WO2021049852A1 - Souche lactobacillus paracasei subsp. tolerans sw1 présentant une capacité d'amélioration des rides cutanées et produisant des métabolites naturels hautement fonctionnels, et son utilisation - Google Patents

Souche lactobacillus paracasei subsp. tolerans sw1 présentant une capacité d'amélioration des rides cutanées et produisant des métabolites naturels hautement fonctionnels, et son utilisation Download PDF

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WO2021049852A1
WO2021049852A1 PCT/KR2020/012118 KR2020012118W WO2021049852A1 WO 2021049852 A1 WO2021049852 A1 WO 2021049852A1 KR 2020012118 W KR2020012118 W KR 2020012118W WO 2021049852 A1 WO2021049852 A1 WO 2021049852A1
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lactobacillus paracasei
tolerans
paracasei subsp
cis
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PCT/KR2020/012118
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English (en)
Korean (ko)
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최용준
정선욱
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서울시립대학교산학협력단
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Publication of WO2021049852A1 publication Critical patent/WO2021049852A1/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
    • A61K8/96—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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/41—Amines
    • 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
    • A61Q19/08—Anti-ageing preparations
    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to a Lactobacillus paracasei subsp. tolerans (Lactobacillus paracasei subsp. tolerans) SW1 strain producing a natural metabolite effective in improving skin wrinkles and its use.
  • Lactobacillus paracasei subsp. tolerans Lactobacillus paracasei subsp. tolerans SW1 strain producing a natural metabolite effective in improving skin wrinkles and its use.
  • Skin protects the body as a primary defense system against external physicochemical hazards, and serves as an indicator of health and beauty. Recently, as modern people's interest in skin health and aesthetics is greatly increased, consumption of functional cosmetics effective for skin whitening, wrinkle improvement, and aging prevention is continuously increasing.
  • Skin aging can be divided into intrinsic aging (natural aging) that occurs naturally with age and photoaging by ultraviolet rays exposed to external activities.
  • the main factor in endogenous aging may be a deficiency of estrogen, a female hormone.
  • Estrogen is well known for promoting collagen synthesis by stimulating fibroblasts in the dermal layer of the skin and inhibiting the expression of collagenase (MMP-1), a collagen-degrading enzyme.
  • MMP-1 collagenase
  • endogenous aging can be characterized by fine lines, pigmentation, dry skin, and decreased skin elasticity.
  • Retinol, alpha-hydroxy acid, and adenosine as representative wrinkle improvement materials are widely used for skin regeneration by increasing collagen synthesis and normalizing the epidermal keratinization cycle of the skin. It is known to be very unstable to light and heat and to be irritating to the skin. Accordingly, there is a need for research and development on materials that have no or very low skin irritation and are effective for anti-aging and wrinkle improvement.
  • microorganisms that exist widely in nature can produce various kinds of natural metabolites through a series of metabolic processes, and the various metabolites they produce are very broad in human life, ranging from food, medicine, feed additives, and cosmetic materials. It is being utilized properly.
  • lactic acid bacteria are widely distributed in natural systems such as skin, digestive tract, intestines, oral cavity, various fermented foods and soil of humans and mammals. These lactic acid bacteria are said to have a positive effect on various physiological activities and immune functions of the human body. It is well known.
  • the present inventors analyzed the metabolite product of the cell extract of the strain Lactobacillus paracasei subspecies Tolerance SW1 (accession number: KACC81096BP) obtained by isolating and culturing lactic acid bacteria from infant skin through gas chromatography.
  • sarcosine and cis-vaccenic acid.
  • Sarcosine is found in muscles and various human tissues, is known as a non-toxic natural amino acid, and is known to regulate UV-blocking efficacy and skin oil.
  • the inventors of the present invention determined that the cell extract of Lactobacillus paracasei subspecies tolerance SW1 containing the two kinds of natural ingredients was compared with the standard substance of sacosine and cis-baxenoic acid and through a control experiment, sacosine was MMP-1 (matrix The present invention was completed by confirming that metalloproteinase-1) inhibition, skin wrinkle improvement effect such as collagen production, and baksen acid are effective in improving inflammation.
  • An object of the present invention is to contain any one or more selected from the group consisting of Lactobacillus paracasei subsp . tolerans (Lactobacillus paracasei subsp. tolerans) SW1 strain, a culture solution of the strain, a concentrate of the culture solution, and a dried product of the culture solution as an active ingredient. It is to provide a cosmetic composition for improving skin wrinkles or for anti-aging.
  • Lactobacillus paracasei subspecies tolerance (Lactobacillus paracasei subsp . tolerans ) It is to provide a health functional food for improving skin wrinkles or for anti-aging containing at least one selected from the group consisting of SW1 strain, a culture medium of the strain, a concentrate of the culture medium, and a dried product of the culture medium as an active ingredient.
  • Another object of the present invention is Lactobacillus paracasei subspecies tolerance (Lactobacillus paracasei subsp . tolerans ) It is to provide a method for producing sarcosine and cis-vaccenic acid, including the step of culturing SW1.
  • the present invention is Lactobacillus paracasei subspecies tolerance (Lactobacillus paracasei subsp . tolerans ) Provides a cosmetic composition for improving skin wrinkles or anti-aging containing any one or more selected from the group consisting of SW1 strain, a culture solution of the strain, a concentrate of the culture solution, and a dried product of the culture solution as an active ingredient.
  • skin wrinkles containing one or more selected from the group consisting of Lactobacillus paracasei subsp. tolerans SW1 strain, a culture medium of the strain, a concentrate of the culture medium, and a dried product of the culture medium as an active ingredient Provides health functional foods for improvement or anti-aging.
  • Lactobacillus paracasei subspecies tolerance (Lactobacillus paracasei subsp . tolerans ) It provides a method for producing sarcosine and cis-vaccenic acid, including the step of culturing SW1.
  • the Lactobacillus paracasei subsp. tolerans SW1 strain of the present invention inhibits MMP-1, promotes collagen synthesis by UV exposure, and improves inflammation, so the cell extract of the strain, and the above
  • a cosmetic composition containing as an active ingredient any one or more selected from the group consisting of the dried product of the extract concentrate may be added to cosmetics and usefully used for preventing and improving endogenous and photogenic skin aging.
  • Figure 1a is a Lactobacillus paracasei subspecies tolerance ( Lactobacillus paracasei subsp. tolerans ) It is a diagram showing the results of gas chromatography analysis of the cell extract of SW1 strain.
  • 1B is a diagram showing the standard peak of sarcosine (peak 1).
  • 1C is a diagram showing a standard peak of cis-vaccenic acid (peak 2).
  • FIG. 2A is a diagram showing the analysis results for inhibition of the production of MMP-1, which inhibits collagen synthesis, by treating the separated microbial cell extracts and standard substances at respective concentrations in fibroblasts.
  • Figure 2b is a diagram showing the analysis results of the amount of collagen produced by UV exposure by treating the skin cells (fibroblast) at each concentration of the separated microbial cell extract and the standard material.
  • FIG. 3 is a diagram showing the analysis results of whether nitric oxide is produced by treating each of Raw264.7 cells with a cell extract and a standard material of the separated microorganism by concentration.
  • Figure 4a is a diagram showing the results of GC-MS analysis of a sample extracted with ethanol from a 3-day culture medium of Lactobacillus paracasei subsp. tolerans SW1 strain.
  • 4B is a diagram showing the results of standard GC-MS analysis of sarcosine.
  • Figure 4c is a diagram showing the results of GC-MS analysis of a sample extracted with ethanol from a 6-day culture medium of Lactobacillus paracasei subsp. tolerans SW1 strain.
  • Figure 4d is a diagram showing the standard GC-MS analysis results of cis-vacceinic acid (cis-vacceinic acid).
  • the present invention relates to the Lactobacillus paracasei subsp. tolerans (Lactobacillus paracasei subsp. tolerans) SW1 strain deposited under the accession number KACC81096BP.
  • SW1 strain The deposited Lactobacillus paracasei subspecies tolerance according to the present invention (Lactobacillus paracasei subsp. tolerans ) SW1 strain (hereinafter, SW1 strain) was identified through isolation and culture from infant skin.
  • the SW1 strain was identified as a novel strain showing 99.18% homology with Lactobacillus paracasei subspecies tolerance JCM1171 in 16S rRNA gene homology search, which is Lactobacillus paracasei subspecies tolerance (Lactobacillus paracasei). subsp. tolerans ) strain.
  • the SW1 strain may have a 16s rRNA sequence of SEQ ID NO: 3.
  • the present inventors used the novel strain Lactobacillus paracasei subspecies tolerance (Lactobacillus paracasei subsp. tolerans ) was named SW1 strain and deposited with the Korea Agricultural Microbial Conservation Center on May 27, 2019, and was given the accession number KACC81096BP.
  • the present invention is a skin containing one or more selected from the group consisting of Lactobacillus paracasei subsp. tolerans SW1 strain, a culture medium of the strain, a concentrate of the culture medium, and a dried product of the culture medium as an active ingredient It provides a cosmetic composition for improving wrinkles.
  • the composition may include sarcosine and cis-vaccenic acid.
  • the culture medium may be an MRS liquid medium obtained by culturing the strain, but is not limited thereto, and may be a conventional microorganism culture medium.
  • the culture solution may be a culture solution obtained by culturing a strain or a culture solution obtained by physically filtering the culture solution, and the culture solution may not be diluted or used to prepare an antibacterial composition.
  • the culture medium exhibits antimicrobial activity, it can be used to prepare an antimicrobial composition through processing such as filtration, concentration, and dilution.
  • the pH of the medium is not significantly limited as long as it is compatible with the SW1 strain, and may preferably be pH 4.5 to 7.0.
  • the culture temperature for culturing the SW1 strain is also not significantly limited as long as it is compatible with the SW1 strain, and may be preferably 25 to 45°C.
  • the present invention contains one or more selected from the group consisting of Lactobacillus paracasei subsp . tolerans (Lactobacillus paracasei subsp. tolerans) SW1 strain, a culture medium of the strain, a concentrate of the culture medium, and a dried product of the culture medium as an active ingredient. It provides a cosmetic composition for anti-aging.
  • the composition may include sarcosine and cis-vaccenic acid.
  • Sarcosine of the present invention has the structure of the following formula (1), is also called N-methyl glycine, is a colorless liquid, has a boiling point of 195.1° C. and a molar mass of 89.094 g/mol.
  • Cis-vaccenic acid also called cis-11-octadecanoic acid, is a cis-type isomer of vaccenic acid. The boiling point is 172°C and the molar mass is 282.25 g/mol.
  • the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the culture of the Lactobacillus paracasei subspecies tolerance SW1 strain, the concentrate of the culture solution, or the dried product of the culture solution as an active ingredient, such as a stabilizer, dissolution Conventional auxiliaries and carriers such as agents, vitamins, pigments and perfumes may be included.
  • the cosmetic composition is a solution, external ointment, cream, foam, nutrient lotion, flexible lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid detergent, bathing agent, sunscreen cream, sun oil, suspension, emulsion, Paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, can be prepared in a formulation selected from the group consisting of a patch and spray, but is not limited thereto.
  • the cosmetic composition may additionally include one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents , Colors, preservatives, fragrances, and the like may be appropriately blended, but are not limited thereto.
  • one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents , Colors, preservatives, fragrances, and the like may be appropriately blended, but are not limited thereto.
  • the cosmetically acceptable carrier included in the cosmetic composition varies depending on the formulation of the cosmetic composition.
  • the formulation of the cosmetic composition is an ointment, paste, cream or gel
  • a carrier component animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide And the like may be used, but is not limited thereto. These may be used alone or in combination of two or more.
  • lactose When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component.
  • additional chlorofluoro It may include a propellant such as hard carbon, propane/butane or dimethyl ether, but is not limited thereto. These may be used alone or in combination of two or more.
  • a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate.
  • a carrier component such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate.
  • Propylene glycol, 1,3-butyl glycol oil, and the like may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid of sorbitan An ester may be used, but is not limited thereto. These may be used alone or in combination of two or more.
  • the formulation of the cosmetic composition 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 may be used, but is not limited thereto. These may be used alone or in combination of two or more.
  • an alkali metal salt of fatty acid a fatty acid hemiester salt, fatty acid protein hydrolyzate, isethionate, lanolin derivative, aliphatic alcohol, vegetable oil, glycerol, sugar, etc.
  • carrier components It may be, but is not limited thereto. These may be used alone or in combination of two or more.
  • the present invention contains one or more selected from the group consisting of Lactobacillus paracasei subsp . tolerans (Lactobacillus paracasei subsp. tolerans) SW1 strain, a culture medium of the strain, a concentrate of the culture medium, and a dried product of the culture medium as an active ingredient. It provides health functional foods for improving skin wrinkles.
  • the health functional food may include sarcosine and cis-vaccenic acid.
  • the present invention contains one or more selected from the group consisting of Lactobacillus paracasei subsp . tolerans (Lactobacillus paracasei subsp. tolerans) SW1 strain, a culture medium of the strain, a concentrate of the culture medium, and a dried product of the culture medium as an active ingredient. It provides health functional foods for anti-aging.
  • the health functional food may include sarcosine and cis-vaccenic acid.
  • Health functional food refers to a food manufactured using nutrients that are liable to be deficient in daily meals or raw materials or ingredients having useful functions for the human body, and refers to foods that help maintain human health, but is not limited thereto. It is not used in a sense that includes all health foods in the usual sense.
  • the form and type of the health functional food is not particularly limited.
  • the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills.
  • the health functional food may contain various flavoring agents, sweetening agents, or natural carbohydrates as additional ingredients.
  • the sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include taumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame.
  • the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.
  • the health functional food of the present invention includes nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pexane and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , Glycerin, alcohol, and the like may be further included. These ingredients can be used independently or in combination.
  • the ratio of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
  • the present invention provides a method for producing sarcosine and cis-vaccenic acid comprising the step of culturing Lactobacillus paracasei subsp . tolerans SW1. .
  • the cultivation may be performed in MRS liquid medium, but is not limited thereto, and may be performed in a conventional microorganism culture medium.
  • the pH of the medium is not greatly limited as long as it is compatible with the SW1 strain, and may preferably be pH 4.5 to 7.0.
  • the culture temperature for culturing the SW1 strain is also not significantly limited as long as it is compatible with the SW1 strain, and may be preferably 25 to 45°C.
  • the cultivation may further include the step of taking a colony confirmed to be pure by spreading and culturing the Lactobacillus paracasei subspecies tolerance SW1 strain on a solid medium to obtain a pure isolated strain.
  • the present inventors confirmed that sarcosine and cis-vaccenic acid were included in the cell extract of Lactobacillus paracasei subspecies Tolerance SW1 strain (see FIG. 1A). ), it was confirmed that it exhibits an activity for improving collagen production and inflammation of skin cells (see FIGS. 2A, 2B, and 3).
  • the SW1 strain can produce sarcosine and cis-vaccenic acid. , It was confirmed that these two substances can be secreted to the outside of the cell (see FIGS. 4A and 4C).
  • composition containing any one or more selected from the group consisting of the strain of the present invention, the cell fluid of the strain, the concentrate of the cell, and the dried product as an active ingredient is added to pharmaceuticals and cosmetics to improve wrinkles and anti-aging of the skin. It can be usefully used.
  • MRS solid medium MRS 1% peptone, 1% beef extract, 0.4% yeast extract, 2% Glucose, 0.5% sodium acetate hydrate, 0.1% tween 80 (polyoxyethylene sorbitan monooleate), 0.2% potassium phosphate monobasic, 0.2% triammonium citrate, 0.02% magnesium sulfate sulfate), 0.005% manganese(II) sulfate monohydrate, 1.5% agar
  • a single colony formed on the MRS solid medium was re-plated and cultured in pure water, and then inoculated into the MRS liquid medium of the same component and cultured with shaking at 30° C. for 3 days. Then, the culture solution was centrifuged at 4,000 rpm and 4° C. for 20 minutes to recover the cells.
  • Genomic DNA was extracted from the collected cells using a DNA isolation kit (Intron lifetechnology), and the extracted DNA was used as a template, and the 16s rRNA nucleotide sequence was amplified using a primer having the nucleotide sequence shown in Table 1 below.
  • PCR was carried out by reacting at 95° C. for 5 minutes, then repeating the reaction process for 30 seconds at 95° C., 30 seconds at 60° C. and 3 minutes at 72° C. and repeating the reaction 25 times.
  • the obtained PCR product was confirmed by electrophoresis for 20 minutes by applying a voltage of 120V on a 1% agarose gel, put it in a 1.5ml tube, and purified using a DNA fragment purification kit (Intron lifetechnology).
  • the nucleotide sequence of the purified gene (SEQ ID NO: 3) was compared with the 16s rRNA nucleotide sequence of the existing strain, and the similarity was determined by NCBI's BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and EZ- It was analyzed using the Taxon database (http://www.eztaxon.org) (Fig. 2).
  • the isolated strain was found to have 99.18% homology with the Lactobacillus paracasei subspecies Tolerance JCM1171 strain (FIG. 1). Accordingly, the strain was named as Lactobacillus paracasei subspecies tolerance SW1 strain, and as of May 27, 2019, it was deposited with the Korea Agricultural Microbial Conservation Center (KACC) under the accession number KACC81096BP.
  • KACC Korea Agricultural Microbial Conservation Center
  • a single colony of the SW1 strain formed on the MRS solid medium was inoculated into the MRS liquid medium of the same component and cultured with shaking at 30° C. for 1 day. Thereafter, 1% of the pre-culture was added to the same liquid medium, followed by shaking culture at 30° C. for 3 days. The cells were recovered by centrifugation at 4,000 rpm and 4° C. for 20 minutes. After removing the supernatant, the strain was washed three times with sterilized distilled water, and then centrifuged under the same conditions to obtain SW1 cells.
  • one colony of Lactobacillus paracasei subspecies tolerance SW1 strain was cultured with shaking in 3 ml MRS liquid medium for 24 hours, and 500 ⁇ l culture was added to 50 ml of MRS liquid medium, followed by additional culture for 3 days. Thereafter, the culture solution was centrifuged at 4,000 rpm and 4° C. for 20 minutes to completely separate the cells, and the supernatant was removed. The collected cells were suspended three times using 25 mL sterile distilled water, and washed by re-centrifugation.
  • SW1 cells were completely suspended using a pipette, transferred to a 2 ml screw cap tube containing 500 ⁇ l of silica beads, and then over a total of 3 times for 1 minute at 6,000 rpm. SW1 cells were disrupted by bead-beating. Then, centrifugation at 13,000 rpm for 15 minutes to separate the cell lysate from silica beads, transfer the supernatant to a new 1.5 ml tube, and then use a polytetrafluoroethylene syringe filter with 0.2 ⁇ m pores. ) (SD13P020NL, Hyundai Micro Corp.).
  • Components contained in the cell extract were analyzed by gas chromatography (Agilent 5977B MSD GC system, Agilent).
  • the column is Agilent 19091S-433UI HP-5ms was used, helium was used as the carrier gas, and the flow rate was 1ml/min, and gradient elution of 10°C/min from 80°C to 320°C for 2 minutes and 80°C to 320°C. ) was applied.
  • the highest concentration of the material was detected at peak retention times of about 15.2 and 17.1 minutes, excluding the extraction solvent peak, and this is a selective ion monitoring mode and Agilent MassHunter workstation software.
  • the peaks detected as a result of analysis through the library of the compounds contained in FIG. 1 b show a high matching rate with sarcosine (peak 1) of FIG. 1 b and cis-vaccenic acid (peak 2) of FIG. 1 c. I got it. Therefore, it was confirmed that the strain of the present invention containing sacosine and cis-baksenic acid in the cell extract has the ability to produce sacosine and cis-baksenic acid (FIG. 1A).
  • MMP-1 collagenase
  • skin fibroblasts were added to a 96-well plate culture dish (96-well plate) at a cell concentration of 1 ⁇ 10 5 CFU/well, and cultured at 37° C. for 24 hours. After removal of the medium, 180 ⁇ l of a new medium was additionally dispensed into each well, and 20 ⁇ l of the dilution of the SW1 strain cell extract was added each to obtain an appropriate final concentration. It was irradiated with 12.5mJ. For each UV-treated experimental group, the cell extract was treated by concentration and cultured for an additional 48 hours, and then the medium and the cells were respectively recovered. The supernatant was taken and the amount of collagen-degrading enzyme present in the medium was measured.
  • a procollagen assay was performed to measure the amount of collagen newly produced in fibroblasts under UV conditions.
  • fibroblasts at a concentration of 1 ⁇ 10 6 CFU/well were placed in a 6-well culture plate and cultured at 37° C. for 24 hours. Thereafter, the medium was removed and a serum-free medium was added to induce starvation of cells for 24 hours, washed twice with PBS, and irradiated with 12.5mJ at a UV (312 nm) wavelength.
  • the cell extract and the standard were treated by concentration and cultured for an additional 48 hours, and then the medium and the cells were respectively recovered. The recovered medium was analyzed for the amount of collagen using a Procollagen Type-I C-Peptide (PIP) EIA kit (Takara Bio, Inc.).
  • PIP Procollagen Type-I C-Peptide
  • RAW 264.7 cells which are mouse macrophages
  • the supernatant was collected and dispensed into a 96-well plate by 100 ul, and the same amount of Grease reagent was added, followed by reaction at room temperature for 15 minutes. After the reaction was completed, the absorbance was measured at 540 nm, and the amount of NO in the medium was measured using the amount of NO produced in the experimental group treated with LPS as 100%.
  • the calibration curve was prepared using sodium nitrate as a standard reagent.
  • the following experiment was performed to analyze the components contained in the cell culture solution of the Lactobacillus paracasei subspecies tolerance SW1 strain.
  • one colony of Lactobacillus paracasei subspecies tolerance SW1 strain was cultured with shaking in 3 ml MRS liquid medium for 24 hours, and 500 ⁇ l culture was added to 50 ml of MRS liquid medium, followed by additional culture for 3 to 6 days. . Thereafter, the culture solution was centrifuged at 4,000 rpm and 4° C. for 20 minutes to completely separate the cells, and the supernatant was recovered. The recovered supernatant was divided into a new 50ml tube by 25ml each, frozen at -80°C for 4 hours, and then freeze-dried until sufficiently dried for 3 days using a freeze dryer.
  • the culture solution of the SW1 strain cultured for 3 days and 6 days was analyzed through the selective ion monitoring mode and the library of the compound included in the Agilent MassHunter workstation software, 3 days.
  • Sarcosine was detected at a peak retention time of 16.162 minutes in the culture broth cultured for 6 days, and cis-vaccenic acid showed a high matching rate at a peak retention time of 18.173 minutes in the culture broth of the SW1 strain cultured for 6 days. It was confirmed (FIGS. 4A to 4D ).
  • the SW1 strain can produce sarcosine and cis-vaccenic acid, and secretion these two substances to the outside of the cell.

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Abstract

La présente invention concerne une composition qui est efficace pour améliorer les rides et l'inflammation cutanées, comprenant, en tant que principe actif, un extrait cellulaire d'une souche de Lactobacillus paracasei subsp. tolerans SW1 nouvellement isolée à partir de la peau de nourrissons et de jeunes enfants et, plus précisément, une composition ayant un effet d'amélioration des rides cutanées et un effet anti-âge, comprenant, en tant que principe actif, un extrait cellulaire d'une souche de Lactobacillus paracasei subsp. tolerans SW1 qui produit de la sarcosine et de l'acide cis-vaccénique en tant que métabolites naturels. Selon la description ci-dessus, l'extrait cellulaire de la souche Lactobacillus paracasei subsp. tolerans SW1 est remarquablement excellente en ce qui concerne l'effet d'augmentation de la production de collagène et d'inhibition de la collagénase (MMP-1) et dans la réduction de la génération d'oxyde nitrique, et est ainsi très adaptée à une application dans des médicaments et des cosmétiques fonctionnels pour améliorer les rides et l'inflammation cutanées.
PCT/KR2020/012118 2019-09-10 2020-09-08 Souche lactobacillus paracasei subsp. tolerans sw1 présentant une capacité d'amélioration des rides cutanées et produisant des métabolites naturels hautement fonctionnels, et son utilisation WO2021049852A1 (fr)

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