CN115181678B - 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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- CN115181678B CN115181678B CN202211011140.XA CN202211011140A CN115181678B CN 115181678 B CN115181678 B CN 115181678B CN 202211011140 A CN202211011140 A CN 202211011140A CN 115181678 B CN115181678 B CN 115181678B
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- inonotus obliquus
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- 241000414067 Inonotus obliquus Species 0.000 title claims abstract description 105
- 238000000855 fermentation Methods 0.000 title claims abstract description 47
- 230000004151 fermentation Effects 0.000 title claims abstract description 47
- 239000006166 lysate Substances 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title abstract description 25
- 239000000284 extract Substances 0.000 claims abstract description 67
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- 239000002131 composite material Substances 0.000 claims abstract description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000008367 deionised water Substances 0.000 claims abstract description 19
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 16
- 238000001914 filtration Methods 0.000 claims abstract description 14
- 241001052560 Thallis Species 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 238000001035 drying Methods 0.000 claims abstract description 8
- 239000012528 membrane Substances 0.000 claims abstract description 4
- 238000010992 reflux Methods 0.000 claims abstract description 4
- 239000000843 powder Substances 0.000 claims description 37
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- 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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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—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
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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
- A61Q19/02—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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- 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
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- 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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- 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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Abstract
The invention discloses a preparation method and application of a 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, a carbon source, a nitrogen source and a growth factor; (2) Filtering the inonotus obliquus fermentation liquor, collecting thalli and drying; (3) Mixing the dried thallus with water, breaking wall, extracting with absolute ethanol solution, filtering, and concentrating under reflux to obtain Inonotus obliquus Kong Junchun extract; (4) And (3) compounding deionized water and dihydric alcohol with the inonotus obliquus Kong Junchun extract, and passing through a microporous filter membrane to obtain an inonotus obliquus fermentation lysate. Compared with wild inonotus obliquus, the cost of the inonotus obliquus lysate obtained by the microbial fermentation method is lower, and the product is more stable.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a preparation method and application of inonotus obliquus fermentation lysate.
Background
Inonotus obliquus @Inonotus obliquus) Is a medicinal fungus growing on birch, its fruiting body shows black blocky morphology similar to carbon, and wild Inonotus obliquus mainly grows in Siberian area of Russian, north sea, finland and Jilin Changbai mountain area of China and Heilongjiang and Xiaoxiangan area. Inonotus obliquus has complex components, and main components comprise polysaccharide, triterpene compounds, sterols, alkaloids and the like, and a large number of pharmacological experiments show that the inonotus obliquus has the effects of resisting oxidation, regulating blood lipid, 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 preventing and treating effects on diseases caused by unreasonable diet in bone.
Inonotus obliquus has certain application in foods, health products and medicines, but in the field of daily chemicals, the main source of the Inonotus obliquus is an Inonotus obliquus extract obtained by extracting through a physical and chemical method, and the Inonotus obliquus is mostly applied to the fields of foods and health products and has few research reports in the field of cosmetics.
With the awareness of people on inonotus obliquus, the demand of inonotus obliquus rises year by year, and the supply and the shortage of wild inonotus obliquus are required. In addition, natural wild inonotus obliquus belongs to rare resources, is high in price and unstable in source, and products in different areas are greatly different, so that the quality of the products is difficult to stabilize. Accordingly, there is a demand for satisfying the growing demand for inonotus obliquus by artificial culture of inonotus obliquus, wherein the production of inonotus obliquus by liquid fermentation technology has been attracting attention in recent years because of the ease of mass production as compared with artificial planting.
Disclosure of Invention
The invention aims to provide a preparation method and application of inonotus obliquus fermentation lysate, so as to solve the problems of the prior art, and compared with the traditional fermentation process, the invention improves the yield and the content of active ingredients; the inonotus obliquus lysate obtained by the invention has better antioxidant and whitening effects.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides an inonotus obliquus strainInonotus obliquus) Inonotus obliquus is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 28 of 2021 and has a preservation address of number 3 of North Chen West Lu No. 1 of the Korean area of Beijing city and a preservation number of CGMCC No.23897.
The invention also provides a method for preparing 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, a carbon source, a nitrogen source and a growth factor;
(2) Filtering the inonotus obliquus fermentation liquor, collecting thalli and drying;
(3) Mixing the dried thallus with water, breaking wall, extracting with absolute ethanol solution, filtering, and concentrating under reflux to obtain Inonotus obliquus Kong Junchun extract;
(4) And (3) compounding deionized water and dihydric alcohol with the inonotus obliquus Kong Junchun extract, and passing through a microporous filter membrane to obtain an inonotus obliquus fermentation lysate.
Preferably, the method further comprises seed liquid culture before fermentation culture, wherein the 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, 0.01-0.1wt% 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 the shaking table is 130-180 rpm.
Preferably, in step (1), the fermentation medium comprises the following components in weight percent: 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 monopotassium phosphate, 0.01-0.1wt% of magnesium sulfate and the balance of water;
the fermentation culture conditions are as follows: the culture temperature is 26-34 ℃, the culture time is 3-6 days, the rotating speed is 200-400rpm, the ventilation is 0.5-3vvm, and the pH is 6-7; and taking the inonotus obliquus fermentation broth with the glucose content less than 2g/L as a culture end point.
Preferably, the composite bark extract is prepared by the following method: crushing birch bark, willow bark and elm bark into powder respectively, and sieving with 100 mesh sieve; adding birch bark powder, willow bark powder and elm bark powder into deionized water, heating at 60-90deg.C for 1-4 hr, filtering to obtain clear solution to obtain composite bark extract; the mass ratio of the birch bark powder to the willow bark powder to the elm bark powder is 5:3:2; 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 1:100.
Preferably, the composite bark extract is prepared by the following method: crushing birch bark, willow bark and elm bark into powder respectively, and sieving with 100 mesh sieve; the birch bark powder, the willow bark powder and the elm bark powder are respectively mixed according to the mass ratio of 1:100 is added into deionized water, heated for 1-4 hours at 60-90 ℃, filtered and taken to obtain clear liquid to obtain birch bark extract, willow bark extract and elm bark extract respectively, and the birch bark extract, the willow bark extract and the elm bark extract are uniformly mixed to obtain composite bark extract; the mass ratio of the birch bark extract, the willow bark extract and the elm bark extract is 5:3:2.
Preferably, in the step (2), the mode of collecting the thalli is centrifugation, the rotating speed is 1500-8000rpm, and the time is 20-40min; the drying mode is drying.
Preferably, in the step (3), the mass ratio of the thalli to the water is 1:1; the wall breaking mode is 100-140mpa high pressure wall breaking; the extraction conditions are as follows: the extraction temperature is 50-65deg.C, and the extraction time is 1-3h; the concentration temperature is 55-65 ℃; the weight of the inonotus obliquus Kong Junchun extract is 1.5 times of the weight of the thallus.
Preferably, in the step (4), the mass ratio of the inonotus obliquus Kong Junchun extract, deionized water and dihydric alcohol is 10:30:1; the dihydric alcohol is 1, 2-pentanediol or 1, 2-hexanediol; the accuracy of the filtration was 0.22 μm, 0.45 μm or 0.9 μm.
The invention also provides application of the inonotus obliquus in preparing 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 has the advantages that:
(1) Compared with the planting technology adopted by the inonotus obliquus culture, the microbial fermentation method is greener, less in pollution, free of pesticide or solvent residues and high in safety;
(2) Compared with wild inonotus obliquus, the cost for culturing inonotus obliquus by a microbial fermentation method is lower, and the product is more stable;
(3) Compared with the existing fermentation process, the invention adopts a plurality of bark phase complexes as the culture medium component of the inonotus obliquus, not only can improve the growth amount of the thalli, but also can lead the thalli to have more biomass and better active matter content, and has better antioxidant and whitening activities of cosmetics.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. 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 invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In the following description, unless otherwise indicated, all methods involved are conventional in the art. Unless otherwise indicated, all materials referred to are those available from published commercial sources.
EXAMPLE 1 isolation of Inonotus obliquus YZS10101
1. Separation and purification
Clean fresh inonotus obliquus fruiting bodies produced in Jilin province white mountain long white valley lands are selected, and impurities such as dust on the surfaces are blown off by dry nitrogen. In the aseptic area of the flame of the alcohol lamp of the aseptic operation table, a plurality of fruiting body blocks with the size of about wheat grains in the fruiting body are cut by a sterilized operating knife, inoculated into inclined-plane test tubes filled with PDA culture medium, and the fruiting body blocks are buried under the surface layer of the culture medium as much as possible, and each fruiting body is separated by 5 test tubes and numbered one by one. Culturing the test tube in a constant temperature incubator at 25 ℃, observing germination conditions and growth characteristics, selecting the test tube with no mixed bacteria pollution, and culturing the test tube with strong 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: glucose 20g/L, yeast extract 1g/L, potassium dihydrogen phosphate 0.2g/L, magnesium sulfate 0.1g/L and water 1L. The 4 Inonotus obliquus strains obtained by separation are respectively inoculated into 5 triangular flasks filled with culture medium according to 2 percent, and are cultured at the rotation speed of a shaking table of 150rpm and the temperature of 25 ℃. After 10 days, checking the appearance of colony in the triangular flask, taking the strain with dense mycelium pellets, robustness, uniformity and no pollution for cross compounding, and screening again to obtain the strain with optimal growth amount.
3. Identification of species
The screened strain is extracted with DNA by CTAB method, PCR amplification is carried out by using primers ITS4 and ITS5, and the PCR reaction product is sequenced after detection by a gel imaging system. The detected ITS segment DNA sequence is searched in a DNA sequence database by adopting GenBank, and the result shows that the Per.
Inonotus obliquus (Inonotus obliquus) strains obtained through separation and screening are preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 12 th month and 28 th year of 2021, and the preservation address is CGMCC No.23897, which is the national institute of sciences of China, national institute of sciences, no. 1, north Chen and West Lu, the Korean area of Beijing.
EXAMPLE 2 preparation of Inonotus obliquus lysate
1. Preparation of seed liquid
(1) Inonotus obliquus YZS10101 is inoculated into 300 g sterile liquid seed culture medium containing 2% of glucose 2wt%, 0.4% wt% of yeast extract, 0.2% wt% of peptone, 0.1% wt% of potassium dihydrogen phosphate and 0.05% wt% of magnesium sulfate, and the inoculation amount is 2%.
(2) The rotation speed of the shaking table is 150rpm, and the culture is carried out for 3 days at the temperature of 31 ℃ to obtain the inonotus obliquus Kong Junchong seed liquid.
2. Preparation of fermentation broths
(1) Preparation of composite bark extract: crushing birch bark, willow bark and elm bark into powder respectively, and sieving with 100 mesh sieve; weighing birch bark powder 10 g, willow bark powder 6 g, elm bark powder 4 g, dissolving in 2000 g deionized water, heating at 90deg.C 2 h, and filtering to obtain bark extract.
(2) 300 mL Inonotus obliquus Kong Junchong seed liquid is inoculated into a fermentation tank containing 3000 g sterile fermentation medium, wherein the fermentation medium comprises: glucose 4 wt%, yeast extract 0.4 wt%, peptone 0.2wt%, potassium dihydrogen phosphate 0.1wt%, magnesium sulfate 0.05 wt%, and bark extract 60 wt%, and culturing at 31deg.C for 4 days under stirring at 200 rpm, ventilation of 1 vvm, and pH of 6 to obtain Inonotus obliquus fermentation broth.
3. Collecting the bacterial cells
The fermentation broth was press-filtered at a pressure of 0.1mpa and a filtration accuracy of 30 μm, and the filtrate was collected. And centrifuging the filtrate at 8000rpm for 20 min, and collecting thalli. 80. Drying the thalli at the temperature of 2 h, and weighing to obtain the dry weight mass of the inonotus obliquus.
4. Concentrated bacterial body
Weighing dried Inonotus obliquus thallus 1 kg, mixing with 1 kg deionized water, uniformly mixing with a homogenizer at 3000 rpm, and crushing under a high pressure homogenizer at wall breaking pressure of 120 mpa. Adding 4 kg absolute ethyl alcohol into the crushed thallus slurry, and uniformly mixing. 60. Extracting at a temperature of 2 hours. Filtering, collecting clear liquid, refluxing and concentrating to obtain the extract of Inonotus obliquus Kong Junchun with the mass of 1.5 and kg.
5. Compounding
Weighing 1.5 kg of the inonotus obliquus Kong Junchun extract, adding into 4.5 kg deionized water, adding 0.1 kg of 1, 2-hexanediol, uniformly mixing, and passing through a filter membrane of 0.45 um to obtain inonotus obliquus lysate.
EXAMPLE 3 preparation of Inonotus obliquus lysate
Inonotus obliquus lysate was prepared as in example 2, except that: the addition amount of the bark extract in the fermentation medium is 80 and wt percent.
EXAMPLE 4 preparation of Inonotus obliquus lysate
Inonotus obliquus lysate was prepared as in example 2, except that: the addition amount of bark extract in the fermentation medium is 90 wt%.
EXAMPLE 5 preparation of Inonotus obliquus lysate
Inonotus obliquus lysate was prepared as in example 2, 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
Inonotus obliquus lysate was prepared as in example 2, except that: the addition amount of bark extract in the fermentation medium is 0.
Comparative example 2 preparation of Inonotus obliquus lysate
Inonotus obliquus lysate was prepared as in example 2, except that: the addition amount of the bark extract in the fermentation medium is 20 wt%.
Comparative example 3 preparation of Inonotus obliquus lysate
Inonotus obliquus lysate was prepared as in example 2, except that: crushing birch bark into powder, and sieving with 100 mesh sieve; the birch bark powder 20, g is weighed and added into 2000, g deionized water, and after heating at 90 ℃ for 2, h, birch bark extract is obtained after filtration and replaces the composite bark extract in example 2.
Comparative example 4 preparation of Inonotus obliquus lysate
Inonotus obliquus lysate was prepared as in example 2, except that: crushing willow bark and elm bark into powder, and sieving with 100 mesh sieve; willow bark powder 12 g and elm bark powder 8 g are weighed respectively, added into deionized water of 2000 g, heated at 90 ℃ for 2 h, filtered to obtain bark extract, and the bark extract is substituted for the composite bark extract in example 2.
Example 6 physical and chemical index detection
1 biomass
The biomass calculating method comprises the following steps:
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 culture medium added with the composite bark extract, the biomass of the inonotus obliquus obtained by culturing is obviously higher than that of comparative example 1 without adding, and the adding amount is positively correlated with the biomass; the composite bark extract obtained by the two processes has little influence on biomass; the single birch bark extract, the composite extract of willow bark and elm bark also has a certain promotion effect on the biomass growth of inonotus obliquus, but the effect is not as good as that of the composite extract of three barks.
Determination of total triterpene content of Fuscoporia obliqua lysate 2
2.1 preparation of control solution
Extracting oleanolic acid, and adding anhydrous methanol to obtain solution containing 0.2. 0.2 mg oleanolic acid per 1. 1 mL methanol.
2.2 drawing of a Standard Curve
Precisely measuring reference solutions 0.1, 0.2, 0.3, 0.4 and 0.5, mL, respectively placing in 15 mL test tubes with plugs, volatilizing, cooling, precisely adding newly prepared vanillin glacial acetic acid solution 0.2 mL and perchloric acid 0.8 mL, shaking, heating in water bath at 70deg.C for 15 min, immediately cooling in ice bath for 5 min, taking out, precisely adding ethyl acetate 4 mL, shaking, taking corresponding reagent as blank, measuring absorbance at 546 nm wavelength by ultraviolet-visible spectrophotometry, and drawing standard curve with absorbance as ordinate and concentration as abscissa. The standard curve equation is obtained as y=0.0451x+0.0136, and the correlation coefficient is r=0.9997, which shows that the oleanolic acid has good linear relation with the absorption value in the experimental concentration range.
2.3 preparation of sample solutions
Taking a proper amount of uniformly mixed samples, precisely weighing, placing the samples into a conical flask with a plug, adding ethanol 50 mL, carrying out ultrasonic treatment for 45 minutes, filtering, placing the filtrate into a measuring flask with a size of 100 mL, washing the filter and filter residues with a proper amount of ethanol, merging the washing liquid into the same measuring flask, adding ethanol to a scale, and shaking uniformly to obtain the product.
2.4 measurement
Precisely measuring the solution of the sample 0.2 and mL, placing the solution in a 15 mL test tube with a plug, operating the method according to the standard curve preparation item from 'volatilizing', measuring absorbance by the same method, reading the content of oleanolic acid in the solution of the sample from the standard curve, and calculating according to a formula 2 to obtain the total triterpene content.
Total triterpene content: w= [ (C-C) 0 )*V*N]Formula/m (2)
Wherein: w is the content of a target object in the sample, and the unit is mg/kg; c, measuring the concentration of a target object in the liquid by using the unit mg/L; c (C) 0 -concentration of target in mg/L in the blank; v-constant volume, unit mL; n-dilution factor; m-the sampling amount of the sample, in g.
TABLE 2 Fuscoporia obliqua lysate Total triterpene content
As can be seen from table 2, the culture medium added with the composite bark extract has significantly higher triterpene content in the cultured inonotus obliquus than in comparative example 1 without addition, and the addition amount is positively correlated with the triterpene content; the three kinds of bark are respectively extracted and then the extracting solutions are mixed to prepare a composite bark extracting solution, which is used for culturing Inonotus obliquus (example 5) with slightly less triterpene content than example 2; the single birch bark extract, the composite extract of willow bark and elm bark also has a certain growth promoting effect on the triterpene content of inonotus obliquus, but the effect is obviously inferior to that of the composite extract of three barks.
3 DPPH free radical clearance test method
The DPPH assay is a simple method for screening radical scavengers and evaluating antioxidant activity. The DPPH ethanol solution developed purple color and had a maximum absorption wavelength of 517 nm. When the free radical scavenger is added into DPPH solution, the lone pair electrons are paired, absorption disappears or weakens, the color of the solution becomes light, yellow or yellowish, the absorbance at 517 and nm becomes small, the change degree and the free radical scavenging intensity are in linear relation, and thus the quantitative analysis can be carried out by a spectrophotometry. The 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 the free radical.
3.1 DPPH（2×10 -4 mol/L) ethanol solution: weighing 20 mg DPPH, adding absolute ethyl alcohol to dissolve and fixIs contained in a 250 mL volumetric flask and stored in a dark place at 0-4 ℃.
3.2 Mixing 3 mL solution to be measured with 3 mL DPPH solution, and measuring 517: 517 nm absorbance (A 1 ) Mixing 3 mL distilled water with 3 mL DPPH solution, and measuring 517: 517 nm absorbance (A 2 ) Mixing distilled water 3 mL with liquid 3 mL, and measuring absorbance 517 nm (A 3 ) Calculating according to a formula 3 to obtain the DPPH free radical scavenging capability of the inonotus obliquus lysate. The clearance rate calculation formula is:
clearance= (a) 2 +A 3 -A 1 ）/A 2 X 100% formula (3)
TABLE 3 Fuscoporia obliqua lysate scavenging ability for DPPH free radical (%)
As can be seen from Table 3, the removal capacity of DPPH free radical of the inonotus obliquus obtained by culturing the culture medium added with the composite bark extract is obviously higher than that of comparative example 1 without adding the extract, and the addition amount of the composite bark extract, the content of the inonotus obliquus lysate and the removal capacity of DPPH free radical are respectively positively correlated; the preparation process of the composite bark extract solution, which is prepared by respectively extracting three kinds of bark and mixing the extract solutions, is used for preparing a lysate of inonotus obliquus (example 5) obtained by a culture medium, and has slightly lower DPPH free radical scavenging capability than that of example 2; the single betulina bark extract, the composite extract of willow bark and elm bark also promotes the DPPH free radical scavenging ability of the inonotus obliquus lysate to a certain extent, but the effect is obviously inferior to that of the composite extract of three barks.
Determination of inhibition ratio of tyrosinase Activity
4.1 preparation of solutions
(1) Disodium hydrogen phosphate solution (0.2 mol/L): weighing 71.63 g of Na 2 HPO 4 ·12H 2 O, dissolved in 1000 mL deionized water.
(2) Sodium dihydrogen phosphate (0.2 mol/L): 31.2 g NaH was weighed out 2 PO 4 ·2H 2 O, dissolved in 1000 mL deionized water.
(3) Phosphate buffered solution (PBS, ph=6.8): accurately measuring 51 mL of the prepared sodium dihydrogen phosphate solution and 49 mL of the disodium hydrogen phosphate solution, mixing to obtain 100 mL of 0.2 mol/L, pH =6.8 of phosphate buffer solution, and storing in a refrigerator at 4 ℃ for later use.
(4) Preparation of L-tyrosine solution (0.15 mmol/L): accurately weighing 0.0272 and g of L-tyrosine, pre-dispersing with the prepared acid salt buffer solution, performing ultrasonic treatment for 30 min, and fixing the volume of the prepared phosphate buffer solution into a 100 mL volumetric flask.
(5) Preparing tyrosinase solution: the tyrosinase powder is precisely weighed to 0.0010 g, dissolved by the prepared phosphate buffer solution with pH=6.8 and then fixed to 20 mL, so that the tyrosinase solution with the volume of 0.05 mg/mL can be prepared, and the prepared tyrosinase solution needs to be stored at the temperature of-4 ℃ in time.
4.2 sample handling
Accurately weighing 0.1000 of sample g, dissolving in 20 mL deionized water, stirring, mixing, precipitating, and detecting the clarified solution. The water agent and the water-soluble gel agent can be directly dissolved; the emulsifying agent is identified before dissolution, and if it is of the emulsified (W/O) type, a inversion dilution method is used to make it of the emulsified (O/W) type.
4.3 instrument reference condition wavelength 475 nm.
4.4 experimental procedure
To each tube, the required buffer solution (ph=6.8, 0.2 mol/L, PBS), sample solution and L-tyrosine solution were sequentially added, the temperature was kept constant for 10 min in a 37 ℃ water bath, then the required tyrosinase solution (0.05 mg/mL) was added, the reaction was further heated in a 37 ℃ water bath for 10 min, the absorbance value of each solution was measured 475 nm, and the experimental reaction system was shown in table 4.
TABLE 4 composition of reaction solution
4.5 sample measurement
Under the instrument conditions shown above, the absorbance of each solution in the experimental system was measured separately, and the readings were recorded.
4.6 results calculation
Tyrosinase inhibition was calculated according to formula 4:
tyrosinase inhibition rate= [1- (T) 2 -T 1 )/(C 2 -C 1 )]X 100% formula (4)
Wherein C is 1 Absorbance for neither sample nor enzyme solution; c (C) 2 Absorbance of the enzyme-added solution for the non-added sample; t (T) 1 Absorbance for the non-enzyme solution with added sample; t (T) 2 There is absorbance of the enzyme-added solution for the added sample.
TABLE 5 inhibition of tyrosinase activity (%)
As can be seen from table 5, the tyrosinase activity inhibition rate of the lysate of the cultured inonotus obliquus is significantly higher than that of comparative example 1, which is not added, and the addition amount of the composite bark extract, the content of the lysate of the inonotus obliquus and the tyrosinase activity inhibition rate are positively correlated; the preparation process of the composite bark extract solution, which is prepared by respectively extracting three kinds of bark and mixing the extract solutions, is used for preparing the lysate of inonotus obliquus (example 5) obtained by the culture medium, and the inhibition rate of tyrosinase activity is slightly lower than that of example 2; the single birch bark extract, the composite extract of willow bark and elm bark also has a certain growth promoting effect on the tyrosinase activity inhibition rate of the inonotus obliquus lysate, but the effect is obviously inferior to that of the composite extract of three barks.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (3)
1. A method for preparing a fermentation lysate by using inonotus obliquus, which is characterized by comprising the following steps:
(1) Fermenting and culturing inonotus obliquus to obtain inonotus obliquus fermentation liquor; wherein 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 monopotassium phosphate, 0.01-0.1wt% of magnesium sulfate and the balance of water;
the conditions of the fermentation culture are as follows: the culture temperature is 26-34 ℃, the culture time is 3-6 days, the rotating speed is 200-400rpm, the ventilation is 0.5-3vvm, and the pH is 6-7; taking the inonotus obliquus fermentation broth with the glucose content less than 2g/L as a culture end point;
(2) Filtering the inonotus obliquus fermentation liquor, collecting thalli and drying;
(3) Mixing the dried thallus with water, breaking wall, extracting with absolute ethanol solution, filtering, and concentrating under reflux to obtain Inonotus obliquus Kong Junchun extract;
the mass ratio of the dried thalli to water is 1:1; the wall breaking mode is 100-140mpa high pressure wall breaking; the mass ratio of the dried thalli to the absolute ethyl alcohol solution is 1:4; the extraction conditions are as follows: the extraction temperature is 50-65deg.C, and the extraction time is 1-3h; the concentration temperature is 55-65 ℃; the mass of the inonotus obliquus Kong Junchun extract is 1.5 times of the mass of the dried thalli;
(4) Mixing the inonotus obliquus Kong Junchun extract with deionized water and dihydric alcohol, and passing through a microporous filter membrane to obtain inonotus obliquus fermentation lysate;
the mass ratio of the inonotus obliquus Kong Junchun extract to deionized water to dihydric alcohol is 10:30:1; the dihydric alcohol is 1, 2-pentanediol or 1, 2-hexanediol;
the inonotus obliquus is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2021, wherein the preservation address is the North Chen Silu No. 1, 3 of the Korean region of Beijing, and the preservation number is CGMCC No. 23897;
the composite bark extract is prepared by the following method: crushing birch bark, willow bark and elm bark into powder respectively, and sieving with 100 mesh sieve; adding birch bark powder, willow bark powder and elm bark powder into deionized water, heating at 90deg.C for 2 hr, and filtering to obtain clear solution to obtain composite bark extract; the mass ratio of the birch bark powder to the willow bark powder to the elm bark powder is 5:3:2; 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 1:100.
2. The method of claim 1, further comprising seed liquor culturing prior to fermentation culturing, wherein the seed culture medium used to obtain the seed liquor comprises the following components in weight percent: 2-3wt% of glucose, 0.1-0.5wt% of yeast extract, 0.1-0.5wt% of peptone, 0.02-0.2wt% of monopotassium phosphate, 0.01-0.1wt% 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 the shaking table is 130-180 rpm.
3. The method according to claim 1, wherein in the step (2), the cells are collected by centrifugation at 1500-8000rpm for 20-40min; the drying mode is drying.
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