Fructus cannabis protein peptide capable of reducing blood sugar and regulating glycometabolism as well as preparation method and application of fructus cannabis protein peptide

Abstract

The invention provides a fructus cannabis protein peptide capable of reducing blood sugar and regulating glycometabolism and a preparation method and application thereof, and belongs to the technical field of functional active peptides, and the preparation method comprises the following steps: performing low-temperature supersonic fluid crushing on fructus cannabis, performing enzymolysis pretreatment on the crushed fructus cannabis, adding compound protease for enzymolysis, adding PNGaseF enzyme for further enzymolysis, and performing freeze drying to obtain the fructus cannabis protein peptide capable of reducing blood sugar and regulating glycometabolism. And carrying out membrane ultrafiltration interception and enrichment on the enzymolysis supernatant, carrying out tangential flow filtration, concentrating, drying and sterilizing to obtain the fructus cannabis protein peptide. Tests prove that the novel fructus cannabis protein peptide has the following remarkable effects that the fasting blood glucose level can be remarkably reduced, and uncomfortable symptoms such as thirst, polydipsia, diuresis and weakness caused by hyperglycemia are relieved; the symptoms of glucose intolerance and insulin resistance caused by hyperglycemia can be obviously relieved, and the onset risk of type 2 diabetes mellitus is reduced; the level of fasting serum insulin and glycosylated hemoglobin can be obviously reduced, and the level of serum GLP-1 can be improved.

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 lowering 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 suitable, 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 a 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 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 lowering 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 lowering 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 lowering 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 to intercept and 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 the 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 grinding comprises the following steps: using a supersonic airflow mill for grinding, 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 crushed 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 amount 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 holding pressure 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 glucose 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] To achieve the above object, the present invention also provides an application of hemp seed protein peptide in food and health products.

[0017] The above technical solutions of the present invention have at least the following beneficial effects: 1. In the present invention, low-temperature 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 high, which is not conducive to the transfer of raw materials. In addition, a large amount of heat is 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 deform and inactivate, 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 less than 10 μm, which not only ensures the natural activity of hemp seed protein but also increases the specific surface area of raw materials, 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 two-step hydrolysis with a composite protease and PNGaseF enzyme. The composite 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 peptide segments. In addition, the fragments generated by enzymatic cleavage are more easily directly absorbed by the body. Further hydrolysis with PNGaseF quickly and effectively removes 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 graph of the fasting blood glucose of the mice in the present invention; Figure 2 It is a graph of the blood glucose change after the mice in the present invention orally take glucose; Figure 3 It is a graph of the AUC value after the mice in the present invention orally take glucose; Figure 4 It is a graph of the blood glucose change after the mice in the present invention inject insulin; Figure 5 It is a graph of the AUC value after the mice in the present invention inject insulin; Figure 6 It is a graph of the serum sugar metabolism related indexes during the experiment of the mice in the present invention.

[0021] In the figure: Figure 1 , 3 , n = 8 in 5 and 6; *p < 0.5; **p < 0.01; ***p < 0.001; Figure 1 , 2 , in 3, 4, 5 and 6, hemp peptide is the abbreviation of hemp seed protein peptide, representing hemp seed protein peptide. Detailed Embodiments

[0022] 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 only a part of the embodiments of the present invention, rather than all of the embodiments. 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.

[0023] Example 1 Take 500 g of hemp seeds, remove the shells, preliminarily grind and crush them, and then put them into a low-temperature supersonic airflow mill for crushing. The temperature of the reaction kettle is -30°C, the feeding speed is 80 Kg / h, the air flow rate is 3 m 3 / min, and the air pressure is 0.5 Mpa.

[0024] The crushed hemp seeds are subjected to enzymatic hydrolysis pretreatment. The crushed hemp seeds are mixed with water at a solid-liquid ratio of 1:15, the pH is adjusted to 3.5, ultrasonic stirring is carried out at a temperature of 4°C, the ultrasonic power is 200w, the ultrasonic time is 120min, and the stirring speed is 60rmp / min. The ultrasonic suspension is homogenized by a high-pressure microfluidizer with a nozzle flow rate of 5L / min and a pressure of 300Mpa to obtain a hemp seed homogenized suspension.

[0025] Compound protease is added to the hemp seed homogenized suspension for the first enzymatic hydrolysis. The compound 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 compound 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, the time of the enzymatic hydrolysis is 3h, the temperature for inactivating the enzyme after the enzymatic hydrolysis is 75°C, and the time is 5min. After the compound protease enzymatic hydrolysis, PNGaseF enzyme is added for the second enzymatic hydrolysis. The addition amount of PNGaseF enzyme is 0.1% of the weight of the product after the compound protease enzymatic hydrolysis. The pH of the enzymatic hydrolysis is 6.0, the temperature of the enzymatic hydrolysis is 35°C, the time of the enzymatic hydrolysis is 2h, the temperature for inactivating the enzyme after the enzymatic hydrolysis is 80°C, and the time is 5min to obtain an enzymatic hydrolysate.

[0026] The enzymatic hydrolysate is separated by a centrifuge at a centrifugal speed of 3000rpm / min for 15min. The supernatant is taken, and the supernatant is ultrafiltered and intercepted using a 500Da ultrafiltration membrane to remove small molecule impurities such as oligosaccharides. Then, through a tangential flow membrane concentrator, the hemp seed protein peptide filtrate obtained using a 2kDa ultrafiltration membrane is concentrated and enriched with a cross-flow pressure of 0.5MPa and a membrane surface flow rate of 3m / s.

[0027] The concentrated and enriched hemp seed protein peptide filtrate is dried into powder using a spray dryer. The inlet air temperature of the spray drying is 15°C, the outlet air temperature is 30°C, the feed rate is 50kg / h, the voltage of the electrostatic generator is 40kV, and the pressure of the atomizer is 15MPa. Hemp seed protein peptide dry powder is obtained and vacuum-sealed and packaged using a sterile aluminum foil bag.

[0028] The finished product of hemp seed protein peptide is sterilized using an ultra-high pressure sterilization autoclave with a working pressure of 100MPa, a working temperature of 25°C, and a holding pressure time of 90s, and stored at room temperature after sterilization.

[0029] Example 2 Take 500g of hemp seeds, remove the shells, and conduct preliminary grinding and crushing. Then put them into a low-temperature supersonic airflow crusher for crushing. The temperature of the reaction kettle is 0°C, the feeding speed is 50Kg / h, the air flow rate is 9m 3 / min, and the air pressure is 1Mpa.

[0030] The crushed hemp seeds are pretreated by enzymatic hydrolysis. The crushed hemp seeds are mixed with water at a material-liquid ratio of 1:25, the pH is adjusted to 1.5, ultrasonic stirring is carried out at a temperature of 15°C, the ultrasonic power is 350 w, the ultrasonic time is 30 min, and the stirring speed is 200 rmp / min. The ultrasonic suspension is homogenized by a high-pressure microfluidizer, the nozzle flow rate is 10 L / min, and the pressure is 350 Mpa to obtain a hemp seed homogenized suspension.

[0031] Compound protease is added to the hemp seed homogenized suspension 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 hemp seed homogenized suspension. 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, PNGaseF enzyme is added 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.

[0032] The enzymatic hydrolysate is separated by a centrifuge at a centrifugal speed of 3000 rpm / min for 15 min. The supernatant is taken, and the supernatant is ultrafiltered and intercepted using a 400 Da ultrafiltration membrane to remove small molecule impurities such as oligosaccharides. Then, through a tangential flow membrane concentrator, the hemp seed protein peptide filtrate obtained using a 1.8 kDa ultrafiltration membrane is concentrated and enriched, with a cross-flow pressure of 2 MPa and a membrane surface flow rate of 7 m / s.

[0033] The concentrated and enriched hemp seed protein peptide filtrate is dried and powdered using a spray dryer. The inlet air temperature of the spray drying is 25°C, the outlet air temperature is 40°C, the feed rate is 150 kg / h, the voltage of the electrostatic generator is 30 kV, and the pressure of the atomizer is 20 MPa. Hemp seed protein peptide dry powder is obtained and vacuum-sealed and packaged using a sterile aluminum foil bag.

[0034] The finished product of hemp seed protein peptide is sterilized using an ultra-high pressure sterilization kettle. The working pressure is 500 MPa, the working temperature is 40°C, the pressure holding time is 20 s, and it is stored at room temperature after sterilization.

[0035] Example 3 Take 500 g of hemp seeds, remove the shells, and carry out preliminary grinding and crushing. Then put them into a low-temperature supersonic airflow crusher for crushing. 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.

[0036] The crushed hemp seeds are pretreated by enzymatic hydrolysis. The crushed hemp seeds are mixed with water, and the solid-liquid ratio is 1:20. The pH is adjusted to 2.5, and ultrasonic stirring is carried out at a temperature of 9 °C, an ultrasonic power of 250 W, an ultrasonic time of 80 min, and a stirring speed of 150 rmp / min. The ultrasonic suspension is homogenized by a high-pressure microfluidizer with a nozzle flow rate of 7 L / min and a pressure of 320 Mpa to obtain a homogenized hemp seed suspension.

[0037] Compound protease is added to the homogenized hemp seed suspension 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 hemp seed suspension. The pH of the enzymatic hydrolysis is 2.5, the temperature of the enzymatic hydrolysis is 50 °C, and the time of the enzymatic hydrolysis is 4 h. After the enzymatic hydrolysis, the temperature for enzyme inactivation is 85 °C and the time is 15 min. After the compound protease enzymatic hydrolysis, PNGaseF enzyme is added 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, and the time of the enzymatic hydrolysis is 2.5 h. After the enzymatic hydrolysis, the temperature for enzyme inactivation is 85 °C and the time is 10 min to obtain an enzymatic hydrolysate.

[0038] The enzymatic hydrolysate is separated by a centrifuge at a centrifugal speed of 3000 rpm / min for 15 min. The supernatant is taken, and the supernatant is ultrafiltered and intercepted with a 600 Da ultrafiltration membrane to remove small molecule impurities such as oligosaccharides. Then, through a tangential flow membrane concentrator, the hemp seed protein peptide filtrate obtained by using a 2.1 kDa ultrafiltration membrane is concentrated and enriched with a cross-flow pressure of 1 MPa and a membrane surface flow rate of 5 m / s.

[0039] The concentrated and enriched hemp seed protein peptide filtrate is dried and powdered by a spray dryer. The inlet air temperature of the spray drying is 20 °C, the outlet air temperature is 35 °C, the feed rate is 100 kg / h, the voltage of the electrostatic generator is 55 kV, and the pressure of the atomizer is 18 MPa. Hemp seed protein peptide dry powder is obtained and vacuum-sealed and packaged using a sterile aluminum foil bag.

[0040] The finished product of hemp seed protein peptide is sterilized by an ultra-high pressure sterilization kettle with a working pressure of 300 MPa, a working temperature of 30 °C, and a holding pressure time of 60 s, and stored at room temperature after sterilization.

[0041] Example 4 Take 500 g of hemp seeds, remove the shells, and conduct preliminary grinding and crushing. Then put them into a low-temperature supersonic airflow crusher for crushing. The temperature of the reaction kettle is -10 °C, the feeding speed is 70 Kg / h, the air flow rate is 8 m 3 / min, and the air pressure is 0.6 Mpa.

[0042] The crushed hemp seeds are subjected to enzymatic hydrolysis pretreatment. The crushed hemp seeds are mixed with water, and the solid-liquid ratio is 1:18. The pH is adjusted to 2, and ultrasonic stirring is carried out at a temperature of 12 °C, an ultrasonic power of 300 W, and an ultrasonic time of 50 min. The stirring speed is 100 rmp / min. The ultrasonic suspension is homogenized by a high-pressure microfluidizer, with a nozzle flow rate of 9 L / min and a pressure of 340 Mpa, to obtain a hemp seed homogenized suspension.

[0043] Compound protease is added to the hemp seed homogenized suspension for the first enzymatic hydrolysis. The compound 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 compound protease is 5.5% of the weight of the hemp seed homogenized suspension. The pH of the enzymatic hydrolysis is 2, the temperature of the enzymatic hydrolysis is 55 °C, and the time of the enzymatic hydrolysis is 4.5 h. After the enzymatic hydrolysis, the temperature for inactivating the enzyme is 82 °C and the time is 9 min. After the compound protease enzymatic hydrolysis, PNGaseF enzyme is added 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, and the time of the enzymatic hydrolysis is 3 h. After the enzymatic hydrolysis, the temperature for inactivating the enzyme is 86 °C and the time is 8 min, to obtain an enzymatic hydrolysate.

[0044] The enzymatic hydrolysate is separated by a centrifuge at a centrifugal speed of 3000 rpm / min for 15 min. The supernatant is taken, and the supernatant is ultrafiltered and intercepted using a 550 Da ultrafiltration membrane to remove small molecule impurities such as oligosaccharides. Then, through a tangential flow membrane concentrator, the hemp seed protein peptide filtrate obtained using a 1.9 kDa ultrafiltration membrane is concentrated and enriched, with a cross-flow pressure of 1.5 MPa and a membrane surface flow rate of 4 m / s.

[0045] The concentrated and enriched hemp seed protein peptide filtrate is dried into powder using a spray dryer. The inlet air temperature of the spray drying is 20 °C, the outlet air temperature is 35 °C, the feed rate is 80 kg / h, the voltage of the electrostatic generator is 45 kV, and the pressure of the atomizer is 17 MPa. Hemp seed protein peptide dry powder is obtained and vacuum-sealed and packaged using a sterile aluminum foil bag.

[0046] The finished product of hemp seed protein peptide is sterilized using an ultra-high pressure sterilization autoclave, with a working pressure of 400 MPa, a working temperature of 35 °C, and a pressure holding time of 40 s. After sterilization, it is stored at room temperature.

[0047] The hemp seed protein peptides prepared in Examples 1 to 4 were subjected to mass spectrometry analysis. The parameters of the mass spectrometry analysis were as follows: a reverse-phase chromatographic column was used, the aqueous phase was a 0.1% formic acid aqueous solution, the organic phase was a mobile phase of acetonitrile, 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.

[0048] Animal experiments were conducted on the hemp seed protein peptides prepared in Example 3 to detect their effects on lowering blood glucose and regulating glucose metabolism.

[0049] 1 Animal experiments 1.1 Mouse treatment and grouping Forty-eight 8-week-old male C57BL / 6 mice were selected and evenly divided into 2 groups according to the principle of body weight balance. 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 high-fat feed 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 feed + normal saline), the low-dose group (high-fat feed + 250 mg / kg hemp seed protein peptide), and the high-dose group (high-fat feed + 500 mg / kg hemp seed protein peptide). Different concentrations of hemp seed protein peptide solutions or normal saline were intragastrically administered to the mice daily according to different groups for 6 consecutive weeks of intragastric 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 (water not restricted) for 12 h, anesthetized, bled, and sacrificed after 12 h. Serum was prepared by centrifugation and stored at -80 °C.

[0050] 1.2 Oral glucose tolerance test On the afternoon of the day before the experiment at 5 o'clock, the mice in each group were transferred to clean cages and fasted 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 mice with a blood glucose meter and blood glucose test strips are shown in Figure 1 ; The blood glucose change graph after mice orally administered glucose is shown in Figure 2 ; The AUC value graph after mice orally administered glucose is shown in Figure 3 .

[0051] The drug AUC value refers to the product of the concentration reached by the drug in the whole body and time. By calculating and analyzing this value, information such as the absorption rate, absorption degree, and action time of the drug in the body can be obtained.

[0052] 1.3 Insulin tolerance test On the afternoon of the day before the experiment at 5 o'clock, the mice in each group were transferred to clean cages and fasted 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 mice intraperitoneally injected insulin is shown in Figure 4 ; The AUC value after mice intraperitoneally injected insulin is shown in Figure 5 .

[0053] 1.4 Detection of mouse serum indexes Use an ELISA kit to measure glycated hemoglobin (HbAlc), insulin (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 glucose metabolism in mouse serum are shown in Figure 6 .

[0054] 2 Test results 2.1 Fasting blood glucose measurement As Figure 1As shown in the figure, the results of fasting blood glucose detection showed that the fasting blood glucose values of the normal group mice were 5.1 mmol / L and 5.2 mmol / L for two times, both within the normal range. The fasting blood glucose values of the high-fat group mice were 7.8 mmol / L and 9.0 mmol / L for two times, indicating that high-fat feeding successfully induced obvious hyperglycemia symptoms in mice. Both high and low doses of hemp seed protein peptide intervention could significantly relieve 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.

[0055] 2.2 Oral Glucose Tolerance Test The oral glucose tolerance test is used to test the fasting blood glucose and glucose tolerance of mice to determine whether the mice have hyperglycemia symptoms and glucose intolerance. The larger the AUC (area under the curve), the higher the blood glucose integral value, indicating worse blood glucose regulation ability.

[0056] 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 was always higher than that of the normal group and the high- and low-dose hemp seed protein peptide groups, indicating that high-fat feeding induced glucose intolerance in mice. The intervention of high- and low-dose hemp seed protein peptide could improve this symptom by regulating the sugar metabolism pathway of mice. As can be seen from Figure 3 , 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 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, which further indicated that the hemp seed protein peptide composition could significantly relieve glucose intolerance caused by high-fat feeding and maintain blood glucose homeostasis.

[0057] 2.3 Insulin Tolerance Test The insulin resistance test can be used to determine whether the mice have hyperglycemia symptoms and insulin resistance. The larger the AUC (area under the curve), the higher the blood glucose integral value, indicating weaker insulin sensitivity of the body, worse blood glucose regulation ability, and stronger insulin resistance.

[0058] 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 . As 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 of mice is always higher than that of the normal group and the high- and low-dose groups of hemp seed protein peptides. This indicates that high-fat feeding induces insulin resistance symptoms in mice, while the intervention of high- and low-dose hemp seed protein peptides can improve this symptom by regulating the glucose metabolism pathway and insulin secretion pathway of mice. From Figure 5 it can be seen that the AUC value of the high-fat group of 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 groups of mice are 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 shows that the hemp seed protein peptide composition can significantly relieve insulin resistance caused by high-fat feeding and maintain blood glucose homeostasis.

[0059] 2.4 Serum glucose metabolism-related indicators Glucose metabolism is an extremely complex system in which each internal pathway affects 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 plays a crucial role in the clinical diagnosis and treatment of hyperglycemia and related diseases of glucose metabolism disorders.

[0060] 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.

[0061] 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 various pathways. For example, it can stimulate insulin secretion and inhibit glucagon release, thereby coordinately regulating blood glucose levels and preventing excessive blood glucose fluctuations.

[0062] The results of experimental studies show that both high-dose and low-dose hemp seed protein peptides can 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, there are often abnormal insulin secretion and elevated glycated hemoglobin levels, while 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.

[0063] 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 the protection scope of the present invention.

Claims

1. A method for preparing hemp seed protein peptide for lowering blood sugar and regulating sugar metabolism, characterized in that: The following steps are involved: The hemp seeds are crushed by low-temperature supersonic fluid, the crushed hemp seeds are pre-treated by enzymatic hydrolysis, a composite protease is added for enzymatic hydrolysis, and then a PNGaseF enzyme is added for further enzymatic hydrolysis to obtain an enzymatic hydrolyzate, the enzymatic hydrolyzate is subjected to solid-liquid separation to obtain a supernatant, the supernatant is subjected to membrane ultrafiltration interception to remove impurities, and then subjected to tangential flow filtration, concentrated, dried, and sterilized to obtain hemp seed protein peptides; the composite protease is pepsin and acid protease.

2. The method for preparing the hemp seed protein peptide for lowering 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 537 protease and 3.350 acidic protease.

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

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

5. The method for preparing the hemp seed protein peptide for lowering blood sugar and regulating sugar metabolism according to claim 1, characterized in that, The low-temperature supersonic fluid pulverization comprises the following steps: using a supersonic airflow pulverizer for pulverization, the temperature of the reactor is -30 to 0°C, the feed rate is 50 to 80 kg / h, and the air flow rate is 3 to 9 m 3 / min, air pressure 0.5~1Mpa.

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

7. The method for preparing the hemp seed protein peptide for lowering blood sugar and regulating sugar metabolism according to claim 1, characterized in that: The membrane ultrafiltration interception uses a 400-600Da ultrafiltration membrane.

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

9. A hemp seed protein peptide obtained by the method for preparing the hemp seed protein peptide for lowering blood sugar and regulating sugar metabolism as claimed in any one of claims 1 to 8, characterized in that: 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.

10. The hemp seed protein peptide as claimed in claim 9 is used in food and health products.

Images