Preparation method of nutmeg non-starch polysaccharide

Abstract

The invention discloses a preparation method of myristica fragrans non-starch polysaccharide. Crushing and sieving the clean myristica fragrans, performing reflux extraction with hot water, and performing suction filtration; taking filtrate, performing enzymolysis step by step by adopting alpha-amylase and amyloglucosidase, and then performing concentration, alcohol precipitation and freeze drying on enzymatic hydrolysate to obtain the myristica fragrans non-starch polysaccharide. The optimization process of the alpha-amylase comprises the following steps: performing enzymolysis for 40 minutes at 70 DEG C according to the addition amount of 0.1% (mL / mL), and inspecting the enzymolysis completion time by using an I-KI reagent; the optimization process of amyloglucosidase comprises the following steps: carrying out enzymolysis for 120 minutes at 60 DEG C according to the addition amount of 2% (mg / mL), inspecting the enzymolysis completion time according to the area change of a glucose peak composed of monosaccharide in HPLC (High Performance Liquid Chromatography), and proving that the polysaccharide yield is 3.09 + / -0.38% by virtue of an infrared spectrum. The oxidation resistance, blood sugar reduction and immunoregulation of the polysaccharide after enzymolysis are all remarkably improved. The nutmeg non-starch polysaccharide has various biological activities and has application potential in development of the medicine field.

Description

Technical Field

[0001] The invention relates to the technical field of extraction and purification of active ingredients of traditional Chinese medicines, in particular to a method for preparing nutmeg non-starch polysaccharide. Background Art

[0002] Exploring the efficient preparation methods and biological activities of nutmeg non-starch polysaccharides is of great significance for the development of new functional foods and drugs.

[0003] Regarding the preparation of non-starch polysaccharides: A non-starch polysaccharide component of arrowhead, and its preparation method and use (CN113801248B), comprising the following steps: washing and slicing fresh arrowhead, drying at 60°C to constant weight, crushing and passing through a 60-mesh sieve to prepare arrowhead powder for later use; adding petroleum ether to defat in the dark for 12 hours, and filtering under reduced pressure to obtain the defatted arrowhead powder; soaking the defatted arrowhead powder in water with a certain liquid-to-solid ratio, and then adjusting the pH value to 5.0±1.0, adding a complex enzyme of α-amylase and cellulase, placing it in a shaker at 50°C to 55°C for incubation, and after the incubation, placing the enzymatic hydrolyzate in a boiling water bath to inactivate the enzyme for 10min to 15min; adjusting the pH value to 7.0±1.0 after cooling, placing the enzymatic hydrolyzate in a microwave-ultrasonic combined synthetic extractor, and treating it for a certain time at different powers under microwave mode; after the end, centrifuging to obtain the supernatant, concentrating the supernatant, adding anhydrous ethanol to precipitate, and freeze-drying the precipitate to obtain the crude non-starch polysaccharide of arrowhead.

[0004] A method for continuously extracting and preparing non-starch polysaccharides from highland barley (CN111378055B) comprises the following enzymatic hydrolysis steps: adding 15 ml of high temperature resistant α-amylase to the filtrate S, and water bathing at 95°C for 3 hours; cooling, adjusting the water bath temperature to 60°C, adding 1 ml of saccharifying enzyme, and water bathing for 30 minutes; adding 10 g of papain, and water bathing for 1 hour; inactivating the enzyme in a water bath at 100°C for 10 minutes; and centrifuging the enzymatic hydrolyzate at 3000 rpm for 10 minutes to obtain a supernatant S1.

[0005] In the existing non-starch polysaccharide preparation methods, there are few reports on the specific parameters of the enzymatic hydrolysis step, and usually excessive enzymatic hydrolysis time is required for enzymatic hydrolysis, and there is no judgment on whether the enzymatic hydrolysis is complete. The present invention provides a method for judging whether the two-step enzymatic hydrolysis is complete by changing the glucose peak area in the I-KI reagent and the monosaccharide composition. The present invention determines the optimal time for the two-step enzymatic hydrolysis through a single factor experiment, and compares the activities of enzymatically hydrolyzed and unenzymatically hydrolyzed polysaccharides, and finds that the activity of the polysaccharide after enzymatic hydrolysis is improved, which is conducive to further development and utilization, and provides a research basis for the research and development of non-starch polysaccharides from other starch-containing plants. Summary of the invention

[0006] The object of the present invention is to provide a preparation of non-starch polysaccharides from nutmeg, using α-amylase and amyloglucosidase for step-by-step enzymolysis to completely remove starch, and comparing the antioxidant, hypoglycemic and immunomodulatory activities of the enzymolyzed polysaccharides prepared by the method with those of the unenzymolyzed polysaccharides, and the steps are as follows:

[0007] (1) Wash the raw material of nutmeg, crush it with a grinder, sieve it, and dry it to constant weight;

[0008] (2) adding water and reflux extraction, filtering, and taking the filtrate to obtain a nutmeg water extract;

[0009] (3) adding an appropriate amount of α-amylase and heating in a water bath for enzymolysis and inactivation;

[0010] (4) continue to add amyloglucosidase, heat in a water bath for enzymolysis and inactivation;

[0011] (5) The enzymatic hydrolysate after the two-step enzymatic hydrolysis is concentrated, an appropriate amount of anhydrous ethanol is added, and the mixture is precipitated overnight, and the residue is filtered and freeze-dried to obtain nutmeg non-starch polysaccharide.

[0012] Preferably, the amount of α-amylase added in step (3) is 0.1% (mL / mL) of the water extract.

[0013] Preferably, the enzymatic hydrolysis time in step (3) is 10, 20, 30, 40, 50, or 60 min, and the enzymatic hydrolysis completion time is measured by I-KI reagent, and is more preferably 40 min.

[0014] Preferably, the enzymatic hydrolysis temperature in step (3) is 70°C.

[0015] Preferably, the amount of amyloglucosidase added in step (4) is 2% (mg / mL) of the enzymatic hydrolysate in the first step.

[0016] Preferably, the enzymatic hydrolysis time in step (4) is 10, 30, 60, 120, or 180 min, and the enzymatic hydrolysis completion time is determined by the monosaccharide composition, and is more preferably 120 min.

[0017] Preferably, the enzymatic hydrolysis temperature in step (4) is 60°C.

[0018] Preferably, the preparation method comprises the following specific steps:

[0019] (1) Wash the raw material of nutmeg, crush it with a grinder, pass it through a 60-mesh sieve, and dry it at 60°C to constant weight;

[0020] (2) Weigh nutmeg powder, add water at a liquid-to-solid ratio of 15:1 mL / g, heat under reflux and extract for 2.5 h, filter and collect the filtrate to obtain a nutmeg water extract;

[0021] (3) adding 0.1% (mL / mL) α-amylase by volume to the aqueous extract, heating in a water bath at 70° C. for 40 min, and then heating at 100° C. for 15 min to inactivate the α-amylase;

[0022] (4) Cool to room temperature, continue to add 2% (mg / mL) glucoamylase by volume to the enzymatic hydrolyzate, heat in a water bath at 60° C. for 120 min, and heat at 100° C. for 15 min to inactivate amyloglucosidase;

[0023] (5) The enzymatic hydrolyzate after the two-step enzymatic hydrolysis was concentrated to 1 / 4 volume, and an appropriate amount of anhydrous ethanol was added to an ethanol concentration of 80%. The precipitated overnight, filtered, and the residue was freeze-dried to obtain nutmeg non-starch polysaccharide. The extraction rates of the enzymatically hydrolyzed polysaccharide and the unenzymatically hydrolyzed polysaccharide were 3.09±0.38% and 9.13±0.59%, respectively, and the purities were 86.52±2.42% and 72.72±0.94%, respectively. The infrared spectra of the enzymatically hydrolyzed polysaccharide and the unenzymatically hydrolyzed polysaccharide were compared to find that the characteristic peak of starch in the enzymatically hydrolyzed polysaccharide disappeared.

[0024] IC values ​​of Vc, enzymatically hydrolyzed polysaccharides and unenzymatically hydrolyzed polysaccharides in ABTS free radical scavenging ability 50 The results of total reducing power were 0.0023±0.00017, 0.47±0.057 and 1.20±0.082 mg / mL respectively. The results of total reducing power were Vc> hydrolyzed polysaccharides> unhydrolyzed polysaccharides. The IC values ​​of acarbose, hydrolyzed polysaccharides and unhydrolyzed polysaccharides in α-glucosidase inhibition activity were 50 The results were 0.058±0.0079, 4.50±0.29 and 8.29±0.54 mg / mL respectively. In terms of immunomodulatory activity, enzymatic polysaccharide > lipopolysaccharide (LPS1μg / mL) > non-enzymatic polysaccharide. In summary, the antioxidant, hypoglycemic and immunomodulatory activities of enzymatic polysaccharides were significantly improved compared with those of direct extraction, indicating that this preparation method can improve the activity of polysaccharides and is conducive to further development and utilization.

[0025] Compared with the prior art, the present invention has the following significant advantages:

[0026] (1) Through the monosaccharide composition, it was found that starch and its hydrolysis products could not be completely removed by α-amylase alone. On this basis, amyloglucosidase was used to continue enzymatic hydrolysis and finally completely remove the starch.

[0027] (2) The changes in the glucose peak in the I-KI reagent and the monosaccharide composition provide a method for determining whether the two-step enzymatic hydrolysis is complete, and infrared spectroscopy is used to assist in verification.

[0028] (3) The time of each enzymatic hydrolysis step is shortened, and thus the overall enzymatic hydrolysis time is also shortened accordingly.

[0029] (4) Enzymatic hydrolysis can improve the purity and activity of non-starch polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the liquid chromatogram of monosaccharide composition at different enzymatic hydrolysis times;

[0031] Figure 2 It is the graph of glucose peak area changing with enzymatic hydrolysis time;

[0032] Figure 3 is the infrared spectra of enzymatically hydrolyzed and unhydrolyzed nutmeg polysaccharides;

[0033] Figure 4 The results of antioxidant activity experiments are shown in Figure 1. (a) DPPH free radical scavenging experiment, (b) ABTS free radical scavenging experiment, (c) OH free radical scavenging experiment, (d) total reducing power (***: p<0.001, ****: p<0.0001);

[0034] Figure 5 is the result of hypoglycemic activity experiment (****: p<0.0001);

[0035] Figure 6 It is a graph showing the results of the immunomodulatory activity experiment (ns: p>0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). DETAILED DESCRIPTION

[0036] Example 1

[0037] This embodiment provides an α-amylase enzymatic hydrolysis process and a detection method thereof.

[0038] 1. Preparation and detection method of I-KI test solution

[0039] Accurately weigh 1.00g of iodine and 10.00g of potassium iodide, add appropriate amount of water and ultrasonically dissolve them, make up to volume in a 50mL volumetric flask, and store in a dark place. When measuring samples, dilute them 10 times for determination.

[0040] Add 3mL of I-KI test solution into a test tube, and then add 1mL of sample. If the test solution turns blue, starch is present. If the test solution does not change color, starch is not contained in the sample.

[0041] 2. Preparation of Nutmeg Water Extract

[0042] Take the original nutmeg material, wash it, crush it with a grinder, pass it through a 60-mesh sieve, and dry it to constant weight; accurately weigh the nutmeg powder, add water at a liquid-to-solid ratio of 15:1 mL / g, extract it under hot reflux at 80°C for 2.5 h, filter it, and take the filtrate to obtain the nutmeg water extract.

[0043] 3. Single Factor Experiment

[0044] The fixed conditions of the single factor experiment are: the amount of α-amylase added is 0.1% (mL / mL) of the volume of the nutmeg water extract, the enzymolysis temperature is 70°C, stirring is uniform, sealing, and whether the enzymolysis can be completely completed at the enzymolysis time of 10, 20, 30, 40, 50, and 60 minutes. The experimental results are shown in Table 1. When the enzymolysis time is 40 minutes, the I-KI test solution does not change color, and the enzymolysis time is determined to be 40 minutes.

[0045] Table 1 α-amylase hydrolysis time experimental results

[0046]

[0047] Example 2

[0048] This embodiment provides an amyloglucosidase enzymatic hydrolysis process and a detection method thereof.

[0049] 1. Monosaccharide composition determination method

[0050] Weigh 5 mg of sample and place it in a hydrothermal synthesis reactor, add 2 mL of 2 mol / L trifluoroacetic acid, hydrolyze at 110 ° C for 5 h, add a small amount of methanol, concentrate in a water bath, and repeat several times until the trifluoroacetic acid is completely removed. Dissolve the hydrolyzed sample in distilled water, take 0.5 mL, add 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) solution 0.5 mL and 0.5 mol / L NaOH solution 0.5 mL, react at 70 ° C for 1 h, add 0.5 mol / L HCl solution 0.5 mL, extract three times with an equal volume of chloroform solution, collect the water layer, filter through a 0.45 μm filter membrane, and determine it with a high performance liquid chromatography.

[0051] Chromatographic conditions: Supersil ODS column (4.6×250 mm2, 5 μm); phosphate buffer solution (pH 6.8): acetonitrile (82:18, v:v); flow rate of 1 mL / min, injection volume of 20 μL; U3000 DAD detector, detection wavelength of 245 nm, column temperature of 30°C.

[0052] The enzymatic hydrolysis is complete when the glucose peak in the monosaccharide composition no longer changes over time.

[0053] 2. Single Factor Experiment

[0054] Take 50 mL of α-amylase hydrolyzed solution, add 2% (mg / mL) of the water extract volume under fixed conditions of amyloglucosidase, set the hydrolysis temperature to 60°C, stir evenly, seal, react for 10, 30, 60, 120, and 180 min, boil for 15 min to inactivate, concentrate, precipitate with alcohol, and freeze-dry to obtain crude polysaccharide, and determine the glucose peak area by the monosaccharide composition.

[0055] Figure 1The liquid phase monosaccharide composition at different time points is shown in Figure 2. Figure 2 is a line graph of the changes in glucose peak area at different time points. Figure 1 and 2 As shown, when the enzymatic hydrolysis time was 120 min, the glucose peak area did not continue to decrease with time, so the enzymatic hydrolysis time was determined to be 120 min.

[0056] Example 3

[0057] This embodiment provides a method for preparing nutmeg non-starch polysaccharide, the steps are as follows:

[0058] Take the original nutmeg material, wash it, crush it in a grinder, pass it through a 60-mesh sieve, dry it at 60℃ to constant weight, add water at a liquid-to-solid ratio of 15:1mL / g, extract it at 80℃ for 2.5h, filter it, and take the filtrate. Add 0.1% (mL / mL) of α-amylase in the extract volume, heat it in a water bath at 70℃ for 40min under closed conditions, and then inactivate it in a boiling water bath for 15min. Continue to add 2% (mg / mL) of amyloglucosidase in the hydrolysis solution volume, heat it in a water bath at 60℃ for 120min under closed conditions, and then inactivate it in a boiling water bath for 15min. Concentrate the hydrolysis after two steps of hydrolysis to one-fourth of the volume, add an appropriate amount of anhydrous ethanol to an ethanol concentration of 80%, precipitate it overnight, filter it, and freeze-dry the residue to obtain nutmeg non-starch polysaccharides, with a calculated yield of 3.09±0.38% and a purity of 86.52±2.42%.

[0059] Example 4

[0060] This example compares the infrared spectra of enzymatically hydrolyzed and unhydrolyzed nutmeg polysaccharides to further illustrate the enzymatic hydrolysis effect.

[0061] 1. Sample Preparation

[0062] Preparation method of enzymatically hydrolyzed polysaccharide: Enzymatically hydrolyzed nutmeg polysaccharide was prepared according to Example 3.

[0063] Preparation method of unhydrolyzed polysaccharide: take the original medicinal material of nutmeg, wash it, crush it in a grinder, pass it through a 60-mesh sieve, dry it at 60°C to constant weight, add water at a liquid-to-solid ratio of 15:1mL / g, extract it under hot reflux at 80°C for 2.5h, filter it, take the filtrate, concentrate it to one-fourth of its volume, add an appropriate amount of anhydrous ethanol to an ethanol concentration of 80%, precipitate it with alcohol overnight, filter it, and freeze-dry the residue to obtain the obtained product. The calculated extraction rate is 9.13±0.59% and the purity is 72.72±0.94%.

[0064] 2. Infrared spectroscopy analysis

[0065] The enzymatically hydrolyzed and unenzymatically hydrolyzed nutmeg polysaccharides were mixed with KBr powder at a ratio of 1:150, and then pressed into tablets.-1 The test results are shown in the following table. Figure 3 shown.

[0066] In the unhydrolyzed nutmeg polysaccharide, the -1 There is 1151.29cm in the range -1 、1081.87cm -1 and 1026.32cm -1 The three absorption peaks are the pyranose structures of glucose in starch. In the nutmeg polysaccharide after enzymatic hydrolysis, the characteristic peaks of starch disappear, indicating that the enzymatic hydrolysis process completely removes the starch.

[0067] Example 5

[0068] In this example, the antioxidant, hypoglycemic and immunomodulatory activities of nutmeg polysaccharides were compared between enzymatically hydrolyzed (prepared by the method of Example 3) and unenzymatically hydrolyzed (prepared by the method of Example 4.1).

[0069] 1. Antioxidant Activity Assay

[0070] (1) Determination of DPPH free radical scavenging ability

[0071] Prepare 0.1mmol / L DPPH solution, take 2mL of samples with polysaccharide concentrations of 4, 2, 1, 0.5, and 0.25mg / mL and 2mL of DPPH solution, mix well, and react for 30min in the dark, measure the absorbance at 517nm, add 2mL of ethanol to 2mL of sample as blank control, and add 2mL of ethanol to 2mL of DPPH solution as A0. Vc is used as positive control.

[0072] DPPH free radical scavenging rate (%) = [1-(A 样品 -A 空白 ) / A0]×100%

[0073] (2) Determination of ABTS free radical scavenging ability

[0074] Take 88 μL of 140 mmol / mL potassium persulfate solution and mix it with 5 mL of 7 mmol / L ABTS. After reacting in the dark for 12 hours, dilute the ABTS solution with distilled water until the absorbance at 734 nm is 0.7 ± 0.02. Take 2 mL of sample solution with polysaccharide concentrations of 2, 1, 0.5, 0.25, and 0.125 mg / mL and mix it with 2 mL of ABTS solution, shake it, keep it in the dark for 6 minutes, and measure the absorbance at 734 nm. The blank group is distilled water instead of the sample solution. Vc is used as a positive control.

[0075] ABTS free radical scavenging rate (%) = [(A 空白 -A样品 ) / A 空白 ]×100%

[0076] (3) Determination of OH radical scavenging ability

[0077] Accurately weigh 0.125 g of Fe2SO4 into a 50 mL volumetric flask and make up to volume with pure water. Accurately weigh 0.1243 g of salicylic acid into a 100 mL volumetric flask and make up to volume with 70% ethanol. Accurately transfer 0.4 mL of 30% H2O2 into a 100 mL volumetric flask and make up to volume with pure water.

[0078] Take 2 mL of samples with polysaccharide concentrations of 2, 1, 0.5, 0.25, and 0.125 mg / mL, add 2 mL of prepared Fe2SO4 solution, 2 mL of salicylic acid-ethanol, and 2 mL of H2O2, mix thoroughly, place in a constant temperature water bath at 37°C for 30 minutes, cool to room temperature, and measure absorbance at 510 nm. The background group is distilled water instead of H2O2, and the blank is distilled water instead of the sample solution. Vc is used as a positive control.

[0079] OH radical scavenging rate (%) = [1-(A 实验 -A 背景 ) / A 空白 ]×100%

[0080] (4) Total reducing power

[0081] Take 1 ml of samples with polysaccharide concentrations of 8, 4, 2, 1, and 0.5 mg / mL respectively, add 2.5 ml of PBS (pH = 6.6) solution, then add 2.5 ml of 1% potassium ferrocyanide, incubate in a 50°C water bath for 20 min, add 2.5 ml of 10% trichloroacetic acid, centrifuge at 3000 r / min for 10 min, take 2.5 ml of supernatant, add 0.5 ml of 0.1% ferric chloride and 2.5 ml of distilled water, shake well, and measure the absorbance of the reaction system at 700 nm.

[0082] (5) Experimental results

[0083] The antioxidant activity of enzymatically hydrolyzed and unenzymatically hydrolyzed nutmeg polysaccharides was compared through experiments in four systems. Since unenzymatically hydrolyzed nutmeg polysaccharides contain macromolecular starch, they are prone to precipitation when mixed with ethanol solution, so the DPPH and OH free radical scavenging ability could not be determined.

[0084] The results of DPPH free radical scavenging ability test are as follows Figure 4 As shown in a, IC of Vc and enzymatically hydrolyzed polysaccharides 50 The results of ABTS free radical scavenging ability test are shown in Figure 4As shown in b, IC values ​​of Vc, enzymatically hydrolyzed polysaccharides and unenzymatically hydrolyzed polysaccharides 50 The results of OH radical scavenging ability test were as follows: Figure 4 As shown in c, IC of Vc and enzymatically hydrolyzed polysaccharides 50 The total reducing power results are as follows: Figure 4 As shown in Figure d, in the range of 0.5-8 mg / mL, the total reducing capacity at the same concentration is Vc> enzymatically hydrolyzed polysaccharides> unenzymatically hydrolyzed polysaccharides. In summary, the enzymatic hydrolysis process has a significant effect on the antioxidant activity and can improve the antioxidant activity.

[0085] 2. Determination of hypoglycemic activity

[0086] Its hypoglycemic ability was determined by α-glucosidase inhibitory activity. Take 100 μL of samples with polysaccharide concentrations of 8, 4, 2, 1, and 0.5 mg / mL, add 600 μL of PBS (pH = 6.8) and 300 μL of α-glucosidase, incubate at 37°C for 15 min, add 100 μL of pNPG, incubate at 37°C for 20 min, add 0.2 mol / L Na2CO3 4mL to terminate the reaction, and measure the absorbance at 400 nm. Use PBS instead of the sample as a negative control, use PBS instead of α-glucosidase as a background control, and use PBS instead of the sample and pNPG as a blank control. Use acarbose as a positive control.

[0087] α-glucosidase inhibition rate (%) = (A 阴性 -A 空白 )-(A 实验 -A 背景 ) / (A 阴性 -A 空白 )×100%

[0088] The experimental results are as follows Figure 5 As shown in Figure 2, the inhibitory effect on α-glucosidase was dose-dependent in the range of 0.5 to 8 mg / mL. IC values ​​of acarbose, enzymatically hydrolyzed polysaccharides, and unenzymatically hydrolyzed polysaccharides 50 They were 0.058±0.0079, 4.50±0.29 and 8.29±0.54 mg / mL respectively.

[0089] 3. Immunomodulatory activity assay

[0090] In the study of the immunomodulatory activity of polysaccharides, the immunomodulatory properties of polysaccharides are generally evaluated by stimulating macrophages to release NO. RAW264.7 cells (3×10 6cells / mL) were pre-incubated in a 24-well plate for 24 h, and enzymatically hydrolyzed polysaccharides and unhydrolyzed polysaccharides (800, 400, 200, 100, and 50 μg / mL) were added, respectively. The culture was continued for 24 h, and the NO concentration in the culture medium was measured. Lipopolysaccharide (1 μg / mL) was used as a positive control.

[0091] like Figure 6 As shown in the results, in the concentration range of 50-800 μg / mL, the hydrolyzed polysaccharides at concentrations of 50 μg / mL and 100 μg / mL had significant stimulatory activity (p<0.0001). They were 9.42±0.70 and 12.14±1.91 μmol / L, respectively, both higher than 9.13±0.43 μmol / L of lipopolysaccharide. The other concentrations of hydrolyzed polysaccharides had different stimulatory activities compared with the blank group. However, there was no obvious stimulatory activity when the concentration of unhydrolyzed polysaccharides was above 200 μg / mL. The results showed that the stimulatory activity was highest when the concentration of hydrolyzed polysaccharides was 100 μg / mL. In summary, the enzymatic hydrolysis process can improve the immunomodulatory activity.

Claims

1. A method for preparing nutmeg non-starch polysaccharide, comprising the following steps: (1) Take the original medicinal material nutmeg, wash it, crush it with a grinder, sieve it, and dry it to constant weight; (2) using a hot reflux extraction method to obtain a nutmeg water extract; (3) using α-amylase to perform a first step of enzymatic hydrolysis on the water extract; (4) Determine the time for enzymatic hydrolysis to be completed using I-KI reagent; (5) performing a second step of enzymolysis on the enzymolysis solution using amyloglucosidase; (6) Determine the time for enzymatic hydrolysis to be completed by monosaccharide composition; (7) The enzymatic hydrolysate is concentrated, precipitated with alcohol, filtered, and freeze-dried to obtain nutmeg non-starch polysaccharide.

2. The method according to claim 1, characterized in that: In step (3), the amount of α-amylase added to the water extract for enzymatic hydrolysis is 0.1% of the volume of the water extract.

3. The method according to claim 1, characterized in that: In step (3), the temperature for enzymatic hydrolysis of the water extract by using α-amylase is 70°C.

4. The method according to claim 1, characterized in that: In step (3), the time for enzymatic hydrolysis of the water extract with α-amylase is 10 to 60 minutes.

5. The method according to claim 1, characterized in that: In step (4), the water extract after enzymatic hydrolysis is tested by I-KI reagent. If the reagent does not change color, the enzymatic hydrolysis is complete. The optimized enzymatic hydrolysis time of α-amylase is 40 minutes.

6. The method according to claim 1, characterized in that: In step (5), the amount of amyloglucosidase added for enzymatic hydrolysis is 2% of the volume of the enzymatic hydrolysis solution.

7. The method according to claim 1, characterized in that: In step (5), the temperature of enzymolysis of the enzymolysis solution using amyloglucosidase is 60°C.

8. The method according to claim 1, characterized in that: In step (5), the enzymatic hydrolysis time of the enzymatic hydrolyzate using amyloglucosidase is 10 to 180 minutes.

9. The method according to claim 1, characterized in that: In step (6), the hydrolyzate after the second step of enzymatic hydrolysis is concentrated, precipitated with alcohol, and freeze-dried, and the glucose peak area of ​​the crude polysaccharide is determined by the monosaccharide composition. If the peak area does not change with time, the enzymatic hydrolysis is complete, and the time for amyloglucosidase enzymatic hydrolysis is further optimized to 120 minutes.

10. The method according to claim 1, characterized in that: After two-step enzymatic hydrolysis, the antioxidant, hypoglycemic and immunomodulatory effects of polysaccharides were significantly improved.

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