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• • 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; 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
• • 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
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from 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
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9728—Fungi, e.g. yeasts
• • 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
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9783—Angiosperms [Magnoliophyta]
  • A61K8/9789—Magnoliopsida [dicotyledons]
• • 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
• • 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
  • 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
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
  • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
• • 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
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
  • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
• • 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
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/02—Monosaccharides
• • 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
  • A61K2800/74—Biological properties of particular ingredients
  • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
  • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/25—Lactobacillus plantarum
• • 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
  • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
• • 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
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/66—Aspergillus
  • C12R2001/69—Aspergillus oryzae

Definitions

• the present invention relates to providing a skin composition, a skin improving agent, and a skin improving agent with moisturizing properties, whose main ingredients are fermented extracts produced by two types of microorganisms, koji mold and lactic acid bacteria.
• the skin improving composition may be one whose main ingredient is Lactococcus cremoris itself or a fermented extract produced by fermenting the same.
• the skin is affected by external factors such as infrared rays, ultraviolet rays, dryness, heat, and various pathogens, as well as internal factors such as natural aging, disease, and viruses. This is known to result in phenomena such as wrinkles, age spots, dryness, roughness, and rough skin. Much research has been conducted to improve and prevent these skin disorders and aging phenomena, and skin preparations have been developed and commercialized.
• Patent Document 1 The pharmacological effects of fermented plant extracts have been reported to include anti-aging effects. Other reported effects include inhibition of tyrosinase (inhibition of melanin production), inhibition of AGE production (anti-glycation effect), and inhibition of TNF- ⁇ and NO production (anti-inflammatory effect) (Patent Document 2).
• Hyaluronic acid synthesis promoters have been reported that contain as active ingredients a lactic acid bacteria fermentation product of pomegranate juice (Patent Document 3) and a lactic acid bacteria fermentation product of bean extract (Patent Document 4). As described above, various beneficial effects on the skin are expected.
• nano-type lactic acid bacteria (Lactbacillus plantarum) are derived from sunki pickles eaten in the Kiso region of Nagano Prefecture, and there is little knowledge about their effects on the skin.
• the inventors have been actively researching the uses and effects of fermented extracts and fermented compositions of various microorganisms. They came up with the working hypothesis that beneficial fermented extracts can be produced by fermenting multiple microorganisms with different properties individually and in stages, which led to the present invention.
• the present invention provides a skin composition containing, as an active ingredient, an extract obtained by fermenting and maturing two types of bacteria, koji mold and lactic acid bacteria.
• the lactic acid bacteria may be of the genus Lactococcus or Lactobacillus.
• the present invention provides a skin composition containing, as an active ingredient, an extract obtained by adding water to Aspergillus oryzae and subjecting it to primary fermentation to saccharification, then adding lactic acid bacteria (Lactococcus cremoris H6 strain) and subjecting it to secondary fermentation, and then adding nano-type lactic acid bacteria (Lactbacillus plantarum).
• an extract obtained by adding water to Aspergillus oryzae and subjecting it to primary fermentation to saccharification, then adding lactic acid bacteria (Lactococcus cremoris H6 strain) and subjecting it to secondary fermentation, and then adding nano-type lactic acid bacteria (Lactbacillus plantarum).
• the Aspergillus oryzae may be sterilized after the primary fermentation, the lactic acid bacteria may be sterilized after the secondary fermentation, and then the extract from which the Aspergillus oryzae and lactic acid bacteria have been removed by filtration may be used as the active ingredient. Aspergillus oryzae is sometimes called koji mold or rice koji.
• the skin composition may be used as a skin improving agent with moisturizing properties as a main component.
• the skin composition may be used as an elastase activity inhibitor as a main component.
• the skin composition may be used as a collagen production promoter as a main component.
• the skin composition may be used as a ⁇ -hexosaminidase activity inhibitor having the skin composition as a main component.
• the skin improvement composition has the skin composition as a main component and has the multifaceted effects of being an elastase activity inhibitor, a collagen production promoter, and a ⁇ -hexosaminidase activity inhibitor.
• composition for skin may be used as a composition for external use on the skin, the composition for skin being the main component.
• This fermented extract and composition is suggested to improve skin function and have preventive effects against skin disorders. It is also predicted that the above-mentioned preventive effects can be enhanced by adding nano-type lactic acid bacteria (Lactbacillus plantarum) after the fermentation process.
• nano-type lactic acid bacteria Lacbacillus plantarum
• FIG. 1 is an explanatory diagram showing the process for producing a rice koji lactic acid bacteria fermentation liquid of the present invention.
• Graph showing elastase activity in rice koji lactic acid bacteria fermentation liquid (various concentrations) of the present invention Graph showing collagen production in the rice koji lactic acid bacteria fermentation liquid of the present invention
• the inventors focused on microorganisms, koji mold and two types of lactic acid bacteria, and obtained a fermented extract (SW01, the subject of the test in this example) as described below.
• FIG. 1 is an explanatory diagram showing the fermentation process.
• the first fermentation about 80 to 99% water and about 1 to 20% koji mold (including culture medium) are added and mixed, and then fermented.
• the koji mold used was Aspergillus oryzae and National Koji Mold (NBRC 100959).
• the fermentation temperature was 50 to 55°C, and the fermentation time was 17 to 22 hours.
• the temperature is raised to approximately 90°C to 120°C and maintained at that temperature for 10 to 20 minutes, killing the koji mold with the high temperature and stopping the fermentation process.
• the medium for the koji mold used in this invention is rice starch (powder).
• starch a medium component (rice starch component) outside the koji mold cell, is broken down into monosaccharides such as glucose using the fungal enzymes, and saccharified.
• the Brix was 0.0 and the glucose concentration was 0.03% before fermentation, but after fermentation this increased to Brix 0.8 and glucose concentration 0.38%.
• the koji mold simply breaks down rice starch, so the number of bacteria itself does not change, but the OD660 was shown to increase from 0.50 to 1.50. This correlates with the increase in Brix value.
• the koji mold converts the medium components (rice starch components) outside the koji mold cell into sugars and breaks them down into monosaccharides such as glucose. These monosaccharides are then used as nutrients for the lactic acid bacteria that are administered, making it possible to produce a fermentation filtrate that does not use ingredients, additives, or additives that are not derived from koji mold or lactic acid bacteria.
• Lactococcus cremoris SW01 (NITE BP-03847) was added to the fermented liquid after fermentation was stopped to promote fermentation by the lactic acid bacteria.
• the dosage of the Lactococcus cremoris SW01 strain was 0.01 to 1% by weight of the total amount.
• the glucose previously saccharified is used as a nutrient source for the lactic acid bacteria.
• the lactic acid bacteria is known to be found in plant-derived cheese.
• the Lactococcus cremoris SW01 strain (SW01) was deposited at the National Institute of Technology and Evaluation, Patent Biological Depositary Center on March 3, 2023 (NITE BP-03847).
• the secondary fermentation using this lactic acid bacteria was carried out at a fermentation temperature of 25°C to 35°C for a fermentation time of 17 to 22 hours. It was shown that the OD660 after the fermentation time had elapsed was 1.00 to 2.00. The OD660 value also showed that the number of lactic acid bacteria administered increased. The OD660 value depends on the number of bacteria, and an increase in the value indicates an increase in the number of bacteria.
• the temperature is raised to approximately 90 to 120°C and maintained at that temperature for 10 to 20 minutes, killing the lactic acid bacteria with the high temperature and stopping the fermentation process.
• the pH of the fermented liquid is 3.5 to 4.5, and insoluble matter such as rice residue is filtered out to increase the transparency of the fermented extract.
• the degree of filtration can be changed.
• filtration aids such as diatomaceous earth derived from fossil minerals or perlite derived from volcanic rocks are used.
• mesh filtration is selected.
• the OD value after filtration changed from 1.00-2.00 to 0.00-0.05.
• this filtration process removes insoluble matter such as residues, and the fermentation filtrate (fermentation extract, fermentation composition) can be obtained.
• nano-type lactic acid bacteria (Lactbacillus plantarum) is added to the filtered fermented liquid to a concentration of 0.01-0.05%.
• This nano-type lactic acid bacteria (Lactbacillus plantarum) is a sterilized lactic acid bacteria (20-25% lactic acid bacteria, the rest is dextrin powder). Lactbacillus plantarum is known to be derived from sunki pickles.
• lactic acid bacteria have the disadvantage of clumping when sterilized.
• nano-type lactic acid bacteria are nano-processed (high-pressure homogenization) to prevent clumping and make them easier to handle as a raw material.
• Nano-type lactic acid bacteria cells walls, nucleic acids, proteins, sugars, etc.
• beneficial effects such as strengthening the immune system of the epithelium (epidermis) and normalizing homeostatic functions.
• the OD660 changes from 0.00-0.05 to 0.25-1.00.
• the fermented extract (fermented composition) was kept at 85-100°C for 10-30 minutes to sterilize the fermented liquid. This was done to prevent spoilage and to prevent contamination due to secondary contamination, etc.
• the dermis located inside the epidermis, is mainly composed of collagen, elastin, and hyaluronic acid.
• Elastin is produced by dermal fibroblasts and plays an important role in maintaining the flexibility and elasticity of the skin.
• Elastin is a "elastic fiber” that is flexible and stretchable (elastic), and has the property of returning to its original shape when pressure is released. There is a symbiotic relationship between the two, with elastin bundling collagen.
• Elastase an elastin-degrading enzyme
• Elastase an elastin-degrading enzyme
• elastin is known to be induced by inflammation, aging, ultraviolet rays, active oxygen, and stimuli caused by stress.
• elastin is broken down by elastase, the cross-linked structure between collagen and elastin becomes brittle, causing the skin to lose elasticity and become the cause of wrinkles. Therefore, inhibiting the activity of elastase can be expected to have the effect of restoring or maintaining the elasticity of the skin's dermis and preventing aging.
• elastase derived from human skin fibroblasts was used to evaluate elastase activity by measuring the amount of nitroaniline produced using N-Succinyl-Ala-Ala-Ala-p-nitroanilide as a substrate. Based on the elastase activity, the skin dermis elasticity maintenance and anti-aging function (activity inhibition rate) of the test subject, rice koji lactic acid bacteria fermentation liquid SW01 (hereinafter SW01), was examined.
• a control reaction was performed using only elastase derived from human skin fibroblasts (NHDF-Ad) and the substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide, and the elastase activity was set at 100%.
• the reaction conditions for this experiment were 37°C and 2 hours.
• administration of 50 nM of phosphoramidon, a standard drug for inhibiting elastase demonstrated elastase inhibition, confirming that this experimental system was functioning effectively.
• SW01 rice koji lactic acid bacteria fermentation liquid
• SW01 was added to human dermal fibroblasts (NHDF-Ad), and the amount of collagen in the culture supernatant after 72 hours was measured using ELISA.
• the amount of collagen produced per cell in the test group to which 500 ⁇ g/mL SW01 was added was 1.5 times higher ( Figure 3). This indicates that rice koji lactic acid bacteria fermentation liquid SW01 promotes collagen production in the dermis layer of the skin.
• rice koji lactic acid bacteria fermentation liquid SW01 (sample) contributes to anti-inflammatory and anti-allergic effects. It is known that when IgE bound to the cell surface of fat cells is cross-linked by an antigen, granulocytes containing histamine and other substances are released outside the cell, which causes an allergic reaction.
• ⁇ -hexosaminidase which is abundantly present in granules. This allows the effect of the test substance on allergic reactions to be evaluated.
• rat basophilic leukemia cells RBL-2H3 release granulocytes containing histamine and other substances outside the cell when IgE bound to the cell surface is cross-linked by an antigen.
• This experimental system is used in exploratory research to screen substances with anti-allergic effects against type I allergies.
• RBL-2H3 cells are stimulated with an antigen, and inhibition of degranulation is evaluated by measuring the activity of ⁇ -hexosaminidase (Figure 4).
• the activity of ⁇ -hexosaminidase was measured by adding various amounts of the rice koji lactic acid bacteria fermentation liquid SW01 of the present invention, which was the test subject, and the activity of ⁇ -hexosaminidase was measured by setting the activity of ⁇ -hexosaminidase as 100% when sterilized water was dropped as a control.
• 50 ⁇ g/ml of quercetin was used as a positive control.
• the activity of ⁇ -hexosaminidase was about 40%, demonstrating the suitability of this experimental system.
• the activity of ⁇ -hexosaminidase was about 50% when 2.5 ⁇ g/ml of rice koji lactic acid bacteria fermentation liquid SW01 was added. It was shown that rice koji lactic acid bacteria fermentation liquid SW01 suppresses the activity of ⁇ -hexosaminidase and exerts anti-allergic and anti-inflammatory effects.
• the p value in Figure 4 is p ⁇ 0.01.
• the rice koji lactic acid bacteria fermentation liquid SW01 of the present invention has various effects on improving the skin, such as inhibiting elastase activity, inhibiting ⁇ -hexosaminidase activity, and promoting collagen production.
• Skin improvement includes preventing minor skin diseases such as itching and inflammation from a hygienic perspective and preventing illness.

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• 2023
  • 2023-10-25 CN CN202380014643.9A patent/CN118647359A/zh active Pending
  • 2023-10-25 KR KR1020247022839A patent/KR20240120740A/ko active Pending
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