A mulberry oligosaccharide, a preparation method thereof and application thereof in blood glucose regulation

Abstract

The present application relates to a mulberry oligosaccharide, a preparation method thereof and application thereof in blood sugar regulation. Through a large number of researches, the present application uses specific enzymolysis, selenium acid modification, plasma treatment and other process steps to treat mulberries to obtain mulberry oligosaccharide, which can obviously improve the stability, significantly improve the inhibition ability to alpha-glucosidase, improve the glucose tolerance condition of diabetic mice, stabilize the blood sugar level, and at the same time, can improve the insulin sensitivity of the body, improve the insulin resistance degree, improve the absorption and utilization of insulin to glucose, improve the SOD activity of the body, and reduce the MDA content, thereby effectively slowing down the development of diabetes. In addition, the present application uses natural mulberries as the main preparation raw material, does not add any harmful chemical components, has low cost, good safety, and has great social significance.

Description

Technical Field

[0001] This invention belongs to the field of natural product applications, specifically relating to a mulberry oligosaccharide, its preparation method, and its application in blood glucose regulation. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia and is one of the most important chronic diseases and leading causes of death in modern society. In recent decades, the prevalence and incidence of diabetes have been on the rise, with approximately 10% of the global population already suffering from or at risk of developing type 2 diabetes. In patients with type 2 diabetes, alpha-glucosidase hydrolyzes starch into glucose during metabolism, leading to a significant increase in postprandial blood glucose. Therefore, screening for effective ingredients with therapeutic potential by conducting alpha-glucosidase inhibition assays on natural plant components has become a common method for developing hypoglycemic drugs with no toxic side effects.

[0003] Plant polysaccharides possess hypoglycemic, hypolipidemic, and anti-inflammatory properties, but their large molecular weight and limited water solubility restrict their applications. Hydrolyzing large polysaccharides into smaller oligosaccharides, followed by appropriate modification to enhance their functionality, can broaden their application areas. Current processes for extracting plant oligosaccharides involve simple mechanical crushing-solvent extraction, hot reflux extraction, or simple enzymatic hydrolysis, making it difficult to extract the active oligosaccharide components and thus hindering their ability to effectively regulate blood sugar. Therefore, refining, extracting, degrading, and modifying plant resources to prepare a highly efficient, stable, and blood sugar-regulating mulberry oligosaccharide has significant market potential.

[0004] Existing technologies for extracting plant oligosaccharides involve simple crushing and mixing of plant resources, or simple mechanical crushing-solvent extraction, hot reflux extraction, or simple enzymatic hydrolysis. This makes it difficult to extract the active oligosaccharide components, thus hindering their ability to effectively regulate blood sugar. Therefore, refining, extracting, degrading, and modifying plant resource components, and finding a highly efficient, stable, and blood sugar-regulating mulberry oligosaccharide, is of paramount importance. Summary of the Invention

[0005] The purpose of this invention is to address the problem in existing technologies that it is difficult to effectively extract active oligosaccharides from mulberries and thus cannot maximize their blood sugar regulation effect. Therefore, this invention provides a method for preparing mulberry oligosaccharides by using specific enzymatic hydrolysis, selenic acid modification, plasma treatment and other process steps to process mulberries, thereby developing a stable, safe, and effective mulberry oligosaccharide that can regulate blood sugar.

[0006] To achieve the above objectives, the present invention is accomplished by the following means:

[0007] The first aspect of this invention provides a method for preparing mulberry oligosaccharides, comprising the following steps:

[0008] (1) After crushing and sieving the mulberry, water extraction was performed. The extract was concentrated and then subjected to alcohol precipitation with ethanol. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide.

[0009] (2) The mulberry polysaccharide obtained in step (1) is first hydrolyzed by glucanase and then by alkaline protease to obtain the hydrolysate;

[0010] (3) Take the enzymatic hydrolysate obtained in step (2) and add vitamin C solution to stir and react. Then add H2O2 and stir to carry out oxidative degradation reaction to obtain mulberry oligosaccharide;

[0011] (4) The mulberry oligosaccharide obtained in step (3) is separated by ultrafiltration and the retentate is taken; sodium selenite solution, nitric acid solution and barium chloride are added to the retentate and the reaction is carried out by water bath heating; after the reaction is completed, the barium chloride is removed, the supernatant is obtained by centrifugation, the pH is adjusted to neutral, the ions are removed by dialysis, the water is changed once after 6-12 hours, and the selenic acid modified mulberry oligosaccharide solid powder is obtained by vacuum freeze drying.

[0012] (5) Place the selenic acid modified mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment to obtain the product.

[0013] Preferably, in step (1), the mulberry is crushed and then passed through a 30-mesh sieve.

[0014] Preferably, the water extraction temperature in step (1) is 70-90℃ and the time is 3-8h.

[0015] Preferably, the ethanol in step (1) is 95% ethanol, the volume of ethanol is 3 times the volume of the concentrate, and the alcohol precipitation time is 12-36h.

[0016] Preferably, in step (2), the temperature for enzymatic hydrolysis with dextranase is 35-45℃, the amount of enzyme added is 200U / mL, and the time is 1-2h; the temperature for enzymatic hydrolysis with alkaline protease is 40-50℃, the amount of enzyme added is 300U / mL, and the time is 2-3h.

[0017] Preferably, the concentration of the vitamin C solution in step (3) is 3-8 mmol / L, and the volume ratio of the vitamin C solution to the enzymatic hydrolysate is 1:150-250.

[0018] Preferably, the temperature of the stirring reaction in step (3) is 35-45℃ and the time is 20-40min.

[0019] Preferably, the concentration of H2O2 in step (3) is 1-4 mmol / L, and the volume ratio of H2O2 to the enzymatic hydrolysate is 1:40-60.

[0020] Preferably, the temperature of the oxidative degradation reaction in step (3) is 35-45℃ and the time is 40-80min.

[0021] Preferably, the ultrafiltration separation in step (4) is as follows: first, the macromolecular substances are removed by passing through an ultrafiltration membrane with a molecular weight of 5 kDa, and the effluent is taken; then, the effluent is taken by passing through an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is taken to obtain a 0.5-5 kDa mulberry oligosaccharide solution.

[0022] Preferably, the concentration of the sodium selenite solution in step (4) is 40-60 mg / mL, and the volume ratio of the sodium selenite solution to the enzymatic hydrolysate in step (3) is 1:50-150.

[0023] Preferably, the volume fraction of the nitric acid solution in step (4) is 0.5-1.5%, and the volume ratio of the nitric acid solution to the enzymatic hydrolysate in step (3) is 1:150-250.

[0024] Preferably, the mass-to-volume ratio (g:mL) of barium sulfate in step (4) to the enzymatic hydrolysate in step (3) is 1:1500-2500.

[0025] Preferably, the water bath heating reaction in step (4) is carried out at a temperature of 50-70°C for 3-8 hours.

[0026] Preferably, the removal of barium chloride in step (4) specifically involves adding a saturated sodium sulfate solution to remove barium chloride.

[0027] Preferably, the centrifugation speed in step (4) is 5000-15000 rpm and the time is 5-15 min.

[0028] Preferably, sodium carbonate solution is used to adjust the pH in step (4).

[0029] Preferably, in step (4), a dialysis bag with a molecular weight of 2000 Da is used for dialysis.

[0030] Preferably, the vacuum degree of the vacuum freeze drying in step (4) is 10-30 Pa and the temperature is -50~-20℃.

[0031] Preferably, the plasma treatment in step (5) is as follows: using dielectric barrier discharge as the low-temperature plasma generation method, using air environment as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 4-6cm, the discharge spacing is set to 6-10mm, the input voltage is 100-130V, the input current is 0.5-1.5A, and the processing time is 0.5-2min.

[0032] A second aspect of the present invention provides mulberry oligosaccharides prepared according to the above-described preparation method.

[0033] A third aspect of the present invention provides the application of mulberry oligosaccharides prepared according to the above preparation method in the preparation of products for regulating blood sugar.

[0034] Preferably, the product is selected from one or more of food, health products, and pharmaceuticals.

[0035] A fourth aspect of the present invention provides a composition for regulating blood sugar, comprising mulberry oligosaccharide prepared according to the above preparation method, and excipients.

[0036] Preferably, the excipients include, but are not limited to, one or more of solvents, fillers, flavoring agents, coloring agents, stabilizers, chelating agents, adhesives, lubricants, and fragrances.

[0037] Mulberries, a plant used both as food and medicine, are rich in various natural bioactive components. Their functional oligosaccharide component—mulberry oligosaccharides—as water-soluble dietary fiber, has been proven to be one of the core active substances in mulberry fruit. Modern pharmacological studies have shown that mulberry oligosaccharides possess multi-target biological activities, such as hypoglycemic, anti-fatty liver, anti-aging, neuroprotective, immune-enhancing, and anti-cancer effects. Particularly noteworthy is that, based on modern pharmacological techniques, mulberry oligosaccharides exert their hypoglycemic effect primarily through mechanisms such as inhibiting intestinal α-glucosidase activity, regulating hepatic glucokinase expression, and improving insulin sensitivity.

[0038] Current industrial-scale preparation processes for plant-derived oligosaccharides largely rely on traditional methods, including mechanical pulverization-solvent extraction, thermal reflux extraction, or simple enzymatic hydrolysis. These methods have limitations, such as the wide molecular weight distribution of unmodified oligosaccharides, limited bioavailability, and difficulty in achieving effective blood glucose regulation. Therefore, refining and extracting mulberry components and appropriately degrading and modifying them to prepare mulberry oligosaccharides that effectively regulate blood glucose is of great significance. This invention, through extensive research, utilizes specific enzymatic hydrolysis, selenic acid modification, and plasma treatment processes to obtain mulberry oligosaccharides. These processes significantly improve their stability, enhance their inhibitory ability against α-glucosidase, improve glucose tolerance in diabetic mice, stabilize blood glucose levels, improve insulin sensitivity, reduce insulin resistance, enhance insulin absorption and utilization of glucose, increase SOD activity, and reduce MDA content, thereby effectively slowing the progression of diabetes. In addition, this invention uses naturally sourced mulberry as the main raw material, without adding any harmful chemical components, making it low in cost, safe, and of great social significance. Detailed Implementation

[0039] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0040] Example 1

[0041] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0042] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 400 mg / mL.

[0043] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 40°C for 1.5 h with glucanase, and then enzymatically hydrolyzed at 45°C for 2.5 h with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0044] (3) Take 1L of the enzymatic hydrolysate obtained in step (2) and add 5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 40℃ for 30min. Then add 20mL of H2O2 with a concentration of 2.5mmol / L and stir at 40℃ for 60min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0045] (4) The supernatant of mulberry oligosaccharide obtained in step (3) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. Then, 10 mL of sodium selenite solution with a concentration of 50 mg / mL, 5 mL of nitric acid solution with a volume fraction of 1% and 0.5 g of barium chloride are added, and the mixture is heated in a water bath at 60℃ for 5 h. After the reaction, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the mixture is centrifuged at 8000 rpm for 10 min to obtain the supernatant. Sodium carbonate solution is added to adjust the pH to 7, and ion removal is achieved by dialysis using a dialysis bag (Mw: 2000 Da). The water is changed once after 8 h, and the mixture is freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30℃ to obtain selenic acid modified mulberry oligosaccharide solid powder.

[0046] (5) Place 5g of selenic acid modified mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: dielectric barrier discharge is used as the low-temperature plasma generation mode, air environment is used as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, the discharge spacing is set to 8mm to ensure uniform discharge, the input voltage is controlled at 110V, the frequency is adjusted so that the input current is 1.0A, the treatment is carried out for 1min, and the sample is collected in a sealed container for storage.

[0047] Example 2

[0048] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0049] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 90℃ for 3 hours. The extract was concentrated to 1 / 3 and then subjected to alcohol precipitation with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 300 mg / mL.

[0050] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 35°C for 2 hours with glucanase, and then enzymatically hydrolyzed at 40°C for 3 hours with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0051] (3) Take 1L of the enzymatic hydrolysate obtained in step (2) and add 4.5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 35℃ for 40min. Then add 18mL of H2O2 with a concentration of 2.5mmol / L and stir at 35℃ for 80min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0052] (4) The supernatant of mulberry oligosaccharide obtained in step (3) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. Then, 7 mL of sodium selenite solution with a concentration of 50 mg / mL, 4 mL of nitric acid solution with a volume fraction of 1% and 0.4 g of barium chloride are added, and the mixture is heated in a water bath at 50℃ for 8 h; after the reaction, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the supernatant is obtained by centrifugation at 15000 rpm for 5 min. Sodium carbonate solution is added to adjust the pH to 7, and ion removal is carried out by dialysis using a dialysis bag (Mw: 2000 Da). After 8 h, the water is changed once, and the selenic acid modified mulberry oligosaccharide solid powder is obtained by vacuum freeze drying at a vacuum degree of 20 Pa and a shelf temperature of -30℃.

[0053] (5) Place 5g of selenic acid modified mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: dielectric barrier discharge is used as the low-temperature plasma generation mode, air environment is used as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, the discharge spacing is set to 8mm to ensure uniform discharge, the input voltage is controlled at 110V, the frequency is adjusted so that the input current is 1.0A, the treatment is carried out for 1min, and the sample is collected in a sealed container for storage.

[0054] Example 3

[0055] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0056] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 70℃ for 8 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 500 mg / mL.

[0057] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 45°C for 1 hour with glucanase, and then enzymatically hydrolyzed at 50°C for 2 hours with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0058] (3) Take 1L of the enzymatic hydrolysate obtained in step (2) and add 6mL of vitamin C solution with a concentration of 5mmol / L. Stir at 45℃ for 20min. Then add 25mL of H2O2 with a concentration of 2.5mmol / L and stir at 45℃ for 40min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0059] (4) The mulberry oligosaccharide supernatant obtained in step (3) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. Then, 15 mL of sodium selenite solution with a concentration of 50 mg / mL, 6 mL of nitric acid solution with a volume fraction of 1% and 0.6 g of barium chloride are added, and the mixture is heated in a water bath at 70℃ for 3 h. After the reaction, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the mixture is centrifuged at 5000 rpm for 15 min to obtain the supernatant. Sodium carbonate solution is added to adjust the pH to 7, and ion removal is achieved by dialysis using a dialysis bag (Mw: 2000 Da). The water is changed once after 8 h, and the mixture is freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30℃ to obtain selenic acid modified mulberry oligosaccharide solid powder.

[0060] (5) Place 5g of selenic acid modified mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: dielectric barrier discharge is used as the low-temperature plasma generation mode, air environment is used as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, the discharge spacing is set to 8mm to ensure uniform discharge, the input voltage is controlled at 110V, the frequency is adjusted so that the input current is 1.0A, the treatment is carried out for 1min, and the sample is collected in a sealed container for storage.

[0061] Comparative Example 1

[0062] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0063] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 400 mg / mL.

[0064] (2) Take 1L of the mulberry polysaccharide solution obtained in step (1) and add 5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 40℃ for 30min. Then add 20mL of H2O2 with a concentration of 2.5mmol / L and stir at 40℃ for 60min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0065] (3) The mulberry oligosaccharide supernatant obtained in step (2) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. Then, 10 mL of sodium selenite solution with a concentration of 50 mg / mL, 5 mL of nitric acid solution with a volume fraction of 1% and 0.5 g of barium chloride are added, and the mixture is heated in a water bath at 60℃ for 5 h. After the reaction, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the mixture is centrifuged at 8000 rpm for 10 min to obtain the supernatant. Sodium carbonate solution is added to adjust the pH to 7, and dialysis is performed using a dialysis bag (Mw: 2000 Da) to remove ions. After 8 h, the water is changed once, and the mixture is freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30℃ to obtain selenic acid modified mulberry oligosaccharide solid powder.

[0066] (4) Place 5g of selenic acid modified mulberry oligosaccharide solid powder obtained in step (3) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: use dielectric barrier discharge as the low-temperature plasma generation mode, use air environment as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, set the discharge spacing to 8mm to ensure uniform discharge, control the input voltage to 110V, adjust the frequency to make the input current 1.0A, process for 1min, collect the sample into a sealed container for storage, and the product is obtained.

[0067] Comparative Example 2

[0068] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0069] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 400 mg / mL.

[0070] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 40°C for 1.5 h with glucanase, and then enzymatically hydrolyzed at 45°C for 2.5 h with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0071] (3) The enzymatic hydrolysate obtained in step (2) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a solution with a molecular weight of 0.5-5 kDa. Then, 10 mL of sodium selenite solution with a concentration of 50 mg / mL, 5 mL of nitric acid solution with a volume fraction of 1% and 0.5 g of barium chloride are added, and the mixture is heated in a water bath at 60℃ for 5 h. After the reaction, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the mixture is centrifuged at 8000 rpm for 10 min to obtain the supernatant. Sodium carbonate solution is added to adjust the pH to 7, and ion removal is achieved by dialysis using a dialysis bag (Mw: 2000 Da). The water is changed once after 8 h, and the mixture is freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30℃ to obtain selenic acid modified mulberry oligosaccharide solid powder.

[0072] (4) Place 5g of selenic acid modified mulberry oligosaccharide solid powder obtained in step (3) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: use dielectric barrier discharge as the low-temperature plasma generation mode, use air environment as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, set the discharge spacing to 8mm to ensure uniform discharge, control the input voltage to 110V, adjust the frequency to make the input current 1.0A, process for 1min, collect the sample into a sealed container for storage, and the product is obtained.

[0073] Comparative Example 3

[0074] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0075] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 400 mg / mL.

[0076] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 40°C for 1.5 h with glucanase, and then enzymatically hydrolyzed at 45°C for 2.5 h with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0077] (3) Take 1L of the enzymatic hydrolysate obtained in step (2) and add 5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 40℃ for 30min. Then add 20mL of H2O2 with a concentration of 2.5mmol / L and stir at 40℃ for 60min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0078] (4) The mulberry oligosaccharide supernatant obtained in step (3) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. The solution is then freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30 °C to obtain mulberry oligosaccharide solid powder.

[0079] (5) Place 5g of mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: use dielectric barrier discharge as the low-temperature plasma generation mode, use air environment as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, set the discharge spacing to 8mm to ensure uniform discharge, control the input voltage to 110V, adjust the frequency to make the input current 1.0A, process for 1min, collect the sample into a sealed container for storage, and the product is obtained.

[0080] Comparative Example 4

[0081] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0082] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 400 mg / mL.

[0083] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 40°C for 1.5 h with glucanase, and then enzymatically hydrolyzed at 45°C for 2.5 h with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0084] (3) Take 1L of the enzymatic hydrolysate obtained in step (2) and add 5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 40℃ for 30min. Then add 20mL of H2O2 with a concentration of 2.5mmol / L and stir at 40℃ for 60min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0085] (4) The mulberry oligosaccharide supernatant obtained in step (3) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. Then, 10 mL of sodium selenite solution with a concentration of 50 mg / mL, 5 mL of nitric acid solution with a volume fraction of 1% and 0.5 g of barium chloride are added, and the mixture is heated in a water bath at 60℃ for 5 h. After the reaction, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the mixture is centrifuged at 8000 rpm for 10 min to obtain the supernatant. Sodium carbonate solution is added to adjust the pH to 7, and dialysis is performed using a dialysis bag (Mw: 2000 Da) to remove ions. After 8 h, the water is changed once, and the mixture is freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30℃ to obtain selenic acid modified mulberry oligosaccharide solid powder. The sample is collected and stored in a sealed container.

[0086] Comparative Example 5

[0087] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0088] (1) After crushing the mulberry and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The polysaccharide concentration was determined by the phenol-sulfuric acid method and then diluted to 400 mg / mL.

[0089] (2) The mulberry polysaccharide obtained in step (1) is first enzymatically hydrolyzed at 40°C for 1.5 h with glucanase, and then enzymatically hydrolyzed at 45°C for 2.5 h with alkaline protease to obtain the enzymatic hydrolysate; wherein the amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0090] (3) Take 1L of the enzymatic hydrolysate obtained in step (2) and add 5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 40℃ for 30min. Then add 20mL of H2O2 with a concentration of 2.5mmol / L and stir at 40℃ for 60min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0091] (4) The mulberry oligosaccharide supernatant obtained in step (3) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. The solution is then freeze-dried under vacuum at a vacuum degree of 20 Pa and a shelf temperature of -30 °C to obtain mulberry oligosaccharide solid powder.

[0092] (5) Place 5g of mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: dielectric barrier discharge is used as the low-temperature plasma generation method, air environment is used as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, the discharge spacing is set to 8mm to ensure uniform discharge, the input voltage is controlled at 110V, and the frequency is adjusted so that the input current is 1.0A for 1min.

[0093] (6) Prepare a 400 mg / mL solution of plasma-treated mulberry oligosaccharide solid powder obtained in step (5). Take 1 L of the solution and add 10 mL of 50 mg / mL sodium selenite solution, 5 mL of 1% nitric acid solution and 0.5 g of barium chloride. Heat the solution in a water bath at 60 °C for 5 h. After the reaction, add an appropriate amount of saturated sodium sulfate solution to remove barium chloride. Centrifuge at 8000 rpm for 10 min to obtain the supernatant. Add sodium carbonate solution to adjust the pH to 7. Dialyze the solution using a dialysis bag (Mw: 2000 Da) to remove ions. Change the water once after 8 h. Freeze dry the solution under vacuum at 20 Pa and shelf temperature at -30 °C to obtain solid powder. Collect the sample in a sealed container for storage.

[0094] Comparative Example 6

[0095] A mulberry oligosaccharide, the preparation method of which specifically includes the following steps:

[0096] (1) After crushing the mulberry into powder and passing it through a 30-mesh sieve, water was extracted at 80℃ for 5 hours. The extract was concentrated to 1 / 3 and then precipitated with 3 times the volume of 95% ethanol for 24 hours. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. The mulberry polysaccharide solid powder was obtained by vacuum freeze drying at a vacuum degree of 20 Pa and a shelf temperature of -30℃.

[0097] (2) Place 5g of mulberry polysaccharide solid powder obtained in step (1) into the groove of the reaction vessel for plasma treatment; the specific conditions for plasma treatment are as follows: dielectric barrier discharge is used as the low-temperature plasma generation method, air environment is used as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 5cm, the discharge spacing is set to 8mm to ensure uniform discharge, the input voltage is controlled at 110V, and the frequency is adjusted so that the input current is 1.0A for 1min.

[0098] (3) Prepare a 400 mg / mL solution of mulberry polysaccharide solid powder obtained by plasma treatment in step (2). Take 1 L of the solution and first enzymatically hydrolyze it with glucanase at 40℃ for 1.5 h, and then enzymatically hydrolyze it with alkaline protease at 45℃ for 2.5 h to obtain the hydrolysate. The amount of glucanase added is 200 U / mL and the amount of alkaline protease added is 300 U / mL.

[0099] (4) Take 1L of the enzymatic hydrolysate obtained in step (3) and add 5mL of vitamin C solution with a concentration of 5mmol / L. Stir at 40℃ for 30min. Then add 20mL of H2O2 with a concentration of 2.5mmol / L and stir at 40℃ for 60min to carry out oxidative degradation reaction to obtain mulberry oligosaccharide.

[0100] (5) The mulberry oligosaccharide supernatant obtained in step (4) is first separated by an ultrafiltration membrane with a molecular weight of 5 kDa to remove macromolecular substances, and the effluent is collected; then it is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected to obtain a mulberry oligosaccharide solution with a molecular weight of 0.5-5 kDa. Then, 10 mL of sodium selenite solution with a concentration of 50 mg / mL, 5 mL of nitric acid solution with a volume fraction of 1% and 0.5 g of barium chloride are added, and the mixture is heated in a water bath at 60℃ for 5 h. After the reaction is completed, an appropriate amount of saturated sodium sulfate solution is added to remove barium chloride, and the mixture is centrifuged at 8000 rpm for 10 min to obtain the supernatant. Sodium carbonate solution is added to adjust the pH to 7, and dialysis is performed using a dialysis bag (Mw: 2000 Da) to remove ions. The water is changed once after 8 h, and the mixture is freeze-dried to obtain a solid powder. The sample is collected and stored in a sealed container.

[0101] Verification Example 1

[0102] Zeta potential is an important indicator of the stability of oligosaccharide particles in solution. When the zeta potential value is high, the particle surface in the solution has a stronger electrostatic repulsion, resulting in better stability and making it easier for oligosaccharides to exert their active effects (see Table 1).

[0103] Table 1 Relationship between Zeta potential and solution stability

[0104] 0-3 The greatest possibility is aggregation and precipitation. 5 The possibility of aggregation and precipitation is relatively strong. 10-15 Critical value of aggregation 16-30 Critical value for fine dispersion 31-40 Medium stability 41-60 Good stability 61-80 Very good stability 81-100 Excellent stability

[0105] Quantitative indicator: The system is highly stable when |Zeta| > 30 mV.

[0106] To this end, samples prepared in Examples 1-3 and Comparative Examples 1-6 were prepared into solutions with a concentration of 1 mg / mL. These solutions were filtered through a 0.22 μm filter membrane before detection to avoid multiple scattering effects. The Zeta potentials of the examples and comparative examples were analyzed using a dynamic light scattering particle size analyzer at 25 °C. Each set of data was tested five times, and the average value was taken. The experimental results are shown in Table 2 below.

[0107] Table 2. Zeta potentials of samples from Examples 1-3 and Comparative Examples 1-6

[0108] Example 1 -88.9 88.9 Example 2 -86.8 86.8 Example 3 -87.5 87.5 Comparative Example 1 -28.6 28.6 Comparative Example 2 -31.5 31.5 Comparative Example 3 -33.4 33.4 Comparative Example 4 -25.3 25.3 Comparative Example 5 -29.4 29.4 Comparative Example 6 -30.5 30.5

[0109] The results above show that the potentials of Examples 1-3 ranged from -86.8 mV to -88.9 mV, and their absolute values ​​were higher than those of Comparative Examples 1-6. This indicates that the oligosaccharides of Examples 1-3 are negatively charged, are anionic oligosaccharides, and have excellent stability in solution.

[0110] Verification Example 2

[0111] Inhibiting α-glucosidase activity can reduce the rate of carbohydrate conversion to glucose, delay the body's absorption of glucose, thereby lowering postprandial blood glucose and controlling blood glucose levels. To investigate this, samples prepared in Examples 1-3 and Comparative Examples 1-6 were used to study their inhibitory ability on α-glucosidase. The specific steps are as follows: 50 μL of 10 mg / mL oligosaccharide samples from the examples and comparative examples and 25 μL of 0.2 U / mL α-glucosidase solution (prepared with 0.1 mol / L phosphate buffer, pH 6.8) were added to a 96-well microplate. After thorough mixing, the mixture was reacted at 37°C for 10 min. Then, 25 μL of 2.5 mmol / L PNPG (prepared with 0.1 mol / L phosphate buffer, pH 6.8) was added, and the mixture was incubated at 37°C for 30 min. Finally, 100 μL of 0.2 mol / L sodium carbonate solution was added to terminate the reaction, and the absorbance was measured at 405 nm. Simultaneously, a background control group and a blank control group were set up. The α-glucosidase inhibition rate was calculated using the following formula:

[0112]

[0113] In the formula: A 空白 The absorbance of the enzyme reaction after adding 0.1 mol / L phosphate buffer solution; A 样品 To increase the absorbance of the sample and enzyme involved in the reaction; A 背景 The absorbance is the result of adding only the sample to be tested without adding the enzyme.

[0114] The results are shown in Table 3. The results show that the α-glucosidase inhibition rate of Examples 1-3 was 80.2-81.4%, which was higher than that of Comparative Examples 1-6, indicating that Example 1 was more effective.

[0115] Table 3. Inhibitory effect of Examples 1-3 and Comparative Examples 1-6 on α-glucosidase

[0116] Example 1 80.5 Example 2 81.4 Example 3 80.2 Comparative Example 1 38.4 Comparative Example 2 41.6 Comparative Example 3 39.4 Comparative Example 4 35.1 Comparative Example 5 40.9 Comparative Example 6 36.5

[0117] Verification Example 3

[0118] Samples prepared in Examples 1-3 and Comparative Examples 1-6 were used to study their effects on type 2 diabetic mice. The specific steps were as follows: 72 mice were placed in a sterile animal room with a temperature of 22-26℃ and a relative humidity of 40-70% for 7 days of acclimatization. During the feeding period, the mice were provided with sufficient sterile water and ordinary feed and the mouse bedding was changed in a timely manner. Seven days later, six mice were randomly selected as the normal group and received no treatment. The remaining 66 mice were fasted for 12 hours but allowed free water, and then injected intraperitoneally with 50 mg / (kg·bw) streptozotocin (STZ, prepared on ice with 0.1 mol / L sodium citrate buffer solution at pH 4.4, and used immediately). Fasting blood glucose (FBG) was measured on days 3 and 7 after injection. Mice with FBG ≤ 8 mmol / L were fasted again for 12 hours but allowed free water, and then injected with 80 mg / (kg·bw) STZ. FBG was measured on day 7 after injection. Mice with FBG ≥ 8.0 mmol / L were considered to have successfully developed type 2 diabetes.

[0119] Type 2 diabetic mice were randomly divided into 11 groups of 6 mice each: a model group, a positive control group, Examples 1-3 groups, and Comparative Examples 1-6 groups. Mice were administered acarbose once daily at the same time. The normal control and model groups were given saline, the positive control group was given 100 mg / (kg·bw) acarbose, and Examples 1-3 and Comparative Examples 1-6 were given 1000 mg / (kg·bw) acarbose. The experiment lasted for 9 weeks. During the experiment, mice were provided with ample sterile water, feed, and clean sterile bedding. After 9 weeks of continuous gavage, the mice were fasted but allowed free water for 12 hours. After anesthesia, blood was collected by enucleation, and the mice were euthanized, dissected, and their serum was collected for later use.

[0120] The glucose tolerance test can detect the body's ability to regulate blood glucose concentration and reflect the functional state of pancreatic islet cells. The smaller the AUC, the faster the blood glucose level recovers. Therefore, the effects of the samples prepared in Examples 1-3 and Comparative Examples 1-6 on glucose tolerance in mice were investigated. The specific steps are as follows: After 9 weeks of continuous treatment, mice were fasted for 12 hours but allowed free access to water. Mice were then orally administered 2 g / (kg·bw) glucose via gavage. Blood glucose levels were measured in the tail vein of each group of mice at 0 min, 30 min, 60 min, and 120 min, and the area under the blood glucose curve (AUC) was calculated using the following formula.

[0121]

[0122] Where a is the blood glucose value at 0 min, b is the blood glucose value at 30 min, c is the blood glucose value at 60 min, and d is the blood glucose value at 120 min.

[0123] The experimental results are shown in Table 4. The results show that ingestion of the samples from Examples 1-3 can effectively improve glucose tolerance and stabilize blood glucose levels in diabetic mice.

[0124] Table 4. Effects of Examples 1-3 and Comparative Examples 1-6 on the Area Under the Glucose Tolerance Curve (AUC) in diabetic mice.

[0125] normal group 21.2 Model group 56.3 Positive control group 29.5 Example 1 32.4 Example 2 31.2 Example 3 32.8 Comparative Example 1 45.1 Comparative Example 2 48.6 Comparative Example 3 47.4 Comparative Example 4 49.4 Comparative Example 5 51.3 Comparative Example 6 50.8

[0126] Under normal physiological conditions, elevated blood glucose levels stimulate pancreatic β-cells to secrete insulin. Insulin binds to specific receptors, thereby accelerating glucose absorption and metabolism. However, diabetic patients develop insulin resistance, resulting in a lower insulin biological effect and requiring the secretion of more insulin to maintain blood glucose homeostasis. Therefore, samples prepared in Examples 1-3 and Comparative Examples 1-6 were used to study their effects on insulin in mice. The specific steps were as follows: Whole blood was allowed to stand at room temperature for 1 hour, then centrifuged at 4°C and 4000 rpm for 10 minutes. Serum was separated and stored at -80°C for later use. Insulin levels were detected according to the instructions of the kit (Nanjing Jiancheng Biotechnology).

[0127] The experimental results are shown in Table 5. The results show that ingestion of the samples from Examples 1-3 can significantly reduce serum insulin levels in diabetic mice, improve insulin sensitivity, reduce insulin resistance, and enhance insulin absorption and utilization of glucose.

[0128] Table 5. Effects of Examples 1-3 and Comparative Examples 1-6 on insulin in diabetic mice.

[0129] normal group 3.52 Model group 6.87 Positive control group 3.68 Example 1 3.71 Example 2 3.76 Example 3 3.73 Comparative Example 1 5.93 Comparative Example 2 6.01 Comparative Example 3 6.12 Comparative Example 4 5.95 Comparative Example 5 5.97 Comparative Example 6 6.06

[0130] Elevated blood glucose levels lead to the production of reactive oxygen species (ROS) and an imbalance between antioxidants and ROS, ultimately resulting in increased oxidative stress in various tissues. Oxidative stress may exacerbate damage to pancreatic β-cells and insulin antibodies in diabetic patients. Superoxide dismutase (SOD) is a key enzyme in scavenging superoxide products and can regulate the level of oxidative damage in the body. Malondialdehyde (MDA) can directly reflect the degree of lipid peroxidation in the body. Therefore, samples prepared in Examples 1-3 and Comparative Examples 1-6 were used to further study their effects on SOD and MDA in mice. The specific steps were as follows: Whole blood was allowed to stand at room temperature for 1 hour, then centrifuged at 4°C and 4000 rpm for 10 minutes. Serum was separated and stored at -80°C for later use. SOD and MDA were detected according to the kit instructions.

[0131] The experimental results are shown in Table 6. The results show that ingestion of the samples from Examples 1-3 significantly increased SOD activity and decreased MDA content in the body. This improved the activity of the antioxidant enzyme system in diabetic mice, reduced lipid peroxidation damage, and thus effectively slowed the progression of diabetes. In summary, the samples prepared in the embodiments of this invention have a significant hypoglycemic effect on diabetic mice.

[0132] Table 6. Effects of Examples 1-3 and Comparative Examples 1-6 on SOD and MDA in diabetic mice.

[0133] normal group 110.36 0.65 Model group 53.21 2.12 Positive control group 102.25 0.84 Example 1 100.68 0.89 Example 2 101.52 0.86 Example 3 99.85 0.92 Comparative Example 1 68.31 1.87 Comparative Example 2 65.14 1.79 Comparative Example 3 71.38 1.85 Comparative Example 4 66.42 1.93 Comparative Example 5 67.56 2.02 Comparative Example 6 64.18 1.95

[0134] In summary, this invention, through extensive research, utilizes specific enzymatic hydrolysis, selenic acid modification, and plasma treatment processes to process mulberry to obtain mulberry oligosaccharides, significantly improving their stability. Mulberry polysaccharides can be rapidly converted into oligosaccharides under the action of enzymes such as glucanase and alkaline protease, achieving precise degradation of the polysaccharides. Oxidative degradation is performed using hydrogen peroxide, where H2O2 decomposes under vitamin C catalysis to generate hydroxyl radicals (·OH). These radicals specifically attack the glycosidic bonds (such as β-1,4 glycosidic bonds) in the oligosaccharide molecules, achieving directional cleavage. This process does not destroy the monosaccharide composition and major functional group structure (such as hydroxyl and carboxyl groups) of the oligosaccharides, ensuring that the degradation products have the biological activity of inhibiting α-glucosidase. Ultrafiltration using a specific process can remove small monosaccharide molecules completely decomposed during enzymatic hydrolysis, small molecules generated during oxidative degradation, and large polysaccharide molecules not decomposed during enzymatic hydrolysis. Organic selenium has higher safety, lower toxicity, and better bioavailability than inorganic selenium. Therefore, selenochemical modification of oligosaccharides can convert inorganic selenium into organic selenium, simultaneously enhancing the functions of both selenium and oligosaccharides. After selenochemical modification, selenium binds to the O6 position of C6 in oligosaccharides with a -Se(O)OH group, resulting in superior blood glucose lowering ability compared to unmodified oligosaccharides. Selenochemical-modified polysaccharides can reduce oxidative stress damage to pancreatic β-cells by increasing superoxide dismutase (SOD) activity and reducing malondialdehyde (MDA) content, thereby scavenging excess free radicals caused by hyperglycemia. This protective effect can directly improve insulin resistance, enhance the biological effect of insulin, and increase the utilization efficiency of glucose in peripheral tissues (such as muscle and fat), thus lowering blood glucose levels and maintaining postprandial blood glucose homeostasis. Furthermore, plasma treatment of oligosaccharides increases the negative charge on the surface of the oligosaccharide particles. This change stems from the interaction between reactive oxygen species (such as hydroxyl radicals) generated during plasma treatment and the polysaccharide molecules, introducing more charged groups (such as -OH and -COOH), thereby increasing the surface charge density and raising the |Zeta| potential. When the zeta potential is high, the surface of the particles in the solution has a stronger electrostatic repulsion, resulting in better stability and making it easier for oligosaccharides to exert their active effects.

[0135] The mulberry oligosaccharides prepared by this invention can significantly enhance the inhibitory effect on α-glucosidase, improve glucose tolerance in diabetic mice, stabilize blood glucose levels, and simultaneously improve insulin sensitivity, reduce insulin resistance, enhance insulin absorption and utilization of glucose, increase SOD activity, and reduce MDA content, thereby effectively slowing the progression of diabetes. Furthermore, this invention uses naturally sourced mulberries as the main raw material, without adding any harmful chemical components, making it low-cost, safe, and of great social significance.

[0136] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. A method for preparing mulberry oligosaccharides, characterized in that, Includes the following steps: (1) After crushing and sieving the mulberry, water extraction was performed. The extract was concentrated and then subjected to alcohol precipitation with ethanol. The ethanol was recovered and the precipitate was collected to obtain mulberry polysaccharide. (2) The mulberry polysaccharide obtained in step (1) is first hydrolyzed by glucanase and then by alkaline protease to obtain the hydrolysate; (3) Take the enzymatic hydrolysate obtained in step (2) and add vitamin C solution to stir and react. Then add H2O2 and stir to carry out oxidative degradation reaction to obtain mulberry oligosaccharide; (4) The mulberry oligosaccharide obtained in step (3) is subjected to ultrafiltration separation. The ultrafiltration separation is as follows: first, the macromolecular substances are removed by passing through an ultrafiltration membrane with a molecular weight of 5 kDa, and the effluent is collected; then, the effluent is passed through an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is collected; sodium selenite solution, nitric acid solution and barium chloride are added to the retentate and heated in a water bath for reaction; after the reaction is completed, the barium chloride is removed, the supernatant is obtained by centrifugation, the pH is adjusted to neutral, ions are removed by dialysis, the water is changed once after 6-12 hours, and the selenic acid modified mulberry oligosaccharide solid powder is obtained by vacuum freeze drying. (5) Place the selenic acid modified mulberry oligosaccharide solid powder obtained in step (4) into the groove of the reaction vessel for plasma treatment to obtain the product.

2. According to the preparation method of claim 1, in step (2), the temperature of the dextranase hydrolysis is 35-45℃, the time is 1-2h, and the enzyme addition amount is 200U / mL; the temperature of the alkaline protease hydrolysis is 40-50℃, the time is 2-3h, and the enzyme addition amount is 300U / mL.

3. The preparation method according to claim 1, characterized in that, The stirring reaction in step (3) is carried out at a temperature of 35-45℃ for 20-40 minutes.

4. The preparation method according to claim 1, characterized in that, The temperature of the oxidative degradation reaction in step (3) is 35-45℃ and the time is 40-80min.

5. The preparation method according to claim 1, characterized in that, The water bath heating reaction in step (4) is carried out at a temperature of 50-70℃ for 3-8 hours.

6. The preparation method according to claim 1, characterized in that, In step (4), a dialysis bag with a molecular weight of 2000 Da is used for dialysis.

7. The preparation method according to claim 1, characterized in that, The plasma treatment in step (5) is specifically as follows: using dielectric barrier discharge as the low-temperature plasma generation method, using air environment as the reaction atmosphere, the diameter of the upper and lower circular quartz plates is 4-6cm, the discharge spacing is set to 6-10mm, the input voltage is 100-130V, the input current is 0.5-1.5A, and the processing time is 0.5-2min.

8. Mulberry oligosaccharides prepared by the preparation method according to any one of claims 1-7.

9. The application of mulberry oligosaccharides prepared by any one of claims 1-7 in the preparation of products for regulating blood sugar.

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