A green extraction method for extracting grifola frondosa polysaccharide from grifola frondosa

Abstract

The present application relates to chemical and chemical technology field, and proposes a kind of green extraction method for extracting grifola frondosa polysaccharide from grifola frondosa, green extraction method for extracting grifola frondosa polysaccharide from grifola frondosa, comprising the following steps: S1, with grifola frondosa fruit body dry product as raw material, after crushing, sieving, add water and mix evenly, continue to mix after adding enzyme, obtain enzymatic hydrolysate by enzymolysis;S2, after centrifugation, take supernatant, obtain mixed solution I;S3, after alcohol extraction, centrifugation, take precipitate, polysaccharide crude product is obtained by washing precipitate with ethanol;Polysaccharide crude product is dispersed uniformly after being added into solvent II, and eluted using adsorption resin, collect eluate, concentrate, dry to obtain grifola frondosa polysaccharide.The grifola frondosa polysaccharide extracted by the green method provided in the present application has good yield and purity, and solves the technical problems of insufficient yield and extraction rate of existing grifola frondosa polysaccharide.

Description

Technical Field

[0001] This invention relates to the field of chemical engineering technology, specifically to a green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa. Background Technology

[0002] Maitake mushroom, also known as Grifola frondosa, is rich in polysaccharides, amino acids, trace elements and other active nutrients. Among them, Grifola frondosa polysaccharide, as its core functional active substance, has broad development and application prospects in functional foods, biomedicine, skin care and health care, and other fields due to its advantages of being safe, non-toxic and having stable efficacy. Currently, the industry primarily uses traditional hot water extraction and organic solvent extraction processes for Grifola frondosa polysaccharides. Hot water extraction, with its simple equipment and convenient operation, is the most widely used method. However, this method suffers from drawbacks such as high extraction temperature, high energy consumption, long extraction time, and low polysaccharide dissolution rate. Prolonged high-temperature treatment can also damage the natural spatial structure and bioactivity of Grifola frondosa polysaccharides, leading to a decline in product quality. While organic solvent extraction can improve the extraction rate to some extent, it leaves residual organic reagents, resulting in insufficient product purity, potential safety hazards for consumption and application, and the generation of large amounts of organic waste liquid, which is inconsistent with the concept of green and low-carbon production. At present, Grifola frondosa polysaccharide extraction processes are difficult to adapt to the requirements of modern, green, efficient, and high-quality large-scale production. Therefore, developing a green extraction method for Grifola frondosa polysaccharides with high yield and high purity is of great significance. Summary of the Invention

[0003] To address the above technical problems, this invention provides a green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa. The Grifola frondosa polysaccharides extracted by the green method provided by this invention have good yield and purity, solving the technical problem of insufficient yield and extraction rate of existing Grifola frondosa polysaccharides.

[0004] The specific technical solution of the present invention is as follows: According to one aspect of the present invention, a green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa is provided, comprising the following steps: S1. Using dried fruiting bodies of Grifola frondosa as raw material, after crushing and sieving, add water and mix evenly, add enzyme and continue mixing, and obtain enzymatic hydrolysate through enzymatic hydrolysis; S2. After centrifuging the enzyme hydrolysate, take the supernatant to obtain mixture I; S3. The mixture I is extracted with alcohol and centrifuged to obtain the precipitate. The precipitate is washed with ethanol to obtain crude polysaccharide. S4. After the crude polysaccharide is added to solvent II and dispersed evenly, it is eluted with adsorption resin, the eluent is collected, concentrated and dried to obtain the Grifola frondosa polysaccharide. In step S1, the enzymatic hydrolysis includes primary enzymatic hydrolysis and secondary enzymatic hydrolysis. The enzymes used in the primary and secondary enzymatic hydrolysis are different. The enzyme used in the primary enzymatic hydrolysis is cellulase, and the enzyme used in the secondary enzymatic hydrolysis is composed of papain and pectinase.

[0005] In the above technical solution, the amount of enzyme added is 1.5wt%~2.5wt% of the dried fruiting body of Grifola frondosa.

[0006] In the above technical solution, the mass ratio of papain to pectinase is 1~3:1.

[0007] In the above technical solution, the specific steps of S1 are as follows: using dried fruiting bodies of Grifola frondosa as raw material, after crushing and passing through a 60-100 mesh sieve, add water and mix evenly, add cellulase under microwave assistance and mix, perform the first enzymatic hydrolysis, then add papain and pectinase for the second enzymatic hydrolysis to obtain the enzymatic hydrolysate.

[0008] In the above technical solution, the temperature of the first enzymatic hydrolysis is 65~75℃ and the time is 1~2 min; the temperature of the second enzymatic hydrolysis is 50~55℃ and the time is 3~5 min.

[0009] In the above technical solution, step S3 includes a first alcohol extraction and a second alcohol extraction, and the solvents for the first alcohol extraction and the second alcohol extraction are different.

[0010] In the above technical solution, the solvent for the first alcohol extraction is ethanol, and the solvent for the second alcohol extraction is ethylene glycol and glycerol.

[0011] In the above technical solution, the volume ratio of ethylene glycol to glycerol is 1.5~2.5:1.

[0012] In the above technical solution, the ethanol is a 70wt% aqueous solution of ethanol.

[0013] In the above technical solution, in step S3, the adsorption resin includes a macroporous adsorption resin.

[0014] Compared with existing technologies, this invention provides a green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa. It employs two enzymatic hydrolysals: the first hydrolyze uses cellulase, while the second hydrolyze uses papain and pectinase, which improve the yield and purity of Grifola frondosa polysaccharides. The cellulase used in the first hydrolyze degrades the cellulose structure in the cell walls of Grifola frondosa fruiting bodies, promoting the full dissolution of polysaccharide components encapsulated within the cells and effectively increasing the polysaccharide extraction rate. The pectinase used in the second hydrolyze further degrades the pectin that acts as an adhesive in the cell walls, making the cell wall structure looser and facilitating polysaccharide dissolution. Papain efficiently hydrolyzes proteins bound to polysaccharides and protein impurities in the cells, reducing the obstruction of polysaccharide-protein complexes to polysaccharide dissolution. These three enzymes optimize the extraction effect from two dimensions: cell wall disruption and sugar release, and impurity removal, effectively solving the problems of low polysaccharide yield and insufficient purity in existing technologies. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention more apparent, the invention is described in detail below. It should be understood that the invention is not limited to the description herein.

[0016] dried fruiting bodies of Grifola frondosa The dried fruiting body of *Grifola frondosa* used in this invention is a known product in the art for extracting *Grifola frondosa* polysaccharides. In the green extraction method for extracting *Grifola frondosa* polysaccharides in this invention, the dried fruiting body is the core base material for extracting *Grifola frondosa* polysaccharides and serves as the source carrier of active polysaccharides.

[0017] Cellulase The cellulase used in this invention is a cellulase known in the art that can be used to extract Grifola frondosa polysaccharides. In this invention, the cellulase can hydrolyze the cellulose skeleton in the cell wall of Grifola frondosa fruiting bodies, destroy the dense structure of the cell wall, and cause the cell wall to have holes or rupture, thereby promoting the initial release of Grifola frondosa polysaccharides from the cells and creating favorable conditions for subsequent second enzymatic hydrolysis and efficient extraction.

[0018] Papain The papain used in this invention is a papain known in the art that can be used to extract Grifola frondosa polysaccharides. In this invention, the papain hydrolyzes proteins bound to Grifola frondosa polysaccharides and protein impurities in cell debris. On the one hand, it removes the obstacle of polysaccharide-protein complex to polysaccharide dissolution, and on the other hand, it achieves preliminary purification in the extraction stage, reducing protein residue in the final product, thereby effectively improving the extraction rate and purity of Grifola frondosa polysaccharides.

[0019] pectinase The pectinase used in this invention is a pectinase known in the art that can be used to extract Grifola frondosa polysaccharides. In this invention, the pectinase can hydrolyze the pectin in the middle layer and intercellular layer of Grifola frondosa cell wall, further disintegrating the matrix structure of the cell wall, making the cell wall tissue loose, thereby improving the dissolution efficiency of Grifola frondosa polysaccharides and the convenience of subsequent separation and purification.

[0020] A green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa. The present invention provides a green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, comprising the following steps: S1. Using dried fruiting bodies of Grifola frondosa as raw material, after crushing and sieving, add water and mix evenly, add enzyme and continue mixing, and obtain enzymatic hydrolysate through enzymatic hydrolysis; S2. After centrifuging the enzyme hydrolysate, take the supernatant to obtain mixture I; S3. After extraction with alcohol and centrifugation of mixture I, the precipitate is collected and washed with ethanol to obtain crude polysaccharide. S4. After the crude polysaccharide is added to solvent II and dispersed evenly, it is eluted with adsorption resin, the eluent is collected, concentrated and dried to obtain Grifola frondosa polysaccharide. In step S1, enzymatic hydrolysis includes primary enzymatic hydrolysis and secondary enzymatic hydrolysis. The enzymes used in primary and secondary enzymatic hydrolysis are different. The enzyme used in primary enzymatic hydrolysis is cellulase, while the enzymes used in secondary enzymatic hydrolysis consist of papain and pectinase.

[0021] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the amount of enzyme added is 1.5wt% to 2.5wt% of the dried fruiting body of Grifola frondosa, preferably 1.5wt% to 2wt%.

[0022] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the mass ratio of papain to pectinase is 1~3:1, preferably 1~2.5:1, more preferably 1.5~2.5:1, and most preferably 1.5~2:1.

[0023] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the specific steps of S1 are as follows: using dried Grifola frondosa fruiting bodies as raw materials, after crushing and passing through a 60-100 mesh sieve, add water and mix evenly, add cellulase under microwave assistance and mix, perform the first enzymatic hydrolysis, then add papain and pectinase for the second enzymatic hydrolysis to obtain the enzymatic hydrolysate.

[0024] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the temperature for the first enzymatic hydrolysis is 65~75℃ and the time is 1~2 min; the temperature for the second enzymatic hydrolysis is 50~55℃ and the time is 3~5 min.

[0025] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, step S3 includes a first alcohol extraction and a second alcohol extraction, with different solvents used for the first and second alcohol extractions.

[0026] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the solvent for the first alcohol extraction is ethanol, and the solvent for the second alcohol extraction is ethylene glycol and glycerol.

[0027] This invention improves the yield and purity of Grifola frondosa polysaccharides by first extracting with ethanol, followed by extraction with ethylene glycol and glycerol. Ethanol effectively reduces the solubility of polysaccharides in aqueous solution, causing most medium-molecular-weight and conventionally structured Grifola frondosa polysaccharides to precipitate. The polysaccharides remaining in the supernatant are mostly small-molecular-weight, highly polar substances that are difficult to precipitate fully with ethanol alone. The subsequent combination of ethylene glycol and glycerol has both good water solubility and precipitation properties, which can specifically capture these small- and medium-molecular-weight polar polysaccharides, thereby increasing the overall yield. At the same time, the stepwise action of the two solvents can retain a large amount of water-soluble impurities such as proteins and pigments in the liquid phase, preventing impurities from precipitating simultaneously with polysaccharides. Combined with subsequent ethanol washing and resin purification steps, the impurity content is further reduced, ultimately achieving simultaneous optimization of Grifola frondosa polysaccharide extraction rate and purity.

[0028] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the volume ratio of ethylene glycol to glycerol is 1.5~2.5:1, preferably 1.5~2:1, and more preferably 1.8~2:1.

[0029] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, the ethanol is a 70 wt% aqueous solution.

[0030] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, in step S3, the adsorption resin includes a macroporous adsorption resin.

[0031] In the green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, in step S4, the eluent used for elution is water.

[0032] To further illustrate the present invention, the following detailed description will be provided through the examples below. The raw materials used in the following examples and comparative examples of the present invention are all commercially available products, including cellulase (model: CEL01); pectinase (model: FDY-2212); papain (model: G8432); and 001×7 type macroporous adsorption resin. Extraction rate of Grifola frondosa polysaccharide (%) = (mass of Grifola frondosa polysaccharide product × purity of Grifola frondosa polysaccharide product) / mass of polysaccharides in Grifola frondosa × 100%.

[0033] Example 1 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product is pulverized and sieved through a 60-mesh sieve. It is then mixed thoroughly with water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g: 20 mL). Under microwave assistance, 0.5 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and mixed at 65°C for 2 minutes for the first enzymatic hydrolysis. After this initial hydrolysis, papain and pectinase are added (the amount of papain and pectinase added is 1 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 1:1). The mixture is then heated at 50°C. The mixture was mixed at ℃ for 5 min for a second enzymatic hydrolysis to obtain the hydrolysate. After centrifugation, the supernatant of the hydrolysate was collected to obtain mixture I. 5 times its mass of 70wt% ethanol aqueous solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was added to water and dispersed evenly, then eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 13.28%, and the purity of Grifola frondosa polysaccharide was 75.2%.

[0034] Example 2 Using dried fruiting bodies of *Grifola frondosa* as raw material, after pulverizing and passing through an 80-mesh sieve, water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g: 20 mL) is added and mixed evenly. Under microwave assistance, 0.8 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and mixed at 70℃ for 1.5 min for the first enzymatic hydrolysis. Then, papain and pectinase (the amount of papain and pectinase added is 1.2 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 1:1) are added. The mixture was mixed at 53℃ for 4 min for a second enzymatic hydrolysis to obtain the hydrolysate. After centrifugation, the supernatant of the hydrolysate was collected to obtain mixture I. 5 times its mass of 70wt% ethanol aqueous solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was added to water and dispersed evenly, then eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 13.46%, and the purity of Grifola frondosa polysaccharide was 75.6%.

[0035] Example 3 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product is pulverized and sieved through a 100-mesh sieve. It is then mixed thoroughly with water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g: 20 mL). Under microwave assistance, 1 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and the mixture is stirred at 75°C for 2 minutes for the first enzymatic hydrolysis. Following this, papain and pectinase are added (the amount of papain and pectinase added is 1.5 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 1:1). The mixture is then stirred at 5... The mixture was mixed at 5℃ for 3 min for a second enzymatic hydrolysis to obtain the hydrolysate. After centrifugation, the supernatant of the hydrolysate was collected to obtain mixture I. Five times its mass of 70wt% ethanol aqueous solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was added to water and dispersed evenly, then eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 13.51%, and the purity of Grifola frondosa polysaccharide was 75.9%.

[0036] Example 4 Using dried fruiting bodies of *Grifola frondosa* as raw material, after pulverizing and passing through an 80-mesh sieve, water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g: 20 mL) is added and mixed evenly. Under microwave assistance, 0.8 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and mixed at 70℃ for 1.5 min for the first enzymatic hydrolysis. Then, papain and pectinase (the amount of papain and pectinase added is 1.2 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 2:1) are added. The mixture was mixed at 53℃ for 4 min for a second enzymatic hydrolysis to obtain the hydrolysate. After centrifugation, the supernatant of the hydrolysate was collected to obtain mixture I. 5 times its mass of 70wt% ethanol aqueous solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was added to water and dispersed evenly, then eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 14.25%, and the purity of Grifola frondosa polysaccharide was 79.9%.

[0037] Example 5 Using dried fruiting bodies of *Grifola frondosa* as raw material, after pulverizing and passing through an 80-mesh sieve, water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g:20 mL) is added and mixed evenly. Under microwave assistance, 0.8 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and mixed at 70℃ for 1.5 min for the first enzymatic hydrolysis. Then, papain and pectinase (the amount of papain and pectinase added is 1.2 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 3:1) are added. The mixture was mixed at 53℃ for 4 min for a second enzymatic hydrolysis to obtain the hydrolysate. After centrifugation, the supernatant of the hydrolysate was collected to obtain mixture I. 5 times its mass of 70wt% ethanol aqueous solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was added to water and dispersed evenly, then eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 13.48%, and the purity of Grifola frondosa polysaccharide was 76.2%.

[0038] Example 6 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product is pulverized, passed through an 80-mesh sieve, and then mixed with water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g: 20 mL). Under microwave assistance, 0.8 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and mixed at 70°C for 1.5 min for the first enzymatic hydrolysis. Then, papain and pectinase (the amount of papain and pectinase added is 1.2 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 1:1) are added and mixed at 53°C for 4 min for the second enzymatic hydrolysis, yielding the enzymatic hydrolysate. The hydrolysate is then centrifuged, and the upper portion is collected. The supernatant was obtained by mixing the supernatant with ethanol. Five times its mass of 70 wt% aqueous ethanol solution was added to mixture I, and centrifugation was performed to obtain a precipitate and supernatant. The precipitate was washed with ethanol to obtain crude polysaccharide I. Three times its mass of ethylene glycol and glycerol (volume ratio 1.5:1) were added to the supernatant, and centrifugation was performed to obtain a precipitate. The precipitate was washed with ethanol to obtain crude polysaccharide II. Crude product I and crude product II were mixed to obtain a crude product. The crude polysaccharide was dispersed evenly in water and eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 14.18%, and the purity of Grifola frondosa polysaccharide was 79.4%.

[0039] Example 7 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product was pulverized and passed through an 80-mesh sieve. Water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water was added and mixed thoroughly. Under microwave assistance, 0.8 wt% cellulase from the dried *Grifola frondosa* fruiting bodies was added and mixed at 70°C for 1.5 min for the first enzymatic hydrolysis. Papain and pectinase (the amount of papain and pectinase added was 1.2 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase was 1:1) were added and mixed at 53°C for 4 min for the second enzymatic hydrolysis, yielding the enzymatic hydrolysate. The hydrolysate was centrifuged and then... The supernatant was used to obtain mixture I. Five times its mass of 70wt% ethanol aqueous solution was added to mixture I, and centrifuged to obtain a precipitate and supernatant. The precipitate was washed with ethanol to obtain crude polysaccharide I. Three times its mass of ethylene glycol and glycerol (volume ratio 2:1) were added to the supernatant, and centrifuged to obtain a precipitate. The precipitate was washed with ethanol to obtain crude polysaccharide II. Crude product I and crude product II were mixed to obtain a crude product. The crude polysaccharide was dispersed evenly in water and eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 14.23%, and the purity of Grifola frondosa polysaccharide was 79.8%.

[0040] Example 8 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product is pulverized, passed through an 80-mesh sieve, and then mixed with water (the mass-to-volume ratio of dried *Grifola frondosa* fruiting bodies to water is 1 g: 20 mL). Under microwave assistance, 0.8 wt% cellulase from the dried *Grifola frondosa* fruiting bodies is added and mixed at 70°C for 1.5 min for the first enzymatic hydrolysis. Then, papain and pectinase (the amount of papain and pectinase added is 1.2 wt% of the dried *Grifola frondosa* fruiting bodies; the mass ratio of papain to pectinase is 1:1) are added and mixed at 53°C for 4 min for the second enzymatic hydrolysis, yielding the enzymatic hydrolysate. The hydrolysate is then centrifuged, and the upper portion is collected. The supernatant was obtained by mixing the supernatant with ethanol. Five times its mass of 70 wt% aqueous ethanol solution was added to mixture I, and centrifugation was performed to obtain a precipitate and a supernatant. The precipitate was washed with ethanol to obtain crude polysaccharide I. Three times its mass of ethylene glycol and glycerol (volume ratio 2.5:1) were added to the supernatant, and centrifugation was performed to obtain a precipitate. The precipitate was washed with ethanol to obtain crude polysaccharide II. Crude product I and crude product II were mixed to obtain a crude product. The crude polysaccharide was dispersed evenly in water and eluted with macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain Grifola frondosa polysaccharide. The extraction rate of Grifola frondosa polysaccharide was 14.20%, and the purity of Grifola frondosa polysaccharide was 79.6%.

[0041] Comparative Example 1 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product was pulverized, passed through an 80-mesh sieve, and then mixed with water (the mass-to-volume ratio of dried fruiting bodies to water was 1 g: 20 mL). Under microwave assistance, 2 wt% cellulase from the dried fruiting bodies was added and the mixture was stirred at 70°C for 5.5 min to obtain an enzymatic hydrolysate. The hydrolysate was centrifuged, and the supernatant was collected to obtain mixture I. Five times its mass (70 wt%) of an aqueous ethanol solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was dispersed evenly in water and eluted using a macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain *Grifola frondosa* polysaccharide. The extraction rate of *Grifola frondosa* polysaccharide was 11.58%, and the purity was 69.1%.

[0042] Comparative Example 2 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product was pulverized, passed through an 80-mesh sieve, and then mixed with water (the mass-to-volume ratio of dried fruiting bodies to water was 1 g: 20 mL). Under microwave assistance, 0.8 wt% cellulase from the dried fruiting bodies was added and mixed at 70°C for 1.5 min for the first enzymatic hydrolysis. Then, 1.2 wt% papain from the dried fruiting bodies was added and mixed at 53°C for 4 min for the second enzymatic hydrolysis, yielding the hydrolysate. The hydrolysate was centrifuged, and the supernatant was collected to obtain mixture I. Five times its mass (70 wt%) of an aqueous ethanol solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was dispersed evenly in water and eluted using a macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain *Grifola frondosa* polysaccharide. The extraction rate of *Grifola frondosa* polysaccharide was 12.12%, and the purity was 70.9%.

[0043] Comparative Example 3 Using dried fruiting bodies of *Grifola frondosa* as raw material, the product was pulverized, passed through an 80-mesh sieve, and then mixed with water (the mass-to-volume ratio of dried fruiting bodies to water was 1 g: 20 mL). Under microwave assistance, 0.8 wt% cellulase from the dried fruiting bodies was added and mixed at 70°C for 1.5 min for the first enzymatic hydrolysis. Then, 1.2 wt% pectinase from the dried fruiting bodies was added and mixed at 53°C for 4 min for the second enzymatic hydrolysis, yielding the hydrolysate. The hydrolysate was centrifuged, and the supernatant was collected to obtain mixture I. Five times its mass (70 wt%) of an aqueous ethanol solution was added to mixture I, and after centrifugation, the precipitate was collected and washed with ethanol to obtain crude polysaccharide. The crude polysaccharide was dispersed evenly in water and eluted using a macroporous adsorption resin. The eluent was collected, concentrated, and dried to obtain *Grifola frondosa* polysaccharide. The extraction rate of *Grifola frondosa* polysaccharide was 12.04%, and the purity was 70.0%.

[0044] The yield and purity of Examples 1-5 were higher than those of Comparative Examples 1-3, indicating that the present invention improved the yield and purity of Grifola frondosa polysaccharide by first hydrolyzing it with cellulase and then with papain and pectinase.

[0045] The yields and purity of Examples 6-8 were higher than those of Example 2, indicating that the present invention first extracts with ethanol, and then with ethylene glycol and glycerol, thereby improving the yield and purity of Grifola frondosa polysaccharide.

[0046] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa, characterized in that, Includes the following steps: S1. Using dried fruiting bodies of Grifola frondosa as raw material, after crushing and sieving, add water and mix evenly, add enzyme and continue mixing, and obtain enzymatic hydrolysate through enzymatic hydrolysis; S2. After centrifuging the enzyme hydrolysate, take the supernatant to obtain mixture I; S3. The mixture I is extracted with alcohol and centrifuged to obtain the precipitate. The precipitate is washed with ethanol to obtain crude polysaccharide. S4. After the crude polysaccharide is added to solvent II and dispersed evenly, it is eluted with adsorption resin, the eluent is collected, concentrated and dried to obtain the Grifola frondosa polysaccharide. In step S1, the enzymatic hydrolysis includes primary enzymatic hydrolysis and secondary enzymatic hydrolysis. The enzymes used in the primary and secondary enzymatic hydrolysis are different. The enzyme used in the primary enzymatic hydrolysis is cellulase, and the enzyme used in the secondary enzymatic hydrolysis is composed of papain and pectinase.

2. The green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 1, characterized in that, The amount of enzyme added is 1.5wt% to 2.5wt% of the dried fruiting body of Grifola frondosa.

3. The green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 1, characterized in that, The mass ratio of papain to pectinase is 1~3:

1.

4. The green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 1, characterized in that, The specific steps of S1 are as follows: using dried fruiting bodies of Grifola frondosa as raw material, after crushing and passing through a 60-100 mesh sieve, add water and mix evenly, add cellulase under microwave assistance and mix, perform the first enzymatic hydrolysis, then add papain and pectinase for the second enzymatic hydrolysis to obtain the enzymatic hydrolysate.

5. A green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 4, characterized in that, The first enzymatic hydrolysis is performed at a temperature of 65-75°C for 1-2 minutes; the second enzymatic hydrolysis is performed at a temperature of 50-55°C for 3-5 minutes.

6. The green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 1, characterized in that, In step S3, the alcohol extraction includes a first alcohol extraction and a second alcohol extraction, and the solvents used in the first alcohol extraction and the second alcohol extraction are different.

7. A green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 6, characterized in that, The solvent for the first alcohol extraction is ethanol, and the solvent for the second alcohol extraction is ethylene glycol and glycerol.

8. The green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 7, characterized in that, The volume ratio of ethylene glycol to glycerol is 1.5~2.5:

1.

9. A green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 7, characterized in that, The ethanol is a 70 wt% aqueous solution of ethanol.

10. A green extraction method for extracting Grifola frondosa polysaccharides from Grifola frondosa according to claim 1, characterized in that, In step S3, the adsorption resin includes a macroporous adsorption resin.