WO2020129888A1 - Agent de soin de la peau contenant un lactobacillus issu d'une plante - Google Patents

Agent de soin de la peau contenant un lactobacillus issu d'une plante Download PDF

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WO2020129888A1
WO2020129888A1 PCT/JP2019/049144 JP2019049144W WO2020129888A1 WO 2020129888 A1 WO2020129888 A1 WO 2020129888A1 JP 2019049144 W JP2019049144 W JP 2019049144W WO 2020129888 A1 WO2020129888 A1 WO 2020129888A1
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skin
strain
casei
care agent
skin care
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PCT/JP2019/049144
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Japanese (ja)
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知也 岡本
善久 中田
健太郎 広瀬
武久 熊谷
真理子 井口
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一丸ファルコス株式会社
亀田製菓株式会社
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Priority to JP2020550194A priority Critical patent/JP6805399B2/ja
Publication of WO2020129888A1 publication Critical patent/WO2020129888A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • 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.
  • lactic acid bacteria have various functions such as immunostimulatory action, and lactic acid bacteria are attracting attention.
  • Lactobacillus paracasei K71 strain which is a plant-derived lactic acid bacterium (Patent Document 1)
  • Patent Document 2 the antioxidant function of a specific strain of Lactobacillus casei of Brassicaceae plants
  • Patent Document 3 the antioxidant function of a specific strain of Lactobacillus casei of Brassicaceae plants
  • Patent Document 3 A collagen production promoter
  • 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)).
  • 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.
  • CE cornified envelope
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1A shows changes in the expression level of ⁇ -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 ⁇ -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. 1A shows changes in the expression level of ⁇ -defensin 3 in epidermal keratinocytes measured by the RT-PCR experiment of Example 1.
  • FIG. 1B also shows the change in the expression
  • 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 ⁇ -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.
  • 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.
  • lactic acid bacteria belonging to the genus Lactobacillus separated from fermented foods made from rice can be used.
  • 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). ..
  • 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.
  • mutant strain 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ⁇ -Defensin 3 present in human epithelial cells is a peptide consisting of 45 amino acid residues.
  • the skin care agent of the present invention 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.
  • 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.
  • 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.
  • the skin care agent of the present invention When 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.
  • humectants such as glycerin, petrolatum, urea, hyaluronic acid, heparin
  • PABA derivatives paraaminobenzoic acid, escarol 507, etc.
  • cinnamic acid derivatives
  • 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, pantothe
  • an extract from various plants or an additive derived from an animal-based material
  • 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,
  • effects such as scenting, deodorization, thickening, antiseptic, and buffering can be expected.
  • the external preparation composition examples 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.
  • 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.
  • a skin care agent which is preferably in the form of an external preparation for skin
  • Continuous 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.
  • 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.
  • 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.
  • 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 (GibcoTM 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.
  • NHEK Differentiation-induced epidermal keratinocytes
  • 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 ⁇ -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 ⁇ -defensin 3 and hyaluronic acid secreted in the culture supernatant was quantified using an ELISA method.
  • NHEK differentiation-induced epidermal keratinocytes
  • 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.
  • RNA was purified 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.
  • TB Green Premix Ex Taq sold by TaKaRa was used.
  • RPS18 ribosome protein S18
  • FIG. 1A the expression level of ⁇ -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).
  • FIG. 1A 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. 1B 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.
  • FIG. 2A similarly, the expression levels of involucrin (FIG. 2A) and filaggrin (FIG.
  • FIG. 2B were increased by adding 0.1% of K-1 strain cells.
  • 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.
  • 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.
  • 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.
  • 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. ⁇ -Defensin 3 and hyaluronic acid were quantified from the culture supernatant after culturing.
  • FIGS. 5 and 6 The results are shown in FIGS. 5 and 6. As shown in FIG. 5, an increase in ⁇ -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).
  • 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 ⁇ -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 122278 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

Le problème à la base de la présente invention est de fournir un promoteur de formation de couche cornée supérieure et un promoteur de synthèse d'acide hyaluronique pour améliorer un effet embellissant par la conservation de la santé de la peau et la prévention de rides. L'invention concerne un agent de soin de la peau, de préférence un promoteur de synthèse d'acide hyaluronique, un promoteur de formation de couche cornée ou un régulateur d'équilibre de flore résidente de la peau. L'agent de soin de la peau comprend, en tant que principe actif, des cellules bactériennes de Lactobacillus casei subsp. casei 327 déposée sous le numéro d'accès NITE PB-0270.
PCT/JP2019/049144 2018-12-17 2019-12-16 Agent de soin de la peau contenant un lactobacillus issu d'une plante WO2020129888A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117298032A (zh) * 2023-11-17 2023-12-29 天津中耀博研生物医药有限公司 一种改变皮肤微生态的后生元面膜及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010270152A (ja) * 2010-09-03 2010-12-02 Toyo Hakko:Kk 抗アレルギー性組成物及び美白組成物、並びにこれらを含有する化粧品及び飲食品
WO2018216744A1 (fr) * 2017-05-23 2018-11-29 一丸ファルコス株式会社 Agent cosmétique et protecteur de la peau contenant une bactérie lactique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010270152A (ja) * 2010-09-03 2010-12-02 Toyo Hakko:Kk 抗アレルギー性組成物及び美白組成物、並びにこれらを含有する化粧品及び飲食品
WO2018216744A1 (fr) * 2017-05-23 2018-11-29 一丸ファルコス株式会社 Agent cosmétique et protecteur de la peau contenant une bactérie lactique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAITO ET AL.: "Effects of intake of Lactobacillus casei subsp. casei 327 on skin conditions: a randomized, double-blind, placebo-controlled, parallelgroup study in women", BIOSCIENCE OF MICROBIOTA, FOOD AND HEALTH, vol. 36, no. 3, 2017, pages 111 - 120, XP055722688, ISSN: 2186-3342 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117298032A (zh) * 2023-11-17 2023-12-29 天津中耀博研生物医药有限公司 一种改变皮肤微生态的后生元面膜及其制备方法

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