1. The extraction method of the inonotus obliquus polysaccharide is characterized by comprising the following steps:

s1 heating the inonotus obliquus fruiting body to 50-100 ℃ through an ionic liquid water solution, and leaching for 1-2 hours to obtain a leaching solution; the mass fraction of the ionic liquid in the ionic liquid aqueous solution is 1% -10%, and the mass volume ratio of the inonotus obliquus sporocarp to the ionic liquid aqueous solution is 1: 10-1: 100;

the ionic liquid aqueous solution is any one or the combination of more than two of 1-ethyl-3-methylimidazole phosphate diethyl ester salt, 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole acetate, 1-amido-3-methylimidazole chloride salt, 1-methyl-3-methylimidazole phosphate dimethyl ester salt, 1-methyl-3-methylimidazole phosphate diethyl ester salt, 1-ethyl-3-methylimidazole phosphate dimethyl ester salt, 1-butyl-3-methylimidazole chloride salt or 1-butyl-3-methylpyridine chloride salt;

s2 enzymolysis: carrying out enzymolysis on the obtained leaching liquor by using a compound enzyme to obtain an enzymolysis extracting solution;

s3, concentrating the obtained enzymolysis extracting solution under reduced pressure to obtain an extract, adding an ethanol solution into the extract for ethanol precipitation, and drying the obtained precipitate to obtain the inonotus obliquus polysaccharide.

2. The method for extracting inonotus obliquus polysaccharide according to claim 1, wherein the enzymatic hydrolysis comprises the following steps: adjusting the pH value of the obtained leaching liquor to 5-7, adding cellulase and lysozyme, and stirring and carrying out enzymolysis for 1-2 hours at 40-50 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml.

3. The method for extracting inonotus obliquus polysaccharide according to claim 2, wherein: the enzymolysis also comprises the following steps: adjusting the pH value of enzymatic hydrolysate of cellulase and lysozyme to 3-7, adding papain with the enzyme activity of 824.6U/ml, stirring and carrying out enzymolysis for 1-2 hours at the temperature of 20-50 ℃, carrying out enzyme deactivation treatment, and centrifuging to obtain supernatant as an enzymolysis extracting solution.

4. The method for extracting inonotus obliquus polysaccharide according to claim 1, wherein: and heating the residue obtained by degreasing the inonotus obliquus sporocarp with an organic solvent by using an ionic liquid aqueous solution.

5. The method for extracting inonotus obliquus polysaccharide according to claim 1, wherein: the mass volume ratio of the organic solvent to the dried inonotus obliquus sporocarp is 5: 1-10: 1; the organic solvent is petroleum ether or/and n-ethane.

6. The method for extracting inonotus obliquus polysaccharide according to claim 1, wherein: the density of the extract is 1-1.5 g/m³The mass fraction of the ethanol solution is 95 percent, and the volume of the ethanol solution is 3 times of the volume of the extract.

7. An Inonotus obliquus polysaccharide extracted by the method for extracting Inonotus obliquus polysaccharide according to any one of claims 1-6.

Background

Inonotus obliquus (Fr.) Pilt is a rare and rare medicinal fungus, which is recorded in the Chinese herbal compilation and named as Fuscoporia obliqua, also named as Inonotus obliquus, Fuscoporia obliqua, Inonotus obliquus, Polyporus fuscus, Fuscoporia obliqua, Inonotus obliquus, and Siberian ganoderma lucidum, belongs to the biological categories of Eumycota, Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae and Aphyllophorales fuscus, is mainly parasitic under the bark of white birch, silver birch, elm, red poplar or on the withered stems after being cut, and is mainly distributed in the northern latitude of 45-50 degrees.

As early as the 12 th century, the Manchu nationalities have taken care of diseases with inonotus obliquus, and the Manchu nationalities are said to rely on inonotus obliquus to treat a variety of diseases in the great snow seal mountain, and the inonotus obliquus has been used for many years in many countries to combat the diseases. It is worth mentioning that no significant toxicity or side effects are found during the treatment of various diseases. The U.S. FDA has certified it as "the KingofHerbs" and is listed as "specific natural substances". Modern researches show that the inonotus obliquus contains the following active ingredients: polysaccharides, triterpenes (fuscoporianol, lanosterol, ergosterol, streptococcic acid, fuscoporian and the like), flavonoids, amino acids, lignins, alkaloids, polyphenols, folic acid derivatives, aromatic substances (vanillic acid, syringic acid, gamma hydroxybenzoic acid and the like), sphingolipid analogues, tripeptides and the like, and has a plurality of pharmacological activities of oxidation resistance, aging resistance, immunity regulation, tumor resistance, blood sugar and blood fat reduction, liver and kidney injury protection, virus resistance, inflammation resistance, fatigue resistance and the like.

The inonotus obliquus has good health-care efficacy and nutritional value, and has gradually received attention of scholars and consumers at home and abroad in recent years. The conventional method for extracting the inonotus obliquus polysaccharide is difficult to industrially produce due to low yield. How to improve the yield of the inonotus obliquus polysaccharide and the water solubility thereof by improving the production process is a difficult problem for preparing the inonotus obliquus polysaccharide.

In the patent publication No. CN202011478246.1, a method for extracting and purifying inonotus obliquus polysaccharide is disclosed, which comprises sequentially performing ethyl acetate and alcohol extraction, ultrasonic treatment and cellulase enzymolysis to remove cellulose, deproteinizing with papain, decolorizing with polyamide resin column and decolorizing with AB-8 resin to increase yield of inonotus obliquus polysaccharide. Those skilled in the art will recognize that: the method adopting polyamide resin column decolorization and AB-8 resin decolorization has very high loss rate of organic matters.

Disclosure of Invention

Technical problem to be solved

In view of the above disadvantages and shortcomings of the prior art, the invention provides an inonotus obliquus polysaccharide and an extraction method thereof, which can effectively improve the yield of the inonotus obliquus polysaccharide, especially can remarkably improve the extraction rate of water-soluble inonotus obliquus polysaccharide, by combining an ionic liquid with a complex enzyme enzymolysis method.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, an embodiment of the present invention provides a method for extracting inonotus obliquus polysaccharide, which includes the following steps:

s1 heating the inonotus obliquus fruiting body to 50-100 ℃ through an ionic liquid water solution, and leaching for 1-2 hours to obtain a leaching solution; the mass fraction of the ionic liquid in the ionic liquid aqueous solution is 1% -10%, and the mass volume ratio of the inonotus obliquus sporocarp to the ionic liquid aqueous solution is 1: 10-1: 100;

the ionic liquid aqueous solution is any one or the combination of more than two of 1-ethyl-3-methylimidazole phosphate diethyl ester salt, 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole acetate, 1-amido-3-methylimidazole chloride salt, 1-methyl-3-methylimidazole phosphate dimethyl ester salt, 1-methyl-3-methylimidazole phosphate diethyl ester salt, 1-ethyl-3-methylimidazole phosphate dimethyl ester salt, 1-butyl-3-methylimidazole chloride salt or 1-butyl-3-methylpyridine chloride salt;

s2 enzymolysis: carrying out enzymolysis on the obtained leaching liquor by using a compound enzyme to obtain an enzymolysis extracting solution;

s3, concentrating the obtained enzymolysis extracting solution under reduced pressure to obtain an extract, adding an ethanol solution into the extract for ethanol precipitation, and drying the obtained precipitate to obtain the inonotus obliquus polysaccharide.

The ionic liquid is a salt which is in a liquid state at or near room temperature and is completely composed of anions and cations, and is also called low-temperature molten salt. The main reason why the ionic liquid is used as an ionic compound and has a low melting point is that ions cannot be regularly accumulated into crystals due to the asymmetry of certain substituents in the structure of the ionic liquid. It is generally composed of an organic cation and an inorganic or organic anion. The ionic liquid in the scheme of the invention can form hydrogen bonds with the inonotus obliquus polysaccharide, can effectively promote the dissolution of wood fibers, has good biocompatibility with enzyme and promotes the dissolution rate of the polysaccharide.

In the scheme of the invention, the ionic liquid can effectively promote the dissolution of wood fiber after being heated, has good biocompatibility with enzyme, and effectively improves the dissolution rate of polysaccharide in the enzymolysis stage of the compound enzyme, thereby improving the extraction rate of the inonotus obliquus polysaccharide. The inonotus obliquus polysaccharide is released and dissolved in water along with the ionic liquid in a composite enzymolysis and degradation mode under the action of strong hydrogen bond with the ionic liquid, so that non-polysaccharide substances can be removed in a centrifugal filtration mode. Wherein, the compound enzyme can be any enzyme combination applied to the field of polysaccharide; preferably a complex enzyme which can degrade the cell wall.

In an optional scheme of the extraction method of the inonotus obliquus polysaccharide, the enzymolysis comprises the following steps: adjusting the pH value of the obtained leaching liquor to 5-7, adding cellulase and lysozyme, and stirring and carrying out enzymolysis for 1-2 hours at 40-50 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml.

The invention preferably adopts cellulase and lysozyme as complex enzyme for enzymolysis, wherein, the invention particularly uses lysozyme, which mainly breaks beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall to decompose insoluble mucopolysaccharide of the cell wall into soluble glycopeptide with biological and pharmacological activity; cellulose is degraded into glucose by cellulase; the application can degrade the cell wall rapidly through the combination of cellulase and lysozyme, generate soluble glycopeptide and polysaccharide simultaneously, do benefit to and separate with macromolecular organic matter, can avoid the too big purification of attrition rate such as column chromatography.

In an optional scheme of the extraction method of the inonotus obliquus polysaccharide, the enzymolysis further comprises the following steps of: adjusting the pH value of enzymatic hydrolysate of cellulase and lysozyme to 3-7, adding papain with the enzyme activity of 824.6U/ml, stirring and carrying out enzymolysis for 1-2 hours at the temperature of 20-50 ℃, carrying out enzyme deactivation treatment, and centrifuging to obtain supernatant as an enzymolysis extracting solution.

The protein can be further degraded by enzymatic hydrolysate of the cellulase and the lysozyme through papain to remove the protein, wherein, preferably, the enzymatic hydrolysate of the cellulase and the lysozyme is added with the papain after being filtered and deslagged; papain degrades residual macromolecular protein and improves the purity of polysaccharide molecules. Under the enzymolysis condition, macromolecular protein and polysaccharide molecules can be separated into insoluble macromolecular polypeptide, and the protein can be effectively removed by a centrifugal method.

In an optional scheme of the extraction method of the inonotus obliquus polysaccharide, residues obtained by degreasing the inonotus obliquus sporocarp with an organic solvent are subjected to heating treatment with an ionic liquid aqueous solution.

Preferably, in step S1, dried inonotus obliquus fruit bodies are taken, pulverized and sieved through a 100-mesh sieve.

In an optional scheme of the extraction method of the inonotus obliquus polysaccharide, the mass volume ratio of the organic solvent to the dried inonotus obliquus sporocarp is 5: 1-10: 1; the organic solvent is petroleum ether or/and n-ethane.

In an optional scheme of the extraction method of the inonotus obliquus polysaccharide, the density of the extract is 1-1.5 g/m³The mass fraction of the ethanol solution is 95 percent, and the volume of the ethanol solution is 3 times of the volume of the extract.

In a second aspect, the present invention also provides the inonotus obliquus polysaccharide obtained in any of the above embodiments.

(III) advantageous effects

The invention has the beneficial effects that:

according to the extraction method of the inonotus obliquus polysaccharide, the extraction rate of the inonotus obliquus polysaccharide can be effectively improved in a mode of heating and leaching of ionic liquid and enzymolysis of compound enzyme, and the polysaccharide content in the obtained dried inonotus obliquus polysaccharide is more than 70%; compared with the traditional ultrasonic-assisted extraction method, microwave-assisted extraction method and hot water extraction method, the extraction rate can be improved by 2-10%; the method is simple, easy to operate and convenient for industrial production;

the ionic liquid can form hydrogen bonds with the polysaccharide, can effectively promote dissolution of the wood fiber, has good biocompatibility with enzyme, and promotes dissolution rate of the polysaccharide.

The invention preferably adopts cellulase and lysozyme which are combined in the ionic liquid to efficiently degrade macromolecules, and breaks glycosidic bonds in cell walls to decompose insoluble mucopolysaccharides in the cell walls into soluble polysaccharides.

Detailed Description

For a better understanding of the present invention, reference will now be made in detail to the present invention by way of specific embodiments thereof.

According to the extraction method of the inonotus obliquus polysaccharide provided by the embodiment of the invention, the inonotus obliquus polysaccharide is released and dissolved in water along with the ionic liquid in a composite enzymolysis degradation mode under the action of strong hydrogen bond with the ionic liquid by a method of heating and leaching the ionic liquid aqueous solution and a method of composite enzyme hydrolysis in sequence, so that non-polysaccharide substances can be removed in a centrifugal filtration mode.

In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. While the following shows exemplary embodiments of the invention, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Example 1

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

s1 oven drying Fuscoporia obliqua fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried Fuscoporia obliqua fruiting body, pulverizing, and sieving with 100 mesh sieve; adding a 5 mass percent aqueous solution of 1-ethyl-3-methylimidazolium diethyl phosphate salt into dried inonotus obliquus fruiting bodies according to the mass-volume ratio of 1: 50, heating to 80 ℃, and leaching for 1 hour to obtain a leaching solution;

s2 enzymolysis: cooling the obtained leaching solution to 45 ℃, adjusting the pH value to 6, adding cellulase and lysozyme, and stirring and performing enzymolysis for 1.5 hours at 40 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml; filtering after enzymolysis to obtain supernatant as an enzymolysis extracting solution;

s3 concentrating the obtained enzymolysis extractive solution under reduced pressure to obtain paste with density of 1g/m³Adding an ethanol solution with the mass fraction of 95% and the volume of 3 times that of the extract into the extract, carrying out alcohol precipitation for 12 hours, repeating the steps for 2 times, collecting precipitates, cleaning the precipitates with acetone for 2 times, drying the precipitates in a freezing spray dryer at the temperature of-30 ℃ and the pressure of not higher than 0.05MPa, freezing for 12 hours, and drying to obtain the inonotus obliquus polysaccharide.

In the embodiment, the inonotus obliquus sporocarp is subjected to heating leaching by an ionic liquid aqueous solution, and enzymolysis by a complex enzyme of cellulase and lysozyme is carried out to obtain the inonotus obliquus polysaccharide with higher purity. The content of polysaccharide in the inonotus obliquus polysaccharide is 70.6%, and the yield is 36.3%.

Example 2

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

s1 oven drying Fuscoporia obliqua fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried Fuscoporia obliqua fruiting body, pulverizing, and sieving with 100 mesh sieve; adding a 5 mass percent aqueous solution of 1-ethyl-3-methylimidazolium diethyl phosphate salt into dried inonotus obliquus fruiting bodies according to the mass-volume ratio of 1: 50, heating to 80 ℃, and leaching for 1 hour to obtain a leaching solution;

s2 enzymolysis: cooling the obtained leaching solution to 45 ℃, adjusting the pH value to 6, adding cellulase and lysozyme, and stirring and performing enzymolysis for 1.5 hours at 40 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml; carrying out enzymolysis to obtain a compound enzymolysis liquid;

s3, adding papain into the compound enzyme enzymolysis liquid according to the enzyme activity of 824.6U/ml, stirring and carrying out enzymolysis for 1.5 hours at the temperature of 40 ℃, heating to 100 ℃ after the enzymolysis is finished, keeping for 2 minutes for carrying out enzyme deactivation treatment, and centrifuging for 10 minutes at 6000r/min to obtain supernatant as an enzymolysis extracting solution;

s4 concentrating the obtained enzymolysis extractive solution under reduced pressure to obtain paste with density of 1g/m³Adding an ethanol solution with the mass fraction of 95% and the volume of 3 times that of the extract into the extract, carrying out alcohol precipitation for 12 hours, repeating the steps for 2 times, collecting precipitates, cleaning the precipitates with acetone for 2 times, drying the precipitates in a freezing spray dryer at the temperature of-30 ℃ and the pressure of not higher than 0.05MPa, freezing for 12 hours, and drying to obtain the inonotus obliquus polysaccharide.

In the embodiment, the inonotus obliquus sporocarp is subjected to heating leaching by an ionic liquid aqueous solution, the polysaccharides are degraded by enzymolysis of a compound enzyme of cellulase and lysozyme, and the proteins are removed by papain, so that the inonotus obliquus polysaccharides with high purity are obtained. The content of polysaccharide in the inonotus obliquus polysaccharide is 76.8%, and the yield is 33.6%.

Example 3

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

s1 oven drying Fuscoporia obliqua fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried Fuscoporia obliqua fruiting body, pulverizing, and sieving with 100 mesh sieve; adding a 5 mass percent aqueous solution of 1-ethyl-3-methylimidazolium diethyl phosphate salt into dried inonotus obliquus fruiting bodies according to the mass-volume ratio of 1: 50, heating to 80 ℃, and leaching for 1 hour to obtain a leaching solution;

s2 enzymolysis: cooling the obtained leaching solution to 45 ℃, adjusting the pH value to 6, adding cellulase and lysozyme, and stirring and performing enzymolysis for 1.5 hours at 40 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml; filtering after enzymolysis to obtain supernatant as complex enzyme enzymolysis liquid;

s3, adding papain into the compound enzyme enzymolysis liquid according to the enzyme activity of 824.6U/ml, stirring and carrying out enzymolysis for 1.5 hours at the temperature of 40 ℃, heating to 100 ℃ after the enzymolysis is finished, keeping for 2 minutes for carrying out enzyme deactivation treatment, and centrifuging for 10 minutes at 6000r/min to obtain supernatant as an enzymolysis extracting solution;

s4 concentrating the obtained enzymolysis extractive solution under reduced pressure to obtain paste with density of 1g/m³Adding ethanol with the mass fraction of 95 percent and the volume of 3 times of the extract into the extractPrecipitating the solution with ethanol for 12 hr, repeating for 2 times, collecting precipitate, cleaning with acetone for 2 times, drying in a freeze spray dryer at-30 deg.C under pressure of not higher than 0.05MPa for 12 hr, and drying to obtain Inonotus obliquus polysaccharide.

The embodiment passes through ionic liquid aqueous solution with inonotus obliquus fruit body and heats the leaching to and the compound enzyme enzymolysis degradation polysaccharide of cellulase and lysozyme, after filtering and getting rid of the macromolecule insoluble substance, and through papain degradation protein to macromolecule polypeptide, and after filtering the macromolecule polypeptide through the centrifugation, obtain the higher inonotus obliquus polysaccharide of purity. The content of polysaccharide in the inonotus obliquus polysaccharide is 79.49%, and the yield is 30.05%.

Example 4

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

s1 drying inonotus obliquus fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried inonotus obliquus fruiting body, pulverizing, sieving with 100 mesh sieve, adding petroleum ether, defatting under reflux twice, defatting under reflux for 1 hr each time, the mass volume ratio of petroleum ether to inonotus obliquus powder is 5:1, filtering to obtain residue, and drying until water content is 8%;

adding 5 mass percent of 1-ethyl-3-methylimidazolium diethyl phosphate salt aqueous solution into the dried residues according to the mass-volume ratio of 1: 50, heating to 80 ℃, and leaching for 1 hour to obtain leaching liquor;

s2 enzymolysis: cooling the obtained leaching solution to 45 ℃, adjusting the pH value to 6, adding cellulase and lysozyme, and stirring and performing enzymolysis for 1.5 hours at 40 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml; filtering after enzymolysis to obtain supernatant as complex enzyme enzymolysis liquid;

s3, adding papain into the compound enzyme enzymolysis liquid according to the enzyme activity of 824.6U/ml, stirring and carrying out enzymolysis for 1.5 hours at the temperature of 40 ℃, heating to 100 ℃ after the enzymolysis is finished, keeping for 2 minutes for carrying out enzyme deactivation treatment, and centrifuging for 10 minutes at 6000r/min to obtain supernatant as an enzymolysis extracting solution;

s4 concentrating the obtained enzymolysis extractive solution under reduced pressure to obtain paste densityIs 1g/m³Adding an ethanol solution with the mass fraction of 95% and the volume of 3 times that of the extract into the extract, carrying out alcohol precipitation for 12 hours, repeating the steps for 2 times, collecting precipitates, cleaning the precipitates with acetone for 2 times, drying the precipitates in a freezing spray dryer at the temperature of-30 ℃ and the pressure of not higher than 0.05MPa, freezing for 12 hours, and drying to obtain the inonotus obliquus polysaccharide.

This embodiment loops through the degreasing of petroleum ether backward flow with the inonotus obliquus fruit body and passes through ionic liquid aqueous solution heating leaching to and the compound enzyme enzymolysis degradation polysaccharide of cellulase and lysozyme, after filtering and getting rid of the macromolecule insoluble substance, and degrade albumen to macromolecular polypeptide through papain, and filter behind the macromolecular polypeptide through the centrifugation, obtain the higher inonotus obliquus polysaccharide of purity. The content of polysaccharide in the inonotus obliquus polysaccharide is 81.05%, and the yield is 28.05%.

Through analysis, the inonotus obliquus polysaccharide obtained in the embodiments 1-4 of the invention contains glycopeptides, and also contains mannose, rhamnose, glucose, galactose, xylose and arabinose.

Comparative example 1

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

the other points are different from example 4 in that: s1 drying inonotus obliquus fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried inonotus obliquus fruiting body, pulverizing, sieving with 100 mesh sieve, adding petroleum ether, defatting under reflux twice, defatting under reflux for 1 hr each time, the mass volume ratio of petroleum ether to inonotus obliquus powder is 5:1, filtering to obtain residue, and drying until water content is 8%; adding distilled water into the dried residue according to the mass-to-volume ratio of 1: 50, heating to 80 ℃, and leaching for 1 hour to obtain a leaching solution.

The content of polysaccharide in the inonotus obliquus polysaccharide obtained in comparative example 1 was 75.78%, and the yield was 20.13%.

Example 5

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

s1 drying inonotus obliquus fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried inonotus obliquus fruiting body, pulverizing, sieving with 100 mesh sieve, adding n-ethane, reflux-defatting twice, reflux-defatting for 1 hr each time, the mass volume ratio of petroleum ether to inonotus obliquus powder is 10:1, filtering to obtain residue, and drying until water content is 8%;

adding 10% by mass of a mixed aqueous solution of 1-ethyl-3-methylimidazolium diethyl phosphate and 1-amido-3-methylimidazolium chloride in a mass ratio of 1: 2 into the dried residue according to a mass-volume ratio of 1: 70, and heating to 50 ℃ for leaching for 1.5 hours to obtain a leaching solution;

s2 enzymolysis: adjusting the pH value of the obtained leaching liquor to 5.5, adding cellulase and lysozyme, and stirring for enzymolysis for 1 hour at 48 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml; filtering after enzymolysis to obtain supernatant as complex enzyme enzymolysis liquid;

s3, adjusting the pH value of the complex enzyme enzymatic hydrolysate to 4, adding papain according to the enzyme activity of 824.6U/ml, stirring and carrying out enzymolysis for 2 hours at the temperature of 35 ℃, heating to 100 ℃ after the enzymolysis is finished, keeping for 3 minutes for carrying out enzyme deactivation treatment, and centrifuging for 15 minutes at 8000r/min to obtain supernatant which is an enzymolysis extracting solution;

s4 concentrating the obtained enzymolysis extractive solution under reduced pressure to obtain paste with density of 1.5g/m³And (2) adding an ethanol solution with the mass fraction of 95% and the volume of 3 times that of the extract into the extract for alcohol precipitation to obtain a precipitate, drying the precipitate in a freeze spray dryer at the temperature of-30 ℃, the pressure of not higher than 0.05MPa and the freezing time of not less than 12 hours, and drying to obtain the inonotus obliquus polysaccharide.

The content of polysaccharide in the inonotus obliquus polysaccharide obtained in the example is 77.18%, and the yield is 26.13%.

Example 6

The extraction method of the inonotus obliquus polysaccharide comprises the following steps:

s1 drying inonotus obliquus fruiting body at 40 deg.C under reduced pressure until water content is 10% to obtain dried inonotus obliquus fruiting body, pulverizing, sieving with 100 mesh sieve, adding petroleum ether, defatting under reflux twice, defatting under reflux for 1 hr each time, the mass volume ratio of petroleum ether to inonotus obliquus powder is 5:1, filtering to obtain residue, and drying until water content is 8%;

adding 7 mass percent of mixed aqueous solution of 1-butyl-3-methylimidazole acetate, 1-amido-3-methylimidazole chloride, 1-butyl-3-methylimidazole chloride or 1-butyl-3-methylpyridine chloride in a mass ratio of 1: 3 into the dried residue according to a mass-volume ratio of 1: 15, and heating to 100 ℃ for leaching for 2 hours to obtain leaching liquor;

s2 enzymolysis: adjusting the pH value of the obtained leaching liquor to 7, adding cellulase and lysozyme, and stirring for enzymolysis for 2 hours at 40 ℃; in the leaching liquor, the activity of the cellulase is 58.8U/ml, and the activity of the lysozyme is 70.5U/ml; the supernatant obtained by filtering after enzymolysis is the complex enzyme enzymolysis liquid,

s3, adjusting the pH value of the complex enzyme enzymatic hydrolysate to 7, adding papain according to the enzyme activity of 824.6U/ml, adding papain according to the enzyme activity of 824.6U/ml, stirring and performing enzymolysis at the temperature of 50 ℃ for 1 hour, heating to 100 ℃ after the enzymolysis is finished, keeping the temperature for 1 minute for enzyme deactivation, and centrifuging at 4000r/min for 5 minutes to obtain supernatant as an enzymolysis extracting solution;

s4 concentrating the obtained enzymolysis extractive solution under reduced pressure to obtain paste with density of 1.3g/m³And (2) adding an ethanol solution with the mass fraction of 95% and the volume of 3 times that of the extract into the extract for alcohol precipitation to obtain a precipitate, drying the precipitate in a freeze spray dryer at the temperature of-30 ℃, the pressure of not higher than 0.05MPa and the freezing time of not less than 12 hours, and drying to obtain the inonotus obliquus polysaccharide.

The content of polysaccharide in the inonotus obliquus polysaccharide obtained in the example is 75.36%, and the yield is 23.89%.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.