Preparation method and application of burdock polysaccharide with anti-fatigue effect

Abstract

The invention relates to the technical field of polysaccharide preparation, and discloses a preparation method and application of burdock polysaccharide with an anti-fatigue efficacy, and the preparation method comprises the following steps: step 1, plasma pretreatment; step 2, acidolysis; step 3, performing enzymolysis; step 4, alkaline ultrasonic treatment; step 5, concentration and ethanol gradient fractional precipitation; and step 6, carrying out ultrafiltration, collecting 3-30kDa trapped fluid, and drying to obtain the burdock polysaccharide. The burdock polysaccharide with the specific molecular weight is obtained through the method, has the advantages of being high in extraction rate, stable in activity, remarkable in anti-fatigue effect and the like, and provides a high-quality raw material source for development of functional food and health care products.

Description

Technical Field

[0001] This application relates to the field of polysaccharide preparation technology, and more specifically, to a method for preparing burdock polysaccharide with anti-fatigue effects and its application. Background Technology

[0002] Burdock is a plant used both as food and medicine. Its roots are rich in polysaccharides, cellulose, polyphenols, and other active ingredients, possessing various biological activities such as antioxidant, immunomodulatory, and anti-inflammatory effects. Among these, burdock polysaccharides, as one of the main active ingredients, have been shown to have immunomodulatory, hypoglycemic, and anti-tumor effects. However, traditional polysaccharide extraction methods typically employ hot water extraction or single enzymatic hydrolysis, which suffer from low extraction efficiency, easy destruction of polysaccharide structures, and significant loss of active ingredients, thus limiting their industrial application.

[0003] Therefore, developing an efficient and controllable method for preparing burdock polysaccharides to obtain high-purity, anti-fatigue burdock polysaccharides within a specific molecular weight range has significant application value and market prospects. Summary of the Invention

[0004] In order to develop an efficient and controllable method for preparing burdock polysaccharide, this invention provides a method for preparing burdock polysaccharide with anti-fatigue effects and its application.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a method for preparing burdock polysaccharide with anti-fatigue effects, comprising the following steps: Step 1: Take fresh burdock root slices and perform plasma pretreatment; Step 2: Acid hydrolyze the pretreated burdock root, centrifuge to obtain the hydrolysate; Step 3: After acid hydrolysis, enzymatic hydrolysis is performed using cellulase and pectinase to obtain the enzymatic hydrolysate; Step 4: After enzymatic hydrolysis, add sodium carbonate solution to adjust the pH to 9-10, and sonicate to obtain the extract. Step 5: Concentrate the extract and use ethanol gradient precipitation with ethanol concentrations of 30%, 50%, and 70% respectively, and collect the 50% ethanol precipitate. Step 6: Dissolve the collected precipitate in deionized water, ultrafilter, collect the 3-30 kDa retentate, and dry it to obtain burdock polysaccharide.

[0006] Furthermore, in step 1, the plasma pretreatment, the power is 40~60W, the treatment time is 4~6min, the gas is argon or nitrogen, and the gas pressure is 50~100Pa.

[0007] Furthermore, in step 2, 0.1~0.5 mol / L citric acid is used for acid hydrolysis, with a material-to-liquid ratio of 1:(10~15).

[0008] Further, the specific steps of step 2 are as follows: acid hydrolysis of the pretreated burdock root at a temperature of 40~55°C, with continuous stirring for 30~60 minutes during the process, and centrifugation at 2000~3000 rpm for 5~10 minutes after acid hydrolysis is completed.

[0009] Furthermore, in step 3, the enzyme activity ratio of cellulase to pectinase is (3-5):1, with a total activity of 600-800 U / g raw material.

[0010] Further, the specific steps of step 3 are as follows: after acid hydrolysis, adjust the pH to 4.8~5.2, add cellulase and pectinase for enzymatic hydrolysis, keep the temperature at 50~60°C and shake for 2~4 hours, after enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10~15 minutes, centrifuge at 6000~8000 rpm for 10~20 minutes to obtain the enzymatic hydrolysate.

[0011] Further, the specific steps of step 4 are as follows: after enzymatic hydrolysis, add 0.3~0.5mol / L sodium carbonate solution, adjust the pH to 9~10, sonicate at 35~40kHz and 50~60°C for 30~40min, and after sonication, centrifuge at 6000~8000rpm for 10~20min to obtain the extract.

[0012] Further, the specific steps of step 5 are as follows: concentrate the extract to 20-30% of the original volume, slowly add 95% ethanol to the final concentration of 30%, let it stand at 4°C for 1-2 hours, centrifuge at 6000-8000 rpm for 10-20 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 8-10 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 2-4 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is sufficient.

[0013] Further, the specific steps of step 6 are as follows: dissolve the collected precipitate in deionized water, pass it through ultrafiltration membranes of 100kDa, 30kDa and 3kDa in sequence, collect the 3~30kDa retentate, freeze-dry it to obtain burdock polysaccharide.

[0014] In a first aspect, this application provides the application of burdock polysaccharide with anti-fatigue effects prepared by any of the preparation methods described in the first aspect in the preparation of health products with anti-fatigue effects.

[0015] In summary, this application has the following beneficial effects: 1. The preparation method of this application adopts plasma pretreatment, which effectively destroys the cell wall structure of burdock, enhances the efficiency of subsequent acid hydrolysis and enzymatic hydrolysis, and improves the polysaccharide dissolution rate.

[0016] 2. The preparation method of this application combines acid hydrolysis and enzymatic hydrolysis for synergistic extraction, which fully degrades cellulose and pectin in the cell wall, resulting in more complete release of polysaccharides and a significant increase in extraction rate.

[0017] 3. The preparation method of this application reduces the degradation of polysaccharides during the extraction process by adjusting pH and ultrasonic treatment, maintains their biological activity, and enhances their anti-fatigue effects. Furthermore, this application uses gradient ethanol precipitation to collect 50% ethanol precipitate fraction (3~30kDa), which has better anti-fatigue activity.

[0018] 4. The preparation method of this application is simple to operate, has a short extraction time, produces high product purity, is easy to scale up, and has good application prospects. Detailed Implementation

[0019] The technical solutions and effects of this application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0020] This application discloses a method for preparing burdock polysaccharide with anti-fatigue effects, comprising the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. During plasma pretreatment, the power is 40-60 W, the treatment time is 4-6 min, the gas is argon, and the pressure is 50-100 Pa. Step 2: At a temperature of 40~55°C, the pretreated burdock root is acid-hydrolyzed using 0.1~0.5 mol / L citric acid at a material-to-liquid ratio of 1:(10~15). During the process, the reaction is continuously stirred for 30~60 min. After the acid hydrolysis is completed, the root is centrifuged at 2000~3000 rpm for 5~10 min. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of (3-5):1, and a total activity of 600-800 U / g raw material. Keep the mixture at 50-60°C with constant shaking for 2-4 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 10-15 minutes, and centrifuge at 6000-8000 rpm for 10-20 minutes to obtain the enzymatic hydrolysate. Step 4: After enzymatic hydrolysis, add 0.3-0.5 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 35-40 kHz and 50-60°C for 30-40 min. After sonication, centrifuge at 6000-8000 rpm for 10-20 min to obtain the extract. Step 5: Concentrate the extract to 20-30% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1-2 hours, centrifuge at 6000-8000 rpm for 10-20 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 8-10 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 2-4 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 6: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa, and 3kDa in sequence. Collect the 3-30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0021] Example 1

[0022] A method for preparing burdock polysaccharide with anti-fatigue effects includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. The power of plasma pretreatment is 50W, the treatment time is 5min, the gas is argon, and the pressure is 75Pa. Step 2: At a temperature of 50°C, the pretreated burdock root was acid-hydrolyzed using 0.3 mol / L citric acid at a material-to-liquid ratio of 1:12. During the process, the reaction was continuously stirred for 45 min. After the acid hydrolysis was completed, the root was centrifuged at 2500 rpm for 7.5 min. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 4:1 and a total activity of 700 U / g raw material. Keep the mixture at 55°C and shake for 3 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 12 minutes and centrifuge at 7000 rpm for 15 minutes to obtain the enzymatic hydrolysate. Step 4: After enzymatic hydrolysis, add 0.4 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 37.5 kHz and 55°C for 35 min. After sonication, centrifuge at 7000 rpm for 15 min to obtain the extract. Step 5: Concentrate the extract to 25% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1.5 hours, centrifuge at 7000 rpm for 15 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 9 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 3 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 6: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa, and 3kDa in sequence. Collect the 3-30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0023] Example 2

[0024] A method for preparing burdock polysaccharide with anti-fatigue effects includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. In the plasma pretreatment, the power is 40W, the treatment time is 4min, the gas is argon, and the pressure is 50Pa. Step 2: At a temperature of 40°C, the pretreated burdock root was acid-hydrolyzed using 0.1 mol / L citric acid at a material-to-liquid ratio of 1:10. During the process, the reaction was continuously stirred for 30 minutes. After the acid hydrolysis was completed, the root was centrifuged at 2000 rpm for 5 minutes. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 3:1 and a total activity of 600 U / g raw material. Keep the mixture at 50°C and shake for 2 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 10 minutes and centrifuge at 6000 rpm for 10 minutes to obtain the enzymatic hydrolysate. Step 4: After enzymatic hydrolysis, add 0.3 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 35 kHz and 50°C for 30 min. After sonication, centrifuge at 6000 rpm for 10 min to obtain the extract. Step 5: Concentrate the extract to 20% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1 hour, centrifuge at 6000 rpm for 10 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 8 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 2 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 6: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa, and 3kDa in sequence. Collect the 3-30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0025] Example 3

[0026] A method for preparing burdock polysaccharide with anti-fatigue effects includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. In the plasma pretreatment, the power is 60W, the treatment time is 6min, the gas is argon, and the pressure is 100Pa. Step 2: At a temperature of 55°C, the pretreated burdock root was acid-hydrolyzed using 0.5 mol / L citric acid at a material-to-liquid ratio of 1:15. During the process, the reaction was continuously stirred for 60 min. After the acid hydrolysis was completed, the root was centrifuged at 3000 rpm for 10 min. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 5:1 and a total activity of 800 U / g raw material. Keep the mixture at 60°C and shake for 4 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 15 minutes and centrifuge at 8000 rpm for 20 minutes to obtain the enzymatic hydrolysate. Step 4: After enzymatic hydrolysis, add 0.5 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 40 kHz and 60°C for 40 min. After sonication, centrifuge at 8000 rpm for 20 min to obtain the extract. Step 5: Concentrate the extract to 30% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 2 hours, centrifuge at 8000 rpm for 20 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 10 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 4 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 6: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa, and 3kDa in sequence. Collect the 3-30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0027] Comparative Example 1 A method for preparing burdock polysaccharide includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. The power of plasma pretreatment is 50W, the treatment time is 5min, the gas is argon, and the pressure is 75Pa. Step 2: At a temperature of 50°C, the pretreated burdock root was acid-hydrolyzed using 0.3 mol / L citric acid at a material-to-liquid ratio of 1:12. During the process, the reaction was continuously stirred for 45 min. After the acid hydrolysis was completed, the root was centrifuged at 2500 rpm for 7.5 min. Step 3: After acid hydrolysis, add 0.4 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 37.5 kHz and 55°C for 35 min. After sonication, centrifuge at 7000 rpm for 15 min to obtain the extract. Step 4: Concentrate the extract to 25% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1.5 hours, centrifuge at 7000 rpm for 15 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 9 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 3 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 5: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa and 3kDa in sequence. Collect the 3~30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0028] Comparative Example 2 A method for preparing burdock polysaccharide includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. The power of plasma pretreatment is 50W, the treatment time is 5min, the gas is argon, and the pressure is 75Pa. Step 2: Adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 4:1 and a total activity of 700 U / g raw material. Keep the mixture at 55°C and shake for 3 hours. After the enzymatic hydrolysis is complete, inactivate the enzyme in a boiling water bath for 12 minutes and centrifuge at 7000 rpm for 15 minutes to obtain the enzymatic hydrolysate. Step 3: After enzymatic hydrolysis, add 0.4 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 37.5 kHz and 55°C for 35 min. After sonication, centrifuge at 7000 rpm for 15 min to obtain the extract. Step 4: Concentrate the extract to 25% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1.5 hours, centrifuge at 7000 rpm for 15 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 9 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 3 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 5: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa and 3kDa in sequence. Collect the 3~30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0029] Comparative Example 3 A method for preparing burdock polysaccharide includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. The power of plasma pretreatment is 50W, the treatment time is 5min, the gas is argon, and the pressure is 75Pa. Step 2: At a temperature of 50°C, the pretreated burdock root was acid-hydrolyzed using 0.3 mol / L citric acid at a material-to-liquid ratio of 1:12. During the process, the reaction was continuously stirred for 45 min. After the acid hydrolysis was completed, the root was centrifuged at 2500 rpm for 7.5 min. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 4:1 and a total activity of 700 U / g raw material. Keep the mixture at 55°C and shake for 3 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 12 minutes and centrifuge at 7000 rpm for 15 minutes to obtain the enzymatic hydrolysate. Step 4: Concentrate the enzymatic hydrolysate to 25% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1.5 hours, centrifuge at 7000 rpm for 15 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 9 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 3 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 5: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa and 3kDa in sequence. Collect the 3~30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0030] Comparative Example 4 A method for preparing burdock polysaccharide includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. The power of plasma pretreatment is 50W, the treatment time is 5min, the gas is argon, and the pressure is 75Pa. Step 2: At a temperature of 50°C, the pretreated burdock root was acid-hydrolyzed using 0.3 mol / L citric acid at a material-to-liquid ratio of 1:12. During the process, the reaction was continuously stirred for 45 min. After the acid hydrolysis was completed, the root was centrifuged at 2500 rpm for 7.5 min. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 4:1 and a total activity of 700 U / g raw material. Keep the mixture at 55°C and shake for 3 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 12 minutes and centrifuge at 7000 rpm for 15 minutes to obtain the enzymatic hydrolysate. Step 4: After enzymatic hydrolysis, add 0.4 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 37.5 kHz and 55°C for 35 min. After sonication, centrifuge at 7000 rpm for 15 min to obtain the extract. Step 5: Concentrate the extract to 25% of its original volume, slowly add 95% ethanol to a final concentration of 50%, let stand at 4°C for 1.5 h, centrifuge at 7000 rpm for 15 min, and collect the target precipitate by centrifugation. Step 6: Dissolve the collected precipitate in deionized water and pass it through ultrafiltration membranes of 100kDa, 30kDa, and 3kDa in sequence. Collect the 3-30kDa retentate and freeze-dry it to obtain burdock polysaccharide.

[0031] Comparative Example 5 A method for preparing burdock polysaccharide includes the following steps: Step 1: Take fresh burdock root slices with a thickness of 2-3 mm and perform plasma pretreatment. The power of plasma pretreatment is 50W, the treatment time is 5min, the gas is argon, and the pressure is 75Pa. Step 2: At a temperature of 50°C, the pretreated burdock root was acid-hydrolyzed using 0.3 mol / L citric acid at a material-to-liquid ratio of 1:12. During the process, the reaction was continuously stirred for 45 min. After the acid hydrolysis was completed, the root was centrifuged at 2500 rpm for 7.5 min. Step 3: After acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, with an enzyme activity ratio of cellulase to pectinase of 4:1 and a total activity of 700 U / g raw material. Keep the mixture at 55°C and shake for 3 hours. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 12 minutes and centrifuge at 7000 rpm for 15 minutes to obtain the enzymatic hydrolysate. Step 4: After enzymatic hydrolysis, add 0.4 mol / L sodium carbonate solution to adjust the pH to 9-10, and sonicate at 37.5 kHz and 55°C for 35 min. After sonication, centrifuge at 7000 rpm for 15 min to obtain the extract. Step 5: Concentrate the extract to 25% of its original volume, slowly add 95% ethanol to a final concentration of 30%, let stand at 4°C for 1.5 hours, centrifuge at 7000 rpm for 15 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 9 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 3 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is complete. Step 6: Dissolve the collected precipitate in deionized water, ultrafilter through a 30kDa ultrafiltration membrane, collect the retentate, freeze-dry it, and obtain burdock polysaccharide.

[0032] Performance testing 1. Physicochemical property testing Yield calculation: polysaccharide mass / raw material dry weight × 100%, results are shown in the table below: Group Yield Example 1 32.4% Example 2 30.2% Example 3 30.5% Comparative Example 1 21.6% Comparative Example 2 22.8% Comparative Example 3 23.2% Comparative Example 4 29.9% Comparative Example 5 30.0% Molecular weight distribution (HPLC-ELSD): Group 3~50kDa Example 1 86.3% Example 2 84.6% Example 3 85.0% Comparative Example 1 65.4% Comparative Example 2 62.6% Comparative Example 3 68.3% Comparative Example 4 70.5% Comparative Example 5 80.1% 2. Fatigue resistance test 1. Forced swimming test in mice (1) Group design (n=12 / group): Control group (physiological saline) Positive group (rhodioloside 200mg / kg) Low, medium, and high dose groups (100 / 200 / 400 mg / kg polysaccharide (prepared in Example 1)) (2) Test steps: After 28 days of continuous gavage, one hour after the last administration, the animals were fed a 5% weight-bearing load on their tails and swam to exhaustion in water at 25±1℃. The swimming time was recorded, and blood samples were taken to measure lactate and blood urea nitrogen. After sacrifice, the liver was harvested to measure glycogen, and the muscle was harvested to measure lactate dehydrogenase (LDH).

[0033] (3) Results Group Swimming time (min) Blood lactate (mmol / L) Liver glycogen (mg / g) LDH (U / mg prot) Blank group 42.5±5.8 8.9±0.8 14.2±1.5 35.2±3.1 positive group 68.3±7.2 5.6±0.6 22.7±2.0 48.6±4.3 low-dose group 75.1±8.6* 4.8±0.5* 26.3±2.4* 53.2±4.8* medium dose group 91.4±9.3** 3.6±0.4** 31.5±3.2** 61.7±5.2** High-dose group 94.2±10.1** 3.3±0.3** 33.8±3.5** 63.4±5.6** Note: *p<0.05, **p<0.01 vs positive group This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing burdock polysaccharide with anti-fatigue effects, characterized in that, Includes the following steps: Step 1: Take fresh burdock root slices and perform plasma pretreatment; Step 2: Acid hydrolyze the pretreated burdock root, centrifuge to obtain the hydrolysate; Step 3: After acid hydrolysis, enzymatic hydrolysis is performed using cellulase and pectinase to obtain the enzymatic hydrolysate; Step 4: After enzymatic hydrolysis, add sodium carbonate solution to adjust the pH to 9-10, and sonicate to obtain the extract. Step 5: Concentrate the extract and use ethanol gradient precipitation with ethanol concentrations of 30%, 50%, and 70% respectively, and collect the 50% ethanol precipitate. Step 6: Dissolve the collected precipitate in deionized water, ultrafilter, collect the 3~30kDa retentate, and dry it to obtain burdock polysaccharide.

2. The preparation method according to claim 1, characterized in that, In step 1, the plasma pretreatment, the power is 40-60W, the treatment time is 4-6min, the gas is argon or nitrogen, and the pressure is 50-100Pa.

3. The preparation method according to claim 1, characterized in that, In step 2, 0.1~0.5 mol / L citric acid is used for acid hydrolysis, and the material-to-liquid ratio is 1:(10~15).

4. The preparation method according to claim 3, characterized in that, The specific steps of step 2 are as follows: acid hydrolysis of the pretreated burdock root at a temperature of 40~55°C, with continuous stirring for 30~60 minutes during the process, and centrifugation at 2000~3000 rpm for 5~10 minutes after acid hydrolysis is completed.

5. The preparation method according to claim 1, characterized in that, In step 3, the enzyme activity ratio of cellulase to pectinase is (3-5):1, with a total activity of 600-800 U / g raw material.

6. The preparation method according to claim 5, characterized in that, The specific steps of step 3 are as follows: after acid hydrolysis, adjust the pH to 4.8-5.2, add cellulase and pectinase for enzymatic hydrolysis, keep the temperature at 50-60°C and shake for 2-4 hours, after enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10-15 minutes, and centrifuge at 6000-8000 rpm for 10-20 minutes to obtain the enzymatic hydrolysate.

7. The preparation method according to claim 1, characterized in that, The specific steps of step 4 are as follows: after enzymatic hydrolysis, add 0.3~0.5mol / L sodium carbonate solution, adjust the pH to 9~10, and sonicate at 35~40kHz and 50~60°C for 30~40min. After sonication, centrifuge at 6000~8000rpm for 10~20min to obtain the extract.

8. The preparation method according to claim 1, characterized in that, The specific steps of step 5 are as follows: concentrate the extract to 20-30% of the original volume, slowly add 95% ethanol to the final concentration of 30%, let it stand at 4°C for 1-2 hours, centrifuge at 6000-8000 rpm for 10-20 minutes, and discard the precipitate to remove macromolecular impurities. Continue adding 95% ethanol to a final concentration of 50%, let stand at 4°C for 8-10 hours, and then centrifuge to collect the target precipitate. Continue adding 95% ethanol to a final concentration of 70%, let stand at 4°C for 2-4 hours, centrifuge to precipitate, and test the residual polysaccharide content of the 70% precipitate. If the residual polysaccharide content is <5%, it indicates that the 50% precipitation step is sufficient.

9. The preparation method according to claim 1, characterized in that, The specific steps of step 6 are as follows: dissolve the collected precipitate in deionized water, pass it through ultrafiltration membranes of 100kDa, 30kDa and 3kDa in sequence, collect the 3~30kDa retentate, freeze dry it to obtain burdock polysaccharide.

10. The use of burdock polysaccharide with anti-fatigue effects prepared by the preparation method according to any one of claims 1-9 in the preparation of health products with anti-fatigue effects.