Acidic sea cucumber glycoprotein with high galactose content and application thereof in reducing postprandial blood sugar

Abstract

The invention relates to the technical field of acid sea cucumber glycoprotein with high galactose content, in particular to acid sea cucumber glycoprotein with high galactose content and application of the acid sea cucumber glycoprotein in postprandial blood sugar reduction, the glycoprotein is simply marked as SCGP, the ratio of the protein content to the total sugar content is 1: 0.85-1: 1.20, and the content of the glycoprotein is 1: 0.85-1: 1.20. The monosaccharide is composed of mannose, rhamnose, glucosamine, glucose, galactose and fucose, the molar ratio of the galactose is 41.8%-46.6%, the SCGP contains 18 amino acids, the SCGP provided by the invention can effectively inhibit the activity of alpha-glucosidase, and can effectively reduce the postprandial blood sugar level of normal mice and prediabetic mice in animal experiments, so that the SCGP can be used for preparing medicines for preventing and treating diabetes mellitus. The sea cucumber glycoprotein provided by the invention realizes high-value utilization of sea cucumber byproducts, and has a good prospect in the field of development of hypoglycemic foods.

Description

Technical Field

[0001] The invention relates to the technical field of acidic sea cucumber glycoproteins with high galactose content, in particular to acidic sea cucumber glycoproteins with high galactose content and application thereof in reducing postprandial blood sugar. Background Art

[0002] Non-insulin-dependent diabetes mellitus, or type II diabetes, is a group of metabolic diseases characterized by hyperglycemia caused by insulin resistance and abnormal insulin production. Blood glucose levels rise rapidly in patients with type II diabetes due to the hydrolysis of starch by pancreatic α-amylase and the absorption of glucose by α-glucosidase in the small intestine. Inhibition of these glycohydrolases can significantly reduce postprandial hyperglycemia after a mixed carbohydrate diet and may be a key strategy for controlling diabetes. However, a major disadvantage of currently used therapeutic α-glucosidase inhibitors (such as acarbose) is their strong α-amylase inhibitory activity, which leads to abnormal bacterial fermentation of undigested starch in the colon, resulting in bloating, flatulence, vomiting, and possible diarrhea. Therefore, the search for natural active substances with low inhibitory activity against α-amylase and strong inhibitory activity against α-glucosidase is used as an effective strategy for treating postprandial hyperglycemia.

[0003] Sea cucumber is a marine invertebrate with medicinal value, belonging to the phylum Echinodermata. As a traditional marine treasure, sea cucumber contains a variety of nutrients and rich bioactive compounds, such as proteins, peptides, polysaccharides, saponins, etc., and is known as the "ginseng in the sea". These compounds have been shown to have a variety of biological functions, including antioxidant, anti-diabetic, and anti-obesity activities. In the actual processing of sea cucumbers, its by-products such as viscera, gonads, intestines and eggs are often discarded, resulting in a large amount of resource waste and environmental pollution. In order to achieve high-value and maximize the development and utilization of sea cucumber resources, sea cucumber processing by-products have become the focus and hotspot of researchers in recent years. However, compared with the body wall, the breadth and depth of research on the development and utilization of sea cucumber processing by-products is far from enough, and related research mainly focuses on the development and utilization of protein resources, while reports on glycoproteins are relatively few. Therefore, the development and preparation of a glycoprotein with hypoglycemic activity is of great significance to achieve high-value utilization of sea cucumber by-products and develop hypoglycemic products. Summary of the invention

[0004] The invention provides a high-galactose acidic sea cucumber glycoprotein and its application in reducing postprandial blood sugar. The sea cucumber glycoprotein is prepared from sea cucumber byproducts. In vitro activity tests show that the polysaccharide has a significant inhibitory effect on α-glucosidase. Animal experiments show that the component effectively reduces the postprandial blood sugar level of normal mice and prediabetic mice and promotes the insulin sensitivity of mice. Therefore, SCGP can effectively reduce postprandial blood sugar levels and has good prospects in the field of developing blood sugar-lowering foods.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an acidic sea cucumber glycoprotein with a high galactose content and its application in lowering postprandial blood sugar. The sea cucumber glycoprotein SCGP provided by the present invention has a ratio of protein content to total sugar content of 1:0.85-1:1.20, a monosaccharide composition of mannose, rhamnose, glucosamine, glucose, galactose, fucose, and a molar ratio of galactose of 41.8%-46.6%, and contains 18 kinds of amino acids.

[0006] Furthermore, the sea cucumber glycoprotein SCGP is prone to undergo β-elimination reaction in NaOH solution, produces obvious ultraviolet absorption at 240nm absorbance, and has O-glycopeptide bonds.

[0007] The preparation method of the sea cucumber glycoprotein SCGP comprises the following steps:

[0008] Step 1: crush the freeze-dried sea cucumber byproducts, pass through a 60-mesh sieve and set aside;

[0009] Step 2: Soak the powder from step 1 in acetone solution 6 times the volume of the powder, centrifuge to remove the acetone, and blow dry with nitrogen;

[0010] Step 3: Add 20-40 times the volume of deionized water to the powder obtained in step 2, mix well, stir and extract at 40-70°C for 2-3h, and remove the precipitate after centrifugation;

[0011] Step 4: Add 1%-3% neutral protease to the supernatant obtained in step 3 and react at 45-60°C for 6-24h. After the enzymolysis is completed, heat in a boiling water bath to inactivate the enzyme, and collect the enzymolysis supernatant after centrifugation;

[0012] Step 5: adding quaternary ammonium salt to the supernatant obtained in step 4 and collecting the precipitate;

[0013] Step 6: Add calcium chloride solution to dissolve the precipitate obtained in step 5, and add 2-4 times the volume of 95% ethanol to precipitate at 4°C overnight, centrifuge to obtain the precipitate and reconstitute it with pure water;

[0014] Step 7: The reconstituted solution obtained in step 6 is subjected to Sevage method to remove free protein, dialyzed using a 10 kDa dialysis bag, and freeze-dried to obtain sea cucumber glycoprotein.

[0015] The present invention has at least the following beneficial effects:

[0016] The sea cucumber glycoprotein SCGP provided by the present invention can effectively inhibit the activity of α-glucosidase, and can effectively reduce the postprandial blood sugar levels of normal mice and diabetic mice in animal experiments, and promote the insulin sensitivity of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the molecular weight distribution diagram of sea cucumber glycoprotein SCGP;

[0018] Figure 2 This is the infrared spectrum of sea cucumber glycoprotein SCGP;

[0019] Figure 3 It is the β-elimination UV spectrum of sea cucumber glycoprotein SCGP;

[0020] Figure 4 The sea cucumber glycoprotein SCGP inhibits α-glucosidase activity in vitro;

[0021] Figure 5 Sea cucumber glycoprotein SCGP improved the postprandial blood glucose level of normal mice (compared with the control group, *P<0.05, **P<0.01, ***P<0.001);

[0022] Figure 6 Sea cucumber glycoprotein SCGP improved the postprandial blood glucose level in prediabetic mice (compared with the normal group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001);

[0023] Figure 7 Sea cucumber glycoprotein SCGP improves insulin tolerance in prediabetic mice (compared with the normal group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001);

[0024] Figure 8 This is the pathological picture of sea cucumber glycoprotein SCGP improving small intestinal tissue;

[0025] Fig. 9 Effect of sea cucumber glycoprotein SCGP on the expression of genes related to small intestinal glucose metabolism. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1: A method for extracting sea cucumber glycoprotein SCGP, comprising the following steps:

[0028] Step 1: freeze-dry the sea cucumber byproducts in a vacuum freeze dryer, crush them and pass them through a 60-mesh sieve for later use;

[0029] Step 2: Weigh 1 g of sea cucumber byproduct powder, add 5 volumes of acetone (w / v), soak at 4°C for 24 h, and blow dry with nitrogen to remove the acetone;

[0030] Step 3: Add 35 mL of distilled water to 1.0 g of defatted powder, stir and extract at 60 °C for 3 h, centrifuge at 6000 r / min for 10 min, and remove the precipitate;

[0031] Step 4: Add 3% neutral protease to the supernatant, react in a 50℃ water bath for 18h, and inactivate at 95℃ for 15min to terminate the reaction. Centrifuge the solution at 6000r / min for 10min;

[0032] Step 5: Take the supernatant, add 3 mL of 100 g / L hexadecyltrimethylammonium bromide solution, let it stand for 24 hours, and then centrifuge at 6000 r / min for 10 minutes;

[0033] Step 6: Discard the supernatant, dissolve the precipitate in 20 mL of 2 mol / L calcium chloride solution, then add 80 mL of 95% ethanol solution and let stand at 4 °C for 12 h;

[0034] Step 7: After centrifugation at 6 000 r / min for 10 min, remove the supernatant and add distilled water to dissolve the precipitate;

[0035] Step 8: Prepare Sevage reagent with a volume ratio of chloroform: n-butanol of 5:1, mix the reconstituted solution with the Sevage reagent and shake vigorously, let it stand and take the supernatant, repeat 6 times, dialyze the supernatant with a 10kDa dialysis bag for 48h, and freeze-dry to obtain the sea cucumber glycoprotein SCGP.

[0036] The total sugar content was determined by the phenol-sulfuric acid method, the protein content was determined by the Kjeldahl method, and the sulfate content was determined by the gelatin-barium chloride method. The results are shown in Table 1;

[0037] Table 1 Total sugar, protein and sulfate content of SCGP

[0038]

[0039] Example 2: Molecular weight, monosaccharide composition and amino acid composition of sea cucumber glycoprotein SCGP

[0040] (1) Molecular weight determination:

[0041] The SCGP and dextran standards in Example 1 were prepared into 2 mg / mL solutions with double distilled water and measured using an Agilent high performance liquid chromatography-evaporative light scattering detector. The chromatographic column was Waters Ultrahydrogel 500-250-120A (300*7.8mm), the mobile phase was deionized water, the carrier gas was N2, the gas flow rate was 1L / min, and the injection volume was 50μL. The logarithm of the relative molecular mass (LgMw) and the retention time (Rt) of the standard were used as the standard curve, and the molecular weight range of SCGP was measured. The results are shown in the figure. Figure 1 According to the standard curve of dextran with different molecular weights (LgMw=-0.4583x+25.13, R2=0.9989), the molecular weight of SCGP was calculated to be 1287 kDa.

[0042] (2) Determination of monosaccharide composition:

[0043] 10 mg of SCGP in Example 1 was weighed and dissolved in 3 mL of 2 mol / L trifluoroacetic acid (TFA) solution, hydrolyzed at 110°C for 8 h, TFA was removed by rotary vacuum evaporator, and the pH of the solution was adjusted to 7.0 with 6 mol / L NaOH. The monosaccharide standard and the acid hydrolyzate of SCGP were added to 1-phenyl-3-methyl-5-pyrazolone (PMP) solution (0.5 mL, 0.5 mol / L) and heated in a 70°C water bath for 60 min. After cooling, 0.2 mol / L NaOH solution was used to adjust to neutrality, and then the excess PMP was removed by extraction with chloroform five times. The supernatant was filtered through a 0.22 μm organic phase filter and analyzed by high performance liquid chromatography. The chromatographic column was a ZORBAX300XDB-C18 column with a wavelength of 245 nm; the detection mobile phase A was acetonitrile, the mobile phase B was 0.05 mol / L phosphate buffer solution, the gradient elution was 0-60 min, the mobile phase A: mobile phase B = 17%: 83%, and the injection volume was 20 μL.

[0044] Results: As shown in Table 2, SCGP1 is composed of six monosaccharides, and the composition and molar mass ratio of each monosaccharide are mannose: rhamnose: glucosamine: glucose: galactose: fucose = 4.24: 11.12: 26.91: 4.30: 41.82: 11.63.

[0045] Table 2 Monosaccharide composition of SCGP

[0046]

[0047] (3) Determination of amino acid composition:

[0048] Weigh 50 mg of SCGP in Example 1, place it in an ampoule, add 10 mL-15 mL 6 mol / L HCl (containing 5% mercaptoethanol), fill with nitrogen and seal the tube, place it in an oven at 110 ° C for hydrolysis for 22 hours. After cooling, dilute the hydrolyzate to 50 mL with 0.02 mol / L HCl, draw 1 mL of the hydrolyzate in a rotary evaporator, add 3-5 drops of ultrapure water and continue to evaporate until dry, repeat three times. Add 2 mL 0.02 mol / L HCl to re-dissolve, and the solution is filtered through a 0.22 μm water filter membrane and then enters the fully automatic amino acid analyzer for determination. Detector type: fluorescence detector; detection wavelength: 440, 570 nm; chromatographic column: cation exchange column, column temperature 70 ° C; injection volume: 10 μL; mobile phase: citric acid buffer, flow rate 0.35 mL / min; column temperature of the post-column reaction column: 135 ° C.

[0049] Results: As shown in Table 3, SCGP contains 18 kinds of amino acids, and the amino acids with higher content include glutamic acid, glycine, aspartic acid, threonine, etc. Among them, essential amino acids account for 26.85%, including threonine, valine, isoleucine, leucine, phenylalanine, and lysine. The results show that SCGP is rich in amino acids, which play an important role in its physiological function activity.

[0050] Table 3 Amino acid composition of SCGP

[0051]

[0052] Example 3: Infrared spectroscopy analysis of sea cucumber glycoprotein SCGP

[0053] A small amount of SCGP prepared in Example 1 was mixed with KBr, ground evenly, and pressed into tablets. -1 Scan detection within range.

[0054] Results: Fourier transform infrared spectroscopy is often used to partially reveal the presence of characteristic functional groups of a substance. Figure 2 As shown, 3369.93cm -1 The strong absorption peak at 2928.35 cm is the OH stretching vibration of the hydroxyl group in the polysaccharide. -1 The absorption peak near is the CH stretching vibration of the alkyl group; at 1662.83cm -1 The obvious absorption peak at 1237cm is caused by the stretching vibration of C=O in the amide carboxyl group; -1 The absorption peak at 1067.76cm is caused by the stretching vibration of the sulfate group S=O, indicating that the sample contains sulfate groups; -1 The stretching vibration peak of CO is at 968.14 cm -1 The absorption peak at 828.13cm is the torsional vibration absorption peak of C=C;-1 The absorption peak of α-pyranose glycosidic bond is at 1400-1600cm -1 The multiple weak absorption peaks may be caused by the stretching vibrations of the direct binding bonds between sugar and protein in SCGP.

[0055] Example 4: Determination of glycopeptide bonds in sea cucumber glycoprotein SCGP

[0056] 2 mg of SCGP in Example 1 was weighed and dissolved in 10 mL of 0.4 mol / L NaOH and distilled water, respectively, and placed in a water bath at 60° C. for 30 min. The wavelength was scanned between 220 and 300 nm, and the type of glycosylamine bond was determined based on the absorbance at 240 nm.

[0057] Results: O-type glycopeptide bonds and N-type glycopeptide bonds, the former is unstable to alkali and is prone to β-elimination reaction in NaOH solution. The serine and threonine on the glycopeptide bond are converted into α-aminoacrylic acid and α-butylacrylic acid under the action of alkali, both of which will produce obvious ultraviolet absorption at 240nm absorbance. Usually, glycoprotein samples are dissolved in NaOH solution for reaction, and samples dissolved in water are used as controls. Full-wavelength ultraviolet scanning is performed, and the type of glycopeptide bonds in glycoproteins is preliminarily judged by judging the changes in the absorbance of the control at 240nm. Figure 3 As shown in the figure, the absorbance of SCGP after NaOH treatment at 240 nm increased significantly, indicating that the glycopeptide bond type of SCGP is O-glycopeptide bond, which also proves that there is a binding state between sugar and protein in SCGP.

[0058] Example 5: Determination of the inhibition of α-glucosidase by sea cucumber glycoprotein SCGP

[0059] 100 μL of SCGP solution of different concentrations was mixed with an equal volume of α-glucosidase solution (0.5 U / mL, pH = 6.9) and incubated at 37°C for 20 min. 100 μL of p-nitrophenyl-α-D-pyranoglucoside (pNPG, 5 mM) was added and incubated at 37°C for 30 min. 1 mL of 1 mol / L Na2CO3 was added to terminate the reaction, and the absorbance was measured at 405 nm. The inhibition rate (%) was calculated according to the following formula, and the half inhibitory concentration (IC) was calculated according to the inhibition curve. 50 ), and acarbose was used as a positive control.

[0060]

[0061] Results: Figure 4 As shown in Figure 2, the inhibitory effect of SCGP on α-glucosidase was dose-dependent. The IC 50 The above results show that SCGP has a significant inhibitory effect on α-glucosidase.

[0062] Example 6: Effect of sea cucumber glycoprotein SCGP on improving postprandial blood glucose in mice

[0063] Six-week-old ICR male mice were selected and raised in an air-conditioned room with a temperature of 25±2℃ and a light / dark cycle of 12h. After one week of adaptive feeding, the mice were divided into 8 groups (n=6). Among them, 4 groups of male ICR mice were fasted for 12h, and each mouse was gavaged with 2.0g / kg sucrose, and 150mg / kg SCGP was orally administered after an interval of 15min. Blood samples were drawn from the tail after administration, and blood glucose levels were measured at 0, 30, 60, 90 and 120min. Blood glucose levels were measured with a blood glucose meter and compared with the control group that did not take the inhibitor. In addition, mice were fed with high sugar and high fat for five weeks to construct a prediabetes model, and the above measurements were repeated. At the same time, insulin injections were performed to determine the effect of SCGP on improving insulin tolerance in prediabetic mice.

[0064] Results: To confirm the α-glucosidase inhibitory activity of the above SCGP and the in vivo relevance of the in vitro results, we used ICR normal mice and prediabetic models for sucrose loading tests. Figure 5 As shown in -A, compared with the control group, SCGP can significantly reduce postprandial hyperglycemia caused by sucrose loading 15 minutes later, with blood glucose dropping from 15.5mmol / L to 9.9mmol / L, and there is no obvious blood glucose peak period, and the change trend is similar to that of acarbose. Figure 6 -A shows that compared with the normal group, after gavage of sucrose and maltose, the blood glucose level of the model group mice continued to rise during the 15-30min period after the meal, and showed a downward trend at 90min, but the blood glucose level was about 10mmol / L after 120min, which was much higher than that of the normal group. The area under the curve values ​​of the normal group and the model group were 16.3 and 23.5mmol / L*h, respectively. The blood glucose level of the acarbose group decreased significantly at 15min after the meal, and the blood glucose level dropped to a level close to the normal level, and the area under the curve decreased to 18.7mmol / L*h, which was not significantly different from the normal group. After intervention in the SCGP group, the postprandial blood glucose reached a peak at 15min, but dropped significantly after 30min, and dropped to a normal level after 120min, which can effectively improve the postprandial blood glucose level. The occurrence of type 2 diabetes is closely related to the increase of postprandial blood glucose. Therefore, reducing the postprandial hyperglycemia response is a key strategy for controlling diabetes.

[0065] Example 7 Effect of sea cucumber glycoprotein SCGP on improving insulin tolerance in prediabetic mice

[0066] After 6 weeks of drug administration, mice in each group were fasted for 12 hours, and blood was collected from the tail vein using a blood glucose meter to measure fasting blood glucose levels. Insulin (0.4 U / kg) was injected intraperitoneally into the mice, and blood glucose levels were measured at 15, 30, 60, and 120 minutes after insulin injection to calculate the oral insulin glucose tolerance of the mice.

[0067] Results: Insulin tolerance is another way to test for insulin resistance. Figure 7 As shown in the figure, the blood glucose level in the normal group dropped rapidly, while the blood glucose level in the model group dropped the slowest. Compared with the model group, the mice in the SCGP group showed a rapid downward trend, and the same results were obtained by calculating the insulin AUC. This shows that long-term intake of SCGP can effectively improve glucose metabolism and increase insulin sensitivity.

[0068] Example 8 Effect of Sea Cucumber Glycoprotein SCGP on the Intestinal Improvement of Prediabetic Mice

[0069] Six weeks after gavage, all mice were killed by cervical dislocation. The mouse intestines were fixed in 4% paraformaldehyde solution for more than 24 hours, then dehydrated and embedded in paraffin. Paraffin-embedded tissue sections were cut, stained with hematoxylin and eosin, and observed under an optical microscope. The small intestinal tissues of each group of mice were quickly frozen and crushed with liquid nitrogen, RNA was extracted with a kit, and reverse transcribed with a cDNA reverse transcription kit. Quantitative polymerase chain reaction (qPCR) was used to quantitatively analyze the expression of genes related to sugar metabolism. All sequences of qPCR primers are shown in Table 4, and SHDA is an internal reference.

[0070] Table 4 qPCR primer sequences

[0071]

[0072] Results: Figure 8 As shown in the figure, the villi of the small intestine of the normal group of mice were arranged neatly, the single layer of columnar epithelium on the surface of the villi was neat and orderly, there were obvious goblet cells, the intestinal crypts were complete, and there were no histological abnormalities. The number of small intestinal villi in the intestine of prediabetic mice was significantly reduced and obvious damage appeared, the number of intestinal crypts was significantly shallower and reduced, the number of goblet cells was significantly reduced, and the mucosal muscle became thinner. After long-term gavage, the state of the small intestinal villi in the SCGP group returned to normal levels, the number of goblet cells and intestinal crypts was significantly restored, and the mucosal muscle thickened.

[0073] Sodium / glucose cotransporter 1 (SGLT1) is a high-affinity, low-capacity transporter that transports glucose into the mesenteric vein when the intraluminal concentration of glucose is below 30 mM, whereas glucose is transported primarily via GLUT2, a low-affinity, high-capacity transporter, when the intraluminal concentration of glucose is higher after a meal. Modulation of SGLT1 activity is an attractive therapeutic target for the treatment of diabetes by controlling glucose uptake and transport. The function of SGLT1 is associated with the activity of phospholipase Cβ2 (PLCβ2) and protein kinase C (PKC), which can increase intracellular Ca 2+ The release of further activates SGLT1 for glucose transport. Glucagon-like peptide (GLP-1) is also secreted by enteroendocrine cells and mainly regulates postprandial glucose excursion by enhancing glucose-stimulated insulin secretion. Incretin function. GLP-1 has been shown to attenuate small intestinal glucose absorption. Fig. 9 As shown in the results, SCGP can inhibit the expression of SGLT1 by downregulating the PLCβ2 / PKCα pathway, inhibit the small intestine's uptake of blood glucose, and help lower postprandial blood glucose. At the same time, SCGP can increase the gene expression of GLP-1 and promote insulin secretion.

[0074] Comparative Example 1:

[0075] The difference between this comparative example and Example 1 is that a composite protease is used for enzymolysis. The protein content and total sugar content of the obtained product are 40.09% and 36.22% respectively, and the monosaccharide composition and molar percentage are mannose: rhamnose: glucose: galactose: arabinose: fucose = 5.89: 21.79: 12.55: 29.12: 20.13: 10.53. After in vitro experiments, the composite protease obtained product has no significant effect on inhibiting α-glucosidase. When the product concentration is 15 mg / mL, the inhibition rate is only 47.25%.

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high galactose content acidic sea cucumber glycoprotein, characterized in that: The ratio of protein content to total sugar content in the sea cucumber glycoprotein is 1:0.85-1:1.20, the monosaccharide composition is mannose, rhamnose, glucosamine, glucose, galactose, fucose, and the molar proportion of galactose is 41.8%-46.6%, and it contains 18 kinds of amino acids.

2. The high galactose content acidic sea cucumber glycoprotein according to claim 1, characterized in that: The sea cucumber glycoprotein is prone to β-elimination reaction in NaOH solution, produces obvious ultraviolet absorption at 240nm absorbance, and has O-glycopeptide bonds.

3. The method for preparing a high-galactose content acidic sea cucumber glycoprotein according to claim 2, characterized in that: The method comprises the following preparation steps: Step 1: crush the sea cucumber byproduct after freeze-drying, and pass it through a 60-mesh sieve for later use; Step 2: Soak the powder from step 1 in acetone solution 6 times the volume of the powder, centrifuge to remove the acetone, and blow dry with nitrogen; Step 3: Add 20-40 times the volume of deionized water to the powder obtained in step 2, mix well, stir and extract at 40-70°C for 2-3h, and remove the precipitate after centrifugation; Step 4: Add 1%-3% neutral protease to the supernatant obtained in step 3 and react at 45-60°C for 6-24h. After the enzymolysis is completed, heat in a boiling water bath to inactivate the enzyme, and collect the enzymolysis supernatant after centrifugation; Step 5: adding quaternary ammonium salt to the supernatant obtained in step 4 and collecting the precipitate; Step 6: Add calcium chloride solution to dissolve the precipitate obtained in step 5, and add 2-4 times the volume of 95% ethanol to precipitate at 4°C overnight, centrifuge to obtain the precipitate and reconstitute it with pure water; Step 7: The reconstituted solution obtained in step 6 is subjected to Sevage method to remove free protein, dialyzed using a 10 kDa dialysis bag, and freeze-dried to obtain sea cucumber glycoprotein.

4. The high galactose content acidic sea cucumber glycoprotein according to claim 1, characterized in that: The sea cucumber glycoprotein is used for reducing postprandial blood sugar. The sea cucumber glycoprotein inhibits α-glucosidase activity, reduces postprandial blood sugar levels of normal mice and diabetic mice in animal experiments, and promotes insulin sensitivity of mice.

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