Wheat peptide with hypoglycemic effect as well as preparation method and application thereof

Abstract

The invention discloses a wheat peptide with a hypoglycemic effect as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a bioactive peptide LPQ or LPQF with a hypoglycemic effect, and animal model experiments prove that the bioactive peptide can effectively reduce the blood sugar level and improve related metabolic indexes, and has no obvious side effects. The invention also provides a wheat peptide rich in oligopeptide containing the peptide fragment LPQ, and animal model experiments prove that the wheat peptide has a remarkable effect in the aspect of reducing blood sugar. The invention provides a safe and effective alternative scheme for development of diabetes treatment drugs, and has good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a wheat peptide with blood sugar lowering effect, and a preparation method and application thereof. Background Art

[0002] Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia, caused by absolute or relative insulin deficiency and utilization disorders. The disease is mainly divided into three types: type 1, type 2 and gestational diabetes. The cause is mainly attributed to the combined effects of genetic and environmental factors, including decreased insulin secretion caused by islet cell dysfunction, or the body's insensitivity to insulin, or both, which prevents glucose in the blood from being effectively utilized and stored. The symptoms of diabetes are mainly manifested as "three mores and one less", namely, polydipsia, polyuria, polyphagia and weight loss, which not only affects the patient's quality of life, but also increases the risk of serious complications such as cardiovascular disease and kidney disease, bringing a heavy economic burden to patients and society.

[0003] In this context, the search for safe and effective blood sugar-lowering ingredients has received increasing attention. Although traditional drug treatments are effective, they are often accompanied by side effects and drug resistance, which makes patients face difficulties in choosing treatment options. Therefore, the development of blood sugar-lowering products based on natural ingredients has become a trend.

[0004] Bioactive peptides are a class of peptide compounds that are beneficial to the life activities of biological organisms or have physiological effects. They are composed of two or more amino acids connected by peptide bonds and have a variety of biological functions. Compared with proteins, bioactive peptides have the advantages of simple spatial structure, high stability, low immunogenicity or no immunogenicity. In recent years, domestic and foreign scholars have continuously isolated polypeptide substances with hypoglycemic function from natural animals and plants and human bodies, and studied their structure and mechanism of action, opening up a new path for the prevention and treatment of diabetes.

[0005] For example, patent document CN119192288A discloses the use of multiple enzymes to enzymolyze defatted peanut meal, and screened out four bioactive peptides with antioxidant and hypoglycemic functions from the enzymolysis products, with amino acid sequences of AFPKFR, PFPIK, APPFDPNKPK, and FATPVPLPK. Patent document CN119019492A discloses the use of neutral protease to enzymolyze spirulina, and screened out bioactive peptides with DPP-IV inhibitory activity from the enzymolysis products, with amino acid sequences of IASY and IAGIDE.

[0006] Wheat peptides are made from wheat protein (gluten powder) through biological enzymolysis, refining, spray drying and other processes. Patent document CN114990180A discloses enzymolysis of gluten powder using alkaline protease, compound enzyme and flavor protease in sequence, decolorization and deodorization after enzyme inactivation, and 0.25μm membrane filtration and 10nm membrane filtration in sequence on the enzymolysis solution, and drying to obtain wheat peptides, which have the effect of assisting in lowering blood sugar. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out high-efficiency peptide segments related to the hypoglycemic effect, it will provide a basis for the development of new hypoglycemic preparations. Summary of the invention

[0007] The purpose of the present invention is to provide a natural small molecule bioactive peptide with hypoglycemic effect, and to apply the peptide to the development of related products for hypoglycemic effect.

[0008] To achieve the above object, the present invention adopts the following technical solution: The present invention utilizes alkaline protease, neutral protease and flavor protease to perform biological enzymolysis on wheat gluten powder in sequence, then utilizes ZGC108DQ gel strong acid cation exchange resin to adsorb the enzymolysis solution, and obtains clarified wheat peptide after desorption using hydrochloric acid solution. The peptide sequence of clarified wheat peptide before and after gastrointestinal simulated digestion is analyzed by LC-MS / MS peptide spectrum analysis technology, and it is found that the clarified wheat peptide is rich in oligopeptides with LPQF peptide segment (amino acid sequence is Leu-Pro-Gln-Phe), and the peptide segment LPQF is generated after gastrointestinal digestion. Molecular docking technology is used to explore the interaction between the peptide segment and dipeptidyl peptidase-IV (DPP-IV), and the results show that the peptide segments LPQ and LPQF have high molecular docking energy with DPP-IV. Further artificially synthesized tripeptide LPQ and tetrapeptide LPQF, functional verification found that the peptide segment has DPP-IV inhibitory activity and hypoglycemic function.

[0009] Therefore, the present invention provides a bioactive peptide with hypoglycemic effect, wherein the amino acid sequence of the bioactive peptide is Leu-Pro-Gln or Leu-Pro-Gln-Phe.

[0010] The present invention also provides a method for preparing the bioactive peptide, which can be prepared by solid phase synthesis. Specifically, an Fmoc solid phase synthesis strategy is adopted, Fmoc-protected amino acids are used as raw materials, Wang resin is selected as a solid phase carrier, glutamine, proline, and leucine residues are introduced in sequence, and the peptide chain is extended from the C-terminus to the N-terminus to synthesize the tripeptide LPQ in solid phase, or phenylalanine, glutamine, proline, and leucine residues are introduced in sequence, and the peptide chain is extended from the C-terminus to the N-terminus to synthesize the tetrapeptide LPQF in solid phase.

[0011] The present invention also provides the use of the bioactive peptide in the preparation of a drug for preventing or treating diabetes or a food for assisting in lowering blood sugar. The present invention shows that the bioactive peptide has a blood sugar lowering effect. In an in vitro experiment, the bioactive peptide exhibits significant DPP-IV inhibitory activity; in a hyperglycemic zebrafish model, the blood sugar lowering effect of the bioactive peptide after intervention is better than that of the positive control metformin. Therefore, it can be applied to the development of related products for lowering blood sugar.

[0012] Furthermore, the diabetes is type 2 diabetes. The symptoms of diabetes include: hyperglycemia and insulin resistance.

[0013] Another object of the present invention is to provide a wheat peptide having a hypoglycemic effect, wherein the wheat peptide is rich in oligopeptides containing a peptide segment LPQ, wherein the amino acid sequence of the peptide segment LPQ is Leu-Pro-Gln; The preparation method of the wheat peptide comprises: (1) Enzymatic hydrolysis: wheat gluten and water are mixed at a mass ratio of 1:15-30, the pH is adjusted to 8.0±0.2, and 0.5% of alkaline protease based on the total weight of the gluten is added for enzymatic hydrolysis for 15-30 min; then, shearing is performed at a speed of 10000-15000 r / min for 15-30 min; then, 0.5%-3.0% of alkaline protease based on the total weight of the gluten and 0.5%-2.0% of neutral protease based on the total weight of the gluten are added for further enzymatic hydrolysis for 4-6 h, and after the end, 0.1%-1.5% of flavor protease based on the total weight of the gluten is added for 15-30 min; (2) Separation: The enzymatic hydrolysate is centrifuged at a speed of 4000-6000 r / min, and the supernatant is taken. A strong acid cationic resin is added to the supernatant, and the pH is adjusted to 3.0. The mixture is stirred for adsorption and then filtered. The filter cake is redissolved in a hydrochloric acid solution with a mass concentration of 3%-5% for desorption. The supernatant is nanofiltered, concentrated, sterilized, and dried to obtain the wheat peptide.

[0014] The wheat peptide provided by the present invention is rich in oligopeptides with a specific amino acid sequence of Leu-Pro-Gln. After systematic research and experimental verification, it is proved that it has a significant effect on lowering blood sugar. In a high-fat diet-induced hyperglycemic mouse model, the wheat peptide can effectively reduce blood sugar levels after intervention, and improve related metabolic indicators, improve the trend of impaired glucose tolerance, reduce the insulin resistance index, and significantly reduce the body weight growth rate and reduce the fat index. The results of the study show that the active ingredients of wheat peptides can play a role in multiple ways such as regulating insulin secretion, improving insulin sensitivity, and lowering inflammatory factors, thereby achieving a comprehensive blood sugar-lowering effect. Therefore, it can be applied to the development of related products for lowering blood sugar.

[0015] Furthermore, the oligopeptide containing the peptide segment LPQ is at least one of the peptide segments having an amino acid sequence of LPQFEELLNR (SEQ ID NO.2), LPQFEELR (SEQ ID NO.3), LPQFAELR (SEQ ID NO.4), LPQFEALR (SEQ ID NO.5), LPQFEELNR (SEQ ID NO.6), LPQFEF (SEQ ID NO.7), and LPQQL (SEQ ID NO.8).

[0016] The present invention provides the use of the wheat peptide in preparing a medicine for preventing or treating diabetes or a food for assisting in lowering blood sugar. The wheat peptide exerts a blood sugar lowering effect after being digested by the gastrointestinal tract.

[0017] The present invention also provides a pharmaceutical composition for preventing or treating diabetes, comprising an effective dose of a bioactive peptide having an amino acid sequence of Leu-Pro-Gln or Leu-Pro-Gln-Phe, or a wheat peptide rich in oligopeptides containing the peptide segment LPQ.

[0018] The tripeptide LPQ or the tetrapeptide LPQF or the wheat peptide rich in oligopeptides containing the peptide segment LPQ in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that exerts a hypoglycemic effect, or can be compounded with other active ingredients having a hypoglycemic function.

[0019] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject.

[0020] Furthermore, the pharmaceutically acceptable carrier includes: one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0021] The present invention uses the tripeptide LPQ or the tetrapeptide LPQF or the wheat peptide rich in the polypeptide having the peptide segment LPQ as the main active ingredient, adds a pharmaceutically acceptable carrier, and prepares a preparation according to the preparation preparation method recorded in pharmaceutics. Further, the preparation form of the pharmaceutical composition can be but not limited to an oral preparation. Specifically, the preparation form can be but not limited to an oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0022] The present invention also provides a health food for assisting in lowering blood sugar, comprising as an active ingredient a bioactive peptide with an amino acid sequence of Leu-Pro-Gln or Leu-Pro-Gln-Phe, or a wheat peptide rich in oligopeptides containing the peptide segment LPQ, and excipients acceptable in food science.

[0023] The food-acceptable excipient is a food-grade excipient that can deliver an effective dose of the active substance of the present invention and does not interfere with the biological activity of the active substance.

[0024] Furthermore, the dosage form of the health food is a beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0025] The present invention has the following beneficial effects: (1) The present invention provides a bioactive peptide LPQ or LPQF with hypoglycemic effect. Animal model experiments have shown that the bioactive peptide can effectively lower blood sugar levels and improve related metabolic indicators without obvious side effects. Therefore, the bioactive peptide can be used to prepare drugs or foods for lowering blood sugar.

[0026] (2) The present invention provides a wheat peptide rich in oligopeptides including the peptide segment LPQ, which has been verified by animal model experiments to have a significant effect in lowering blood sugar. The present invention provides a safe and effective alternative for the development of diabetes treatment drugs, and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the secondary mass spectrum of peptide LPQF, in which the ordinate Intensity (%) represents relative abundance; L / P / Q / F represents the tetrapeptide sequence; y1 represents the first fragment ion produced by the cleavage of the C-terminus of the peptide; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; y2-H2O represents the second dehydrated fragment ion produced by the cleavage of the C-terminus of the peptide; y2-NH3 represents the second deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; b3-H2O represents the third dehydrated fragment ion produced by the cleavage of the N-terminus of the peptide; y3-NH3 represents the third deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error (Da) represents the deviation.

[0028] Figure 2It is the secondary mass spectrum of peptide LPQFE, in which the ordinate Intensity (%) represents relative abundance; L / P / Q / F / E represents the pentapeptide sequence; y1 represents the first fragment ion produced by the cleavage of the C-terminus of the peptide; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; b3 represents the third fragment ion produced by the cleavage of the N-terminus of the peptide; y3 represents the third fragment ion produced by the cleavage of the C-terminus of the peptide; y3-H2O represents the third dehydrated fragment ion produced by the cleavage of the C-terminus of the peptide; y3-NH3 represents the third deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; b4 represents the fourth fragment ion produced by the cleavage of the N-terminus of the peptide; y4 represents the fourth fragment ion produced by the cleavage of the C-terminus of the peptide; pre[1+] represents charge [1+]; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error(Da) represents the deviation.

[0029] Figure 3 This is the secondary mass spectrum of peptide LPQQL. The ordinate Intensity (%) represents relative abundance; L / P / Q / Q / L represents the pentapeptide sequence; y2-H2O represents the second dehydrated fragment ion produced by the cleavage of the C-terminus of the peptide; y2-NH3 represents the second deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; b3 represents the third fragment ion produced by the cleavage of the N-terminus of the peptide; y4 represents the fourth fragment ion produced by the cleavage of the C-terminus of the peptide; pre[1+] represents charge [1+]; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error(Da) represents the deviation.

[0030] Figure 4 This is the secondary mass spectrum of the peptide LPQFEELLNR. The ordinate Intensity (%) in the figure represents relative abundance; L / P / Q / F / E / E / L / L / N / R represents the ten-peptide sequence; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; b3 represents the third fragment ion produced by the cleavage of the N-terminus of the peptide; y3 represents the third fragment ion produced by the cleavage of the C-terminus of the peptide; y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide; y6 represents the sixth fragment ion produced by the cleavage of the C-terminus of the peptide; y7 represents the seventh fragment ion produced by the cleavage of the C-terminus of the peptide; y9-NH3 represents the ninth deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; y9 represents the ninth fragment ion produced by the cleavage of the C-terminus of the peptide; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error (Da) represents the deviation.

[0031] Figure 5This is the secondary mass spectrum of peptide LPQFEELR, in which the ordinate Intensity (%) represents relative abundance; L / P / Q / F / E / E / L / R represents the octapeptide sequence; y1 represents the first fragment ion produced by the cleavage of the C-terminus of the peptide; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; y3 represents the third fragment ion produced by the cleavage of the C-terminus of the peptide; y4 represents the fourth fragment ion produced by the cleavage of the C-terminus of the peptide; y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide; y6 represents the sixth fragment ion produced by the cleavage of the C-terminus of the peptide; y7 represents the seventh fragment ion produced by the cleavage of the C-terminus of the peptide; bMax represents the maximum value at the N-terminus; yMax represents the maximum value at the C-terminus; Error (Da) represents the deviation.

[0032] Figure 6 This is the secondary mass spectrum of peptide LPQFAELR, in which the ordinate Intensity (%) represents relative abundance; L / P / Q / F / A / E / L / R represents the octapeptide sequence; y1 represents the first fragment ion produced by the cleavage of the C-terminus of the peptide; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; y3 represents the third fragment ion produced by the cleavage of the C-terminus of the peptide; y4 represents the fourth fragment ion produced by the cleavage of the C-terminus of the peptide; y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide; y6-H2O represents the sixth dehydrated fragment ion produced by the cleavage of the C-terminus of the peptide; y6 represents the sixth fragment ion produced by the cleavage of the C-terminus of the peptide; y7 represents the seventh fragment ion produced by the cleavage of the C-terminus of the peptide; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error (Da) represents the deviation.

[0033] Figure 7This is the secondary mass spectrum of peptide LPQFEALR, in which the ordinate Intensity (%) represents relative abundance; L / P / Q / F / EA / L / R represents the octapeptide sequence; y1 represents the first fragment ion produced by the cleavage of the peptide C-terminus; b2 represents the second fragment ion produced by the cleavage of the peptide N-terminus; y2 represents the second fragment ion produced by the cleavage of the peptide C-terminus; b3-H2O represents the third dehydrated fragment ion produced by the cleavage of the peptide N-terminus; y4 represents the fourth fragment ion produced by the cleavage of the peptide C-terminus; y5 represents the fifth fragment ion produced by the cleavage of the peptide C-terminus; y6-NH3 represents the sixth deaminated fragment ion produced by the cleavage of the peptide C-terminus; y6 represents the sixth fragment ion produced by the cleavage of the peptide C-terminus; y7 represents the seventh fragment ion produced by the cleavage of the peptide C-terminus; bMax represents the maximum value at the N-terminus; yMax represents the maximum value at the C-terminus; Error (Da) represents the deviation.

[0034] Figure 8 This is the secondary mass spectrum of the peptide LPQFEELNR. The ordinate Intensity (%) in the figure represents relative abundance; L / P / Q / F / E / E / L / NR represents the nine-peptide sequence; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; y3 represents the third fragment ion produced by the cleavage of the C-terminus of the peptide; y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide; y6 represents the sixth fragment ion produced by the cleavage of the C-terminus of the peptide; y8 represents the eighth fragment ion produced by the cleavage of the C-terminus of the peptide; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error (Da) represents the deviation.

[0035] Fig. 9 This is the secondary mass spectrum of peptide LPQFEF, in which the ordinate Intensity (%) represents relative abundance; L / P / Q / F / E / F represents the hexapeptide sequence; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; b3 represents the third fragment ion produced by the cleavage of the N-terminus of the peptide; b4 represents the fourth fragment ion produced by the cleavage of the N-terminus of the peptide; b5 represents the fifth fragment ion produced by the cleavage of the N-terminus of the peptide; y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide; pre[1+] represents charge [1+]; bMax represents the maximum value of the N-terminus; yMax represents the maximum value of the C-terminus; Error(Da) represents the deviation.

[0036] Fig.10 Schematic diagram of the binding interaction between the tripeptide LPQ and DPP-IV.

[0037] Fig.11 Schematic diagram of the binding interaction between the tetrapeptide LPQF and DPP-IV.

[0038] Fig.12 is the DPP-IV inhibition rate of the tripeptide LPQ and the tetrapeptide LPQF.

[0039] Fig.13 The hypoglycemic effect of tripeptide LPQ and tetrapeptide LPQF in the hyperglycemic zebrafish model. The * in the figure indicates a significant difference compared with the blank control group (NC). p <0.05, **** means p <0.0001; # indicates significant difference compared with the model group (MC), ## indicates p <0.01, ### indicates p <0.001, #### indicates p <0.0001.

[0040] Fig.14 To compare the DPP-IV inhibition rate (A) and hypoglycemic effect (B) of LPQ / LPQF and LP, the letters a, b, and c in A represent significant differences between the groups, the same letters represent no significant differences between the groups, and different letters represent significant differences between the groups. p <0.05; * in B indicates significant difference compared with blank control group (NC), ** indicates p <0.01, *** indicates p <0.001, # indicates significant difference compared with the model group (MC), ## indicates p <0.01.

[0041] Fig.15 To compare the DPP-IV inhibition rate (A) and hypoglycemic effect (B) of LPQ / LPQF and FP / VP, the letters a, b, and c in A represent significant differences between the groups, the same letters represent no significant differences between the groups, and different letters represent significant differences between the groups. p <0.05; * in B indicates significant difference compared with blank control group (NC), *** indicates p <0.001, **** means p <0.0001, # indicates significant difference compared with the model group (MC), ## indicates p <0.01.

[0042] Fig.16 To compare the DPP-IV inhibition rate (A) and hypoglycemic effect (B) of LPQ / LPQF and LPT, the letters a, b, and c in A represent significant differences between the groups, the same letters represent no significant differences between the groups, and different letters represent significant differences between the groups. p<0.05; * in B indicates significant difference compared with blank control group (NC), *** indicates p <0.001, **** means p <0.0001, # indicates significant difference compared with the model group (MC), ## indicates p <0.01.

[0043] Fig.17 To compare the hypoglycemic effects of LPQ / LPQF and IPI, * indicates significant difference compared with the blank control group (NC), **** indicates p <0.0001, # indicates significant difference compared with the model group (MC), ## indicates p <0.01.

[0044] Fig.18 Figure 2 shows the hypoglycemic effect of wheat peptide containing peptide LPQ in a hyperglycemic mouse model, where A is the experimental design, B is energy intake, C is body weight, D is the size of adipocytes, and E is the H&E staining result of epididymal fat; * indicates a significant difference compared with the normal group, **** indicates p <0.0001, # indicates significant difference compared with the modeling group, # indicates p <0.05.

[0045] Fig.19 Figure 2 shows the hypoglycemic effect of wheat peptide containing peptide segment LPQ in a hyperglycemic mouse model, where A is oral glucose tolerance, B is the area under the blood glucose curve, C is fasting blood glucose, and D is the insulin resistance index; * in the figure indicates a significant difference compared with the normal group, and * indicates p <0.05,** indicates p <0.01, **** means p <0.0001, # indicates significant difference compared with the modeling group, # indicates p <0.05, ## indicates p <0.01, ### indicates p <0.001. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0048] Wheat protein (gluten powder) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme (Yichang) Co., Ltd.

[0049] The amino acid sequence is from N-terminus to C-terminus from left to right.

[0050] Example 1 Preparation and analysis of clarified wheat peptides 1. Preparation of clarified wheat peptides by enzymatic hydrolysis: Wheat protein (gluten) and water are put into the enzymatic hydrolysis reaction tank at a ratio of 1:20, the pH of the feed solution is adjusted to 8.0±0.2, and 0.5% alkaline protease (derived from Bacillus licheniformis) of the total weight of gluten is added for enzymatic hydrolysis for 30 min, and then the shearing machine is turned on with a shearing speed of 10000 r / min for 30 min.

[0051] After the shearing was completed, the pH of the feed solution was adjusted to 8.0±0.2, and 2.5% of the total weight of gluten by alkaline protease (derived from Bacillus licheniformis) and 1.0% of the total weight of gluten by neutral protease (derived from Bacillus subtilis) were added to continue enzymatic hydrolysis for 4 h. After the end, 0.8% of the total weight of gluten by flavor protease was used for 30 min.

[0052] After the enzymatic hydrolysis, the supernatant was retained by a disc centrifuge (speed 6000 r / min).

[0053] ZGC108DQ gel strong acid cation exchange resin was added to the supernatant after butterfly separation, the pH was adjusted to 3.0, and the mixture was stirred and adsorbed for 2 h. After the adsorption, the filter cake was passed through a plate and frame filter, and the filter cake was redissolved in a 3% hydrochloric acid solution for desorption for 60 min. The supernatant was nanofiltered, concentrated, sterilized, and spray-dried to obtain wheat peptide (glutamine peptide) powder.

[0054] 2. Analysis of wheat peptides (glutamine peptides) The main components and molecular weight distribution range of the wheat peptide (glutamine peptide) prepared in step 1 were determined, and the results are shown in Tables 1 to 3: Table 1. Content of main components of glutamine peptide Element content(%) Moisture 4.02 Ash 4.21 Protein content (dry basis) 83.12 Degree of hydrolysis 12.38 Peptide content (dry basis) 50.31 Table 2. Relative molecular weight distribution of glutamine peptides Relative molecular weight range, Da Wheat peptides (glutamine peptides), % >10000 2.61 5000-10000 6.60 5000-3000 3.53 3000-2000 3.72 2000-1000 9.89 1000-500 18.62 500-180 38.84 <180 16.20 Table 3. Amino acid composition of glutamine peptide

[0055] Example 2 Active peptide screening In this example, we simulated the gastrointestinal digestion of wheat peptides in vitro and identified the related peptides before and after digestion by LC-MS / MS, used molecular docking technology to explore their specific mechanism of action with dipeptidyl peptidase-IV (DPP-IV), and screened out highly effective DPP-IV inhibitory peptides.

[0056] 1. In vitro simulated digestion: Take 10 mL of the sample solution of Example 1 (preheated in a water bath at 37 °C), add 9 mg / mL NaCl solution (physiological saline, preheated in a water bath at 37 °C), adjust the pH value to 2 with 1 mol / L HCl, add 4 mL of gastric juice (0.4 g pepsin dissolved in 0.01 mol / L HCl solution) to the system, and complete the simulation of gastric digestion after shaking at 37 °C water bath and 120 r / min for 2 h. The pH value of the digested sample was pre-adjusted to 6.5. Pipette 2 mL of the gastric digested sample into a centrifuge tube, add 4 mL of intestinal juice (0.02 g trypsin and 0.12 g pig bile salt were dissolved in 10 mL 0.1 mol / L NaHCO3 solution, respectively, to obtain 2 mg / mL trypsin solution and 12 mg / mL bile solution, pancreatic juice and bile were mixed 1:1), adjust the pH to 7.0, and complete the simulation of intestinal digestion after shaking at 37 °C water bath and 120 r / min for 2 h. All experiments were performed in the absence of light and oxygen, and the simulated digested samples were lyophilized and stored at −80 °C until analysis.

[0057] 2. LC-MS / MS identification of active peptides of wheat peptides before and after digestion The sample prepared in Example 1 and the digested sample prepared in step 1 above were dissolved in NH4HCO3 solution, dithiothreitol solution was added, and the mixture was reduced in a 56 ℃ water bath for 1 h. Then iodoacetamide solution was added and the mixture was reacted for 40 min in the dark. After desalting, the solvent was evaporated, and then 10 μL of mobile phase A (0.1% formic acid) was used to dissolve the sample into a liquid injection bottle. LC-MS / MS analysis was then performed.

[0058] Chromatographic conditions: analytical column (Acclaim PepMap RPLC C18, 150×150 mm, 3 μm); mobile phase A (0.1% formic acid); mobile phase B (0.1% formic acid and 80% acetonitrile); flow rate (600 nL / min). Gradient elution program: 0-2 min, 4% B -8% B; 2-45 min, 8% B-40% B; 45-55 min, 40% B-60% B; 55-56 min, 60% B-95% B; 56-66 min, 95% B.

[0059] Mass spectrometry conditions: Full scan MS using Orbitrap for primary scanning, scan range (100-1500 m / z), resolution (70,000), maximum ion introduction time (100 ms), automatic gain control (3×10 6 ); The top 20 parent ions that met the MS / MS fragmentation conditions were fragmented using high-energy collision dissociation and scanned using Orbitrap, with a resolution of 17500, a maximum ion introduction time of 50 ms, and an automatic gain control of 1×10 5 The raw data obtained by mass spectrometry were analyzed for peptide sequence using the De novo (de novo sequencing) software of PEAKS Studio.

[0060] Analysis of the peptide spectra of wheat peptides before and after digestion revealed that wheat peptides were rich in multiple peptides containing LPQ sequences before and after digestion, which are shown in Table 4 after sorting.

[0061] Table 4. LPQ amino acid sequences in wheat peptides

[0062] The secondary mass spectrum of the above peptide is as follows Figure 1-Figure 9 shown.

[0063] 3. The molecular docking score of LPQ / LPQF and DPP-Ⅳ is high Dipeptidyl peptidase-IV (DPP-IV) can weaken the hypoglycemic effect of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) by degrading them. Inhibiting DPP-IV activity can maintain the activity of these incretin hormones, thereby promoting insulin secretion, inhibiting glucagon release, and achieving blood sugar control. DPP-IV inhibitors selectively block the activity of this enzyme and become a core drug in the treatment of type 2 diabetes that lowers blood sugar by enhancing the function of the endogenous incretin system. Therefore, in this example, wheat peptide segments are molecularly docked with DPP-IV to screen small molecule peptide segments that may have hypoglycemic effects.

[0064] First, the crystal structure of DPP-IV (1WCY) was downloaded from the PDB protein database. The active center was defined by removing water molecules and adding hydrogen atoms from the receptor target using DiscoveryStudio software. The peptide LPQ / LPQF was molecularly docked with dipeptidyl peptidase-IV (DPP-IV).

[0065] The 2D and 3D images of the molecular docking of tripeptide LPQ and DPP-IV are shown in Fig.10As shown. Analysis of chemical bonds revealed that the tripeptide LPQ was mainly bound to DPP-IV through van der Waals forces, hydrogen bonds (conventional hydrogen bonds) and electrostatic interactions (salt bridges), with a docking energy of -32.50 kcal / mol. The tripeptide LPQ formed two van der Waals forces with amino acid residues SER458 and SER460, two hydrogen bonds with ARG471 and VAL459, and two electrostatic interactions with GLU408 and ARG471.

[0066] The 2D and 3D images of the molecular docking of tetrapeptide LPQF with DPP-IV are shown in Fig.11 As shown. Analysis of chemical bonds revealed that the tetrapeptide was mainly bound to DPP-IV through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), hydrophobic interactions (alkyl groups), and electrostatic interactions (including salt bridges and attractive charges), with a docking energy of -52.58 kcal / mol. The tetrapeptide LPQF formed seven van der Waals forces with amino acid residues SER473, TYR456, SER458, GLY406, VAL459, ILE418, and LEU57, three hydrogen bonds with ARG471 and SER59, two hydrophobic interactions with ILE405, and three electrostatic interactions with ARG471 and GLU408.

[0067] The molecular docking results show that the tripeptide LPQ and the tetrapeptide LPQF can bind to DPP-IV and then inhibit DPP-IV, thereby exerting a hypoglycemic effect.

[0068] We commissioned Jier Biochemical (Shanghai) Co., Ltd. to synthesize peptides LPQ and LPQF with a purity of ≥98% for subsequent functional verification.

[0069] Example 3 In vitro DPP-Ⅳ inhibition rate of LPQ / LPQF The DPP-IV inhibition rate of the peptide was determined using the Cayman (manufacturer) DPP-IV inhibitor kit. The assay principle is as follows: The test uses the fluorescent substrate gly-pro-aminomethylcoumarin (AMC) to measure the activity of DPP-IV. DPP cleaves the peptide bond to release the free AMC group, generating fluorescence with an excitation wavelength of 350-360 nm and an emission wavelength of 450-465 nm.

[0070] The DPP-IV inhibition rates of tripeptide LPQ and tetrapeptide LPQF at 1000 µM and 500 µM were determined. The determination method is as follows: (1) 100% Initial Activity Wells - Add 30 µL of dilution buffer, 10 µL of DPP-IV, and 10 µL of pure water to three wells.

[0071] (2) Background wells - Add 40 µL of diluted buffer and 10 µL of pure water to three wells.

[0072] (3) Inhibitor Wells - Add 30 µL of diluted assay buffer, 10 µL of diluted DPP-IV, and 10 µL of inhibitor (intervention sample, 1000 µM and 500 µM concentrations of LPQ and LPQF) to three wells.

[0073] The specific operation is as follows: first, add 30 µL buffer, 10 µL DPP-IV enzyme and 10 µL sample to a 96-well plate and mix well, add 50 µL substrate solution to start the reaction, and then incubate at 37 °C for 30 min, and read the fluorescence with an excitation wavelength of 350-360 nm and an emission wavelength of 450-465 nm. Detect the fluorescence intensity and record the result as sample A. The result measured by using buffer instead of DPP-IV is recorded as blank A, and the result measured by using buffer instead of sample is recorded as control A. The calculation formula of DPP-IV inhibition rate is as follows: inhibition rate (%) = (control A-sample A) / (control A-blank A) × 100%.

[0074] The results are as follows Fig.12 As shown, the DPP-IV inhibition rates of tripeptide LPQ and tetrapeptide LPQF were both higher than 85% at 1000 µM and 500 µM, indicating that tripeptide LPQ and tetrapeptide LPQF have DPP-IV inhibitory potential.

[0075] Example 4 Hypoglycemic effect of LPQ / LPQF in a hyperglycemic zebrafish model The hyperglycemic zebrafish model was used to determine the hypoglycemic effects of the tripeptide LPQ and the tetrapeptide LPQF. Wild AB zebrafish of 4 days old were placed in a six-well plate, with 3 parallel wells per group and 10 fish per well. The group interventions were as follows: (1) Blank control group (NC): system water; (2) Model group (MC): 333 μM alloxan + 2.67% glucose + system water; (3) Metformin positive control group (Met): 333 μM alloxan + 2.67% glucose + 5 μg / mL metformin + system water; (4) Tripeptide LPQ intervention group (LPQ-5, LPQ-1, LPQ-0.1): 333 μM alloxan + 2.67% glucose + 5 / 1 / 0.1 μg / mL tripeptide LPQ + system water.

[0076] (5) Tetrapeptide LPQF intervention group (LPQF-5, LPQF-1, LPQF-0.1): 333 μM alloxan + 2.67% glucose + 5 / 1 / 0.1 μg / mL tetrapeptide LPQF + system water.

[0077] After 24 h of intervention with the above intervention scheme, 4-day-old zebrafish were washed three times with PBS solution to clean the sugar solution on the surface of zebrafish. Use a disposable dropper to transfer the zebrafish in the six-well plate to a 1.5 mL centrifuge tube, remove the excess liquid in the centrifuge tube, add 0.1 mL of anhydrous ethanol, place in a cool place for 15 min, move into an oven for 120 min (60 ℃) until dried (crushed), and finally add 5 µL of ultrapure water to the centrifuge tube, shake it ultrasonically for 10 min, and draw 2 µL to measure the glucose value (S) using a blood glucose meter.

[0078] The results are as follows Fig.13 As shown in the figure, compared with the blank control group (NC group), the blood glucose level of the model group (MC group) after 24 hours of modeling with 333 μM alloxan + 2.67% glucose was significantly increased ( p <0.0001), indicating that the hyperglycemia model was successfully established. After intervention with the positive drug 5 μg / mL metformin (Met group), there was a significant downregulation effect compared with the MC group ( p <0.01). The tripeptide LPQ and the tetrapeptide LPQF at the concentrations of 5 μg / mL, 1 μg / mL and 0.1 μg / mL could significantly reduce the blood glucose level compared with the MC group ( p <0.001), and was downregulated to no significant difference with the NC group ( p >0.05), and the differences between LPQ / LPQF and MC groups at the tested doses (5 μg / mL, 1 μg / mL and 0.1 μg / mL) were significant ( p <0.001) was stronger than the significant difference between the positive control metformin and MC group ( p <0.05), indicating that the tripeptide LPQ and the tetrapeptide LPQF have the effect of lowering blood sugar.

[0079] Example 5 In vitro DPP-Ⅳ inhibition rate of dipeptide with P at the second position and its hypoglycemic effect in zebrafish The above studies show that both the tripeptide LPQ and the tetrapeptide LPQF have potential DPP-IV inhibition and hypoglycemic effects. In order to explore whether the reduction of one more amino acid still has the potential for DPP-IV inhibition and hypoglycemic effects, Jier Biochemical (Shanghai) Co., Ltd. was commissioned to synthesize the peptides LP (amino acid sequence is Leu-Pro), FP (amino acid sequence is Phe-Pro), and VP (amino acid sequence is Val-Pro) with a purity of ≥98% for subsequent functional verification experiments.

[0080] 1. LPQ / LPQF is better than LP The DPP-IV inhibition experimental method was the same as that of “In vitro DPP-IV inhibition rate of LPQ / LPQF”, and the DPP-IV inhibition rates of LPQ / LPQF and LP at 1000 µM and 500 µM were compared, respectively.

[0081] The results are as follows Fig.14 As shown in A, at 1000 µM and 500 µM, the DPP-IV inhibition rate of dipeptide LP was significantly lower than that of LPQ / LPQF ( p <0.0001).

[0082] The experimental method for studying blood sugar reduction in zebrafish model was the same as that of “blood sugar reduction effect of LPQ / LPQF in hyperglycemic zebrafish model”, and the blood sugar reduction effects of LPQ / LPQF and LP at a dose of 1 μg / mL were compared.

[0083] The results are as follows Fig.14 As shown in Figure B, the tripeptide LPQ and the tetrapeptide LPQF at a concentration of 1 μg / mL can significantly reduce blood glucose levels compared with the MC group ( p <0.001), and was downregulated to no significant difference with the NC group ( p >0.05), but there was no significant difference between the dipeptide LP group and the MC group after intervention, indicating that the tripeptide LPQ and the tetrapeptide LPQF have stronger hypoglycemic effects than the dipeptide LP.

[0084] 2. LPQ / LPQF is better than FP / VP In vitro DPP-Ⅳ inhibition rate and zebrafish hypoglycemic results are as follows Fig.15 As shown, the hypoglycemic effect of the tripeptide LPQ and the tetrapeptide LPQF is stronger than that of the dipeptide FP / VP.

[0085] Example 6 In vitro DPP-Ⅳ inhibition rate and hypoglycemia in zebrafish of other tripeptides with P at the second position The above studies show that both the tripeptide LPQ and the tetrapeptide LPQF have potential DPP-IV inhibitory and hypoglycemic effects. In order to explore whether the third amino acid still has DPP-IV inhibitory and hypoglycemic potential, Jier Biochemical (Shanghai) Co., Ltd. was commissioned to synthesize the peptide LPT (amino acid sequence is Leu-Pro-Thr) with a purity of ≥98% for subsequent functional verification experiments.

[0086] The DPP-IV inhibition experimental method was the same as that of “In vitro DPP-IV inhibition rate of LPQ / LPQF”, and the DPP-IV inhibition rates of LPQ / LPQF and LPT at 1000 µM and 500 µM were compared, respectively.

[0087] The results are as follows Fig.16 As shown in A, at 1000 µM and 500 µM, the DPP-IV inhibition rate of the tripeptide LPT was significantly lower than that of LPQ / LPQF ( p <0.001, p <0.0001).

[0088] The experimental method for studying the hypoglycemic effect of zebrafish model was the same as that of “hyperglycemic effect of LPQ / LPQF in hyperglycemic zebrafish model”, and the hypoglycemic effects of LPQ / LPQF and LPT at a dose of 1 μg / mL were compared.

[0089] The results are as follows Fig.16 As shown in Figure B, the tripeptide LPQ and the tetrapeptide LPQF at a concentration of 1 μg / mL can significantly reduce blood glucose levels compared with the MC group ( p <0.001), and was downregulated to no significant difference with the NC group ( p >0.05), but there was no significant difference between the MC group and the tripeptide LPT intervention group, indicating that the tripeptide LPQ and the tetrapeptide LPQF have stronger hypoglycemic effects than the tripeptide LPT.

[0090] Example 7 Comparison of hypoglycemic efficacy between LPQ / LPQF and IPI IPI has been reported to have DPP-IV inhibitory effects. This example compares the hypoglycemic effects of LPQ / LPQF and IPI. Jier Biochemical (Shanghai) Co., Ltd. was commissioned to synthesize the peptide IPI (amino acid sequence is Ile-Pro-Ile) with a purity of ≥98% for subsequent functional verification experiments.

[0091] The experimental method for studying blood sugar reduction in zebrafish model was the same as that of “blood sugar reduction effect of LPQ / LPQF in hyperglycemic zebrafish model”, and the blood sugar reduction effects of LPQ / LPQF and IPI at a dose of 1 μg / mL were compared.

[0092] The results are as follows Fig.17 As shown in the figure, the tripeptide LPQ and the tetrapeptide LPQF at a concentration of 1 μg / mL can significantly reduce blood glucose levels compared with the MC group ( p <0.01), and was downregulated to no significant difference with the NC group ( p >0.05), IPI intervention can also significantly reduce blood sugar ( p <0.01). From the significance level, the hypoglycemic effects of tripeptide LPQ and tetrapeptide LPQF were comparable to those of IPI, and from the average values, the hypoglycemic effects of tripeptide LPQ and tetrapeptide LPQF were superior to those of IPI.

[0093] Example 8 Hypoglycemic effect of wheat peptide (prepared in Example 1) in a hyperglycemic mouse model A high-fat diet-induced hyperglycemic mouse model was used to investigate whether wheat peptides have the effect of lowering blood sugar. The experiment is as follows: (1) Mice and materials Male C57BL / 6 mice aged 6 to 8 weeks (certificate number: 20230524Abzz0100018521, ethics approval number: MG20230531082) were purchased from Hangzhou Medical College, production license number: SCXK (Zhejiang) 2019-0042. Mouse basal feed was purchased from Shenzhen Maohua Biotechnology Co., Ltd. 45% purified high-fat feed (MD12032) was provided by Jiangsu Medison Biological Co., Ltd.

[0094] (2) Animal grouping and feeding 60 6-8 week old C57BL / 6 male mice were housed in the Animal Experiment Center of Zhejiang University of Technology at a temperature of 23±1℃ and a 12 h day and night cycle. After purchase, the mice were fed with normal diet for one week, and then divided into a normal diet group and a high-fat diet modeling group. The normal group was fed with normal diet, and the high-fat group was fed with 45% kcal high-fat diet for 8 consecutive weeks. After the eighth week, high, medium and low doses of LPQ / LPQF wheat peptides were used for intervention for 8 weeks. The specific experimental design is shown in Fig.18 A in the figure. The food intake and body weight of mice were recorded weekly. After 16 weeks, mice were fasted for 16 h, blood was collected from the eye sockets, and epididymal fat and other tissues were collected, frozen with liquid nitrogen, transported on dry ice, and stored in a -80 °C refrigerator for later use. Blood samples were centrifuged at 3000 rpm for 10 min to obtain serum. A small portion of epididymal fat tissue was taken and fixed in 4% neutral formaldehyde.

[0095] (3) Hematoxylin-eosin (H&E) staining of fat sections After fixation with 4% neutral paraformaldehyde for 24 h, the epididymal fat and liver of mice were dehydrated, embedded in paraffin, sliced ​​(3 μm thickness), baked for 2 h, dewaxed and hydrated, stained with H&E, and finally dehydrated and sealed with neutral plastic. The fat sections were observed and analyzed under an optical microscope, and the fat sections were photographed in a random field of view. The size of epididymal adipocytes in each section was statistically analyzed using Image J software.

[0096] (4) OGTT test in mice The day before the experiment, mice were fasted but not watered for 16 h, and 2 g / kg of glucose was gavaged into the stomach, and the gavage time was recorded. Blood glucose levels were measured at 0 min, 30 min, 60 min, and 120 min using a blood glucose meter. The blood glucose value-time curve was drawn, and the area under the curve of each experimental group was calculated.

[0097] (5) Determination of biochemical related indicators and hormone levels in serum The Nanjing Jiancheng kit was used to measure the glucose content in serum (fasting blood sugar). The Gene Beauty ELISA kit was used to measure the insulin content in mouse serum. The insulin resistance index was calculated based on the fasting blood sugar and fasting insulin values ​​of the mice. The calculation formula is as follows: Insulin resistance index = mouse fasting blood glucose value (mmol / L) × mouse fasting insulin value (μU / mL) / 22.5.

[0098] (6) Results analysis a. The hypoglycemic effect of wheat peptides in a hyperglycemic mouse model - body weight and fat After 8 weeks of intervention of high-fat diet-induced insulin resistance mice with the wheat peptide prepared in Example 1 at doses of 1000 mg / kg (high dose), 250 mg / kg (medium dose) and 50 mg / kg (low dose), the food intake, body weight and epididymal fat shape of the mice were as follows: Fig.18 As shown. Fig.18 As shown in B, there was no significant difference in energy intake among the normal group, model control group, high-dose wheat peptide intervention group, medium-dose wheat peptide intervention group, and low-dose wheat peptide intervention group ( p >0.05). Fig.18 As shown in C, in terms of weight gain, the weight growth rate of the modeling control group was significantly higher than that of the normal group ( p <0.0001), after intervention with 1000 mg / kg (high dose) and 250 mg / kg (medium dose) of wheat peptide, the body weight growth rate was significantly downregulated compared with the model control group ( p <0.05), indicating that wheat peptides have the effect of reducing weight gain. The results of H&E staining of epididymal fat are as follows Fig.18 As shown in E in Figure 1, the size of fat cells was statistically analyzed, and the results are as follows: Fig.18 As shown in D, it can be seen that the high-fat diet model significantly enlarges the adipocytes ( p <0.0001), and after intervention with 250 mg / kg dose of wheat peptide, the size of adipocytes in the wheat peptide group was significantly reduced compared with the model control group ( p <0.01), indicating that wheat peptide has lipid-lowering effect.

[0099] b. The hypoglycemic effect of wheat peptides in a hyperglycemic mouse model: blood sugar and insulin resistance The results of the oral glucose tolerance test (OGTT) are as follows: Fig.19As shown in A. The blood glucose levels of the four groups of mice fed with high-fat diets were higher than those of the normal group at each time point after oral glucose administration, and the blood glucose levels of the mice in the model control group at 0 min, 30 min, 90 min and 120 min were significantly higher than those of the mice in the model control group. Statistical analysis of the area under the blood glucose curve showed that ( Fig.19 B), 16 weeks of high-fat diet feeding significantly increased the area under the blood glucose curve of mice, and the intake of wheat peptides at doses of 1000 mg / kg, 250 mg / kg and 50 mg / kg significantly improved the trend of impaired glucose tolerance caused by high-fat diet ( p <0.01), indicating that wheat peptides may have a significant effect on alleviating high blood sugar induced by high-fat diet. In addition, from the fasting blood sugar results ( Fig.19 C) It can also be seen that compared with the model control group, the intervention of wheat peptide can significantly adjust the fasting blood glucose value ( p <0.05). In terms of insulin resistance, Fig.19 As shown in D, the insulin resistance index of the model control group mice was significantly higher than that of the normal group ( p <0.05), indicating that mice fed a high-fat diet had already developed significant insulin resistance. Compared with the model control group, wheat peptides at three intervention doses, high, medium and low, could significantly reduce the high-fat diet-induced insulin resistance index ( p <0.05), indicating that wheat peptides can significantly improve insulin resistance induced by a high-fat diet.

Claims

1. A bioactive peptide having a blood sugar lowering effect, characterized in that: The amino acid sequence of the bioactive peptide is Leu-Pro-Gln or Leu-Pro-Gln-Phe.

2. The method for preparing the bioactive peptide according to claim 1, characterized in that: The bioactive peptide is prepared by solid phase synthesis.

3. The use of the bioactive peptide according to claim 1 in the preparation of a drug for preventing or treating diabetes or a food for assisting in lowering blood sugar, characterized in that: The bioactive peptide has a blood sugar lowering effect.

4. The use according to claim 3, characterized in that The diabetes is type 2 diabetes.

5. A wheat peptide having a blood sugar lowering effect, characterized in that: The wheat peptide is rich in oligopeptides including the peptide segment LPQ, and the amino acid sequence of the peptide segment LPQ is Leu-Pro-Gln; The preparation method of the wheat peptide comprises: (1) Enzymatic hydrolysis: wheat gluten and water are mixed at a mass ratio of 1:15-30, the pH is adjusted to 8.0±0.2, and 0.5% of alkaline protease based on the total weight of the gluten is added for enzymatic hydrolysis for 15-30 min; then, shearing is performed at a speed of 10000-15000 r / min for 15-30 min; then, 0.5%-3.0% of alkaline protease based on the total weight of the gluten and 0.5%-2.0% of neutral protease based on the total weight of the gluten are added for further enzymatic hydrolysis for 4-6 h, and after the end, 0.1%-1.5% of flavor protease based on the total weight of the gluten is added for 15-30 min; (2) Separation: The enzymatic hydrolysate is centrifuged at a speed of 4000-6000 r / min, and the supernatant is taken. A strong acid cationic resin is added to the supernatant, and the pH is adjusted to 3.

0. The mixture is stirred for adsorption and then filtered. The filter cake is redissolved in a hydrochloric acid solution with a mass concentration of 3%-5% for desorption. The supernatant is nanofiltered, concentrated, sterilized, and dried to obtain the wheat peptide.

6. The wheat peptide according to claim 5, characterized in that The oligopeptide containing the peptide segment LPQ is at least one of the peptide segments having the amino acid sequences of LPQFEELLNR, LPQFEELR, LPQFAELR, LPQFEALR, LPQFEELNR, LPQFEF, and LPQQL.

7. Use of the wheat peptide according to claim 5 or 6 in the preparation of a drug for preventing or treating diabetes or a food for assisting in lowering blood sugar.

8. A pharmaceutical composition for preventing or treating diabetes, characterized in that: The pharmaceutical composition comprises an effective dose of a bioactive peptide with an amino acid sequence of Leu-Pro-Gln or Leu-Pro-Gln-Phe, or a wheat peptide rich in oligopeptides containing the peptide segment LPQ, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is in the form of an oral preparation.

10. A health food for assisting in lowering blood sugar, characterized in that: The invention comprises a bioactive peptide with an amino acid sequence of Leu-Pro-Gln or Leu-Pro-Gln-Phe as an active ingredient, or a wheat peptide rich in oligopeptides containing the peptide segment LPQ, and excipients acceptable in food science.

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