An active oligopeptide for maintaining healthy blood glucose level and its preparation method and application

Abstract

This invention relates to an active oligopeptide that maintains healthy blood glucose levels, its preparation method, and its application. The active oligopeptide is selected from any one or more combinations of active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3, whose amino acid sequences are shown in SEQ ID NO. 1-3, respectively. Compared with the prior art, active oligopeptide P1, active oligopeptide P2, active oligopeptide P3, and oat enzymatic hydrolysate containing active oligopeptides P1, P2, and P3 can significantly stimulate intestinal endocrine cells to secrete GLP-1 and lower blood glucose. Furthermore, they are safe, non-toxic, and easily absorbed by the body, thus more readily exerting their physiological regulatory functions.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an active oligopeptide that maintains healthy blood glucose levels, its preparation method, and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a complex metabolic disease, essentially caused by insulin secretion defects or insulin resistance, leading to an imbalance in glycemic homeostasis. Persistent hyperglycemia can trigger a variety of serious complications, such as cardiovascular disease, kidney damage, and neurological dysfunction. Therefore, finding effective strategies to maintain healthy glycemic levels has become a top priority in current research.

[0003] In the body's own glucose regulation system, glucagon-like peptide-1 (GLP-1), an incretin hormone secreted by intestinal L cells, plays a crucial role. It can efficiently and safely lower blood glucose by promoting insulin secretion, inhibiting glucagon release, delaying gastric emptying, and increasing satiety in a glucose concentration-dependent manner. However, endogenous GLP-1 has significant drawbacks: its half-life in vivo is only 2-3 minutes, and its degradation product, GLP-1(9-36), acts as a GLP-1 receptor antagonist, further hindering the function of active GLP-1. Furthermore, patients with type 2 diabetes mellitus (T2DM) generally exhibit a weakened incretin effect, with postprandial GLP-1 secretion reduced by 40-50% compared to healthy individuals, exacerbating insufficient insulin secretion and glycemic disturbances. Given the important role of GLP-1 and the challenges it faces, enhancing its activity through exogenous means has become an effective strategy for intervening in glucose metabolism.

[0004] Existing research has shown that GLP-1 secretion is regulated by dietary factors. Therefore, promoting intestinal GLP-1 secretion through diet has become a potentially important strategy for improving or alleviating type 2 diabetes mellitus (T2DM) and related chronic diseases. Food-derived peptides, with their small molecular weight, easy absorption, and high safety, show great potential in the functional food field. Therefore, developing bioactive peptides that can effectively promote GLP-1 secretion and have hypoglycemic functions has significant application value and development prospects for the prevention and adjunctive management of type 2 diabetes. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem that insufficient GLP-1 secretion exacerbates insufficient insulin secretion and blood glucose disorders, and to provide an active oligopeptide that can maintain healthy blood glucose levels, its preparation method, and its application.

[0006] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide an active oligopeptide that maintains healthy blood glucose levels, selected from any one of active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3. The amino acid sequence of the active oligopeptide P1 is shown in SEQ ID NO.1; The amino acid sequence of the active oligopeptide P2 is shown in SEQ ID NO.2; The amino acid sequence of the active oligopeptide P3 is shown in SEQ ID NO.3.

[0007] The second technical solution of the present invention is to provide a nucleic acid molecule, wherein the nucleic acid molecule is encoding any one or more of the active oligopeptides P1, P2, and P3 as described in one of the above technical solutions.

[0008] The third technical solution of the present invention is to provide an oat protein hydrolysate that maintains healthy blood sugar levels, wherein the oat protein hydrolysate contains any one or more of the active oligopeptides P1, P2, and P3 as described in one of the above technical solutions.

[0009] The fourth technical solution of the present invention is to provide a method for preparing oat protein hydrolysate as described in the third technical solution above, which includes obtaining oat protein hydrolysate by biological enzymatic hydrolysis.

[0010] In some specific embodiments, the bioenzyme is a complex flavor protease extracted by fermentation of Aspergillus oryzae. The mass ratio of biological enzyme to oat protein is 1:(10-100). The enzymatic hydrolysis conditions are: 37℃ for 1-4 hours.

[0011] In some specific embodiments, the oat protein is selected from any one or a combination of two of oat gliadin and oat 12S seed storage globulin 1.

[0012] The fifth technical solution of the present invention is to provide a method for preparing the active oligopeptide described in one of the above technical solutions, selected from any one of the following methods 1) to 3): 1) By enzymatically hydrolyzing oat protein, oat protein hydrolysate is obtained. The oat protein hydrolysate is then separated and purified to obtain active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3. 2) Artificially synthesize active oligopeptides P1, P2, and P3 using genetic engineering methods; 3) Active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3 were prepared by chemical synthesis.

[0013] In some specific implementations, method 1) specifically includes the following steps: S1. Extracting oat protein from oats; S2. The oat protein from step S1 is hydrolyzed using flavor enzymes to obtain oat protein hydrolysate. S3. Separate and purify the oat protein hydrolysate obtained in step S2 to obtain active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3.

[0014] In some specific embodiments, step S1, the method for extracting oat protein from oats, is as follows: Oat flour was ground, defatted, and mixed with water. Aminoglycosides and cellulase were added and pretreated at 45°C and pH 4.5 for 0.5-2 h. After adjusting the pH to 11, protein was extracted. The supernatant was collected by centrifugation. The pH of the supernatant was adjusted to 4.5, allowed to stand, centrifuged again, and washed with water. The oat protein was then dried.

[0015] In some specific embodiments, step S3, the method for separating and purifying the enzymatic hydrolysis products, is as follows: Oat protein hydrolysates were separated using a C18 reversed-phase column. Different fractions were collected by using deionized water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution and anhydrous ethanol as eluents. The effects of different fractions on GLP-1 secretion in enteroendocrine cells were detected, and the fraction with the highest activity in stimulating GLP-1 secretion was selected as the target fraction containing active oligopeptides. The target components were separated using a C18 reversed-phase column. A 0.1% formic acid aqueous solution was used as mobile phase A, and a 0.1% formic acid / 80% acetonitrile solution was used as mobile phase B. Gradient elution was used to obtain active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3.

[0016] In some specific embodiments, step S3, the method for separating and purifying the enzymatic hydrolysis products, is as follows: Oat protein hydrolysates were separated using a YMC ODS C18 column with gradient elution at a flow rate of 2 mL / min. The gradient elution program was as follows: 0–60 min, deionized water; 61–100 min, 10% ethanol aqueous solution; 101–160 min, 30% ethanol aqueous solution; 161–220 min, 50% ethanol aqueous solution; 221–280 min, 70% ethanol aqueous solution; 281–340 min, anhydrous ethanol. Different fractions were collected. The effects of different fractions on GLP-1 secretion in enteroendocrine cells were examined, and the fraction with the highest activity in stimulating GLP-1 secretion was selected as the target fraction containing active oligopeptides. The target components were separated using an Acclaim PepMap RPLC C18 reversed-phase chromatography column. A 0.1% formic acid aqueous solution was used as mobile phase A, and a 0.1% formic acid / 80% acetonitrile solution was used as mobile phase B. Gradient elution was performed at a flow rate of 600 nL / min. The gradient elution program was as follows: 0–2 min, 4–8% mobile phase B; 2–45 min, 8–40% mobile phase B; 45–55 min, 40–60% mobile phase B; 55–56 min, 60–95% mobile phase B; 56–66 min, 95% mobile phase B, yielding active oligopeptides P1, P2, and P3.

[0017] In some specific embodiments, method 2) is a method for artificially synthesizing active oligopeptides P1, P2, and P3 through genetic engineering. This method can be based on DNA recombination technology, using a suitable DNA template to control the sequence synthesis of the oligopeptides.

[0018] In some specific embodiments, the DNA template is selected from DNA encoding any one or a combination of two of oat gliadin and oat 12S seed storage globulin 1.

[0019] In some specific embodiments, the method of preparing active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3 by chemical synthesis in method 3) can be the traditional solid-phase synthesis method.

[0020] The sixth technical solution of the present invention is to provide the application of the active oligopeptide as described in one of the above technical solutions or the oat protein hydrolysate as described in the third of the above technical solutions in the preparation of functional products, wherein the functional products have at least one function of promoting GLP-1 secretion, improving insulin sensitivity, and improving and restoring blood lipid levels in serum and liver.

[0021] In some specific implementations, the functional products include food, health products, and pharmaceuticals.

[0022] In some specific implementations, the improvement in insulin sensitivity is manifested as an increase in restored insulin and an alleviation of the insulin resistance index.

[0023] In some specific embodiments, the improvement in blood lipid levels in serum and liver is manifested as a decrease in restored triglycerides, total cholesterol, and low-density lipoprotein, and an increase in restored high-density lipoprotein and liver glycogen.

[0024] Compared with the prior art, the present invention has the following advantages: (1) This invention provides oat-derived active oligopeptides P1, P2 and P3 that promote the secretion of intestinal GLP-1 and maintain healthy blood glucose levels. Active oligopeptides P1, P2 and P3 are all derived from natural oat protein, with well-defined sequences and novel structures, and have not been publicly reported to date.

[0025] (2) The active oligopeptides P1, P2 and P3 of the present invention can be prepared by enzymatic hydrolysis technology. The process is simple and the conditions are mild, making it easy to achieve industrial-scale production and possessing good conversion potential.

[0026] (3) The active oligopeptides P1, P2, P3 and the oat enzymatic hydrolysate containing active oligopeptides P1, P2, P3 can not only significantly stimulate intestinal endocrine cells to secrete GLP-1 and maintain healthy blood sugar levels, but also have the characteristics of being safe and free of toxic side effects, resistant to gastrointestinal digestive enzyme hydrolysis and easily absorbed by the body, thus making it easier to exert its physiological regulatory function.

[0027] (4) The active oligopeptides P1, P2, P3 and oat enzymatic hydrolysate containing active oligopeptides P1, P2, P3 of the present invention significantly increase serum GLP-1 levels, reduce blood glucose, enhance insulin sensitivity, and thus effectively regulate blood lipid metabolism and control weight, showing great potential in the development of functional foods and preparations to help maintain healthy blood glucose levels. Attached Figure Description

[0028] Figure 1 The effects of active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3 on the viability and GLP-1 secretion of STC1 cells were investigated.

[0029] Figure 2 This is a chromatogram of oat protein hydrolysate separated by a YMC ODS C18 column.

[0030] Figure 3 Cellular activity of each component isolated from oat protein hydrolysate (a), and the effect of oat protein hydrolysate and isolated components on GLP-1 secretion by STC-1 cells (b).

[0031] Figure 4 The intraperitoneal glucose tolerance and AUC values ​​of oat protein hydrolysates administered by gavage were determined.

[0032] Figure 5 The effect of oat protein hydrolysate on serum lipid levels.

[0033] Figure 6 The effects of oat protein hydrolysate on blood lipid levels and liver glycogen content in the liver.

[0034] Figure 7 The effect of oat protein hydrolysate on serum insulin levels and insulin resistance index.

[0035] Figure 8 The effect of oat protein hydrolysate on serum glucagon and glucagon-like peptide-1 levels. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0038] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0039] Example 1 This embodiment provides a chemical synthesis method for synthesizing active oligopeptides P1, P2, and P3. Specifically, the peptides were synthesized by Hefei Hesheng Biotechnology Co., Ltd. using a peptide solid-phase synthesis method. The purity of the synthesized peptides was verified to be greater than 95% by high-performance liquid chromatography and mass spectrometry.

[0040] The amino acid sequence of the active oligopeptide P1 is shown in SEQ ID NO.1: QLLQPQLQ; The amino acid sequence of the active oligopeptide P2 is shown in SEQ ID NO.2: LQAFEPLR; The amino acid sequence of the active oligopeptide P3 is shown in SEQ ID NO.3: RADTYNPR.

[0041] Example 2 The active oligopeptides P1, P2, and P3 synthesized in Example 1 were tested for their effects on STC 1 cell activity and GLP-1 secretion. 1. Tests on the effect on STC 1 cell viability (1) Culture of STC 1 cells STC 1 cells are an intestinal secretin tumor cell line. STC 1 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 100 U / mL penicillin, and 0.1 mg / mL streptomycin. Cells were incubated at 37°C in a cell culture incubator containing 5% CO2, and passaged by trypsin digestion when they reached 80-90% confluence.

[0042] (2) Cell viability assay Cell viability was determined using the CCK-8 colorimetric method. Cells were inoculated at 5 × 10⁶ cells / mL. 3 Cells / well were seeded at a density of 96 wells and cultured in a 5% CO2 incubator for 24 h, after which the culture medium was changed. 5 mg / mL of active peptide solution was added to the sample group, while an equal volume of D-Hank's solution was added to the control group. Six parallel wells were set up for each group. After culturing for another 2 h, 10 μL of CCK-8 reagent was added to each well, and the absorbance was measured at 450 nm using a microplate reader after 2 h of reaction. Cell viability was calculated using the following formula: (Experimental group OD / Control group OD × 100%).

[0043] The results are as follows Figure 1 As shown in Figure A, at the tested concentration (5 mmol / L), the viability of STC1 cells did not change significantly compared to the control group, indicating that the active oligopeptides P1 (QLLQPQLQ), P2 (LQAFEPLR), and P3 (RADTYNPR) were non-toxic to cells.

[0044] (3) Determination of hormone secretion content in STC1 cells Active oligopeptides P1 (QLLQPQLQ), P2 (LQAFEPLR), and P3 (RADTYNPR) were prepared into peptide solutions with molar concentrations of 0.2, 2, and 5 mM, respectively, using Hank's buffer. STC1 cells were cultured in 24-well plates at a concentration of 1.25 × 10⁻⁶ mM. 5 Cells were seeded at a density of [number] cells. When cells reached 80%-90% confluence, they were washed twice with Hank's buffer to remove the culture medium. Peptide solutions were added to STC1 cells, and the cells were incubated at 37°C for 2 hours. After incubation, the cells were centrifuged at 1000 g for 20 minutes, and the supernatant was collected. GLP-1 levels were determined using a GLP-1 assay kit from the Japan Immunobiology Institute. The total protein content of the supernatant measured using the Beyotime Biotechnology BCA kit was used as a reference to minimize batch-to-batch errors. The calculation formula is as follows: C GLP-1 / (C BCA ×100)(×10 7 ), where CGLP-1 The concentration of GLP-1 in the supernatant is expressed as pg / mL, C BCA This represents the total protein concentration measured in the supernatant minus the measured peptide concentration (mg / mL).

[0045] The effects of active oligopeptide P1 (QLLQPQLQ), active oligopeptide P2 (LQAFEPLR), and active oligopeptide P3 (RADTYNPR) on GLP-1 secretion in STC1 cells are shown in the table below. Figure 1 B. It can be seen that active oligopeptide P1 (QLLQPQLQ), active oligopeptide P2 (LQAFEPLR), and active oligopeptide P3 (RADTYNPR) can all significantly increase the secretion of GLP-1.

[0046] Example 3 This embodiment provides a method for preparing oat enzymatic hydrolysate by biological enzymatic hydrolysis.

[0047] Oat flour was milled and passed through an 80-sieve, then defatted with n-hexane. The defatted oat flour was soaked in distilled water at a mass ratio of 1:21 in a beaker, and the pH was adjusted to 4.5 with 1 mol / L HCl. The mixture was then treated with saccharifying enzyme and cellulase (both 1% of the defatted oat flour mass) at 45°C for 1 h. The pH was then adjusted to 11.0 with 1 mol / L NaOH, and the mixture was stirred with a magnetic stirrer for 2 h before centrifugation to collect the supernatant. The pH of the supernatant was adjusted to its isoelectric point (pH 4.5) with 1 mol / L HCl, allowed to stand for 1 h, and then centrifuged. The precipitate was washed with water until neutral, reconstituted with a small amount of distilled water, and then freeze-dried to obtain oat protein, which was stored at 4°C for later use.

[0048] 1 g of lyophilized protein powder was dissolved in 20 mL of an aqueous solution containing 40 mg of freshly prepared flavor enzyme (a compound flavor protease extracted from Aspergillus oryzae fermentation, Dongheng Huadao enzyme preparation). The pH of the solution was adjusted to 7.0, and the mixture was incubated at 37°C for 4 h. NaOH solution (0.5 mol / L) was added dropwise at intervals to adjust the pH to the optimal pH of 7.5. After incubation, the supernatant was collected by centrifugation and freeze-dried to obtain oat protein hydrolysate (named Fla12).

[0049] Oat protein hydrolysate was separated and purified using a YMC ODS C18 column. During separation, ultrapure water was used as the initial mobile phase, and the sample was loaded after equilibration to a baseline of 4 column volumes. The oat protein hydrolysate sample (100 mg / mL) was filtered through a 0.45 μm microporous membrane and centrifuged at 4℃ and 10,000 g for 5 min to completely remove particulate matter. Sample separation was performed using this C18 reversed-phase chromatography column at a constant flow rate of 2 mL / min. The gradient elution program was as follows: 0% ethanol solution for the initial 0-60 minutes, switching to 10% ethanol solution for 61-100 minutes, adjusting to 30% ethanol solution for 101-160 minutes, using 50% ethanol solution for 161-220 minutes, increasing to 70% ethanol solution for 221-280 minutes, and finally using 100% ethanol solution for elution from 281-340 minutes. The absorbance signal at 220 nm was monitored in real time using an HD-A chromatography system during elution. Separation chromatogram of oat protein hydrolysate is shown below Figure 2 As can be seen, the C18 column separates the protein hydrolysate into four peptide fractions (F1, F2, F3, and F4). The collected eluent is rapidly transferred to -80°C for pre-freezing, and then freeze-dried to remove the solvent, yielding a lyophilized powder.

[0050] The cytotoxicity and GLP-1 secretion-stimulating activity of each component (5 mg / mL) were evaluated using the STC-1 cell model system (refer to Example 2). It was found that among the four peptide components, the F1 component exhibited the best ability to stimulate GLP-1 secretion in STC-1 cells. This component was used for subsequent peptide identification, such as... Figure 3 As shown.

[0051] Example 4 In this embodiment, the F1 component prepared in Example 3 is further separated to obtain active oligopeptide P1 (QLLQPQLQ), active oligopeptide P2 (LQAFEPLR), and active oligopeptide P3 (RADTYNPR).

[0052] The sample was dissolved in distilled water to prepare a 1 mg / mL sample. Separation was performed using a reversed-phase column (150 μm id. × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid / 80% acetonitrile solution. Gradient elution was performed at a flow rate of 600 nL / min. The separation gradient was as follows: 0–2 min, 4–8% mobile phase B; 2–45 min, 8–40% mobile phase B; 45–55 min, 40–60% mobile phase B; 55–56 min, 60–95% mobile phase B; 56–66 min, 95% mobile phase B. The mass spectrometry ion source was an electrospray ionization (ESI) source, positive ion scan mode, ionization voltage 2200 V, and capillary temperature 270 °C. Level 1 mass spectrometry parameter settings: scan range 100-2000 m / z, maximum resolution 70000, automatic gain parameter 3000000. Level 2 mass spectrometry parameter settings: scan range 50-2000 m / z, maximum resolution 17500, automatic gain parameter 100000.

[0053] Mass spectrometry analysis revealed that the main ion peaks in fraction F1 were m / z = 416.68, z = 2; m / z = 484.28, z = 2; and m / z = 331.5, z = 3. Further secondary mass spectrometry analysis of the molecular ion peaks, matched to a database, identified the following peptides: Gln-Leu-Leu-Gln-Pro-Gln-Leu-Gln (QLLQPQLQ), representing active peptide P1; Leu-Gln-Ala-Phe-Glu-Pro-Leu-Arg (LQAFEPLR), representing active peptide P2; and Arg-Ala-Asp-Thr-Val-Asn-Pro-Arg (RADTYNPR), representing active peptide P3.

[0054] Example 5 Oat protein mainly consists of glutenin and prolamins. This embodiment also provides a specific source of oat protein: active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3. The bioactive peptide P1 can be derived from oat Avenin protein (Uniprot protein accession number: F4MJY2), positions 120-127 and 129-136.

[0055] The bioactive peptide P2 can be derived from oat 12S seed storage globulin 1 (Uniprot protein accession number: P12615), positions 50-57.

[0056] The bioactive peptide P3 can be derived from oat 12S seed storage globulin 1 (Uniprot protein accession number: P12615), positions 337-344.

[0057] The amino acid sequence of oat avenin protein (Uniprot protein accession number: F4MJY2) is shown in SEQ ID NO.4: LTLLAMAATM ATAQFDPSEQ YQPYPEQQQP FLQQQPLLQQ 40 QQQLLQVLQQ QLNPCRQFLV QQCSPVAEVP FLRSQILQQS 80 SCQVMKQQCC QQLAQIPEQV RCPAIHSVVQ AIILQKLQQ Q 120 LLQPQLQ Q QL LQPQLQ QQLL QAQLQQQLLQ AQLQQQLLQA 160 QLQQQLLQPQ VQQQLQQQLI QPQLQQVFIP PQLQQVFQPQ 200 QQAQFEGMRA FALQALPAMC DVYVP 225 The amino acid sequence of oat 12S seed storage globulin 1 (Uniprot protein accession number: P12615) is shown in SEQ ID NO.5: MHERAVWIFL CDLGGQNMPF EGSIPVEIGN LVNLFSLGME 40 GLRGCKFDR L QAFEPLR QVR SQAGITEYFD EQNEQFRCAG 80 VSVIRRVIE PQGLLLPQYH NAPGLVYILQ GRGFTGLTFPG 120 CPATFQQQFQ QFDQARFAQG QSKSQNLKDE HQRVHHIKQG 160 DVVALPAGIV HWCYNDGDAP IVAVYVFDVN NNANQLEPRQ 200 KEFLLAGNNK REQQFGQNIF SGFSVQLLSE ALGISQQAAQ 240 KIQSQNDQRG EIIRVSQGLQ FLKPFVSQQG PVEHQAYQPI 280 QSQQEQSTQY QVGQSPQYQE GQSTQYQSGQ SWDQSFNGLE 320 ENFCSLEARQ NIENPK RADT YNPR AGRITH LNSKNFPTLN 360 LVQMSATRVN LYQNAILSPY WNINAHSVMH MIQGRARVQV 400 VNNHGQTVFND ILRRGQLLII PQHYVVLKKA EREGCQYIS 440 FKTTPNSMVS YIAGKTSILR ALPVDVLANA YRISRQESQN 480 LKNNRGEEFG AFTPKFAQTG SQSYQDEGES SSTEKASE 518 Example 6 This embodiment aims to further determine the ability of oat protein hydrolysates to maintain healthy blood glucose levels.

[0058] 1. Mouse modeling: This embodiment uses C57BL / 6 mice to establish a type 2 diabetes mellitus (T2DM) model. After one week of acclimatization to a standard environment, the experimental animals were randomly divided into a control group (n=12) and a model group (n=36). The control group was fed a maintenance diet, while the model group was fed a high-fat diet. After 4 weeks of high-fat dietary intervention, the model group mice were intraperitoneally injected with streptozotocin (STZ, 110 mg / kg) while fasting, while the control group was injected with an equal volume of buffer solution. Fasting blood glucose was measured 48 hours later. Individuals with a blood glucose level ≥11.1 mmol / L were confirmed as successful models, and those that did not reach the target level were supplemented with streptozotocin until the target level was reached. After successful model establishment, all diabetic mice continued to maintain a high-fat diet, while the control group was always fed a standard diet.

[0059] 2. The procedure for grouping and processing experimental animals is as follows: Thirty-six T2DM model mice were randomly divided into three groups according to their fasting blood glucose levels: the model group (DM), the positive drug control group (Met), and the oat protein hydrolysate intervention group (OPH), with 12 mice in each group.

[0060] The control group and the DM group were given the same volume of normal saline by gavage daily; the Met group was given metformin (120 mg / kg bw) by gavage; and the OPH group was given oat protein hydrolysate (500 mg / kg bw) by gavage. During the experiment, mice had free access to food and water. Fasting blood glucose and body weight were monitored weekly, and the dosage was adjusted accordingly. After four weeks of continuous intervention, mice were fasted for 12 hours, and ocular blood was collected 30 minutes after the last administration. Immediately after euthanasia, the liver, pancreas, and intestines were dissected. Some tissues were flash-frozen in liquid nitrogen and stored at -80°C for molecular biological analysis, while other tissues were fixed in 10% formalin for pathological examination. All sample collection was performed on ice to ensure biological activity.

[0061] 3. Intraperitoneal glucose tolerance test: Animals were administered intraperitoneal glucose tolerance tests before grouping and 27 days after modeling. Fifteen minutes after intraperitoneal injection of 20% glucose solution (i.e., 500 mg / kg bw), different doses of oat protein hydrolysate samples (0.25 g / kg bw, 0.50 g / kg bw, and 1.0 g / kg bw) were administered to each group via gavage. Blood glucose changes before and after intraperitoneal injection (at -15, 0, 15, 30, 60, 90, and 120 min) were recorded via tail vein sampling.

[0062] The area under the blood glucose versus time curve (AUC) is calculated as follows: like Figure 4 As shown in A and 4B, in the short-term intraperitoneal glucose tolerance test, both medium-dose (0.5 g / kg) and high-dose (1.0 g / kg) oat protein hydrolysate intervention significantly reduced blood glucose levels in mice on a high-fat diet after 30 minutes, and restored blood glucose to normal levels at 120 minutes. There was no significant difference in hypoglycemic effect between the medium- and high-dose groups.

[0063] 4. Serum, liver biochemical indicators and organ index measurements The levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in mouse serum and liver were determined using a kit.

[0064] like Figure 5 and 6 As shown, diabetic mice in the DM model group exhibited typical dyslipidemia, characterized by significantly elevated levels of TC, TG, and LDL-C in serum and liver. After intervention with oat protein hydrolysate (OPH group), TC, TG, and LDL-C levels significantly decreased compared to the diabetic model group. Simultaneously, as... Figure 6 As shown in E, intervention with oat protein hydrolysate increased liver glycogen reserves in diabetic mice by 38.4%, effectively improving abnormal glucose metabolism.

[0065] These results indicate that oat protein hydrolysate has a dual effect of improving diabetes-related lipid metabolism disorders and enhancing liver glycogen reserves.

[0066] 5. Insulin and Insulin Resistance (HOMA-IR) Index Serum insulin levels were measured using a relevant ELISA kit, and the measurement method was performed according to the kit instructions.

[0067] like Figure 7 As shown in A and 7B, the serum insulin level in the DM model mice was significantly lower than that in the normal control group by 39.7%, and severe insulin resistance was observed. However, after 4 weeks of intervention with metformin (Met group) and oat protein hydrolysate (OPH group), the insulin levels in the two groups recovered to 48.2% and 40.3%, respectively, and the HOMA-IR index decreased significantly by 58% and 52% compared with the DM model group.

[0068] The above results indicate that oat protein hydrolysate can effectively improve insulin sensitivity and alleviate insulin resistance in diabetic mice.

[0069] 6. Glucagon and GLP-1 release Serum GLP-1 and glucagon levels were measured using relevant ELISA kits, and the measurement methods were performed according to the kit instructions.

[0070] like Figure 8 As shown in Figure A, glucagon levels in the DM model group were significantly elevated to 378.3 ± 49.5 pg / mL, while oat protein hydrolysate intervention (OPH group) significantly reduced them to 277.9 ± 23.0 pg / mL. Simultaneously, the OPH group exhibited the strongest GLP-1 secretion-promoting effect. Figure 8 B).

[0071] The above results indicate that oat protein hydrolysate exerts its glycemic regulatory effect through a dual pathway of inhibiting excessive glucagon secretion and promoting GLP-1 release.

[0072] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An active oligopeptide that maintains healthy blood glucose levels, characterized in that, The active oligopeptide is selected from any one of active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3. The amino acid sequence of the active oligopeptide P1 is shown in SEQ ID NO.1; The amino acid sequence of the active oligopeptide P2 is shown in SEQ ID NO.2; The amino acid sequence of the active oligopeptide P3 is shown in SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is a combination of one or more of the active oligopeptides P1, P2, and P3 as described in claim 1.

3. An oat protein hydrolysate that helps maintain healthy blood sugar levels, characterized in that, The oat protein hydrolysate is a combination of any one or more of the active oligopeptides P1, P2, and P3 as described in claim 1.

4. The method for preparing oat protein hydrolysate as described in claim 3, characterized in that, This includes obtaining oat protein hydrolysate by enzymatically hydrolyzing oat protein.

5. The preparation method according to claim 4, characterized in that, The bioenzyme is a complex flavor protease extracted by fermentation of Aspergillus oryzae. The mass ratio of biological enzyme to oat protein is 1:(10-100). The enzymatic hydrolysis conditions are: 37℃ for 1-4 hours.

6. The preparation method according to claim 4, characterized in that, The oat protein is selected from any one or a combination of two of oat gliadin and oat 12S seed storage globulin 1.

7. A method for preparing the active oligopeptide as described in claim 1, characterized in that, Choose any one of the following methods 1) to 3): 1) By enzymatically hydrolyzing oat protein, oat protein hydrolysate is obtained. The oat protein hydrolysate is then separated and purified to obtain active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3. 2) Artificially synthesize active oligopeptides P1, P2, and P3 using genetic engineering methods; 3) Active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3 were prepared by chemical synthesis.

8. The preparation method according to claim 7, characterized in that, Method 1) specifically includes the following steps: S1. Extracting oat protein from oats; S2. The oat protein from step S1 is hydrolyzed using flavor enzymes to obtain oat protein hydrolysate. S3. Separate and purify the oat protein hydrolysate obtained in step S2 to obtain active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3.

9. The preparation method according to claim 8, characterized in that, Step S1, the method for extracting oat protein from oats is as follows: Oat flour was ground, defatted, and mixed with water. Aminoglycosides and cellulase were added and pretreated at 45°C and pH 4.5 for 0.5-2 h. After adjusting the pH to 11, protein was extracted. The supernatant was collected by centrifugation. The pH of the supernatant was adjusted to 4.5, allowed to stand, centrifuged again, washed with water, and dried to obtain oat protein. In step S2, the bio-enzyme is a complex flavor protease extracted by fermentation of Aspergillus oryzae, and the mass ratio of the bio-enzyme to oat protein is 1:(10-100); the enzymatic hydrolysis conditions are: 37℃ for 1-4 h. Step S3, the method for separating and purifying the enzymatic hydrolysis products is as follows: Oat protein hydrolysates were separated using a C18 reversed-phase column. Different fractions were collected by using deionized water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, 50% ethanol aqueous solution, 70% ethanol aqueous solution and anhydrous ethanol as eluents. The effects of different fractions on GLP-1 secretion in enteroendocrine cells were detected, and the fraction with the highest activity in stimulating GLP-1 secretion was selected as the target fraction containing active oligopeptides. The target components were separated using a C18 reversed-phase column. A 0.1% formic acid aqueous solution was used as mobile phase A, and a 0.1% formic acid / 80% acetonitrile solution was used as mobile phase B. Gradient elution was used to obtain active oligopeptide P1, active oligopeptide P2, and active oligopeptide P3.

10. The application of the active oligopeptide as described in claim 1 or the oat protein hydrolysate as described in claim 3 in the preparation of functional products, wherein the functional products have at least one function of promoting GLP-1 secretion, improving insulin sensitivity, and improving and restoring blood lipid levels in serum and liver.

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