WO2021017449A1 - 发酵的桦树汁及其生产方法及其在皮肤外用组合物中的用途 - Google Patents

发酵的桦树汁及其生产方法及其在皮肤外用组合物中的用途 Download PDF

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WO2021017449A1
WO2021017449A1 PCT/CN2020/073351 CN2020073351W WO2021017449A1 WO 2021017449 A1 WO2021017449 A1 WO 2021017449A1 CN 2020073351 W CN2020073351 W CN 2020073351W WO 2021017449 A1 WO2021017449 A1 WO 2021017449A1
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fermentation
birch
birch sap
fermented
sap
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PCT/CN2020/073351
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English (en)
French (fr)
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段颖异
王昕悦
洪涛
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浙江养生堂天然药物研究所有限公司
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Priority to KR1020217001641A priority Critical patent/KR20220039644A/ko
Priority to EP20816371.7A priority patent/EP4006161A4/en
Priority to US17/256,522 priority patent/US20210177734A1/en
Priority to AU2020320370A priority patent/AU2020320370B2/en
Priority to CA3134197A priority patent/CA3134197A1/en
Publication of WO2021017449A1 publication Critical patent/WO2021017449A1/zh

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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/347—Phenols
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • 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
    • 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
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening 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
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • 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
    • 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 present invention relates to fermented birch sap and a production method thereof, wherein the fermented birch sap is obtained by fermentation using Inonotus obliquus as a strain and birch sap as a substrate; the method includes using Inonotus obliquus Bacteria are used as strains and birch sap is used as a substrate for fermentation.
  • Birch is a deciduous tree of the birch family. There are currently about 100 species in the world, mainly distributed in the northern temperate zone and the cold temperate zone. Among them, there are about 29 varieties in my country, mainly distributed in the northeast, northwest, north and southwest. Birch trees are mostly grown in remote mountainous areas with little human intervention and no industrial pollution. Birch sap (also called birch sap) is the fresh sap from the birch bark being cut or the trunk is drilled. It is colorless or light yellow, free of precipitation and impurities, and has a light birch fragrance. The birch sap contains a lot of sugars, amino acids, vitamins, biotin, cytokinins, trace mineral elements, aromatic oils, betulin, saponin and other compounds. It has good moisturizing, anti-inflammatory, anti-wrinkle and whitening properties. And other skin care effects.
  • birch sap in existing fermentation processes is limited to beverages and wines.
  • CN107586640A discloses a birch sap wine fermentation process
  • CN108676666A discloses a birch sap distilled wine and its production process
  • CN108676666A discloses a birch sap beer and its brewing method, in which brewer's yeast is used to ferment birch
  • the sap is used to produce alcoholic beverages
  • CN109055170A discloses a birch sap vinegar and a preparation method thereof, wherein the birch sap undergoes alcohol fermentation and acetic acid fermentation to obtain birch sap vinegar with a total acid of >3%.
  • CN109077960A discloses a white birch sap anti-aging and whitening composition, in which although fermented birch sap is used as a cosmetic raw material, the fermentation bacteria, functional ingredients and efficacy of the raw material are unknown, and the fermentation cycle is as long as 30 days. Problems such as low efficiency.
  • Inonotus obliquus (Inonotus obliquus), commonly known as Chaga, belongs to the genus Basidiomycotina, Layer Fungi, Non-Gallomycetes, Polyporaceae, and Inonotus obliquus. Inonotus obliquus contains more than ten kinds of trace elements such as carbon, potassium, nitrogen, calcium and aluminum, as well as active substances such as protein, polysaccharides, flavonoids, polyphenol compounds, sterols, alkaloids, peptides and organic acids. These active substances produced by Inonotus obliquus have biological activities such as antioxidant, anti-inflammatory, and immune regulation, and have good application prospects.
  • Liquid submerged fermentation technology can shorten the growth cycle of Inonotus obliquus, obtain a large amount of mycelium in a short time, and enable Inonotus obliquus to accumulate a large amount of intracellular and extracellular products.
  • the fermentation filtrate of Inonotus obliquus contains active ingredients such as polyphenols and polysaccharides (Zhu Jinwei et al., 2011, Journal of Zhejiang Sci-Tech University, Vol. 28, Issue 4, pp.
  • CN104956925A discloses a production method of Inonotus obliquus continuous submerged fermentation broth and powder, in which the fermented filtrate is concentrated, which has a relatively high process cost
  • CN106434755A discloses the submerged fermentation product of Inonotus obliquus and its use, wherein Ethanol is used to elute fermentation products, and the use of organic solvents is likely to cause environmental pollution and increase processing energy consumption.
  • the present invention attempts to expand the application of fermented birch sap and improve the current use of the fermentation filtrate of Inonotus obliquus.
  • the inventors have discovered through research that by selecting Inonotus obliquus as the strain and fermenting with birch sap as the sole or main substrate, a fermented birch sap product with improved performance can be obtained, which has the activity of birch sap itself.
  • the ingredients are also rich in active ingredients such as polysaccharides and polyphenols, so that they can be advantageously used as active materials for skin external compositions.
  • the present invention relates to a method for producing fermented birch sap, which includes the steps of using Inonotus obliquus as a strain and birch sap as a substrate for fermentation.
  • the method further comprises in-situ thermal extraction in the fermentor to obtain the fermentation product in the fermentation step, so that the intracellular polysaccharides and polyphenols of Inonotus obliquus are released into the fermentation broth.
  • the in-situ thermal extraction step includes heating the fermentation product to about 60-95°C, preferably about 65-90°C, more preferably about 70-85°C, and maintaining it for about 30-150 minutes, It is preferably about 40-100 minutes, more preferably about 50-80 minutes; then it is cooled to about 18-30°C, preferably about 20-28°C.
  • the production method specifically includes the following steps:
  • seed liquid is known in the art.
  • Inonotus obliquus scraped from a solid medium plate can be inoculated into a shake flask containing a certain amount (for example, 50 mL) of seed culture medium at a culture temperature of 25-30°C, a shaker speed of 150-180 rpm, and culture 4 -6 days to obtain the first-level seed solution; under aseptic conditions, according to the 5-10% inoculum amount, transfer the first-level seed solution to a shake flask containing a certain amount (for example, 400 mL) of the seed culture medium.
  • a certain amount for example, 400 mL
  • the preferred solid medium is yeast powder glucose agar medium, which includes 1000 mL of deionized water, 0.6% potato powder, 2% glucose and 1.5% agar.
  • a preferred seed medium is an oat germ bran medium, which includes 1000 mL of deionized water, 3% glucose, 1% oat germ bran powder, 1% yeast powder, 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, Natural pH.
  • the birch sap medium is prepared with birch sap as the sole substrate, and a carbon source, a nitrogen source, an inorganic salt and/or a pH regulator is optionally added to it.
  • the birch sap is obtained from the genus Betula, Betula alba, Betula pubescens, Betula Pendula, and Asian white birch (Betula platyphylla).
  • the birch sap is a colorless, transparent, no-sediment-free, no debris, and nutrient-rich sap that is obtained by manually drilling a hole at the base of the birch trunk between thawing and early spring.
  • the birch sap is commercially available and used as it is, for example, it can be purchased from Daxinganling Chaoyue Wild Berry Development Co., Ltd.
  • the birch sap may be a raw birch sap or a concentrated birch sap, wherein the concentration of the concentrated birch sap is 1.2-6 times, preferably 1.5-4 times.
  • the concentrated birch sap is obtained by concentrating the above-mentioned commercially available birch sap.
  • Concentration methods are known in the art, such as heating concentration, low-temperature vacuum concentration, membrane concentration and the like.
  • the concentration is preferably carried out by a low-temperature freeze concentration or membrane concentration process.
  • the commercially available birch juice stock solution is input into a low-temperature drying equipment, the temperature is lowered to -40°C to -70°C, and the vacuum is applied to 0.1-30Pa. Concentrated in vacuum at low temperature to obtain concentrated birch sap with different concentration times.
  • the birch sap culture medium may use birch sap as the sole substrate, without adding any additional ingredients to it, including no additional water, but not excluding water inherently contained in each component .
  • a carbon source, a nitrogen source, an inorganic salt and/or a pH adjuster can be optionally added to the birch sap as needed.
  • the carbon source is known in the art, including but not limited to glucose, glycerin, molasses, lactose, mannose, maltose, corn starch, sucrose, fructose, and the like. In the present invention, glucose is preferably used.
  • the content of the carbon source in the birch sap medium is usually about 0-3.5%, preferably about 0.5-3%, based on the total weight of the birch sap medium.
  • the nitrogen source is known in the art, including but not limited to whey protein, vegetable protein, cereal powder, hydrolyzed vegetable polypeptide, yeast powder, tryptone and the like.
  • the preferred whey protein is isolated whey protein powder
  • the preferred vegetable protein is pea flour and hydrolyzed pea protein
  • the preferred cereal flour is rye flour, hydrolyzed oat flour, and hydrolyzed brown rice flour.
  • the content of the nitrogen source in the birch sap medium is generally about 0-3%, preferably about 0.3-2.5%, based on the total weight of the birch sap medium.
  • the inorganic salt is known in the art, including but not limited to potassium dihydrogen phosphate, magnesium sulfate, sodium dihydrogen sulfate, calcium chloride, and the like. In the present invention, potassium dihydrogen phosphate and anhydrous magnesium sulfate are preferred.
  • the content of the inorganic salt in the birch sap medium is generally about 0-0.3%, preferably about 0.05-0.1%, based on the total weight of the birch sap medium.
  • pH adjusters are known in the art and include, but are not limited to, citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide, and the like. In the present invention, citric acid and sodium citrate are preferred. According to the initial pH of the birch sap raw material, optionally, a pH adjusting agent is used to adjust the pH of the birch sap medium to about 5.0-6.5.
  • birch sap medium In the case of containing carbon source, nitrogen source, inorganic salt and/or pH regulator, adding these substances to birch sap can prepare birch sap medium.
  • the content of birch sap in the medium is about 90% or more, preferably about 93% or more, based on the birch sap medium total weight.
  • step (3) inoculation fermentation is known in the art.
  • the volume shall prevail, and the secondary seed liquid shall be inserted into the fermentor under aseptic conditions according to the inoculum amount of about 5-10%, at a temperature of about 25-30°C, a stirring speed of 180-250rmp and a stirring speed of 1.2-2.0vvm
  • step (4) in situ thermal extraction of the fermentation product involves the in situ thermal extraction of the fermentation product obtained in step (3) in a fermentor to release the intracellular polysaccharides and polyphenols of Inonotus obliquus into the fermentation broth.
  • step (4) includes heating the fermentation product obtained in step (3) to about 60-95°C, preferably about 65-90°C, more preferably about 70-85°C, and holding it for about 30-150 minutes , Preferably about 40-100 minutes, more preferably about 50-80 minutes; then cool to about 18-30°C, preferably about 20-28°C.
  • centrifugation and filtration are known in the art, and are usually carried out at 6000-10000 rpm for 15-30 minutes.
  • the centrifugation step removes the bacteria from the fermentation product to obtain the supernatant, and the supernatant is filtered to obtain the fermented birch juice filtrate product.
  • the method of the present invention may further include subjecting the obtained filtrate product to ultra-high temperature instantaneous sterilization at a sterilization temperature of (115 ⁇ 5)°C and a time of 10-30 seconds. Then, the sterilized fermented birch juice filtrate product is transferred to a storage tank for storage.
  • the fermented birch juice filtrate obtained by the above method (also referred to as fermented birch juice) is light in color and transparent, and not only contains the nutrients of the birch juice substrate itself (including B vitamins, trace elements, amino acids, fatty acids, etc.) ), it also contains about 100 mg/L or more, preferably about 130 mg/L or more, more preferably about 150 mg/L or more total phenols, and about 1.3 g/L or more, preferably 1.8 g/L or more, more preferably about 3.0 g/L or more Of polysaccharides.
  • the total phenol determination method is the Folin colorimetric method
  • the determination method of the extracellular polysaccharide is the anthrone sulfate colorimetric method.
  • the total phenols tested mainly include gallic acid, ferulic acid, naringin, quercetin and kaempferol.
  • the method of the present invention has the following advantages and effects:
  • the resulting fermented birch sap filtrate has more active components such as total phenols and polysaccharides than the raw birch sap, so that the birch sap has Excellent tyrosinase activity inhibition rate and antioxidant activity, so it has better skin external use effects, such as skin care effects, which expands the application range of fermented birch sap, and is no longer limited to applications only as beverages and wines ;
  • the obtained fermented birch juice filtrate can be used directly without going through the concentration process, which improves the utilization rate of raw materials and reduces the production cost;
  • the formula of the birch sap culture medium is optimized. Glucose, vegetable protein, etc. are used as the carbon and nitrogen sources required for the fermentation of Inonotus obliquus. The raw materials are easily available and the cost is low. The color of the fermentation filtrate obtained from the optimized culture medium is used. It is shallow and transparent, and has better properties than the fermentation product of Inonotus obliquus reported in the prior art.
  • the present invention also relates to the use of fermented birch sap in an extra-skin composition, wherein the fermented birch sap is obtained by fermentation using Inonotus obliquus as a strain and birch sap as a substrate.
  • the fermented birch juice filtrate product contains 100 mg/L or more, preferably 130 mg/L or more, more preferably 150 mg/L or more total phenols, and 1.3 g/L or more, preferably 1.8 g/L Above, more preferably 3.0 g/L or above polysaccharide.
  • the present invention further relates to a skin external composition
  • a skin external composition comprising (A) fermented birch sap, wherein the fermented birch sap uses Inonotus obliquus as a strain and birch sap as a substrate. It is obtained by fermentation.
  • the fermented birch juice filtrate product contains 100 mg/L or more, preferably 130 mg/L or more, more preferably 150 mg/L or more total phenols, and 1.3 g/L or more, preferably 1.8 g/L Above, more preferably 3.0 g/L or above polysaccharide.
  • the content of the fermented birch sap in the skin external composition can vary within a wide range. For example, about greater than 0 to less than 100%, preferably about 80-95%, based on the total weight of the skin external composition.
  • the external skin composition includes a pharmaceutical composition or a cosmetic composition, especially a skin care cosmetic composition, a whitening cosmetic composition and an anti-aging cosmetic composition.
  • the skin topical composition may optionally include (B) ingredients commonly used in skin topical compositions, including but not limited to vehicles, active ingredients and excipients Wait.
  • Component (B) is known in the art, and those skilled in the art can select its type and amount as required. For example, the content of component (B) is about 2-82%, based on the total amount of the skin external composition weight.
  • the vehicle includes, for example, a diluent, a dispersant, or a carrier, and examples thereof include, but are not limited to, ethanol, dipropylene glycol, butylene glycol, and the like.
  • the content of the vehicle in the skin topical composition is known in the art, for example, it usually accounts for 0.5-20% of the total weight of component (B).
  • the active ingredients include, for example, emollients, moisturizers, whitening active ingredients, anti-aging active ingredients and the like.
  • emollient examples include, but are not limited to, olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, white flower seed oil, safflower seed oil, Dogtooth rose hip oil, argan tree kernel oil, jojoba seed oil, sunflower seed oil, palm tree fruit oil, squalane, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutylene, isotene Hexane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosine, isononyl isononanoate, hydrogenated polydecene, glycerol tri(ethylhexanoate) , Cetyl alcohol ethyl hexanoate, bis-diethoxy diethylene glycol cyclohexane
  • solid emollients include, but are not limited to, cetyl alcohol, stearyl alcohol, cetearyl alcohol, behenyl alcohol, scylitol, lauric acid, myristic acid, palmitic acid, stearic acid, beeswax, candelilla Tree wax, carnauba wax, lanolin, ozokerite, jojoba seed wax, paraffin wax, microcrystalline wax, hydrogenated rice bran wax, hydrogenated coconut oil glycerides, glyceryl behenate/eicosanate, myristyl alcohol One or more of myristate, bis-diglyceride polyacyl adipate-2, shea butter, and muluxing palm seed butter.
  • the content of the emollient in the external skin composition is known in the art, for example, it usually accounts for 1-50% of the total weight of the component (B).
  • moisturizer examples include, but are not limited to, glycerin, diglycerin, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, 1,2-pentanediol, polyethylene glycol-8, polyethylene glycol Alcohol-32, methylglucitol-10, methylglucitol-20, PEG/PPG-17/6 copolymer, glycerol-7, glycerol-26, glycerol glucoside, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, PEG/PPG/polybutylene glycol-8/5/3 glycerin, sucrose, trehalose, rhamnose, mannose, raffinose, Betaine, erythritol, xylitol, urea, glyceryl polyether-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, acetyl
  • the whitening active ingredients include, but are not limited to, kojic acid, ascorbyl glucoside, arbutin, tranexamic acid, nicotinamide, plant sterols, plant sterols/behenyl alcohol/octyldecanol lauroyl glutamate, phenyl ethyl Resorcinol, turmeric root extract, birch bark extract, ceramide 2, ceramide 3, acetyl sphingosine, resveratrol, palm tree bark extract, coleus forskohlii root Extract, pepper seed extract, ubiquinone, cholesterol, cholesterol stearate, ascorbic acid, ascorbyl dipalmitate, tocopherol (vitamin E), tocopherol acetate, bisabolol, tetraisoascorbate Palmitate, pyridoxine dicaprylate, pyridoxine dipalmitate, retinyl palmitate, phytosterol/octyl
  • anti-aging active ingredients include, but are not limited to, tocopherol (vitamin E), retinol, retinol palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydroturmeric Ellagic acid, ubiquinone, whey protein, peptides, acetylhexapeptide-8, palmitoyl pentapeptide-4, salicyl sphingosine, birch sap, silymarin, silk protein, tocopherol Sodium phosphate, ribonucleic acid (RNA), dipeptide diaminobutyrylbenzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide -7, VITIS VINIFERA seed extract, PTEROCARPUS MARSUPIUM bark extract,
  • the auxiliary materials include, for example, emulsifiers, thickeners, preservatives, perfumes and the like.
  • emulsifier examples include, but are not limited to, cetearyl oleate, sorbitan oleate, polysorbate-60, polysorbate-80, methylglucose sesquistearic acid Ester, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butanol-26, PEG-4 polyglycerol-2 stearate, PEG-60 hydrogenated Castor oil, steareth-2, steareth-21, PPG-13-decyltetradeceth-24, cetearyl glucoside, PEG-100 stearate, glycerin Stearate, Glyceryl Stearate SE, Coco Glucoside, Ceteareth-25, PEG-40 Stearate, Polyglyceryl-3 Methyl Glucose Distearate, Glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyce
  • the thickener examples include, but are not limited to, carbomers, acrylic acid (ester) and its derivatives, xanthan gum, gum arabic, polyethylene glycol-14M, polyethylene glycol-90M, succinyl poly One or more of high molecular polymers such as sugar, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.
  • the content of the thickener in the external skin composition is known in the art, for example, it usually accounts for 0.1-10% of the total weight of the component (B).
  • Examples of the preservative include, but are not limited to, methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis(hydroxymethyl)imidazolidinylurea, potassium sorbate, sodium benzoate, chlorobenzene Glycerol, sodium dehydroacetate, caprylic hydroxamic acid, 1,2-hexanediol, 1,2-pentanediol, p-hydroxyacetophenone, caprylyl glycol, glyceryl caprylate, undecylenic acid One or more of glycerides, sorbitan caprylate, ethylhexylglycerol, tree peony root extract, etc.
  • the content of the preservative in the external skin composition is known in the art, for example, it usually accounts for 0.01-2% of the total weight of the component (B).
  • the fermented birch sap of (A) can be mixed with other pharmaceutical or cosmetic ingredients to obtain a pharmaceutical composition or cosmetic composition Things.
  • the other pharmaceutical ingredients or cosmetic ingredients are the ingredients commonly used in (B) skin external compositions as described above.
  • the external skin composition can be made into various dosage forms, such as solutions, suspensions, ointments, creams, emulsions, gels, powders or sprays.
  • the Inonotus obliquus strain used in Examples 1-5 is deposited in the China Forestry Microbial Culture Collection and Management Center (strain code cfcc 6584).
  • the Saccharomyces cerevisiae strain used in Comparative Examples 5-8 was from Angel Yeast Co., Ltd. (strain number ANGEL 1021).
  • the culture temperature is 28°C
  • the shaker speed is 150 rpm
  • the culture is for 4 days to obtain the first-level seed liquid
  • the medium formulation includes 1000mL deionized water, 3% glucose, 1% oat germ bran powder, 1% yeast powder, 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, natural pH;
  • birch sap stock (brix 0.875) collected in the Northeast Daxinganling as the substrate, add 2% glucose as the carbon source, 0.3% whey protein (WPI90, purchased from New Zealand Fonterra) as the nitrogen source, and add 0.1% Potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, using 1M citric acid aqueous solution to adjust the pH of the birch sap medium to 5.5;
  • step (2) Add the birch sap culture medium prepared in step (2) to a 300L fermentor and sterilize at 121°C for 30 minutes according to the 60% (v/v) volume of liquid volume; With an inoculum of 5%, connect the secondary seed liquid into the fermentor under aseptic conditions, and continue to ferment for 7 days under the conditions of 30°C, 200 rpm and 1.8 vvm aeration, and stop the tank to obtain the fermentation product;
  • the fermentation product obtained in step (3) was heated to 90°C in situ in a tank, kept for 60 minutes, and then cooled to 28°C, and then centrifuged to remove the bacteria.
  • the rotation speed was 6000rpm and the time was 30 minutes;
  • the supernatant obtained by centrifugation is filtered to obtain the birch juice filtrate fermented by Inonotus obliquus;
  • the obtained filtrate is further sterilized by an ultra-high temperature instant sterilization machine, where the sterilization temperature is 120°C and the time is 30 seconds, and the sterilized fermented birch
  • the sap filtrate is transferred to the storage tank for storage;
  • the preparation process of the seed liquid is the same as in Example 1;
  • the birch sap stock solution (brix 1.3) collected in Finland was used as the substrate, 1.5% glucose was added as the carbon source, 1% pea flour (purchased from Cargill Cereals and Oils (Nantong) Co., Ltd.) was added as the nitrogen source, and 0.1% phosphoric acid was added. Potassium dihydrogen and 0.05% anhydrous magnesium sulfate, using 1M sodium citrate aqueous solution to adjust the pH of the birch sap medium to 5.8;
  • step (2) Add the birch sap culture medium prepared in step (2) to a 100L fermentor according to the 65% (v/v) volume of the liquid, and sterilize at 121°C for 30 minutes; subject to the volume of the fermentation medium, follow Connect the secondary seed liquid to the fermenter under aseptic conditions with an inoculum of 7%, and continue to ferment for 7 days under the aeration conditions of 30°C, 200rpm and 1.6vvm, and stop the tank to obtain the fermentation product;
  • step (3) The fermentation product obtained in step (3) is heated to 80°C in situ in the tank, kept for 90 minutes, and then cooled to 25°C, and then centrifuged, filtered and heat sterilized in the same manner as in Example 1;
  • the preparation method of the seed liquid is the same as in Example 1;
  • birch sap juice concentrate concentrate (concentrated 3 times, brix 3.58) from the Northeast Xiaoxing'an Mountains as a substrate, 0.5% glucose was added as a carbon source, and 3% hydrolyzed oat protein (purchased from Xiamen Granbell Biotechnology Co., Ltd.)
  • As a nitrogen source add 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, use 1M sodium citrate aqueous solution to adjust the pH of the birch sap medium to 6.0;
  • step (2) Add the birch sap culture medium prepared in step (2) to a 500L fermentor and sterilize at 121°C for 30 minutes according to the 65% (v/v) volume of the liquid volume; the volume of the fermentation medium is subject to 10% Connect the secondary seed liquid to the fermentor under aseptic conditions, and continue to ferment for 7 days under the conditions of 30°C, 220rmp and 1.8vvm aeration, and stop the tank to obtain the fermentation product;
  • step (3) The fermentation product obtained in step (3) was heated in situ in the tank to 65°C, kept for 120 minutes, and then cooled to 28°C, and then centrifuged, filtered and heat sterilized in the same manner as in Example 1.
  • the preparation method of the seed liquid is the same as in Example 1;
  • the birch sap juice concentrate (concentrated 1.5 times, brix 1.8) from the Northeast Xiaoxing'an Mountains was used as the substrate, 1.5% glucose was added as a carbon source, and 3% hydrolyzed pea protein (purchased from Roquette (China) Fine Chemical Co., Ltd.) )
  • As a nitrogen source add 0.1% potassium dihydrogen phosphate and 0.05% anhydrous magnesium sulfate, use 1M sodium citrate aqueous solution to adjust the pH of the birch sap medium to 6.0;
  • the preparation method of the seed liquid is the same as in Example 1;
  • birch sap concentrate concentrate (concentrated 3 times, brix3.58) from the Northeast Xiaoxinganling in Example 3 as a raw material, without adding carbon source, nitrogen source, inorganic salt and pH regulator;
  • the birch sap culture medium is not inoculated and fermented, and the preparation method of the unfermented birch sap filtrate is the same as step (4) in Example 1;
  • the birch sap culture medium is not inoculated and fermented, and the preparation method of the unfermented birch sap filtrate is the same as step (4) in Example 2;
  • the preparation method of birch sap culture medium is the same as in Example 3;
  • the birch sap culture medium is not inoculated and fermented, and the preparation method of the unfermented birch sap filtrate is the same as step (4) in Example 3;
  • the birch sap medium is not inoculated and fermented, and the preparation method of the unfermented birch sap filtrate is the same as step (4) in Example 4;
  • yeast freeze-dried powder according to the added amount of 30g/HL (hectares), add it to 1L sterile saline and disperse evenly to obtain yeast seed liquid; then inoculate the yeast seed liquid into a 300L fermenter, Under the conditions of °C, 200rpm and 1.2vvm ventilation, fermentation was continued for 48h, and the tank was stopped to obtain a yeast fermentation product; then the yeast fermentation birch juice filtrate was obtained in the same manner as step (4) of Example 1;
  • Table 1 The total phenol and polysaccharide content of the fermented birch juice filtrate obtained in Examples 1-5 and the birch juice filtrate obtained in Comparative Examples 1-8
  • the fermented birch juice filtrate produced by Inonotus obliquus contains extremely high levels of total phenols and polysaccharides compared with the unfermented birch juice filtrate and the birch juice filtrate fermented with conventional yeasts. Content, which greatly enhances the activity of the product, thereby giving it the possibility of being used in skin external compositions.
  • Example 6 Whitening effect test of fermented birch juice filtrate
  • tyrosinase (EC.1.14.18.1) is the key enzyme for the synthesis of melanin in organisms, which catalyzes the hydroxylation of L-tyrosine to form L-dopa, and then oxidizes L -Dopa forms dopaquinone, which forms melanin after a series of enzymatic and non-enzymatic reactions. Therefore, by inhibiting the activity of tyrosinase, the production of melanin in the organism can be regulated.
  • PBS buffer (0.2M, pH6.8): Measure 51mL 0.2M sodium dihydrogen phosphate solution and 49mL 0.2M disodium hydrogen phosphate solution, and mix;
  • L-Tyrosine 1.5mM: Weigh 0.0272g of L-Tyrosine, pre-disperse with the above PBS buffer, sonicate for 30 minutes, transfer to a 100mL volumetric flask, and add to the mark with PBS buffer;
  • Tyrosinase 250U: Dissolve tyrosinase (purchased from sigma aldrich) in the above PBS buffer, transfer to a 100mL volumetric flask, dilute to the mark with PBS buffer, and configure to 250U/ mL of use solution.
  • Inhibition rate of tyrosinase activity% ((C-A)-(D-B))/(C-A)*100%
  • Table 2 Comparison of the inhibition rate of tyrosinase activity between the fermented birch juice filtrate obtained in Examples 1-5 and the birch juice filtrate obtained in Comparative Examples 1-8
  • Example 7 Antioxidant efficacy test of fermented birch juice filtrate
  • reaction reagents including (A) 75 ⁇ L 0.8mM FeCl 3 +75 ⁇ L 4mM EDTA+600 ⁇ L 4mM KH 2 PO 4 -KOH buffer + 2.0565mL distilled water + 43.5 ⁇ L 3% H 2 O 2 +75 ⁇ L 112mM deoxyribose+75 ⁇ L 4mM ascorbic acid; (B)75 ⁇ L 0.8mM FeCl 3 +75 ⁇ L 4mM EDTA+600 ⁇ L 4mM KH 2 PO 4 -KOH buffer+2.0565mL sample solution+43.5 ⁇ L 3% H 2 O 2 +75 ⁇ L deoxyribose + 75 ⁇ L 4mM 112mM ascorbic acid; (C) 75 ⁇ L 0.8mM FeCl 3 + 75 ⁇ L 4mM EDTA + 600 ⁇ L4 mM KH 2 PO 4 -KOH buffer + 2.0565mL sample liquid + 43.5 ⁇ L 3% H 2 O 2 + 75 ⁇ L
  • reaction mixtures of each group were incubated in a constant temperature water bath at 37°C for 1 hour, and then 2mL 0.6% TBA was added to the mixture, and the mixture was heated in a boiling water bath for 15 minutes. After rapid cooling, it was centrifuged, and the absorbance of the supernatant was measured at 450nm, 532nm, and 600nm. value.
  • Hydroxyl radical scavenging rate (%) [1-(B-C)/A]*100%
  • DPPH radical is a single-electron radical, which has a characteristic absorption at 517nm.
  • the radical scavenger can pair with the one-electron of DPPH radical to make the absorption at 517nm disappear, and its fading degree is similar to that
  • the number of electrons accepted is a quantitative relationship, and the scavenging ability of the free radical scavenger can be inferred from this.
  • reaction reagents including (A) 1mL distilled water + 3mL 0.1mM DPPH reaction solution; (B) 1mL sample solution + 3mL 0.1mM DPPH reaction solution; (C) 1mL sample Liquid liquid + 3mL ethanol. Each group was shaken well and reacted for 30 minutes in the dark at room temperature, and then the absorbance value at 517nm was measured.
  • Table 3 Hydroxyl radical scavenging rate and DPPH free radical scavenging rate of the fermented birch juice filtrate obtained in Examples 1-5 and the birch tree juice filtrate obtained in Comparative Examples 1-8
  • the fermented birch juice filtrate prepared in Example 1 is used to prepare whitening lotion, and the formula is as follows:
  • the preparation method of the lotion is: mixing the fermented birch juice filtrate, sodium benzoate and pentanediol and then filtering it.
  • the fermented birch juice filtrate prepared in Example 3 is used to prepare a face cream, and the formula is as follows:
  • the above cream is prepared as follows: the oil phase and water phase components in the above ingredients are mixed separately, heated to 75°C to form an oil phase and a water phase respectively; then the obtained oil phase and water phase are added to a vacuum homogenizer to homogenize Emulsify, cool to 45°C, add flavor and sodium benzoate, and stir well to get.

Abstract

发酵的桦树汁及其制备方法及其在皮肤外用组合物中的用途，所述发酵的桦树汁采用桦褐孔菌为菌株、以桦树汁为底物发酵获得。

Description

[根据细则37.2由ISA制定的发明名称]　发酵的桦树汁及其生产方法及其在皮肤外用组合物中的用途 技术领域
本发明涉及发酵的桦树汁及其生产方法，其中所述发酵的桦树汁是采用桦褐孔菌作为菌株、以桦树汁作为底物通过发酵获得的；所述方法包括采用桦褐孔菌作为菌株、以桦树汁作为底物进行发酵的步骤。
背景技术
桦树为桦木科落叶乔木，目前全球大约有100个品种，主要分布于北温带和寒温带。其中，我国境内约有29个品种，主要分布在东北、西北、华北和西南等地。桦树大多生长于人为干涉较少、且没有工业污染的边远山区。桦树汁(也称桦树液)是桦树树皮被划开或树干钻孔流出的新鲜汁液，无色或浅黄色，无沉淀及杂质，具有淡淡的桦树清香。桦树汁内含大量的糖类、氨基酸、维生素、生物素、细胞分裂素、微量的矿质元素、芳香油、桦树醇、皂角甙等化合物，具有良好的保湿、抗炎、抗皱、美白等护肤功效。
现有发酵工艺对桦树汁的利用局限于饮料和酒类领域。例如，CN107586640A公开了一种桦树液酒发酵工艺，CN108676666A公开了一种桦树汁蒸馏酒及其生产工艺，CN108676666A公开了一种桦树汁啤酒及其酿造方法，其中均使用酿酒酵母发酵桦树汁来生产含酒精饮料；CN109055170A公开了一种白桦树液醋及其制备方法，其中桦树汁经过酒精发酵和醋酸发酵获得总酸>3％的桦树液醋。因此，现有发酵桦树汁的工艺目的都在于增加桦树汁中的乙醇或乙酸含量，不能使桦树汁具有其他活性成分，从而限制了发酵桦树汁的应用。CN109077960A公开了一种白桦树汁抗衰美白组合物，其中虽然使用了发酵桦树汁作为化妆品原料，但该原料的发酵菌种、功效成分和功效不明，且存在发酵周期长达30天、生产效率低等问题。
桦褐孔菌(Inonotus obliquus)俗称白桦茸，属担子菌亚门、层菌纲、非褶菌目、多孔菌科、褐卧孔菌属。桦褐孔菌含有碳、钾、氮、钙、铝等十余种微量元素，以及蛋白质、多糖类、黄酮类、多酚类化合物、甾醇、生物碱、多肽和有机酸等活性物质。桦褐孔菌产生的这些活性物质，具有抗氧化、抗炎、免疫调节等生物活性，具有良好的应用前景。
液体深层发酵技术能够缩短桦褐孔菌的生长周期，在短时间内获得大量菌丝体，同时使桦褐孔菌积累大量的胞内、胞外产物。尽管桦褐孔菌发酵滤液中含有多酚、多糖等活性成分(朱金卫等，2011，浙江理工大学学报，第28卷第4期，616-620页；陈才法等，2007，中草药，第3期，358-361页)，但现有的桦褐孔菌液体深层发酵工艺都集中在菌丝体生物量和胞内产物(三萜、多糖等)的获得，例如，参见CN160978465A，其公开了一种提高总三萜产量的发酵方法。
此外，现有工艺对桦褐孔菌发酵滤液常使用有机溶剂或浓缩处理。例如，CN104956925A公开了桦褐孔菌连续深层发酵发酵液和发酵粉的生产方法，其中将发酵滤 液浓缩处理，具有较高的工艺成本；CN106434755A公开了桦褐孔菌深层发酵产物及其用途，其中使用乙醇对发酵产物进行洗脱，有机溶剂的使用容易造成环境污染、增加处理能耗。
基于现有技术的现状，本发明尝试拓展发酵桦树汁的应用，并改善桦褐孔菌发酵滤液的使用现状。
发明内容
本发明人经研究发现，选择桦褐孔菌作为菌株、以桦树汁作为唯一或者主要底物进行发酵，可以得到一种具有改进性能的发酵桦树汁产物，其在具有桦树汁本身活性成分的同时还富含多糖和多酚等活性成分，从而可有利地作为皮肤外用组合物的活性原料。
因此，在一方面，本发明涉及一种生产发酵的桦树汁的方法，其包括采用桦褐孔菌作为菌株、以桦树汁作为底物进行发酵的步骤。
在优选的实施方案中，所述方法还包括在发酵罐中原位热提取在发酵步骤中得到发酵产物，以使桦褐孔菌的胞内多糖和多酚释放到发酵液中。
在一个实施方案中，所述原位热提取步骤包括将所述发酵产物加热至约60-95℃，优选约65-90℃，更优选约70-85℃，并保温约30-150分钟，优选约40-100分钟，更优选约50-80分钟；而后冷却至约18-30℃，优选约20-28℃。
通常，所述生产方法具体地包括下述步骤：
(1)制备种子液，
(2)制备桦树汁培养基，
(3)接种发酵，得到发酵产物，
任选地(4)原位热提取所述发酵产物,和
(5)离心除去菌体，过滤上清液，得到发酵的桦树汁滤液产物。
上述步骤(1)种子液的制备是本领域已知的。例如，可将从固体培养基平板上刮下的桦褐孔菌接种到装有一定量(例如50mL)种子培养基的摇瓶中，培养温度25-30℃，摇床转速150-180rpm，培养4-6天，得到一级种子液；在无菌条件下，按照5-10％的接种量，将一级种子液转接到装有一定量(例如400mL)种子培养基的摇瓶中，在25-30℃温度和150-180rpm的摇床转速下，恒温培养2-3天，得到二级种子液。
上述步骤(1)中，优选的固体培养基为酵母粉葡萄糖琼脂培养基，其包括1000mL去离子水、0.6％马铃薯浸粉、2％葡萄糖和1.5％琼脂。优选的种子培养基为燕麦胚芽麸皮培养基，其包括1000mL去离子水、3％葡萄糖、1％燕麦胚芽麸粉、1％酵母粉、0.1％磷酸二氢钾和0.05％无水硫酸镁，自然pH。
上述步骤(2)中，制备桦树汁培养基是以桦树汁为唯一底物，任选地向其中添加碳源、氮源、无机盐和/或pH调节剂。
所述桦树汁得自桦木科桦树属，其可来自白桦(Betula alba)、柔毛桦(Betula pubescens)、垂枝桦(Betula Pendula)和亚洲白桦(Betula platyphylla)等品种。所述桦树汁为在解冻至早 春发叶之间，人工在桦树的树干基部钻孔收集而得的无色透明、无沉淀、无杂物，具有桦树清香营养丰富的汁液。所述桦树汁可商购获得并原样采用，例如可购自大兴安岭超越野生浆果开发有限责任公司。
所述桦树汁可为桦树汁原液或浓缩的桦树汁，其中浓缩的桦树汁的浓缩倍数为1.2-6倍，优选1.5-4倍。
所述浓缩的桦树汁是将上述商购桦树汁浓缩得到的。浓缩方法是本领域已知的，例如加热浓缩、低温真空浓缩、膜浓缩等。在本发明中，优选通过低温冷冻浓缩或膜浓缩工艺进行浓缩，例如，将商购的桦树汁原液输入低温干燥设备，降温至-40℃至-70℃，抽真空至0.1-30Pa而进行低温真空浓缩，从而得到不同浓缩倍数的浓缩桦树汁。
在一个实施方案中，桦树汁培养基可以桦树汁为唯一底物，不向其中添加任何额外的成分，其中包括不添加任何额外的水，但不排除各组分中固有地包含的水分。
在一个实施方案中，可以根据需要，任选地向桦树汁中添加碳源、氮源、无机盐和/或pH调节剂。
所述碳源是本领域已知的，包括但不限于葡萄糖、甘油、糖蜜、乳糖、甘露糖、麦芽糖、玉米淀粉、蔗糖和果糖等。在本发明中，优选采用葡萄糖。所述碳源在所述桦树汁培养基中的含量通常为约0-3.5％，优选约0.5-3％，基于桦树汁培养基的总重量。
所述氮源是本领域已知的，包括但不限于乳清蛋白、植物蛋白、谷物粉、水解植物多肽、酵母粉和胰蛋白胨等。在本发明中，优选的乳清蛋白为分离乳清蛋白粉，优选的植物蛋白为豌豆粉、水解豌豆蛋白，优选的谷物粉为黑麦粉、水解燕麦粉、水解糙米粉。所述氮源在所述桦树汁培养基中的含量通常为约0-3％，优选约0.3-2.5％，基于桦树汁培养基的总重量。
所述无机盐是本领域已知的，包括但不限于磷酸二氢钾、硫酸镁、硫酸二氢钠和氯化钙等。在本发明中，优选磷酸二氢钾和无水硫酸镁。所述无机盐在所述桦树汁培养基中的含量通常为约0-0.3％，优选约0.05-0.1％，基于桦树汁培养基的总重量。
pH调节剂是本领域已知的，包括但不限于柠檬酸、柠檬酸钠、乳酸、乳酸钠和氢氧化钠等。在本发明中，优选柠檬酸和柠檬酸钠。根据桦树汁原料的初始pH，任选地，使用pH调节剂将桦树汁培养基的pH调节为约5.0-6.5。
在包含碳源、氮源、无机盐和/或pH调节剂的情况下，将这些物质加入桦树汁中即可制备桦树汁培养基。在包含碳源、氮源、无机盐和/或pH调节剂的情况下，桦树汁在所述培养基中的含量为约90％以上，优选约93％以上,基于桦树汁培养基的总重量。
上述步骤(3)接种发酵是本领域已知的。例如，按照约60-70％(v/v)装液量，将步骤(2)的桦树汁培养基加入100-12000L的发酵罐中，121℃灭菌20-40分钟；以发酵培养基体积为准，按照约5-10％的接种量将二级种子液在无菌条件下接入发酵罐中，于约25-30℃的温度、180-250rmp的搅拌转速和1.2-2.0vvm的通气量条件下，连续发酵7-9天，停罐，即完成发酵过程，得到发酵产物。
上述步骤(4)原位热提取发酵产物涉及将步骤(3)中所得发酵产物在发酵罐中原位热提 取，以使桦褐孔菌的胞内多糖和多酚释放到发酵液中。在一个实施方案中，步骤(4)包括将步骤(3)中所得发酵产物加热至约60-95℃，优选约65-90℃，更优选约70-85℃，并保温约30-150分钟，优选约40-100分钟，更优选约50-80分钟；而后冷却至约18-30℃，优选约20-28℃。
上述步骤(5)离心和过滤是本领域已知的，通常在6000-10000rpm下进行15-30分钟。所述离心步骤从发酵产物中去除菌体后得到上清液，过滤所述上清液，即得到所述发酵的桦树汁滤液产物。
本发明的方法可进一步包括将所得滤液产物经超高温瞬时杀菌，杀菌温度(115±5)℃，时间10-30秒。然后，将杀菌过的发酵桦树汁滤液产物转至存储罐存储。
通过上述方法获得的发酵桦树汁滤液产物(也简称为发酵的桦树汁)色浅、透明，不但含有桦树汁底物自身的营养成分(包括B族维生素、微量元素、氨基酸、脂肪酸等)，还含有约100mg/L以上、优选约130mg/L以上、更优选约150mg/L以上的总酚，和约1.3g/L以上、优选1.8g/L以上、更优选约3.0g/L以上的多糖。其中，所述总酚测定方法为福林酚比色法，胞外多糖的测定方法为硫酸蒽酮比色法。其中，所述总酚经检测主要包括没食子酸、阿魏酸、柚皮苷、槲皮素和山奈酚。
本发明的方法相对于现有技术具有如下的优点和效果：
(1)以桦褐孔菌作为发酵出发菌株，以桦树汁为主要发酵底物，所得发酵桦树汁滤液比原料桦树汁增加了总酚、多糖等活性成分，从而使桦树汁具有优良的酪氨酸酶活性抑制率和抗氧化活性，因此具有更好的皮肤外用功效，例如护肤功效，这拓展了发酵桦树汁的应用范围，不再局限于仅仅作为饮料和酒类的应用；
(2)对发酵的桦树汁滤液的处理过程中不使用有机溶剂，绿色环保，可操作性强；
(3)所得发酵桦树汁滤液可不经过浓缩工艺，直接使用，提高原料利用率，缩减生产成本；
(4)对桦树汁培养基的配方进行了优化，使用葡萄糖、植物蛋白等作为桦褐孔菌发酵所需碳氮源，原料易得、成本低，使用优化后的培养基所得发酵滤液色浅透明，比现有技术中报道的桦褐孔菌发酵产物具有更好的性状。
在又一方面，本发明还涉及发酵的桦树汁在皮肤外组合物中的用途，其中所述发酵的桦树汁是采用桦褐孔菌作为菌株、以桦树汁作为底物通过发酵获得的。在优选的实施方案中，所述发酵的桦树汁滤液产物包含100mg/L以上、优选130mg/L以上、更优选150mg/L以上的总酚，和1.3g/L以上、优选1.8g/L以上、更优选3.0g/L以上的多糖。
在再一方面，本发明进一步涉及一种皮肤外用组合物，其包含(A)发酵的桦树汁，其中所述发酵的桦树汁是采用桦褐孔菌作为菌株、以桦树汁作为底物通过发酵获得的。
在优选的实施方案中，所述发酵的桦树汁滤液产物包含100mg/L以上、优选130mg/L以上、更优选150mg/L以上的总酚，和1.3g/L以上、优选1.8g/L以上、更优选3.0g/L以上的多糖。
所述发酵的桦树汁在皮肤外用组合物中的含量可以在宽范围内变化。例如约大于0至小于100％，优选为约80-95％，基于所述皮肤外用组合物的总重量。
所述皮肤外用组合物包括药物组合物或者化妆品组合物，尤其是护肤化妆品组合物、美白化妆品组合物和抗衰老化妆品组合物。
除了所述(A)发酵的桦树汁滤液外，所述皮肤外用组合物还可以任选地包含(B)皮肤外用组合物中常用的成分，其包括但不限于媒介物、活性成分和辅料等。组分(B)是本领域已知的，本领域技术人员可根据需要选择其类型和用量，例如，组分(B)的含量为约2-82％，基于所述皮肤外用组合物的总重量。
所述媒介物包括例如稀释剂、分散剂或载体等，其实例包括但不限于乙醇、双丙甘醇、丁二醇等。所述媒介物在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.5-20％。
所述活性成分包括例如润肤剂、保湿剂、美白活性成分、抗衰老活性成分等。
所述润肤剂的实例包括但不限于橄榄油、澳洲坚果油、甜杏仁油、葡萄籽油、鳄梨油、玉米油、芝麻油、大豆油、花生油、白池花籽油、红花籽油、狗牙蔷薇果油、刺阿干树仁油、霍霍巴籽油、向日葵籽油、毛瑞榈果油、角鲨烷、棕榈酸乙基己酯、肉豆蔻酸异丙酯、氢化聚异丁烯、异十六烷、异十二烷、碳酸二乙基己酯、碳酸二辛酯、月桂酰肌氨酸异丙酯、异壬酸异壬酯、氢化聚癸烯、甘油三(乙基己酸)酯、鲸蜡醇乙基己酸酯、双-二乙氧基二甘醇环己烷1,4-二羧酸酯、辛酸/癸酸甘油三酯、油醇芥酸酯、辛基十二醇肉豆蔻酸酯、辛基十二醇、聚二甲基硅氧烷、辛基聚甲基硅氧烷、鲸蜡基聚二甲基硅氧烷、环五聚二甲基硅氧烷等的一种或多种。固体润肤剂的实例包括但不限于鲸蜡醇、硬脂醇、鲸蜡硬脂醇、山嵛醇、鲨肝醇、月桂酸、肉豆蔻酸、棕榈酸、硬脂酸、蜂蜡、小烛树蜡、巴西棕榈蜡、羊毛脂、地蜡、霍霍巴籽蜡、石蜡、微晶蜡、氢化米糠蜡、氢化椰油甘油酯类、甘油山嵛酸酯/二十酸酯、肉豆蔻醇肉豆蔻酸酯、双-二甘油多酰基己二酸酯-2、牛油果树果脂、木鲁星果棕籽脂等中的一种或多种。所述润肤剂在所述皮肤外用组合物中的含量是本领域已知的，例如其通常占组分(B)总重量的1-50％。
所述保湿剂的实例包括但不限于甘油、双甘油、丁二醇、丙二醇、1,3-丙二醇、双丙甘醇、1,2-戊二醇、聚乙二醇-8、聚乙二醇-32、甲基葡糖醇聚醚-10、甲基葡糖醇聚醚-20、PEG/PPG-17/6共聚物、甘油聚醚-7、甘油聚醚-26、甘油葡糖苷、PPG-10甲基葡糖醚、PPG-20甲基葡糖醚、PEG/PPG/聚丁二醇-8/5/3甘油、蔗糖、海藻糖、鼠李糖、甘露糖、棉子糖、甜菜碱、赤藓醇、木糖醇、尿素、甘油聚醚-5乳酸酯、透明质酸钠、水解透明质酸钠、乙酰化透明质酸钠、聚谷氨酸钠、水解小核菌胶、出芽短梗酶多糖、银耳多糖、酸豆籽多糖等中的一种或多种。所述保湿剂在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的1-30％。
所述美白活性成分包括但不限于曲酸、抗坏血酸葡糖苷、熊果苷、传明酸、烟酰胺、植物甾醇、植物甾醇/山嵛醇/辛基癸醇月桂酰谷氨酸酯、苯乙基间苯二酚、姜黄根提取物、白桦树皮提取物、神经酰胺2、神经酰胺3、乙酰植物鞘氨醇、白藜芦醇、花榈木树皮提取物、毛喉鞘蕊花根提取物、胡椒籽提取物、泛醌、胆甾醇、胆甾醇硬脂酸酯、抗坏血酸、抗坏血酸二棕榈酸酯、生育酚(维生素E)、生育酚乙酸酯、红没药醇、抗坏血酸四异棕榈 酸酯、吡哆素二辛酸酯、吡哆素二棕榈酸酯、视黄醇棕榈酸酯、植物甾醇/辛基十二醇月桂酰谷氨酸酯、双-山嵛醇/异硬脂醇/植物甾醇二聚亚油醇二聚亚油酸酯、植物甾醇澳洲坚果油酸酯、各种肽类、各种植物提取物等中的一种或多种。所述美白活性成分在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.01-30％。
所述抗衰老活性成分的实例包括但不限于生育酚(维生素E)、视黄醇、视黄醇棕榈酸酯、水解胶原、水解弹性蛋白、尿囊素、酵母提取物、谷维素、四氢姜黄素、鞣花酸、泛醌、乳清蛋白、多肽、乙酰基六肽-8、棕榈酰五肽-4、水杨酰植物鞘氨醇、浓缩桦树汁、水飞蓟素、蚕丝胶蛋白、生育酚磷酸酯钠、核糖核酸(RNA)、二肽二氨基丁酰苄基酰胺二乙酸盐、棕榈酰三肽-5、寡肽-1、六肽-9、棕榈酰寡肽、棕榈酰四肽-7、葡萄(VITIS VINIFERA)籽提取物、花榈木(PTEROCARPUS MARSUPIUM)树皮提取物、茶(CAMELLIA SINENSIS)多酚、葡萄酒提取物、苹果籽提取物、欧洲水青冈(FAGUS SYLVATICA)芽提取物、水解猴面包树(ADANSONIA DIGITATA)提取物、卤虫(ARTEMIA)提取物、香根鸢尾(IRIS FLORENTINA)根提取物、橙皮苷、人参皂甙、丹参(SALVIA MILTIORRHIZA)提取物、烟酰胺、熊果酸、透明质酸钠、乙酰化透明质酸钠、水解透明质酸钠、番茄红素、咖啡(COFFEA ARABICA)提取物、二肽-2、乳酸、超氧化物歧化酶(SOD)、月见草(OENOTHERA BIENNIS)油、神经酰胺、二棕榈酰羟脯氨酸、羟基硬脂酸、水杨酸、麦角硫因、溶血卵磷脂、肌肽、脱羧肌肽HCL、硫辛酸、腺苷、糖原、白藜芦醇、阿魏酸、二裂酵母发酵产物溶胞物、乳酸菌发酵溶胞产物等中的一种或多种。所述抗衰老活性成分在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.01-10％。
所述辅料包括例如乳化剂、增稠剂、防腐剂、香料等。
所述乳化剂的实例包括但不限于鲸蜡硬脂醇橄榄油酸酯、山梨坦橄榄油酸酯、聚山梨醇酯-60、聚山梨醇酯-80、甲基葡糖倍半硬脂酸酯、PEG-20甲基葡糖倍半硬脂酸酯、PEG-40氢化蓖麻油、PPG-26-丁醇聚醚-26、PEG-4聚甘油-2硬脂酸酯、PEG-60氢化蓖麻油、硬脂醇聚醚-2、硬脂醇聚醚-21、PPG-13-癸基十四醇聚醚-24、鲸蜡硬脂基葡糖苷、PEG-100硬脂酸酯、甘油硬脂酸酯、甘油硬脂酸酯SE、椰油基葡糖苷、鲸蜡硬脂醇聚醚-25、PEG-40硬脂酸酯、聚甘油-3甲基葡糖二硬脂酸酯、甘油硬脂酸酯柠檬酸酯、聚甘油-10硬脂酸酯、聚甘油-10肉豆蔻酸酯、聚甘油-10二油酸酯、聚甘油-10月桂酸酯、聚甘油-10异硬脂酸酯、聚甘油-10油酸酯、聚甘油-10二异硬脂酸酯、聚甘油-6月桂酸酯、聚甘油-6肉豆蔻酸酯、蔗糖硬脂酸酯、蔗糖多硬脂酸酯等中的一种或多种。所述乳化剂在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.5-10％。
所述增稠剂的实例包括但不限于卡波姆类、丙烯酸(酯)类及其衍生物、黄原胶、阿拉伯胶、聚乙二醇-14M、聚乙二醇-90M、琥珀酰聚糖、羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素等高分子聚合物中的一种或多种。所述增稠剂在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.1-10％。
所述防腐剂的实例包括但不限于羟苯甲酯、羟苯丙酯、苯氧乙醇、苯甲醇、苯乙醇、 双(羟甲基)咪唑烷基脲、山梨酸钾、苯甲酸钠、氯苯甘醚、脱氢乙酸钠、辛酰羟肟酸、1,2-己二醇、1,2-戊二醇、对羟基苯乙酮、辛甘醇、甘油辛酸酯、十一碳烯酸甘油酯、山梨坦辛酸酯、乙基己基甘油、牡丹根提取物等中的一种或多种。所述防腐剂在所述皮肤外用组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.01-2％。
可根据皮肤外用组合物(药物组合物或化妆品组合物)工业中已知的任何方法，将(A)所述发酵的桦树汁与其它药物成分或者化妆品成分混合，得到药物组合物或者化妆品组合物。其中所述其它的药物成分或者化妆品成分如上所述为(B)皮肤外用组合物中常用的成分。
根据需要，可以将所述皮肤外用组合物可制成各种剂型，例如溶液、悬浮液、膏、霜、乳液、凝胶、粉末或喷剂等。
实施例
以下结合实施例，对本发明进行进一步详细说明。但是，应当理解为，这些实施例、对比例仅仅是用于更详细地说明本发明，而不应理解为用于以任何形式限制本发明所附权利要求书的范围。
在下述实施例中，实施例1-5中采用的桦褐孔菌(Inonotus obliquus)菌株保藏于中国林业微生物菌种保藏管理中心(菌株编码cfcc 6584)。对比例5-8中采用的酵母(Saccharomyces cerevisiae)菌株来自安琪酵母股份有限公司(菌株编号ANGEL 1021)。
实施例1：制备发酵的桦树汁滤液
(1)种子液的制备
从固体PDA培养基平板上刮下桦褐孔菌，接种到装有50mL种子培养基的摇瓶中，培养温度28℃，摇床转速150rpm，培养4天，得到一级种子液；在无菌条件下，按照10％的接种量，将一级种子液转接到装有400mL种子培养基的摇瓶中，在28℃和150rpm条件下，恒温培养2天，得到二级种子液；其中种子培养基的配方包括1000mL去离子水、3％葡萄糖、1％燕麦胚芽麸粉、1％酵母粉、0.1％磷酸二氢钾和0.05％无水硫酸镁，自然pH；
(2)桦树汁培养基的制备
以东北大兴安岭采集的桦树汁原液(brix 0.875)为底物，向其中添加2％葡萄糖作为碳源，添加0.3％乳清蛋白(WPI90，购自新西兰恒天然集团)作为氮源，添加0.1％磷酸二氢钾和0.05％无水硫酸镁，使用1M柠檬酸水溶液调整桦树汁培养基的pH为5.5；
(3)接种发酵
按照60％(v/v)体积装液量，将步骤(2)所配制的桦树汁培养基加入到300L发酵罐中，121℃灭菌30分钟；以发酵培养基的体积为准，按照5％的接种量将二级种子液在无菌条件下接入发酵罐中，于30℃、200rmp和1.8vvm通气量的条件下，连续发酵7天，停罐，得到发酵产物；
(4)发酵产物滤液的制备
将步骤(3)所得发酵产物在罐中原位加热至90℃，保温60分钟，而后冷却至28℃，然 后离心去除菌体，其中转速为6000rpm和时间为30分钟；使用硅藻土板框将离心所得上清液过滤，即得到桦褐孔菌发酵的桦树汁滤液；进一步将所得滤液经超高温瞬时杀菌机杀菌，其中杀菌温度为120℃和时间为30秒，将杀菌过的发酵桦树汁滤液转至存储罐存储；
(5)取发酵桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
实施例2：制备发酵的桦树汁滤液
(1)种子液的制备
种子液制备过程与实施例1相同；
(2)桦树汁培养基的制备
以芬兰采集的桦树汁原液(brix 1.3)为底物，向其中添加1.5％葡萄糖作为碳源，添加1％豌豆粉(购自嘉吉粮油(南通)有限公司)作为氮源，添加0.1％磷酸二氢钾和0.05％无水硫酸镁，使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为5.8；
(3)接种发酵
按照65％(v/v)体积装液量，将步骤(2)所配制的桦树汁培养基加入到100L发酵罐中，121℃灭菌30分钟；以发酵培养基的体积为准，按照7％的接种量将二级种子液在无菌条件下接入发酵罐中，于30℃、200rmp和1.6vvm的通气量条件下，连续发酵7天，停罐，得到发酵产物；
(4)发酵产物滤液的制备
将步骤(3)所得发酵产物在罐中原位加热至80℃，保温90分钟，而后冷却至25℃，然后与实施例1相同地进行离心、过滤和热杀菌步骤；
(5)取发酵桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
实施例3：制备发酵的桦树汁滤液
(1)种子液的制备
种子液的制备方法与实施例1相同；
(2)桦树汁培养基的制备
以来自东北小兴安岭的桦树汁浓缩汁(浓缩3倍，brix 3.58)为底物，向其中添加0.5％葡萄糖作为碳源，添加3％水解燕麦蛋白(购自厦门格兰贝尔生物科技有限公司)作为氮源，添加0.1％磷酸二氢钾和0.05％无水硫酸镁，使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为6.0；
(3)接种发酵
按照65％(v/v)体积装液量，将步骤(2)所配制的桦树汁培养基加入500L发酵罐中，121℃灭菌30分钟；以发酵培养基体积为准，按照10％的接种量将二级种子液在无菌条件下接入发酵罐中，于30℃、220rmp和1.8vvm通气量的条件下，连续发酵7天，停罐，得到发酵产物；
(4)发酵产物滤液的制备
将步骤(3)所得发酵产物在罐中原位加热至65℃，保温120分钟，而后冷却至28℃，然后与实施例1相同地进行离心、过滤和热杀菌步骤；
(5)取发酵桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
实施例4：制备发酵的桦树汁滤液
(1)种子液的制备
种子液的制备方法与实施例1相同；
(2)桦树汁培养基的制备
来自东北小兴安岭的桦树汁浓缩汁(浓缩1.5倍，brix 1.8)为底物，向其中添加1.5％葡萄糖作为碳源，添加3％水解豌豆蛋白(购自罗盖特(中国)精细化工有限公司)作为氮源，添加0.1％磷酸二氢钾和0.05％无水硫酸镁，使用1M柠檬酸钠水溶液调整桦树汁培养基的pH为6.0；
(3)接种发酵
接种发酵的过程与实施例1相同；
(4)发酵产物滤液的制备
不经过罐内热提取工艺，直接将步骤(3)所得发酵产物进行与实施例1相同的离心、过滤和热杀菌步骤；
(5)取发酵桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
实施例5：制备发酵的桦树汁滤液
(1)种子液的制备
种子液的制备方法与实施例1相同；
(2)桦树汁培养基的制备
以实施例3中的东北小兴安岭的桦树汁浓缩汁(浓缩3倍，brix3.58)为原料，不添加碳源、氮源、无机盐和pH调节剂；
(3)接种发酵
接种发酵的过程与实施例3相同；
(4)发酵产物滤液的制备
发酵桦树汁滤液的制备过程与实施例3相同；
(5)取发酵桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例1：制备未发酵的桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例1相同；
(2)未发酵的桦树汁滤液的制备
不对桦树汁培养基接种发酵，未发酵的桦树汁滤液的制备方法与实施例1中的步骤(4)相同；
(3)取未发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例2：制备未发酵的桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例2相同；
(2)未发酵的桦树汁滤液的制备
不对桦树汁培养基接种发酵，未发酵的桦树汁滤液的制备方法与实施例2中的步骤(4)相同；
(3)取未发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例3：制备未发酵的桦树汁滤液
(1)桦树汁培养基的制备与灭菌
桦树汁培养基的制备方法与实施例3相同；
(2)未发酵的桦树汁滤液的制备
不对桦树汁培养基接种发酵，未发酵的桦树汁滤液的制备方法与实施例3中的步骤(4)相同；
(3)取未发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例4：制备未发酵的桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例4相同；
(2)未发酵的桦树汁滤液的制备
不对桦树汁培养基接种发酵，未发酵的桦树汁滤液的制备方法与实施例4中的步骤(4)相同；
(3)取未发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例5：制备酵母发酵的桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例1相同；
(2)酵母发酵桦树汁及其滤液的制备
按照30g/HL(百升)的添加量称取90g酵母冻干粉，添加到1L无菌生理盐水中分散均匀，得到酵母菌种子液；然后将酵母种子液接种到300L发酵罐中，于30℃、200rmp和1.2vvm通气量的条件下，连续发酵48h，停罐，得到酵母发酵产物；然后以与实施例1的步骤(4)相同的方式获得酵母发酵的桦树汁滤液；
(3)取酵母发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例6：制备酵母发酵的桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例2相同；
(2)酵母发酵桦树汁及其滤液的制备
按照30g/HL(百升)的添加量称取30g酵母冻干粉，添加到350mL无菌生理盐水中分散均匀，得到酵母菌种子液；然后将酵母种子液接种到100L发酵罐中，于30℃、180rmp和1.2vvm通气量的条件下，连续发酵48h，停罐，得到酵母发酵产物；然后以与实施例2的步骤(4)相同的方式获得酵母发酵的桦树汁滤液；
(3)取酵母发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例7：制备酵母发酵的桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例3相同；
(2)酵母发酵桦树汁及其滤液的制备
按照30g/HL(百升)的添加量称取150g酵母冻干粉，添加到1.8L无菌生理盐水中分散均匀，得到酵母菌种子液；然后将酵母种子液接种到500L发酵罐中，于30℃、200rmp和1.2vvm通气量的条件下，连续发酵48h，停罐，得到酵母发酵产物；然后以与实施例3的步骤(4)相同的方式获得酵母发酵的桦树汁滤液；
(3)取酵母发酵桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
对比例8：制备酵母发酵桦树汁滤液
(1)桦树汁培养基的制备
桦树汁培养基的制备与灭菌方法与实施例4相同；
(2)酵母发酵桦树汁及其滤液的制备
按照30g/HL(百升)的添加量称取90g酵母冻干粉，添加到1L无菌生理盐水中分散均匀，得到酵母菌种子液；然后将酵母种子液接种到300L发酵罐中，于30℃、180rmp和1.2vvm通气量的条件下，连续发酵48h，停罐，得到酵母发酵产物；然后以与实施例4的步骤(4)相同的方式获得酵母发酵的桦树汁滤液；
(3)取酵母发酵的桦树汁滤液，测定其中的总酚和多糖含量，结果如表1所示。
表1:实施例1-5中获得的发酵的桦树汁滤液和对比例1-8中获得的桦树汁滤液的总酚和多糖含量
Figure PCTCN2020073351-appb-000001
上述结果表明，与未发酵的桦树汁滤液和采用常规酵母菌发酵的桦树汁滤液相比，采用桦褐孔菌生产的发酵的桦树汁滤液产物含有极其显著高的总酚和多糖含量，这使得所述产物的活性大大提升，从而赋予了其可在皮肤外用组合物中应用的可能。
实施例6：发酵桦树汁滤液的美白功效测试
在该实施例中，根据酪氨酸酶活性抑制率，评价实施例1-5中获得的发酵桦树汁滤液和对比例1-8中获得的桦树汁滤液的美白功效，具体如下。
(1)酪氨酸酶活性抑制率评价原理：酪氨酸酶(EC.1.14.18.1)是生物体合成黑色素的关键酶，催化L-酪氨酸羟基化形成L-多巴，再氧化L-多巴形成多巴醌，多巴醌经一系列的酶促和非酶促反应后，形成黑色素。因此通过抑制酪氨酸酶的活性可以调控生物体黑色素的生成量。
(2)试剂：
(a)PBS缓冲液(0.2M，pH6.8)：量取51mL0.2M的磷酸二氢钠溶液与49mL0.2M的磷酸氢二钠溶液，混匀；
(b)L-酪氨酸(1.5mM)：称取L-酪氨酸0.0272g，用上述PBS缓冲液预分散，超声30分钟，转移至100mL容量瓶，用PBS缓冲液加至刻度；
(c)酪氨酸酶(250U)：将酪氨酸酶(购自sigma aldrich)溶解于上述PBS缓冲液中，转移至100mL容量瓶中，用PBS缓冲液定容到刻度，配置成250U/mL的使用液。
(3)实验方法：取A、B、C、D四只离心管，分别加入L-酪氨酸0.25mL，PBS0.25mL。B和D管中另加入0.25mL样品溶液。A和C管另中加入0.25mL PBS溶液。将4支管涡旋混匀，37℃恒温10分钟后，于C和D管中加入0.25mL酶液，A和B管加入相同体积的PBS缓冲液补足测试样体积。将四只管子置于37℃继续保温反应20分钟。取200微升上述试液加入96孔板中，用酶标仪检测475nm处吸光值。
(4)酪氨酸活性抑制率计算：
酪氨酸酶活性抑制率％＝((C-A)-(D-B))/(C-A)*100％
得到的结果如下表2所示。
表2：实施例1-5中获得的发酵桦树汁滤液和对比例1-8中获得的桦树汁滤液的酪氨酸酶活性抑制率对比
Figure PCTCN2020073351-appb-000002
上述结果表明，与未发酵的桦树汁滤液和采用常规酵母菌发酵的桦树汁滤液相比，采 用桦褐孔菌生产的发酵的桦树汁滤液产物的酪氨酸酶活性抑制率极其显著地提高，这说明所述产物的美白功效显著，从而使得其可应用于具有美白功效的皮肤外用组合物，尤其是美白化妆品组合物。
实施例7：发酵桦树汁滤液的抗氧化功效测试
在该实施例中，根据羟基自由基清除率和DPPH自由基清除率，评价实施例1-5中获得的发酵桦树汁滤液和对比例1-8中获得的桦树汁滤液的的抗氧化功效，具体如下。
1.羟基自由基清除率
(1)测定原理：已知FeCl 3-EDTA的混合物、过氧化氢和抗坏血酸三者在pH7.4条件下会反应产生羟自由基，羟自由基可以降解脱氧核糖形成丙二醛；在低pH下，产物丙二醛(MDA)和硫代巴比妥酸(TBA)加热产生粉红色发色团，依据粉红色发色团的含量可以判断羟自由基含量。
(2)测定方法：配制三个组别的反应试剂，包括(A)75μL 0.8mM FeCl 3+75μL 4mM EDTA+600μL 4mM KH 2PO 4-KOH缓冲液+2.0565mL蒸馏水+43.5μL3％H 2O 2+75μL 112mM脱氧核糖+75μL 4mM抗坏血酸；(B)75μL 0.8mM FeCl 3+75μL 4mM EDTA+600μL 4mM KH 2PO 4-KOH缓冲液+2.0565mL样品液+43.5μL 3％H 2O 2+75μL 112mM脱氧核糖+75μL 4mM抗坏血酸；(C)75μL 0.8mM FeCl 3+75μL 4mM EDTA+600μL4 mM KH 2PO 4-KOH缓冲液+2.0565mL样品液+43.5μL 3％H 2O 2+75μL 4mM抗坏血酸。将各组别反应混合物在37℃下恒温水浴培育1h，而后向混合液中加入2mL0.6％TBA，沸水浴15分钟，迅速冷却后离心，测上清液于450nm，532nm，600nm下的吸光值。
(3)计算方法
羟基自由基清除率(％)＝[1-(B-C)/A]*100％
2.DPPH自由基清除率
(1)测定原理：DPPH自由基是单电子自由基，其在517nm处有特征吸收，自由基清除剂能够与DPPH自由基的单电子配对而使其在517nm处的吸收消失，其褪色程度与其接受的电子数量成定量关系，据此推断自由基清除剂的清除能力。
(2)测定方法：配制三个组别的反应试剂并混合，包括(A)1mL蒸馏水+3mL 0.1mM DPPH反应液；(B)1mL样品液+3mL0.1mM DPPH反应液；(C)1mL样品液液+3mL乙醇。各组别充分摇匀后于室温下避光反应30分钟，而后测定其在517nm处的吸光值。
(3)计算方法
DPPH自由基清除能力(％)＝[1-(B-C)/A]＊100％
得到的结果如下表3所示。
表3:实施例1-5中获得的发酵桦树汁滤液和对比例1-8中获得的桦树汁滤液的羟基自由基清除率和DPPH自由基清除率
项目 羟基自由基清除率 DPPH自由基清除率
实施例1 54.7％ 72.5％
实施例2 48.8％ 70.8％
实施例3 52.1％％ 71.5％
实施例4 50.5％ 69.0％
实施例5 40.7％ 45.1％
对比例1 7.0％ 11.2％
对比例2 5.4％ 10.4％
对比例3 5.3％ 8.3％
对比例4 6.1％ 8.4％
对比例5 6.4％ 12.0％
对比例6 5.5％ 9.7％
对比例7 5.7％ 8.7％
对比例8 3.3％ 9.0％
上述结果表明，与未发酵的桦树汁滤液和采用常规酵母菌发酵的桦树汁滤液相比，采用桦褐孔菌生产的发酵的桦树汁滤液产物的羟基自由基清除率和DPPH自由基清除率极其显著地提高，这说明所述产物的抗氧化功效显著，从而使得其可应用于具有抗氧化功效的皮肤外用组合物，尤其是抗氧化的化妆品组合物。
实施例8：美白化妆水的制备
在该实施例中，采用实施例1制备的发酵桦树汁滤液来制备美白化妆水，其配方如下：
成分 含量(质量％)
发酵桦树汁滤液 95.65
苯甲酸钠 0.35
1,2-戊二醇 4
所述化妆水的制备方法为：将所述发酵的桦树汁滤液、苯甲酸钠和戊二醇混合后过滤即得。
实施例9：抗氧化面霜的制备
在该实施例中，采用实施例3中制备的发酵桦树汁滤液来制备面霜，其配方如下：
成分 含量(质量％)
发酵桦树汁滤液 83.45
辛酸癸酸甘油三酯 3
乳木果油 5
C16醇 2
聚乙二醇醚混合物 3
甲基异噻唑啉酮 0.1
氨基酸保湿剂 3.0
苯甲酸钠 0.3
香精 0.15
上述面霜如下制备：将上述成分中的油相成分和水相成分分别混合，加热到75℃，分别制成油相和水相；而后将所得油相和水相加入真空均质机中均质乳化，冷却到45℃，加入香精、苯甲酸钠，搅拌均匀即得。
以上所述实施例的技术方案是本发明优选实施方式，在不脱离本发明原理的前提下还可以进行若干改进和变换，这些改进和变化也应视为在本发明的保护范围内。

Claims (14)

  1. 一种发酵的桦树汁，其是采用桦褐孔菌作为菌株、以桦树汁作为底物通过发酵获得的。
  2. 权利要求1的发酵的桦树汁，其含有100mg/L以上、优选130mg/L以上、更优选150mg/L以上的总酚，和1.3g/L以上、优选1.8g/L以上、更优选3.0g/L以上的多糖。
  3. 一种发酵的桦树汁在皮肤外组合物中的用途，其中所述发酵的桦树汁是采用桦褐孔菌作为菌株、以桦树汁作为底物通过发酵获得的。
  4. 权利要求3的用途，其中所述发酵的桦树汁含有100mg/L以上、优选130mg/L以上、更优选150mg/L以上的总酚，和1.3g/L以上、优选1.8g/L以上、更优选3.0g/L以上的多糖。
  5. 一种皮肤外用组合物，其包含(A)发酵的桦树汁，其中所述发酵的桦树汁是采用桦褐孔菌作为菌株、以桦树汁作为底物通过发酵获得的。
  6. 权利要求5的皮肤外用组合物，其中所述发酵的桦树汁含有100mg/L以上、优选130mg/L以上、更优选150mg/L以上的总酚，和1.3g/L以上、优选1.8g/L以上、更优选3.0g/L以上的多糖。
  7. 权利要求5或6的皮肤外用组合物，其中发酵的桦树汁在所述皮肤外用组合物中的含量为大于0至小于100％，优选为80-95％，基于所述皮肤外用组合物的总重量。
  8. 权利要求5-7任一项的皮肤外用组合物，其是护肤化妆品组合物、美白化妆品组合物或抗衰老化妆品组合物。
  9. 一种生产发酵的桦树汁的方法，其包括采用桦褐孔菌作为菌株、以桦树汁作为底物进行发酵的步骤。
  10. 权利要求9的方法，其中所述方法还包括在发酵罐中原位热提取在发酵步骤中得到的发酵产物的步骤。
  11. 权利要求10的方法，其中所述原位热提取步骤包括将所述发酵产物加热至60-95℃，优选65-90℃，更优选70-85℃，并保温30-150分钟，优选40-100分钟，更优选50-80分钟；而后冷却至18-30℃，优选20-28℃。
  12. 权利要求9-11任一项的方法，其中在发酵步骤中，以桦树汁为唯一底物，不向其中添加任何额外的成分。
  13. 权利要求9-11任一项的方法，其中在发酵步骤中，向桦树汁中添加碳源、氮源、无机盐和/或pH调节剂；在这种情况下，桦树汁的含量为90％以上，优选93％以上,基于发酵步骤中采用的培养基的总重量。
  14. 权利要求9-13任一项的方法，其中所述方法包括下述步骤：
    (1)制备种子液，
    (2)制备桦树汁培养基，
    (3)接种发酵，得到发酵产物，
    (4)原位热提取所述发酵产物,和
    (5)离心除去菌体，过滤上清液，得到发酵的桦树汁滤液产物。
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