Composition for improving skin function containing enterococcus faecalis as active ingredient

Abstract

The present invention relates to a composition for improving skin function, containing Enterococcus faecalis as an active ingredient and, particularly, to a composition for skin whitening, skin barrier amelioration, skin wrinkle amelioration and skin elasticity enhancement, or skin soothing, containing, as an active ingredient, an Enterococcus faecalis EF -2001 strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, or a dead cell thereof.

Description

Composition for improving skin function containing Enterococcus faecalis as an active ingredient

[0001] The present invention relates to a composition for improving skin function containing Enterococcus faecalis as an active ingredient, and more particularly, to a composition for whitening skin, improving skin barrier, improving skin wrinkles, and increasing skin elasticity, or soothing skin, containing Enterococcus faecalis EF-2001 strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried substance thereof, or a killed cell thereof as an active ingredient.

[0002]

[0003] The skin is the outermost organ of the human body, detecting various external stimuli to maintain homeostasis and protect the body. It is also known to function as an immune system, preventing the invasion of foreign substances from the outside. Among the four layers of the epidermis—the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale—intercellular lipids between keratinocytes in the stratum corneum form the skin barrier. It is known that external stimuli such as ultraviolet rays and fine dust, as well as internal factors such as metabolites produced by intestinal microorganisms, can accelerate the decline in normal skin function, loss of elasticity, wrinkle formation, and excessive melanin production. However, there are few solutions to control these conditions.

[0004]

[0005] In this regard, primary cilia are non-motile organelles that are present in almost all eukaryotic cells, including skin cells, and are antenna-like structures that protrude from the cell surface. These primary cilia detect external stimuli or various sensory signals (visual, olfactory, auditory, kinesthetic, etc.) and mediate signaling processes that are crucial for maintaining tissue homeostasis in the body, such as Hedgehog (Hh), Transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), AMP-activated protein kinase (AMPK), and autophagy.

[0006]

[0007] In particular, recent studies have reported that primary cilia activity is essential for skin cell growth, differentiation, and strengthening the skin barrier, preventing excessive melanin production, and maintaining tissue homeostasis in the skin epidermis.

[0008]

[0009] Accordingly, research and development on materials for activating these primary cilia is insufficient, and in the cosmetics field, it is important to not cause toxicity or irritation to the skin, so research using natural substances for activating primary cilia is required.

[0010]

[0011] In this regard, microorganisms of the genus Enterococcus are widely found in nature and utilize carbohydrates aerobically. In general, bacteria such as Enterococcus microorganisms are known to prevent damage caused by pathogenic microorganisms through in vivo antagonism or secreted antimicrobial substances. Among these, Enterococcus faecalis EF-2001 was identified through a screening of the intestinal flora of a two-year-old girl. This Enterococcus faecalis EF-2001 killed cell was obtained by heat treatment to kill Enterococcus faecalis EF-2001 and recovering the bacterial components.

[0012]

[0013] Studies reported on the physiological activity of Enterococcus faecalis EF-2001 showed that mice suffering from colitis induced by DSS (Dextran sulfate sodium) showed an alleviating effect on DSS in mice fed Enterococcus faecalis EF-2001, and that the proliferation of transplanted sarcoma cancer cells (Sarcoma-180) was reduced and NK cells were activated (Tadano et al., J. Japan Mibyou System association, 2011). In addition, regarding the inhibition of harmful bacteria and the effect of intestinal regulation, Enterococcus faecalis EF-2001 shows the effect of improving and preventing symptoms by inhibiting the activity of Candida albicans, the cause of leukoplakia (Ishijima et al., Med. Mycol. J, 2014), and the effect of rapidly proliferating beneficial bacteria and inhibiting harmful bacteria compared to the intestinal control group in mice administered antibiotics has been disclosed (Simohashi et al., Medicine and biology, 2002). Meanwhile, various physiological activities are shown in relation to the killed cells of Enterococcus faecalis EF-2001, and this is based on the fact that the killed cells are not affected by heat and pH due to their characteristics, so they can be processed into various forms of preparations (Kan, Food industry, 2001). In addition, Enterococcus faecalis EF-2001 has a cell content of 7.5 trillion cells per gram, so a large amount of lactic acid bacteria can be consumed even in a small amount.

[0014]

[0015] However, the specific effects of Enterococcus faecalis on improving skin function, such as inhibition of melanin production, enhancement of skin elasticity, improvement of skin barrier function, and skin soothing, are not specifically known.

[0016]

[0017] Against this backdrop, the inventors of the present invention have made efforts to develop a material capable of improving overall skin functions, such as controlling excessive melanin production, enhancing skin elasticity, reducing inflammatory responses, and improving the skin barrier by activating primary cilia. As a result, they have confirmed that Enterococcus faecalis EF-2001, its lysate, its culture, its concentrate, its dried product, and its killed cells are effective in improving overall skin functions, such as controlling excessive melanin production, enhancing skin elasticity, reducing inflammatory responses, and improving the skin barrier by activating primary cilia, and have completed the present invention.

[0018]

[0019] [Prior Art Literature]

[0020] [Patent Document]

[0021] (Patent Document 1) Korean Patent No. 10-2503163

[0022] (Patent Document 2) Korean Patent Publication No. 10-2022-0079092

[0023]

[0024] The purpose of the present invention is to provide a composition for improving skin function, which comprises at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an effective ingredient.

[0025] Another object of the present invention is to provide a composition for improving skin function, which suppresses excessive melanin production, increases skin elasticity, improves skin barrier function, and has a skin soothing effect by activating primary cilia.

[0026] Another object of the present invention is to provide a cosmetic for improving skin function, comprising at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an active ingredient.

[0027] Another object of the present invention is to provide a health functional food for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells.

[0028] Another object of the present invention is to provide a food additive for improving skin function, which comprises as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells.

[0029] Another object of the present invention is to provide an external preparation for improving skin function, which comprises at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an active ingredient.

[0030] Another object of the present invention is to provide a pharmaceutical composition for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells.

[0031]

[0032] In order to achieve the above purpose, the present invention provides a composition for improving skin function, comprising at least one selected from the group consisting of Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product, and its dead cells as an effective ingredient.

[0033] In addition, the present invention provides a composition for improving skin function, characterized in that the strain is Enterococcus faecalis EF-2001 strain.

[0034] In addition, the present invention provides a composition for improving skin function, characterized in that the composition activates primary cilia.

[0035] In addition, the present invention provides a composition for improving skin function, characterized in that the composition inhibits melanin production.

[0036] In addition, the present invention provides a composition for improving skin function, characterized in that the composition promotes skin elasticity.

[0037] In addition, the present invention provides a composition for improving skin function, characterized in that the composition has an activity for improving skin barrier function.

[0038] In addition, the present invention provides a composition for improving skin function, characterized in that the composition has a skin soothing effect.

[0039] In addition, the present invention provides a cosmetic for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product, and its dead cells.

[0040] In addition, the present invention provides a health functional food for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product, and its dead cells.

[0041] In addition, the present invention provides a food additive for improving skin function, which comprises at least one selected from the group consisting of Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product, and its dead cells as an effective ingredient.

[0042] In addition, the present invention provides an external preparation for improving skin function, which comprises at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an effective ingredient.

[0043] In addition, the present invention provides a pharmaceutical composition for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product, and its dead cells.

[0044]

[0045] A composition for improving skin function, comprising at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products and their killed cells as an active ingredient, has a skin whitening effect by activating primary cilia, inhibits melanin production, improves skin barrier function by improving the expression of Claudin-1 protein, inhibits the expression of MMP-1, a collagen-decomposing enzyme, and inhibits the activation of Nuclear factor-κB (NF-κB), thereby exhibiting skin wrinkle improvement and soothing effects.

[0046]

[0047] Therefore, the composition for improving skin function can be usefully used in cosmetics, health functional foods, food additives, external preparations, and pharmaceutical compositions for improving skin function.

[0048]

[0049] Figure 1 shows the number of primary cilia-forming cells and the length of primary cilia by treatment of the culture medium and dead cells of Enterococcus faecalis EF-2001 strain according to Experimental Example 1 of the present invention.

[0050] Figure 2 shows the number of primary cilia forming cells and the length of primary cilia when dead cells of the Enterococcus faecalis EF-2001 strain were treated with a stress-inducing substance according to Experimental Example 1 of the present invention.

[0051] Figure 3 shows the results of measuring the melanin content by treating the culture solution and dead cells of Enterococcus faecalis EF-2001 strain according to Experimental Example 2 of the present invention.

[0052] Figure 4 is an image showing Claudin-1 fluorescence expression and skin barrier formation by treatment of dead cells of Enterococcus faecalis EF-2001 strain according to Experimental Example 3 of the present invention.

[0053] Figure 5 shows the expression of Claudin-1 protein by treatment of dead cells of Enterococcus faecalis EF-2001 strain according to Experimental Example 3 of the present invention.

[0054] Figure 6 shows the expression of MMP-1 by treatment of dead cells of Enterococcus faecalis EF-2001 strain according to Experimental Example 4 of the present invention.

[0055] Figure 7 shows the activation of NF-κB by treatment of dead cells of Enterococcus faecalis EF-2001 strain according to Experimental Example 4 of the present invention.

[0056]

[0057] Hereinafter, preferred embodiments of the present invention will be described in detail so that a person having ordinary skill in the art to which the present invention pertains can easily practice the present invention.

[0058]

[0059] Various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, specific embodiments may be practiced with one or more of these specific details, or with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of "one embodiment" or "an embodiment" in various places throughout this specification does not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0060]

[0061] Unless otherwise specifically defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0062]

[0063] The present invention, as one embodiment, provides a composition for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells. It is preferable to use the Enterococcus faecalis strain registered as Enterococcus faecalis EF-2001 (Domestic Accession Number: KCTC SD1560), but the present invention is not limited thereto.

[0064]

[0065] In the present invention, the term "crusted material" means a product obtained by crushing the Enterococcus faecalis strain of the present invention by a known method.

[0066]

[0067] The term "cultivation" in the present invention refers to growing the Enterococcus faecalis strain of the present invention under appropriately controlled environmental conditions. The culturing process of the present invention can be performed using suitable media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be batch, continuous, or fed-batch, but is not limited thereto.

[0068]

[0069] For example, the culture of the Enterococcus faecalis strain of the present invention may be performed in a nutrient medium (NA medium) under one or more of the following conditions: a temperature of 25°C to 40°C, an agitation speed of 50 rpm to 200 rpm, and a pH of 6 to 9, but is not limited thereto. In addition, the culture may be performed under a relative humidity condition of 40% to 50% based on a 24-hour relative humidity, or a temperature condition of 30°C to 40°C, but is not limited thereto.

[0070]

[0071] In the present invention, the term "culture medium" means a culture medium obtained by culturing the Enterococcus faecalis strain, and may be a culture medium containing the strain or a cell-free culture medium from which the bacterial cells have been removed.

[0072]

[0073] In one specific example of the present invention, the culture may be a culture medium obtained by culturing Enterococcus faecalis EF-2001 strain in a liquid culture medium, and specifically, the culture solution of Enterococcus faecalis EF-2001 strain may be a culture stock solution including bacterial cells, or may be bacterial cells obtained by removing or concentrating the culture supernatant. The composition of the culture solution may additionally include components necessary for typical Enterococcus culture, as well as components that act synergistically on the growth of Enterococcus, and the composition accordingly may be easily selected by a person having ordinary skill in the art. In addition, the properties of the culture may be liquid or solid, and are not limited to either.

[0074]

[0075] In the present invention, the term "concentrate" refers to a culture of the strain concentrated using a conventional method. The concentrate may be a concentrate obtained by concentrating a liquid culture medium in which the Enterococcus faecalis EF-2001 strain is cultured using a conventional method; however, the nature of the culture is not limited to a liquid.

[0076]

[0077] In the present invention, the term "dry product" or "dry product of culture" refers to a culture of the strain dried by a conventional method. The dried product of the culture may be a concentrate of a liquid culture medium in which the Enterococcus faecalis EF-2001 strain is cultured by a conventional method, but the nature of the culture is not limited to a liquid.

[0078]

[0079] In addition, in the present invention, the Enterococcus faecalis strain or its dead cells may be any of those commercially available or those manufactured using a known dead cell manufacturing method, and are non-toxic and harmless to the human body.

[0080]

[0081] Additionally, the above-mentioned dead cells can be manufactured by heat-treating the corresponding living cells or treating them with formalin or other sterilizing agents, and the dead cells can be used even if they are substantially dead.

[0082] In addition, the above-mentioned dead cells may be obtained by washing a strain obtained by culturing it by a conventional method, dehydrating it by centrifugation, repeating washing and dehydration as needed, suspending it in distilled water, saline solution, etc., and heating the suspension at, for example, 80 to 115°C for 30 minutes to 3 seconds, thereby obtaining a dead cell suspension or a dried product thereof, or by irradiating the above-mentioned dead cell suspension with gamma rays or neutron rays, thereby obtaining a dead cell suspension or a dried product thereof.

[0083] The drying means for the above-mentioned dead cell suspension is not particularly limited as long as it is a known drying means, and examples thereof include spray drying and freeze drying. In some cases, enzyme treatment, surfactant treatment, grinding / pulverizing treatment may be performed before or after sterilization treatment by heating or the like, or before or after drying treatment, and the products obtained by such treatments are also included in the dead cell suspension of the present invention.

[0084]

[0085] In addition, the above-mentioned dead cells can be manufactured by the following methods, but are not limited thereto:

[0086] 1) A step of culturing Enterococcus faecalis as a starter at a pH of 4.0 to 9.0 and a temperature of 15 to 45°C, more preferably at a pH of 5.0 to 8.0 and a temperature of 20 to 40°C;

[0087] 2) A step of drying and powdering the Enterococcus faecalis cultured in the above step 1) after heat treatment at a temperature of 60 to 140°C for 1 to 40 minutes, more preferably at a temperature of 70 to 130°C for 5 to 30 minutes.

[0088]

[0089] As another embodiment of the present invention, the composition for improving skin function may have the effect of activating primary cilia.

[0090]

[0091] In this regard, primary cilia are non-motile organelles present in almost all eukaryotic cells, including skin cells, and are antenna-like structures protruding from the cell surface. These primary cilia detect external stimuli or various sensory signals (visual, olfactory, auditory, kinesthetic, etc.) and mediate signaling processes crucial for maintaining tissue homeostasis in the body, such as Hedgehog (Hh), Transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), AMP-activated protein kinase (AMPK), and Autophagy. Recent studies have reported that primary cilia activity promotes the growth and differentiation of skin cells and strengthens the skin barrier, and is essential for preventing excessive melanin production and maintaining tissue homeostasis of the skin epidermis. Therefore, the composition for improving skin function of the present invention can achieve the intended effect of the present invention by activating primary cilia.

[0092]

[0093] Additionally, according to one embodiment of the present invention, the activation of the primary cilia can be confirmed by measuring the primary cilia formation rate and the length of the primary cilia.

[0094]

[0095] As another embodiment of the present invention, the composition for improving skin function may have the effect of inhibiting melanin production.

[0096] As another embodiment of the present invention, the composition for improving skin function may have an effect of increasing skin elasticity.

[0097] As another embodiment of the present invention, the composition for improving skin function may have an activity for improving skin barrier function.

[0098] As another embodiment of the present invention, the composition for improving skin function may have a skin soothing effect.

[0099]

[0100] In order to stably preserve the Enterococcus faecalis EF-2001 strain of the present invention for a long period of time, the cells may be dissolved in a storage solution prepared by mixing a certain amount of glycerol component in water and stored at -70°C, or suspended in sterilized 10% skim milk and freeze-dried, but this is not limited thereto, and a long-term preservation form may be achieved by various known methods.

[0101]

[0102] Meanwhile, the concentration of Enterococcus faecalis EF-2001 strain in the above composition is not limited thereto, but is 10 3 CFU / ml to 10 10 CFU / ml, 10 3 CFU / ml to 10 9 CFU / ml, 10 3 CFU / ml to 10 8 CFU / ml, 10 3 CFU / ml to 10 7 CFU / ml, 10 3 CFU / ml to 10 6 CFU / ml, 10 3 CFU / ml to 10 5 CFU / ml, 10 3 CFU / ml to 10 4 CFU / ml, 10 4 CFU / ml to 10 10 CFU / ml, 10 5 CFU / ml to 10 10 CFU / ml, 10 6 CFU / ml to 10 10 CFU / ml, 10 7 CFU / ml to 10 10CFU / ml, 10 8 CFU / ml to 10 10 CFU / ml, 10 9 CFU / ml to 10 10 CFU / ml, 10 4 CFU / ml to 10 9 CFU / ml, 10 5 CFU / ml to 10 8 CFU / ml, or 10 6 CFU / ml to 10 7 It can be CFU / ml.

[0103]

[0104] The composition may further comprise a cryoprotectant or excipient. The cryoprotectant or excipient may be, but is not limited to, a non-naturally occurring substance or a naturally occurring substance. In another specific example, the cryoprotectant or excipient may be, but is not limited to, a substance with which the Enterococcus faecalis EF-2001 strain does not naturally come into contact, or a substance that is not naturally contained together with the strain. In another specific example, the composition may further comprise one or more cryoprotectants selected from the group consisting of glycerol, trehalose, maltodextrin, skimmed milk powder, and starch; and / or one or more excipients selected from the group consisting of glucose, dextrin, and skimmed milk. The cryoprotectant of the present invention may be included in an amount of 0.01 wt% to 20 wt%, or 0.01 wt% to 10 wt%, based on the total weight of the composition. Specifically, the glycerol may be included in an amount of 5 wt% to 20 wt%, the trehalose may be included in an amount of 2 wt% to 10 wt%, the maltodextrin may be included in an amount of 2 wt% to 10 wt%, the skimmed milk powder may be included in an amount of 0.5 wt% to 2 wt%, and the starch may be included in an amount of 0.1 wt% to 1 wt%. In addition, the excipient may be included in an amount of 75 wt% to 95 wt%, or 85 wt% to 95 wt%, based on the total weight of the composition.

[0105]

[0106] In addition, a method for producing a composition comprising the Enterococcus faecalis EF-2001 strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, or killed cells thereof may include a step of mixing at least one selected from the group consisting of the Enterococcus faecalis EF-2001 strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and killed cells thereof with an additive. The additive may be the aforementioned cryoprotectant or excipient.

[0107]

[0108] The composition may be a cosmetic composition, a food composition, a health functional food composition, a food additive composition, a composition for external application, or a pharmaceutical composition.

[0109]

[0110] The Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product or its dead cells contained in the composition for improving skin function of the present invention have excellent melanin production inhibition effect, skin elasticity promotion effect, skin function improvement activity and skin soothing effect, and can be usefully used in cosmetics for improving skin function, health functional foods for improving skin function, food additives for improving skin function, external preparations for improving skin function or pharmaceutical compositions for improving skin function.

[0111]

[0112] As one embodiment of the present invention, the present invention provides a cosmetic or external preparation comprising at least one selected from the group consisting of the Enterococcus faecalis strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof as an active ingredient.

[0113]

[0114] In addition, since the method for obtaining the Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product or its dead cells is the same as that described in the composition for improving skin function, the specific description is based on the above-mentioned content, and only the unique composition of the external preparation and cosmetics will be described below.

[0115]

[0116] The ingredients included in the cosmetics or external preparations of the present invention may include, in addition to the above-mentioned effective ingredients, ingredients commonly used in cosmetics or external preparations, and include, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0117]

[0118] In order to formulate the effective ingredient of the present invention, it can be easily formulated by performing the process according to the commercial method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspending agents, and other commercially available auxiliary agents can be appropriately used.

[0119]

[0120] The cosmetic or external preparation of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream, and spray, but is not limited thereto. More specifically, it can be manufactured into the formulation of an emollient toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, cleansing water, a pack, a spray, or a powder.

[0121]

[0122] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0123]

[0124] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester can be used.

[0125]

[0126] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0127]

[0128] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0129]

[0130] In addition, as an embodiment of the present invention, the present invention provides a health functional food or food additive comprising at least one selected from the group consisting of the Enterococcus faecalis strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof as an effective ingredient.

[0131]

[0132] Since the method for obtaining the above Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product or its dead cells and the content described in the composition for improving skin function are the same as those described above, the specific description is based on the above content, and only the unique composition of the health functional food and food additive will be described below.

[0133]

[0134] The method for obtaining the Enterococcus faecalis strain, its culture medium and its killed cells and the type of obesity or metabolic syndrome induced by obesity are the same as those described in the pharmaceutical composition for improving or treating obesity or metabolic syndrome induced by obesity, which comprises the Enterococcus faecalis strain, its culture medium or its killed cells as an active ingredient. Therefore, the specific description refers to the above contents, and only the unique composition of the health functional food is described below.

[0135]

[0136] There is no particular limitation on the type of food to which the Enterococcus faecalis of the present invention is added. Examples of the food include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, alcoholic beverages, and vitamin complexes, and includes all health foods and health functional foods in the conventional sense.

[0137]

[0138] The amount of Enterococcus faecalis mixed according to the present invention can be appropriately determined depending on its intended use (prevention or improvement). Generally, the amount of Enterococcus faecalis in health foods can be added in an amount of 0.001 to 50 wt% of the total food weight. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may also be used in an amount exceeding the above range.

[0139]

[0140] The health functional composition of the present invention contains the Enterococcus faecalis as an essential ingredient in the indicated proportions, and has no particular limitations on other ingredients, and may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0141]

[0142] In addition to the above, the food or food additive of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the Enterococcus faecalis of the present invention may contain fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratio of these additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of Enterococcus faecalis of the present invention.

[0143]

[0144] In addition, as one embodiment of the present invention, the present invention provides a pharmaceutical composition comprising at least one selected from the group consisting of the Enterococcus faecalis strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof as an active ingredient.

[0145]

[0146] The method for obtaining the above Enterococcus faecalis strain, its lysate, its culture, its concentrate, its dried product or its dead cells is the same as that described in the composition for improving skin function, so the specific description is based on the above content.

[0147]

[0148] The composition of the present invention contains 10 strains as an active ingredient based on the total weight of the composition. 6 10 inland 13It may contain a content of cfu / g, or may contain a lysate, culture, concentrate, dried product, or dead cells having an equivalent number of live cells. In addition, one or more pharmaceutically acceptable conventional carriers or one or more additives may be selected from the effective amount of the Enterococcus faecalis strain of the present invention, the culture solution thereof, or the dead cells thereof, which are the main components, to prepare a composition in a conventional dosage form.

[0149]

[0150] The carrier may be selected from one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and the additive may be selected from one or more of flavorings, vitamins, and antioxidants.

[0151]

[0152] In the present invention, any pharmaceutically acceptable carrier and additive may be used. Specifically, as a diluent, lactose monohydrate, trehalose, cornstarch, soybean oil, microcrystalline cellulose, or D-mannitol is preferable, as a lubricant, magnesium stearate or talc is preferable, and as a binder, polyvinyl pyrrolidone (PVP) or hydroxypropylcellulose (HPC) is preferably selected. In addition, it is preferable to select from among carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, polacrylin potassium, or cross-linked polyvinylpyrrolidone as a disintegrant, white sugar, fructose, sorbitol, or aspartame as a sweetener, carboxymethylcellulose sodium (Na-CMC), β-cyclodextrin, white bee's wax, or xanthan gum as a stabilizer, and methyl p-hydroxy benzoate (methylparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate as a preservative. It is preferable to select from among sorbate, but is not limited thereto.

[0153]

[0154] The pharmaceutical composition may be administered to a patient as a single dose, or may be administered via a fractionated treatment protocol in which multiple doses are administered over a long period of time. The term "pharmaceutically effective amount" as used herein refers to an amount that elicits a response greater than that of a negative control group, and preferably refers to an amount sufficient to prevent or treat an inflammatory disease. In addition, the pharmaceutically effective amount may be appropriately varied depending on various factors, such as the disease and its severity, the patient's age, weight, health status, sex, route of administration, and treatment period.

[0155]

[0156] The composition of the present invention can be formulated in various ways according to the route of administration using a method known in the art together with the pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans, and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions. The composition of the present invention can be formulated in various ways according to the route of administration using a method known in the art together with the pharmaceutically acceptable carrier. The route of administration is not limited thereto, but may be administered orally or parenterally.

[0157]

[0158] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that the following examples are provided solely for the purpose of more concretely illustrating the present invention, and that the scope of the present invention is not construed as being limited by these examples.

[0159]

[0160] Manufacturing Example 1. Preparation of culture solution of Enterococcus faecalis EF-2001

[0161]

[0162] Enterococcus faecalis EF-2001 (Korea Berum Co., Ltd., Korea) was cultured aerobically or anaerobically in a medium used for general lactic acid bacteria culture, and then cultured for 1 to 3 days while maintaining pH 5.0 to 8.0 and 20 to 40℃ to obtain a dry weight (DW) of 7.5 × 10 12 The main culture was performed to reach a cell count of 10 cfu / g or more. Afterwards, the supernatant was separated by centrifugation at 8,000 rpm for 10 minutes. The separated supernatant was diluted 10-fold (10%, v / v) to prepare a culture solution of Enterococcus faecalis EF-2001.

[0163]

[0164] Manufacturing Example 2. Manufacturing of killed cells of Enterococcus faecalis EF-2001

[0165]

[0166] Enterococcus faecalis EF-2001 cultured in the above manufacturing example 1 was heat treated at 70 to 130°C for 5 to 30 minutes to kill the cells, and then the cells were separated and recovered using a continuous centrifuge, followed by freeze-drying and powderization.

[0167]

[0168] Experimental Example 1. Evaluation of Primary Cilia Activation

[0169] 1-1. Cell line preparation and culture

[0170] Human fibroblast from skin (GM00969 Corriell Institute), keratinocytes (HaCat), and mouse melanoma cell line B16F1 (mouse melanoma, ATCC) cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 15% or 10% (v / v) fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen, CA, USA) at 37°C in a 5% (v / v) CO2 incubator.

[0171]

[0172] 1-2. Treatment with Enterococcus faecalis EF-2001

[0173] To confirm the activity of primary cilia formation by EF-2001 treatment in the above cell line, 8 X 10 cells per well were seeded in a 12-well culture plate (SPL Life Sciences, South Korea). 4 After culturing the cells for 24 hours, from the time point when the cells were approximately 90% confluent, the cells were treated with a control group containing only the medium, a stress-inducing substance (fine dust) particulate matter 2.5 (PM2.5; 30 μg / ml) alone, or a culture solution of each test substance, EF-2001 (0.1, 1, 5%) and dead cells (0.01, 0.1, 1 mg / ml), in a medium containing the stress-inducing substance PM2.5 at each concentration for 24 hours.

[0174]

[0175] 1-3. Evaluation of primary cilia activation of Enterococcus faecalis EF-2001

[0176] To confirm the primary cilia activation effect of EF-2001, human skin-derived fibroblasts were cultured in simple medium without EF-2001 (labeled Medium), culture medium of EF-2001 (labeled Sub.), and cell culture medium containing killed EF-2001 cells (labeled Bacteria) alone or together with the stress-inducing substance PM2.5 (30 μg / ml) at various concentrations for 24 hours. After immunostaining with ARL13B (17711-1-AP, 1:1000 dilution, Proteintech, Chicago, IL), a major primary cilia marker, and Hoechst dye for nuclear staining, the primary cilia formation rate and primary cilia length were measured under a fluorescence microscope (IX71, Olympus, Japan).

[0177] Here, the production rate of primary cilia is expressed as the percentage of the number of cells with primary cilia out of the total number of cells, and the length of primary cilia is a measurement of the actual length of primary cilia observed under a microscope.

[0178]

[0179] As a result, as shown in Fig. 1, it was confirmed that primary cilia activity was increased even with treatment of the culture medium and dead cells of EF-2001 at each concentration. In addition, when the stress-inducing substance (fine dust) PM2.5 was treated, primary cilia formation was inhibited, but when the dead cells of EF-2001 were treated together with the stress-inducing substance PM2.5, it was confirmed that primary cilia formation was statistically significantly restored to a normal level or higher, as shown in Fig. 2 (**p < 0.01, ***p < 0.005, ****p < 0.001).

[0180]

[0181] Experimental Example 2. Evaluation of Melanin Production Inhibition Efficacy

[0182] The mouse-derived melanoma cell line B16F1 (ATCC, USA) cells cultured above were pretreated with 0.5 μM of melanin-producing hormone α-MSH (alpha-melanocyte-stimulating hormone, Sigama-Aldrich) for 48 hours, and then additionally treated with a culture solution (0.1, 1, 5%) of EF-2001 according to Example 1 and killed cells (0.01, 0.1, 1 mg / ml) according to Example 2 for 24 hours, and then the melanin content was measured. Here, the melanin content was analyzed by measuring the absorbance after dissolving the prepared cells in an aqueous NaOH solution.

[0183]

[0184] At this time, melasolv (10 ppm) was used as a positive control substance for the whitening effect through inhibition of melanin production. Melanin content was analyzed as a % compared to the control group by measuring absorbance at a wavelength of 475 nm using a spectrophotometer after dissolving the treated B16F1 cells in an aqueous NaOH solution.

[0185]

[0186] As a result, as shown in Fig. 3, it was confirmed that the amount of melanin production, which was increased by about three times by α-MSH, a melanin production hormone, was significantly suppressed under the conditions of EF-2001 culture solution and EF-2001 dead cells treatment. Therefore, it was confirmed that the EF-2001 culture solution and EF-2001 dead cells according to the present invention are useful for skin whitening.

[0187]

[0188] Experimental Example 3. Evaluation of Improvement in Skin Barrier Function

[0189] A damaged skin barrier is considered a major risk factor for skin sensitization, and among them, Claudin-1 is known to be an essential factor for the barrier function of the epidermis.

[0190]

[0191] In order to evaluate the effect of EF-2001 according to the present invention on improving the skin barrier, human-derived keratinocytes were treated together with the stress-inducing substance PM2.5 (30 μg / ml) and dead cells of EF-2001 at different concentrations (0.01, 0.1, 1 mg / ml), and after 24 hours, the expression of Claudin-1 (#37-4900, 1:1000 dilution, Thermo-Fisher), a major membrane protein of the epithelial barrier, was confirmed through fluorescent immunostaining.

[0192]

[0193] Specifically, all samples prepared from the above cells were prepared for Western blotting by dissolving in 2× Laemmli sample buffer [62.5 mM Tris-HCl, pH 6.8, 25% (v / v) glycerol, 2% (w / v) sodium dodecyl sulfate (SDS), 5% (v / v) β-mercaptoethanol, and 0.01% (w / v) bromophenol blue (Bio-Rad, Hercules, CA, USA)]. All cellular proteins were quantified using Bradford solution (BioRad) according to the manufacturer's instructions. Afterwards, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride membrane (PVDF, Bio-Rad), blocked with 4% (w / v) skim milk in Tris-buffered saline and Tween [TBST; 25 mM Tris, 140 mM sodium chloride, and 0.05% (v / v) Tween 20], and then reacted overnight at 4°C with the following specific primary antibodies;

[0194] Actin (ACTA1; 1:10,000 dilution, MAB1501, Millipore, MA, USA), Claudn-1 (1:3,000 dilution, #37-4900, Thermo Fisher, MA, USA), phospho-NF-κB p65 (1:1,000 dilution, #3031, Cell Signaling Technology, MA, USA), and MMP-1 (1:3,000 dilution, NB600-1192, Novous Biologicals, CO, USA).

[0195]

[0196] For protein detection, horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) were used for reaction at room temperature for 2 hours, and the luminescence signal was measured using Clarity Western ECL substrate (ATTO, Moto Asakusa, Tokyo, Japan).

[0197] The expression level of each protein was confirmed using the AE-9300 Ez-Capture MG Hours Image Saver HR image capture tool (WSE-7120L, ATTO, Tokyo, Japan).

[0198]

[0199] The human keratinocytes treated above were fixed with 4% (w / v) paraformaldehyde, permeabilized with phosphate-buffered saline (PBS) containing 0.1% (v / v) Triton X-100 (PBS-T), blocked with 1% (v / v) bovine serum albumin (BSA), and incubated overnight at 4°C with a primary antibody against Claudin-1 (#37-4900, 1:1000 dilution, Thermo-Fisher), an important marker substance for the cell barrier. After washing with PBS, Alexa Fluor 555 goat anti-mouse (Invitorgen) was diluted 1:1000 with the PBS mentioned above and incubated for 2 hours at room temperature as a secondary antibody, and the nuclei of the cells were stained with 1 μg / ml of Hoechst33342 dye (H3570, Thermo-Fisher) for 20 minutes at room temperature, and images of barrier formation were obtained using a fluorescence microscope IX71 (IX71, Olympus, Japan).

[0200]

[0201] As a result, as shown in Fig. 4, when treated with the stress-inducing substance PM2.5, the expression of Claudin-1 was significantly reduced and the barrier was formed abnormally, whereas when the dead cells of EF-2001 according to the present invention were added, it was confirmed that the barrier formation was noticeably improved along with an increase in the fluorescence expression of Claudin-1.

[0202]

[0203] Furthermore, this trend was confirmed in Western blot analysis, as shown in Figure 5, where it was confirmed that the expression of Claudin-1 protein, which was reduced by the stress-inducing substance PM2.5, was restored by treatment with EF-2001 dead cells. Considering these results, it can be confirmed that EF-2001 according to the present invention is effective in improving skin barrier function.

[0204]

[0205] Experimental Example 4. Evaluation of Wrinkle Improvement and Anti-Inflammatory Effects

[0206] The above human skin-derived fibroblasts were treated with dead cells of EF-2001 at various concentrations (0.01, 0.1, 1 mg / ml) together with the stress-inducing substance PM2.5 (30 μg / ml), and after 24 hours, cell lysates were prepared, and after protein quantification, protein changes were measured using a matrix metalloproteinase-1 (MMP-1) specific antibody by Western blot.

[0207]

[0208] As a result, as shown in Fig. 6, when the cells were treated with the stress-inducing substance PM2.5 (30 μg / ml), the expression of MMP-1, a collagen-decomposing enzyme, increased compared to the control group, whereas when the dead cells of EF-2001 according to the present invention were treated, the expression of MMP-1, which was increased by the stress-inducing substance PM2.5, was confirmed to be reduced to the control group level.

[0209]

[0210] In addition, Nuclear factor -κB (NF-κB) p65 is known as a major inflammatory transcription factor that regulates inflammatory responses, and the effect of EF-2001 according to the present invention on the activation of NF-κB in cells treated with PM2.5, a stress-inducing substance, was additionally confirmed.

[0211]

[0212] As a result, as shown in Fig. 7, it was confirmed that when the dead cells of EF-2001 according to the present invention were treated, the activation of NF-κB by the stress-inducing substance PM2.5 in human-derived fibroblasts was suppressed.

[0213]

[0214] In summary, these results suggest that EF-2001 according to the present invention can have an effect of improving wrinkles and soothing by activating the formation of primary cilia in fibroblasts and suppressing the expression of collagenase and the activation of inflammatory factors increased by the stress-inducing substance PM2.5.

[0215]

[0216] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their killed cells.

2. In paragraph 1, A composition for improving skin function, characterized in that the strain is Enterococcus faecalis EF-2001 strain.

3. In paragraph 1, A composition for improving skin function, characterized in that the composition activates primary cilia.

4. In paragraph 1, The composition above is a composition for improving skin function, characterized by inhibiting melanin production.

5. In paragraph 1, The composition above is a composition for improving skin function, characterized by promoting skin elasticity.

6. In paragraph 1, A composition for improving skin function, characterized in that the composition has an activity for improving skin barrier function.

7. In paragraph 1, The composition above is a composition for improving skin function, characterized in that it has a skin soothing effect.

8. A cosmetic for improving skin function, comprising at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their killed cells as an effective ingredient.

9. A health functional food for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their killed cells.

10. A food additive for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their killed cells.

11. An external preparation for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their killed cells.

12. A pharmaceutical composition for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Enterococcus faecalis strains, their lysates, their cultures, their concentrates, their dried products, and their killed cells.

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