WO2021006549A1 - Procédé de préparation d'une composition de bloc de dermobiotiques ayant des fonctions d'hydratation et de régénération de la peau - Google Patents

Procédé de préparation d'une composition de bloc de dermobiotiques ayant des fonctions d'hydratation et de régénération de la peau Download PDF

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WO2021006549A1
WO2021006549A1 PCT/KR2020/008699 KR2020008699W WO2021006549A1 WO 2021006549 A1 WO2021006549 A1 WO 2021006549A1 KR 2020008699 W KR2020008699 W KR 2020008699W WO 2021006549 A1 WO2021006549 A1 WO 2021006549A1
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dermobiotics
skin
lactic acid
paraprobiotics
block
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PCT/KR2020/008699
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Korean (ko)
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최동환
인정훈
이창완
최지휘
차소윤
이수연
이승훈
신송석
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주식회사 현대바이오랜드
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Priority to CN202080048730.2A priority Critical patent/CN114072128B/zh
Publication of WO2021006549A1 publication Critical patent/WO2021006549A1/fr

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    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0208—Tissues; Wipes; Patches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0212—Face masks
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241—Containing particulates characterized by their shape and/or structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241—Containing particulates characterized by their shape and/or structure
    • A61K8/025—Explicitly spheroidal or spherical shape
    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/736—Chitin; Chitosan; Derivatives thereof
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • 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
    • A61Q19/007—Preparations for dry skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

Definitions

  • the present invention relates to a Dermobiotics block composition that maximizes the effect of skin moisturizing and skin regeneration of dermal cells by trapping a fermented product of lactic acid bacteria having functions of skin moisturizing and skin regeneration with hyaluronic acid.
  • the present invention fermented Dermobiotics, which is a functional lactic acid bacteria for skin moisturizing and skin regeneration, in a restricted medium containing hyaluronic acid or chitosan, and then heated and filtered. It relates to a method for producing a dermobiotics block composition in which a fermented product of lactic acid bacteria is collected with hyaluronic acid by inducing the formation of block particles.
  • This composition is hyaluronic acid with high skin affinity, and by trapping the fermented product of lactic acid bacteria having skin moisturizing and skin regeneration function, it can maximize the effect of skin moisturizing and skin regeneration by efficiently delivering the fermented product of lactic acid bacteria to skin dermal cells.
  • the skin is composed of a multi-layered structure of three layers: the stratum corneum, the epidermis and the dermis.
  • the outermost stratum corneum in the skin structure is formed as a very thin layer with a thickness of about 0.5mm.
  • the stratum corneum composed of 25 to 30 dead skin cells, protects our skin, and the skin keratin that has finished its role falls off by itself, and after the new skin cells come up as new flesh, it repeats the process of becoming keratin. Protect and organize.
  • the epidermal layer is just below the stratum corneum of the skin and is responsible for the functions of penetration barrier, innate immunity, protection against ultraviolet rays, wound recovery, and synthesis of vitamin D.
  • the dermal layer supplies nutrients to the epidermis and is responsible for skin regeneration, and functions to inhibit pathogen invasion, repair wounds, and maintain the structure and elasticity of the skin.
  • Probiotics means'for life' in Latin, meaning mainly live cells, and means microorganisms that help host health when ingested in an appropriate amount (WHO/FAO, 2002). ).
  • Microorganisms such as Lactobacillus sp., Bifidobacterium sp., and Streptococcus sp. are used as representative probiotics and are isolated from the intestine. These lactic acid bacteria have high affinity with the intestinal mucosa in the intestine, and after adhesion, interact with immune cells present in the intestinal mucosa to improve the immunity of each organ of the body and prevent disease.
  • probiotics function is confirmed, research results have been announced that it is effective not only for intestinal health but also for skin moisturizing. Using this, Streptococcus genus, Lactobacillus genus, Lactococcus genus, etc. are fermented, filtered or extracted, and used in the cosmetic field.
  • probiotics in 2002 has been proposed as partially revised academically.
  • results of studies that show that probiotics can exhibit the effect of probiotics even if they are not necessarily living bacteria against probiotics defined through conceptual definition in 2002 in WHO/FAO have been systematized, and a new concept of paraprobiotics emerged (paraprobiotics + fermented organic acid). Taverniti & Guglielmetti, 2011; Ananta & Knorr, 2009).
  • Hyaluronic acid salts and hyaluronic acid are negatively charged glycosaminoglycan polysaccharides, which are present throughout the body, especially in large amounts in the skin.
  • Hyaluronic acid which exists as a part stored in the matrix of cells that also contains collagen and other proteins surrounding cells such as epidermal keratinocytes and dermal fibroblasts, is abundant in both the dermis and epidermis.
  • Hyaluronic acid plays a very important role on the skin. It improves the skin's moisturizing power, maintains skin elasticity, and reduces damage to the lower layers of the skin when the skin is damaged, and acts like a lubricant to smooth the movement of collagen, a major component of the skin, between cells. Do it.
  • the lactic acid bacteria fermented product is collected with hyaluronic acid so that the lactic acid bacteria having functions of skin moisturizing and skin regeneration different from the skin flora and physiologically/ecologically different can be transplanted without rejection.
  • the lactic acid bacteria fermented product Dermobiotics block composition having the function of skin moisturizing and skin regeneration was prepared.
  • Patent Document 1 Korean Patent Registration No. 10-1967809 (Name of the invention: Cosmetic composition for skin wrinkle improvement or skin moisturizing containing nanoized kimchi lactic acid bacteria, Applicant: Biogenix Korea Co., Ltd. and one other, registration date: April 2019 April 04)
  • Patent Document 2 Republic of Korea Patent Registration No. 10-1764412 (Name of the invention: Functional hydrated hyaluronic acid and a method for producing coated lactic acid bacteria with excellent intestinal mucosa adhesion ability and selective antagonism using the same, Applicant: Ildong Bioscience Co., Ltd. , Registration date: July 27, 2017)
  • Patent Document 3 Republic of Korea Patent Registration No. 10-1280232 (Name of invention: manufacturing method of 4-coated lactic acid bacteria and 4-coated lactic acid bacteria produced by the method, Applicant: Ildong Pharmaceutical Co., Ltd., registration date: June 25, 2013 )
  • Patent Document 4 Republic of Korea Patent Registration No. 10-1742542 (Name of the invention: composition for skin moisturizing or wrinkle improvement containing the dead cells of Tindalhwa lactic acid bacteria as an active ingredient, Applicant: Korean Institute of Oriental Medicine, registration date: May 26, 2017 Work)
  • Patent Document 5 Korean Patent Registration No. 10-1106077 (Name of invention: moisturizer, Applicant: House Wellness Food Corporation, registration date: January 9, 2012)
  • Patent Document 6 Republic of Korea Patent Publication No. 10-2018-0108209 (Name of the invention: Composition for treating skin damage containing Lactobacillus rhamnosus JC1225 strain, Applicant: Immune Biolab Co., Ltd. and one other, published Date: October 4, 2018)
  • Patent Document 7 Japanese Published Patent No. 2006-199608 (Name of invention: external preparation for skin, Applicant: ACE BIO PRODUCT KK, Publication date: January 19, 2005)
  • Non-Patent Document 1 Hyaluronic Acid Coated Chitosan Nanoparticles Reduced the Immunogenicity of the Formed Protein Corona, Abdulaziz Almalik, SCIENTIFIC REPORTS, 7: 10542, DOI:10.1038/s41598-017-10836-7 (www.nature.com)
  • Non-Patent Document 2 The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept), 2011, Genes Nutr. 6: 261-274
  • Non-Patent Document 3 Comparison of inactivation pathways of thermal or high pressure inactivatd Lactobacillus rhamnosus ATCC 53103 by flow cytometry analysis, E. Ananta, D. Knorr, ScienceDirect, Volume 26, Issue 5, August 2009, Pages 542-556
  • the present invention is a spherical block through a mixture reaction according to pH of the fermented product after heating each fermented product produced by fermenting Dermobiotics, a functional lactic acid bacteria for skin moisturizing and skin regeneration, with a restricted medium containing hyaluronic acid or chitosan.
  • a method for producing a dermobiotics block composition in which a fermented product of skin functional lactic acid bacteria is collected with hyaluronic acid by inducing the formation of This composition is hyaluronic acid with high skin affinity, and by trapping the fermented product of lactic acid bacteria having skin moisturizing and skin regeneration function, it can maximize the effect of skin moisturizing and skin regeneration by efficiently delivering the fermented product of lactic acid bacteria to skin dermal cells.
  • the dermobiotics block composition may be provided as a material in the form of various cosmetic compositions, skin patches, and mask packs.
  • the method of manufacturing the mobiotics block composition of the present invention includes the following steps.
  • (first step) preparing a first restriction medium containing hyaluronic acid and glucose and a second restriction medium containing chitosan and glucose;
  • (Fourth step) preparing a dermobiotics block composition by dropwise mixing hyaluronic acid paraprobiotics and chitosan paraprobiotics; may include.
  • the first restriction medium is an aqueous solution containing 0.1 to 1.0 (w/v)% of medium-molecular hyaluronic acid (1,500 to 2,000 kDa) and 0.1 to 2.0 (w/v)% of glucose
  • the second restriction medium is characterized in that it is an aqueous solution containing 0.1 to 1.0 (w/v)% of chitosan and 0.1 to 2.0 (w/v)% of glucose.
  • Each restriction medium is preferably sterilized and used by a conventional medium preparation method. In more detail, it is recommended to sterilize for 10 to 60 minutes at 1.1 to 1.3 atmospheres at 110 to 125°C. In the case of using such a restricted medium, fermentation odor that inevitably occurs during culturing of lactic acid bacteria can be drastically reduced.
  • the molecular weight of hyaluronic acid in the first restriction medium is less than 1,500 kDa or exceeds 2,000 kDa, lactic acid bacteria may not be well cultured or the dermobiotic block may not be formed well, which is not preferable.
  • the molecular weight of chitosan in the second restriction medium exceeds 100 kDa, lactic acid bacteria may not be well cultured or the dermobiotics block may not be formed well, which is not preferable.
  • the functional lactic acid bacteria of the skin of the second step are Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp. ( Enterococcus sp.), Pediococcus sp., Leuconostoc sp., and Weissella sp. It may be selected from the group consisting of, but is not limited thereto.
  • lactobacillus genus Lactobacillus casei Lactobacillus casei
  • Lactobacillus paracasei Lactobacillus paracasei
  • Lactobacillus brevis Lactobacillus brevis
  • Lactobacillus culbatus Lactobacillus culvatus
  • Lactobacillus sakei Lactobacillus plantarum
  • Lactobacillus gasseri and the like, but are not limited thereto.
  • the lactic acid bacteria of the genus Bifidobacterium include, but are not limited to, Bifidobacterium longum , Bifidobacterium bifidum , and the like.
  • the lactic acid bacteria of the genus Streptococcus include, but are not limited to, Streptococcus thermophilus , Streptococcus entericus , and the like.
  • the lactic acid bacteria of the genus Lactococcus include, but are not limited to, Lacotococcus lactis , Lacotococcus garvieae , and the like.
  • the lactic acid bacteria of the genus Enterococcus include, but are not limited to, Enterococcus faecium , Enterococcus faecalis , and the like.
  • the lactic acid bacteria of the genus Pediococcus are Pediococcus pentosaceus , Pediococcus acidilactici , Pediococcus damnosus , Pediococcus cellicola And the like, but is not limited thereto.
  • the lactic acid bacteria of the genus Leuconostoc include, but are not limited to, Leuconostoc lactis .
  • the lactic acid bacteria of the genus Bisella include, but are not limited to, Weissella confusa , Weissella paramensenteroides , and the like.
  • the heat treatment of the fermented lactic acid bacteria for the production of each paraprobiotic is preferably performed at 90 to 100°C for 5 to 15 minutes (most preferably at 100°C for 10 minutes). At this time, if the heat treatment condition is less than 90°C or exceeds 100°C, or the heating time is less than 5 minutes or more than 15 minutes, the subsequent themobiotics block may not be formed well.
  • the fourth step when mixing hyaluronic acid paraprobiotics and chitosan paraprobiotics, it may be carried out by dropping chitosan paraprobiotics while stirring the hyaluronic acid paraprobiotics. Alternatively, it may be carried out by dropping hyaluronic acid paraprobiotics while stirring chitosan paraprobiotics. In this case, if the paraprobiotics are mixed immediately without using the dropping method, the yield of forming the demobiotics block is significantly lowered, which is not preferable.
  • the present invention can also provide a cosmetic composition containing the dermobiotics block prepared by the above method.
  • the cosmetic composition may have excellent functions of skin moisturizing and skin regeneration, and more preferably, it may improve the functions of skin moisturizing and skin regeneration of skin dermal cells.
  • the cosmetic composition may contain all commonly used ingredients. For example, it may contain general auxiliary ingredients such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, and fragrances.
  • the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica as carrier components , Talc or zinc oxide may be used.
  • the formulation of the cosmetic composition of the present invention is a powder or a spray
  • lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component.
  • chlorofluorohydro Propellants such as carbon, propane-butane or dimethyl ether.
  • a solvent, a solvating agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
  • the formulation of the cosmetic composition of the present invention is a suspension
  • a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
  • the formulation of the cosmetic composition of the present invention is a surfactant containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acetionate, imidazolinium derivative, methyltaurate, sarcosinate , Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty diethanolamide, vegetable oil, linoline derivative or ethoxylated glycerol fatty acid ester, and the like may be used.
  • the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrophobic substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreen agents, vitamins, plant extracts, etc. .
  • the amount of the ingredients may be selected within a range that does not impair the inherent effect of the cosmetic composition according to the formulation or purpose of use.
  • the amount of the ingredients added may be, for example, 0.1 to 10% by weight, preferably 0.1 to 6% by weight, based on the total weight of the composition, but is not limited thereto.
  • the type of formulation of the cosmetic composition is not particularly limited, and for example, skin care cosmetics such as lotion, emulsion, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body scrub, soap, oil, Makeup cosmetics such as lipstick and foundation, cosmetics for hair, and the like, and the formulation is not particularly limited.
  • the present invention can also provide a skin patch, an external skin preparation, a mask pack, and the like containing the dermobiotics block composition.
  • the skin patch or external preparation for skin may be prepared by mixing with various excipients contained in a conventional patch composition or composition for external application.
  • the hyaluronic acid or chitosan lactic acid bacteria fermented product produced by fermenting skin functional lactic acid bacteria at each pH in a restriction medium containing hyaluronic acid or chitosan is heat-treated, prepared with paraprobiotics, and reacted to produce paraprobiotics with hyaluronic acid. It is possible to secure the collected spherical dermobiotics block composition.
  • the Dermobiotics block composition maximizes AQP-3 expression in epidermal cells of paraprobiotics, HAS-2 expression in dermal cells, and wound healing effect to improve skin moisturizing and skin regeneration functions.
  • the mobiotics block composition formed as described above exhibits a beneficial effect on skin moisturizing and skin regeneration, so it can be effectively used as a high-tech bio material such as a functional cosmetic composition, a functional skin patch, and a functional mask pack.
  • Example 1 is a scanning microscope photograph taken by centrifugation and lyophilization of a Dermobiotics block composition by dropping hyaluronic acid paraprobiotics of pH 4.0 while stirring chitosan paraprobiotics of pH 5.5 of Example 1-3-2. .
  • Example 2 is a skin moisturization of epidermal cells of hyaluronic acid paraprobiotics of pH 4.0 in Example 1-1, chitosan paraprobiotics of pH 5.5 in Example 1-2, and Dermobiotics block composition of Example 1-3-2 It is a graph showing the effect on.
  • Example 3 is a skin moisturization of dermal cells of hyaluronic acid paraprobiotics of pH 4.0 of Example 1-1 and chitosan paraprobiotics of pH 5.5 of Example 1-2 and Dermobiotics block composition of Example 1-3-2 It is a graph showing the effect on.
  • Example 4 is a graph showing the effect of the Dermobiotics block composition of Example 1-3-2 and the dermal cell of Example 2 on skin moisturizing.
  • Example 5 to 12 are various block compositions such as hyaluronic acid paraprobiotics of pH 4.0 in Example 1-1, chitosan paraprobiotics of pH 5.5 in Example 1-2, and themobiotics of Example 1-3-2 ( This is a measurement photograph (confirmation of cell density) of the wound healing effect related to skin regeneration in (including comparative conditions).
  • a culture of pH 3.0-4.5 having a certain number of lactic acid bacteria is prepared with a medium of hyaluronic acid and glucose, and as another method, a certain number of lactic acid bacteria is used as a restriction medium of chitosan and glucose.
  • a culture of pH 5.0 to 6.5 having a pH of 5.0 to 6.5 each culture having a different pH is mixed to use the principle that hyaluronic acid is formed into a sphere by ionic bonding.
  • the carboxyl group (pka: 3.19) of hyaluronic acid in the fermentation product cultured in the hyaluronic acid and glucose restriction medium is negatively charged
  • the chitosan (pka: 6.5) in the fermentation product cultured in the restriction medium of chitosan and glucose is positively charged. It is charged to induce ionic bonding of the two materials to collect paraprobiotic cultures, thereby producing a spherical dermobiotics block.
  • Example 1-1 Preparation of hyaluronic acid paraprobiotics of pH 3.0-4.5
  • Lactobacillus ( L. paracasei SKB1192) lactic acid bacteria were inoculated in MRS medium and then cultured at 37° C. for 20 hours to obtain a number of bacteria of 10 9-10 10 CFU/ml. Lactobacillus ( L. paracasei SKB1192) lactic acid bacteria cultured in MRS medium were recovered by centrifugation, and washed twice with sterile water.
  • Example 1-2 Preparation of chitosan paraprobiotics of pH 5.0-6.5
  • Lactobacillus ( L. paracasei SKB1192) lactic acid bacteria were inoculated in MRS medium and then cultured at 37° C. for 20 hours to obtain a number of bacteria of 10 9-10 10 CFU/ml. Lactobacillus ( L. paracasei SKB1192) lactic acid bacteria cultured in MRS medium were recovered by centrifugation, and washed twice with sterile water.
  • Example 1-3 Confirmation of optimal conditions for mixing hyaluronic acid paraprobiotics and chitosan paraprobiotics according to pH for the formation of Dermobiotics block
  • Example 1-3-1 Yield Characteristics of Dermobiotics Blocks Formed by Dropping Chitosan Paraprobiotics at pH 3.0 ⁇ 4.5 Hyaluronic Acid Paraprobiotics
  • Example 1-1 Each hyaluronic acid paraprobiotic prepared in Example 1-1 to pH 3.0 to 4.5 was stirred at 100 rpm, and each chitosan paraprobiotic prepared in Example 1-2 was added dropwise (dripping). Through this, a dermobiotics block formation reaction was caused, and the dermobiotics block formed through centrifugation at 4,000 rpm for 20 minutes was recovered and the yield was measured. Optimal conditions were found to have a maximum yield of 42% when chitosan paraprobiotics of pH 6.0 were added dropwise to hyaluronic acid paraprobiotics of pH 4.0 [Table 1]. Dermobiotics block yield was calculated as a percentage by dividing the total solids of the Dermobiotics block recovered through centrifugation by the total solids of the Dermobiotics block reaction solution (%, w/w).
  • Example 1-3-2 Yield characteristics of dermobiotics blocks formed when pH 3.0-4.5 hyaluronic acid paraprobiotics are added to pH 5.0-6.5 chitosan paraprobiotics
  • Example 1-1 Each chitosan paraprobiotic of pH 3.0 to 4.5 prepared in Example 1-1 was added dropwise (dripping) while stirring each chitosan paraprobiotic of pH 5.0 to 6.5 prepared in Example 1-2 at 100 rpm. Through this, a dermobiotics block formation reaction was caused, and the dermobiotics block formed through centrifugation at 4,000 rpm for 20 minutes was recovered and the yield was measured. Optimal conditions were confirmed to have a maximum yield of 58% when hyaluronic acid paraprobiotics of pH 4.0 were added dropwise to chitosan paraprobiotics of pH 5.5 [Table 2]. Dermobiotics block yield was calculated as a percentage by dividing the total solids of the Dermobiotics block recovered through centrifugation by the total solids of the Dermobiotics block reaction solution (%, w/w).
  • Example 1-1 While stirring each chitosan paraprobiotics of pH 5.0 to 6.5 prepared in Example 1-2 at 100 rpm, each hyaluronic acid paraprobiotics of pH 3.0 to 4.5 prepared in Example 1-1 were quickly mixed, and the mobiotics block A formation reaction occurred. As a result, it was confirmed that the agglomeration phenomenon occurred and the mobiotics block formation was impossible.
  • Example 2-1 Bifidobacterium long gum ( Bifidobacterium longum The Mobiotics Block Composition Preparation Using)
  • Hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared with Bifidobacterium longum were mixed with the optimized Dermobiotics block composition in Example 1-3-2 (pH 5.5 pH 4.0 while stirring chitosan paraprobiotics) Hyaluronic acid paraprobiotics were added dropwise, hereinafter the same), to prepare a Dermobiotics block composition.
  • Example 2-2 Streptococcus Thermophilus ( Streptococcus thermophilus The Mobiotics Block Composition Preparation Using)
  • Example 1-3-2 The hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared from Streptococcus thermophilus were optimized in Example 1-3-2 to prepare a Dermobiotics block composition, thereby preparing a Dermobiotics block composition.
  • Example 2-3 Lactococcus lactis ( Lacotococcus lactis The Mobiotics Block Composition Preparation Using)
  • the hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared with Lacotococcus lactis were prepared with the dermobiotics block composition optimized in Example 1-3-2, to prepare a dermobiotics block composition.
  • Example 2-4 Enterococcus faecium ( Enterococcus faecium The Mobiotics Block Composition Preparation Using)
  • Hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared with Enterococcus faecium were optimized in Example 1-3-2 to prepare a Dermobiotics block composition, and a Dermobiotics block composition was prepared.
  • Example 1-3-2 The hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared with Pediocuccus pentosaceus were optimized in Example 1-3-2 to prepare a Dermobiotics block composition, to prepare a Dermobiotics block composition. .
  • Example 2-6 Leukonostock lactis ( Leuconostoc lactis The Mobiotics Block Composition Preparation Using)
  • Hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared with Leuconostoc lactis were optimized in Example 1-3-2 to prepare a Dermobiotics block composition, to prepare a Dermobiotics block composition.
  • Example 2-7 Vicella Confussa ( Weisslla confusa The Mobiotics Block Composition Preparation Using)
  • Hyaluronic acid paraprobiotics and chitosan paraprobiotics prepared by Weisslla confusa were optimized in Example 1 to prepare a Dermobiotics block composition, to prepare a Dermobiotics block composition.
  • hyaluronic acid first restriction medium and chitosan second restriction medium were prepared and used as comparative examples.
  • Chitosan 0.4% (w/v) and glucose 2.0% (w/v) were dissolved in tertiary distilled water, adjusted to pH 5.5 with lactic acid, and then sterilized (121° C., 15 minutes) to prepare a second limited chitosan medium.
  • Aquaporin is a membrane pore protein that acts as a water channel that selectively induces the entry of water molecules in the skin while interfering with the passage of other ions or solutes.
  • AQP-3 is aquaglyceroporin by passing glycerol together with water. It is also called and exists in human keratinocytes and prevents the loss of water molecules, and functions such as skin hydration, wound healing, and homeostasis. Therefore, it is possible to confirm the moisturizing effect of the raw material through increased expression of AQP-3.
  • human normal keratinocytes (gibco) were inoculated into a culture dish, cultured for 24 hours (37°C, CO 2 5%), and then replaced with Serum-free medium (Epilife, gibco), and hyaluronic acid of Example 1-1.
  • Hyaluronic acid paraprobiotics (pH 4.0), chitosan paraprobiotics of Example 1-2 (pH 5.5), Dermobiotics block composition of Example 1-3-2 (pH 5.5 chitosan paraprobiotics, pH 4.0 while stirring)
  • Example 1-3-2 pH 5.5 chitosan paraprobiotics, pH 4.0 while stirring
  • Comparative Example 2-1 Comparative Example 2-2, and Comparative Example 3 were treated at a concentration of 3% (w/v) in the same manner, and 50nM retinoic acid was used as a positive control group.
  • Aquaporin(AQP)-3 coding gene base sequence division Base sequence Aquaporin(AQP)-3 Forward 5’- GGC TGA AGC AGG AGA ATC AC-3’ Reverse 5’-GCT GAG TCC CAG CTG TTT TT-3’ ⁇ -Actin Forward 5’- GGC ACC CAG CAC AAT GAA G -3’ Reverse 5’- CCG ATC CAC ACG GAG TAC TTG -3’
  • the dermobiotics block composition of the present invention is a composition capable of improving skin hydration effect by activating a water channel in the epidermal cells of the skin and contributing to the moisturizing maintenance of not only the epidermis but also the dermal cells.
  • HAS Hydrophilicity Agent Synthase
  • human normal fibroblast (gibco) was inoculated into a culture dish, cultured for 24 hours (37° C., CO 2 5%), and then replaced with Serum-free medium (Epilife, gibco), and hyaluronic acid of Example 1-1.
  • Hyaluronic acid paraprobiotics (pH 4.0), chitosan paraprobiotics of Example 1-2 (pH 5.5), Dermobiotics block composition of Example 1-3-2 (pH 5.5 chitosan paraprobiotics, pH 4.0 while stirring)
  • Example 1-3-2 pH 5.5 chitosan paraprobiotics, pH 4.0 while stirring
  • Comparative Example 1-1 Comparative Example 1-2, and Comparative Example 2 were treated at a concentration of 3% (w/v) in the same manner, and 50nM retinoic acid was used as a positive control group.
  • Examples 2-1 to 2-7 were treated at a concentration of 3% (w/v) in the same manner.
  • 50nM retinoic acid was used as a positive control.
  • Hyaluronic acid synthase (HAS-2) coding gene base sequence division Base sequence Hyaluronic acid synthase-2 (HAS-2) Forward 5'- ACT TCC CGC CAA GAT GTT TG -3' Reverse 5’- TTC CTT CCT GAT GTG CCC -3’ ⁇ -Actin Forward 5’- GGC ACC CAG CAC AAT GAA G -3’ Reverse 5’- CCG ATC CAC ACG GAG TAC TTG -3’
  • HAS-2 expression was remarkably compared to Comparative Examples and Examples in which the Dermobiotics block composition of Example 1-3-2 proceeded at the same concentration as in [Fig. 3] It was confirmed that it was induced.
  • the Dermobiotics block composition of the present invention is an excellent composition that promotes skin moisturizing effect through the skin regeneration process of synthesizing hyaluronic acid in dermal cells.
  • Comparative Example 2-1, Comparative Example 2-2, and Comparative Example 3 were treated at a concentration of 3% (w/v) in the same manner, and 7 ⁇ g/ml of adenosine was used as a positive control group.
  • the cells of Example 1-3-2 Themobiotics block in comparison to the control group and the comparative example It was confirmed that the proliferation was more densely increased, and it was confirmed that the cell density was similar to that of the positive control, adenosine 7 ⁇ g/ml. It was confirmed that Example 1-3-2 Dermobiotics block induces proliferation of damaged skin cells, thereby improving skin regeneration effect.

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Abstract

Selon la présente invention, il est possible d'obtenir une composition de bloc de dermobiotiques sphérique dans laquelle, en faisant réagir au goutte à goutte, à chaque pH, de l'acide hyaluronique et des para-probiotiques de chitosane, qui sont produits par fermentation de bactéries lactiques dans un milieu défini contenant de l'acide hyaluronique ou du chitosane, les para-probiotiques sont capturés avec de l'acide hyaluronique. Le bloc de dermobiotiques améliore l'expression de AQP-3 et HAS-2 par les cellules de la peau lorsqu'il est appliqué sur la peau, présentant ainsi une fonction d'amélioration de l'hydratation et une fonction de régénération de la peau qui favorise la croissance des cellules de la peau, et, en particulier, l'obtention de ces fonctions dans les cellules dermiques peut être présumée. À ce titre, les dermobiotiques formés sous forme de bloc présentent des effets sains d'hydratation et de régénération de la peau, et peuvent ainsi être efficacement utilisés dans des compositions cosmétiques et des bio-cosmétiques tels que des timbres transdermiques et des enveloppements de masque, qui comprennent les dermobiotiques en tant que composant fonctionnel.
PCT/KR2020/008699 2019-07-05 2020-07-03 Procédé de préparation d'une composition de bloc de dermobiotiques ayant des fonctions d'hydratation et de régénération de la peau WO2021006549A1 (fr)

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