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WO2020177390A1 - 一种猴头菌发酵制备含麦角硫因的化妆品原液的方法 - Google Patents

一种猴头菌发酵制备含麦角硫因的化妆品原液的方法 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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English (en)
French (fr)
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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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Publication of WO2020177390A1 publication Critical patent/WO2020177390A1/zh

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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

一种猴头菌发酵获得含麦角硫因的化妆品原液的制备方法，包括如下步骤：步骤1：将猴头菌菌丝接种到液体种子培养基中培养，得到种子液；步骤2：将种子液接种到发酵培养基中发酵并添加麦角硫因前体物质，发酵至终点，得到发酵液；步骤3：对含有菌丝体的发酵液进行处理，得到含麦角硫因的发酵原液作为含麦角硫因的化妆品原液，该原液产品含有300mg/L以上的麦角硫因，具有较强抗氧化、防辐射、抗炎功效，可作为原料添加到水、乳、膏霜等中，广泛应用于化妆品领域中。

Description

一种猴头菌发酵制备含麦角硫因的化妆品原液的方法 技术领域
本发明涉及化妆品原料领域，特别涉及一种猴头菌发酵制备麦角硫因化妆品原液的方法以及由其制备的发酵原液。
背景技术
猴头菌隶属于担子菌门、猴头菌科，是著名的药食兼用菌。其子实体和菌丝体均可入药，性平味甘，具有助消化、利五脏的功能，营养丰富，素有“山珍猴头，海味燕窝”之说。猴头菌的药效成分主要有多糖、低聚糖、甾醇、脂肪酸、猴头菌素和猴头菌酮等，具有保肝护胃、降血糖、保护神经、增强人体免疫力、抗癌、抗氧化等功效，除此之外，猴头菌中还含有丰富的硫元素，而硫是对人体不可或缺的重要的营养元素，在猴头菌中硫元素主要以麦角硫因的形式存在，猴头菌中的麦角硫因一旦进入人体，绝大部分最终成为无机硫酸盐、硫酸酯及中性硫进入血液循环，功效之一就是健脾益胃，增加胃黏膜屏障机能，对各种慢性胃炎均有较好的治疗作用，麦角硫因在护肤品领域还能抗辐射、促进胚胎生长发育、抗疲劳、提高机体生存能力等作用。麦角硫因(L-Ergothioneine，EGT)，化学名为2-巯基组氨酸三甲基内盐，是至今唯一为人们所知的天然2-硫代咪唑氨基酸，它具有明确的抗氧化及抗炎功效，应用到化妆品中则具有抗炎、抗衰老、防辐射的作用，国外众多化妆品已将麦角硫因作为配方当中的主要功效成分，早在2014年，麦角硫因就被国家食品药品监督管理局列入已使用化妆品原料目录，化妆品中EGT的含量达到0.01％～2％，即可起到较好效果。多种美白化妆品中均添加有EGT，膏状类、乳霜类和化妆水类的EGT的添加量范围分别在0.01％～1.0％、0.01％～2.0％和0.01％～3.0％。美国OXIS公司推出了两款以EGT为原料的化妆品：Reverge和Prograce。前者作为润肤剂可改善衰老的肤质，后者能有效地祛除皱纹，促进新肌肤的再生，雅诗兰黛更是将此成分作为镇牌之宝，添加在倩碧、悦木之源、海蓝之谜等各线产品中。
虽有如此卓越的功效作用但是该物质人体自身无法合成只能从外界食物中摄取。在食物中尤以食用菌中含量最高，在人们通常认识中，食用真菌多以种植栽培为主，尽管不少菇类产品已经达成商业生产的规模，但是子实 体的培养需要消耗大量劳力、空间设备、时间等。另外，有些食用菌的栽培很容易吸收土壤中的重金属离子，所以如何进一步积累菌丝体是能否高效合成麦角硫因的关键。利用液态发酵技术进行食用菌发酵培养可以大大降低发酵成本，缩短发酵周期同时利用发酵代谢调控以麦角硫因为目标产物对发酵过程进行控制，能够大幅度提高该活性产物的含量。
专利文献1中公开了一种“含有麦角硫因的制品及制备方法和蕈菌胞外发酵液的用途”，其特征在于蕈菌进行发酵并将发酵液中的菌丝体去除后获得胞外发酵液或该胞外发酵液的浓缩物提供，其缺点在于该过程中富含高浓度麦角硫因的菌丝体被去除，造成活性成分的流失，而菌丝体中除了麦角硫因外还含有多糖、甾醇等活性物质，均可以作为化妆品中重要的功效成分。
专利文献2中公开了一种“制备麦角硫因的方法”，其特征在于通过利用发酵代谢调控方法对糙皮侧耳发酵工艺进行优化，之后通过热水浸提将菌丝中的麦角硫因浸提到发酵液中，其缺点在于纯化工艺中采用热水浸提方式，作为化妆品原料，产品外观、颜色、气味十分重要，而该方式一方面会使发酵液颜色较深并产生异味，另一方面发酵液中其他热敏性活性成分如真菌多糖也存在降解失活的可能，特别是会对麦角硫因产生一定降解作用，影响产品功效，难以作为化妆品原料应用。
专利文献3中公开了“一种制备麦角硫因的方法”，其特征在于以蘑菇为原料通过乙醇提取再经树脂分离纯化获得麦角硫因提取物。其缺点在于依靠纯植物提取工艺，效率较低，另外工艺中涉及有机溶剂，存在有机溶剂的残留风险，同时对环境可能造成污染。
专利文献4中公开了“微生物麦角硫因生物合成”，其特征在于通过基因手段将合成麦角硫因相关基因转录到大肠杆菌中进行发酵从而获得麦角硫因。其缺点在于大肠杆菌为致病菌，在化妆品中难以应用。
专利文献5中公开了“富含麦角硫因功能性口服剂的制备方法”，其特征在于将富含麦角硫因的食用菌菌丝体与水混合，在80℃～100℃搅拌浸取，浓缩得浓缩液，浓缩液加上食品可接受的添加剂制成液体口服制剂。其产品主要应用于食品保健品领域，无法直接作为化妆品原料应用。
综上所述，目前开发食用菌麦角硫因的化妆品原料主要存在以下不足：
1.猴头菌多应用于食品领域，而对其发酵或提取的活性成分尚无在化妆品领域中应用的专利报道。
2.针对麦角硫因发酵液所应用的现有的发酵、提取工艺，不足以制备出符合化妆品规范要求的产品。
3.现有研究中利用菌株发酵获得的麦角硫因含量普遍不高。
现有技术文献
专利文献
专利文献1:CN 107708443A
专利文献2:CN 105296559
专利文献3:CN 106831596 A
专利文献4:CN 106661585 A
专利文献5:CN 103181933
发明内容
针对现有技术中存在的问题，本发明旨在提供一种猴头菌发酵制备麦角硫因化妆品原液的方法，该方法工艺简单、物理状态好、产品稳定、活性物质含量高、安全健康符合化妆品原料的要求。本发明首次采用猴头菌发酵，发酵稳定且产量较高，可达300mg/L以上，属国内外领先水平。
为实现上述目的，本发明采用如下技术方案。
1.一种猴头菌发酵制备含麦角硫因的发酵原液的方法，其特征在于，包括以下步骤：
步骤1：将猴头菌菌丝接种到液体种子培养基中培养，得到种子液；
步骤2：将种子液接种到发酵培养基中发酵并添加麦角硫因前体物质，发酵至终点，得到发酵液；
步骤3：对含有菌丝体的发酵液进行处理，得到含麦角硫因的发酵原液作为含麦角硫因的化妆品原液。
2.根据项1所述的方法，其特征在于，所述猴头菌(Hericium erinaceus)的保藏编号为CCTCC NO：M 2018567。
3.根据项1所述的方法，其特征在于，所述种子培养基包括以下组分：1～10质量％的碳源、1～5质量％的有机氮源和0.01～1质量％的无机盐。
4.根据项1所述的方法，其特征在于，步骤1中，在15～30℃、pH 4.0～7.0、50～300rpm、优选100rpm～200rpm振荡培养5～10天，得到种子液。
5.根据项1所述的方法，其特征在于，所述发酵培养基包括以下组分： 1～5质量％的碳源、1～10质量％的有机氮源、0.01～1质量％的无机盐、0.0001～0.001质量％的微量元素和0.0001～0.001质量％的辅酶。
6.根据项1所述的方法，其特征在于，步骤2中，在15～30℃、pH 4.0～7.0、50～300rpm、优选100rpm～200rpm振荡发酵7～15天，发酵至终点，获得发酵液。
7.根据项1所述的方法，其特征在于，步骤2中，所述前体物质为天冬氨酸、谷氨酰胺和甜菜碱中的至少一种，添加量为1～20mM。
8.根据项1所述的方法，其特征在于，步骤2中，种子液接种到发酵液中接种量为1～10体积％。
9.根据项1所述的方法，其特征在于，步骤2中，所述发酵终点为发酵液中残糖≤0.5质量％。
10.根据项1所述的方法，其特征在于，步骤3中，所述处理包括均质、超声和活性炭吸附中的至少一种。
11.根据项10所述的方法，其特征在于，所述均质的条件为主轴转速1000r/min～3000r/min，均质时间10～30min。
12.根据项10所述的方法，其特征在于，所述超声的条件为超声功率为100w～1000w，超声时间为5～60min，超声终点为麦角硫因的含量不再增加。
13.根据项10所述的方法，其特征在于，所述活性炭吸附中所添加活性炭的量为发酵液体积的0.1～1％，吸附时间为10～60min。
14.根据项1所述的方法，其特征在于，还包括步骤4：除去处理后的发酵液中杂质。
15.根据项14所述的方法，其特征在于，步骤4中，去除杂质是通过将发酵液以1000-5000rpm离心10～30min后，将得到的上清液用0.22μm～0.45μm孔径的滤芯过滤而进行的。
16.根据项1-15中任一项所述的方法，其特征在于，所得到的含麦角硫因的化妆品原液中麦角硫因的含量为300mg/L以上。
17.根据项1-15中任一项所述的方法，其特征在于，所得到的含麦角硫因的化妆品原液含有2.5g/L以上的真菌多糖和总量为5.0mg/100mL以上的氨基酸，优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以 上。
18.根据项1-15中任一项所述的方法，其特征在于，所得到的含麦角硫因的化妆品原液的0.5v/v％-2v/v％的稀释液的1,1-二苯基-2-三硝基苯肼DPPH的清除率为30％以上，
Figure PCTCN2019118949-appb-000001
19.根据项1-15中任一项所述的方法，其特征在于，经所得到的含麦角硫因的化妆品原液的0.5v/v％-2v/v％的稀释液处理的细胞经紫外线损伤后的相对增殖率为70％以上。
20.一种含麦角硫因的发酵原液，其特征在于，含有：0.3g/L以上的麦角硫因、2.5g/L以上的真菌多糖和总量为5.0mg/100mL以上的氨基酸，优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以上。
21.根据项20所述的含麦角硫因的发酵原液，其特征在于，其是通过采用项1-19中任一项所述的方法获得的。
22.根据项20或21所述的含麦角硫因的发酵原液，其特征在于，所述发酵原液的0.5v/v％-2v/v％的稀释液的1,1-二苯基-2-三硝基苯肼DPPH的清除率为30％以上，
Figure PCTCN2019118949-appb-000002
23.根据项20或21所述的含麦角硫因的发酵原液，其特征在于，经发酵原液的0.5v/v％-2v/v％的稀释液处理的细胞经紫外线损伤后的相对增殖率为70％以上。
24.一种由项1-19中任一项所述的方法获得的含麦角硫因的发酵原液或项20-23中任一项所述的含麦角硫因的发酵原液在化妆品中的应用。
25.一种用于生产含麦角硫因的化妆品原液的发酵培养基组合物，其特征在于，包括以下组分：1～5质量％碳源、1～10质量％的有机氮源、0.01～1质量％的无机盐、0.0001～0.001质量％的微量元素和0.0001～0.001质量％的辅酶。
26.一种含麦角硫因的化妆品原液组合物，其特征在于，包括300mg/L以上的麦角硫因、2.5g/L以上的真菌多糖和5.0mg/100mL以上的氨基酸， 优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以上。
本发明具有以下优点：
本发明的一个方面采用猴头菌菌丝体发酵法，将猴头菌发酵活性产物的应用范围延伸到了化妆品领域；
本发明的另一方面采用物理破碎细胞方式最大限度的将食用菌菌丝体胞内麦角硫因等活性物保留并释放到了胞外且无任何物理特性及生物学功效活性上的破坏。
本发明的又一个方面采用温和脱色工艺将在化妆品中影响产品物理特性的气味及色素均较好的去除，更能满足不同化妆品配方需求。
本发明经发酵含有较高含量的麦角硫因以外，还含有其他多种活性成分，如真菌多糖，小分子氨基酸等。
附图说明
图1为麦角硫因含量测定的HPLC图谱，其中，a)为标准品(麦角硫因含量浓度为100mg/L)的HPLC图谱，b)为实施例5的发酵液的HPLC图谱，c)为实施例6的发酵液的HPLC图谱，e)为实施例7的发酵液的HPLC图谱，d)为实施例8的发酵液的HPLC图谱；
图2为发酵原液促进细胞修复的结果图，其中，a为对照处理组初始，b为对照处理组24h，c为S0组初始，d为S0处理24h，e为S1处理组初始，f为S1处理组24h。
发明的具体实施方式
以下将对本发明做以详细说明。
本发明提供的猴头菌发酵制备含麦角硫因的化妆品原液的方法，包括以下步骤：
步骤1：将猴头菌菌丝接种到液体种子培养基中培养，得到种子液；
步骤2：将种子液接种到发酵培养基中发酵并添加麦角硫因前体物质，发酵至终点，得到发酵液；
步骤3：对含有菌丝体的发酵液进行处理，得到含麦角硫因的发酵原液作为含麦角硫因的化妆品原液。
本发明采用猴头菌菌丝体发酵法，将猴头菌发酵活性产物的应用范围延伸到了化妆品领域。该方法工艺简单、物理状态好、产品稳定、活性物质含量高、安全健康符合化妆品原料的要求。
步骤1
步骤1：将猴头菌菌丝接种到液体种子培养基中培养，得到种子液。
其中，猴菇菌为真菌类担子菌纲多孔菌目齿菌科猴头菌Hericium erinaceus(Rull ex F.)Pers.，是一种腐生菌，也是一种著名的食用菌。全形似刺猬或猴头，故俗称猴头菇或猴菇。其中，猴头菌优选为猴头菌(Hericium erinaceus)CCTCC NO：M 2018567，已于2018年8月23日在中国典型培养物保藏中心(简称CCTCC)保藏，该菌株是本申请人发现的新的猴头菌种，而且发现其可用于麦角硫因的生物合成。
单一丝网状细胞称为菌丝,包括营养菌丝和气生菌丝，菌丝集合在一起构成一定的宏观结构称为菌丝体。
在一个具体的实施方式中，将猴头菌菌丝从试管中取约5*5cm大小菌块接种液体种子培养基，可以在15～30℃、pH 4.0～7.0、50～300rpm、优选100rpm～200rpm摇床振荡培养5～10天，得到种子液。
在一个具体的实施方式中，上述种子培养基包括以下组分：1～10质量％碳源、1～5质量％的有机氮源和0.01～1质量％的无机盐。
上述碳源为微生物常用碳源，没有特别限定，可以包括以下的一种或多种的组合：甘油、蔗糖、果糖、葡萄糖、麦芽糖等，优选为蔗糖，
上述氮源为微生物培养常用氮源，没有特别限定，可以包括以下的一种或多种的组合：牛肉膏、蛋白胨、酵母粉、豆饼粉等，优选为豆饼粉，
上述无机盐为微生物培养常用无机盐，没有特别限定，可以包括以下的一种或多种的组合：氯化钠、磷酸二氢钠、磷酸氢二钠、氯化钾、硫酸钠等，优选为磷酸二氢钠和硫酸钠。
种子培养基的pH调节可以用常见有机酸或无机酸，如磷酸、盐酸、醋酸、乳酸，优选为醋酸。通过在上述条件下培养猴头菌，更适合菌种的生长，能够获得活性好的菌种。
步骤2
步骤2：将种子液接种到发酵培养基中发酵并添加麦角硫因前体物质，发酵至终点，得到发酵液。
发酵指人们借助微生物在有氧或无氧条件下的生命活动来制备微生物菌体本身、或者直接代谢产物或次级代谢产物的过程。
在一个具体的实施方式中，在15～30℃、pH 4.0～7.0、50～300rpm、优选100rpm～200rpm摇床振荡发酵7～15天，发酵至终点，获得发酵液。
发酵培养基的pH调节可以用常见有机酸或无机酸，如磷酸、盐酸、醋酸、乳酸，优选为醋酸。
在一个具体的实施方式中，发酵培养基包括以下组分：1～5质量％碳源、1～10质量％的有机氮源、0.01～1质量％的无机盐、0.0001～0.001质量％的微量元素和0.0001～0.001质量％的辅酶。
上述碳源为微生物常用碳源，没有特别限定，可以包括以下的一种或多种的组合：甘油、蔗糖、果糖、葡萄糖、麦芽糖等，优选为甘油。
上述氮源为微生物培养常用氮源，没有特别限定，可以包括以下的一种或多种的组合：牛肉膏、蛋白胨、酵母粉、豆饼粉等，优选为牛肉膏。
上述无机盐为微生物培养常用无机盐，没有特别限定，可以包括以下的一种或多种的组合：氯化钠、磷酸二氢钠、磷酸氢二钠、氯化钾、硫酸钠等，优选为磷酸二氢钠和硫酸钠。
上述微量元素，可以包括以下的一种或多种的组合：硫酸镁、氯化亚铁、氯化锌等，优选为氯化锌。
上述辅酶，可以包括以下的一种或多种的组合：生物素、烟酸、磷酸吡哆醛、甜菜碱、维生素B12，核黄素等，优选为磷酸吡哆醛。
本发明通过对发酵培养基中的碳源、氮源、辅酶等因素进行优化，可提高麦角硫因的产量。
在一个具体的实施方式中，种子液接种到发酵液中接种量为1～10体积％。
在一个具体的实施方式中，上述前体物质为天冬氨酸、谷氨酰胺和甜菜碱中的至少一种，添加量为1～20mM。通过添加前体物质，能够对猴头菌提供更好的营养，利于其发酵生产麦角硫因。
在一个具体的实施方式中，上述发酵终点为发酵液中残糖≤0.5质量％。优选为发酵液中无葡萄糖，上述葡萄糖含量测定可采用常用的化学法，如DNS法、斐林试剂法、间接碘量法或者旋光法等。
步骤3
步骤3：对含有菌丝体的发酵液进行处理，得到含麦角硫因的发酵原液作为含麦角硫因的化妆品原液。
在一个具体的实施方式中，上述处理包括物理破碎处理和脱色处理中的至少一种。物理破碎处理可以为均质和/或超声，脱色处理可以采用活性炭吸附。通过对发酵液进行均质、超声使得发酵液中的猴头菌菌丝体充分破碎。通过在发酵液中添加活性炭使发酵液充分吸附脱色。
上述均质的条件为主轴转速1000r/min～3000r/min，均质时间10～30min。
上述超声的条件为超声功率为100w～1000w，超声时间为5～60min，超声终点为发酵液中的麦角硫因含量不再增加，优选用高效液相来检测发酵液中的麦角硫因含量。
上述活性炭吸附中所添加活性炭的量为发酵液体积的0.1～1％，吸附时间为10～60min。
本发明通过采用物理破碎细胞方式，最大限度的将食用菌菌丝体胞内麦角硫因等活性物保留并释放到了胞外且无任何物理特性及生物学功效活性上的破坏。本发明通过采用温和脱色工艺，将在化妆品中影响产品物理特性的气味及色素均较好的去除，更能满足不同化妆品配方需求。
步骤4
本发明的方法根据需要，还可以包括步骤4：除去处理后的发酵液中杂质。
在一个具体的实施方式中，去除杂质是通过将发酵液以1000-5000rpm离心10～30min后，将得到的上清液用0.22μm～0.45μm孔径的滤芯过滤而进行的。上述离心可以除去菌体细胞碎片及培养基中杂质，上述过滤可以除去微生物。通过采用离心和过滤，能够获得性能更好、满足不同化妆品配方需求的含麦角硫因的化妆品原液。
本发明还提供一种含麦角硫因的发酵原液(化妆品原液)，上述麦角硫因发酵原液(化妆品原液)中麦角硫因含量优选为300mg/L以上，所述发酵原液在化妆品中具有抗氧化、细胞修复功效。
在一个优选的实施方式中，含麦角硫因的发酵原液含有：300mg/L以上的麦角硫因、2.5g/L以上的真菌多糖和总量为5.0mg/100mL以上的氨基酸。
在一个具体的实施方式中，本发明的含麦角硫因的发酵原液是通过上述本发明的猴头菌发酵制备含麦角硫因的化妆品原液的方法获得的。通过本发 明的猴头菌发酵制备含麦角硫因的化妆品原液的方法所得到的含麦角硫因的化妆品原液中麦角硫因的含量为300mg/L以上。而且所得到的含麦角硫因的化妆品原液除了含有较高含量的麦角硫因以外，还含有其他多种活性成分，如真菌多糖，小分子氨基酸等。例如含有2.5g/L以上的真菌多糖和总量为5.0mg/100mL以上的氨基酸。其中，氨基酸包括天冬氨酸、苏氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、半胱氨酸、缬氨酸、甲硫氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、赖氨酸、组氨酸、精氨酸等。进一步优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以上
所得到的含麦角硫因的化妆品原液还具有抗氧化、防辐射的功效。所得到的含麦角硫因的化妆品原液的0.5v/v％-2v/v％的稀释液的1,1-二苯基-2-三硝基苯肼DPPH的清除率为30％以上，
Figure PCTCN2019118949-appb-000003
经所得到的含麦角硫因的化妆品原液的0.5v/v％-2v/v％的稀释液处理的细胞经紫外线损伤后的相对增殖率为70％以上。
本发明的化妆品原液具有极高的抗氧化性，对紫外线损伤具有较好的防护作用，同时具有对受损细胞进行修复的良好效果。
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例
下述实施例中所使用的实验方法如无特殊要求，均为常规方法。
下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
(1)试管保藏菌种取菌块接种到100ml种子培养基中，150rpm摇床，25℃培养5天；
种子培养基成分如下：
蔗糖20g/L，豆饼粉15g/L，硫酸钠0.5g/L，磷酸氢二钠0.5g/L，醋酸调pH 5.0。
(2)种子培养基接种到1L发酵培养基中，200rpm摇床，25℃发酵培养10天至无残糖；
发酵培养基成分如下：
甘油25g/L，牛肉膏20g/L，硫酸钠1g/L，磷酸氢二钠1g/L，氯化锌0.005g/L，磷酸吡哆醛0.001g/L，醋酸调pH 5.0，天冬氨酸10mM，谷氨酰胺5mM，甜菜碱10Mm；
(3)发酵液经均质30min，均质转速为3000rpm/min，再经超声15min，超声功率为500w，可使得麦角硫因及其他活性成分释放到发酵液中得初步发酵液。
实施例2
(1)试管保藏菌种取菌块接种到200ml种子培养基中，200rpm摇床，25℃培养7天；
种子培养基成分如下：
葡萄糖25g/L，豆饼粉15g/L，硫酸钠0.5g/L，磷酸氢二钠0.5g/L，醋酸调pH 5.5
(2)种子培养基接种到2L发酵培养基中，200rpm摇床，25℃发酵培养9天至无残糖；
发酵培养基成分如下：
甘油25g/L，牛肉膏20g/L，硫酸钠0.75g/L，磷酸氢二钠1g/L，氯化锌0.005g/L，磷酸吡哆醛0.001g/L，醋酸调pH 5.5，天冬氨酸8mM，谷氨酰胺5mM，甜菜碱15Mm；
(3)发酵液经均质40min，均质转速为3000rpm/min，再经超声30min，超声功率为800w，可使得麦角硫因及其他活性成分释放到发酵液中得初步发酵液。
实施例3：
(1)试管保藏菌种取菌块接种到500ml种子培养基中，150rpm摇床，30℃培养8天；
种子培养基成分如下：
麦芽糖50g/L，牛肉膏35g/L，氯化钾0.75g/L，磷酸氢二钠0.5g/L，乳酸调pH 4.5。
(2)种子培养基接种到5L发酵培养基中，100rpm摇床，30℃发酵培养15天至无残糖；
发酵培养基成分如下：
甘油50g/L，蛋白胨30g/L，磷酸二氢钠1g/L，磷酸氢二钠1g/L，氯化亚铁0.0075g/L，维生素B12 0.002g/L，醋酸调pH 4.5，天冬氨酸10mM，谷氨酰胺5mM，甜菜碱15Mm；
(3)发酵液经均质30min，均质转速为3000rpm/min，再经超声45min，超声功率为500w，可使得麦角硫因及其他活性成分释放到发酵液中得初步发酵液。
实施例4：
(1)试管保藏菌种取菌块接种到300ml种子培养基中，200rpm摇床，20℃培养9天；
种子培养基成分如下：
葡萄糖35g/L，酵母粉15g/L，氯化钾0.75g/L，磷酸氢二钠0.5g/L，盐酸调pH 4.2。
(2)种子培养基接种到2L发酵培养基中，150rpm摇床，20℃发酵培养15天至无残糖；
发酵培养基成分如下：
葡萄糖40g/L，蛋白胨25g/L，磷酸二氢钠1g/L，磷酸氢二钠1g/L，氯化锌0.0075g/L，维生素B12 0.002g/L，生物素0.001g/L，醋酸调pH 4.3，天冬氨酸15mM，谷氨酰胺10mM，甜菜碱5Mm；
(3)发酵液经均质20min，均质转速为3000rpm/min，再经超声60min，超声功率为1000w，可使得麦角硫因释放到发酵液中得初步发酵液。
实施例5
(1)向实施例1制备好的1L发酵液中添加5g活性炭，吸附脱色30min。
(2)发酵液经3000rpm离心30min，取出沉淀活性炭及菌体碎片，再经 0.22μm孔径滤芯，过滤除菌即可制得终产品。
(3)向步骤(2)中获得的1L发酵液终产品中添加9毫升苯氧乙醇，1毫升乙基己基甘油，50毫升1,3-丁二醇作为防腐剂混匀后无菌包装，液相检测麦角硫因含量为319.8mg/L
实施例6
(1)向实施例2制备好的2L发酵液中添加20g活性炭，吸附脱色45min。
(2)发酵液经3000rpm离心30min，取出沉淀活性炭及菌体碎片，再经0.22μm孔径滤芯，过滤除菌即可制得终产品。
(3)向步骤(2)中获得的2L发酵液终产品中添加18毫升苯氧乙醇，2毫升乙基己基甘油，100毫升1,3-丁二醇作为防腐剂混匀后无菌包装，液相检测麦角硫因含量为308.5mg/L。
实施例7
(1)向实施例3制备好的500mL发酵液中添加4g活性炭，吸附脱色30min。
(2)发酵液经4000rpm离心30min，取出沉淀活性炭及菌体碎片，再经0.22μm孔径滤芯，过滤除菌即可制得终产品。
(3)向步骤(2)中获得的500mL发酵液终产品中添加5毫升苯氧乙醇，0.5毫升乙基己基甘油，25毫升1,3-丁二醇作为防腐剂混匀后无菌包装，液相检测麦角硫因含量为331.1mg/L。
实施例8
(1)向实施例4制备好的300mL发酵液中添加1g活性炭，吸附脱色15min。
(2)发酵液经5000rpm离心30min，取出沉淀活性炭及菌体碎片，再经0.22μm孔径滤芯，过滤除菌即可制得终产品。
(3)向步骤(2)中获得的300mL发酵液终产品中添加2毫升苯氧乙醇，0.3毫升乙基己基甘油，15毫升1,3-丁二醇作为防腐剂混匀后无菌包装，液相检测麦角硫因含量为300.5mg/L。
实施例9理化性质检测
1.麦角硫因含量
将实施例5～8中样品分别命名为S1、S2、S3和S4，采用外标法检测。HPLC条件：Hypersil ODS C18柱(250mm×4.6mm，粒径5μm)；流动相：乙腈-水(3∶97)；流速：1.0mL/min；柱温：30℃；检测波长：254nm；进样量：20μL。色谱图如图1所示，其中，a)为标准品(麦角硫因含量浓度为100mg/L)，b)为S1，c)为S2，d)S3，e)S4结果如表1所示。
表1实施例5～8产品中麦角硫因的含量
  S1 S2 S3 S4
含量(mg/L) 319.8 308.5 331.1 300.5
2.真菌多糖
本发明采用苯酚硫酸法检测多糖含量。
(1)仪器：可见-紫外分光光度计、分析天平(精度0.0001g)、漩涡混合器(2)试剂：
2.1标准溶液：精确称取干燥恒重的葡萄糖(分析纯)100mg至容量瓶中，加水定容至250mL，摇匀后精确吸取10mL该溶液，加水定容至100mL。
2.2 80％苯酚液的配制：准确移取重蒸酚80mL，加蒸馏水定容至100mL，即得80％苯酚液，棕色瓶中避光保存。
2.3 6％苯酚液的配制：将80％苯酚液加水稀释至6％，临用现配。
2.4浓硫酸(优级纯)
(3)检测：
3.1制作标准曲线：分别吸取葡萄糖标准液0.0，0.4，0.8，1.2，1.6，2.0mL于具塞试管中，各加水补至2.0mL。分别加入6％苯酚溶液1mL，混合均匀后快速加入浓硫酸5.0mL(浓硫酸时悬空加入，不能贴壁)，即刻摇匀，室温反应20min后于490nm测吸光度，以0管做空白对照，纵坐标为多糖浓度，横坐标为吸光度，得标准曲线。
3.2样品制备：分别称取实施例5～8中的样品S1-S4各0.2ml待置于50mL容量瓶中，加适量水，完全溶解后加水定容至刻度作为贮备液，摇匀。用前量取贮备液5mL，置50mL容量瓶中，加水至刻度，再以相同方法稀释10倍。取2mL于具塞试管中，按上述“加入6％重蒸酚1.0mL”起，同法 操作，由标准曲线得待测样品多糖浓度，根据稀释倍数计算多糖含量。
表2.实施例5～8的多糖含量：
样品编号 多糖含量(g/L)
实施例5(S1) 2.92
实施例6(S2) 2.74
实施例7(S3) 2.94
实施例8(S4) 2.87
3.氨基酸
以实施例5～8中样品S1～S4为实验样品，进行氨基酸含量测定，采用氨基酸自动分析仪进行，结果如下表所示。
表3.实施例5～8的氨基酸含量
Figure PCTCN2019118949-appb-000004
Figure PCTCN2019118949-appb-000005
为说明发酵产品特有高活性功效选择S0作为对比例。
为进一步说明本工艺所制备的发酵液产品活性是由多种活性物质共同作用，取350mg麦角硫因纯品(含量大于等于98％)，添加1L纯化水充分溶解配制成麦角硫因溶液S0作为对比例。
4.抗氧化活性
分别精密量取DPPH(1,1-二苯基-2-三硝基苯肼)溶液5.0mL和不同浓度S0-S4样品溶液5.0mL置具塞试管中，混匀。以等体积的水与95％乙醇混合溶液为空白对照。室温放置30分钟，于523nm处分别测定溶液吸光度值。另设一组分别精密量取DPPH溶液5.0mL和纯化水5.0mL混合，操作同上。计算方法如下：
Figure PCTCN2019118949-appb-000006
选取S1作为检测样品，结果如表3所示。由表中数据可知，在添加0.5％-2％的浓度梯度下，样品S1具有较高的清除自由基的能力，2％浓度下可达70％，有极高的抗氧化性。
表4样品S0～S4清除DPPH自由基活性
原液浓度(％) 0.5 1 2
S1清除率(％) 31％ 54％ 70％
S2清除率(％) 28％ 46％ 61％
S3清除率(％) 39％ 57％ 76％
S4清除率(％) 27％ 44％ 60％
S0清除率(％) 17％ 35％ 51％
5.对UVA与UVB的防护作用
5.1样品溶液的配制：将实施例5～8得到的样品S1-S4和样品S0溶解于无血清培养基，配制终浓度为2.0％的溶液，0.22μm滤膜过滤除菌，使用前用无血清培养基稀释到指定浓度。
5.2铺板：取处于对数生长期的HaCaT细胞(人永生化表皮细胞)，胰酶 消化后，调细胞密度1×10 5/mL，接种于平底96孔细胞培养板，每孔100μL细胞悬液，置二氧化碳培养箱37℃、5％CO 2常规培养过夜。
5.3加药：弃去培养液，试验分组如下，试验组每孔加入100μL浓度为0.5％、1.0％、2.0％的样品溶液，正常组和模型组加入等量的无血清培养基，放入培养箱中继续培养24h。
表5样品S1-S4和S0对UVA与UVB的防护作用
Figure PCTCN2019118949-appb-000007
5.4照射：采用7.2J/cm 2UVA加126mJ/cm 2UVB照射HaCaT细胞。
照射后，弃掉旧培养液，加入无血清培养液，继续培养24h后，每孔加入10μL WST-1，放入细胞培养箱中继续孵育4h。于450nm波长处用酶标仪测定光吸收值。
由表6可知，经UVA与UVB联合照射损伤后，与正常组(100％)相比，模型组的HaCaT细胞增殖率下降了45.58％，表明造模成功。在UV照射之前用不同浓度的试验组预处理HaCaT细胞，与未经处理的模型组相比，细胞增殖率都有显著提高，表明该发酵原液对紫外线损伤具有较好的防护作用，且防护效果随着发酵液浓度的增加而增强，同时紫外防护效果发酵液组S1明显好于只含有麦角硫因溶液的S0组。
表6经不同浓度样品预处理的HaCaT细胞再经过紫外线损伤后的相 对增殖率(％)
Figure PCTCN2019118949-appb-000008
6.促进细胞修复效果
细胞培养液：含10％FBS的DMEM培养液。样品溶液：用细胞培养液将S1和S0配制成4％的母液。
将HaCaT细胞以5×10 4个/mL的密度接种于24孔板，37℃、5％CO 2条件下培养24h。在接近融合的单层细胞上，用200μL枪头在24孔板的每个孔内垂直划线。弃去孔中液体，加入样品溶液(最终血清含量2.5％)，继续培养24h后拍照观察。
结果如图2所示，其中，a为对照处理组初始，b为对照处理组24h，c为S0组初始，d为S0处理24h，e为S1组初始，f为S1处理组24h。在浓度为2.0％时与受损细胞接触24h后S1组和S0组均能够较好地促进细胞的迁移与自我修复，且两者相比S1组效果好于只含有麦角硫因溶液的S0组。
如上所述，利用本发明方法获得的化妆品原液，由于同时含有高含量的麦角硫因、真菌多糖和氨基酸，因此具有良好的抗氧化活性、对紫外线损伤具有加好的保护作用，以及具有对受损细胞进行修复的良好效果。
本发明的以上实施例仅用来说明实现本发明的具体实施方式，这些实施方式不能理解为是对本发明的限制。其他的任何在未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均视为等效的置换方式，落在本发明的保护范围之内。

Claims (26)

  1. 一种猴头菌发酵制备含麦角硫因的化妆品原液的方法，其特征在于，包括以下步骤：
    步骤1：将猴头菌菌丝接种到液体种子培养基中培养，得到种子液；
    步骤2：将种子液接种到发酵培养基中发酵并添加麦角硫因前体物质，发酵至终点，得到发酵液；
    步骤3：对含有菌丝体的发酵液进行处理，得到含麦角硫因的发酵原液作为含麦角硫因的化妆品原液。
  2. 根据权利要求1所述的方法，其特征在于，所述猴头菌(Hericium erinaceus)的保藏编号为CCTCC NO：M 2018567。
  3. 根据权利要求1所述的方法，其特征在于，所述种子培养基包括以下组分：1～10质量％的碳源、1～5质量％的有机氮源和0.01～1质量％的无机盐。
  4. 根据权利要求1所述的方法，其特征在于，步骤1中，在15～30℃、pH 4.0～7.0、50～300rpm振荡培养5～10天，得到种子液。
  5. 根据权利要求1所述的方法，其特征在于，所述发酵培养基包括以下组分：1～5质量％的碳源、1～10质量％的有机氮源、0.01～1质量％的无机盐、0.0001～0.001质量％的微量元素和0.0001～0.001质量％的辅酶。
  6. 根据权利要求1所述的方法，其特征在于，步骤2中，在15～30℃、pH 4.0～7.0、50～300rpm振荡发酵7～15天，发酵至终点，获得发酵液。
  7. 根据权利要求1所述的方法，其特征在于，步骤2中，所述前体物质为天冬氨酸、谷氨酰胺和甜菜碱中的至少一种，添加量为1～20mM。
  8. 根据权利要求1所述的方法，其特征在于，步骤2中，种子液接种到发酵液中接种量为1～10体积％。
  9. 根据权利要求1所述的方法，其特征在于，步骤2中，所述发酵终点为发酵液中残糖≤0.5质量％。
  10. 根据权利要求1所述的方法，其特征在于，步骤3中，所述处理包括均质、超声和活性炭吸附中的至少一种。
  11. 根据权利要求10所述的方法，其特征在于，所述均质的条件为主轴转速1000r/min～3000r/min，均质时间10～30min。
  12. 根据权利要求10所述的方法，其特征在于，所述超声的条件为超 声功率为100w～1000w，超声时间为5～60min，超声终点为麦角硫因的含量不再增加。
  13. 根据权利要求10所述的方法，其特征在于，所述活性炭吸附中所添加活性炭的量为发酵液体积的0.1～1％，吸附时间为10～60min。
  14. 根据权利要求1所述的方法，其特征在于，还包括步骤4：除去处理后的发酵液中杂质。
  15. 根据权利要求14所述的方法，其特征在于，步骤4中，去除杂质是通过将发酵液以1000-5000rpm离心10～30min后，将得到的上清液用0.22μm～0.45μm孔径的滤芯过滤而进行的。
  16. 根据权利要求1-15中任一项所述的方法，其特征在于，所得到的含麦角硫因的化妆品原液中麦角硫因的含量为300mg/L以上。
  17. 根据权利要求1-15中任一项所述的方法，其特征在于，所得到的含麦角硫因的化妆品原液含有2.5g/L以上的真菌多糖和总量为5.0mg/100mL以上的氨基酸，优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以上。
  18. 根据权利要求1-15中任一项所述的方法，其特征在于，所得到的含麦角硫因的化妆品原液的0.5v/v％-2v/v％的稀释液的1,1-二苯基-2-三硝基苯肼DPPH的清除率为30％以上，
    Figure PCTCN2019118949-appb-100001
  19. 根据权利要求1-15中任一项所述的方法，其特征在于，经所得到的含麦角硫因的化妆品原液的0.5v/v％-2v/v％的稀释液处理的细胞经紫外线损伤后的相对增殖率为70％以上。
  20. 一种含麦角硫因的发酵原液，其特征在于，含有：300mg/L以上的麦角硫因、2.5g/L以上的真菌多糖和总量为5.0mg/100mL以上的氨基酸，优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以上。
  21. 根据权利要求20所述的含麦角硫因的发酵原液，其特征在于，其是通过采用权利要求1-19中任一项所述的方法获得的。
  22. 根据权利要求20或21所述的含麦角硫因的发酵原液，其特征在于， 所述发酵原液的0.5v/v％-2v/v％的稀释液的1,1-二苯基-2-三硝基苯肼DPPH的清除率为30％以上，
    Figure PCTCN2019118949-appb-100002
  23. 根据权利要求20或21所述的含麦角硫因的发酵原液，其特征在于，经发酵原液的0.5v/v％-2v/v％的稀释液处理的细胞经紫外线损伤后的相对增殖率为70％以上。
  24. 一种由权利要求1-19中任一项所述的方法获得的含麦角硫因的发酵原液或权利要求20-23中任一项所述的含麦角硫因的发酵原液在化妆品中的应用。
  25. 一种用于生产含麦角硫因的化妆品原液的发酵培养基组合物，其特征在于，包括以下组分：1～5质量％的碳源、1～10质量％的有机氮源、0.01～1质量％的无机盐、0.0001～0.001质量％的微量元素和0.0001～0.001质量％的辅酶。
  26. 一种含麦角硫因的化妆品原液组合物，其特征在于，包括300mg/L以上的麦角硫因、2.5g/L以上的真菌多糖和5.0mg/100mL以上的氨基酸，优选多糖含量在2.6g/L以上，进一步优选多糖含量在2.7g/L以上，优选氨基酸的总含量在5.5mg/100mL以上，进一步优选在5.7mg/100mL以上。
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