents, disintegrants, surfactants and the like.

Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations may include at least one excipient such as starch, calcium carbonate, water, or the like in the pharmaceutical composition of the present invention. Can be prepared by mixing cross (Lucose) or lactose (Lactose), gelatin and the like.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical formulation of the present invention may include an external preparation for skin.

Enterococcus faecium L-15 KCTC13498BP, its culture or its extract as an external preparation for skin, further contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, Suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes , Pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredient commonly used in external preparations for skin, may contain adjuvants commonly used in the field of dermatology. The ingredients may also be introduced in amounts generally used in the field of dermatology.

Enterococcus faecium L-15 KCTC13498BP, the culture or its extract, when provided in a topical skin formulation, is not limited thereto and may have a formulation such as an ointment, patch, gel, cream or spray. Can be.

In addition, the composition can be used for preparing cosmetic formulations.

The cosmetic formulation may be in the form of a general emulsion formulation and solubilization formulation. For example, lotions such as flexible lotions or nourishing lotions, emulsions such as facial lotions, body lotions, creams such as nourishing creams, moisture creams, eye creams, essences, cosmetic ointments, sprays, gels, packs, sunscreens, makeup bases, liquids Formulations such as foundations, powders, cleansing creams, cleansing lotions, makeup removers such as cleansing oils, cleansing foams, soaps, body washes and the like.

In addition, the cosmetics are Enterococcus faecium L-15 (Enterococcus faecium L-15) KCTC13498BP, the culture or extracts thereof, in addition to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents , Stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments , Hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredient commonly used in cosmetics, may contain adjuvant commonly used in the cosmetic field.

The cosmetic formulation is a relatively high concentration of enterococcus faecium L-15 in the case of wash-off type cosmetics such as a makeup remover and a detergent, in which an active ingredient stays on the skin within a short period of time. KCTC13498BP , The culture or extract thereof. On the other hand, in the case of leave-on type cosmetics such as lotion, emulsion, cream, essence, etc., in which the active ingredient stays on the skin for a long time, enterococcus pasium L-15 is lower than the wash-off type cosmetics. (Enterococcus faecium L-15) KCTC13498BP, its culture or its extract may be included. Although not limited thereto, in one embodiment of the present invention, the composition comprises Enterococcus faecium L-15 KCTC13498BP, its culture, or its extract, from 0.0001% to 10% by weight of the total composition. % (Preferably 0.0001% to 1% by weight). When the composition of the present invention comprises less than 0.0001% by weight of Enterococcus faecium L-15 KCTC13498BP, the culture or the extract thereof, sufficient skin regeneration effect is not expected, more than 10% by weight If it is included to prevent unwanted reactions such as allergies or skin safety problems may occur.

In the present invention, the term 'effective amount' refers to an amount of Enterococcus faecium L-15 KCTC13498BP, a culture thereof, or an extract thereof, which may exhibit the effects described above. When the composition of the present invention comprises an effective amount of Enterococcus faecium L-15 KCTC13498BP, its culture or its extract, it can provide the desired effect. Although the effective amount of Enterococcus faecium L-15 KCTC13498BP, the culture or the extract thereof included in the composition of the present invention may vary depending on the form in which the composition is commercialized, the daily dosage may be, for example, entero Enterococcus faecium L-15 KCTC13498BP, may be 0.0001 to 100 mg / kg based on the amount of the culture or its extract.

The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

The present invention also relates to a dietary supplement comprising Enterococcus faecium L-15 KCTC13498BP, a culture thereof, or an extract thereof.

The health functional food of the present invention may have health functionalities (prevention or improvement) such as skin regeneration, promoting cartilage cell differentiation, preventing or improving degenerative arthritis, and promoting hair growth.

The formulation of the health functional food of the present invention is not particularly limited, but may be, for example, formulated into a liquid such as tablets, granules, pills, powders, capsules, drinks, caramels, gels, bars, tea bags, and the like. The food composition or health food composition of each formulation can be appropriately selected and formulated by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredients, depending on the formulation or purpose of use, and synergistic effect when applied simultaneously with other raw materials Can happen.

The health functional food of the present invention can be administered by various methods such as simple intake, drinking, injection, spray or squeeze.

In the dietary supplement of the present invention, the dosage determination of the active ingredient is within the level of those skilled in the art, and its daily dosage may be, for example, 0.00001 mg / kg / day to 15 mg / kg / day, The present invention is not limited thereto and may vary depending on various factors such as age, health condition, and complications of the subject to be administered.

The health functional food of the present invention may be, for example, various functional foods such as chewing gum, caramel products, candy, ice cream, confectionery, beverage products such as soft drinks, mineral water, alcoholic drinks, vitamins and minerals, and the like. have.

The health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

The health functional food of the present invention may include a pulp for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is typically included in the range of 0 to about 50 parts by weight per 100 parts by weight of the composition according to one aspect of the invention.

Hereinafter, the present invention will be described in detail with reference to Examples.

Preparation example. Preparation of Enterococcus faecium L-15 Extract

1.Isolation and Identification of L15 (Enterococcus faecium)

To isolate candidate beneficial bacteria, rice fermented foods containing flat fish were added to PBS solution for sufficient vortexing. It was plated on TSB (tryptic soy broth) solid medium and incubated at 37 ° C. for 18 hours in an incubator. The grown candidate colonies were inoculated again in TSB liquid medium, and then incubated for 18 hours at 37 ° C., and stored at −80 ° C. by adding 15% glycerol. The stored candidate beneficial bacteria were selected through initial experiments, and finally the selected beneficial bacteria were identified through API identification kit and 16S sequencing.

2. L15 extract production

L15 bacteria stored for preculture were inoculated in 5 mL of TSB liquid medium and incubated for 18 hours at 37 ° C. in an incubator. After inoculating 1% of L15 precultured in 200 mL of sterile TSB liquid medium, the cells were incubated at 37 ° C for 18 hours. The liquid medium incubated with L15 was centrifuged at 4 ° C., 10,000 rpm for 15 minutes at high speed, and the supernatant was removed. The L15 pellet was resuspended in PBS to remove TSB media components, followed by high speed centrifugation at 4 ° C., 10,000 rpm for 15 minutes, and removal of the supernatant. This process was repeated three times. The L15 pellet was resuspended in third sterile distilled water, and then centrifuged at 4 ° C., 10,000 rpm for 15 minutes at high speed, and the supernatant was removed. After resuspension with third sterile distilled water, ultrasonic grinding was performed for 30 minutes using an ultrasonic grinder. The supernatant was transferred after centrifugation at 4 ° C., 10,000 rpm for 15 minutes and rapidly frozen at −80 ° C. The freeze-dried L15 extract was freeze-dried using a lyophilizer, and the L15 extract in powder form was dissolved in PBS to a desired concentration and filtered through a 0.2 μm filter. Thereafter, aliquots were stored at -20 ° C or -80 ° C.

Example

1. Promotes regeneration and growth of skin stem cells

(1) This experiment measured the growth rate of murine skin-derived precursor cells (SKP, skin stem cells) by water-soluble tetrazolium-1 assay method. Referring to Figure 1 Enterococcus faecium L-15 extract was treated with the highest increase rate at 50 μg / ml it can be seen that induce the regeneration and growth of the skin stem cells. A 50 μg / ml concentration was selected and used for later experiments.

(2) FIG. 2 is a photograph of SKP spheres treated with Enterococcus faecium L-15 extract for 72 hours, and FIG. 3 shows the number of such spheres. It can be seen that the size of the spheres also increased by increasing the proliferation rate in the group treated with the extract.

(3) Western blot confirmed the mechanism by which the proliferation of SKP treated with Enterococcus faecium L-15 extract for 72 hours was activated (FIG. 4). In the extract treated group, activation of the PI3K / Akt signal, which is involved in cell proliferation, was confirmed by increased expression of phosphorylated Akt. And it was confirmed that the expression of Cyclin D1 that regulates the S phase during the cell cycle is also increased.

(4) Based on the data of increased expression of Cyclin D1, the cell cycle was analyzed between the control and extract treated groups by flow cytometry (FIG. 5). In the group treated with the extract, it was confirmed that the number of cells corresponding to the S phase was increased.

(5) Enterococcus faecium L-15 extract was added to the SKP culture medium, followed by culture for 72 hours, and RNA was extracted to confirm the expression change of the pluripotency marker. Referring to FIG. 6, the expression of Oct4 was not significantly different between the control group and the treated group, but Sox2 and Klf4 were significantly increased than the control group in the extract-treated group and were known as oncogenes capable of inducing pluripotency markers and cancer. Expression of -Myc was found to be significantly decreased in the group treated with the extract.

(6) Enterococcus faecium L-15 extract was added to SKP culture medium, followed by culture for 72 hours, and RNA was extracted to confirm the expression of fat, bone, and cartilage differentiation markers. Referring to FIG. 7, expression of both adipose differentiation markers (C / EBPa, PPARg) and bone differentiation markers (Runx2, ALP) were significantly decreased in the extract-treated group, and expression of Sox9 and Acan, which are cartilage differentiation markers, also decreased. Acan did not show any significant difference. It can be seen that Enterococcus faecium L-15 extract is involved in maintaining the undifferentiated state of SKP by increasing the expression of some pluripotency markers.

(7) Enterococcus faecium L-15 extract was added to SKP culture medium, followed by incubation for 72 hours, spheres were isolated into single cells, seeded on plates, and then differentiated in adipose differentiation medium. Referring to FIG. 8, RNA was extracted on days 3 and 5 during adipose differentiation, and the expression of adipose differentiation markers C / EBPa and PPARg was analyzed by PCR. Expression of C / EBPa was significantly increased in the 3rd and 5th day samples, and PPARg was significantly increased on the 3rd day and increased on the 5th day, but the significance was not confirmed (see FIG. 5). It was confirmed that Enterococcus faecium L-15 extract can promote differentiation during adipose differentiation by enhancing the quality of SKP as a stem cell.

2. Promote differentiation of human dental pulp stem cells into cartilage cells

(1) The cytotoxicity of Enterococcus faecium L-15 was confirmed by water-soluble tetrazolium-1 assay. Human dental pulp stem cells were treated with Enterococcus faecium L-15 extract and found to be cytotoxic from 100 μg / ml (FIG. 9). A concentration of 50 μg / ml without cytotoxicity was selected and used for cartilage differentiation.

(2) Enterococcus feacium L-15 extract was added to the cartilage differentiation medium to proceed cartilage differentiation. Cartilage differentiation medium was composed of 50 ㎍ / ml ascorbic acid 2-phosphate, 40 ㎍ / ml l-proline, 1 μM dexamethasone, 10% ITS + Pre-mix Tissue Culture Supplement, 10 ㎍ / ml transforming growth factor beta 1 (TGF-β1) was added and used. RNA was extracted at 3, 5, 7, 10, and 14 days during cartilage differentiation, and real-time PCR analysis of COL10A1, RUNX2, and MMP13 with SOX9, COL2A1, ACAN and hypertrophic markers as early cartilage markers (Fig. 10).

Early cartilage differentiation markers showed high expression on all days in the group treated with Enterococcus faecium L-15 extract, especially on day 5. Among the hypertrophic markers, COL10A1 and RUNX2 showed high expression in the group treated with Enterococcus faecium L-15 extract on the 5th day and became similar to that of the control group as 7 days 10 days and 14 days.

MMP13 is known to degrade the proteoglycans of chondrocytes. The expression of MMP13 was also higher at Day 5 in the group treated with Enterococcus faecium L-15 extract, but at 7 days 10 days and 14 days than the control group.

This result indicates that Enterococcus faecium L-15 extract promotes differentiation into chondrocytes, especially early in differentiation, and inhibits hypertrophic chondrocytes differentiated by decreasing MMP13 expression in later stages.

(3) Alcian blue staining is a staining method that specifically reacts to proteoglycans and is used to identify chondrocyte differentiation. Alcian blue staining was performed to confirm chondrocyte differentiation and quantified due to the difficulty in distinguishing the degree of staining with the naked eye. On day 3, day 5, day 7, day 10 and day 14, it was confirmed that staining was better in the group treated with Enterococcus faecium L-15 extract (FIG. 11).

Enterococcus faecium L-15 extract promotes cartilage differentiation during cartilage differentiation.

(4) Cartilage is mostly composed of extracellular matrix. Since glycosaminoglycan (GAG) is an essential component of cartilage in the extracellular matrix, the amount of GAG was measured by GAG assay during chondrocyte differentiation. 3 days 5 days 7 days 10 days 14 days all the Enterococcus faecium L-15 extract treated group was confirmed that the amount of GAG more (Fig. 12).

Enterococcus faecium L-15 extract promotes cartilage differentiation during cartilage differentiation.

Claims (6)

Enterococcus faecium L-15 KCTC13498BP strain.
The method according to claim 1, wherein the strain is to promote the regeneration or growth of skin stem cells; Or a strain having a differentiation promoting ability of stem cells into chondrocytes.
A composition for skin regeneration comprising the strain of claim 1 or a culture thereof.
Composition for inducing differentiation of stem cells into chondrocytes comprising the strain of claim 1 or a culture thereof.
The composition of claim 4, wherein the stem cells are pulp stem cells.
Cosmetic composition comprising the strain of claim 1 or its culture.

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