A method for extracting hericium erinaceus polysaccharide

Abstract

The application relates to the technical field of polysaccharide extraction, and discloses an extraction method of Hericium erinaceus polysaccharide, which comprises the following steps: preparing a Hericium erinaceus polysaccharide crude extract; constructing a three-phase system; three-phase distribution; phase separation and polysaccharide recovery; and polysaccharide purification. In the application scheme, specific three-phase distribution systems can be used to more effectively separate polysaccharides, proteins and fat-soluble impurities, and the extraction efficiency and the purity of the Hericium erinaceus polysaccharide can be improved by optimizing the extraction process.

Description

A method for extracting polysaccharides from Hericium erinaceus. Technical Field

[0001] This application relates to the field of polysaccharide extraction technology, and mainly to a method for extracting polysaccharides from Hericium erinaceus. Background Technology

[0002] Traditional extraction of Hericium erinaceus polysaccharides typically employs water extraction and alcohol precipitation. The resulting polysaccharide solution contains numerous impurities, such as proteins, pigments, small-molecule sugars, and inorganic salts. These impurities not only affect the purity of the polysaccharides but may also interfere with their biological activity. Therefore, purification of the crude extract is necessary. Traditional separation and purification methods, such as alcohol precipitation, activated carbon adsorption, ion exchange resin methods, and ultrafiltration, while capable of removing some impurities to a certain extent, have several limitations. Alcohol precipitation is not ideal for removing proteins, easily leading to excessive protein content in the polysaccharide product. Activated carbon adsorption, while removing impurities such as pigments, has poor selectivity, easily adsorbing some polysaccharides, resulting in product loss, and the regeneration and disposal of activated carbon is also problematic. Ion exchange resin methods are complex to operate and costly. Ultrafiltration easily clogs the membrane, affecting separation efficiency. These traditional purification methods suffer from high cost, low efficiency, and severe environmental pollution. Therefore, developing an efficient, economical, and environmentally friendly method for extracting and purifying Hericium erinaceus polysaccharides is of great significance. Aqueous two-phase extraction (APE), as a novel separation technique, boasts advantages such as simple operation, low cost, and environmental friendliness, and holds broad application prospects in the field of bioseparation. However, when using APE for the purification of Hericium erinaceus polysaccharides, challenges remain, including selecting a suitable phase system and optimizing extraction conditions, requiring further in-depth research.

[0003] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a method for extracting Hericium erinaceus polysaccharide, which aims to improve the extraction efficiency of Hericium erinaceus polysaccharide.

[0005] The technical solution of this application is as follows:

[0006] A method for extracting polysaccharides from Hericium erinaceus, comprising the following steps:

[0007] Preparation of crude extract of Hericium erinaceus polysaccharide;

[0008] Constructing a three-phase system: PEG and sulfate were added to the crude extract of Hericium erinaceus polysaccharide to achieve final concentrations of 5-15 w / w% and 10-20 w / w, respectively; chloroform was added to achieve a final concentration of 5-15 w / w%; and the mixture was thoroughly mixed.

[0009] Three-phase separation: Place the three-phase system in a separatory funnel and allow it to stand at 20-30℃ for 1.5-2.5 hours to separate into layers;

[0010] Phase separation and polysaccharide recovery: Separating the three phases and collecting the upper phase:

[0011] Polysaccharide purification: The superphase was precipitated with anhydrous ethanol, and the precipitate was collected by centrifugation; the precipitate was dissolved in deionized water, dialyzed, and freeze-dried to obtain the Hericium erinaceus polysaccharide.

[0012] In this application, by adopting a specific three-phase partitioning system, polysaccharides, proteins and fat-soluble impurities can be separated more effectively, and by optimizing the extraction process, the extraction efficiency and the purity of Hericium erinaceus polysaccharides can be improved.

[0013] The method for extracting Hericium erinaceus polysaccharides, wherein the process of preparing the crude extract of Hericium erinaceus polysaccharides includes the following steps:

[0014] The dried Hericium erinaceus was pulverized and passed through a 50-80 mesh sieve. Deionized water was added at a material-to-liquid ratio of 1g:15-20mL. Microwave power was set to 250-350W, duty cycle to 50%, and frequency to 1Hz. Extraction was carried out at 75-85℃ for 1.5-2.5 hours, and the extraction was repeated twice. The extracts were combined and centrifuged to remove the residue, thus obtaining the crude extract of Hericium erinaceus polysaccharides.

[0015] In this step, microwave radiation can accelerate the rupture of cell walls and promote the release of Hericium erinaceus polysaccharides, thereby improving extraction efficiency while ensuring that the polysaccharides are fully dissolved.

[0016] The method for extracting Hericium erinaceus polysaccharide, wherein the PEG is one of PEG6000, PEG4000, PEG8000, and PEG10000;

[0017] The sulfate is one of ammonium sulfate, potassium sulfate, and magnesium sulfate.

[0018] The method for extracting Hericium erinaceus polysaccharides, wherein the PEG is modified PEG, and the preparation method of the modified PEG includes the following steps:

[0019] PEG was mixed with octenyl succinic anhydride and added to dichloromethane. The catalyst 4-dimethylaminopyridine was added, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was washed three times with ethyl acetate and dried under vacuum to obtain the modified PEG.

[0020] The molar ratio of PEG to octenyl succinic anhydride is 1:0.1;

[0021] The amount of dichloromethane used is such that the concentration of the PEG is 10 w / v%.

[0022] The molar ratio of PEG to 4-dimethylaminopyridine is 1:0.01;

[0023] The PEG is one of PEG6000, PEG4000, PEG8000, and PEG10000;

[0024] The sulfate is one of ammonium sulfate, potassium sulfate, and magnesium sulfate.

[0025] The method for extracting Hericium erinaceus polysaccharides, wherein the process of constructing the three-phase system further includes the following steps:

[0026] Add stabilizer Span 80 to achieve a final concentration of 0.1-0.3 w / w%.

[0027] The method for extracting Hericium erinaceus polysaccharide, wherein, before purifying the polysaccharide, the method further includes the following steps:

[0028] Combine the mesophase and chloroform phase, add an equal volume of PEG aqueous solution to the upper phase, perform three-phase partitioning again, collect the upper phase, repeat the extraction of the mesophase and chloroform phase 1-3 times, and combine all the upper phases.

[0029] The PEG aqueous solution is obtained by mixing PEG with deionized water, and the concentration of PEG in the PEG aqueous solution is 5 w / w.

[0030] The method for extracting Hericium erinaceus polysaccharides, wherein the process of precipitating the superphase with anhydrous ethanol includes the following steps:

[0031] Add the anhydrous ethanol to the upper phase to make the final concentration of ethanol 40 v / v%, let it stand at 4°C for 2 hours, centrifuge to remove the precipitate, and retain the supernatant;

[0032] Continue to add anhydrous ethanol to the supernatant to make the final concentration of ethanol 70 v / v%, let it stand at 4°C for 2 hours, and then collect the precipitate by centrifugation.

[0033] The precipitate was washed several times with a 70% ethanol solution.

[0034] The method for extracting Hericium erinaceus polysaccharides, wherein a 3500 Da dialysis membrane is used during the dialysis process.

[0035] The method for extracting Hericium erinaceus polysaccharides, wherein the centrifugation process is performed by centrifuging at 3500-4500 r / min for 10-15 min.

[0036] The method for extracting Hericium erinaceus polysaccharides, wherein the process of centrifuging to remove residue is centrifuging at 3500-4000 r / min for 10-15 min.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] In this application, by adopting a specific three-phase partitioning system, polysaccharides, proteins and fat-soluble impurities can be separated more effectively, and by optimizing the extraction process, the extraction efficiency and the purity of Hericium erinaceus polysaccharides can be improved. Detailed Implementation

[0039] To facilitate understanding of this application, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of this application.

[0040] This application provides a method for extracting polysaccharides from Hericium erinaceus, mainly targeting the extraction of Hericium erinaceus polysaccharides, including the following steps:

[0041] Step 1: Prepare crude extract of Hericium erinaceus polysaccharide.

[0042] First, the polysaccharides of Hericium erinaceus are initially extracted through water extraction.

[0043] Specifically, the dried Hericium erinaceus is pulverized, passed through a 50-80 mesh sieve, and deionized water is added at a material-to-liquid ratio of 1:15-20 (g / mL). The mixture is placed in a microwave extractor, and the microwave power is set to 250-350W. Extraction is carried out at 75-85℃ for 1.5-2.5 hours, and the extraction is repeated twice. The extracts are combined and centrifuged (3500-4000 r / min, 10-15 min) to remove the residue, thus obtaining a crude extract of Hericium erinaceus polysaccharides.

[0044] In this step, water bath extraction at 75-85℃ can effectively dissolve polysaccharides. Microwave extraction for 1.5-2.5 hours can improve extraction efficiency while ensuring full dissolution of polysaccharides. Extraction twice can increase the extraction rate. Centrifugation at 4000r / min for 10-15 minutes can effectively remove residues.

[0045] In this step, microwave radiation accelerates cell wall rupture and promotes the release of Hericium erinaceus polysaccharides. Preferably, the microwave extractor is set to pulsed microwave mode with a microwave power of 300W, a duty cycle of 50%, and a frequency of 1Hz. Using pulsed microwave mode and optimizing the duty cycle and frequency reduces the risk of localized overheating. Pulsed microwave mode provides microwave energy intermittently, lowering the average temperature of the extraction system, reducing the risk of localized overheating, and minimizing Hericium erinaceus polysaccharide degradation. Here, a 50% duty cycle means that the microwave on and off times are equal, and a 1Hz frequency means that the microwave is turned on and off once per second. By optimizing the duty cycle and frequency, the risk to Hericium erinaceus polysaccharides can be minimized while ensuring extraction efficiency.

[0046] Step 2: Construct a three-phase system.

[0047] Specifically, step 2 includes the following steps:

[0048] Modified PEG and ammonium sulfate were added to the crude extract of Hericium erinaceus polysaccharide to achieve final concentrations of 5-15% (w / w) and 10-20% (w / w), respectively.

[0049] Add chloroform to bring the final concentration to 5-15% (w / w);

[0050] Add stabilizer Span 80 to a final concentration of 0.1-0.3% (w / w);

[0051] Mix thoroughly and evenly.

[0052] The main purpose of step 2 is to form a three-phase system capable of separating polysaccharides, proteins, and lipid-soluble impurities. Modified PEG and ammonium sulfate form the aqueous phase, while chloroform forms the organic phase. The core of this process lies in utilizing three-phase partitioning technology to achieve efficient separation and purification of Hericium erinaceus polysaccharides. Compared to aqueous two-phase extraction, three-phase partitioning can more effectively separate polysaccharides, proteins, and lipid-soluble impurities. Polysaccharides are mainly partitioned into the upper phase rich in modified PEG, lipid-soluble impurities and proteins are mainly partitioned into the chloroform phase, and small molecule impurities are mainly partitioned into the intermediate phase.

[0053] The separation effect can be further improved by optimizing the ratio of modified PEG, ammonium sulfate, chloroform and stabilizer Span 80.

[0054] In this application, the concentration of modified PEG is preferably 5-15% (w / w). A higher concentration of modified PEG is beneficial to the dissolution of polysaccharides, but an excessively high concentration of modified PEG will increase the viscosity of the system and affect mass transfer.

[0055] In this application, the concentration of ammonium sulfate is preferably 10-20% (w / w). A higher concentration of ammonium sulfate is beneficial for protein precipitation, but an excessively high concentration of ammonium sulfate will reduce the solubility of polysaccharides.

[0056] In this application, the concentration of chloroform is preferably 5-15% (w / w). The proportion of chloroform also needs to be optimized; too low a proportion cannot effectively remove fat-soluble impurities and proteins, while too high a proportion will increase costs. Chloroform is a commonly used organic solvent that can dissolve fat-soluble impurities and proteins. In this application, chloroform is introduced to form a three-phase system, which can effectively remove fat-soluble impurities and proteins, thereby improving the purity of polysaccharides.

[0057] In this application, the stabilizer Span 80 is added, and the concentration of Span 80 is preferably 0.1-0.3% (w / w). Span 80 is a nonionic surfactant that can reduce interfacial tension, prevent interfacial blurring, and improve the stability of the three-phase system. Span 80 has a low hydrophilic-lipophilic balance (HLB) value, which is beneficial for its adsorption at the oil-water interface, forming a stable emulsion and preventing phase separation.

[0058] In this application, modified PEG is used instead of traditional PEG. Specifically, the preparation method of modified PEG includes the following steps:

[0059] Commercially available PEG6000 was mixed with octenyl succinic anhydride and added to dichloromethane. The catalyst 4-dimethylaminopyridine (DMAP) was added, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was washed three times with ethyl acetate and dried under vacuum to obtain modified PEG (OSA-PEG).

[0060] The molar ratio of PEG6000 to octenyl succinic anhydride is 1:0.1; the amount of dichloromethane used is such that the concentration of PEG6000 is 10% (w / v); and the molar ratio of PEG to DMAP is 1:0.01.

[0061] Octenyl succinic anhydride is a food-grade emulsifier. Modifying PEG with octenyl succinic anhydride imparts a degree of hydrophobicity. Compared to ordinary polyethylene glycol, using OSA-PEG can improve the selectivity and stability of the three-phase system, enhance the distribution of polysaccharides in the upper phase, and promote the precipitation of impurities such as proteins in the lower phase.

[0062] Besides PEG6000, polyethylene glycol can also be replaced by PEG4000, PEG8000, and PEG10000.

[0063] Besides ammonium sulfate, potassium sulfate and magnesium sulfate can also be used as substitutes for sulfates.

[0064] Step 3: Three-phase distribution.

[0065] Specifically, the constructed three-phase system is placed in a separatory funnel and allowed to stand at 20-30℃ for 1.5-2.5 hours to allow it to separate into layers.

[0066] The main purpose of step 3 is to ensure the full distribution of polysaccharides, proteins, and lipid-soluble impurities in the three phases. In this step, maintaining the temperature at 20-30°C ensures the activity of the polysaccharides, and allowing the phases to stand for 1.5-2.5 hours ensures complete separation of the three phases.

[0067] Step 4: Phase separation and polysaccharide recovery.

[0068] Specifically, the three phases are separated, and the upper phase is collected. The upper phase (rich in modified PEG) mainly contains polysaccharides, the middle phase mainly contains small molecule impurities, and the lower phase (chloroform phase) mainly contains lipid-soluble impurities and proteins.

[0069] The main purpose of step 4 is to separate the upper phase, which is rich in polysaccharides, the intermediate phase, which is rich in small molecule impurities, and the lower phase, which is rich in lipid-soluble impurities and proteins. Care must be taken during this step to avoid mixing of the three phases and ensure effective separation.

[0070] Step 5: Multi-stage extraction.

[0071] Specifically, the mesophase and chloroform phase are combined, and an aqueous modified PEG solution with an equal volume to the upper phase is added. The three-phase partitioning is performed again, the upper phase is collected, and the mesophase and chloroform phase are extracted repeatedly 1-3 times. All upper phases are then combined.

[0072] The modified PEG aqueous solution was obtained by mixing modified PEG with deionized water, and the concentration of modified PEG was 5% (w / w).

[0073] After the three-phase partitioning, some polysaccharides remain in the intermediate phase and chloroform phase. Preferably, by performing multiple extractions on these two phases, the residual polysaccharides can be redistributed to the modified PEG-rich upper phase, thereby improving the polysaccharide recovery rate and reducing losses.

[0074] Step 5: Polysaccharide purification.

[0075] Specifically, the upper phase is precipitated with anhydrous ethanol, and the precipitate is collected by centrifugation.

[0076] The precipitate was dissolved in a small amount of deionized water, and small molecule impurities were removed by dialysis. The precipitate was then freeze-dried to obtain purified Hericium erinaceus polysaccharide.

[0077] The main purpose of step 5 is to further remove residual impurities and obtain high-purity polysaccharides. Alcohol precipitation can precipitate polysaccharides, dialysis can remove small molecule impurities, and freeze-drying can obtain dry polysaccharide powder.

[0078] In this step, the process of precipitating the upper phase with anhydrous ethanol is called gradient alcohol precipitation, which includes the following steps:

[0079] First precipitation: Add anhydrous ethanol to the upward phase to make the final concentration of ethanol 40% (v / v), let stand at 4°C for 2 hours, centrifuge to remove the precipitate, and retain the supernatant;

[0080] Second precipitation: Add anhydrous ethanol to the supernatant to make the final concentration of ethanol 70% (v / v), let stand at 4°C for 2 hours, and collect the precipitate by centrifugation;

[0081] Washing: Wash the precipitate several times with a 70% ethanol solution.

[0082] In this step, two ethanol precipitation processes are employed. The first precipitation removes some impurities (such as proteins), while the second precipitation enriches polysaccharides, improving separation efficiency. Utilizing the difference in solubility of ethanol in modified PEG, chloroform, and polysaccharides, a gradient precipitation method is used to remove modified PEG and any present chloroform from the upper phase. The first precipitation uses an ethanol concentration of 40% (v / v) to precipitate some impurities, while the second precipitation uses an ethanol concentration of 70% (v / v) to precipitate polysaccharides. Precipitation is carried out at 4°C to minimize polysaccharide loss. Subsequent dialysis removes residual modified PEG and small molecule salt impurities.

[0083] In this step, centrifugation can be performed at 3500-4500 rpm for 10-15 minutes. During dialysis, a 3500 Da dialysis membrane can be used to remove small molecule impurities. When dissolving the precipitate with a small amount of deionized water, 1-5 mL of deionized water can be used per gram of precipitate. There is no specific limit to the amount of deionized water used in this step; simply ensure the precipitate is completely dissolved.

[0084] Compared with traditional methods such as alcohol precipitation, dialysis, and activated carbon adsorption, this process has the following advantages:

[0085] 1. Good separation effect: Three-phase partitioning can more effectively separate polysaccharides, proteins and fat-soluble impurities, and improve the purity of polysaccharides.

[0086] 2. Simple operation: The three-phase partitioning operation is simple and easy to implement for large-scale industrial application. Furthermore, the use of OSA-PEG improves the selectivity of the three-phase system and enhances the partitioning of polysaccharides in the upper phase; the addition of Span 80 stabilizer improves the stability of the three-phase system and enhances phase separation.

[0087] 3. Higher recovery rate: Multi-stage extraction is used to improve the recovery rate of polysaccharides and reduce product loss.

[0088] 4. Higher extraction efficiency: Intermittent shaking promotes mass transfer balance and improves extraction efficiency.

[0089] 5. Wide range of applications: This process can be applied to extracts of Hericium erinaceus polysaccharides from different sources and with different purities.

[0090] The present application will be further described below through specific embodiments.

[0091] Example 1:

[0092] The extraction method of Hericium erinaceus polysaccharide in this embodiment includes the following steps:

[0093] Step 1: Prepare crude extract of Hericium erinaceus polysaccharide.

[0094] 50g of dried Hericium erinaceus was crushed and passed through an 80-mesh sieve. Deionized water was added at a material-to-liquid ratio of 1:20 (g / mL). The mixture was placed in a microwave extractor with a microwave power of 300W, a duty cycle of 50%, and a frequency of 1Hz. The mixture was extracted in an 80℃ water bath for 2 hours. The extraction was repeated twice. The extracts were combined and centrifuged (4000r / min, 10min) to remove the residue, thus obtaining a crude extract of Hericium erinaceus polysaccharides.

[0095] Step 2: Construct a three-phase system.

[0096] Preparation of modified PEG: 100 g of PEG6000 (purchased from Sigma-Aldrich) and 0.35 g of octenyl succinic anhydride (purchased from Sigma-Aldrich, purity ≥98%) were mixed at a molar ratio of 1:0.1, added to dichloromethane, and brought to a final volume of 1000 mL (PEG6000 concentration 10% (w / v)). 20.4 mg of catalyst 4-dimethylaminopyridine (PEG to DMAP molar ratio 1:0.01) was added, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was washed three times with ethyl acetate and dried under vacuum to obtain modified PEG.

[0097] OSA-PEG and ammonium sulfate were added to 100g of Hericium erinaceus polysaccharide crude extract to achieve final concentrations of 10% (w / w) and 15% (w / w), respectively. Chloroform was then added to achieve a final concentration of 8% (w / w). Finally, Span 80 stabilizer was added to achieve a final concentration of 0.2% (w / w). The mixture was thoroughly mixed.

[0098] Step 3: Three-phase distribution.

[0099] The constructed three-phase system was placed in a separatory funnel and allowed to stand at 25°C for 2 hours to separate into layers.

[0100] Step 4: Phase separation and polysaccharide recovery.

[0101] Carefully separate the three phases and collect the upper phase.

[0102] Step 5: Multi-stage extraction.

[0103] Combine the mesophase and chloroform phase, add an equal volume of modified PEG aqueous solution (final concentration of modified PEG 5% (w / w)) to the upper phase, perform three-phase partitioning again, collect the upper phase, and repeat the extraction of the mesophase and chloroform phase twice. Combine all upper phases.

[0104] Step 6: Polysaccharide purification.

[0105] Anhydrous ethanol was added to the combined supernatant to bring the final ethanol concentration to 40% (v / v). The mixture was allowed to stand at 4°C for 2 hours, then centrifuged (4000 r / min, 10 min) to remove the precipitate, retaining the supernatant. Anhydrous ethanol was then added to the supernatant to bring the final ethanol concentration to 70% (v / v). The mixture was allowed to stand at 4°C for 2 hours, then centrifuged (4000 r / min, 10 min) to collect the precipitate. The precipitate was washed twice with a 70% ethanol solution. The precipitate was dissolved in a small amount of deionized water, dialyzed (molecular weight cutoff 3500 Da) to remove small molecule impurities, and freeze-dried to obtain 0.416 g of purified Hericium erinaceus polysaccharide.

[0106] Example 2:

[0107] The extraction method of Hericium erinaceus polysaccharide in this embodiment includes the following steps:

[0108] Step 1: Prepare crude extract of Hericium erinaceus polysaccharide. Same as in Example 1.

[0109] Step 2: Construct a three-phase system.

[0110] Preparation of modified PEG: Same as in Example 1.

[0111] OSA-PEG and ammonium sulfate were added to 100g of Hericium erinaceus polysaccharide crude extract to achieve final concentrations of 8% (w / w) and 18% (w / w), respectively. Chloroform was then added to achieve a final concentration of 6% (w / w). Finally, Span 80 stabilizer was added to achieve a final concentration of 0.3% (w / w). The mixture was thoroughly mixed.

[0112] Step 3: Three-phase distribution.

[0113] The constructed three-phase system was placed in a separatory funnel and allowed to stand at 25°C for 2 hours to separate into layers.

[0114] Step 4: Phase separation and polysaccharide recovery.

[0115] Carefully separate the three phases and collect the upper phase.

[0116] Step 5: Multi-stage extraction.

[0117] Combine the mesophase and chloroform phase, add an equal volume of modified PEG aqueous solution (final concentration of modified PEG 5% w / w) to the upper phase, perform three-phase partitioning again, collect the upper phase, and repeat the extraction of the mesophase and chloroform phase twice. Combine all upper phases.

[0118] Step 6: Polysaccharide purification.

[0119] Anhydrous ethanol was added to the upward phase to bring the final ethanol concentration to 40% (v / v). The mixture was allowed to stand at 4°C for 2 hours, and then centrifuged (4000 r / min, 10 min) to remove the precipitate. Anhydrous ethanol was added to the supernatant to bring the final ethanol concentration to 70% (v / v). The mixture was allowed to stand at 4°C for 2 hours, and then centrifuged (4000 r / min, 10 min) to collect the precipitate. The precipitate was washed twice with a 70% ethanol solution. The precipitate was dissolved in a small amount of deionized water, dialyzed (molecular weight cutoff 3500 Da) to remove small molecule impurities, and then freeze-dried to obtain 0.420 g of purified Hericium erinaceus polysaccharide.

[0120] Comparative Example 1 (Three-phase preparation using conventional PEG)

[0121] The extraction method of Hericium erinaceus polysaccharide in this comparative example includes the following steps:

[0122] Step 1: Prepare crude extract of Hericium erinaceus polysaccharide. Same as in Example 1.

[0123] Step 2: Construction of the three-phase system.

[0124] Add polyethylene glycol PEG6000 and ammonium sulfate to 100g of Hericium erinaceus polysaccharide crude extract to achieve final concentrations of 10% (w / w) and 15% (w / w), respectively. Then add chloroform to achieve a final concentration of 8% (w / w). Mix thoroughly.

[0125] Step 3: Three-phase distribution.

[0126] The constructed three-phase system was placed in a separatory funnel and allowed to stand at 25°C for 2 hours to separate into layers.

[0127] Step 4: Phase separation and polysaccharide recovery.

[0128] Carefully separate the three phases and collect the upper phase.

[0129] Step 5: Polysaccharide purification.

[0130] The upper phase was precipitated with 4 times its volume of anhydrous ethanol, allowed to stand overnight at 4°C, and centrifuged (4000 r / min, 10 min) to collect the precipitate. The precipitate was dissolved in a small amount of deionized water, dialyzed (molecular weight cutoff 3500 Da) to remove small molecule impurities, and freeze-dried to obtain 0.363 g of purified Hericium erinaceus polysaccharide.

[0131] Comparative Example 2

[0132] Step 1: Preparation of crude extract of Hericium erinaceus polysaccharide. Same as in Example 1.

[0133] Step 2: Construction of the three-phase system.

[0134] Add polyethylene glycol PEG6000 and ammonium sulfate to 100g of Hericium erinaceus polysaccharide crude extract to achieve final concentrations of 8% (w / w) and 18% (w / w), respectively. Then add chloroform to achieve a final concentration of 6% (w / w). Mix thoroughly.

[0135] Step 3: Three-phase distribution.

[0136] The constructed three-phase system was placed in a separatory funnel and allowed to stand at 25°C for 2 hours to separate into layers.

[0137] Step 4: Phase separation and polysaccharide recovery.

[0138] Carefully separate the three phases and collect the upper phase.

[0139] Step 5: Polysaccharide purification.

[0140] The combined upper phases were precipitated with 4 times the volume of anhydrous ethanol, allowed to stand overnight at 4°C, and centrifuged (4000 r / min, 10 min) to collect the precipitate. The precipitate was dissolved in a small amount of deionized water, dialyzed (molecular weight cutoff 3500 Da) to remove small molecule impurities, and freeze-dried to obtain 0.387 g of purified Hericium erinaceus polysaccharide.

[0141] Comparative Example 3

[0142] Polysaccharides from Hericium erinaceus were extracted using a water extraction and alcohol precipitation method.

[0143] 1. Preparation of crude extract of Hericium erinaceus polysaccharide: Same as in Example 1.

[0144] 2. Alcohol precipitation: Add 100g of crude extract of Hericium erinaceus polysaccharide to 3 times the volume of 95% ethanol, let stand at 4℃ for 12 hours, and centrifuge (4000r / min, 10min) to collect the precipitate.

[0145] 3. Dialysis: Dissolve the precipitate in a small amount of deionized water, dialyze (molecular weight cutoff 3500 Da) to remove small molecule impurities, freeze dry to obtain 0.343 g of purified Hericium erinaceus polysaccharide.

[0146] The polysaccharides from Hericium erinaceus prepared in Examples 1-2 and Comparative Examples 1-3 were tested using the following methods:

[0147] 1. Purity determination of Hericium erinaceus polysaccharide: The purity of polysaccharides in the purified Hericium erinaceus polysaccharide was determined by the phenol-sulfuric acid method according to the Chinese Pharmacopoeia 2020.

[0148] 2. Protein content determination: The protein content in the purified Hericium erinaceus polysaccharide was determined by the Coomassie brilliant blue method.

[0149] 3. Recovery rate determination: The recovery rate was calculated by measuring the polysaccharide content in the crude extract and the polysaccharide content in the purified Hericium erinaceus polysaccharide.

[0150] 4. PEG residue determination: PEG residue was determined using the iodine-potassium iodide method.

[0151] 5. Chloroform residue determination: Chloroform residue was determined by gas chromatography.

[0152] Table 1

[0153] Purity, Protein Content, Recovery Rate, PEG Residue, Chloroform Residue: Example 1: 82.5%, 1.3%, 85.7%, 0.03%, <60ppm; Example 2: 84.1%, 1.9%, 88.2%, 0.04%, <60ppm; Comparative Example 1: 75.2%, 3.5%, 68.3%, 0.12%, >60ppm; Comparative Example 2: 73.9%, 3.2%, 71.5%, 0.16%, >60ppm; Comparative Example 3: 65.5%, 8.5%, 56.2%, -- surface

[0154] This application presents an improved version of Comparative Examples 1-2. A comparison of Examples 1-2 and Comparative Examples 1-2 shows that the Hericium erinaceus polysaccharide prepared using the optimized method of Examples 1-2 has significantly higher purity, significantly lower protein content, and significantly higher recovery rate than Comparative Examples 1-3. PEG residue is also significantly reduced, and chloroform residue meets the pharmacopoeia requirements for solvent residue. This indicates that the optimized method of Examples 1-2 effectively improves the separation and purification of Hericium erinaceus polysaccharide, demonstrating significant advantages.

[0155] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A method for extracting polysaccharides from Hericium erinaceus, characterized in that, Includes the following steps: A crude extract of Hericium erinaceus polysaccharide was prepared. A three-phase system was constructed: modified PEG and sulfate were added to the crude extract to achieve final concentrations of 5-15 w / w % and 10-20 w / w %, respectively; chloroform was added to achieve a final concentration of 5-15 w / w %; and Span 80 was added to achieve a final concentration of 0.1-0.3 w / w. %; Mix thoroughly and evenly; Three-phase distribution: Place the three-phase system in a separatory funnel and allow it to stand at 20-30℃ for 1.5-2.5 hours to separate into layers; Phase separation and polysaccharide recovery: Separate the three phases and collect the upper phase; Polysaccharide purification: Precipitate the upper phase with anhydrous ethanol and collect the precipitate by centrifugation; Dissolve the precipitate in deionized water, dialyze, and freeze-dry to obtain the Hericium erinaceus polysaccharide; The preparation method of the modified PEG includes the following steps: Mix PEG with octenyl succinic anhydride, add it to dichloromethane, add the catalyst 4-dimethylaminopyridine, and stir the reaction at room temperature for 24 hours; After the reaction, wash several times with ethyl acetate and vacuum dry to obtain the modified PEG; The molar ratio of PEG to octenyl succinic anhydride is 1:0.1; The amount of dichloromethane used is to make the concentration of PEG 10 w / v The molar ratio of PEG to 4-dimethylaminopyridine is 1:0.01; the PEG is one of PEG6000, PEG4000, PEG8000, and PEG10000; the sulfate is one of ammonium sulfate, potassium sulfate, and magnesium sulfate; before purifying the polysaccharide, the following steps are also included: merging the intermediate phase and the chloroform phase, adding an aqueous PEG solution with a volume equal to that of the upper phase, performing three-phase partitioning again, collecting the upper phase, repeating the extraction of the intermediate phase and the chloroform phase 1-3 times, and merging all the upper phases; the aqueous PEG solution is obtained by mixing the modified PEG with deionized water, and the concentration of modified PEG in the aqueous PEG solution is 5 w / w %; the process of precipitating the upper phase with anhydrous ethanol includes the following steps: adding anhydrous ethanol to the upper phase to make the final concentration of ethanol 40 v / v %, letting it stand at 4°C for 2 hours, centrifuging to remove the precipitate, and retaining the supernatant; continuing to add anhydrous ethanol to the supernatant to make the final concentration of ethanol 70 The precipitate was collected by centrifugation after standing at 4°C for 2 hours with v / v % at 4°C. The precipitate was then washed several times with a 70% ethanol solution.

2. The method for extracting Hericium erinaceus polysaccharides according to claim 1, characterized in that, The process for preparing the crude extract of Hericium erinaceus polysaccharide includes the following steps: pulverizing dried Hericium erinaceus, passing it through a 50-80 mesh sieve, adding deionized water at a material-to-liquid ratio of 1g:15-20mL, setting the microwave power to 250-350W, duty cycle to 50%, and frequency to 1Hz, and extracting at 75-85℃ for 1.5-2.5 hours, extracting twice, combining the extracts, centrifuging to remove residue, and obtaining the crude extract of Hericium erinaceus polysaccharide; a frequency of 1Hz means turning the microwave on and off once per second.

3. The method for extracting Hericium erinaceus polysaccharides according to claim 1, characterized in that, A 3500Da dialysis membrane is used during the dialysis process.

4. The method for extracting Hericium erinaceus polysaccharides according to claim 1, characterized in that, The centrifugation process involves centrifuging at 3500-4500 r / min for 10-15 min.

5. The method for extracting Hericium erinaceus polysaccharides according to claim 2, characterized in that, The process of centrifuging to remove residue involves centrifuging at 3500-4000 r / min for 10-15 min.