CN116970665A - Extraction method and application of tremella aurantialba polysaccharide fermented by tremella aurantialba - Google Patents
Extraction method and application of tremella aurantialba polysaccharide fermented by tremella aurantialba Download PDFInfo
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- CN116970665A CN116970665A CN202311226231.XA CN202311226231A CN116970665A CN 116970665 A CN116970665 A CN 116970665A CN 202311226231 A CN202311226231 A CN 202311226231A CN 116970665 A CN116970665 A CN 116970665A
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- tremella aurantialba
- polysaccharide
- tremella
- fermentation
- golden
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- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 2
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- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 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
- 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
- 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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- 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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- 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
- C12N1/36—Adaptation or attenuation of cells
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- 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 application provides an extraction method of tremella aurantialba polysaccharide by fermenting tremella aurantialba, which comprises the specific steps of preparing strains, preparing seed liquid, inoculating and fermenting, filtering, drying and other post-treatment steps. The application carries out fermentation by the golden fungus, and reasonably controls the processing technology, so that the molecular weight of golden fungus polysaccharide can be reduced, and the golden fungus polysaccharide is more convenient to absorb in cosmetic application. The extraction method is simple, the extraction conditions are mild, various organic reagents are not used, the safety and the environmental protection are realized, and the influence of external factors on the biological activity is reduced; the tremella aurantialba polysaccharide obtained by extraction has high purity and small molecular weight, and can effectively maintain the bioactivity of the polysaccharide; can be widely used in cosmetic products, has excellent antioxidation and moisturizing effects, promotes the development of tremella aurantialba deep processing industry, and creates economic benefits for people.
Description
Technical Field
The application relates to the technical field of microbial extraction, in particular to an extraction method of tremella aurantialba polysaccharide by fermenting tremella aurantialba and application thereof.
Background
The tremella (Tremella aurantialba) belongs to the class of Basidiomycetes, the order of tremella, the family of tremella and the genus tremella, and is also called as a precious edible and medicinal fungus, namely a special strain in China, because of its golden color, which is also called as golden tremella, and is similar to human brain, which is also called as auricularia auricula, huang Er, tremella and the like. The tremella aurantialba contains rich fat, protein, phosphorus, sulfur, manganese, iron and other trace elements, and contains 18 amino acids, so that the tremella aurantialba has very high nutritive value.
Eurotium cristatum (Eurotium cristatum) is a beneficial fungus belonging to the genus Eurotium of the order Eurotiaceae, its ascospores are golden yellow, like Milan, visible to naked eyes in the finished Fuzhuan brick, and is a dominant fungus producing "golden flowers" in the production process of Fuzhuan tea, so that the fungus is commonly called "golden flowers". Meanwhile, eurotium cristatum is also a main factor for forming the unique quality of Fuzhuan tea. The color, the aroma and the taste of the Fuzhuan tea are different from those of other tea due to the effect of the Eurotium cristatum, and the Fuzhuan tea has various newly added substances after fermentation, so that the Fuzhuan tea has various effects, such as antioxidation, digestion promotion, lipid reduction, weight loss, bacteriostasis, anticancer and the like. The new food raw material receiving system of the national defense and accounting Committee receives the new food raw material application of Eurotium cristatum (CGMCC NO. 8730) on 12 months and 7 days of 2016.
The Eurotium cristatum produces extracellular enzymes such as polyphenol oxidase, pectase, cellulase, protease and the like, and catalyzes oxidation, polymerization, degradation and conversion of substances in tremella aurantialba; can convert macromolecular substances such as cellulose and polysaccharide into micromolecular substances.
Researches show that edible fungi and polysaccharide thereof can simultaneously prevent and treat various human diseases, and a great deal of researches and reports on health care effects of edible fungi polysaccharide are provided, wherein the health care effects are related to bioactive compounds of the edible fungi, including polysaccharide. The active polysaccharide has potential pharmacological actions of resisting obesity, diabetes, cancer, microorganism and virus. Their related antioxidant, anti-inflammatory and immunomodulatory activities are discussed at the adipocyte, rodent and human levels. The mechanism of action of polysaccharides also includes an effect on the intestinal microbiota, which can act as a prebiotic in the digestive system. The tremella aurantialba polysaccharide has the effects of regulating immunity, resisting radiation, resisting ulcer and inflammation, and can reduce blood sugar, blood fat and the like of organisms. The variety of tremella aurantialba processing products in the current market is less, the tremella aurantialba industry is slow to develop, and the research and development of tremella aurantialba polysaccharide related products can increase the added value of tremella aurantialba and promote the development of tremella aurantialba industry. The polysaccharide extraction method mainly comprises a water extraction and alcohol precipitation method, an acid-base extraction method, an enzyme method, an ultrasonic method, a microwave method, a supercritical fluid extraction method and the like, but the problems of high polysaccharide bioactivity and high polysaccharide molecular weight and difficult absorption are difficult to maintain. Chinese patent CN115746158A discloses a tremella polysaccharide, its preparation method and application, the polysaccharide has high yield and simple and feasible process, but the patent uses water extraction and alcohol precipitation method, uses a large amount of organic solvent to extract polysaccharide component, and is more difficult to maintain the bioactivity of polysaccharide and has large molecular weight.
Disclosure of Invention
Aiming at the problems, the application provides an extraction method of tremella aurantialba polysaccharide by fermenting tremella aurantialba, which overcomes the defects of the existing extraction method, maintains the bioactivity of tremella aurantialba polysaccharide and reduces the molecular weight of tremella aurantialba polysaccharide.
The application provides an extraction method of tremella aurantialba polysaccharide by fermenting tremella aurantialba, which comprises the following specific steps:
step S1: preparing strains: separating, purifying and domesticating golden flower bacteria growing in Fuzhuan tea to obtain domesticated bacterial strains;
step S2: preparing seed liquid: inoculating the domesticated strain into liquid culture medium, and culturing for 2-5 days;
step S3: inoculating and fermenting: pulverizing dried golden fungus, sieving with 40-80 mesh sieve, placing in a fermentation tank, adding water with a feed-liquid ratio of 1:60-100, sterilizing, inoculating golden fungus seed liquid into the fermentation tank, fermenting at 25-35deg.C and stirring speed of 100-300r/min for 2-7 days to obtain fermentation liquor;
step S4: post-treatment: centrifuging the fermentation liquor, taking supernatant, filtering and drying to obtain the tremella aurantialba polysaccharide.
As a preferred technical scheme, in the step S1, the specific steps of separation and purification are as follows: cutting Fuzhuan tea sample, picking a piece of tea with golden flower spores, inoculating to PDA culture medium under aseptic condition, selecting golden flower bacterial strain for subculture until only golden flower bacteria are in culture medium, and purifying.
As a preferred technical scheme, the PDA culture medium comprises the following raw materials: 200.0g of potato, 20.0g of glucose, 20.0g of agar and 1000mL of distilled water.
The golden flower fungus obtained through separation and purification can be identified by using a conventional identification method, and is determined to be golden flower fungus. Examples of the identification method include: morphological identification and molecular biological identification.
According to the application, through domestication in the step S1, the golden flower fungus is quickly adapted to a culture medium mainly composed of golden fungus components from a PDA culture medium stage, and the golden flower fungus can grow on golden fungus more quickly.
As a preferable technical scheme, the domesticating operation in the step S1 includes the following steps: inoculating purified golden flower fungus on PDA culture medium into aqueous golden ear culture medium containing sucrose, culturing at 28-30deg.C, and allowing golden flower fungus to grow on whole plate.
As a preferable technical scheme, the raw materials of the tremella aurantialba aqueous solution culture medium comprise: 200g of tremella aurantialba, 1000ml of distilled water, 30g of agar and sucrose.
Preferably, the mass content of the sucrose is 0% -2% of the total amount of the raw materials of the culture medium. For example, there may be mentioned: 0%, 0.5%, 1.0%, 1.5%, 2.0%. The present application is not limited to the recited values, and other values not recited in the range of values are equally applicable.
The preparation method of the tremella aurantialba aqueous solution culture medium comprises the following steps: mixing Auricularia with distilled water, boiling, filtering, adding agar, adding sucrose, sterilizing at 115deg.C for 20min, pouring into a flat plate, and cooling.
As a more preferable technical scheme, the domesticating operation in the step S1 includes the following steps:
(1) Inoculating purified golden flower fungus on PDA culture medium into golden ear water solution culture medium containing 2% sucrose, culturing at 28-30deg.C until golden flower fungus grows over the whole plate; (2) Inoculating the golden flower fungus into a golden fungus water solution culture medium containing 1.5% of sucrose, and culturing at 28-32 ℃ until the golden flower fungus grows on the whole plate; (3) Inoculating the golden fungus into a golden fungus aqueous solution culture medium containing 1% of sucrose, and culturing at 28-32 ℃ until the golden fungus grows on the whole plate; (4) Inoculating the golden flower fungus into a golden fungus water solution culture medium containing 0.5% of sucrose, and culturing at 28-32 ℃ until the golden flower fungus grows on the whole plate; (5) Inoculating the golden fungus into a golden fungus aqueous solution culture medium without sucrose, and culturing at 28-32 ℃ until the golden fungus grows on the whole plate.
The inventor finds that the golden flower fungus obtained by separation, purification and identification is directly inoculated into a fermentation tank containing golden fungus, and the golden flower fungus is difficult to grow and needs at least 1 month to grow. According to the application, through domestication treatment of the golden flower fungus, the golden flower fungus strain is gradually adapted to the fermentation environment without sucrose, so that the fermentation is faster, the fermentation time is reduced, the domesticated golden flower fungus only needs 5 days under the same condition, the growth time is greatly reduced, and the preparation efficiency is improved.
As a preferable technical scheme, in the step S2, the inoculation amount of the domesticated strain is 1% -3%.
When the strain inoculation is carried out, if the inoculation amount is too small, the hypha grows slowly, the generation of the hypha group grows more, the difference of young and aging degree among various bodies in the group is large, and when the offspring is propagated, synchronous growth is not easy to approach, and the product yield is affected. Excessive inoculation amount can cause rapid early metabolism, early aging of hyphae, insufficient post-synthesis strength of the product, low later unit, and excessive concentration of hyphae, destroy physical properties of fermentation broth, and influence product synthesis.
As a preferred technical scheme, in the step S2, the preparation method of the liquid culture medium is as follows: mixing 200g of tremella aurantialba and 1000ml of distilled water, boiling, filtering, sterilizing at 115 ℃ for 20min in a sterilizing pot, pouring into a flat plate, and cooling to obtain the tremella aurantialba wine.
As a more preferable technical solution, in the step S2, the specific steps for preparing the seed solution are as follows: filling 100ml/300ml of liquid culture medium, sterilizing at 115 ℃ for 20min, cooling for later use, picking up the strain on the surface of the lawn of the domesticated golden flower fungus strain, inoculating into the sterilized liquid culture solution, and performing shake culture at a shake fermentation temperature of 28 ℃ at a rotational speed of 140r/min for 2-5 days to obtain golden flower strain seed liquid.
As a preferable technical scheme, the concentration of the golden flower fungus seed liquid obtained in the step S2 is 1 multiplied by 10 5 ～8 cfu/ml。
As a preferable technical scheme, in the step S3, the sterilization condition is 62-65 ℃ and the sterilization is kept for 30min.
As a preferable technical scheme, in the step S3, the inoculation amount of the golden flower strain seed liquid is 5-10%.
As a preferable technical scheme, in the step S4, the centrifugation speed is 8000 r/min.
As a preferable embodiment, in the step S4, the filter pore size used for the filtration is 0.1-1. Mu.m.
In order to better maintain the structure and activity of the polysaccharide component, as a preferred technical scheme, in the step S4, the drying is vacuum freeze drying. Vacuum degree is less than 10Pa, temperature is-50deg.C, and drying time is 12-24 h. The drying time of the application can be reasonably adjusted according to the amount of materials.
In the application, vacuum freeze drying is adopted, so that the influence of high-temperature drying on the structural components and activity of the polysaccharide is avoided, and the water content of the product is lower.
In another aspect, the application provides an application of tremella aurantialba polysaccharide in cosmetics. The tremella aurantialba polysaccharide is used in cosmetics, has the effects of resisting oxidization and preserving moisture, promotes the development of tremella aurantialba deep processing industry, and creates economic benefits for people.
The raw materials and equipment used in the present application are common raw materials and equipment used in the art and are commercially available products unless otherwise specified. The methods used in the present application are conventional in the art unless otherwise specified.
Advantageous effects
The application uses the golden flower fungus to ferment and extract golden fungus polysaccharide, the extraction method is simple, the extraction condition is mild, various organic reagents are avoided, the application is safe and environment-friendly, and the influence of external factors on the biological activity is reduced; the tremella aurantialba polysaccharide obtained by extraction has high purity and small molecular weight, and can effectively maintain the bioactivity of the polysaccharide; can be widely used in cosmetic products, has excellent antioxidation and moisturizing effects, promotes the development of tremella aurantialba deep processing industry, and creates economic benefits for people.
Drawings
FIG. 1 is a diagram of a sample of auricularia auricula polysaccharide according to example 3 of the present application;
FIG. 2 is a diagram of a sample of auricularia auricula polysaccharide according to example 3 of the present application;
FIG. 3 is a graph of glucose standard curve when the sulfuric acid phenol method of the present application is used to test polysaccharide content;
FIG. 4 is a graph of protein standards for measuring protein content by Coomassie Brilliant blue method according to the present application.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Examples
Example 1
The embodiment provides an extraction method of tremella aurantialba polysaccharide by fermenting tremella aurantialba, which comprises the following specific steps:
step S1: preparing strains: separating, purifying and domesticating golden flower bacteria growing in Fuzhuan tea to obtain domesticated bacterial strains;
cutting a Fuzhuan tea sample, picking a piece of tea with golden flower spores from the Fuzhuan tea sample, inoculating the tea to a PDA culture medium under the aseptic condition, and selecting golden flower bacterial strains for subculturing until only golden flower bacteria exist in the culture medium, thereby achieving the aim of purification; the golden flower fungus obtained through separation and purification is identified by using a conventional identification method, and is determined to be golden flower fungus.
The PDA culture medium comprises the following raw materials: 200.0g of potato, 20.0g of glucose, 20.0g of agar and 1000mL of distilled water;
the specific steps of domestication are as follows: (1) Inoculating purified golden flower fungus on PDA culture medium into golden ear water solution culture medium containing 2% sucrose, culturing at 30deg.C until golden flower fungus grows over the whole plate; (2) Inoculating the golden flower fungus into a golden fungus water solution culture medium containing 1.5% of sucrose, and culturing at 30 ℃ until the golden flower fungus grows over the whole plate; (3) Inoculating the golden flower fungus into a golden fungus water solution culture medium containing 1% of sucrose, and culturing at 30 ℃ until the golden flower fungus grows over the whole plate; (4) Inoculating the golden flower fungus into a golden fungus water solution culture medium containing 0.5% of sucrose, and culturing at 30 ℃ until the golden flower fungus grows over the whole plate; (5) Inoculating the golden fungus into a golden fungus water solution culture medium without sucrose, and culturing at 30deg.C until the golden fungus grows over the whole plate.
Step S2: preparing seed liquid: filling 100ml/300ml of liquid culture medium, sterilizing at 115 ℃ for 20min, cooling for later use, picking up strains on the surface of a lawn of the domesticated golden flower fungus strain, inoculating 1% of the strains into the sterilized liquid culture solution, carrying out shake culture at a shake fermentation temperature of 28 ℃ at a rotating speed of 140r/min for 4 days to obtain golden flower strain seed solution;
the preparation method of the liquid culture medium comprises the following steps: mixing 200g of tremella aurantialba and 1000ml of distilled water, boiling, filtering, sterilizing at 115 ℃ for 20min in a sterilizing pot, pouring into a flat plate, and cooling to obtain the tremella aurantialba wine.
Step S3: inoculating and fermenting: pulverizing dried golden fungus, sieving with 60 mesh sieve, placing in a fermentation tank, adding water with a feed-liquid ratio of 1:60, heating to 65deg.C, sterilizing for 30min, cooling, inoculating golden fungus seed liquid into the fermentation tank with an inoculum size of 5%, and fermenting at 30deg.C and stirring rotation speed of 200r/min for 5 days to obtain fermentation broth.
Step S4: post-treatment: centrifuging the fermentation liquor for 30min at 8000 r/min, collecting supernatant, filtering with a filter with aperture of 1 μm, and vacuum freeze drying to obtain the auricularia auricula polysaccharide with vacuum degree less than 10Pa, temperature of-50deg.C, and drying time of 20 h.
Example 2
The specific implementation manner of the extraction method of the tremella aurantialba polysaccharide by fermenting tremella aurantialba in the embodiment is the same as that of the embodiment 1, and the difference between the specific implementation manner and the embodiment 1 is that in the inoculation fermentation of the step S3, the feed-liquid ratio is 1:70.
example 3
The specific implementation manner of the extraction method of the tremella aurantialba polysaccharide by fermenting tremella aurantialba in the embodiment is the same as that of the embodiment 1, and the difference between the specific implementation manner and the embodiment 1 is that in the inoculation fermentation of the step S3, the feed-liquid ratio is 1:80.
as shown in fig. 1 and 2, the tremella polysaccharide prepared in this example is a pale yellow powder.
Example 4
The specific implementation manner of the extraction method of the tremella aurantialba polysaccharide by fermenting tremella aurantialba in the embodiment is the same as that of the embodiment 1, and the difference between the specific implementation manner and the embodiment 1 is that in the inoculation fermentation of the step S3, the feed-liquid ratio is 1:100.
example 5
The embodiment of the present application provides a method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba, which is different from embodiment 3 in that the seed liquid inoculation amount in the inoculation fermentation of step S3 is 6% from embodiment 3.
Example 6
The embodiment of the present application provides a method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba, which is different from embodiment 3 in that seed liquid inoculation amount is 8% in inoculation fermentation of step S3.
Example 7
The embodiment of the present application provides a method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba, which is different from embodiment 3 in that seed liquid inoculation amount is 10% in inoculation fermentation of step S3.
Comparative example 1
Pulverizing dried tremella aurantialba, sieving with a 60-mesh sieve, adding deionized water according to a feed-liquid ratio of 1:55, transferring into an extraction tank, and extracting at 95 ℃ for 3.5h; centrifuging at 6000-10000r/min for 20-30min, collecting supernatant, and filtering with 0.1-1 μm filter to obtain filtrate. And performing vacuum freeze drying treatment on the filtrate to obtain the product.
Comparative example 2
The specific implementation manner of the extraction method of the tremella aurantialba polysaccharide by fermenting tremella aurantialba in the embodiment is the same as that of the embodiment 3, and the difference between the specific implementation manner and the embodiment 3 is that in the inoculation fermentation of the step S3, the feed-liquid ratio is 1:40.
comparative example 3
The specific implementation manner of the extraction method of the tremella aurantialba polysaccharide by fermenting tremella aurantialba in the embodiment is the same as that of the embodiment 3, and the difference between the specific implementation manner and the embodiment 3 is that in the inoculation fermentation of the step S3, the feed-liquid ratio is 1:110.
comparative example 4
The embodiment of the present application provides a method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba, which is different from embodiment 3 in that the seed liquid inoculation amount in the inoculation fermentation of step S3 is 12% from embodiment 3.
Comparative example 5
The embodiment of the present application provides a method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba, which is different from embodiment 3 in that the seed liquid inoculation amount in the inoculation fermentation of step S3 is 3% from embodiment 3.
Performance testing
Polysaccharide content determination by sulfuric acid phenol method
1.00mL of sample to be tested is taken and placed in a test tube, 5.00mL of color development liquid is added, and the mixture is uniformly oscillated. Placing the mixture in a boiling water bath for heat preservation for 30-35 min, taking out, placing the mixture in a cold water bath, and cooling the mixture to room temperature. The absorbance was measured at 490 nm.
Determination of glucose standard curve (see fig. 3): 0.1 mg/mL glucose solutions of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mL are respectively taken and placed in test tubes with plugs, distilled water is respectively used for supplementing 1mL, then 5.00mL of developing solution is respectively added, and the vibration is uniform. Placing the mixture in a boiling water bath for heat preservation for 30-35 min, taking out, placing the mixture in a cold water bath, and cooling the mixture to room temperature. The absorbance was measured at 490 nm.
Molecular weight testing: high performance gel permeation chromatography
The molecular weight of the tremella aurantialba polysaccharide is determined by high performance liquid gel permeation chromatography (HPGPC).
The test conditions were: shimadzu LC-2010A high performance liquid chromatograph; differential refractive detector (RID); TSK-Gel G4000 SWXL (7.8 mm X300 mm) Gel chromatography column; the mobile phase is 0.1 mol/L NaN3; the temperature of the column box is 30 ℃ at the flow rate of 0.7 mL/min; the sample injection amount was 15. Mu.L. Determination of molecular weight of standard: dextran standards (10, 40, 70, 150, 270 and 410 kDa) of different molecular weights were dissolved in ultrapure water to prepare standard solutions with a concentration of 2mg/mL, and after filtration through a 0.22 μm aqueous filter membrane, the standard solutions were tested on-line according to HPGPC test conditions, and the retention times were recorded. And drawing a standard curve by taking the retention time (min) as an abscissa x and taking a logarithmic value (LogMW) of the molecular weight of the glucan standard substance as an ordinate y, and obtaining the standard curve by fitting a regression equation.
Determination of the molecular weight of tremella aurantialba polysaccharide: dissolving 4 mg golden fungus polysaccharide sample in 2 mL ultrapure water to prepare polysaccharide solution with concentration of 2mg/mL, filtering with 0.22 μm water phase filter membrane, analyzing on machine according to HPGPC test condition, recording chromatogram of each bamboo shoot polysaccharide component, and calculating molecular weight according to retention time of each polysaccharide sample on chromatogram against dextran standard molecular weight standard curve.
Coomassie brilliant blue method for measuring protein content
The coomassie brilliant blue method for determining protein content is based on the principle of protein-dye combination. Coomassie brilliant blue G-250 dye was combined with protein in an acidic solution, the position of the maximum absorption peak of the dye was changed from 465nm to 595nm, and the color of the solution was changed from brownish black to blue. The amount of protein bound thereto was determined by measuring the increase in light absorption at 595 nm.
Determination of protein standard curve (see fig. 4): accurately sucking standard protein solutions of 0, 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06. 0.06 mL into a 1mL stoppered test tube, supplementing 0.1mL with 0.15mol/L NaCl solution, adding 5 mL Coomassie brilliant blue G-250 solution respectively, shaking thoroughly, standing at room temperature for 10 min, and measuring absorbance at wavelength 595 nm.
Determination of sample protein content: taking a 0.5 mL sample solution, adding 0.15mol/L NaCl solution to complement to 1mL, operating according to standard curve preparation steps, measuring absorbance values and calculating protein content.
The tremella polysaccharide prepared in examples 1 to 7 and comparative examples 1 to 5 were subjected to the above polysaccharide content test and protein content test, and the tremella polysaccharide prepared in example 3 and comparative example 1 was subjected to the molecular weight test, and the results are shown in table 1 below.
TABLE 1
| Examples | Polysaccharide content | Molecular weight | Protein content |
| Example 1 | 86% | / | 2% |
| Example 2 | 95% | / | 0.5% |
| Example 3 | 98% | 7.6×10 5 | 0.1% |
| Example 4 | 97% | / | 0.5% |
| Example 5 | 89% | / | 1.2% |
| Example 6 | 86% | / | 1.3% |
| Example 7 | 85% | / | 2.5% |
| Comparative example 1 | 80% | 1.35×10 6 | 3.0% |
| Comparative example 2 | 86% | / | 2.8% |
| Comparative example 3 | 82% | / | 1.8% |
| Comparative example 4 | 92% | / | 0.5% |
| Comparative example 5 | 83% | / | 3.0% |
As shown in the table 1, the polysaccharide content of the tremella aurantialba polysaccharide prepared by the method is 85% -98%; the protein content is 0.1% -3%; obviously, the tremella aurantialba polysaccharide prepared by the method is high in purity and has the characteristics of high polysaccharide content, low protein content and low molecular weight. The application carries out fermentation by the Eurotium cristatum, and reasonably controls the processing technology, so that the molecular weight of the tremella aurantialba polysaccharide can be reduced, and the tremella aurantialba polysaccharide is more convenient to absorb in cosmetic application.
Moisture retention performance test
The water loss rates of the auricularia auricula polysaccharide and the sodium hyaluronate were measured after 75 hours by placing a 0.5% aqueous solution of the auricularia auricula polysaccharide and the sodium hyaluronate in a constant temperature low humidity (dry silica gel) dryer.
The auricularia auricula polysaccharide of example 3 was subjected to a moisturizing property test, and the results are shown in table 2 below.
TABLE 2
| Auricularia auricula polysaccharide | Sodium hyaluronate | |
| Initial value | 30.05 | 30 |
| After 75 hours | 22.98 | 23.56 |
| Rate of water loss | 23.5% | 24.47% |
Moisture absorption test
The auricularia auricula polysaccharide, the sodium hyaluronate powder and 0.5g of glycerin which are lyophilized to constant weight in vacuum are placed in a drier with constant temperature and humidity (25 ℃,43% relative humidity). The remaining powder was placed at room temperature and humidity, and the mass of absorbed moisture after 72 hours was measured.
The auricularia auricula polysaccharide of example 3 was subjected to a moisture absorption property test, and the results are shown in Table 3 below.
TABLE 3 Table 3
| Auricularia auricula polysaccharide | Sodium hyaluronate | Glycerol | |
| Initial value | 0.5 | 0.5 | 0.5 |
| After 75 hours | 0.55 | 0.55 | 0.65 |
| Water absorption rate | 10% | 10% | 22% |
As is clear from Table 3, the hygroscopicity of the auricle polysaccharide was comparable to that of sodium hyaluronate and tremella polysaccharide, and it was found that the auricle polysaccharide can absorb 10% of water by itself after 72 hours at low humidity (43%) and was slightly lower than that of glycerin (22%)
Antioxidant effect test
(1) Preparation of sample solutions
0.5g of tremella aurantialba polysaccharide is weighed, dissolved in distilled water, filtered and precipitated, and the precipitate is fixed in a 100mL volumetric flask.
Sample measurement
1.0mL of sample solution is precisely measured, placed in a 10mL test tube with a stopper scale, and added with ABTS + 3.0mL of free radical working solution and is placed in a dark place for 6min. The absorbance was measured at 734nm using PBS buffer as a blank.
Calculation of the Oxidation resistance of auricularia auricula polysaccharide
The oxidation resistance of the tremella aurantialba polysaccharide is calculated according to a formula (1):
free radical scavenging rate………………（1）
Wherein:
A 0 1mLPBS solution with 3.0mL ABTS + Absorbance after mixing of free radical working solution
A-1 mL sample solution and 3.0mL ABTS + Absorbance after mixing of free radical working solution
The auricularia auricula polysaccharide of example 3 was subjected to an antioxidant effect test, and the results are shown in table 4 below.
TABLE 4 Table 4
| Auricularia auricula polysaccharide | Diluting 250 times of VC | Blank control | |
| Free radical scavenging rate | 91.22% | 99.71% | 0.00% |
As can be seen from Table 4 above, the auricularia auricula polysaccharide has good oxidation resistance, and the oxidation resistance is equivalent to that of VC at the same concentration of 1/314.
It will be evident to those skilled in the art that the application is not limited to the details of the foregoing illustrative embodiments, and that the present application may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (10)
1. The extraction method of the tremella aurantialba polysaccharide by fermenting the tremella aurantialba is characterized by comprising the following specific steps of:
step S1: preparing strains: separating, purifying and domesticating golden flower bacteria growing in Fuzhuan tea to obtain domesticated bacterial strains;
step S2: preparing seed liquid: inoculating the domesticated strain into liquid culture medium, and culturing for 2-5 days;
step S3: inoculating and fermenting: pulverizing dried golden fungus, sieving with 40-80 mesh sieve, placing in a fermentation tank, adding water with a feed-liquid ratio of 1:60-100, sterilizing, inoculating golden fungus seed liquid into the fermentation tank, fermenting at 25-35deg.C and stirring speed of 100-300r/min for 2-7 days to obtain fermentation liquor;
step S4: post-treatment: centrifuging the fermentation liquor, taking supernatant, filtering and drying to obtain the tremella aurantialba polysaccharide.
2. The method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba according to claim 1, wherein in the step S1, the specific steps of separation and purification are as follows: cutting Fuzhuan tea sample, picking a piece of tea with golden flower spores, inoculating to PDA culture medium under aseptic condition, selecting golden flower bacterial strain for subculture until only golden flower bacteria are in the culture medium.
3. The method for extracting tremella aurantialba polysaccharide from fermentation of tremella aurantialba according to claim 2, wherein the PDA culture medium comprises the following raw materials: 200.0g of potato, 20.0g of glucose, 20.0g of agar and 1000mL of distilled water.
4. The method for extracting tremella aurantialba polysaccharide by fermenting tremella aurantialba according to claim 2, wherein the domesticating operation in the step S1 is as follows: inoculating purified golden flower fungus on PDA culture medium into aqueous golden ear culture medium containing sucrose, culturing at 28-30deg.C, and allowing golden flower fungus to grow over the whole plate.
5. The method for extracting tremella aurantialba polysaccharide from tremella aurantialba fermentation according to claim 4, wherein the raw materials of the tremella aurantialba aqueous solution culture medium comprise: 200g of tremella aurantialba, 1000ml of distilled water, 30g of agar and sucrose.
6. The method for extracting tremella aurantialba polysaccharide through fermentation of tremella aurantialba according to claim 4, wherein the mass content of sucrose is 0% -2% of the total amount of the culture medium raw materials.
7. The method for extracting tremella aurantialba polysaccharide through fermentation of tremella aurantialba according to claim 1, wherein in the step S2, the inoculation amount of the domesticated strain is 1% -3%.
8. The method for extracting tremella aurantialba polysaccharide from tremella aurantialba fermentation according to claim 1, wherein the inoculation amount of tremella aurantialba seed liquid in the step S3 is 5-10%.
9. The method for extracting tremella aurantialba polysaccharide by fermentation of tremella aurantialba according to claim 1, wherein in the step S4, the drying is vacuum freeze drying; vacuum degree is less than 10Pa, temperature is-50deg.C, and drying time is 12-24 h.
10. The use of tremella aurantialba polysaccharides extracted by the extraction method of tremella aurantialba polysaccharides fermented by tremella aurantialba according to any one of claims 1 to 9 in cosmetics.
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