A protein peptide from hemp seeds for lowering blood sugar and regulating glucose metabolism, its preparation method and applications

Abstract

The present invention provides a hemp seed protein peptide for reducing blood sugar and regulating glucose metabolism, its preparation method and application, belonging to the technical field of functional active peptides, and comprising the following steps: The hemp seeds are subjected to low-temperature supersonic fluid pulverization, the pulverized hemp seeds are subjected to enzymatic pretreatment, compound protease is added for enzymatic hydrolysis, PNGaseF enzyme is added for further enzymatic hydrolysis to obtain an enzymatic hydrolysate, the supernatant of the enzymatic hydrolysis is enriched by membrane ultrafiltration interception, and then concentrated, dried and sterilized by tangential flow filtration to obtain the hemp seed protein peptide. The new hemp seed protein peptide in the present invention has been experimentally proven to have the following remarkable effects: It can significantly reduce the fasting blood sugar level and relieve the discomfort symptoms such as thirst, polydipsia, polyuria and fatigue caused by hyperglycemia; It can significantly relieve the symptoms of glucose intolerance and insulin resistance caused by hyperglycemia and reduce the risk of type 2 diabetes; It can significantly reduce the levels of fasting serum insulin and glycated hemoglobin and increase the level of serum GLP-1.

Description

Technical Field

[0001] The present invention relates to the technical field of functional active peptides, and particularly relates to a hemp seed protein peptide for reducing blood sugar and regulating glucose metabolism, a preparation method thereof, and an application thereof. Background Art

[0002] Hemp has a long planting history in China and has long been regarded as a nutritious food ingredient. The amino acid composition ratio of hemp seed protein peptide obtained by directional enzymatic hydrolysis technology is appropriate, and the content of arginine is quite high. At the same time, numerous research results show that hemp seed protein peptide has biological activities such as antioxidant and blood pressure lowering, and these activities imply its potential application value in the health field.

[0003] Chinese patent application with publication number CN119220622A, titled "A Hemp Seed Protein Peptide, Granules for Inhibiting α-Glucosidase, and Preparation Method and Application Thereof", discloses that the hemp seed protein peptide for inhibiting α-glucosidase includes at least one of polypeptides with amino acid sequences of FY, SPVI, TGLGR, and FR. The hemp seed protein peptide provided in the above technical solution has a very high α-glucosidase inhibition rate and has a good blood sugar lowering effect. However, the hemp seed protein peptide in the above technical solution only has an inhibitory effect on α-glucosidase, and the blood sugar lowering and glucose metabolism regulation effects of hemp seeds still need to be further developed. Summary of the Invention

[0004] In view of this, the present invention provides a hemp seed protein peptide for reducing blood sugar and regulating glucose metabolism, a preparation method thereof, and an application thereof, to obtain a new hemp seed protein peptide with the effects of reducing blood sugar and regulating glucose metabolism.

[0005] To achieve the above object, the present invention provides a preparation method of a hemp seed protein peptide for reducing blood sugar and regulating glucose metabolism, including the following steps: the hemp seeds are subjected to low-temperature supersonic fluid pulverization, the pulverized hemp seeds are subjected to enzymatic pretreatment, compound protease is added for enzymatic hydrolysis, and then PNGaseF enzyme is added for further enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is subjected to solid-liquid separation to obtain a supernatant. The supernatant is subjected to membrane ultrafiltration and cut-off to remove impurities, and then concentrated, dried, and sterilized through tangential flow filtration to obtain hemp seed protein peptide; the compound protease is pepsin and acidic protease.

[0006] Optionally, the mass ratio of pepsin to acidic protease is 1:1; the acidic protease is one or a combination of two of 537 protease and 3.350 acidic protease.

[0007] Optionally, the complex protease hydrolysis comprises the following steps: the addition amount of the complex protease is 5-8% of the weight of the product after enzymatic hydrolysis pretreatment, the pH of hydrolysis is 1.5-3.5, the temperature of hydrolysis is 40-65 °C, the time of hydrolysis is 3-5 h, the temperature for inactivating the enzyme after hydrolysis is 75-85 °C, and the time is 5-15 min.

[0008] Optionally, the addition amount of the PNGaseF enzyme is 0.1-0.25% of the weight of the product after complex protease hydrolysis, the pH of hydrolysis is 6.0-8.5, the temperature of hydrolysis is 35-40 °C, the time of hydrolysis is 2-3.5 h, the temperature for inactivating the enzyme after hydrolysis is 80-90 °C, and the time is 5-15 min.

[0009] Optionally, the low-temperature supersonic fluid pulverization comprises the following steps: pulverizing using a supersonic airflow pulverizer, the temperature of the reaction kettle is -30 to 0 °C, the feeding speed is 50-80 Kg / h, the air flow rate is 3-9 m 3 / min, and the air pressure is 0.5-1 Mpa.

[0010] Optionally, the enzymatic hydrolysis pretreatment includes mixing the pulverized hemp seeds with a solvent, the solid-liquid ratio is 1:15-25, adjusting the pH to 1.5-3.5, performing ultrasonic-assisted dissolution, the ultrasonic temperature is 4-15 °C, the ultrasonic power is 200-350 w, the ultrasonic time is 30-120 min, and the stirring speed is 60-200 r / min; the mixture after ultrasonic treatment is subjected to microfluidic homogenization, and the flow rate of the microfluidic homogenization is 5-10 L / min and the pressure is 300-350 Mpa.

[0011] Optionally, a 400-600 Da ultrafiltration membrane is used during membrane ultrafiltration interception.

[0012] Optionally, a 1.8-2.1 KDa ultrafiltration membrane is used during tangential flow filtration, the cross-flow pressure is 0.5-2 MPa, and the membrane surface flow rate is 3-7 m / s.

[0013] Optionally, the drying is low-temperature electrostatic spray drying, the inlet air temperature is 15-25 °C, the outlet air temperature is 30-40 °C, the feeding rate is 50-150 kg / h, the voltage of the electrostatic generator is 30-55 kV, and the pressure of the atomizer is 15-20 MPa.

[0014] Optionally, the sterilization is ultra-high pressure sterilization, the sterilization working pressure is 100-500 MPa, the working temperature is 25-40 °C, and the pressure holding time is 20-90 s.

[0015] To achieve the above object, the present invention provides a hemp seed protein peptide for reducing blood sugar and regulating sugar metabolism, which is obtained by a preparation method of hemp seed protein peptide, and includes the following short peptides: SEQ ID NO: 1: Val-Val-Thr-Pro-Pro-Pro-Ile, SEQ ID NO: 2: Ile-Gln-Ala-Thr-Thr-Thr-His-His-Gln-Ile, SEQ ID NO: 3: Met-Thr-Asn-Leu-Ala-Phe, SEQ ID NO: 4: Met-Gln-Glu-Ile-Ile-Lys, SEQ ID NO: 5: Met-Ile-Phe-Arg.

[0016] The above technical solution of the present invention has at least the following beneficial effects:

[0017] 1. In the present invention, cryogenic supersonic fluid milling is used, while the existing hemp protein and peptide extraction technology uses ordinary wall-breaking milling technology for milling. However, due to the high water and oil content of hemp, the viscosity of the milled material is relatively large, which is not conducive to the transfer of raw materials; in addition, a large amount of heat will be generated during the milling process of the ordinary milling technology using a rotary cutter for milling, which will cause the active protein in hemp seeds to be deformed and inactivated, and is not conducive to the preparation of peptide products with high biological activity. The present invention uses ultra-high pressure gas to generate supersonic fluid to deeply mill hemp seed powder in a low-temperature environment, and the milling diameter can be lower than 10um, which not only ensures the natural activity of hemp seed protein but also increases the specific surface area of the raw material, facilitating subsequent degreasing and enzymatic hydrolysis extraction.

[0018] 2. The present invention performs enzymatic hydrolysis pretreatment on the milled hemp seeds. On the one hand, it increases the dissolution rate of proteins in hemp seeds, and on the other hand, it improves the homogenization stability of the solution; the present invention uses a combination of compound protease and PNGaseF enzyme for two-step hydrolysis. The compound enzyme hydrolysis uses a combination of pepsin and acidic protease. The acidic protease has rich cleavage sites, ensuring the low molecular weight and fragment diversity of the peptide segments. Moreover, the fragments generated by enzymatic cleavage are more easily directly absorbed by the body. Further, PNGaseF hydrolysis is used to quickly and effectively cleave the sugar chains of glycoproteins, effectively reducing the carbohydrate content in hemp seed protein peptides.

[0019] 3. The hemp seed protein peptide obtained in the present invention can reduce the fasting blood glucose value of hyperglycemic mice by 10-25% at different doses; significantly relieve the glucose intolerance of hyperglycemic mice and reduce the OGTTAUC value by 15-25%; significantly relieve the insulin resistance symptoms of hyperglycemic mice and reduce the ITTAUC value by 10-20%; significantly reduce the insulin level in the serum of mice, which can be reduced by 10-19.5% compared with the model group; significantly reduce the glycated hemoglobin level in the serum of mice, which can be reduced by 4-8.7% compared with the model group; significantly increase the GLP-1 level in the serum of mice, which can be increased by 50-85% compared with the model group. Description of the Drawings

[0020] Figure 1 It is a detection result diagram of the fasting blood glucose of mice in the present invention;

[0021] Figure 2 It is a diagram of the blood glucose change of mice in the present invention after oral administration of glucose;

[0022] Figure 3 It is a diagram of the AUC value of mice in the present invention after oral administration of glucose;

[0023] Figure 4 It is a diagram of the blood glucose change of mice in the present invention after injection of insulin;

[0024] Figure 5 It is a diagram of the AUC value of mice in the present invention after injection of insulin;

[0025] Figure 6 It is a diagram of the serum glucose metabolism-related indexes of mice in the present invention during the experiment.

[0026] In the figure: Figure 1 and 3 In 5 and 6, n = 8; *p < 0.5; **p < 0.01; ***p < 0.001;

[0027] Figure 1 and 2 In 3, 4, 5, and 6, hemp peptide is the abbreviation of hemp seed protein peptide, representing hemp seed protein peptide. Detailed Embodiments

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention fall within the scope of protection of the present invention.

[0029] Example 1

[0030] Take 500 g of hemp seeds, remove the shells, and conduct preliminary grinding and pulverization. Then put them into a low-temperature supersonic airflow pulverizer for pulverization. The temperature of the reaction kettle is -30°C, the feeding speed is 80 Kg / h, and the air flow rate is 3 m 3 / min, and the air pressure is 0.5 Mpa.

[0031] Conduct enzymatic pretreatment on the pulverized hemp seeds. Mix the pulverized hemp seeds with water, and the ratio of material to liquid is 1:15. Adjust the pH to 3.5, perform ultrasonic stirring at a temperature of 4°C, with an ultrasonic power of 200 w and an ultrasonic time of 120 min, and the stirring speed is 60 rmp / min. Homogenize the ultrasonicated suspension through a high-pressure microfluidizer, with a nozzle flow rate of 5 L / min and a pressure of 300 Mpa to obtain a hemp seed homogenized suspension.

[0032] Add complex protease to the hemp seed homogenized suspension for the first enzymatic hydrolysis. The complex protease is a mixture of pepsin and 537 protease, and the weight ratio of pepsin to 537 protease is 1:1. The addition amount of the complex protease is 5% of the weight of the hemp seed homogenized suspension. The pH of the enzymatic hydrolysis is 1.5, the temperature of the enzymatic hydrolysis is 40°C, and the time of the enzymatic hydrolysis is 3 h. After the enzymatic hydrolysis, the temperature for inactivating the enzyme is 75°C and the time is 5 min. After the complex protease enzymatic hydrolysis, add PNGaseF enzyme for the second enzymatic hydrolysis. The addition amount of PNGaseF enzyme is 0.1% of the weight of the product after the complex protease enzymatic hydrolysis. The pH of the enzymatic hydrolysis is 6.0, the temperature of the enzymatic hydrolysis is 35°C, and the time of the enzymatic hydrolysis is 2 h. After the enzymatic hydrolysis, the temperature for inactivating the enzyme is 80°C and the time is 5 min to obtain an enzymatic hydrolysate.

[0033] Separate the enzymatic hydrolysate using a centrifuge at a centrifugal speed of 3000 rpm / min for 15 min. Take the supernatant and use a 500 Da ultrafiltration membrane to ultrafilter and intercept the supernatant to remove small molecule impurities such as oligosaccharides. Then pass through a tangential flow membrane concentrator and use a 2 kDa ultrafiltration membrane to concentrate and enrich the hemp seed protein peptide filtrate, with a cross-flow pressure of 0.5 MPa and a membrane surface flow rate of 3 m / s.

[0034] Dry and powder the concentrated and enriched hemp seed protein peptide filtrate using a spray dryer. The inlet air temperature of the spray drying is 15°C, the outlet air temperature is 30°C, the feeding rate is 50 kg / h, the voltage of the electrostatic generator is 40 kV, and the pressure of the atomizer is 15 MPa. Obtain dry powder of hemp seed protein peptide and use a sterile aluminum foil bag for vacuum sealing packaging.

[0035] Sterilize the finished product of hemp seed protein peptide using an ultra-high pressure sterilization kettle. The working pressure is 100 MPa, the working temperature is 25°C, and the pressure holding time is 90 s. After sterilization, store at room temperature.

[0036] Example 2

[0037] Take 500 g of hemp seeds, remove the hulls, and conduct preliminary grinding and pulverization. Then, put them into a low-temperature supersonic airflow pulverizer for pulverization. The temperature of the reaction kettle is 0°C, the feeding speed is 50 Kg / h, the air flow rate is 9 m 3 / min, and the air pressure is 1 Mpa.

[0038] Perform enzymatic pretreatment on the pulverized hemp seeds. Mix the pulverized hemp seeds with water, with a solid-liquid ratio of 1:25. Adjust the pH to 1.5, perform ultrasonic stirring at a temperature of 15°C, an ultrasonic power of 350 w, an ultrasonic time of 30 min, and a stirring speed of 200 rmp / min. Homogenize the ultrasonicated suspension through a high-pressure microfluidizer, with a nozzle flow rate of 10 L / min and a pressure of 350 Mpa to obtain a homogenized suspension of hemp seeds.

[0039] Add compound protease to the homogenized suspension of hemp seeds for the first enzymatic hydrolysis. The compound protease is a mixture of pepsin, 537 protease, and 3.350 acidic protease. The weight ratio of pepsin:(537 protease:3.350 acidic protease) is 1:(0.5:0.5). The addition amount of the compound protease is 8% of the weight of the homogenized suspension of hemp seeds. The pH of the enzymatic hydrolysis is 3.5, the temperature of the enzymatic hydrolysis is 65°C, the time of the enzymatic hydrolysis is 5 h, the temperature for inactivating the enzyme after the enzymatic hydrolysis is 80°C, and the time is 10 min. After the compound protease enzymatic hydrolysis, add PNGaseF enzyme for the second enzymatic hydrolysis. The addition amount of PNGaseF enzyme is 0.25% of the weight of the product after the compound protease enzymatic hydrolysis. The pH of the enzymatic hydrolysis is 8.5, the temperature of the enzymatic hydrolysis is 40°C, the time of the enzymatic hydrolysis is 3.5 h, the temperature for inactivating the enzyme after the enzymatic hydrolysis is 90°C, and the time is 15 min to obtain an enzymatic hydrolysate.

[0040] Separate the enzymatic hydrolysate using a centrifuge at a centrifugal speed of 3000 rpm / min for 15 min. Take the supernatant and perform ultrafiltration retention on the supernatant using a 400 Da ultrafiltration membrane to remove small molecule impurities such as oligosaccharides. Then, pass it through a tangential flow membrane concentrator and concentrate and enrich the filtrate of hemp seed protein peptides obtained using a 1.8 kDa ultrafiltration membrane, with a cross-flow pressure of 2 MPa and a membrane surface flow rate of 7 m / s.

[0041] Dry and powder the concentrated and enriched filtrate of hemp seed protein peptides using a spray dryer. The inlet air temperature of the spray drying is 25°C, the outlet air temperature is 40°C, the feeding rate is 150 kg / h, the voltage of the electrostatic generator is 30 kV, and the pressure of the atomizer is 20 MPa. Obtain dry powder of hemp seed protein peptides and perform vacuum sealing packaging using a sterile aluminum foil bag.

[0042] Sterilize the finished product of hemp seed protein peptides using an ultra-high pressure sterilization kettle, with a working pressure of 500 MPa, a working temperature of 40°C, and a pressure holding time of 20 s. Store at room temperature after sterilization.

[0043] Example 3

[0044] Take 500 g of hemp seeds, remove the shells, and conduct preliminary grinding and pulverization. Then put them into a low-temperature supersonic airflow pulverizer for pulverization. The temperature of the reaction kettle is -15°C, the feeding speed is 60 Kg / h, and the air flow rate is 6 m 3 / min, and the air pressure is 0.8 Mpa.

[0045] Conduct enzymatic pretreatment on the pulverized hemp seeds. Mix the pulverized hemp seeds with water, with a solid-liquid ratio of 1:20. Adjust the pH to 2.5, perform ultrasonic stirring at a temperature of 9°C, with an ultrasonic power of 250 w and an ultrasonic time of 80 min, and a stirring speed of 150 rmp / min. Homogenize the ultrasonicated suspension through a high-pressure microfluidizer, with a nozzle flow rate of 7 L / min and a pressure of 320 Mpa to obtain a homogenized suspension of hemp seeds.

[0046] Add compound protease to the homogenized suspension of hemp seeds for the first enzymatic hydrolysis. The compound protease is a mixture of pepsin and 3.350 acidic protease, and the weight ratio of pepsin to 3.350 acidic protease is 1:1. The addition amount of the compound protease is 6.5% of the weight of the homogenized suspension of hemp seeds. The pH of the enzymatic hydrolysis is 2.5, the temperature of the enzymatic hydrolysis is 50°C, the time of the enzymatic hydrolysis is 4 h, and the temperature for inactivating the enzyme after the enzymatic hydrolysis is 85°C for 15 min. After the compound protease enzymatic hydrolysis, add PNGaseF enzyme for the second enzymatic hydrolysis. The addition amount of PNGaseF enzyme is 0.15% of the weight of the product after the compound protease enzymatic hydrolysis. The pH of the enzymatic hydrolysis is 7, the temperature of the enzymatic hydrolysis is 38°C, the time of the enzymatic hydrolysis is 2.5 h, and the temperature for inactivating the enzyme after the enzymatic hydrolysis is 85°C for 10 min to obtain an enzymatic hydrolysate.

[0047] Separate the enzymatic hydrolysate using a centrifuge at a centrifugation speed of 3000 rpm / min for 15 min. Take the supernatant and use a 600 Da ultrafiltration membrane to ultrafilter and intercept the supernatant to remove small molecule impurities such as oligosaccharides. Then, through a tangential flow membrane concentrator, use a 2.1 kDa ultrafiltration membrane to concentrate and enrich the filtrate of hemp seed protein peptides, with a cross-flow pressure of 1 MPa and a membrane surface flow rate of 5 m / s.

[0048] Dry and powder the concentrated and enriched filtrate of hemp seed protein peptides using a spray dryer. The inlet air temperature of the spray drying is 20°C, the outlet air temperature is 35°C, the feeding rate is 100 kg / h, the voltage of the electrostatic generator is 55 kV, and the pressure of the atomizer is 18 MPa. Obtain the dry powder of hemp seed protein peptides and use a sterile aluminum foil bag for vacuum sealing and packaging.

[0049] Sterilize the finished product of hemp seed protein peptides using an ultra-high pressure sterilization kettle. The working pressure is 300 MPa, the working temperature is 30°C, and the holding pressure time is 60 s. After sterilization, store at room temperature.

[0050] Example 4

[0051] Take 500 g of hemp seeds, remove the shells, and grind them preliminarily. Then put them into a low-temperature supersonic airflow mill for pulverization. The temperature of the reaction kettle is -10°C, the feeding speed is 70 Kg / h, and the air flow rate is 8 m 3 / min, and the air pressure is 0.6 Mpa.

[0052] Perform enzymatic pretreatment on the pulverized hemp seeds. Mix the pulverized hemp seeds with water, with a solid-liquid ratio of 1:18. Adjust the pH to 2, perform ultrasonic stirring at a temperature of 12°C, an ultrasonic power of 300 w, an ultrasonic time of 50 min, and a stirring speed of 100 rmp / min. Homogenize the ultrasonicated suspension through a high-pressure microfluidizer, with a nozzle flow rate of 9 L / min and a pressure of 340 Mpa to obtain a homogenized suspension of hemp seeds.

[0053] Add compound protease to the homogenized suspension of hemp seeds for the first enzymatic hydrolysis. The compound protease is a mixture of pepsin and 537 protease, with a weight ratio of pepsin:537 protease of 1:1. The addition amount of the compound protease is 5.5% of the weight of the homogenized suspension of hemp seeds. The pH of the enzymatic hydrolysis is 2, the temperature of the enzymatic hydrolysis is 55°C, the time of the enzymatic hydrolysis is 4.5 h, the temperature for inactivating the enzyme after the enzymatic hydrolysis is 82°C, and the time is 9 min. After the compound protease enzymatic hydrolysis, add PNGaseF enzyme for the second enzymatic hydrolysis. The addition amount of the PNGaseF enzyme is 0.2% of the weight of the product after the compound protease enzymatic hydrolysis. The pH of the enzymatic hydrolysis is 6.5, the temperature of the enzymatic hydrolysis is 36°C, the time of the enzymatic hydrolysis is 3 h, the temperature for inactivating the enzyme after the enzymatic hydrolysis is 86°C, and the time is 8 min to obtain an enzymatic hydrolysate.

[0054] Separate the enzymatic hydrolysate using a centrifuge at a centrifugal speed of 3000 rpm / min for 15 min. Take the supernatant and perform ultrafiltration retention on the supernatant using a 550 Da ultrafiltration membrane to remove small molecule impurities such as oligosaccharides. Then, pass it through a tangential flow membrane concentrator and concentrate and enrich the hemp seed protein peptide filtrate obtained using a 1.9 kDa ultrafiltration membrane, with a cross-flow pressure of 1.5 MPa and a membrane surface flow rate of 4 m / s.

[0055] Dry and powder the concentrated and enriched hemp seed protein peptide filtrate using a spray dryer. The inlet air temperature of the spray drying is 20°C, the outlet air temperature is 35°C, the feeding rate is 80 kg / h, the voltage of the electrostatic generator is 45 kV, and the pressure of the atomizer is 17 MPa. Obtain dry powder of hemp seed protein peptide and perform vacuum-sealed packaging using a sterile aluminum foil bag.

[0056] Sterilize the finished product of hemp seed protein peptide using an ultra-high pressure sterilization kettle, with a working pressure of 400 MPa, a working temperature of 35°C, and a holding pressure time of 40 s. Store it at room temperature after sterilization.

[0057] By performing mass spectrometry analysis on the hemp seed protein peptides prepared in Examples 1 to 4, the parameters of the mass spectrometry analysis were as follows: using a reverse-phase chromatographic column, with an aqueous phase of 0.1% formic acid aqueous solution and an organic phase of acetonitrile as the mobile phase, the flow rate was 0.2 - 1.0 ml / min, the column temperature was 25 - 40 °C, the ESI spray voltage was 2.5 - 5 kV, the gas was nitrogen, the gas pressure was 1 - 3 Pa, and the collision energy was 10 - 50 eV. The following peptide segments were obtained: SEQ ID NO: 1: Val-Val-Thr-Pro-Pro-Pro-Ile, SEQ ID NO: 2: Ile-Gln-Ala-Thr-Thr-Thr-His-His-Gln-Ile, SEQ ID NO: 3: Met-Thr-Asn-Leu-Ala-Phe, SEQ ID NO: 4: Met-Gln-Glu-Ile-Ile-Lys, SEQ ID NO: 5: Met-Ile-Phe-Arg.

[0058] An animal experiment was conducted on the hemp seed protein peptide prepared in Example 3 to detect its effects on reducing blood glucose and regulating glucose metabolism.

[0059] 1 Animal Experiment

[0060] 1.1 Mouse Treatment and Grouping

[0061] Forty-eight 8-week-old male C57BL / 6 mice were selected. According to the principle of body weight balance, the mice were evenly divided into 2 groups. The first group had 12 mice, and the second group had 36 mice. The first group was the control group, fed with normal feed, and the second group was fed with a high-fat diet for 8 consecutive weeks until the body weight difference between the control group mice and the high-fat group mice was more than 20%. The high-fat group was randomly divided into 3 groups, with 12 mice in each group, namely the model group (high-fat diet + normal saline), the low-dose group (high-fat diet + 250 mg / kg hemp seed protein peptide), and the high-dose group (high-fat diet + 500 mg / kg hemp seed protein peptide). According to different groups, different concentrations of hemp seed protein peptide solution or normal saline were intragastrically administered daily for 6 consecutive weeks of intervention. After the intervention period, fasting blood glucose measurement, oral glucose tolerance, and insulin resistance tests were performed on the mice. After the experiment ended, the mice were fasted (without water restriction) for 12 h. After 12 h, the mice were anesthetized, blood was taken, and the mice were sacrificed. Serum was prepared by centrifugation and stored at -80 °C.

[0062] 1.2 Oral Glucose Tolerance Test

[0063] On the afternoon of the day before the experiment at 5 o'clock, the mice in each group were transferred to clean cages for fasting for 16 h. During the fasting period, the mice maintained normal drinking water. Weigh each mouse and mark the serial number at the root of the mouse's tail with a marker pen. Take the mouse out of the cage, cut off the end of the mouse's tail with scissors, squeeze the mouse's tail, measure the fasting blood glucose with a blood glucose meter and blood glucose test strips, and set it as the blood glucose value at 0 min. After the mouse is stable for 30 min, intragastrically administer a glucose solution at a dose of 2 g / kg body weight according to the mouse's body weight, and intragastrically administer 0.01 mL per gram of body weight. Start timing from the end of intragastric administration, and measure the blood glucose value of each mouse at 30 min, 60 min, and 120 min. The results of measuring the fasting blood glucose of the mice with a blood glucose meter and blood glucose test strips are shown in Figure 1 ; The blood glucose change graph of the mice after oral glucose administration is shown in Figure 2 ; The AUC value graph of the mice after oral glucose administration is shown in Figure 3 .

[0064] The drug AUC value refers to the product of the concentration and time reached by the drug in the whole body. By calculating and analyzing this value, information about the drug's absorption rate, absorption degree, and action time in the body can be obtained.

[0065] 1.3 Insulin tolerance test

[0066] On the afternoon of the day before the experiment at 5 o'clock, the mice in each group were transferred to clean cages for fasting for 16 h. During the fasting period, the mice maintained normal drinking water. Weigh each mouse and mark the serial number at the root of the mouse's tail with a marker pen. Take the mouse out of the cage, cut off the end of the mouse's tail with scissors, squeeze the mouse's tail, measure the fasting blood glucose with a blood glucose meter and blood glucose test strips, and set it as the blood glucose value at 0 min. After the mouse is stable for 30 min, intraperitoneally inject an insulin solution at a dose of 0.5 U / kg body weight according to the mouse's body weight, and intraperitoneally inject 0.01 mL per gram of body weight. Start timing from the end of injection, and measure the blood glucose value of each mouse at 30 min, 60 min, 90 min, 120 min, and 150 min. The blood glucose change after the mice were intraperitoneally injected with insulin is shown in Figure 4 ; The AUC value after the mice were intraperitoneally injected with insulin is shown in Figure 5 .

[0067] 1.4 Detection of mouse serum indexes

[0068] Use an ELISA kit to measure glycated hemoglobin (HbAlc), insulin, and glucagon-like peptide (GLP-1) in the serum of mice in each group. The operation steps are carried out according to the kit instructions. The related indexes of mouse serum glucose metabolism are shown in Figure 6 .

[0069] 2 Test results

[0070] 2.1 Fasting blood glucose measurement

[0071] As Figure 1 shown, the fasting blood glucose test results indicate that the two fasting blood glucose values of the normal group mice, 5.1 mmol / L and 5.2 mmol / L, are both within the normal range. The two fasting blood glucose values of the high-fat group mice are 7.8 mmol / L and 9.0 mmol / L respectively, which indicates that high-fat feeding successfully induced obvious hyperglycemia symptoms in mice. Both high and low doses of hemp seed protein peptide intervention can significantly alleviate the hyperglycemia caused by high-fat diet (P < 0.05 or P < 0.01). Compared with the high-fat group, the blood glucose value of the low-dose hemp seed protein peptide group mice decreased by up to 15.9%, while the blood glucose of the high-dose hemp seed protein peptide group mice decreased by up to 22.7% compared with the high-fat group.

[0072] 2.2 Oral glucose tolerance test

[0073] The oral glucose tolerance test is used to test the fasting blood glucose and glucose tolerance of mice to determine whether mice have hyperglycemia symptoms and glucose intolerance. The larger the AUC (area under the curve), the higher the blood glucose integral value, indicating poorer blood glucose regulation ability.

[0074] As Figure 2 , Figure 3 shown, it can be intuitively seen from Figure 2 that within 0 - 120 min, the blood glucose change curve of the high-fat group mice is always higher than that of the normal group and the high and low dose hemp seed protein peptide groups, which indicates that high-fat feeding induced glucose intolerance in mice. The intervention of high and low doses of hemp seed protein peptide can improve this symptom by regulating the sugar metabolism pathway of mice. It can be seen from Figure 3 that the AUC value of the high-fat group mice is extremely significantly higher than that of the normal group (P < 0.01), while the AUC values of the high and low dose hemp seed protein peptide group mice are both extremely significantly lower than that of the high-fat group (P < 0.01), decreasing by 11.4% and 14.7% respectively compared with the high-fat group. This further shows that the hemp seed protein peptide composition can significantly alleviate the glucose intolerance caused by high-fat feeding and maintain blood glucose homeostasis.

[0075] 2.3 Insulin tolerance test

[0076] The insulin resistance test can be used to determine whether mice have hyperglycemia symptoms and insulin resistance. The larger the AUC (area under the curve), the higher the blood glucose integral value, indicating weaker sensitivity of the body to insulin, poorer blood glucose regulation ability, and stronger insulin resistance.

[0077] The blood glucose change curves of each group of mice after intraperitoneal injection of insulin are shown in Figure 4 , and the AUC values are shown in Figure 5 . It can be seen from Figure 4It can be intuitively seen that within 0 - 150 min, the blood glucose change curve of the high-fat group mice was always higher than that of the normal group and the high- and low-dose hemp seed protein peptide groups. This indicates that high-fat feeding induced insulin resistance symptoms in mice, while the intervention of high- and low-dose hemp seed protein peptides could improve this symptom by regulating the glucose metabolism pathway and insulin secretion pathway of mice. From Figure 5 it can be known that the AUC value of the high-fat group mice was extremely significantly higher than that of the normal group (P < 0.01), while the AUC values of the high- and low-dose hemp seed protein peptide group mice were both extremely significantly lower than that of the high-fat group (P < 0.01), decreasing by 13.5% and 18.3% respectively compared with the high-fat group. This further illustrates that the hemp seed protein peptide composition can significantly relieve insulin resistance caused by high-fat feeding and maintain blood glucose homeostasis.

[0078] 2.4 Serum glucose metabolism-related indicators

[0079] Glucose metabolism is an extremely complex system where each internal pathway influences each other. In this system, glycated hemoglobin has special indicative significance. It can reflect the blood glucose level of the body during a specific past period, so it occupies a crucial position in the clinical diagnosis and treatment of hyperglycemia and related disorders of glucose metabolism.

[0080] Insulin is also an essential key regulatory factor in the process of glucose metabolism. It plays a very important role in maintaining blood glucose homeostasis. Through a variety of complex molecular mechanisms, it promotes the uptake and utilization of glucose by cells, thereby effectively reducing blood glucose concentration to ensure the normal physiological function of the body.

[0081] GLP-1, namely glucagon-like peptide, is a substance that plays an important role in the glucose metabolism regulation network. It is secreted by intestinal endocrine cells and can affect glucose metabolism through multiple pathways. For example, it can stimulate insulin secretion and inhibit glucagon release, thereby coordinately regulating blood glucose levels and preventing excessive blood glucose fluctuations.

[0082] The results of experimental studies showed that both high-dose and low-dose hemp seed protein peptides could significantly alleviate the abnormal increase in insulin and glycated hemoglobin in the serum of mice caused by high-fat feeding (P < 0.05 or P < 0.01). Compared with the high-fat group, the insulin and glycated hemoglobin in the hemp seed protein peptide intervention group decreased by up to 19.5% and 8.7% respectively. In mouse models with hyperglycemia or glucose metabolism disorders, abnormal insulin secretion and an increase in glycated hemoglobin levels often occur, and the intervention of hemp seed protein peptides can reverse this abnormal trend to a certain extent. It is worth noting that high-dose hemp seed protein peptides also have a significant effect on increasing the serum GLP-1 level in mice (P < 0.01). Compared with the high-fat group, the GLP-1 in the hemp seed protein peptide intervention group increased by up to 85%. This result indicates that hemp seed protein peptides may maintain the balance of the glucose metabolism network by regulating the function of insulin-secreting cells, enhancing their sensitivity to blood glucose changes, and promoting the activity of GLP-1-secreting cells.

[0083] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A preparation method of hemp seed protein peptide for reducing blood sugar and regulating glucose metabolism, characterized in that, It includes the following steps: Hemp seeds are subjected to low-temperature supersonic fluid grinding. The ground hemp seeds are subjected to enzymatic pretreatment by adding compound protease for enzymatic hydrolysis, and then PNGaseF enzyme is added for further enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is subjected to solid-liquid separation to obtain a supernatant. The supernatant is subjected to membrane ultrafiltration to intercept and remove impurities, and then concentrated, dried, and sterilized by tangential flow filtration to obtain hemp seed protein peptides. The compound protease is pepsin and acidic protease. The hemp seed protein peptides include the following short peptides: SEQ ID NO: 1: Val-Val-Thr-Pro-Pro-Pro-Ile, SEQ ID NO: 2: Ile-Gln-Ala-Thr-Thr-Thr-His-His-Gln-Ile, SEQ ID NO: 3: Met-Thr-Asn-Leu-Ala-Phe, SEQ ID NO: 4: Met-Gln-Glu-Ile-Ile-Lys, SEQ ID NO: 5: Met-Ile-Phe-Arg.

2. The preparation method of the hemp seed protein peptide for reducing blood sugar and regulating sugar metabolism according to claim 1, characterized in that, The mass ratio of pepsin to acidic protease is 1:

1. The acidic protease is one or a combination of two of 537 protease and 3.350 acidic protease.

3. The preparation method of the hemp seed protein peptide for reducing blood sugar and regulating sugar metabolism according to claim 1, characterized in that, The compound protease enzymatic hydrolysis includes the following steps: The addition amount of the compound protease is 5-8% of the weight of the product after enzymatic pretreatment. The pH of enzymatic hydrolysis is 1.5-3.5, the temperature of enzymatic hydrolysis is 40-65 °C, the time of enzymatic hydrolysis is 3-5 h, the temperature for inactivating the enzyme after enzymatic hydrolysis is 75-85 °C, and the time is 5-15 min.

4. The preparation method of the hemp seed protein peptide for lowering blood sugar and regulating sugar metabolism according to claim 1, characterized in that, The addition amount of PNGaseF enzyme is 0.1-0.25% of the weight of the product after compound protease enzymatic hydrolysis. The pH of enzymatic hydrolysis is 6.0-8.5, the temperature of enzymatic hydrolysis is 35-40 °C, the time of enzymatic hydrolysis is 2-3.5 h, the temperature for inactivating the enzyme after enzymatic hydrolysis is 80-90 °C, and the time is 5-15 min.

5. The preparation method of the hemp seed protein peptide for reducing blood sugar and regulating glucose metabolism according to claim 1, characterized in that, The low-temperature supersonic fluid pulverization includes the following steps: pulverizing using a supersonic airflow pulverizer, with the temperature of the reaction kettle being -30 to 0 °C, the feeding speed being 50 to 80 Kg / h, the air flow rate being 3 to 9 m 3 / min, and the air pressure being 0.5 to 1 Mpa.

6. The preparation method of the hemp seed protein peptide for reducing blood sugar and regulating sugar metabolism according to claim 1, characterized in that, The enzymatic pretreatment includes mixing the ground hemp seeds with a solvent, with a solid-liquid ratio of 1:15-25, adjusting the pH to 1.5-3.5, performing ultrasonic assisted dissolution, with an ultrasonic temperature of 4-15 °C, an ultrasonic power of 200-350 w, an ultrasonic time of 30-120 min, and a stirring speed of 60-200 r / min. The mixture after ultrasonic treatment is subjected to microfluidic homogenization, and the flow rate of the microfluidic homogenization is 5-10 L / min, and the pressure is 300-350 Mpa.

7. The preparation method of the hemp seed protein peptide for reducing blood sugar and regulating sugar metabolism according to claim 1, characterized in that A 400-600 Da ultrafiltration membrane is used during membrane ultrafiltration interception.

8. The preparation method of the hemp seed protein peptide for reducing blood sugar and regulating sugar metabolism according to claim 1, characterized in that, A 1.8-2.1 KDa ultrafiltration membrane is used during tangential flow filtration, with a cross-flow pressure of 0.5-2 MPa and a membrane surface flow rate of 3-7 m / s.

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