CN115181678A - Preparation method and application of inonotus obliquus fermentation lysate - Google Patents
Preparation method and application of inonotus obliquus fermentation lysate Download PDFInfo
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- CN115181678A CN115181678A CN202211011140.XA CN202211011140A CN115181678A CN 115181678 A CN115181678 A CN 115181678A CN 202211011140 A CN202211011140 A CN 202211011140A CN 115181678 A CN115181678 A CN 115181678A
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- inonotus obliquus
- bark
- extract
- fermentation
- powder
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- Granted
Links
- 241000414067 Inonotus obliquus Species 0.000 title claims abstract description 112
- 238000000855 fermentation Methods 0.000 title claims abstract description 46
- 230000004151 fermentation Effects 0.000 title claims abstract description 46
- 239000006166 lysate Substances 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 239000000284 extract Substances 0.000 claims abstract description 67
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000002131 composite material Substances 0.000 claims abstract description 29
- 238000001914 filtration Methods 0.000 claims abstract description 21
- 239000008367 deionised water Substances 0.000 claims abstract description 20
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 17
- 238000012258 culturing Methods 0.000 claims abstract description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 241001052560 Thallis Species 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 8
- 238000013329 compounding Methods 0.000 claims abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 4
- 239000012528 membrane Substances 0.000 claims abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- 238000010992 reflux Methods 0.000 claims abstract description 4
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- 239000003102 growth factor Substances 0.000 claims abstract description 3
- 239000000843 powder Substances 0.000 claims description 42
- 235000018185 Betula X alpestris Nutrition 0.000 claims description 27
- 235000018212 Betula X uliginosa Nutrition 0.000 claims description 27
- 241000124033 Salix Species 0.000 claims description 25
- 241001106462 Ulmus Species 0.000 claims description 25
- 235000019441 ethanol Nutrition 0.000 claims description 20
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
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- 239000002537 cosmetic Substances 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 230000003078 antioxidant effect Effects 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 4
- 230000002087 whitening effect Effects 0.000 claims description 4
- 239000003963 antioxidant agent Substances 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 229940015975 1,2-hexanediol Drugs 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000000469 ethanolic extract Substances 0.000 claims description 2
- FHKSXSQHXQEMOK-UHFFFAOYSA-N hexane-1,2-diol Chemical compound CCCCC(O)CO FHKSXSQHXQEMOK-UHFFFAOYSA-N 0.000 claims description 2
- WCVRQHFDJLLWFE-UHFFFAOYSA-N pentane-1,2-diol Chemical compound CCCC(O)CO WCVRQHFDJLLWFE-UHFFFAOYSA-N 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 abstract description 5
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 4
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- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 4
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 4
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- 239000008055 phosphate buffer solution Substances 0.000 description 4
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 4
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
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- 241000233866 Fungi Species 0.000 description 2
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
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- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
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- 235000012141 vanillin Nutrition 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
-
- 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
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- 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
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- 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
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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
- 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
-
- 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
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- 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
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- General Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
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- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Gerontology & Geriatric Medicine (AREA)
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Abstract
The invention discloses a preparation method and application of inonotus obliquus fermentation lysate, and belongs to the technical field of microbial fermentation. The preparation method of the inonotus obliquus fermentation lysate mainly comprises the following steps: (1) Fermenting and culturing inonotus obliquus to obtain inonotus obliquus fermentation liquor; wherein the fermentation medium comprises composite bark extract, carbon source, nitrogen source and growth factor; (2) Filtering the inonotus obliquus fermentation liquor, collecting thalli, and drying; (3) Mixing the dried thallus with water, breaking cell wall, adding into anhydrous ethanol solution, extracting, filtering, reflux concentrating to obtain Inonotus obliquus ethanol extractive solution; (4) Compounding the inonotus obliquus alcohol extract with deionized water and dihydric alcohol, and filtering with a microporous filter membrane to obtain an inonotus obliquus fermentation lysate. Compared with wild inonotus obliquus, the inonotus obliquus lysate obtained by the microbial fermentation method is lower in cost and more stable in product.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a preparation method and application of a fuscoporia obliqua fermentation lysate.
Background
Inonotus obliquus (Inonotus) is a medicinal fungus growing on birch, the fruiting body of the fungus presents black block shape similar to carbon, and wild inonotus obliquus mainly grows in Siberian area of Russia, northern Hakkai of Japan, finland and Changbai mountain area of Jilin of China and in big and small Khingan areas of Heilongjiang river. The inonotus obliquus has complex components, and the main components of the inonotus obliquus comprise polysaccharide, triterpenoid, sterol, alkaloid and the like, and a large number of pharmacological experiments show that the inonotus obliquus has the effects of resisting oxidation, regulating blood fat, resisting cancer, treating diabetes, resisting virus, resisting aging, enhancing immunity and the like. In addition, inonotus obliquus has certain therapeutic effects on emesis, diarrhea, gastrointestinal dysfunction, gastric and duodenal ulcer, hepatitis, gastritis and nephritis. Has certain prevention and treatment effects on diseases of sclerotin caused by unreasonable diet.
The inonotus obliquus has a certain application in foods, health-care products and medicines, but in the field of daily chemical products, the main source of the inonotus obliquus is an inonotus obliquus extract obtained by physical and chemical extraction, the inonotus obliquus is mostly applied to the fields of foods and health-care products, and research reports in the field of cosmetics are few.
With the cognition of people on the inonotus obliquus, the demand of the inonotus obliquus is increased year by year, and the supply of wild inonotus obliquus is insufficient. In addition, the natural wild inonotus obliquus belongs to rare resources, is expensive and unstable in source, and has great product difference in different regions, so that the product quality is difficult to stabilize. Therefore, it is expected that the increasing demand of inonotus obliquus is satisfied by artificial culture of inonotus obliquus, wherein the liquid fermentation technology for producing inonotus obliquus is more concerned in recent years because it is easier to realize mass production than artificial cultivation.
Disclosure of Invention
The invention aims to provide a preparation method and application of a inonotus obliquus fermentation lysate, which aim to solve the problems in the prior art, and improve the yield and the content of active ingredients compared with the traditional fermentation process; the inonotus obliquus lysate obtained by the method has better cosmetic antioxidation and whitening effects.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a strain of Inonotusobliquus, which is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 28 days in 2021, wherein the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No.23897.
The invention also provides a method for preparing a fermentation lysate by using the inonotus obliquus, which comprises the following steps:
(1) Fermenting and culturing inonotus obliquus to obtain inonotus obliquus fermentation liquor; wherein the fermentation medium comprises composite bark extract, carbon source, nitrogen source and growth factor;
(2) Filtering the inonotus obliquus fermentation liquor, collecting thalli, and drying;
(3) Mixing the dried thallus with water, breaking cell wall, adding into anhydrous ethanol solution, extracting, filtering, reflux concentrating to obtain Inonotus obliquus ethanol extractive solution;
(4) Compounding the inonotus obliquus alcohol extract with deionized water and dihydric alcohol, and filtering with a microporous filter membrane to obtain an inonotus obliquus fermentation lysate.
Preferably, before fermentation culture, the method further comprises seed liquid culture, wherein a seed culture medium for obtaining the seed liquid comprises the following components in percentage by weight: 2-3wt% of glucose, 0.1-0.5wt% of yeast extract, 0.1-0.5wt% of peptone, 0.02-0.2wt% of monopotassium phosphate, 0.01-0.1% of magnesium sulfate and the balance of water;
the seed liquid culture conditions are as follows: the temperature is 26-34 deg.C, the culture time is 2-4 days, and the rotation speed of shaking table is 130-180 r/min.
Preferably, in step (1), the fermentation medium comprises the following components in percentage by weight: 60-90wt% of composite bark extract, 3-5wt% of glucose, 0.1-0.5wt% of yeast extract, 0.1-0.5wt% of peptone, 0.02-0.2wt% of potassium dihydrogen phosphate, 0.01-0.1wt% of magnesium sulfate and the balance of water;
the fermentation culture conditions are as follows: culturing at 26-34 deg.C for 3-6 days at 200-400rpm with ventilation amount of 0.5-3vvm and pH of 6-7; taking the glucose content in the inonotus obliquus fermentation liquor less than 2g/L as a culture end point.
Preferably, the composite bark extract is prepared by the following method: respectively crushing birch bark, willow bark and elm bark into powder, and sieving with a 100-mesh sieve for later use; adding birch bark powder, willow bark powder and elm bark powder into deionized water, heating at 60-90 deg.C for 1-4 hr, filtering to obtain clear liquid to obtain composite bark extract; the mass ratio of the birch bark powder to the willow bark powder to the elm bark powder is (5); the mass ratio of the total mass of the birch bark powder, the willow bark powder and the elm bark powder to the deionized water is 1.
Preferably, the composite bark extract is prepared by the following method: respectively crushing birch bark, willow bark and elm bark into powder, and sieving with a 100-mesh sieve for later use; respectively mixing the birch bark powder, the willow bark powder and the elm bark powder according to the mass ratio of 1:100 adding into deionized water, heating at 60-90 deg.C for 1-4 hr, filtering to obtain clear liquid, respectively to obtain birch bark extract, willow bark extract and elm bark extract, and mixing to obtain composite bark extract; the mass ratio of the birch bark extract to the willow bark extract to the elm bark extract is (5).
Preferably, in the step (2), the bacteria collection mode is centrifugation, the rotating speed is 1500-8000rpm, and the time is 20-40min; the drying mode is drying.
Preferably, in step (3), the mass ratio of the cells to water is 1; the wall breaking method is 100-140mpa high pressure wall breaking; the extraction conditions are as follows: the extraction temperature is 50-65 ℃, and the extraction time is 1-3h; the concentration temperature is 55-65 ℃; the mass of the inonotus obliquus alcohol extract is 1.5 times of that of the thalli.
Preferably, in the step (4), the mass ratio of the inonotus obliquus ethanol extract to the deionized water to the dihydric alcohol is 10; the dihydric alcohol is 1, 2-pentanediol or 1, 2-hexanediol; the filtration precision is 0.22 μm, 0.45 μm or 0.9. Mu.m.
The invention also provides application of the inonotus obliquus in preparation of cosmetics with antioxidant and/or whitening effects.
The invention discloses the following technical effects:
compared with the prior art, the preparation method of the inonotus obliquus fermentation lysate provided by the invention has the advantages that:
(1) Compared with the planting technology adopted by the inonotus obliquus culture, the microbial fermentation method is greener, has less pollution, has no problems of pesticide or solvent residue and the like, and has high safety;
(2) Compared with wild inonotus obliquus, the cost for culturing the inonotus obliquus by a microbial fermentation method is lower, and the product is more stable;
(3) Compared with the existing fermentation process, the method adopts a plurality of bark phases to be compounded as the components of the culture medium of the inonotus obliquus, so that the growth amount of thalli can be increased, the thalli has more biomass and better active matter content, and the inonotus obliquus has better antioxidant and whitening activities of cosmetics.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
In the following description, all methods involved are conventional in the art unless otherwise specified. All the materials referred to are those which are commercially available from the public unless otherwise specified.
Example 1 isolation of Fuscoporia obliqua YZS10101
1. Separating and purifying
Selecting clean and fresh inonotus obliquus fruiting bodies produced from Changbai mountain valley land of mountain city of Jilin province, and blowing off impurities such as dust on the surfaces by using dry nitrogen. In the sterile area of alcohol burner flame of sterile operation table, cutting several fruiting body blocks with wheat grain size from the fruiting body with sterilized scalpel, inoculating into slant test tube containing PDA culture medium, embedding the fruiting body blocks as much as possible under the surface layer of the culture medium, separating 5 test tubes from each fruiting body, and numbering one by one. And (3) placing the test tube in a constant-temperature incubator at 25 ℃ for culture, observing the germination condition and the growth characteristics of the test tube, selecting the test tube without mixed bacteria pollution and with stout and vigorous hypha growth for transfer culture, and obtaining 4 purified strains with better growth vigor after continuous generations.
2. Strain screening
Preparing a culture medium comprising the following components in mass concentration: 20g/L glucose, 1g/L yeast extract, 0.2g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate and 1L water. Inoculating the 4 isolated Inonotus obliquus strains into 5 triangular flasks containing culture medium according to 2%, respectively, and culturing at 25 deg.C at 150rpm of shaking table. And checking the appearance of the bacterial colony in the triangular flask after 10 days, taking the dense, strong, uniform and pollution-free bacterial strains of the mycelium pellet, performing cross compounding, and screening again to obtain the bacterial strain with the optimal growth amount.
3. Strain identification
Extracting DNA from the screened strain by a CTAB method, carrying out PCR amplification by using primers ITS4 and ITS5, detecting by a gel imaging system, and sequencing a PCR reaction product. The determined ITS segment DNA sequence is searched in a DNA sequence database by using GenBank and is compared and analyzed, the result shows that Per. Ident of the strain compared with Inonotus obliquus respectively reaches 99.31 percent, and the strain is judged to be Inonotus obliquus by combining the morphological characteristics.
The inonotus obliquus (Inonotus) strain obtained by separation and screening is preserved in the China general microbiological culture Collection center at 28 days 12 months in 2021, the preservation address is the microbiological research institute of China academy of sciences No. 3 of Xilu No. 1 of Beijing republic of Chaoyang, and the preservation number is CGMCC No.23897.
Example 2 preparation of Inonotus obliquus lysate
1. Preparation of seed liquid
(1) Inonotus obliquus YZS10101 is inoculated into 300g of sterile liquid seed culture medium containing 2wt% of glucose, 0.4wt% of yeast extract, 0.2wt% of peptone, 0.1wt% of potassium dihydrogen phosphate and 0.05wt% of magnesium sulfate, wherein the inoculation amount is 2%.
(2) Culturing at 31 deg.C for 3 days with shaking table rotation speed of 150rpm to obtain Inonotus obliquus seed solution.
2. Preparation of fermentation broth
(1) Preparing a composite bark extracting solution: respectively crushing birch bark, willow bark and elm bark into powder, and sieving with a 100-mesh sieve for later use; weighing birch bark powder 10g, willow bark powder 6g and elm bark powder 4g, dissolving in deionized water 2000g, heating at 90 deg.C for 2 hr, and filtering to obtain bark extract.
(2) 300mL of inonotus obliquus seed solution was inoculated into a fermenter containing 3000g of sterile fermentation medium, which included: glucose 4wt%, yeast extract 0.4wt%, peptone 0.2wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.05wt%, and bark extract 60wt%, and culturing at 31 deg.C for 4 days under conditions of stirring rotation speed of 200rpm, ventilation amount of 1vvm, and pH of 6 to obtain Inonotus obliquus fermentation liquid.
3. Collecting the thallus
Filter-pressing the fermentation liquor with the pressure of 0.1mpa and the filtering precision of 30 mu m, and collecting the filtrate. The filtrate was centrifuged at 8000rpm for 20min to collect the cells. Drying the thalli at 80 ℃ for 2h, and weighing to obtain the dry weight of the inonotus obliquus.
4. Concentrated bacteria
Weighing 1kg of dried Inonotus obliquus thallus, mixing with 1kg of deionized water, mixing uniformly under a homogenizer of 3000rpm, and then crushing under a high-pressure homogenizer with wall breaking pressure of 120mpa. 4kg of absolute ethyl alcohol is added into the crushed thallus slurry and evenly mixed. Extracting at 60 deg.C for 2 hr. Filtering, collecting clear liquid, refluxing and concentrating to obtain the alcohol extract of the inonotus obliquus with the mass of 1.5 kg.
5. Compounding
Weighing 1.5kg of the above Inonotus obliquus ethanol extractive solution, adding into 4.5kg of deionized water, adding 0.1kg1, 2-hexanediol, mixing well, and filtering with 0.45um filter membrane to obtain Inonotus obliquus lysate.
Example 3 preparation of Inonotus obliquus lysate
An inonotus obliquus lysate was prepared as in example 1, except that: the addition amount of the bark extract in the fermentation medium was 80wt%.
Example 4 preparation of Inonotus obliquus lysate
An Inonotus obliquus lysate was prepared as in example 1, except that: the addition amount of the bark extract in the fermentation medium was 90wt%.
Example 5 preparation of Inonotus obliquus lysate
An Inonotus obliquus lysate was prepared as in example 1, except that: extracting birch bark powder, willow bark powder and elm bark powder with deionized water respectively, and mixing the extractive solutions to obtain composite bark extractive solution; the proportion of each formula is unchanged.
Comparative example 1 preparation of Inonotus obliquus lysate
An inonotus obliquus lysate was prepared as in example 1, except that: the addition amount of the bark extract in the fermentation medium was 0.
Comparative example 2 preparation of Inonotus obliquus lysate
An Inonotus obliquus lysate was prepared as in example 1, except that: the addition amount of the bark extract in the fermentation medium was 20wt%.
Comparative example 3 preparation of Inonotus obliquus lysate
An Inonotus obliquus lysate was prepared as in example 1, except that: only birch bark is smashed into powder and is screened by a 100-mesh screen for standby; weighing birch bark powder 20g, adding into deionized water 2000g, heating at 90 deg.C for 2 hr, and filtering to obtain birch bark extract instead of the composite bark extract in example 1.
Comparative example 4 preparation of Inonotus obliquus lysate
An inonotus obliquus lysate was prepared as in example 1, except that: respectively crushing willow bark and elm bark into powder, and sieving with a 100-mesh sieve for later use; weighing willow bark powder 12g and elm bark powder 8g respectively, adding into deionized water 2000g, heating at 90 deg.C for 2 hr, and filtering to obtain bark extract instead of the composite bark extract in example 1.
Example 6 physical and chemical index detection
1 Biomass
The biomass was calculated as:
biomass = inonotus obliquus dry weight/mass of fermentation broth x 100% formula (1)
TABLE 1 Inonotus obliquus Biomass
As can be seen from Table 1, the biomass of the inonotus obliquus cultured by the culture medium added with the composite bark extract is obviously higher than that of the culture medium not added in comparative example 1, and the addition amount is positively correlated with the biomass; the composite bark extract obtained by the two processes has little influence on the biomass; the single birch bark extract and the willow bark and elm bark compound extract also have certain promotion effect on the biomass growth of the inonotus obliquus, but the effect is not as good as that of the three bark compound extracts.
2 determination of total triterpene content of Inonotus obliquus lysate
2.1 preparation of control solutions
Collecting oleanolic acid, and adding anhydrous methanol to obtain solution containing 0.2mg oleanolic acid per 1mL methanol.
2.2 drawing of Standard Curve
Precisely measuring 0.1 mL, 0.2mL, 0.3 mL, 0.4 mL and 0.5mL of reference substance solution, respectively placing the reference substance solution in a 15mL test tube with a plug, volatilizing, cooling, precisely adding 0.2mL of newly prepared vanillin glacial acetic acid solution and 0.8mL of perchloric acid, shaking up, heating in a 70 ℃ water bath for 15min, immediately placing in an ice bath for cooling for 5min, taking out, precisely adding 4mL of ethyl acetate, shaking up, taking corresponding reagents as blanks, measuring absorbance at a 546nm wavelength by using an ultraviolet-visible spectrophotometry method, and drawing a standard curve by using the absorbance as a vertical coordinate and the concentration as a horizontal coordinate. The obtained standard curve equation is y =0.0451x +0.0136, the correlation coefficient is r =0.9997, and the oleanolic acid shows a good linear relation with the absorption value in the experimental concentration range.
2.3 preparation of test solutions
Taking a proper amount of a uniformly mixed sample, precisely weighing, placing in a conical flask with a plug, adding 50mL of ethanol, carrying out ultrasonic treatment for 45 minutes, filtering, placing filtrate in a 100mL measuring flask, washing the filter and filter residue by times with a proper amount of ethanol, merging the washing solution into the same measuring flask, adding ethanol to a scale, and shaking up to obtain the traditional Chinese medicine composition.
2.4 determination of
Precisely measuring 0.2mL of test solution, placing the test solution in a 15mL test tube with a plug, measuring the absorbance by the same method from the 'volatilizing', reading the content of oleanolic acid in the test solution from the standard curve, and calculating according to a formula 2 to obtain the total triterpene content according to the method under the preparation item of the standard curve.
Total triterpene content: w = [ (C-C) 0 )*V*N]/m formula (2)
In the formula: w is the content of the target object in the sample, and the unit is mg/kg; c is the concentration of the target object in the sample measuring solution, and the unit is mg/L; c 0 -the concentration of target in the blank in mg/L; v is volume of constant volume, unit mL; n-dilution factor; m is the sample size in g.
TABLE 2 Total triterpene content of Inonotus obliquus lysate
As can be seen from Table 2, the medium containing the extract of composite bark has a significantly higher triterpene content in Inonotus obliquus than the non-added comparative example 1, and the addition amount is positively correlated with the triterpene content; a preparation process of a composite bark extract obtained by respectively extracting three kinds of barks and mixing the extracts is used for obtaining a culture medium, wherein the content of triterpene in inonotus obliquus (example 4) is slightly less than that in example 1; the single birch bark extract and the willow bark and elm bark compound extract also have a certain growth promoting effect on the triterpene content of the inonotus obliquus, but the effect is obviously inferior to that of the three bark compound extracts.
3DPPH free radical clearance rate test method
DPPH assay is a simple method for screening free radical scavengers and evaluating antioxidant activity. The ethanol solution of DPPH showed purple color with a maximum absorption wavelength of 517nm. When a free radical scavenger is added into a DPPH solution, the lone pair of electrons is paired, the absorption disappears or is weakened, so that the color of the solution becomes light and is yellow or faint yellow, the absorbance at 517nm is reduced, and the change degree of the solution and the free radical scavenging intensity are in a linear relation, so that the quantitative analysis can be carried out by a spectrophotometer method. This method can be expressed in terms of clearance, with greater clearance indicating greater clearance of the substance. The stable free radical DPPH provides a simple and ideal pharmacological model for detecting the activity of scavenging free radicals.
3.1DPPH(2×10 -4 mol/L) preparation of ethanol solution: weighing 20mgDPPH, adding absolute ethyl alcohol to dissolve, fixing the volume in a 250mL volumetric flask, and storing at 0-4 ℃ in the dark.
3.2 mixing 3mL of the solution to be measured with 3mL of the solution of LDPPH, and measuring the absorbance at 517nm (A) 1 ) Mixing 3mL of distilled water with 3mL of the solution of LDPPH, and measuring the absorbance (A) at 517nm 2 ) Mixing 3mL of distilled water with 3mL of the solution to be detected, and measuring the absorbance (A) at 517nm 3 ) The DPPH radical scavenging ability of the Inonotus obliquus lysate is obtained by calculation according to the formula 3. The clearance rate is calculated by the formula:
clearance = (a) 2 +A 3 -A 1 )/A 2 X 100% formula (3)
TABLE 3DPPH radical scavenging capacity (%)
As can be seen from table 3, the clearance of the lysate of the inonotus obliquus cultured in the medium added with the composite bark extract is significantly higher than that of the ethanol solution of the lysate of the inonotus obliquus cultured in the comparative example 1 without the added lysate, and the addition amount of the composite bark extract, the content of the inonotus obliquus lysate and the clearance of the DPPH free radicals are respectively and positively correlated; a process for preparing an extract solution of composite bark in which three types of bark are extracted separately and the extract solutions are mixed, which is used for a lysate of Inonotus obliquus (example 4) obtained from a culture medium, and which has a slightly lower DPPH radical scavenging ability than that of example 1; the single birch bark extract and the willow bark and elm bark compound extract also promote the DPPH free radical scavenging capacity of the inonotus obliquus lysate to a certain extent, but the effect is obviously inferior to that of the three bark compound extracts.
Determination of the inhibition Rate of 4 tyrosinase Activity
4.1 preparation of the solution
(1) Disodium hydrogen phosphate solution (0.2 mol/L): weighing 71.63gNa 2 HPO 4 ·12H 2 O, dissolved in 1000mL deionized water.
(2) Phosphoric acid dihydrogen esterSodium (0.2 mol/L): weighing 31.2g of NaH 2 PO 4 ·2H 2 O, dissolved in 1000mL deionized water.
(3) Phosphate buffered solution (PBS, pH = 6.8): 51mL of the prepared sodium dihydrogen phosphate solution and 49mL of the disodium hydrogen phosphate solution are precisely measured and mixed to obtain 100mL0.2mol/L phosphate buffer solution with the pH =6.8, and the phosphate buffer solution is stored in a refrigerator at 4 ℃ for later use.
(4) Preparation of L-tyrosine solution (0.15 mmol/L): accurately weighing 0.0272g of L-tyrosine, predispersing with the prepared acid salt buffer solution, carrying out ultrasonic treatment for 30min, and metering the volume of the prepared phosphate buffer solution into a 100mL volumetric flask.
(5) Preparing a tyrosinase solution: accurately weighing 0.0010g of tyrosinase powder, dissolving the tyrosinase powder by using a prepared phosphate buffer solution with the pH =6.8, and then fixing the volume to 20mL to obtain a tyrosinase solution with the concentration of 0.05mg/mL, wherein the prepared tyrosinase solution needs to be stored at the temperature of-4 ℃ in time.
4.2 sample treatment
Accurately weighing 0.1000g of a sample, dissolving the sample in 20mL of deionized water, stirring, uniformly mixing, precipitating and the like, and removing a clear solution part for detection. The aqua and the water-soluble gel can be directly dissolved; the emulsifier is identified before dissolution, and in the case of emulsion (W/O) type, it is converted into emulsion (O/W) type by dilution method.
4.3 Instrument reference wavelength is 475nm.
4.4 Experimental procedures
The required buffer solution (pH =6.8,0.2mol/L, PBS), sample solution and L-tyrosine solution were added to each tube in sequence, the temperature was maintained in a 37 ℃ water bath for 10min, then the required tyrosinase solution (0.05 mg/mL) was added, the reaction was heated in a 37 ℃ water bath for 10min, and the absorbance value of 475nm solution was measured, and the experimental reaction system is shown in Table 4.
TABLE 4 composition of the reaction solution
4.5 sample determination
Under the instrument conditions indicated above, the absorbance of each solution in the test system was measured separately and the readings recorded.
4.6 results calculation
Tyrosinase inhibition was calculated according to equation 4:
tyrosinase inhibition rate = [1- (T) 2 -T 1 )/(C 2 -C 1 )]X 100% formula (4)
In the formula, C 1 The absorbance of the sample and the enzyme solution is not added; c 2 The absorbance of the enzyme solution added to the sample is not added; t is 1 The absorbance of the solution with the sample and without the enzyme is shown; t is 2 The absorbance of the enzyme-added solution was measured as the presence of the sample.
TABLE 5 tyrosinase activity inhibition ratio (%)
As can be seen from Table 5, the tyrosinase activity inhibition rate of the Inonotus obliquus lysate obtained by culturing the medium added with the composite bark extract is obviously higher than that of the non-added comparative example 1, and the addition amount of the composite bark extract, the content of the Inonotus obliquus lysate and the tyrosinase activity inhibition rate are in positive correlation; the preparation process of the composite bark extract by respectively extracting the three barks and mixing the extracts is used for lysate of the inonotus obliquus (example 4) obtained by a culture medium, and the tyrosinase activity inhibition rate of the lysate is slightly lower than that of the lysate of the inonotus obliquus (example 1); the single birch bark extract and the willow bark and elm bark composite extract also have certain growth promotion effect on the tyrosinase activity inhibition rate of the inonotus obliquus lysate, but the effect is obviously inferior to that of the three bark composite extracts.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A strain of Inonotus obliquus (Inonotus obliquus) is characterized in that the Inonotus obliquus is preserved in the common microorganism center of China general microbiological culture Collection center in 2021 year, 12 months and 28 days, the preservation address is No. 3 of Xilu No. 1 of Beijing, chaoyang district, and the preservation number is CGMCC No.23897.
2. A method of preparing a fermentation lysate using inonotus obliquus according to claim 1, comprising the steps of:
(1) Fermenting and culturing inonotus obliquus to obtain inonotus obliquus fermentation liquor; wherein the fermentation medium comprises composite bark extract, carbon source, nitrogen source and growth factor;
(2) Filtering the inonotus obliquus fermentation liquor, collecting thalli, and drying;
(3) Mixing the dried thallus with water, breaking cell wall, adding into anhydrous ethanol solution, extracting, filtering, reflux concentrating to obtain Inonotus obliquus ethanol extractive solution;
(4) Compounding the inonotus obliquus alcohol extract with deionized water and dihydric alcohol, and filtering with a microporous filter membrane to obtain an inonotus obliquus fermentation lysate.
3. The method of claim 2, further comprising a seed liquid culture before the fermentation culture, wherein a seed culture medium for obtaining the seed liquid comprises the following components in percentage by weight: 2-3wt% of glucose, 0.1-0.5wt% of yeast extract, 0.1-0.5wt% of peptone, 0.02-0.2wt% of monopotassium phosphate, 0.01-0.1% of magnesium sulfate and the balance of water;
the seed liquid culture conditions are as follows: the temperature is 26-34 ℃, the culture time is 2-4 days, and the rotation speed of a shaker is 130-180 r/min.
4. The method according to claim 2, wherein in step (1), the fermentation medium comprises the following components in percentage by weight: 60-90wt% of composite bark extract, 3-5wt% of glucose, 0.1-0.5wt% of yeast extract, 0.1-0.5wt% of peptone, 0.02-0.2wt% of potassium dihydrogen phosphate, 0.01-0.1wt% of magnesium sulfate and the balance of water;
the fermentation culture conditions are as follows: culturing at 26-34 deg.C for 3-6 days at 200-400rpm with ventilation amount of 0.5-3vvm and pH of 6-7; taking the glucose content in the inonotus obliquus fermentation liquor less than 2g/L as a culture end point.
5. The method as claimed in claim 4, wherein the extract of composite bark is prepared by: respectively crushing birch bark, willow bark and elm bark into powder, and sieving with a 100-mesh sieve for later use; adding birch bark powder, willow bark powder and elm bark powder into deionized water, heating at 60-90 deg.C for 1-4 hr, filtering to obtain clear liquid to obtain composite bark extract; the mass ratio of the birch bark powder to the willow bark powder to the elm bark powder is 5; the mass ratio of the total mass of the birch bark powder, the willow bark powder and the elm bark powder to the deionized water is 1.
6. The method as claimed in claim 4, wherein the extract of composite bark is prepared by: respectively crushing birch bark, willow bark and elm bark into powder, and sieving with a 100-mesh sieve for later use; respectively mixing the birch bark powder, the willow bark powder and the elm bark powder in a mass ratio of 1:100 adding into deionized water, heating at 60-90 deg.C for 1-4 hr, filtering to obtain clear liquid, respectively to obtain birch bark extract, willow bark extract and elm bark extract, and mixing to obtain composite bark extract; the mass ratio of the birch bark extract to the willow bark extract to the elm bark extract is 5.
7. The method according to claim 2, wherein in the step (2), the bacteria collection mode is centrifugation, the rotating speed is 1500-8000rpm, and the time is 20-40min; the drying mode is drying.
8. The method according to claim 2, wherein in the step (3), the mass ratio of the bacteria to the water is 1; the wall breaking method is 100-140mpa high pressure wall breaking; the extraction conditions are as follows: the extraction temperature is 50-65 ℃, and the extraction time is 1-3h; the concentration temperature is 55-65 ℃; the mass of the inonotus obliquus alcohol extract is 1.5 times of that of the thalli.
9. The method according to claim 2, wherein in the step (4), the mass ratio of the inonotus obliquus ethanol extract to the deionized water to the glycol is 10; the dihydric alcohol is 1, 2-pentanediol or 1, 2-hexanediol.
10. Use of Inonotus obliquus according to claim 1 or a lysate obtained by a method according to any one of claims 2 to 9 for the preparation of a cosmetic product with antioxidant and/or whitening effects.
Priority Applications (1)
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