Composition for improving skin function containing lactobacillus plantarum belp1 as active ingredient

Abstract

The present invention relates to a composition for improving skin function, containing Lactobacillus plantarum as an active ingredient and, specifically, to a composition having the effects of activating primary cilia, improving skin barrier function, soothing skin, reducing skin wrinkles, whitening skin, and inhibiting melanin production, the composition containing a Lactobacillus plantarum beLP1 strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, or dead cells thereof as an active ingredient.

Description

Composition for improving skin function containing Lactobacillus plantarum beLP1 as an active ingredient

[0001] The present invention relates to a composition for improving skin function containing Lactobacillus plantarum as an active ingredient, and more particularly, to a composition having primary cilia activation, skin barrier function improvement, skin soothing, skin wrinkle improvement, skin whitening and melanin production inhibition effects, which contains Lactobacillus plantarum beLP1 strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof or a killed cell thereof as an active ingredient.

[0002] The skin, the outermost organ of the human body, detects various external stimuli, maintains homeostasis, and protects the body. It is also known to function as an immune system, protecting the body from foreign substances.

[0003] Among the constituent layers of the skin, the epidermis prevents moisture loss from the body. Among the four layers of the epidermis - the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale - the intercellular lipids between the 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 skin function, loss of elasticity, wrinkle formation, and excessive melanin production. However, there are few solutions to control these issues.

[0004] 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.

[0005] These primary cilia detect external stimuli and 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.

[0006] 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.

[0007] 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.

[0008] In this regard, in the cosmetics field, lactic acid bacteria cultures have been used since their commercialization in 1955, and research is actively being conducted on raw material forms such as direct fermentation and filtration or extraction using Streptococcus, Lactobacillus, Lactococcus, Leuconostoc, and Bifidobacterium genera, and fermentation and then filtration or extraction by inoculating active materials.

[0009] Among them, microorganisms of the genus Lactobacillus are lactic acid bacteria that undergo homotypic or heterotypic fermentation and are commonly found in the intestines of animals, including humans, and in the fermentation process of dairy products and vegetables. Microorganisms of the genus Lactobacillus are known to maintain an acidic intestinal pH, inhibiting the growth of harmful bacteria such as E. coli and Clostridium, improving diarrhea and constipation, and playing a role in vitamin synthesis, anticancer effects, and lowering serum cholesterol [Michael and Philippe, Probiotics and prebiotics: Effects on diarrhea, The journal of nutrition, Volume 137, March 2007, pages 803S-811S; Roberfroid, Prebiotics and probiotics: Are they functional foods, American journal of clinical nutrition, Volume 71, June 2000, pages 1682S-1687S].

[0010] Technologies that utilize Lactobacillus spp. and are being applied to various purposes include Korean Patent Publication No. 10-1846796, “Cosmetics for improving elasticity or moisturizing containing whey obtained by fermentation with Korean-type lactic acid bacteria,” Korean Patent Publication No. 10-1492003, “Novel Lactobacillus plantarum HY7714 having skin wrinkle improvement and moisturizing effects and products containing the same as an active ingredient,” and Korean Patent Publication No. 10-1998067, “Bifidobacterium spp. GFC-B09 and fermented products manufactured using the same.”

[0011] However, the specific effects of Lactobacillus plantarum on improving skin function, such as activation of primary cilia, improvement of skin barrier function, skin soothing, improvement of skin wrinkles, skin whitening, and inhibition of melanin production, are not specifically known.

[0012] Against this backdrop, the inventors of the present invention have made efforts to develop a material capable of improving overall skin functions, such as improving skin barrier function, soothing skin, improving skin wrinkles, and having skin whitening effects, by activating primary cilia. As a result, they have confirmed that Lactobacillus plantarum beLP1 strain, its lysate, its culture, its concentrate, its dried product, or its dead cells improve skin barrier function, soothing skin, improving skin wrinkles, and having skin whitening effects by activating primary cilia, and have completed the present invention.

[0013] [Prior Art Literature]

[0014] (Patent Document 0001) Korean Patent Registration No. 10-1846796

[0015] (Patent Document 0002) Korean Patent Registration No. 10-1998067

[0016] 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 Lactobacillus plantarum strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an effective ingredient.

[0017] Another object of the present invention is to provide a composition for improving skin function that activates primary cilia, improves skin barrier function, has skin soothing effects, skin wrinkle improvement effects, skin whitening effects, and has the effect of inhibiting melanin production.

[0018] Another object of the present invention is to provide a cosmetic for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof.

[0019] Another object of the present invention is to provide a food for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of Lactobacillus plantarum strain, its lysate, its culture, its concentrate, its dried product, and its dead cells.

[0020] Another object of the present invention is to provide a food additive for improving skin function, which comprises at least one selected from the group consisting of Lactobacillus plantarum strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an effective ingredient.

[0021] The purpose of the present invention is not limited to what has been described above, and other purposes not mentioned will be clearly understood by those skilled in the art from the description below.

[0022] In order to achieve the above purpose, the present invention provides a composition for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof.

[0023] In addition, the present invention provides a composition for improving skin function, characterized in that the strain is a Lactobacillus plantarum beLP1 strain deposited under the deposit number KCTC15902BP.

[0024] In addition, the present invention provides a composition for improving skin function, characterized in that the strain is a Lactobacillus plantarum beLP1 strain including a DNA sequence encoding a 16S rRNA sequence of sequence number 1.

[0025] In addition, the present invention provides a composition for improving skin function, characterized in that the composition activates primary cilia.

[0026] In addition, the present invention provides a composition for improving skin function, characterized in that the composition improves skin barrier function.

[0027] In addition, the present invention provides a composition for improving skin function, characterized in that the composition has a skin soothing effect.

[0028] In addition, the present invention provides a composition for improving skin function, characterized in that the composition has a skin wrinkle improvement effect.

[0029] In addition, the present invention provides a composition for improving skin function, characterized in that the composition has a skin whitening effect.

[0030] In addition, the present invention provides a composition for improving skin function, characterized in that the composition inhibits melanin production.

[0031] 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 a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof.

[0032] In addition, the present invention provides a food for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof.

[0033] In addition, the present invention provides a food additive for improving skin function, which comprises as an effective ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof.

[0034] A composition for improving skin function, comprising as an active ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof, according to the present invention, exhibits the efficacy of activating primary cilia.

[0035] In addition, a composition for improving skin function, comprising as an active ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof, according to the present invention, exhibits effects of improving skin barrier function, soothing skin, and improving skin wrinkles.

[0036] In addition, a composition for improving skin function, comprising at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof, as an active ingredient, exhibits a skin whitening effect by inhibiting melanin production by inhibiting the activity of a melanin production enzyme.

[0037] Therefore, the composition for improving skin function can be usefully used in cosmetics for improving skin function, foods for improving skin function, health functional foods for improving skin function, and food additives for improving skin function.

[0038] Figure 1 shows the morphology of the colony of the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0039] Figure 2 shows scanning electron microscope (SEM) photographs of the live cell form and heat-treated dead cell form of the Lactobacillus plantarum beLP1 strain (accession number: KCTC15902BP) according to the present invention.

[0040] Figure 3 is a graph showing the cell growth curve of the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0041] Figure 4 is a graph showing the heat lethality curve of the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0042] Figure 5 is an image showing the hemolysis of the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0043] Figure 6 is an image showing whether or not biogenic amines are produced by the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0044] Figure 7 shows the sugar utilization confirmed using the API CHL50 kit (50300, bioMerieux) for the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0045] Figure 8 is an image showing whether the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention decomposes bile salts.

[0046] Figure 9 is a graph showing the cytotoxicity of the Lactobacillus plantarum beLP1 strain (Accession No.: KCTC15902BP) according to the present invention.

[0047] Figure 10 shows the production rate of primary cilia and the length of primary cilia by treatment of the culture solution and dead cells of Lactobacillus plantarum beLP1 according to Experimental Example 1 of the present invention.

[0048] Figure 11 shows the expression of Claudin-1 protein by treatment of a culture solution and dead cells of Lactobacillus plantarum beLP1 according to Experimental Example 2 of the present invention.

[0049] Figure 12 shows the expression of p-NF-κ MMP-1 and ACTA1 by treatment of a culture solution and dead cells of Lactobacillus plantarum beLP1 according to Experimental Example 3 of the present invention.

[0050] Figure 13 shows the activity of tyrosinase by measuring the amount of DOPA chrome produced by treating the culture solution, culture lysate, and dead cells of Lactobacillus plantarum beLP1 according to Experimental Example 4 of the present invention.

[0051] 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.

[0052] 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.

[0053] Unless otherwise specifically defined in the specification, 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.

[0054] The present invention provides, as an embodiment, a composition for improving skin function, comprising as an effective ingredient at least one selected from the group consisting of a Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof.

[0055] In the present invention, the term “crushed product” means a product obtained by crushing the Lactobacillus plantarum strain of the present invention by a known method.

[0056] The term "cultivation" in the present invention refers to growing the Lactobacillus plantarum strain of the present invention under appropriately controlled environmental conditions. The culturing process of the present invention can be performed using appropriate 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.

[0057] For example, the cultivation of the Lactobacillus plantarum strain of the present invention may be performed 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 in a nutrient medium (NA medium), but is not limited thereto. In addition, the cultivation 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.

[0058] In the present invention, the term "culture medium" means a culture medium obtained by culturing the Lactobacillus plantarum strain, and may be a culture medium containing the strain or a cell-free culture medium from which the bacterial cells have been removed.

[0059] In one specific example of the present invention, the culture may be a culture medium obtained by culturing the Lactobacillus plantarum beLP1 strain in a liquid culture medium, and specifically, the culture solution of the Lactobacillus plantarum beLP1 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 general Lactobacillus culture, as well as components that synergistically affect the growth of Lactobacillus, and the composition accordingly may be easily selected by a person having ordinary skill in the art. In addition, the form of the culture may be liquid or solid, and is not limited to either.

[0060] In the present invention, the term "concentrate" refers to a culture of the strain concentrated by a conventional method. The concentrate may be a concentrate obtained by concentrating a liquid culture medium in which the Lactobacillus plantarum beLP1 strain is cultured by a conventional method, but the nature of the culture is not limited to a liquid.

[0061] 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 obtained by drying a liquid culture medium in which the Lactobacillus plantarum beLP1 strain is cultured by a conventional method, but the nature of the culture is not limited to a liquid.

[0062] In addition, in the present invention, the Lactobacillus plantarum strain or its dead cells may be any one manufactured by a known manufacturing method, and are non-toxic and harmless to the human body.

[0063] In addition, the above-described dead cells can be manufactured by heat-treating corresponding live cells or treating them with formalin or other sterilizing agents, and the dead cells can be used even if they are substantially dead. In addition, the above-described dead cells can be obtained by washing a strain obtained by culturing it by a conventional method, dehydrating it by centrifugation, repeating washing and dehydration as necessary, and then suspending it in distilled water, saline solution, etc., and heating the suspension at, for example, 60 to 140°C for 1 to 60 minutes, thereby obtaining a dead cell suspension or a dried product thereof, or by irradiating the above-described dead cell suspension with gamma rays or neutron rays, thereby obtaining a dead cell suspension or a dried product thereof.

[0064] The drying method for the above-mentioned dead cell suspension is not particularly limited as long as it is a known drying method, and examples thereof include spray drying and freeze drying. In some cases, enzyme treatment, surfactant treatment, and grinding / pulverizing treatment may be performed before or after sterilization treatment by heating or the like, or before or after drying treatment, and products obtained through such treatments are also included in the dead cell suspension of the present invention. In addition, the dead cell suspension may be manufactured by the following methods, but is not limited thereto:

[0065] 1) A step of culturing Lactobacillus plantarum beLP1 as a starter culture, and then culturing the main culture 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;

[0066] 2) A step of drying and powdering the Lactobacillus plantarum beLP1 cultured in the above step 1) by heat treating it at a temperature of 60 to 140°C for 1 to 60 minutes, more preferably at a temperature of 80 to 115°C for 5 to 30 minutes.

[0067] As another embodiment of the present invention, the Lactobacillus plantarum strain may be the Lactobacillus plantarum beLP1 strain deposited under the deposit number KCTC15902BP.

[0068] The above strain is a strain having the biochemical properties of the sugar fermentation pattern of Table 7 and the 16S rDNA of sequence number 1, and is a strain newly identified by the present inventors who confirmed that there is no identical strain as a result of 16S rDNA examination.

[0069] The term "Lactobacillus" in the present invention refers to a microorganism belonging to the genus Lactobacillus, which is aerobic or facultatively anaerobic, Gram-positive bacilli, widely distributed in nature. Microorganisms belonging to the genus Lactobacillus include Lactobacillus plantarum. The present inventors have deposited and provided a novel strain belonging to Lactobacillus plantarum under the accession number KCTC15902BP, and this is named Lactobacillus plantarum beLP1. This is a probiotic strain, is harmless to the human body, and can be used without side effects.

[0070] In the present invention, the term "probiotics" refers to live bacteria that enter the body and have a beneficial effect on health. Most probiotics known to date have been consumed as fermented milk products made using lactic acid bacteria such as Lactobacillus, but recently, fermented milk, granules, powders, etc. containing some strains such as Bifidobacterium and Enterococcus in addition to Lactobacillus have been sold. The Lactobacillus plantarum beLP1 of the present invention may also be used in the form of fermented milk, granules, powders, etc., but is not limited thereto.

[0071] The Lactobacillus plantarum beLP1 strain of the present invention was obtained by isolation from "kimchi." The morphological characteristics of the strain of the present invention are that it is a Gram-positive rod and has a 16s rDNA base sequence of sequence number 1. Analysis of the base sequence revealed that it had 99% homology with Lactobacillus plantarum. Accordingly, the inventors of the present invention deposited the newly isolated Lactobacillus plantarum beLP1 strain with the Korea Research Institute of Bioscience and Biotechnology (KRIBB), a depository institution under the Budapest Treaty, on May 7, 2024, under the accession number KCTC15902BP.

[0072] The Lactobacillus plantarum beLP1 strain of the present invention can be cultured using a conventional Lactobacillus strain culture method. A natural medium or a synthetic medium can be used as the medium. For example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used as a carbon source of the medium, and peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate, and other organic or inorganic nitrogen-containing compounds can be used as a nitrogen source, but are not limited to these components. Inorganic salts included in the medium can be magnesium, manganese, calcium, iron, potassium, etc., but are not limited to these. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and related compounds can be added to the medium. The culture temperature conditions for the novel isolated strain of the present invention can be cultured at a temperature range of 20 to 40°C for 12 hours to 4 days.

[0073] Specifically, the culture medium of the newly isolated strain may be a culture stock solution containing the cells, or may be a cell obtained by removing or concentrating the culture supernatant. The composition of the culture medium may additionally include components that act synergistically on the growth of the lactobacillus in addition to the components required for the typical lactobacillus culture, and the composition accordingly may be easily selected by a person skilled in the art.

[0074] In addition, the state of the strain may be a liquid state or a dry state, and the drying method may include, but is not limited to, ventilation drying, natural drying, spray drying, and freeze drying.

[0075] The concentration of Lactobacillus plantarum beLP1 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 3CFU / 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 10 CFU / 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.

[0076] In order to stably preserve the Lactobacillus plantarum beLP1 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.

[0077] As another embodiment of the present invention, the composition for improving skin function may have the effect of activating primary cilia.

[0078] 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.

[0079] Additionally, according to one embodiment of the present invention, the activation of the primary cilia can be confirmed by measuring the primary cilia production rate and the length of the primary cilia.

[0080] Specifically, when the culture solution or dead cells of Lactobacillus plantarum of the present invention were treated to human skin-derived fibroblasts treated with TNF-α, it was confirmed that the production rate of primary cilia and the length of primary cilia were restored to a normal level or higher, thereby indicating the efficacy of activating primary cilia by the Lactobacillus plantarum strain of the present invention.

[0081] As another embodiment of the present invention, the composition for improving skin function may have the effect of improving skin barrier function.

[0082] 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. Therefore, improvement in skin barrier function can be confirmed by measuring the expression of Claudin-1, a major membrane protein of the epithelial barrier.

[0083] Specifically, when the culture solution or dead cells of Lactobacillus plantarum of the present invention were treated to human skin-derived keratinocytes treated with TNF-α, it was confirmed that barrier formation was noticeably improved along with an increase in Claudin-1 fluorescence expression, and from this, the efficacy of improving skin barrier function by the Lactobacillus plantarum strain of the present invention could be known.

[0084] As another embodiment of the present invention, the composition for improving skin function may have a skin soothing effect. Furthermore, the composition for improving skin function may have a skin wrinkle-improving effect.

[0085] Nuclear factor -κB (NF-κB) p65 is known as a major inflammatory transcription factor that regulates inflammatory responses. Therefore, the skin soothing effect of Lactobacillus plantarum according to the present invention can be confirmed through the activation of NF-κB in TNF-α-treated cells. In addition, the wrinkle improvement effect of Lactobacillus plantarum according to the present invention can be confirmed through the expression of MMP-1, a collagen-decomposing enzyme.

[0086] Specifically, when the culture solution of Lactobacillus plantarum or the killed cells of Lactobacillus plantarum according to the present invention is treated, it can be confirmed that the activation of NF-κB by TNF-α in human-derived fibroblasts is suppressed, thereby indicating the skin soothing effect of the Lactobacillus plantarum strain of the present invention. In addition, when the culture solution of Lactobacillus plantarum or the killed cells of Lactobacillus plantarum according to the present invention is treated, it can be confirmed that the expression of MMP-1 increased by TNF-α is reduced, thereby indicating the skin wrinkle improvement effect of the Lactobacillus plantarum strain of the present invention.

[0087] As another embodiment of the present invention, the composition for improving skin function may have a skin whitening effect and may also inhibit melanin production.

[0088] When the tyrosinase enzyme in skin melanocytes becomes active due to ultraviolet rays or the surrounding environment, tyrosine (or DOPA) changes into a melanin polymer through an auto-oxidation reaction through the intermediate DOPA chrome production pathway. By measuring the production of the intermediate DOPA chrome using L-DOPA as a substrate, melanin production and skin whitening efficacy can be confirmed.

[0089] Specifically, when the culture solution, culture lysate or dead cells of Lactobacillus plantarum according to the present invention are treated, it can be confirmed that the amount of DOPA chrome produced is reduced in a concentration-dependent manner, and from this, the melanin production inhibition effect and skin whitening effect by the Lactobacillus plantarum strain of the present invention can be known.

[0090] In addition, a method for producing a composition comprising the Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof or a killed cell thereof may include a step of mixing at least one selected from the group consisting of the Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof and a killed cell thereof with an additive.

[0091] The above composition may be a cosmetic composition, a food composition, a health functional food composition, or a food additive composition.

[0092] The Lactobacillus plantarum 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 primary cilia activation effect, skin barrier improvement effect, skin soothing effect, skin wrinkle improvement effect, skin whitening effect and melanin production inhibition effect, and can be usefully used in cosmetics for improving skin function, foods for improving skin function, health functional foods for improving skin function, or food additives for improving skin function.

[0093] As one embodiment of the present invention, the present invention provides a cosmetic comprising at least one selected from the group consisting of the Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof as an active ingredient.

[0094] In addition, since the method for obtaining the Lactobacillus plantarum 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 cosmetic is described below.

[0095] The ingredients included in the cosmetics 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.

[0096] 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.

[0097] The cosmetic of the present invention can be manufactured in any formulation commonly manufactured in the art, and for example, can be formulated as 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 in the form 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] In addition, as an embodiment of the present invention, the present invention provides a food for improving skin function, a health functional food for improving skin function, or a food additive comprising at least one selected from the group consisting of the Lactobacillus plantarum strain, a lysate thereof, a culture thereof, a concentrate thereof, a dried product thereof, and a dead cell thereof as an effective ingredient.

[0103] Since the method for obtaining the above Lactobacillus plantarum 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, and only the unique composition of food, health functional food and food additive is described below.

[0104] There is no particular limitation on the type of food to which the Lactobacillus plantarum 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.

[0105] The amount of Lactobacillus plantarum mixed according to the present invention can be appropriately determined depending on its intended use (prevention or improvement). Generally, the amount of Lactobacillus plantarum in a food 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.

[0106] The health functional composition of the present invention contains the Lactobacillus plantarum 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.

[0107] 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 Lactobacillus plantarum of the present invention may contain fruit pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion 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 the Lactobacillus plantarum of the present invention.

[0108] 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.

[0109] <Isolation and selection of strains>

[0110] 1-1. Sample acquisition and strain isolation

[0111] The target strain used in the present invention, Lactobacillus plantarumbeLP1, is characterized as a novel strain of Lactobacillus plantarum isolated and identified from “kimchi,” a traditional Korean food.

[0112] Kimchi samples were roughly filtered through a 160 μm sieve, and the filtrate was diluted with sterile saline. The obtained samples were serially diluted and inoculated onto MRS solid medium (288210, Difco® Lactobacilli MRS Agar), and the plates were incubated at 37°C for 48 hours.

[0113] A single colony was picked from the plate and streaked onto a new MRS agar plate. The plate was incubated at 37°C for 36 h. Each colony was then subcultured up to three times for bacterial purification. Isolated colonies were used for Gram staining, catalytic assays, and genetic identification using 16S rRNA. Lactobacillus plantarum beLP1 strain was further cultured in MRS broth (Laboratorios Conda, Spain) and stored in a 20% (v / v) glycerol solution at -80°C until further use.

[0114] The morphology of the colonies of the strains cultured above is shown in Figure 1.

[0115] Specifically, the shape of the colony of the cultured strain is circular, the elevation is convex, the surface is smooth and glossy, and the color is milky white.

[0116] 1-2. Strain morphology

[0117] The live cell form and heat-treated dead cell form of Lactobacillus plantarumbeLP1 (Lactobacillus plantarumbeLP1), the target strain of the present invention, were subjected to SEM (Scanning Electron Microscope) measurement at the Chuncheon Center of the Korea Basic Science Institute, and the cell surface and size of the strain were confirmed.

[0118] The specific form is as shown in Fig. 2.

[0119] As described above, it was confirmed that the Lactobacillus plantarumbeLP1 strain of the present invention has a rounded end and a straight shape, and a length of 2-7 μm.

[0120] That is, the morphological characteristics of the Lactobacillus plantarumbeLP1 strain were as follows: Gram staining showed that it was Gram positive, and electron microscopy confirmed that it was a rod-shaped bacterium (Fig. 2).

[0121] 1-3. Strain identification

[0122] To more accurately identify the strain, a molecular phylogenetic method using DNA base sequences was performed. The strain was identified by performing 16S rRNA sequence analysis on colonies grown on MRS solid medium (Difco™ Lactobacilli MRS Agar) to MACROGEN Co., Ltd. The primers used were 785F (5'-GGA TTA GAT ACC CTG GTA-3'), 907R (5'-CCG TCA ATT CMT TTR AGT TT-3'), 27F (5'-AGA GTT TGA TCM TGG CTC AG-3'), and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3)'. As a result of base sequence analysis, it was confirmed that the strain has up to 99% homology with strains of the genus Lactobacillus, and the base sequence was confirmed as follows.

[0123] Using the Megablast program, a BLAST search was performed between the type material base sequence of the rRNA / ITS database (16S ribosomal RNA sequences) and the 16S rRNA gene base sequence of the experimental strain.

[0124] The experimental strain that had 99.67% identical 16S rRNA base sequences to L. plantarum type strain KCKM 1170 (Accession No. OR436370.1) was designated as beLP1, and the taxonomic type of L. plantarum beLP1 is shown in Table 1.

[0125] Taxonomic group Classification Bacteria Phylum Sclerotomycota Class Bacilli Order Lactobacillus Family Lactobacillus Family Genus Lactobacillus Species Lactiplantibacillus plantarum Strain Lactiplantibacillus plantarumbeLP1

[0126] The isolated strain was named "Lactobacillus plantarum beLP1". The novel microorganism thus identified was deposited with the Korea Research Institute of Bioscience and Biotechnology, an international depository under the Budapest Treaty, as Lactobacillus plantarum beLP1 on May 7, 2024, and was assigned the accession number KCTC15902BP. * Base sequence of Lactobacillus plantarum beLP1 strain

[0127] CTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCA GTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTAC AACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTTGAAGTCGC

[0128] 1-4. Cell growth curve

[0129] For the purpose of preculture, colonies grown on MRS solid medium were cultured overnight (37°C, 100 rpm) with shaking in MRS liquid medium (Difco™ Lactobacilli MRS Broth) and the OD value was adjusted to 1.0. New MRS liquid medium was added to a dedicated tube for RTS-1C (Biosan), and 1% of the preculture solution was inoculated. Cell growth of the strain was confirmed for 24 hours using RTS-1C (Biosan).

[0130] The cell growth curve confirmed above is as shown in Fig. 3.

[0131] 1-5. Thermal Death Curve

[0132] The heat lethality test was performed using a heat treatment method. 10 6 beLP1 with double density ® The cultured bacteria were heat-treated at 115°C for 30 minutes, the conditions for the cell-killing process. During the process, samples were collected four times at 10-minute intervals, at 0, 10, 20, and 30 minutes, spread on agar plates, and cultured for 24 hours to determine the number of colonies formed (CFU).

[0133] To obtain the thermal death rate (D value), the following procedure was followed. Lactobacillus plantarum beLP1 was cultured on MRS medium at 37°C for 24 hours, centrifuged at 6,700×g for 10 minutes, and the obtained bacteria were cultured in phosphate-buffered saline (PBS) at 50, 60, 70, 80, and 90°C for 3 hours. After inoculating each sample onto MRS agar medium, the medium was cultured at 37°C for 48 hours, and the number of surviving bacteria was counted at 10-minute intervals. At this time, 2.5×10 per square -4 mm 3The bacteria were directly counted using a Neubauer counting chamber (Superior, Germany) having a size of . The results are shown in Table 2 and Fig. 4.

[0134] Heat treatment time (min) Viable cell count, CFU / mL10 1 10 2 10 3 10 4 10 5 10 6 10 7 0TNTCTNTCTNTCTNTCTNTCTNTC231TNTCTNTCTNTC32NGNGNG2TNTCTNTCTNTC45NGNGNG437NGNGNGNGNGNG638NGNGNGNGNGNG885NGNGNGNGNGNGNG1098NGNGNGNGNGNGNG150NGNGNGNGNGNGNG200NGNGNGNGNGNG250NGNGNGNGNGNG300NGNGNGNGNGNGNG

[0135] * Only use media showing colonies in the range of 25-250* TNTC, difficult to count* NG, no colonies found

[0136] Decimal reduction time (D value) means the time for sterilization to reach 90% at a specific temperature, D 115 = t × (logN0- logN f ) can be obtained through the formula. At this time, D 115 is the D value at 115℃, t is the time of exposure to heat (minutes), N0 is the initial amount of bacteria, N f is the number of surviving bacteria after exposure to heat. The D value of beLP1 was calculated starting from the sample before heat treatment, and D 115 =1.862 minutes (100.02 seconds) was obtained. Meanwhile, log 10 The value decreased significantly from 8.3 to 2.9 over 10 minutes, and no surviving bacteria were found after 10 minutes.

[0137] 1-6. Antibiotic sensitivity test

[0138] Lactobacillus plantarum beLP1 live cells stored at -80℃ in 50% glycerol were cultured at 37℃ for 48 hours. Before conducting the sensitivity test, a single colony was selected, inoculated into MRS liquid medium, and cultured at 37℃ for 24 hours.

[0139] Products to be used for sensitivity testing E-strips (ETEST) ® The antibiotic sensitivity ranges of ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tylosin, tetracycline, and chloramphenicol were measured using strips; bioMιrieux Inc.

[0140] The results are shown in Table 3 below. Specifically, the MIC (minimum inhibitory concentration) values ​​of Lactobacillus plantarum beLP1 were the same or lower than the values ​​indicated by EFSA, except for vancomycin, gentamicin, kanamycin, and streptomycin, which were measured to be high (see Table 3). Although live bacteria harbor antibiotic resistance, it was confirmed that heat-treated Lactobacillus plantarum beLP1 did not have any resistance to antibiotics.

[0141] AntibioticMIC (μg / mL)EFSA Cut-off value (μg / ml)Ampicillin0.192Vancomycin*>10242Gentamycin>102432Kanamycin>10241024Streptomycin>1024128Erythromycin24Clindamycin1.54Tylosin44Tetracycline34Chloramphenicol616

[0142] 1-7. Hemolytic test

[0143] To evaluate the hemolytic activity of Lactobacillus plantarum beLP1 under controlled laboratory conditions, Lactobacillus plantarum beLP1 was cultured in MRS broth for 20 h and then streaked on 5% sheep blood agar plates (MB cell, Seoul, Korea). The standard strains Staphylococcus aureus ATCC 6538 and Streptococcus pneumoniae ATCC 6305 were used as a hemolytic positive control and α-hemolytic positive control, respectively. β-, α-, or γ-hemolytic activity was determined after 48 h of incubation at 37°C.

[0144] As a result, L. plantarum beLP1 inoculated onto a 5% sheep blood agar plate exhibited γ-hemolysis, while the control strain, ATCC 6538, exhibited β-hemolysis and colorless areas around the colonies (see Fig. 5). Likewise, amino acid decarboxylase was not detected in Lactobacillus plantarum beLP1 under the test conditions.

[0145] 1-8. Biogenic amine production test

[0146] Excessive intake of biogenic amines can cause various adverse effects in the human body, and the level of biogenic amine contamination in foods is often used as an indicator of microbial spoilage caused by enzymatic amino acid decarboxylation. Therefore, we investigated whether Lactobacillus plantarum beLP1 produces biogenic amines through amino acid decarboxylation.

[0147] To induce decarboxylase before this experiment, the test bacteria were cultured on six types of MRS solid media, and 1% of precursor amino acids (L-arginine, L-histidine, L-lysine, L-ornithine, L-tryptophan, and L-tyrosine) were added to each medium. Once activated, the test bacteria were streaked onto new decarboxylase media with 1% of each precursor amino acid added, and cultured at 37℃ for 96 hours under aerobic conditions while observing the color change of the solid media. Media without added amino acids were used as a negative control. When biogenic amines are produced and react, the media turn purple.

[0148] As a result, it was confirmed that the Lactobacillus plantarum beLP1 strain did not produce biogenic amines under the test conditions (see Fig. 6).

[0149] 1-9. Investigation of biochemical characteristics of strains

[0150] In order to analyze the biochemical characteristics of the Lactobacillus plantarumbeLP1 strain, Lactobacillus plantarumbeLP1 was cultured at 37°C for 48 hours using the API 50CHL kit (50300, bioMerieux), and the color change of the reagent was confirmed to confirm the sugar utilization of Lactobacillus plantarumbeLP1 (Fig. 7).

[0151] Specifically, the bacteria cultured overnight on MRS medium were centrifuged and mixed well with 10 mL of API 50 CHL medium, and the bacterial density was 2.0 x 10 8 After standardizing to CFU / mL, the samples were divided into individual sugar groups. Fermentation patterns were observed for 48 hours at 37°C, and color changes due to sugar fermentation were systematically evaluated.

[0152] As a result, the utilization of monosaccharides (D-glucose, D-lactose, D-fructose, D-mannose, D-arabinose, D-ribose), sugar alcohols (D-mannitol, D-sorbitol), and disaccharides (D-lactic acid, D-cellobiose, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-gentibiose, D-turanose) of Lactobacillus plantarum beLP1 was confirmed (see Fig. 7).

[0153] 1-10. Evaluation of bile salt decomposition

[0154] Bile salt degradation is an indicator of the potential response of bacteria inhabiting the human digestive tract. This test examines the ability of Lactobacillus plantarum beLP1 to hydrolyze bile salts by culturing it on MRS agar medium supplemented with 0.5% taurodeoxycholic acid (TDCA).

[0155] Specifically, Lactobacillus plantarum beLP1 strains were streaked onto MRS solid medium supplemented with 0.5% taurodeoxycholic acid and then cultured at 37℃ in an anaerobic environment mimicking the conditions inside the gastrointestinal tract for 24 hours. The medium allowed us to determine whether white colonies were formed by Lactobacillus plantarum beLP1 strains, and by measuring the diameter of the formed colonies in detail, we could determine how much bile salt decomposition had progressed.

[0156] According to the test results, it was confirmed that no white colonies were found around Lactobacillus plantarum beLP1 on MRS solid medium supplemented with 0.5% taurodeoxycholic acid. In other words, it was confirmed that Lactobacillus plantarum beLP1 has no bile salt decomposition ability (negative) (see Figure 8).

[0157] 1-11. D-lactic acid production evaluation

[0158] D-lactic acid forms a stereoisomer with L-lactic acid, and unlike L-lactic acid, it is very difficult for the human body to metabolize. The higher the D-lactic acid content in the gastrointestinal tract, the more likely it is to cause intestinal microbiota imbalances and acidosis, including abdominal pain, vomiting, and diarrhea. D-lactic acid is particularly prone to accumulating in people with certain health conditions, such as short bowel syndrome or gastrointestinal diseases. The D-lactate Assay Kit (Colorimetric) was used to analyze lactic acid produced by Lactobacillus plantarum beLP1.

[0159] As shown in Table 4, we were able to confirm that the Lactobacillus plantarum beLP1 strain produced significantly lower levels of D-lactic acid (average 0.29 nmol / μL). In other words, we were able to confirm that, under certain conditions, Lactobacillus plantarum beLP1 did not produce D-lactic acid at levels that could be harmful to the human body.

[0160] Sample L-lactic acid [nmol] D-lactic acid [nmol / uL] Mean standard deviation beLP1 A 2 7.86 0.28 0.29 0.02 beLP1 B 3 0.88 0.31

[0161] 1-12. Genetic stability test

[0162] Lactobacillus plantarum beLP1 was inoculated and cultured in 10 mL of MRS liquid medium and cultured under the same conditions from the first to the 25th generation. A genetic stability test was performed on the corresponding generations, and whole genome sequencing (WGS) was analyzed using the de novo assembly method only using the Illumina platform (Dierckxsens, Mardulyn et al., 2017). The Orthologous Average Nucleotide Identity (OrthoANI) value of the first and 25th generations showed 99.92% similarity (see Table 5), confirming that Lactobacillus plantarum beLP1 is genetically stable.

[0163] Taxonomic name L. plantarum (beLP1) strain ID1 st G25 th G Status Draft Draft Genome completeness 9999 Genome size (bp) 3,155,686 3,177,108 GC content (%) 44.62 44.71 Contig count 3274 CDS count 2924 2924 RNA gene count 5858 Homology between the 1st and 25th generations of beLP1 through OrthoANI analysis 99.92%

[0164] 1-13. Cell safety testing

[0165] Lactate dehydrogenase (LDH) is an enzyme present in the cytoplasm. Normally, it cannot pass through the cell membrane and is not released from the cell. However, when the cell membrane is damaged or the cell dies, it is released into the medium. Accordingly, the amount of LDH in the medium is proportional to the number of dead or damaged cells. Based on this characteristic of LDH, the LDH assay can confirm cytotoxicity by measuring the absorbance at 490 nm of LDH released from cells using the water-soluble tetrazolium dye WST-8.

[0166] Quantified HT-29 cells were seeded at 1× 10 in a 96-well plate. 5 Cells / well were dispensed and cultured in an incubator at 37°C and 5% CO2, and then Lactobacillus plantarum beLP1 strain (10 7 ~ 10 9 CFU) were inoculated and cultured for 24 hours. The experimental groups were positive control group: Cell lysis solution treatment group, negative control group: Vehicle treatment group, beLP1 strain (10 7 ~ 10 9The cells were treated with 100 CFU (CFU) group, and cytotoxicity was calculated using the Quanti-LDH™ PLUS Cytotoxicity Assay Kit (BCT-LDHP500, BIOMAX). The cytotoxicity was calculated using the following formula. The results are shown in Fig. 9.

[0167] Cytotoxicity(%) = [(Test sample - Negative control) / (Positive control - Negative control)] × 100

[0168] From the above experimental results (Fig. 9), it was confirmed that the Lactobacillus plantarum beLP1 strain had no cytotoxicity.

[0169] <Manufacturing Example>

[0170] Manufacturing Example 1. Preparation of culture solution of Lactobacillus plantarum beLP1

[0171] Lactobacillus plantarum beLP1 live bacteria (Berm 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 3.0 × 10 12 The main culture was performed to reach a cell count of ≥ cfu / g. 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 Lactobacillus plantarum beLP1.

[0172] Manufacturing Example 2. Production of dead cells of Lactobacillus plantarum beLP1

[0173] Lactobacillus plantarum beLP1 cultured in the above manufacturing example 1 was heat treated at 80 to 115°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.

[0174] <Experimental Example>

[0175] Experimental Example 1. Evaluation of Primary Cilia Activation

[0176] 1-1. Cell line preparation and culture

[0177] Human fibroblasts from skin (GM00969 Corriell Institute) and keratinocytes (HaCat) were cultured in Dulbecco's modified Eagle's medium (DMEM) 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.

[0178] 1-2. Treatment with Lactobacillus plantarum beLP1

[0179] To confirm the activity of primary cilia formation by Lactobacillus plantarum beLP1 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). 4After culturing the cells for 24 hours, from the time point when the cells reached approximately 90% confluence, the cells were treated with a control group containing only the medium, Tumor Necrosis Factor-alpha (TNF-α; 20 ng / ml) alone, or a culture solution (0.1, 1, 5%) of Lactobacillus plantarum beLP1 and killed cells (0.01, 0.1, 1 mg / ml) at each concentration in the medium containing TNF-α for 24 hours. 2-isopropylmalic acid (2-IPMA, 100 μM) was used as a positive control.

[0180] 1-3. Evaluation of primary cilia activation by Lactobacillus plantarum beLP1

[0181] To confirm the primary cilia activation effect of Lactobacillus plantarum beLP1, human skin-derived fibroblasts were treated with simple medium without Lactobacillus plantarum beLP1 (labeled L-Media), culture medium of Lactobacillus plantarum beLP1 (labeled L-Sub.), and cell culture medium containing killed Lactobacillus plantarum beLP1 cells (labeled L-Powder), alone or together with TNF-α, at various concentrations for 24 h. 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).

[0182] Here, the production rate of primary cilia was 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 was measured as the actual length of primary cilia observed under a microscope.

[0183] As a result, as shown in Fig. 10, when TNF-α is treated, primary cilia formation is inhibited, whereas when the culture or dead cells of Lactobacillus plantarum beLP1 are treated together with TNF-α, it was confirmed that primary cilia formation is restored to a normal level or higher, as shown in Fig. 10.

[0184] Experimental Example 2. Evaluation of Improvement in Skin Barrier Function

[0185] 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.

[0186] In order to evaluate the effect of Lactobacillus plantarum beLP1 according to the present invention on improving the skin barrier, human keratinocytes were treated with TNF-α (20 ng / ml) and a culture solution of Lactobacillus plantarum beLP1 (denoted as L-Sub.) and killed cells of Lactobacillus plantarum beLP1 (denoted as L-Powder) at various concentrations, 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.

[0187] 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.

[0188] 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).

[0189] Other strains were used as experimental groups along with the beLP1 strain (Table 6).

[0190] Classification Contents Example 1 beLP1 culture L. plantarum Use the supernatant after sonication and centrifugation of the beLP1 strain culture Example 2 beLP1 dead cells Use the dead cells after heat treatment, centrifugation, and drying of the beLP1 strain culture Comparative Example 1 L. plantarum Use a commercially available L. plantarum strain Comparative Example 2 L. paracasei Use a strain from another species of Lactobacillus Comparative Example 3 B. longum Use a Bifidobacterium strain

[0191] As a result, as shown in Fig. 11, when TNF-α was treated, the expression of Claudin-1 was significantly reduced and the barrier was formed abnormally, whereas when the culture solution or dead cells of Lactobacillus plantarum beLP1 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 (Table 7).

[0192] Claudin-1 fluorescence expression intensity (relative value, % compared to control) Negative control (TNF-α treatment only) 100% Example 1 152% ± 7.4 Example 2 149% ± 6.9 Comparative example 1 18% ± 5.2 Comparative example 2 12% ± 5.0 Comparative example 3 110% ± 4.6

[0193] Referring to Table 7, in the cell groups treated with Example 1 (beLP1 culture supernatant) and Example 2 (beLP1 dead cell supernatant), the expression of Claudin-1 was significantly increased compared to the TNF-α only treatment group, and both treatment groups showed similar levels of fluorescence intensity.

[0194] On the other hand, although Claudin-1 expression increased somewhat in the treatment groups of Comparative Examples 1, 2, and 3, the effect was significantly lower than in Examples 1 and 2.

[0195] The above results suggest that cultures and dead cells derived from the Lactobacillus plantarumbeLP1 strain can effectively restore the expression of Claudin-1 in the skin barrier damaged by TNF-α, thereby exhibiting superior physiological activity compared to conventional lactic acid bacteria.

[0196] Experimental Example 3. Evaluation of skin anti-inflammatory and wrinkle improvement effects.

[0197] The above human skin fibroblasts were treated with a culture solution of Lactobacillus plantarum beLP1 or dead cells of Lactobacillus plantarum beLP1 at various concentrations together with TNF-α, and after 24 hours, cell lysates were prepared, proteins were quantified, and changes in proteins were measured using phospho-Nuclear factor-κB (NF-κB) p65 and matrix metalloproteinase-1 (MMP-1) specific antibodies by Western blot.

[0198] 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;

[0199] Actin (ACTA1; 1:10,000 dilution, MAB1501, Millipore, 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).

[0200] 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).

[0201] 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).

[0202] Nuclear factor -κB (NF-κB) p65 is known as a major inflammatory transcription factor that regulates inflammatory responses. The effect of Lactobacillus plantarum beLP1 according to the present invention on the activation of NF-κB in TNF-α treated cells was confirmed.

[0203] The experimental group was set up the same way as in Experimental Example 2 (Table 6).

[0204] As a result, as shown in FIG. 12 and Table 8, it was confirmed that when the culture solution of Lactobacillus plantarum beLP1 or the dead cells of Lactobacillus plantarum beLP1 according to the present invention were treated, the activation of NF-κB by TNF-α in human-derived fibroblasts was inhibited.

[0205] In addition, as shown in Fig. 12, when the cells were treated with TNF-α, the expression of MMP-1, a collagen-decomposing enzyme, increased compared to the control group, whereas when the culture solution of Lactobacillus plantarum beLP1 or the dead cells of Lactobacillus plantarum beLP1 according to the present invention were treated, it was confirmed that the expression of MMP-1, which was increased by TNF-α, decreased.

[0206] Phospho-NF-κp65 expression (% compared to TNF-α) MMP-1 expression (% compared to TNF-α) TNF-α treatment group (control) 100% 100% Example 145% ± 3.8 43% ± 4.1 Example 248% ± 4.2 46% ± 3.9 Comparative example 179% ± 5.0 76% ± 5.2 Comparative example 285% ± 4.7 82% ± 4.5 Comparative example 388% ± 4.9 84% ± 5.0

[0207] In summary of the above results, these results confirm that Lactobacillus plantarum beLP1 according to the present invention is effective in soothing skin and improving wrinkles by activating the formation of primary cilia in fibroblasts, thereby inhibiting the activation of inflammatory factors increased by TNF-α and the expression of collagenase.

[0208] In addition, compared to the control group treated under the same conditions, the culture and dead cells derived from Lactobacillus plantarumbeLP1 more effectively suppressed the expression of phospho-NF-κp65 and MMP-1, and the recovery of Claudin-1 expression was also significantly superior to the control group.

[0209] The above results clearly demonstrate that the Lactobacillus plantarumbeLP1 strain is an effective strain with specific and enhanced physiological activity in skin barrier protection, inflammation relief, and anti-aging effects compared to general lactic acid bacteria, thereby supporting the technical differentiation and practical efficacy of the present invention.

[0210] Experimental Example 4. Evaluation of the inhibitory effect of melanin production enzyme activity.

[0211] When the tyrosinase enzyme in skin melanocytes becomes activated by ultraviolet rays or the surrounding environment, tyrosine (or DOPA) changes into a melanin polymer through an auto-oxidation reaction through the intermediate production step of DOPA chrome.

[0212] In the present invention, the production of DOPA chrome, an intermediate step, was measured using L-DOPA as a substrate.

[0213] A 96-well plate was filled with 40 μl of samples of L-ascorbic acid (100 μg / ml) as a positive control, culture medium or culture lysate of Lactobacillus plantarum beLP1 (1, 10, 20%), and dead cells of Lactobacillus plantarum beLP1 (1000, 2000, 5000 μg / ml), and 120 μl of 8.3 mM L-DOPA dissolved in 50 mM sodium phosphate buffer (pH 6.8). 40 μl of mushroom tyrosinase (125 unit / ml) was mixed, and the mixture was incubated at 37°C for 20 minutes under light-shielding conditions. The absorbance was measured at 490 nm using a microplate reader.

[0214] The experimental group was set up as shown in Table 9.

[0215] ClassificationExperimental group DescriptionTreatment Sample typeTreatment method SummaryExample 1 beLP1 cultureCulture supernatantStrain culture → centrifugation (culture supernatant with live cells removed)Example 2 beLP1 culture homogenateCultureStrain culture → sonication (disintegration) (culture containing culture and bacterial lysate)Example 3 beLP1 dead cellsDead cell supernatantStrain culture → heat treatment → centrifugation and medium removal → drying (dead cell powder)Comparative Example 1 Commercial L. plantarum culture supernatantCommercial strain powder → culture → culture supernatant obtained after processing in the same mannerComparative Example 2 L. paracasei culture supernatantProcessed in the same mannerComparative Example 3 B. longum culture supernatantProcessed in the same manner

[0216] Referring to Fig. 13, vc represents L-ascorbic acid, and the culture solution of Lactobacillus plantarum beLP1 represents a solution in which live bacteria are removed after culturing Lactobacillus plantarum (beLP1).

[0217] In addition, in the case of the culture lysate of Lactobacillus plantarum beLP1, it refers to a solution obtained by culturing Lactobacillus plantarum (beLP1) and then homogenizing the culture and live cells with a homogenizer. The dead cells of Lactobacillus plantarum beLP1 refer to a dried powder-like substance obtained by heat-treating Lactobacillus plantarum (beLP1).

[0218] Classification Tyrosinase activity (%) Inhibition rate (%) Negative control (NO / C) 1000 Positive control (L-AA 100 μg / ml) 7030 Example 1 (beLP1 culture solution 20%) 3565 Example 2 (beLP1 culture lysate 20%) 3070 Example 3 (beLP1 dead cells 5000 μg / ml) 8515 Comparative example 1 (commercial L. plantarum) 8713 Comparative example 2 (L. paracasei) 8515 Comparative example 3 (B. longum) 8317

[0219] Referring to Fig. 13 and Table 10, it was confirmed that the amount of DOPA chrome produced decreased in a concentration-dependent manner in the culture medium and culture lysate groups of Lactobacillus plantarum beLP1, and it was also confirmed that the dead cell group of Lactobacillus plantarum beLP1 decreased in a concentration-dependent manner.

[0220] From this, it can be confirmed that Lactobacillus plantarum beLP1 is effective in skin whitening by inhibiting the activity of melanin production enzyme.

[0221] In particular, the culture medium and culture lysate of Lactobacillus plantarumbeLP1 showed a significantly superior tyrosinase activity inhibition effect compared to existing general lactic acid bacteria strains (L. plantarum, L. paracasei, B. longum), suggesting that metabolites or effective ingredients specifically produced in the Lactobacillus plantarumbeLP1 strain directly contribute to the inhibition of melanin biosynthesis.

[0222] In addition, since a certain level of inhibitory effect was observed even in dead cells derived from Lactobacillus plantarumbeLP1, the present invention can exhibit a certain level of efficacy not only in the form of live cells but also in the form of dead cells, thereby providing advantageous application possibilities in terms of formulation stability and preservation.

[0223] The above results clearly support the technological differentiation of the beLP1 strain-derived culture solution or culture lysate as an effective ingredient for skin whitening functional compositions, demonstrating a significant superiority in melanin production inhibition efficacy compared to existing strains.

[0224] 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.

[0225] [Accession number]

[0226] Name of depositor: Korea Research Institute of Bioscience and Biotechnology (KCTC)

[0227] Accession number: KCTC15902BP

[0228] Date of acceptance: 20240507

[0229] [Correction pursuant to Rule 91, July 30, 2025]

Claims

1. A composition for improving skin function, comprising at least one selected from the group consisting of Lactobacillus plantarum strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an active ingredient.

2. In paragraph 1, A composition for improving skin function, characterized in that the strain is a Lactobacillus plantarum beLP1 strain deposited under the deposit number KCTC15902BP.

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

4. In paragraph 1, The above composition is a composition for improving skin function, characterized in that it improves skin barrier function.

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

6. In paragraph 1, The composition is a composition for improving skin function, characterized in that it has the effect of improving skin wrinkles.

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

8. In paragraph 1, The above composition is a composition for improving skin function, characterized in that it inhibits melanin production.

9. A cosmetic for improving skin function, comprising at least one selected from the group consisting of Lactobacillus plantarum strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells as an active ingredient.

10. A food for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Lactobacillus plantarum strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells.

11. A food additive for improving skin function, comprising as an active ingredient at least one selected from the group consisting of Lactobacillus plantarum strains, their lysates, their cultures, their concentrates, their dried products, and their dead cells.

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