WO2020129888A1 - Skin care agent containing plant-derived lactobacillus - Google Patents

Abstract

The present invention addresses the problem of providing a superior stratum corneum formation promoter and hyaluronic acid synthesis promoter for enhancing a beautifying effect by maintaining skin health and preventing wrinkles. Provided is a skin care agent, preferably a hyaluronic acid synthesis promoter, a stratum corneum formation promoter, or a skin resident flora balance regulator. The skin care agent includes, as an active ingredient, bacterial cells of Lactobacillus casei subsp. casei 327 deposited under accession number NITE PB-0270.

Description

Skin care agent containing plant lactic acid bacteria Cross reference

This application claims priority based on Japanese Patent Application No. 2018-235695 filed in Japan on December 17, 2018, and all contents described in the application are incorporated herein by reference in their entirety. Is used by. Further, the contents described in all patents, patent applications and documents cited in the present application are incorporated herein by reference in their entirety.

The present invention relates to a skin care agent containing a plant lactic acid bacterium, more specifically, a hyaluronic acid synthesis promoter containing a 327 strain of Lactobacillus casei subsp. casei, a horny layer formation promoter, and And/or a skin care agent such as a balance-adjusting agent for skin flora.

Recently, it has become clear that lactic acid bacteria have various functions such as immunostimulatory action, and lactic acid bacteria are attracting attention. For example, the anti-allergic effect of Lactobacillus paracasei K71 strain, which is a plant-derived lactic acid bacterium (Patent Document 1), the antioxidant function of a specific strain of Lactobacillus casei of Brassicaceae plants (Patent Document 2), microalgae or its treatment A collagen production promoter (Patent Document 3) and the like containing a fermented product obtained by culturing a specific lactic acid bacterium in a medium containing a product as an active ingredient have been reported. As a skin care action of lactic acid bacteria, the present inventors have also reported that a specific strain derived from Lactobacillus casei has a function of improving skin moisture by ingestion (see Non-Patent Document 1).

━ Hyaluronic acid is used as an ingredient in cosmetics and pharmaceuticals for the purpose of smoothing the skin and smoothing out wrinkles. Hyaluronic acid is a high molecular polysaccharide present in the epidermis and dermis of the skin, cartilage, synovial fluid, etc., retention of water in the intercellular space, retention of cells based on the formation of a jelly-like matrix in tissues, It has many functions such as maintaining lubricity and flexibility of tissues, resistance to external force such as mechanical damage, and prevention of bacterial infection. Hyaluronic acid, together with collagen, is present in large amounts in the extracellular matrix in the skin, particularly in the dermis, and has a very high ability to retain water, and is deeply involved in the freshness, firmness and elasticity of the skin. Hyaluronic acid is produced by hyaluronic acid synthase (Hyaluronan Synthase (HAS)). It is known that HAS1, HAS2, and HAS3 exist as mammalian hyaluronan synthase. HAS2 is a major hyaluronan synthase in the human dermis and is said to be involved in the synthesis of high molecular weight hyaluronan with high moisturizing power.

Filaggrin is a type of basic protein produced in epidermal granule cells. Filaggrin plays an important role with keratin in forming the stratum corneum, which is essential for the barrier function of the skin. Involucrin is a highly reactive, water-soluble protein present in keratinocytes and epidermis and serves as a substrate for transglutaminase. It first appears in the cytoplasm and then undergoes cross-linking polymerization under the action of transglutaminase. Eventually, it becomes an insoluble protein and contributes to the formation of a cornified envelope (CE). The cornified envelope is an organelle that plays a major role in the barrier function of the horny layer. Thus, filaggrin and involucrin are known as important proteins in the skin barrier function.

Japanese Patent No. 5150722 JP, 2005-156832, A JP, 2005-117234, A

The object of the present invention is to provide, for example, a better horny layer formation promoter and hyaluronic acid synthesis promoter for maintaining the health of the skin and suppressing wrinkles to enhance the cosmetic effect.

In order to achieve the above object, the present inventors examined the function of plant lactic acid bacteria using epidermal keratinocytes, and promoted the synthesis of hyaluronic acid by adding a specific lactic acid bacterium, and a horny layer forming factor. The present invention has been completed by finding that the expression is promoted. That is, the present invention includes the following embodiments.
(1) A skin care agent containing, as an active ingredient, cells of Lactobacillus casei subsp. casei strain 327 deposited under the deposit number NITE BP-02707.
(2) The skin care agent according to (1), which is a hyaluronic acid synthesis promoter, a stratum corneum formation promoter, or a balance-adjusting agent for skin flora.
(3) The skin care agent according to (1) or (2), wherein the cells are dead cells.
(4) The skin care agent according to any one of (1) to (3), which is in the form of a skin external preparation.
(5) To prevent or improve the formation of wrinkles, the increase of skin pH and/or the decrease of skin water content, which comprises applying the skin care agent according to any one of (1) to (4) to the skin. Non-therapeutic method.
(6) A method for improving skin condition, which is intended for a composition containing as an active ingredient a bacterium of Lactobacillus casei subsp. casei strain 327 deposited under accession number NITE BP-02707 A method comprising applying to the skin of a person.
(7) The method according to (6), wherein the improvement of the skin condition is promotion of hyaluronic acid synthesis, promotion of stratum corneum formation, or balance adjustment of skin-indigenous bacteria.
(8) The method according to (6) or (7) for preventing or improving the formation of wrinkles on the skin of the subject, the increase in skin pH, and/or the decrease in skin water content.
(9) Lactobacillus casei subspecies deposited under accession number NITE BP-02707 for producing a composition for promoting the synthesis of hyaluronic acid or keratinization in the skin, or for adjusting the balance of skin indigenous bacteria Use of Lactobacillus casei subsp. casei strain 327 strains.
(10) Lactobacillus casei deposited under Accession No. NITE BP-02707 for producing a composition for preventing or improving skin wrinkle formation, skin pH increase and/or skin moisture decrease. Use of strain 327 strain Lactobacillus casei subsp. casei strain.

FIG. 1A shows changes in the expression level of β-defensin 3 in epidermal keratinocytes measured by the RT-PCR experiment of Example 1. FIG. 1B also shows the change in the expression level of β-defensin 3 depending on the addition concentration of lactic acid bacteria. FIG. 2A shows changes in the expression level of involucrin in epidermal keratinocytes measured by the RT-PCR experiment of Example 1. FIG. 2B also shows changes in the expression level of filaggrin. FIG. 3A shows changes in the expression level of HAS2 in epidermal keratinocytes measured by the RT-PCR experiment of Example 1. FIG. 3B also shows the change in the expression level of HAS2 depending on the addition concentration of lactic acid bacteria. FIG. 4A shows changes in the expression level of HAS3 in epidermal keratinocytes measured by the RT-PCR experiment of Example 1. FIG. 4B also shows the change in the expression level of HAS3 depending on the addition concentration of lactic acid bacteria. FIG. 5 shows changes in the production amount of β-defensin 3 depending on the addition concentration of lactic acid bacteria in the culture supernatant of epidermal keratinocytes measured by the ELISA method of Example 1. FIG. 6 shows changes in the amount of hyaluronic acid synthesized in the culture supernatant of epidermal keratinocytes measured by the ELISA method of Example 1. FIG. 7 shows the effect of lactic acid bacteria on the growth of Staphylococcus epidermidis measured in Example 2.

Hereinafter, preferred embodiments of the present invention will be described in the following order.
(I) Vegetable lactic acid bacterium (II) Skin care agent and its action (III) Use (IV) Cosmetic method

(I) Plant lactic acid bacterium The plant lactic acid bacterium used in the present invention is isolated from plant-derived ones including processed foods such as miso, soy sauce, pickles, bran, grass, rice, wheat and malt, and utilizes sugars and the like. It is a lactic acid bacterium that produces lactic acid. Preferably, lactic acid bacteria belonging to the genus Lactobacillus separated from fermented foods made from rice can be used. For example, a plurality of plant lactic acid bacteria which have been isolated from rice and processed rice products and have antimutagenicity have been reported (Journal of Food Science and Engineering, Vol. 48, No. 9, pages 693 to 696, 2001). .. Among these, the plant lactic acid bacterium used as the active ingredient of the present invention is most preferably the subsp. casei strain 327 or a mutant strain thereof. The term "mutant strain" as used herein refers to a specific strain mutated by a method well known to those skilled in the art within a range that does not change its properties, or a person skilled in the art would say that it is equivalent thereto. It is meant to include what can be confirmed.

In addition, Lactobacillus casei subspecies casei (Lactobacillus casei subsp. casei) 327 strains are independent administrative agency product evaluation technology foundation mechanism, patent microorganism deposit center with May 8, 2018 (Hara deposit day) It has been deposited at (Rooms 2-5-8, 122, Kazusa, Kamasa, Kisarazu, Chiba Prefecture, 292-0818, Japan). The deposit number is NITE BP-02707. (Hereinafter, this strain is referred to as "K-1 strain").

The lactic acid bacterium used as an active ingredient of the present invention may be either live or dead bacterium, but dead bacterium is preferably used in consideration of prevention of offensive odor due to growth of live bacterium, for example. More preferably, a heat-sterilized bacterium obtained by sterilizing the lactic acid bacterium by a known heat treatment means is used. The heat-sterilized bacterial cells are obtained by culturing the lactic acid bacteria using an MRS medium (manufactured by Difco) according to a conventional method, for example, by collecting the bacterial cells by a method such as filtration or centrifugation, After washing with water, suspension in water or the like, heat treatment at 120° C. or lower (preferably 80 to 120° C.) for 30 minutes (3 seconds to 30 minutes), and then concentration and drying if necessary can be performed. Alternatively, the bacterial cells may be prepared by baking and steaming (for example, 170° C. or lower for 60 minutes or less). The lactic acid bacterium of the K-1 strain is available as "plant lactic acid bacterium K-1" from Rice Research Institute of Kameda Seika Co., Ltd. Therefore, in the present invention, such a commercially available product may be used as the lactic acid bacterium used as the active ingredient.

(II) Skin Care Agent and Its Action In the present specification, the “skin care agent” means a preparation or composition having a skin care action, containing the bacterial cells of the K-1 strain as an active ingredient. Here, "skin care" typically refers to skin as a barrier against environmental effects such as dust, chemicals, microorganisms, and loss of endogenous substances such as water, natural fats and electrolytes in skin tissues. Refers to the enhancement or reproduction of natural functions. In the present invention, more specific embodiments of the "skin care agent" include "hyaluronic acid synthesis promoter", "corneal layer formation promoter" and "skin resident flora balance modifier" and the like. Not limited. In addition, the dead cells used in the preferred embodiment can not only expect the effects such as the above-mentioned skin care action, but in the case of live cells, there is a possibility that morphological changes occur at the time of delivery and display after the product is manufactured. On the other hand, killed cells that do not cause further morphological changes can be preferably used.

The "hyaluronic acid synthesis promoter" is not limited to the mechanism thereof, as long as it is a preparation or composition that promotes the synthesis of hyaluronic acid mainly in dermal fibroblasts and epidermal keratinocytes, or synovial cells, etc. It is preferable to promote the synthesis of hyaluronan by promoting the expression of genes encoding HAS2 and HAS3, which are animal hyaluronan synthases. Therefore, the skin care agent of the present invention can be used not only as a hyaluronic acid synthesis promoter, but also as a hyaluronan synthase gene expression promoter, particularly, as an expression promoter of HAS2 gene or HAS3 gene. it can. INDUSTRIAL APPLICABILITY The hyaluronic acid synthesis promoter of the present invention can prevent the functional deterioration of various disorders, diseases, diseases and the like due to the decrease of hyaluronic acid in the body, especially in the skin. For example, it promotes moisturization of the skin to improve firmness and elasticity of the skin and moisturizes it, prevents or improves skin troubles such as rough skin, wrinkles, and roughness, and prevents joint disorders and joint ointment damage. It may have an improving effect. In particular, the hyaluronic acid synthesis promoter of the present invention is a main enzyme that synthesizes dermal hyaluronic acid, and promotes the gene expression of HAS2, which is said to produce high molecular hyaluronic acid having relatively high moisturizing property among hyaluronic acid synthases. By doing so, a high beauty effect can be achieved.

The term "horny layer formation promoter" promotes keratinization of epidermal cells, promotes the formation of a healthy horny layer, and acts to keep the horny layer barrier function to protect external stimuli and the living body healthy. As a result, it is considered that the preparation or composition has the effect of improving the water-retaining ability of the skin, the prevention of the conspicuous pores, the prevention of the skin aging such as the formation of wrinkles and the reduction of the texture pattern. The stratum corneum formation-promoting agent of the present embodiment promotes stratum corneum formation via promotion of involucrin expression and/or filaggrin expression, and thus can be used as an expression promoter of these proteins or genes.

“A skin balance flora balance regulator” is a preparation or composition that maintains the skin flora present in healthy skin and imparts a barrier function to prevent the invasion of pathogens from the outside. When the skin flora is out of balance, overgrowth of indigenous bacteria and invasion and proliferation of other harmful bacteria occur, and various skin symptoms are induced. Staphylococcus epidermidis, which is one of the indigenous bacteria of the skin, has an antagonistic action against pathogenic bacteria, such as Staphylococcus aureus, and can prevent the growth of such harmful bacteria. The action of promoting the growth of staphylococci is considered to be one of the functions as a balance adjusting agent for the skin flora. Further, it may be a preparation or composition containing a substance that does not affect the growth of Staphylococcus epidermidis and the like and has an antibacterial action that suppresses the growth of harmful Staphylococcus aureus and the like. An example of such antibacterial substance is an antimicrobial peptide called defensin. This peptide, which consists of 18 to 45 amino acids and contains 6 to 8 conserved cysteine residues, is present in cells of the immune system such as neutrophils and epithelial cells, and functions by binding to the cell membrane of microorganisms. It is said. Β-Defensin 3 present in human epithelial cells is a peptide consisting of 45 amino acid residues. It has a broad spectrum antibacterial spectrum and is effective against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, as well as vancomycin-resistant Enterococcus faecalis.

(III) Applications (various compositions)
When the skin care agent of the present invention is used, for example, in the form of various compositions for external preparations for skin (external medicines, cosmetics, etc.), cells of the K-1 strain as an active ingredient are cultured according to a conventional method for lactic acid bacterium culture, What is separated from the obtained culture by means such as centrifugation for collecting cells can be used as it is. Further, the culture/fermentation solution (culture supernatant), a crudely purified product or a purified product of the culture, a freeze-dried product thereof, or a cytoplasm or a cell wall fraction obtained by treating bacterial cells with an enzyme or a physical means. Can be used.

In the composition as one embodiment, the bacterial cell or its culture as an active ingredient can be used as it is, but it is prepared in the form of an external preparation for skin or the like by appropriately mixing a pharmaceutically acceptable carrier and the like. Is preferred.

Note that the composition of the preferred embodiment may contain lactic acid bacteria in a killed cell form, and when commercializing the composition, conditions other than heating such as pressurization may be appropriately used.

The amount of the K-1 strain bacterial cells to be added to the composition of the present embodiment is generally around 10 ⁸ to 10 ¹¹ cells in 100 g of the composition (they do not have to be viable cells). In the case of containing dead cells, the number of viable cells before sterilization is to be counted. The same applies hereinafter), and can be appropriately selected. The viable cell count is calculated by applying a diluted sample to an agar medium for bacterial culture, culturing at 37° C., and counting the number of grown colonies. Since the viable cell count and turbidity correlate, if the correlation between the viable cell count and turbidity is obtained in advance, the viable cell count is counted by measuring the turbidity instead of measuring the viable cell count. it can. The form of each composition will be specifically described below.

(External skin preparation)
When the skin care agent of the present invention is used as an external preparation composition for cosmetics, external medicines, quasi drugs, etc., it is generally prepared by using a suitable pharmaceutically acceptable pharmaceutical carrier together with the active ingredient of the present invention. It is prepared in the form of an external preparation composition for practical use. Such pharmaceutical carriers include, for example, humectants such as glycerin, petrolatum, urea, hyaluronic acid, heparin; PABA derivatives (paraaminobenzoic acid, escarol 507, etc.), cinnamic acid derivatives (neoheliopan, parsol MCX, Sungard B, etc.), UV light such as salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (parsol A, parsol DAM, etc.), heterocyclic derivatives (tinuvin series, etc.), titanium oxide, etc. Absorbent/scattering agent; disodium edetate, trisodium edetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, gluconic acid, and other sequestering agents; salicylic acid Sebum inhibitors such as sulfur, caffeine and tannin; bactericidal and disinfectant such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate; diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizinic acid and its salts , Anti-inflammatory agents such as glycyrrhizic acid derivatives and glycyrrhetinic acid; vitamin A, vitamin B group (B1, B2, B6, B12, B15), folic acid, nicotinic acids, pantothenic acids, biotin, vitamin C, vitamin D group (D2, Vitamins such as D3), vitamin E, ubiquinones, and vitamin K (K1, K2, K3, K4); aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, Amino acids such as pyrrolidonecarboxylic acid and derivatives thereof; Retinol, tocopherol acetate, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, ellagic acid, placental extract and other whitening agents; butylhydroxytoluene, butylhydroxyanisole, gallic acid Antioxidants such as propyl acid; astringents such as zinc chloride, zinc sulfate, zinc carbonate, zinc oxide, potassium aluminum sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, lactitol; Licorice, chamomile, horse chestnut, Yukinoshita, peony, karin, various plant extracts such as sardine, saccharum, sedum, sardine, ginkgo leaves, etc., oily components, surfactants, thickeners, alcohols, powder components, pigments, etc. Is mentioned.

These are the types of products that are to be added, and the processes that are normally performed according to the form (for example, crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, pulverization). , Granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc. are arbitrarily selected and combined, and various materials may be arbitrarily selected and used.

In addition, when an extract (crude medicine) from various plants or an additive derived from an animal-based material is applied to the external preparation of the present embodiment, it includes protection of skin and hair, moisturizing, improvement of touch/feel, and flexibility. Applying, relieving irritation, relieving stress caused by fragrance, activating cells (preventing cell aging), suppressing inflammation, improving skin quality/hair quality, preventing rough skin and improving it, hair growth, hair growth, hair loss prevention, gloss impartation, In addition to the cleansing effect, fatigue reduction, blood flow promotion, hot bath effect, and other cosmetic effects, effects such as scenting, deodorization, thickening, antiseptic, and buffering can be expected.

In addition to this, in addition to the skin care effect which is the objective of the present invention, it is expected that various raw material materials that have been known so far have various cosmetic and medicinal effects, and by combining these, a multifunctional effect is obtained. It is also possible to make a product that expects a positive effect.

Specific examples of the external preparation composition include cosmetic creams, emulsions, lotions, packs, skin milk (emulsion), gels, powders, lip balms, lipsticks, undermake-ups, foundations, sun care, bath agents, Examples thereof include body shampoo, body rinse, soap, cleansing foam, ointment, patch, jelly and aerosol.

(IV) Cosmetic Method In another embodiment of the present invention, a non-therapeutic method for preventing or ameliorating wrinkles, which comprises applying to the skin a skin care agent containing the bacterial cells of the K-1 strain as an active ingredient. A method is provided. This cosmetic method includes applying a skin care agent, which is preferably in the form of an external preparation for skin, to the skin, and is preferably applied continuously for at least 1 week or more. By using the cosmetic method according to the present embodiment, it becomes possible to more effectively reduce wrinkles when the skin care agent of the present invention is used. “Continuously applied” means applied so that the bacterial cells of the K-1 strain remain at least on and/or in the skin, and may be applied once to several times a day or It also includes a method of applying every few days.

In the cosmetic method according to the present embodiment, the skin care agent according to the present invention needs to be continuously applied for at least one week, but it is also possible to apply continuously for a longer period. As described above, the skin care agent according to the present invention is a composition mainly containing plant lactic acid bacteria, and thus has high safety and is excellent in long-term continuous use.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit% of the numerical value indicating the added amount of the K-1 strain bacterial cells means% by mass.

[Example 1] Functional evaluation test of K-1 strain bacterial cells using epidermal keratinocytes (outline of test method)
Differentiation-induced epidermal keratinocytes (NHEK) were cultured using EpiLife (Thermo Fisher Scientific) supplemented with an antibiotic (Gibco™ Antibiotic-Antimycotic (100X)) and supplement S7. K-1 strain was cultivated in MRS medium at 37°C for 24 hours, and then centrifugally washed (3,000 rpm, 10 minutes) with physiological saline under cooling at 4°C four times. A 10% by mass suspension was prepared and sterilized by an autoclave. K-1 strain cells were mixed with the differentiation-induced epidermal keratinocytes (NHEK) at a predetermined concentration, and after culturing for 24 hours, DNA was extracted from the collected epidermal keratinocytes. Changes in the expression levels of β-defensin 3, corneal factor, and hyaluronan synthase were analyzed by RT-PCR. On the other hand, the cell supernatant after culturing was collected, and the amount of synthesized β-defensin 3 and hyaluronic acid secreted in the culture supernatant was quantified using an ELISA method.

(RT-PCR experimental method)
Epidermal keratinocytes (NHEK cells) were cultured in a 24-well plate at 37° C. in a CO ₂ incubator until the cell density became confluent. Calcium was added to this so that the final concentration would be 1 mM, and differentiation was induced for 24 hours. Lactic acid bacteria K-1 cells were added to the cells after the induction of differentiation so that the mass ratio was 0.004%, 0.02%, and 0.1%, respectively, and the cells were further cultured for 24 hours. MRNA was purified from the cultured cells. For the purification of mRNA, QIAshreder and RNeasy Mini Kit sold by QIAGEN were used.

Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix sold by TaKaRa to synthesize cDNA. RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 1 below, and changes in the expression level were analyzed by relative quantification. For the RT-PCR, TB Green Premix Ex Taq sold by TaKaRa was used. In addition, the amplification result of RPS18 (ribosome protein S18) was used as a reference for the relative amount change analysis.

The results are shown in Figures 1-4. As shown in FIG. 1, the expression level of β-defensin 3 was significantly increased by adding 0.1% of the K-1 strain bacterial cells (FIG. 1A). It was dependent on body concentration (see FIG. 1B). In addition, FIG. The value shown by 1A was 1.839813 in the 0.1% K-1 strain bacterial cell concentration group, while the control was 1. FIG. The values shown in 1B are 1.45807 for the control group of 0.004% K-1 strain cell concentration, 1.589222 for the 0.02% K-1 strain cell concentration group, and 0. The 1% K-1 strain bacterial cell concentration group was 1.594858. In FIG. 2, similarly, the expression levels of involucrin (FIG. 2A) and filaggrin (FIG. 2B) were increased by adding 0.1% of K-1 strain cells. In addition, FIG. The value shown by 2A was 1.31613 for the 0.1% K-1 strain cell concentration group, while the control was 1. FIG. The value shown by 2B was 1.316234 in the 0.1% K-1 strain cell concentration group, while control was 1.

On the other hand, as shown in FIGS. 3 and 4, it was found that the expression of HAS2 and HAS3 was increased by adding 0.1% of K-1 strain cells. In addition, FIG. The value shown by 3A was 1.335735 for the 0.1% K-1 strain cell concentration group, while the control was 1. FIG. The values shown in 3B are 1.07819 for the 0.1% K-1 strain cell concentration group, 1.073654 for the 0.02% K-1 strain cell concentration group, and 0. The 1% K-1 strain cell concentration group was 1.289125. FIG. The value shown by 4A was 2.034563 for the 0.1% K-1 strain cell concentration group, while the control was 1. FIG. The values shown by 4B are 1.0 control for the 0.004% K-1 strain cell concentration group, 1.2045497 for the 0.02% K-1 strain cell concentration group, and 0. The 1% K-1 strain bacterial cell concentration group was 1.344873.

(Quantification of β-defensin 3 and hyaluronic acid)
Epidermal keratinocytes (NHEK cells) were cultured in a 24-well plate at 37° C. in a CO ₂ incubator until the cell density became confluent. Calcium was added to this so that the final concentration would be 1 mM, and differentiation was induced for 24 hours. Lactic acid bacteria K-1 was added to the cells after differentiation induction at a mass ratio of 0.004%, 0.02%, and 0.1%, respectively, and the cells were further cultured for 24 hours. Β-Defensin 3 and hyaluronic acid were quantified from the culture supernatant after culturing. For detection of β-defensin 3, an ELISA kit sold by PEPROTECH, Human BD-3 Mini ELISA Development Kit, was used, and the experimental method was performed according to the instruction manual. For quantification of hyaluronic acid released in the cell culture supernatant, Hyaluronan Quantification Kit sold by Cosmo Bio Co., Ltd. was used. The experimental method was according to the instruction manual.

The results are shown in FIGS. 5 and 6. As shown in FIG. 5, an increase in β-defensin 3 production was observed depending on the bacterial cell concentration of the added K-1 strain. The values (pg/ml) shown in FIG. 5 are 332.782 (pg/ml) for control, and 432.162 (pg/ml), 0 for the 0.004% K-1 strain bacterial concentration group. The 0.02% K-1 strain bacterial cell concentration group was 587.386 (pg/ml), and the 0.1% K-1 strain bacterial cell concentration group was 590.719 (pg/ml). In FIG. 6, the concentration of hyaluronic acid in the culture supernatant was significantly increased by adding 0.1% of the K-1 strain. The value (ng/ml) shown in FIG. 6 is 106.179 (ng/ml) for the 0.1% K-1 strain bacterial concentration group, while the control is 70.937 (pg/ml). It was
From these results, the K-1 strain plays a role of protection against the infection of pathogenic microorganisms from the skin by the antibacterial action of β-defensin 3 and also regulates the balance of the indigenous flora of the skin. It is considered to have an action of maintaining the barrier function. Further, it is considered to have the action of promoting the production of hyaluronic acid in the skin to maintain the freshness, firmness and elasticity of the skin to extend wrinkles.

[Example 2] Effect of strain K-1 on growth of Staphylococcus epidermidis Staphylococcus epidermidis standard strain (ATCC 12228) was mixed with bacterial cells of strain K-1 at various concentrations, and 37 After culturing at 24° C. for 24 hours, a part of the culturing medium was used as Nutribouillon No. 2 (Nissui Pharmaceutical Co., Ltd.) plates were seeded and cultured at 37° C. for 2 days. The number of colonies formed on the plate was counted, and the assimilation of the K-1 strain by Staphylococcus epidermidis was examined. For the cells of the K-1 strain, 10% by mass of dead cell suspension was prepared in the same manner as in Example 1, and the final concentrations were 0.000%, 0.025%, and 0.02% by mass, respectively. It was diluted with pure water so as to be 050% and 0.100%. For Staphylococcus epidermidis, a frozen sample having a concentration of 2.67×10 ⁹ CFU/mL was thawed and diluted with water so that the final concentration was 3×10 ⁶ CFU/mL.

4 types of concentration x 3 sets of bacterial cells of K-1 strain and ultrapure water were dispensed into a 24-well microplate. The diluted Staphylococcus epidermidis was dispensed into this and cultured at 37° C. for 1 day. After 24 hours from the start of the culture, each of the 12 samples was pipetted into 1.5 mL of Tube. After diluting this 50 times with pure water, 20 μL of each was sampled to obtain Nutrient Broth No. The mixture was dropped on 2 plates, spread with a conradi stick, and cultured at 37° C. for 2 days. The results of counting the number of colonies formed on the plate after culturing are shown in Table 2 below. FIG. 7 shows the viable cell concentration of Staphylococcus epidermidis after 24 hours of culture after mixing with K-1 strain cells.

The viable cell concentration (×10 ⁵ CFU/mL) shown in FIG. 7 was calculated as follows.
First, the average number of Staphylococcus epidermidis colonies in each concentration group of K-1 strains shown in Table 2 (the average number of colonies of Staphylococcus epidermidis counted in sets 1 to 3) was calculated. 0.000% K-1 strain cell concentration group is 0, 0.025% K-1 strain cell concentration group is 9.3, 0.050% K-1 strain cell concentration group is 24.3 , And the 0.100% K-1 strain bacterial cell concentration group was 104.7.

Next, the viable cell concentration shown in FIG. 7 was calculated by the following formula.
X=(2.5×10 ³ )×N (Equation 1)
(X is the viable cell concentration shown in FIG. 7, N is the average number of Staphylococcus epidermidis colonies in each K-1 strain cell concentration group)

The viable cell concentration (×10 ⁵ CFU/mL) shown in FIG. 7 is 0.23±0.09×10 ⁵ CFU/mL for the 0.025% K-1 strain cell concentration group when the control is 0. The 0.05% K-1 strain cell concentration group was 0.61±0.17×10 ⁵ CFU/mL, and the 0.1% K-1 strain cell concentration group was 2.62±0.70×10. It was ⁵ CFU/mL. Values of ± (±0.09, ±0.17, ±0.70) are standard deviation values.

In addition, Formula 1 was set as follows. First, after culturing a solution containing Staphylococcus epidermidis for 24 hours, the concentration of Staphylococcus epidermidis contained in the solution was defined as X (CFU/mL). Next, the concentration of X (CFU/mL) was diluted to 1/50. The concentration after dilution was X/50 (CFU/mL). 2×10 ⁻² mL of a solution containing this diluted Staphylococcus epidermidis was prepared. The number of Staphylococcus epidermidis contained in this prepared solution was (X/50)×2×10 ⁻² (CFU). Supposing that N colonies were obtained by spreading this number of Staphylococcus epidermidis on the medium, (X/50)×2×10 ⁻² =N, that is, X=(2.5×10 ³ )×N became.

From these results, it was found that the K-1 strain bacterial cells promote the growth of S. epidermidis in a concentration-dependent manner.

[Example 3] Evaluation of skin condition improving action by human monitor test (test method)
This monitor test was conducted on 20 healthy men aged 27 to 60 years. The test was performed by setting 10 subjects as the K-1 application group (average age 45 years) and 10 subjects as the placebo application group (average age 44 years). From the start of the test (0th day), a total of 20 test subjects washed their faces in the morning and evening every day with the facial cleanser having the composition shown in Table 3. After each face wash, the subjects in the K-1 application group applied 0.8 g of the gel having the composition shown in Table 4 to the face, and the subjects in the placebo application group applied the gel 0. 8g was applied to the face.

On the test start day (0th day) and the fourth week (4w) from the test start day, the subjects (total 20 people) washed their faces with the facial cleansers having the compositions shown in Table 3 and measured the humidity at 20° C. at 50° C. before the following measurement. % Acclimation in a room for 20 minutes, followed by skin moisture measurement using a Corneometer (Courage+Khazaka electronic) and skin pH measurement using a Skin-pH-Meter (Courage+Khazaka electronic).

(result)
The results are shown in Tables 6 and 7. In Table 6 showing the results of skin pH measurement, the pH at week 0 of each administration group (each site: left cheek, right cheek, forehead) is 0, and the value of pH at 4 weeks is shown as a relative value. The pH at week 0 was within the range of 5.7 to 6.1. The pH of healthy human skin is generally considered to be weakly acidic (about pH 4.5 to 6.0), but the pH of the K-1 application group does not change even after 4 weeks compared to 0 days. However, an increase in pH was confirmed in the placebo application group.

Table 7 shows the skin water content measurement results. In Table 7, the measurement result of week 0 of each administration group (each site: left cheek, right cheek) is set as 100, and the value of 4 weeks is shown as a relative value. At least in the placebo-applied group, a decrease in skin water content was confirmed as compared with day 0, as compared with the K-1 application group in which it was confirmed that the skin was kept moisturized.

Claims (10)

1. A skin care agent containing 327 strains of Lactobacillus casei subsp. casei deposited as deposit number NITE BP-02707 as an active ingredient.
2. The skin care agent according to claim 1, which is a hyaluronic acid synthesis promoter, a stratum corneum formation promoter, or a balance adjusting agent for skin flora.
3. The skin care agent according to claim 1 or 2, wherein the bacterial cells are dead bacterial cells.
4. The skin care agent according to any one of claims 1 to 3, which is in the form of a skin external preparation.
5. Non-treatment for preventing or improving the formation of wrinkles, the increase of skin pH and/or the decrease of skin water content, which comprises applying the skin care agent according to any one of claims 1 to 4 to the skin. Method.
6. A method of improving skin condition,
   A method comprising the step of applying to the skin of a subject a composition containing, as an active ingredient, a Lactobacillus casei subsp. casei strain 327 strain deposited under accession number NITE BP-02707.
7. The method according to claim 6, wherein the improvement of the skin condition is promotion of hyaluronic acid synthesis, promotion of stratum corneum formation, or balance adjustment of skin indigenous bacteria.
8. The method according to claim 6 or 7, for preventing or improving the formation of wrinkles on the skin of the subject, the increase in skin pH, and/or the decrease in skin water content.
9. Lactobacillus casei subspecies casei deposited under accession number NITE BP-02707 for producing a composition for promoting the synthesis or keratinization of hyaluronic acid in the skin, or for adjusting the balance of skin-indigenous bacteria use of casei subsp. casei strain 327 strains.
10. Lactobacillus casei subsp. casei deposited under accession number NITE BP-02707 for producing a composition for preventing or improving the formation of skin wrinkles, increase in skin pH and/or decrease in skin water content ( Use of Lactobacillus casei subsp. casei) strain 327 strain.
    
    

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