WO2020177390A1 - Méthode de préparation de solution mère cosmétique contenant de la l-ergothionéine au moyen de la fermentation d'hericium erinaceus - Google Patents

Méthode de préparation de solution mère cosmétique contenant de la l-ergothionéine au moyen de la fermentation d'hericium erinaceus Download PDF

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WO2020177390A1
WO2020177390A1 PCT/CN2019/118949 CN2019118949W WO2020177390A1 WO 2020177390 A1 WO2020177390 A1 WO 2020177390A1 CN 2019118949 W CN2019118949 W CN 2019118949W WO 2020177390 A1 WO2020177390 A1 WO 2020177390A1
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fermentation
stock solution
ergothioine
mass
ergothioneine
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PCT/CN2019/118949
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Chinese (zh)
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陆震
魏玉洁
贾玉倩
孙元军
石艳丽
郭学平
栾贻宏
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华熙生物科技股份有限公司
山东华熙海御生物医药有限公司
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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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • 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
    • A61Q19/08—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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • 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

Definitions

  • the invention relates to the field of cosmetic raw materials, in particular to a method for preparing ergothioine cosmetic stock solution by Hericium erinaceus fermentation and a fermentation stock solution prepared therefrom.
  • Hericium erinaceus belongs to the phylum Basidiomycota and Hericium erinaceus, and is a famous medicinal and edible fungus. Both the fruit body and the mycelium can be used as medicine, the nature is flat and the taste is sweet, it has the functions of helping digestion and benefiting the five internal organs, and is rich in nutrition. It is known as "mountain hericium, seafood bird's nest".
  • the medicinal ingredients of Hericium erinaceus are mainly polysaccharides, oligosaccharides, sterols, fatty acids, hericin and hericin, etc., which can protect the liver and stomach, lower blood sugar, protect nerves, enhance human immunity, and fight cancer. Antioxidant and other functions.
  • Hericium is also rich in sulfur, and sulfur is an important nutrient element that is indispensable to the human body.
  • sulfur is mainly present in the form of thioneine Once the ergothioine in Hericium enters the human body, most of it will eventually become inorganic sulfate, sulfate and neutral sulfur into the blood circulation. One of the effects is to invigorate the spleen and stomach, increase the gastric mucosal barrier function, and affect various Chronic gastritis has a good therapeutic effect.
  • ergothioneine can also resist radiation, promote embryonic growth and development, resist fatigue, and improve the survival ability of the body.
  • Ergothioneine (L-Ergothioneine, EGT), whose chemical name is 2-mercaptohistidine trimethyl inner salt, is the only natural 2-thioimidazole amino acid known so far. It has clear antioxidant and anti-oxidant properties. Inflammation, when applied to cosmetics, it has anti-inflammatory, anti-aging and radiation protection effects. Many foreign cosmetics have used ergothioine as the main functional ingredient in their formulations. As early as 2014, ergothioine was under the National Food and Drug Supervision The Administration is listed in the list of used cosmetic raw materials, and the EGT content in cosmetics reaches 0.01% to 2%, which can achieve better results.
  • EGT is added to various whitening cosmetics, and the addition amount of EGT for creams, creams and lotions ranges from 0.01% to 1.0%, 0.01% to 2.0%, and 0.01% to 3.0%, respectively.
  • American OXIS company has launched two cosmetics based on EGT: Reverge and Prograce.
  • the former can be used as an emollient to improve aging skin, the latter can effectively remove wrinkles and promote the regeneration of new skin.
  • Estee Lauder uses this ingredient as a treasure of the town, adding it to Clinique, the source of wood, and the mystery of the sea And other lines of products.
  • Patent Document 1 discloses a "product containing ergothioneine and its preparation method and use of mushroom extracellular fermentation broth", which is characterized in that the mushroom is fermented and the mycelium in the fermentation broth is removed to obtain extracellular The fermentation broth or the concentrate of the extracellular fermentation broth is provided.
  • the disadvantage is that the mycelium rich in high concentration of ergothione is removed during the process, resulting in the loss of active ingredients, and the mycelium is in addition to ergothione. It contains active substances such as polysaccharides and sterols, which can be used as important functional ingredients in cosmetics.
  • Patent Document 2 discloses a "method for preparing thioneine", which is characterized by optimizing the fermentation process of Pleurotus ostreatus by using a method of fermentation metabolism regulation, and then leaching the thioneine in the mycelia by hot water extraction
  • the disadvantage is that hot water extraction is used in the purification process.
  • the appearance, color and smell of the product are very important.
  • this method will make the fermentation broth darker and produce peculiar smells.
  • heat-sensitive active ingredients in the fermentation broth such as fungal polysaccharides
  • ergothione may also be degraded and inactivated, especially ergothione may be degraded to a certain extent, which affects the efficacy of the product and is difficult to be used as a cosmetic raw material.
  • Patent Document 3 discloses "a method of preparing thioneine", which is characterized in that mushrooms are used as a raw material to obtain thioneine extract through ethanol extraction and resin separation and purification.
  • the disadvantage is that it relies on a pure plant extraction process, which is low in efficiency.
  • organic solvents are involved in the process, and there is a risk of residual organic solvents and may cause environmental pollution.
  • Patent Document 4 discloses "microbial thioneine biosynthesis", which is characterized in that genes related to synthetic thioneine are transcribed into E. coli by genetic means to be fermented to obtain thioneine.
  • the disadvantage is that Escherichia coli is a pathogenic bacterium, which is difficult to apply in cosmetics.
  • Patent Document 5 discloses a "preparation method of a functional oral agent rich in thiothioine", which is characterized by mixing edible fungus mycelium rich in thiothioine with water, and stirring and leaching at 80°C to 100°C. Concentrated to obtain a concentrated liquid, the concentrated liquid is added with food-acceptable additives to make a liquid oral preparation. Its products are mainly used in the field of food and health products, and cannot be directly used as cosmetic raw materials.
  • Hericium erinaceus is mostly used in the food field, and the active ingredients fermented or extracted from it have not been reported in the field of cosmetics.
  • Patent Document 1 CN107708443A
  • Patent Document 2 CN 105296559
  • Patent Document 3 CN 106831596 A
  • Patent Document 4 CN 106661585 A
  • Patent Document 5 CN 103181933
  • the present invention aims to provide a method for preparing ergothione cosmetic stock solution by fermentation of Hericium erinaceus.
  • the method is simple in process, good in physical condition, stable in product, high in active substance content, safe and healthy. Raw material requirements.
  • the invention adopts Hericium erinaceus for the first time to ferment, with stable fermentation and high output, which can reach more than 300mg/L, which is a leading level at home and abroad.
  • the present invention adopts the following technical solutions.
  • a method for preparing a fermentation stock solution containing ergothioneine by fermentation of Hericium erinaceus characterized in that it comprises the following steps:
  • Step 1 Inoculate the hyphae of Hericium erinaceus into the liquid seed medium and cultivate to obtain the seed liquid;
  • Step 2 Inoculate the seed liquid into the fermentation medium for fermentation and add ergothione precursor materials, and ferment to the end to obtain the fermentation liquid;
  • Step 3 The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution.
  • seed culture medium comprises the following components: 1-10% by mass of carbon source, 1-5% by mass of organic nitrogen source and 0.01-1% by mass of inorganic salt.
  • step 1 shaking culture is performed at 15-30°C, pH 4.0-7.0, 50-300 rpm, preferably 100 rpm-200 rpm for 5-10 days to obtain seed liquid.
  • the fermentation medium comprises the following components: 1 to 5 mass% of carbon source, 1 to 10 mass% of organic nitrogen source, 0.01 to 1 mass% of inorganic Salt, 0.0001 to 0.001% by mass of trace elements and 0.0001 to 0.001% by mass of coenzyme.
  • step 2 shaking fermentation is performed at 15-30°C, pH 4.0-7.0, 50-300rpm, preferably 100rpm-200rpm, for 7-15 days, and fermented to the end to obtain fermentation liquid.
  • step 2 the precursor substance is at least one of aspartic acid, glutamine and betaine, and the addition amount is 1-20 mM.
  • step 2 the inoculation amount of the seed liquid into the fermentation liquid is 1-10% by volume.
  • step 2 The method according to item 1, characterized in that, in step 2, the fermentation end point is residual sugar in the fermentation broth ⁇ 0.5% by mass.
  • step 3 the treatment includes at least one of homogenization, ultrasound, and activated carbon adsorption.
  • the method according to item 10 characterized in that the conditions of the homogenization are a spindle speed of 1000 r/min to 3000 r/min, and a homogenization time of 10 to 30 min.
  • the method according to item 10 characterized in that the ultrasound conditions are that the ultrasound power is 100w to 1000w, the ultrasound time is 5 to 60 min, and the ultrasound endpoint is that the content of ergothioneine no longer increases.
  • the method according to item 10 characterized in that the amount of activated carbon added in the activated carbon adsorption is 0.1 to 1% of the volume of the fermentation broth, and the adsorption time is 10 to 60 minutes.
  • step 4 removing impurities in the processed fermentation broth.
  • step 4 the removal of impurities is performed by centrifuging the fermentation broth at 1000-5000 rpm for 10 to 30 minutes, and then using the resulting supernatant with a filter element with a pore size of 0.22 ⁇ m to 0.45 ⁇ m Filtered.
  • the obtained ergothioine-containing cosmetic stock solution contains 2.5 g/L or more of fungal polysaccharides and a total amount of 5.0 mg/100 mL or more of amino acids
  • the polysaccharide content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, preferably the total amino acid content is 5.5 mg/100 mL or more, and more preferably 5.7 mg/100 mL or more.
  • a fermentation stock solution containing ergothioneine characterized in that it contains: 0.3g/L or more ergothioine, 2.5g/L or more fungal polysaccharides, and a total amount of 5.0mg/100mL or more of amino acids, preferably polysaccharides
  • the content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, and the total amino acid content is preferably 5.5 mg/100 mL or more, and more preferably 5.7 mg/100 mL or more.
  • the ergothioine-containing fermentation stock solution according to item 20 characterized in that it is obtained by using the method according to any one of items 1-19.
  • a fermentation medium composition for the production of ergothioine-containing cosmetic stock solution characterized in that it comprises the following components: 1 to 5 mass% carbon source, 1 to 10 mass% organic nitrogen source, 0.01 to 1% by mass of inorganic salts, 0.0001 to 0.001% by mass of trace elements, and 0.0001 to 0.001% by mass of coenzyme.
  • a cosmetic stock solution composition containing ergothioneine characterized in that it comprises ergothioine above 300mg/L, fungal polysaccharides above 2.5g/L and amino acids above 5.0mg/100mL, preferably the polysaccharide content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, preferably the total amino acid content is 5.5 mg/100 mL or more, more preferably 5.7 mg/100 mL or more.
  • One aspect of the present invention adopts the Hericium erinaceus mycelium fermentation method to extend the application scope of the Hericium erinaceus fermentation active product to the field of cosmetics;
  • Another aspect of the present invention is to physically disrupt cells to maximize the retention and release of intracellular ergothioneine and other active substances of edible fungus mycelium to the outside of the cell without any damage to physical properties and biological functions.
  • a mild decolorization process is adopted to better remove the odor and pigment that affect the physical characteristics of the product in cosmetics, and can better meet the requirements of different cosmetic formulations.
  • the present invention also contains other active ingredients, such as fungal polysaccharides, small molecular amino acids and the like.
  • Figure 1 is the HPLC chart of the determination of thioneine content, in which a) is the HPLC chart of the standard product (the concentration of thioneine is 100mg/L), b) is the HPLC chart of the fermentation broth of Example 5, and c) is The HPLC profile of the fermentation broth of Example 6, e) is the HPLC profile of the fermentation broth of Example 7, and d) is the HPLC profile of the fermentation broth of Example 8;
  • Figure 2 is a graph showing the results of fermentation stock promoting cell repair, where a is the initial control treatment group, b is the control treatment group 24h, c is the S0 group initial, d is the S0 treatment 24h, e is the S1 treatment group initial, f is S1 Treatment group 24h.
  • the method for preparing ergothioine-containing cosmetic stock solution by fermentation of Hericium erinaceus includes the following steps:
  • Step 1 Inoculate the hyphae of Hericium erinaceus into the liquid seed medium and cultivate to obtain the seed liquid;
  • Step 2 Inoculate the seed liquid into the fermentation medium for fermentation and add ergothione precursor materials, and ferment to the end to obtain the fermentation liquid;
  • Step 3 The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution.
  • the invention adopts the Hericium erinaceus mycelium fermentation method to extend the application scope of the Hericium erinaceus fermentation active product to the field of cosmetics.
  • the method has simple process, good physical condition, stable product, high active substance content, safety and health and meets the requirements of cosmetic raw materials.
  • Step 1 Inoculate the hyphae of Hericium erinaceus into a liquid seed culture medium to obtain a seed liquid.
  • Hericium erinaceus is a fungus, Basidiomycetes, Polyporaceae, Hericium erinaceus (Rull ex F.) Pers., which is a saprophytic fungus and a famous edible fungus. It looks like a hedgehog or hericium, so it is commonly called hericium or monkey mushroom.
  • Hericium erinaceus is preferably Hericium erinaceus CCTCC NO: M 2018567, which has been deposited at the China Type Culture Collection (CCTCC) on August 23, 2018. This strain is a new strain discovered by the applicant. Hericium erinaceus species, and found that it can be used for the biosynthesis of thioneine.
  • a single wire mesh cell is called hyphae, including vegetative hyphae and aerial hyphae.
  • the hyphae gather together to form a certain macroscopic structure called mycelium.
  • the Hericium erinaceus mycelium is taken from a test tube with a size of about 5*5cm to inoculate the liquid seed culture medium, which can be inoculated at 15-30°C, pH 4.0-7.0, 50-300rpm, preferably 100rpm ⁇ Shake culture on a 200 rpm shaker for 5-10 days to obtain seed liquid.
  • the above-mentioned seed culture medium includes the following components: 1-10% by mass carbon source, 1-5% by mass organic nitrogen source, and 0.01-1% by mass inorganic salt.
  • carbon sources are commonly used carbon sources for microorganisms, and are not particularly limited, and may include one or a combination of the following: glycerol, sucrose, fructose, glucose, maltose, etc., preferably sucrose,
  • the above-mentioned nitrogen source is a commonly used nitrogen source for microbial cultivation, and is not particularly limited, and may include one or more of the following combinations: beef extract, peptone, yeast powder, soybean meal powder, etc., preferably soybean meal powder,
  • inorganic salts are commonly used inorganic salts for microbial cultivation, and are not particularly limited, and can include one or more of the following combinations: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, etc., preferably Sodium dihydrogen phosphate and sodium sulfate.
  • Common organic or inorganic acids such as phosphoric acid, hydrochloric acid, acetic acid, and lactic acid, can be used to adjust the pH of the seed culture medium, preferably acetic acid.
  • Step 2 Inoculate the seed liquid into the fermentation medium for fermentation and add the ergothione precursor material to ferment to the end to obtain the fermentation liquid.
  • Fermentation refers to the process in which people prepare microbial cells themselves, or direct or secondary metabolites through the life activities of microorganisms under aerobic or anaerobic conditions.
  • shake fermentation at 15-30° C., pH 4.0-7.0, 50-300 rpm, preferably 100 rpm-200 rpm, for 7-15 days, and ferment to the end to obtain fermentation broth.
  • the pH of the fermentation medium can be adjusted with common organic or inorganic acids, such as phosphoric acid, hydrochloric acid, acetic acid, and lactic acid, preferably acetic acid.
  • common organic or inorganic acids such as phosphoric acid, hydrochloric acid, acetic acid, and lactic acid, preferably acetic acid.
  • the fermentation medium includes the following components: 1-5% by mass carbon source, 1-10% by mass organic nitrogen source, 0.01-1% by mass inorganic salt, 0.0001-0.001% by mass trace Element and 0.0001 to 0.001 mass% of coenzyme.
  • the carbon source mentioned above is a carbon source commonly used by microorganisms, and is not particularly limited, and may include one or a combination of the following: glycerol, sucrose, fructose, glucose, maltose, etc., preferably glycerol.
  • the above-mentioned nitrogen source is a commonly used nitrogen source for microbial cultivation, and is not particularly limited, and may include one or more combinations of the following: beef extract, peptone, yeast powder, soybean cake powder, etc., preferably beef extract.
  • inorganic salts are commonly used inorganic salts for microbial cultivation, and are not particularly limited, and can include one or more of the following combinations: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, etc., preferably Sodium dihydrogen phosphate and sodium sulfate.
  • the above-mentioned trace elements may include one or a combination of the following: magnesium sulfate, ferrous chloride, zinc chloride, etc., preferably zinc chloride.
  • the aforementioned coenzyme may include one or a combination of the following: biotin, niacin, pyridoxal phosphate, betaine, vitamin B12, riboflavin, etc., preferably pyridoxal phosphate.
  • the present invention optimizes the carbon source, nitrogen source, coenzyme and other factors in the fermentation medium to increase the yield of ergothioneine.
  • the amount of seed liquid inoculated into the fermentation liquid is 1-10% by volume.
  • the aforementioned precursor substance is at least one of aspartic acid, glutamine and betaine, and the addition amount is 1-20 mM.
  • the fermentation end point is that the residual sugar in the fermentation broth is ⁇ 0.5% by mass.
  • the above-mentioned glucose content can be determined by common chemical methods, such as DNS method, Fehling reagent method, indirect iodometry or optical rotation method.
  • Step 3 The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution.
  • the above-mentioned treatment includes at least one of physical crushing treatment and decolorization treatment.
  • the physical crushing treatment can be homogenization and/or ultrasound, and the decolorization treatment can be activated carbon adsorption.
  • the mycelium of Hericium erinaceus in the fermentation broth is fully broken by homogenizing and sonicating the fermentation broth.
  • the fermentation broth is fully adsorbed and decolorized by adding activated carbon to the fermentation broth.
  • the above-mentioned homogenization conditions are the spindle speed of 1000r/min ⁇ 3000r/min, and the homogenization time of 10 ⁇ 30min.
  • the above-mentioned ultrasonic conditions are that the ultrasonic power is 100w-1000w, the ultrasonic time is 5-60min, and the end point of the ultrasonic is that the content of thioneine in the fermentation broth no longer increases.
  • high-performance liquid phase is used to detect the content of thioneine in the fermentation broth.
  • the amount of activated carbon added in the above activated carbon adsorption is 0.1 to 1% of the volume of the fermentation broth, and the adsorption time is 10 to 60 minutes.
  • the present invention By adopting the method of physically disrupting cells, the present invention retains and releases edible fungus mycelium intracellular ergothioneine and other active substances to the outside of the cell to the greatest extent without any damage to physical properties and biological effects.
  • the present invention can better remove the odor and pigment that affect the physical characteristics of the product in cosmetics, and can better meet the requirements of different cosmetic formulations.
  • the method of the present invention may further include step 4: removing impurities in the fermented broth after treatment.
  • the removal of impurities is performed by centrifuging the fermentation broth at 1000-5000 rpm for 10-30 minutes, and then filtering the resulting supernatant with a filter element with a pore size of 0.22 ⁇ m to 0.45 ⁇ m.
  • the centrifugation can remove bacterial cell debris and impurities in the culture medium, and the filtration can remove microorganisms.
  • the present invention also provides a fermentation stock solution (cosmetic stock solution) containing ergothioneine.
  • the ergothioneine fermentation stock solution (cosmetic stock solution) preferably contains more than 300 mg/L of ergothioneine, and the fermentation stock solution has antioxidant properties in cosmetics. , Cell repair effect.
  • the ergothioine-containing fermentation stock solution contains: ergothioine above 300 mg/L, fungal polysaccharides above 2.5 g/L, and amino acids with a total amount of 5.0 mg/100 mL or above.
  • the ergothioine-containing fermentation stock solution of the present invention is obtained by the method for preparing the ergothioine-containing cosmetic stock solution by fermentation of the Hericium erinaceus of the present invention.
  • the content of ergothioine in the ergothioine-containing cosmetic stock obtained by the method for preparing the ergothioine-containing cosmetic stock solution by fermentation of Hericium erinaceus of the present invention is 300 mg/L or more.
  • the obtained ergothioine-containing cosmetic stock solution contains not only a higher content of ergothioine, but also other active ingredients, such as fungal polysaccharides, small molecular amino acids, and the like.
  • amino acids include aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine Acid, phenylalanine, lysine, histidine, arginine, etc.
  • the polysaccharide content is 2.6g/L or more, and the polysaccharide content is more preferably 2.7g/L or more, and the total amino acid content is preferably 5.5mg/100mL or more, and even more preferably 5.7mg/100mL or more
  • the obtained ergothioneine-containing cosmetic stock solution also has the effects of anti-oxidation and radiation protection.
  • the 1,1-diphenyl-2-trinitrophenylhydrazine DPPH clearance rate of the 0.5v/v%-2v/v% dilution of the obtained ergothioine-containing cosmetic stock solution is more than 30%,
  • the relative proliferation rate of the cells treated with the 0.5v/v%-2v/v% dilution of the obtained ergothioneine-containing cosmetic stock solution after being damaged by ultraviolet rays is 70% or more.
  • the cosmetic stock solution of the present invention has extremely high oxidation resistance, has a good protective effect on UV damage, and has a good effect of repairing damaged cells.
  • Test tube preservation of bacterial strains inoculate the bacterial clumps into 100ml seed culture medium, shaker at 150rpm, culture at 25°C for 5 days;
  • composition of the seed medium is as follows:
  • Seed culture medium was inoculated into 1L fermentation culture medium, 200rpm shaker, 25°C fermentation culture for 10 days until no residual sugar;
  • the fermentation medium components are as follows:
  • Glycerin 25g/L, beef extract 20g/L, sodium sulfate 1g/L, disodium hydrogen phosphate 1g/L, zinc chloride 0.005g/L, pyridoxal phosphate 0.001g/L, acetic acid to adjust pH 5.0, aspartame Acid 10mM, Glutamine 5mM, Betaine 10Mm;
  • the fermentation broth is homogenized for 30 minutes, the homogenization speed is 3000 rpm/min, and the ultrasonic power is 500w for 15 minutes, which can release ergothioneine and other active ingredients into the fermentation broth to obtain a preliminary fermentation broth.
  • composition of the seed medium is as follows:
  • Seed culture medium was inoculated into 2L fermentation culture medium, 200rpm shaker, 25°C fermentation culture for 9 days until no residual sugar;
  • the fermentation medium components are as follows:
  • Glycerin 25g/L, beef extract 20g/L, sodium sulfate 0.75g/L, disodium hydrogen phosphate 1g/L, zinc chloride 0.005g/L, pyridoxal phosphate 0.001g/L, acetic acid to adjust pH 5.5, Asparagus Acid 8mM, glutamine 5mM, betaine 15Mm;
  • the fermentation broth is homogenized for 40 minutes, the homogenization speed is 3000 rpm/min, and then ultrasonic for 30 minutes, the ultrasonic power is 800w, which can release ergothioneine and other active ingredients into the fermentation broth to obtain a preliminary fermentation broth.
  • Test tube preservation of bacterial strains inoculate the bacterial clumps into 500ml seed culture medium, shake at 150rpm, culture at 30°C for 8 days;
  • composition of the seed medium is as follows:
  • Seed culture medium was inoculated into 5L fermentation culture medium, 100 rpm shaker, 30 °C fermentation culture for 15 days to no residual sugar;
  • the fermentation medium components are as follows:
  • the fermentation broth is homogenized for 30 minutes, the homogenizing speed is 3000 rpm/min, and then ultrasonicated for 45 minutes with an ultrasonic power of 500w, which can release ergothioneine and other active ingredients into the fermentation broth to obtain a preliminary fermentation broth.
  • composition of the seed medium is as follows:
  • Seed culture medium was inoculated into 2L fermentation culture medium, 150rpm shaker, 20°C fermentation culture for 15 days until no residual sugar;
  • the fermentation medium components are as follows:
  • the fermentation broth is homogenized for 20 minutes at a homogenizing speed of 3000 rpm/min, and then ultrasonicated for 60 minutes with an ultrasonic power of 1000w, which can release ergothione into the fermentation broth to obtain a preliminary fermentation broth.
  • the fermentation broth is centrifuged at 3000 rpm for 30 minutes, the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 ⁇ m pore size filter element to filter and sterilize the final product.
  • the fermentation broth is centrifuged at 3000 rpm for 30 min, and the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 ⁇ m pore size filter element to obtain the final product.
  • the fermentation broth is centrifuged at 4000 rpm for 30 min, and the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 ⁇ m pore size filter element to obtain the final product.
  • step (3) Add 5 ml of phenoxyethanol, 0.5 ml of ethylhexyl glycerol, and 25 ml of 1,3-butanediol as preservatives to the 500 ml final product of fermentation broth obtained in step (2), and then mix them aseptically.
  • the content of ergothioneine detected by liquid phase was 331.1mg/L.
  • the fermentation broth is centrifuged at 5000 rpm for 30 min, and the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 ⁇ m pore size filter element to filter and sterilize the final product.
  • the samples in Examples 5 to 8 were named S1, S2, S3, and S4, respectively, and were detected by the external standard method.
  • HPLC conditions Hypersil ODS C18 column (250mm ⁇ 4.6mm, particle size 5 ⁇ m); mobile phase: acetonitrile-water (3:97); flow rate: 1.0mL/min; column temperature: 30°C; detection wavelength: 254nm; injection Quantity: 20 ⁇ L.
  • the chromatogram is shown in Figure 1, where a) is a standard product (the concentration of ergothioneine is 100mg/L), b) is S1, c) is S2, d) S3, and e) S4 results are shown in Table 1. .
  • the present invention adopts phenol sulfuric acid method to detect polysaccharide content.
  • Standard solution accurately weigh 100mg of dry and constant-weight glucose (analytical purity) into a volumetric flask, add water to make the volume up to 250mL, shake well, draw up 10mL of this solution, and add water to make the volume up to 100mL.
  • the activity of the fermentation broth product prepared by this process is a combination of multiple active substances
  • 350 mg of pure ergothione (content greater than or equal to 98%) is taken, and 1L purified water is added to fully dissolve the thioneine solution S0. Comparative example.
  • sample solution Dissolve the samples S1-S4 and sample S0 obtained in Examples 5-8 in serum-free medium, prepare a solution with a final concentration of 2.0%, filter and sterilize with a 0.22 ⁇ m filter membrane, and use serum-free before use The medium is diluted to the specified concentration.
  • HaCaT cells human immortalized epidermal cells
  • trypsinization adjust the cell density to 1 ⁇ 10 5 /mL
  • inoculate the flat bottom 96-well cell culture plate, 100 ⁇ L cell suspension per well Place a carbon dioxide incubator at 37°C and 5% CO 2 for regular culture overnight.
  • test groups Discard the culture solution, and the test groups are as follows. Each well of the test group is added with 100 ⁇ L of sample solution with a concentration of 0.5%, 1.0%, and 2.0%. The normal group and the model group are added with the same amount of serum-free medium and placed in the culture Continue to incubate for 24h in the box.
  • Cell culture medium DMEM culture medium containing 10% FBS.
  • Sample solution S1 and S0 are prepared into 4% mother liquor with cell culture solution.
  • HaCaT cells were seeded in a 24-well plate at a density of 5 ⁇ 10 4 cells/mL, and cultured at 37°C and 5% CO 2 for 24 hours. On the monolayer of cells close to confluence, use a 200 ⁇ L pipette tip to scribble vertically in each well of the 24-well plate. Discard the liquid in the well, add the sample solution (final serum content 2.5%), continue to incubate for 24 hours and take pictures for observation.
  • a is the initial control treatment group
  • b is the control treatment group 24h
  • c is the S0 group initial
  • d is the S0 treatment 24h
  • e is the S1 group initial
  • f is the S1 treatment group 24h.
  • the S1 group and the S0 group can better promote cell migration and self-repair after contacting damaged cells for 24 hours, and the effect of the S1 group is better than that of the S0 group containing only ergothioine solution.
  • the cosmetic stock solution obtained by the method of the present invention contains high content of ergothioneine, fungal polysaccharides and amino acids at the same time, so it has good antioxidant activity, better protection against UV damage, and has a Good effect of repairing damaged cells.

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Abstract

L'invention concerne une méthode de préparation d'une solution mère cosmétique contenant de la L-ergothionéine au moyen de la fermentation d'Hericium erinaceus, la méthode comprenant les étapes suivantes : étape 1 : inoculation d'hyphes d'Hericium erinaceus dans un milieu de culture de graines liquide pour culture pour obtenir une solution de graine ; étape 2 : inoculation de la solution de graine dans un milieu de culture de fermentation pour fermentation et ajout d'une substance précurseur de L-ergothionéine, et fermentation jusqu'à un point final pour obtenir un bouillon de fermentation ; étape 3 : traitement du bouillon de fermentation contenant du mycélium pour obtenir une solution mère de fermentation contenant de la L-ergothionéine et utilisation de celle-ci en tant que solution mère cosmétique contenant de la L-ergothionéine. Le produit de solution mère décrit contient plus de 300 mg/L de L-ergothionéine, et a de forts effets antioxydants, anti-radiations et anti-inflammatoires ; en outre, la solution mère décrite peut être ajoutée en tant que matière première dans de l'eau, du lait, des crèmes, etc, et est largement appliquée dans le domaine des cosmétiques.
PCT/CN2019/118949 2019-03-01 2019-11-15 Méthode de préparation de solution mère cosmétique contenant de la l-ergothionéine au moyen de la fermentation d'hericium erinaceus WO2020177390A1 (fr)

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