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WO2020204579A1 - Procédé de préparation d'un conjugué de minéral dérivé d'eau de mer de lave et de nucléotide dérivé de dermabiotiques, et composition cosmétique de dermabiotiques fonctionnels l'utilisant - Google Patents

Procédé de préparation d'un conjugué de minéral dérivé d'eau de mer de lave et de nucléotide dérivé de dermabiotiques, et composition cosmétique de dermabiotiques fonctionnels l'utilisant Download PDF

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Publication number
WO2020204579A1
WO2020204579A1 PCT/KR2020/004412 KR2020004412W WO2020204579A1 WO 2020204579 A1 WO2020204579 A1 WO 2020204579A1 KR 2020004412 W KR2020004412 W KR 2020004412W WO 2020204579 A1 WO2020204579 A1 WO 2020204579A1
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Prior art keywords
dermabiotics
mineral
lava seawater
derived
cultured
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English (en)
Korean (ko)
Inventor
이창완
양서진
김두성
김경민
김예향
차소윤
최지휘
이승훈
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Hyundai Bioland Co Ltd
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SK Bioland Co Ltd
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Priority claimed from KR1020190039918A external-priority patent/KR102084350B1/ko
Priority claimed from KR1020200022437A external-priority patent/KR102305076B1/ko
Priority claimed from KR1020200022439A external-priority patent/KR102133689B1/ko
Application filed by SK Bioland Co Ltd filed Critical SK Bioland Co Ltd
Priority to CN202080027218.XA priority Critical patent/CN113692273B/zh
Publication of WO2020204579A1 publication Critical patent/WO2020204579A1/fr
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • 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
    • 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

Definitions

  • the present invention uses useful skin microorganisms (dermabiotics) as cultivation water to solve the intrinsic and extrinsic factors of skin aging at the same time.
  • a mineral-nucleotide conjugate having excellent thermal stability through a thermo-pressure extraction method.
  • the present invention provides a method of purifying mineral-nucleotides by selectively removing cell wall components (cell walls, glycolipids, lipoproteins, etc.) that cause inflammatory reactions of skin cells through cooling centrifugation after hot-pressure extraction through membrane filtration. It is suggested that the dermabiotics-derived mineral-nucleotide conjugate cultivated in lava seawater thus prepared has thermal stability, expresses functional proteins related to skin barrier strengthening, and regulates the skin microbiome, a third skin layer. do.
  • the skin is composed of a multi-layered structure of three layers: the epidermal layer, the dermis layer, and the stratum corneum layer, and the epidermal layer is just below the stratum corneum layer and is responsible for the function of penetration barrier, innate immunity, protection against ultraviolet rays, wound recovery, and synthesis of vitamin D.
  • the dermal layer is the epidermis. It is a layer that supplies nutrients to the skin and is responsible for skin regeneration, and functions to inhibit pathogen invasion, repair wounds, and maintain the structure and elasticity of the skin.
  • 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 differentiation of keratinocytes is the division of basal cells-synthesis in polar cells-self-degradation in granular cells-in keratinocytes. It consists of 4 steps of the reconstruction process and forms a skin barrier. The process of forming this skin barrier takes about 4 weeks of cell regeneration based on young skin.
  • Skin protects the human body from physical and chemical stimuli from the outside, but continuous exposure to ultraviolet rays, a representative external factor of skin aging, damages the DNA of skin cells and causes reactive oxygen species (ROS). It is produced, inhibiting the synthesis of collagen in the skin and promoting decomposition, aging the skin and creating wrinkles. It is accepted as the orthodox of natural aging that the intrinsic factor of skin aging varies depending on the genetic factors inherited from parents, but the rate and condition of skin aging according to the change of the skin microbiome called the third skin layer. Is different.
  • the skin microbiome which has been in the spotlight as a topic of recent research, refers to the ecosystem of the flora distributed in the skin and is mainly present in the skin of the genus Staphylococcus and the genus Propionibacterium . It is reported that microorganisms maintain a healthy skin condition by maintaining a balance through interaction with skin cells.
  • the flora of the skin microbiome is different depending on the age group, women and men, race, and the region where they live, and in particular, it is reported that the dominant skin species by age group maintains skin health.
  • microorganisms are manufactured in the form of lysates and used in cosmetics to avoid skin penetration problems due to the size of microorganisms, death problems due to preservatives, and competitive inhibition problems. It was attempted to improve skin barriers by reacting directly with immune receptors.
  • Bifidobacterium Bifidobacterium
  • Lactobacillus bacteria Lactobacillus
  • peptidoglycan, lipopolysaccharide, and lipoprotein which are known to cause an immune reaction with the skin, are present in the cell wall. If these components are excessive, skin rash, atopy , It can cause skin problems such as erythema.
  • thermal stability of microbial nucleotides, which is an active ingredient in the cytoplasm is 70 to 105°C based on the Tm value (the temperature at which the DNA double helix is unwound), it is not possible to apply an active ingredient above 121°C, a temperature that can completely crush the cell wall. It becomes denatured and does not help improve the skin barrier.
  • one of the problems in the application of microbial materials for skin protection is that when cell debris is applied to the skin, it is not a selective growth factor for the superficial flora, so if harmful bacteria use it as a growth nutrient source, healthy skin microflora The balance of the biome is broken. Since various microbial cell debris, which are currently listed as international cosmetics raw materials, contain a large amount of components of a modulator unit that can affect the human skin microbiome, the problem of imbalance in the skin microbiome is not resolved.
  • Lava seawater is a water that has been aged for a long period of time as seawater flows into the strata as it is naturally filtered through the basalt and sandy layers, and is a unique water resource possessed by Jeju Island.Since the 1980s, lava seawater in Jeju is characterized by the low temperature and cleanliness of lava seawater. It has been actively used as breeding water for flounder farms, and from 5 to 6 years ago, it has been used as water for saunas in terms of health and beauty, and has been in the spotlight. Depending on the purpose of use, desalted lava seawater from which salt has been removed is used or as it is collected.
  • Lava seawater contains more essential minerals such as sodium, magnesium, calcium, and potassium, as well as general useful minerals (iron, manganese, zinc, molybdenum, selenium, etc.) than general seawater, deep water, and Samdawater.
  • vanadium which is known to stabilize insulin secretion or improve diabetes and hyperlipidemia, promotes blood circulation, enhances immunity
  • germanium which has anticancer activity, inhibits oxidation of fat, synergistic effect to maintain the heart and liver, scavenging radicals.
  • the content of selenium which has the effect of improving ability, anticancer, infertility, aging and cholesterol levels, is a characteristic of lava seawater that has never been reported in deep ocean water.
  • lava seawater is a clean groundwater resource in which E. coli, nitrate nitrogen, phosphate phosphorus, phenols, etc. are not detected, and harmful components such as arsenic, mercury, cadmium, etc. are not detected, or lead is detected in a very small amount, which is an obstacle to industrial application. There is no clean raw material.
  • minerals such as magnesium and calcium contained in lava seawater are essential elements for the proliferation of dermabiotics, and human consumption is recommended as an essential nutrient.
  • the eluted DNA has a melting temperature (Tm) among the decomposition elements, which refers to the intermediate value in the temperature change at which double stranded DNA becomes single stranded DNA.
  • Tm melting temperature
  • the eluted DNA is decomposed into nucleotides under this temperature condition with known Tm values of 70 to 105°C, and these are composed of purines and pyrimidines to stabilize between nucleotides by internal hydrogen bonding.
  • the phosphate group among the constituents has a strong negative charge and thus has repulsive power.
  • the nucleotides In order to extract nucleotides having these characteristics, the nucleotides must remain stable even under the hot pressure condition of 121°C, which is higher than the Tm value of 70 ⁇ 105°C, to disrupt the cell wall and cytoplasm.
  • the present inventors confirmed that mineral-nucleotides isolated from dermabiotics cultured in lava seawater are remarkably superior in skin barrier strengthening and skin damage inhibition functions compared to dermabiotics-derived nucleotides cultured in an existing general medium.
  • Patent Document 1 Republic of Korea Registered Patent No. 10-1347694 (Name of the invention: a method of manufacturing a fermented product of desalted lava seawater, a fermented product obtained by the method, and a cosmetic composition using the fermented product, Applicant: Jeju Techno Park, Registration date: December 27, 2013)
  • Patent Document 2 Republic of Korea Patent Registration No. 10-1693574 (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: January 2, 2017 Work)
  • Non-Patent Document 1 Zakostelska Z, Kverka M, Klimesova K, Rossmann P, Mrazek J, et al. (2011) Lysate of Probiotic Lactobacillus casei DN-114 001 Ameliorates Colitis by Strengthening the Gut Barrier Function and Changing the Gut Microenvironment. PLoS ONE 6(11)
  • An object of the present invention is to induce the generation of mineral-nucleotide conjugates by accumulating natural minerals derived from lava seawater in the cytoplasm of Dermabiotics by using lava seawater rich in natural mineral content as cultivation water to proliferate Dermabiotics without separate pretreatment. It is in providing technology.
  • the mineral-nucleotide conjugate is manufactured through the thermal pressure extraction process of Dermabiotics and has excellent thermal stability.Through this, it has a skin barrier reinforcement function and a protective effect of skin cells due to ultraviolet rays, and by controlling the skin microbiome. Improves skin condition.
  • the present invention (first step) obtaining a primary culture solution by culturing Dermabiotics in a medium using lava seawater as culture water;
  • It relates to a method for producing a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater comprising a.
  • the medium of the first step is a medium in which glucose, yeast extract, soypeptone, and casein are included as constituents, and the constituents are dissolved in water containing 30 to 70% (v/v) of lava seawater and sterilized. It features.
  • the medium of the first step is based on a total volume of 1 liter, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soypeptone 0.5-5% (w/v), casein It may be a medium for growth culture prepared by dissolving each component in water containing 30-70% (v/v) lava seawater and sterilizing it so that it becomes 0.5-3% (w/v).
  • a method of maintaining a pH of 6.0 to 7.5 during cultivation by dropping a mixed solution of 10 to 50% (w/v) glucose and 10 to 50% (w/v) sodium hydroxide can be used.
  • the mixed solution may be obtained by mixing a glucose solution and a sodium hydroxide solution in a volume ratio of 1:0.5 to 1:2, respectively.
  • Dermabiotics applied to the first stage culture include Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., and Lactococcus sp. Enterococcus sp., Pediococcus sp., Weissella sp., Saccharomyces sp., Bacillus sp., Staphylococcus sp. It may be one or more strains from the group consisting of the genus Staphylococcus sp. and Propionibacterium sp.
  • the lava seawater mineral enriched water of the second step is characterized in that it is a mineral water obtained by vacuum concentration or osmosis concentration of lava seawater so that calcium 220mg or more, magnesium 1,130mg or more, and selenium 0.013mg or more are contained per 1L.
  • the hot-pressure extraction in the third step may be performed for 15 to 200 minutes at 120 to 130°C and 0.12 to 0.30 MPa.
  • the cooling purification of the third step can be performed by centrifuging for 10 to 30 minutes at 3,000 to 8,000 rpm while maintained at 3 to 5°C, and then, nanoparticles having a pore size of 200 nm or less.
  • the mineral-nucleotide conjugate can be collected using a membrane-based filtration process.
  • the pore size of the nanomembrane may be preferably 20 to 200 nm.
  • the concentration range of nucleotides in the supernatant of the third step may be 200 to 2,000 ⁇ g/ml.
  • the mineral-nucleotides in the supernatant of the third step contain 50 to 200 mg/L of calcium, 300 to 800 mg/L of magnesium, and 0.001 to 0.01 mg/L of selenium.
  • the present invention provides a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater prepared by the above method, and provides a cosmetic composition for strengthening skin barriers comprising the conjugate.
  • the conjugate or the cosmetic composition containing the same is characterized in that it strengthens the skin barrier by enhancing the expression of Serine palmitolytransferase (SPT), Filaggrin, and Claudin-1.
  • SPT Serine palmitolytransferase
  • Filaggrin Filaggrin
  • Claudin-1 Claudin-1
  • the conjugate or the cosmetic composition containing the same is characterized in that it has more effect on the recovery of skin cells due to UV damage.
  • the cosmetic composition is characterized in that it has a recovery effect of skin cells by enhancing the expression of PGC-1 ⁇ (Peroxisome proliferator-activated receptor gamma coactivator 1 ⁇ ).
  • the conjugate or the cosmetic composition containing the same may further have an effect of controlling the microbiome of the skin.
  • the skin microbiome there is an effect of increasing the number of bacteria or maintaining homeostasis of Staphylococcus epidermidis and Cutibacterium acnes .
  • the present invention relates to a method for preparing a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater, and a cosmetic composition having excellent skin barrier reinforcing function, skin microbiome control function, and thermal stability prepared by containing the conjugate.
  • the non-desalted lava seawater was applied as the primary culture water for the cultivation of Dermabiotics to induce the high-concentration growth of Dermabiotics through the inherent natural minerals.
  • the dermabiotics cells were recovered and the lava seawater mineral concentrated water was used.
  • Second culture As a result, it is possible to accumulate minerals in the cytoplasm of Dermabiotics and increase the amount of nucleotides and constructs, such as DNA, RNA, ribosomes, and endoplasmic reticulum, which are cell activating substances present in the cytoplasm, and obtain a conjugate of mineral ions and nucleotides in lava seawater.
  • mineral-nucleotide conjugates having excellent thermal stability can be collected at high concentration.
  • Dermabiotics' cell wall, cytoplasm, protein and protein salt components that affect the toxicity of skin and microbiome and the production of fermentation odor of raw materials through filtration using a nanomembrane filter with a pore size of 200 nanometers or less.
  • this cosmetic composition is non-toxic and capable of regenerating skin cells, strengthening skin barriers, skin soothing effects, and maintaining skin flora. It can be provided as an external skin preparation for its medicines.
  • FIG. 1 is a flow chart showing a method for preparing a mineral-nucleotide conjugate derived from dermabiotics cultured in lava seawater according to the present invention.
  • Example 2 is a graph showing the results of comparing the correlation between the number of dermabiotics in the culture medium cultured in the lava seawater concentration medium of Example 1 and Comparative Example 1 and the nucleotides extracted from the dermabiotics.
  • thermo-pressure extraction time of Dermabiotics obtained after performing the second culture by differentiating the first culture solution cultured in 30 (v/v)% lava seawater as a culture water with normal constant water or lava seawater mineral concentrated water It is a graph of the result of comparing the concentration of nucleotides in the supernatant obtained according to.
  • Figure 4 is a graph showing the results confirming that the dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater through MTT assay has no cytotoxicity up to a treatment concentration of 5 (v/v)%.
  • Figure 6 is a graph confirming the result of increasing the expression of PGC-1 ⁇ , which is reduced during skin aging by dermabiotics-derived mineral-nucleotide conjugates cultured in lava seawater, in fibroblast cells treated with UVB.
  • SPT Serine palmitolytransferase
  • FIG. 9 is a photograph of a mass production sample of a nucleotide (Preparation Example 2) in a cell lysate using a conventional technique and a mineral-nucleotide conjugate derived from dermabiotics cultured in lava seawater.
  • 10A and 10B are results of measuring the purity of nucleotides (Preparation Example 2) in the cell lysate using the prior art and the dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater using UV values.
  • 11 is a result of confirming the skin barrier strengthening effect through comparison of filaggrin gene expression of dermabiotics-derived mineral-nucleotide conjugates cultured in lava seawater prepared with various strains applicable to dermabiotics.
  • the present invention relates to a method for preparing a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater.
  • the recovered cells were suspended in a restricted medium containing only lava seawater mineral concentrated water (including calcium 220 mg/L, magnesium 1,130 mg/L, selenium 0.013 mg/L or more), and cultured.
  • the secondary culture medium is crushed under hot pressure extraction conditions of 0.12 MPa or more to elute mineral-nucleotides, and the cell walls of the crushed cells, proteins and glycoprotein components in the cytoplasm are cooled and precipitated at 3 ⁇ 5°C, centrifuged and filtered through a nanomembrane. It consists of preparing a dermabiotics-derived mineral-nucleotide conjugate cultured in high-purity lava seawater.
  • the medium of the step (a) may preferably be any medium capable of culturing the dermabiotics strain, but more preferably, as a constituent component, glucose, yeast extract, soypeptone, casein are included, and the above configuration It may be a culture medium prepared by dissolving the ingredient in water containing 30-70% (v/v) lava seawater and sterilizing it.
  • the medium of the first step is based on a total volume of 1 liter, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soypeptone 0.5-5% (w/v) ), casein 0.5 ⁇ 3% (w / v) each component dissolved in water containing 30 ⁇ 70% (v / v) lava seawater and sterilized may be prepared for growth culture medium.
  • the medium sterilization is preferably performed at 121 ⁇ 123 °C for 30 minutes.
  • Dermabiotics applied to the first stage culture include Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., and Lactococcus sp. Enterococcus sp., Pediococcus sp., Weissella sp., Saccharomyces sp., Bacillus sp., Staphylococcus sp. It is one or more bacteria from the group consisting of the genus Staphylococcus sp.
  • Propionibacterium sp. preferably Lactobacillus acidophilus , Lactobacillus bulgaricus , lactobacillus bulgaricus Bacillus Kasei (Lactobacillus casei), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus is Serena (Lactobacillus gasseri), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus zone soniyi (Lactobacillus johnsonii ), Lactobacillus plantarum , Lactobacillus reuteri , Bifidobacterium bifidum , Bifidobacterium breve , Bifidobacterium breve , Bifidobacterium infantis infantis ), Bifidobacterium lactis , Bif
  • lactis Enterococcus faecalis , Enterococcus faecium , Pediococcus acidolacticii , Pediococcus pentosaceus , Leukonostock carnosum ( Leuconostoc carnosum ), Leuconostoc citreum , Leuconostoc gasicomitatum , Leuconostoc gellidum , Leuconostoc inhae , Leuconostoc inhae , Leuconostoc kimchii), current Kono Stock lactis (Leuconostoc lactis), current Kono Stock mesen teroyi death (Leuconostoc mesenteroides subsp.
  • the cells may be recovered by centrifugation, ultrafiltration, etc., but it is preferable to perform centrifugation at 5,000 to 8,000 rpm for 10 to 30 minutes.
  • the recovered cells are washed twice with sterile distilled water and suspended in the final lava seawater mineral concentrated water (restricted medium). Thereafter, the cells suspended in the restriction medium are cultured for a second time at 30 to 45° C. for 15 to 20 hours to form mineral-nucleotides. More preferably, the cells are cultured at 37° C. for 16 hours after inoculation.
  • restriction medium refers to a medium that does not sufficiently contain nutrients required for growth and has limited components
  • a restriction medium lava seawater concentrated by a reduced pressure or osmotic concentration method was sterilized and used as a restriction medium.
  • any functional liquid or powder biomaterial that can be used in the cosmetic powder field, polymer and low molecular weight functional biomaterial, etc. can be used for the restricted medium.
  • the hot pressure extraction conditions are 15 to 200 minutes of crushing dermabiotics cells to recover the formed mineral-nucleotides, a temperature of 120 to 130°C, and a pressure of 0.12 to 0.30 MPa. do.
  • the protein components in the cell wall and cytoplasm other than the lava seawater mineral-nucleotide conjugate are thermally denatured, and the thermally denatured component is cooled and precipitated at 3 to 5°C and centrifuged at 3,000 to 8,000 rpm for 10 to 30 minutes.
  • -A dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater is prepared by recovering nucleotides and removing residual components using a nanomembrane.
  • the centrifugation is a condition capable of removing the pellets contained in the cell wall and cytoplasm through the hot-pressure extraction conditions.For example, 3,000 to 8,000 rpm may be used, and the nanomembrane is filtered with a membrane filter of 200 nanometers or less. Thus, it provides a manufacturing method for purifying only dermabiotics-derived mineral-nucleotides.
  • a dermabiotics cosmetic composition for skin improvement characterized in that it has skin barrier reinforcement, skin vitality, and skin wound healing.
  • the present invention can provide a dermabiotic-derived mineral-nucleotide conjugate cultured in lava seawater prepared by the method of the present invention.
  • the cosmetic may contain all commonly used ingredients.
  • 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 amount of the ingredients may be selected within a range that does not impair the inherent effect of the cosmetic.
  • the amount of the ingredients added may be, for example, 0.1 to 100% by weight, preferably 0.1 to 30% by weight based on the total weight of the composition, but is not limited thereto.
  • the type of cosmetic is not particularly limited, and for example, lotion, emulsion, gel, cream, essence, pack, ampoule, lotion, detergent, soap, body products, skin care cosmetics such as soap, oil, lipstick, Makeup cosmetics such as foundations, hair cosmetics, etc., and the formulation is not particularly limited.
  • Dermabiotics seed culture solution was inoculated into the sterilized basic medium of the fermentation tank in this sterilized general constant water medium, and cultured for 20 hours under conditions of 100 rpm and 37°C, but 40% (w/v) glucose and 40% (w/v) An aqueous solution in which sodium hydroxide was mixed in a volume ratio of 1:1 (hereinafter, referred to as glucose-sodium hydroxide solution) was instilled at regular time intervals and cultured while maintaining the pH at 5.0-7.5 (Fed-batch culture performed).
  • glucose-sodium hydroxide solution lactic acid bacteria Lactobacillus plantarum strain was cultured in Lactobacilli MRS Broth (BD) at 37°C for 24 hours, hereinafter referred to as dermabiotics seed culture solution.
  • the dermabiotics culture solution obtained in Preparation Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterilization constant (distilled water), and this was repeated once.
  • the cells were suspended in sterilized water and cultured for a second time at 37° C. for 16 hours. Afterwards, the dermabiotics cells were crushed by hot-pressure extraction for 120 minutes at a temperature of 121°C and a pressure of 0.15 MPa, and after cooling to 4°C, the dermabiotics lysate was centrifuged (6,000rpm, 20 minutes). Dermabiotics-derived nucleotides were recovered.
  • the dermabiotics culture solution obtained in Preparation Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterilization constant (distilled water), and this was repeated once.
  • the cells were suspended in sterilized water and cultured for a second time at 37° C. for 16 hours.
  • the dermabiotics cells were crushed by hot-pressure extraction for 120 minutes at a temperature of 121°C and a pressure of 0.15 MPa, and after cooling to 4°C, the dermabiotics lysate was centrifuged (6,000 rpm, 20 minutes) to the final supernatant. Bay was recovered. The recovered supernatant was finally filtered through a nanomembrane filter of 200 nanometers or less to recover the nucleotide purified product derived from Dermabiotics.
  • Lava seawater mixture containing 10 to 90% (v/v) of lava seawater or lava seawater itself (100%) was prepared as cultivation water, and water (lava seawater 0) under the conditions for preparing the basic medium using the cultivation water in Preparation Example 1. % State) was used instead.
  • the medium was sterilized as in Preparation Example 1 using culture water containing lava seawater at each concentration.
  • the dermabiotics seed culture solution was inoculated into the sterilized lava seawater medium as in Preparation Example 1, and the pH was dropped at intervals of 5.0 to 7.5 using a glucose-sodium hydroxide solution for 20 hours at 100 rpm and 37°C. While maintaining the pH at 5.0-7.5 (Fed-batch culture performed)
  • the dermabiotics culture solution cultured in lava seawater of Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterilization constant (distilled water), and this was repeated once. Thereafter, the cells were suspended in mineral enriched water (calcium 220 mg, magnesium 1,130 mg, selenium 0.013 mg or more) prepared by concentrating lava seawater to 1/10 volume or more, followed by secondary culture at 37° C. for 16 hours. Afterwards, the dermabiotics cells were crushed by hot-pressure extraction for 120 minutes at a temperature of 121°C and a pressure of 0.15 MPa, and after cooling to 4°C, the dermabiotics lysate was centrifuged (6,000rpm, 20 minutes). Only the final supernatant was recovered. The recovered supernatant was finally filtered through a nanomembrane filter of 200 nanometers or less to recover dermabiotics-derived mineral-nucleotides.
  • mineral enriched water calcium 220 mg, magnesium 1,130 mg
  • the culture solutions of Preparation Example 1 and Example 1 were taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed into Petridish, and 20 ml of sterilized Lactobacilli MRS Agar (BD, USA) were mixed and solidified.
  • the number of dermabiotics viable cells was confirmed by counting colonies cultured for 48 hours in a 37°C stationary incubator, and this is shown in FIG. 2 (hereinafter, referred to as dermabiotics viable cell number measurement method).
  • the culture solutions of Preparation Example 1 and Example 1 were centrifuged (6,000 rpm, 20 minutes), only the cells were recovered, washed with sterilization constant (distilled water), and this was repeated once. Thereafter, the cells were suspended in sterilized water and cultured for a second time at 37° C. for 16 hours. Thereafter, the dermabiotics cells were crushed by hot-pressure extraction for 120 minutes at a temperature of 121°C and a pressure of 0.15 MPa, and the dermabiotics lysate was centrifuged (6,000 rpm, 20 minutes) after cooling to 4°C, The nucleotides were identified through final filtration using a nanomembrane filter of 200 nanometers or less as the residual component.
  • the UV wavelength absorbance (230nm, 260nm, 280nm) of the nucleotides prepared in the dermabiotics culture medium for each lava seawater concentration was confirmed, and the nucleotide concentration was shown in FIG.2 (hereinafter referred to as dermabiotics-derived nucleotide measurement method).
  • the concentration of 0% lava seawater means the number of dermabiotics viable cells or dermabiotics-derived nucleotides cultured using the culture medium obtained in Preparation Example 1.
  • dermabiotics were recovered from the dermabiotics culture broth cultured using lava seawater as culture water through centrifugation, and then the amount of nucleotides recovered according to the subsequent secondary culture conditions was compared.
  • the dermabiotics culture solution cultured at a concentration of 30 (v/v)% of lava seawater obtained in Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered and washed with sterilization constant (distilled water). And this was repeated once. Thereafter, the cells were suspended in general constant water and lava seawater mineral enriched water (calcium 220 mg/L, magnesium 1,130 mg/L, selenium 0.013 mg/L or more), respectively, followed by secondary culture at 37° C. for 16 hours. Thereafter, the hot-pressure extraction was performed at a temperature of 121°C and a pressure of 0.15 MPa, but the hot-pressure extraction time was divided into 30 minutes and 120 minutes in order to check the optimum hot-pressure extraction conditions.
  • FIG. 3 shows the concentration of nucleotide content derived from Dermabiotics according to the secondary fermentation water and hot pressure extraction time by checking the UV wavelength absorption using Nanodrop 2000 (Thermo, USA) for each condition.
  • the mineral content of the sample prepared by the above method was analyzed in order to confirm the concentration of minerals introduced from lava seawater and bound to nucleotides.
  • nitric acid nitric acid
  • an inorganic (calcium, magnesium, selenium) standard reagent in the range of about 0.1 to 1 g, it was taken and used as a standard solution.
  • the sample prepared by the above method was diluted with nitric acid.
  • Second culture method Mineral content (mg/L) 30min General constant secondary culture calcium 10 magnesium 120 Selenium 0 Lava seawater mineral enriched water secondary culture calcium 72 magnesium 221 Selenium 0.002 120min General constant secondary culture calcium 22 magnesium 232 Selenium 0 Lava seawater mineral enriched water secondary culture calcium 132 magnesium 532 Selenium 0.006
  • mineral-nucleotides derived from Dermabiotics obtained by primary culture of 30 (v/v)% lava seawater and secondary culture with mineral enriched lava seawater were used as a representative sample of Example 2, and'Lava Seawater Culture Dermabio It is referred to as a mineral-nucleotide conjugate derived from Tix' or a mineral-nucleotide conjugate derived from dermabiotics cultured in lava seawater.
  • nucleotides obtained in Preparation Example 2 are referred to as "nucleotides derived from dermabiotics cultured in general constant water” or “nucleotides derived from dermabiotics cultured in general constant water”.
  • MTT assay is an experimental method that is widely used for cell proliferation and toxicity by measuring the number of living cells. Living cells are water-soluble in the mitochondria, and the yellow salt, MTT(3-[4,5-dimethlythiazole-2-yl]-2,5 -diphenyltetrazolium bromide) is reduced to water-insoluble blue formazan derivatives by succinate dehydrogenase or mitochondrial dehydrogenase.
  • fibroblasts and human keratinocytes were diluted in a 24 well plate at a density of 1.5 ⁇ 10 5 cells/well, and DMEM (Dulbecco's Modified Eagle) containing 10% (v/v) bovine serum.
  • DMEM Dulbecco's Modified Eagle
  • medium fetal bovine serum
  • Welgene, Korea cultured for 1 day in a 37°C, 5% CO 2 incubator with medium. After cultivation, all of the medium was removed, replaced with a DMEM medium containing no bovine serum, and the test samples were added for each concentration, followed by incubation for 1 day.
  • DMSO dimethyl sulfoxide
  • FIGS. 4A and 4B The results are shown in FIGS. 4A and 4B, and cytotoxicity was not confirmed up to 5 (v/v)% of the dermabiotics-derived mineral-nucleotide conjugate preparation solution (supernatant) cultured in lava seawater, but 200 nanometers. Dermabiotics-derived nucleotides cultured in normal water that was not filtered with a nanomembrane filter below a meter (nanometer) showed cytotoxicity from the 3% (v/v) treatment group. ⁇ The sample concentration% in FIGS. 4 to 8 all means (v/v)%.
  • the sample of Preparation Example 2 contains the cell walls, cytoplasm, protein, and protein salt components, which are cell structures that cause skin inflammation, without purification, in the evaluation of basic skin toxicity, in both skin fibroblasts and skin keratinocytes 3% ( It can be seen that apoptosis occurs when the sample is processed above v/v).
  • the inflammation inducing factors were removed through a nanomembrane filter of 200 nanometers or less, and thus up to 5% (v/v). Skin safety was confirmed by not showing cytotoxicity even if each cell of the skin was treated.
  • the dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater exhibited cytotoxicity despite the production of dermabiotics viable bacteria at a high concentration of 2.0x10 10 CFU/ml through 30% (v/v) lava seawater cultivation. As it is manufactured in a form that is not shown, it is possible to expect the maximization of skin health effects through the implementation of more usage.
  • Preparation Example 2 In order to compare samples before/after the nanomembrane filtration process, Preparation Example 2, Preparation Example 3, and Example 2 were treated on skin fibroblasts to confirm cytotoxicity as well, and are shown in Table 2 below. For comparison, a sample without performing a nanomembrane filter of 200 nanometers or less among the manufacturing methods of Example 2 was prepared and used as a comparative group.
  • fibroblasts were dispensed into a 35 mm plate and stabilized for 24 hours, then replaced with a serum-free medium, and samples (Example 2 and Preparation Example 3) were treated.
  • nucleotide obtained in the following Preparation Example 3 is referred to as "a nucleotide purified product derived from dermabiotics cultured in a normal constant water” or a “a nucleotide purified product derived from dermabiotics cultured in a general constant water”.
  • saline buffer solution HBSS
  • HBSS saline buffer solution
  • the saline buffer solution was added to the plate as much as the cell culture solution, and irradiated until the total irradiation amount reached 50 mJ/cm 2 of UVB.
  • the medium was replaced with a Serum-free medium, and the samples (Example 2 and Preparation Example 3) were treated and cultured in a 37°C, 5% CO 2 incubator for 24 hours.
  • 1 ⁇ g/ml of JC-1 solution was treated and reacted in a 37°C, 5% CO 2 incubator without light for 2 hours, and then the medium was removed and observed with a fluorescence microscope.
  • the ratio of red:green is confirmed to be 1:1 if it is a healthy mitochondria, but if a stress that can induce mitochondrial dysfunction is applied or if damage or death of cells is induced, red There is no change in the green value, such as the :green ratio of 0.5:1, and the pure red value decreases remarkably.
  • the dermabiotics-derived mineral-nucleotide conjugate treated group cultured in lava seawater recovers cells with increased mitochondrial damage due to UVB irradiation, and makes mitochondria healthy at the level of the control group not treated with UVB. It is confirmed to keep it in the state.
  • the nucleotide purified products derived from dermabiotics cultured in the general constant did not increase the value up to the control level, indicating that the mitochondrial recovery state was slow.
  • Fibroblasts human fibroblast
  • HBSS salt buffer solution
  • UVB 50 mJ/cm 2 UVB 50 mJ/cm 2 was irradiated.
  • samples (Example 2 and Preparation Example 3) were treated and cultured in a 37°C, 5% CO 2 incubator for 24 hours. After culture, the cells are collected with 1 ml of lysis reagent (Qiagen), and RNA is extracted using chloroform, 2-propanol, and ethanol.
  • a high capacity RNA-to-cDNA kit (Applied Biosystems, USA) was used, and an experiment was performed by the method of the kit.
  • a kit qPCRBIO SyGreen Blu Mic Lo-ROX, PCRBIOSYSTEMS, London, UK
  • the primers used for PCR are shown in Table 3, and were synthesized and used by Cosmo Genetech (KOREA).
  • the expression of PGC-1 ⁇ decreased during skin aging by dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater to recover damaged cells from UVB-treated cells by 5 (v/v)%.
  • Dermabiotics-derived nucleotide conjugate cultured in lava seawater increased by 16% compared to the purified nucleotides derived from dermabiotics cultured at the same concentration in normal water. Is expected.
  • Human keratinocytes are inoculated into cell culture dishes and cultured for 24 hours. Then, it was replaced with a serum-free Epilife (gibco) medium, and the samples (Example 2 and Preparation Example 3) were treated and cultured for 3 days. After culture, the cells are collected with 1 ml of QIAzolTM Lysis reagent (Qiagen), and RNA is extracted using chloroform, 2-propanol, and ethanol. After adding purified water treated with DEPC, the extracted RNA was quantified using a Qubit florometer (Invitrogen, USA) and a Qubit RNA BR Assay kit (Invitrogen, USA), and then cDNA was synthesized and real-time PCR was performed.
  • QIAzolTM Lysis reagent Qiagen
  • RNA is extracted using chloroform, 2-propanol, and ethanol. After adding purified water treated with DEPC, the extracted RNA was quantified using a Qubit flor
  • RNA-to-cDNA kit (Applied Biosystems, USA) was used, and an experiment was performed by the method of the kit.
  • kit qPCRBIO SyGreen Blu Mic Lo-ROX, PCRBIOSYSTEMS, London, UK
  • the amplification product was quantitatively analyzed.
  • the primers used for PCR are shown in Table 4, and were synthesized and used by Cosmo Genetech (KOREA).
  • SPT Serine palmitoyl-transferase
  • Human normal keratinocytes were inoculated into cell culture dishes, cultured for one day, and replaced with serum free Epilife (gibco) medium, and samples (Examples 2 and 3) were treated at different concentrations and cultured for 3 days.
  • 4% (w/v) formaldehyde solution is treated, followed by 0.1% (v/v) Triton X-100 solution, and incubated for 10 minutes. Then, it was replaced with 1% BSA solution, and after 1 hour, 2 ⁇ g/ml of filaggrin antibody (Invitrogen) was treated and reacted. Then, Alexa Fluor® 488 conjugate antibody (Invitrogen) was treated to react, and DAPI (sigma) 1 ⁇ g/ml was added to take a picture.
  • the dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater regulates the passage of water and ions and expresses claudin-1 gene for tight junction protein (TJ), which is involved in skin barrier strengthening function.
  • TJ tight junction protein
  • Each sample was mass-produced at a scale of 100 L by the manufacturing method of dermabiotics-derived mineral-nucleotide conjugate cultured in the lava seawater of Example 2 and the manufacturing method of Preparation Example 2, and a photograph of the result is disclosed in FIG. .
  • the purity was measured by the Dermabiotics Nucleotide Analysis method and shown in FIG. 10.
  • the supernatant (microbial lysate) containing nucleotides using Preparation Example 2 which is a conventional technique, contains various cell wall components, lipoproteins, glycoproteins, etc., so that caramelization is performed between hot-pressure extraction. The color changed to dark brown, and an abnormal fermentation smell was also shown.
  • the method of manufacturing dermabiotics-derived mineral-nucleotide conjugates cultured in lava seawater according to the present invention lava seawater culture, microbial crushing, centrifugation, membrane separation process
  • the purity of the dermabiotics-derived mineral-nucleotide conjugates cultivated in mass-produced lava seawater is considered pure if the absorption ratio 260/280 is 2.0-2.2 and the absorption ratio 260/230 is 1.8-2.0.
  • the concentration at each UV wavelength was high as the nucleotide was concentrated at a high concentration, and the purity was confirmed through the ratio.
  • the unity of the graph could be maintained by removing impurities such as proteins mixed in the manufacturing process.
  • a flexible lotion (skin, 100 g) containing the supernatant containing the mineral-nucleotide conjugate of Example 2 was prepared according to a conventional method.
  • the supernatant of the supernatant containing the mineral-nucleotide conjugate of Example 2 was extracted with a 70 (v/v)% ethanol aqueous solution to obtain a concentrate from which the solvent was removed, and a nutrient lotion (lotion, 100 g) containing this concentrate as shown in Table 8 was usually used. It was prepared according to the method of.
  • a nutrient cream (100 g) containing the supernatant containing the mineral-nucleotide conjugate of Example 2 was prepared according to a conventional method.

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Abstract

La présente invention concerne un procédé de préparation d'un conjugué de minéral et nucléotide dérivé de dermabiotiques cultivés dans de l'eau de mer de lave. Le conjugué minéral-nucléotide est exempt de toxicité pour la peau et présente des fonctions de régénération des cellules cutanées, de renfort de la barrière cutanée, d'apaisement de la peau, et de préservation du microbiote de la peau, et peut de ce fait être utilisé dans une composition cosmétique fonctionnelle ou un agent à usage externe, etc.
PCT/KR2020/004412 2019-04-05 2020-03-31 Procédé de préparation d'un conjugué de minéral dérivé d'eau de mer de lave et de nucléotide dérivé de dermabiotiques, et composition cosmétique de dermabiotiques fonctionnels l'utilisant Ceased WO2020204579A1 (fr)

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KR1020200022437A KR102305076B1 (ko) 2020-02-24 2020-02-24 용암해수 유래 천연 미네랄 코팅 프로바이오틱스의 제조방법 및 이를 이용한 용암해수 유래 천연 미네랄 코팅 프로바이오틱스 분무건조 제제
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KR20170096712A (ko) * 2016-02-17 2017-08-25 주식회사 씨앤비코스메틱 마가목추출물의 발효물을 유효성분으로 함유하는 피부노화방지 및 피부주름개선용 화장료 조성물

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WO2009037698A1 (fr) * 2007-09-17 2009-03-26 Ramot At Tel-Aviv University Ltd. Procédé permettant la culture et la reproduction de tissus coralliens
KR20130046082A (ko) * 2011-10-27 2013-05-07 재단법인 제주테크노파크 탈염 용암해수의 발효물의 제조 방법과 그 방법에 의하여 얻어진 발효물 및 그 발효물을 이용한 화장료 조성물
KR101463012B1 (ko) * 2012-12-13 2014-11-18 재단법인 제주테크노파크 용암해수 미네랄을 이용한 피부 외용제 조성물
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KR20170096712A (ko) * 2016-02-17 2017-08-25 주식회사 씨앤비코스메틱 마가목추출물의 발효물을 유효성분으로 함유하는 피부노화방지 및 피부주름개선용 화장료 조성물

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