Preparation and application of whey protein source hypoglycemic peptide with GLP-1 receptor agonist activity

Abstract

The invention provides a hypoglycemic peptide which is prepared by taking bovine whey protein as a raw material and is stable in gastrointestinal digestion and absorption. In an in-vitro cell experiment, the peptide fragment can improve the uptake of insulin-resistant HepG2 hepatocytes and synthesize glycogen by using glucose, can promote intestinal endocrine cells NCI-H716 to secrete GLP-1, has a certain synergistic effect, and can further play the effects of promoting GLP-1 secretion and activating a GLP-1 receptor through combined use. An insulin resistance mouse model is constructed by utilizing high-fat diet, and the peptide fragment is adopted for intervention, so that the hypoglycemic peptide is further proved to have obvious effects on losing weight, reducing lipid accumulation and assisting in reducing blood fat, and meanwhile, the hypoglycemic peptide can be used for improving insulin resistance related symptoms. The milk peptide provided by the invention has gastrointestinal digestion stability and hypoglycemic activity, can be used as a functional milk base material for developing food, medicines or health care products with the effect of regulating blood sugar, and is wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the field of food and medicine, and in particular to the preparation and application of a whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by elevated blood glucose levels, primarily caused by impaired insulin metabolism or insufficient secretion. According to data released by the International Diabetes Federation (IDF), the number of people with diabetes worldwide reached 537 million in 2021, a number projected to reach 643 million in 2030 and 783 million in 2045. Type 2 diabetes is the most common type of diabetes, accounting for approximately 90% of all diabetics. Factors such as obesity, genetics, lifestyle, and aging contribute to the increasing incidence and prevalence of T2DM.

[0003] Glucagon-like peptide-1 (GLP-1) is a gut hormone produced and released by enteroendocrine L cells. GLP-1 stimulates insulin biosynthesis and secretion in a glucose-dependent manner, thereby increasing glucose sensitivity. Furthermore, GLP-1 enhances insulin secretion from pancreatic β-cells, inhibits glucagon secretion, attenuates gastric emptying, reduces appetite, and improves insulin resistance. These physiological effects make GLP-1 a promising target for the treatment of type 2 diabetes. However, the endogenous enzyme dipeptidyl peptidase-4 (DPP-IV) rapidly metabolizes GLP-1 by cleaving its N-terminal dipeptide residue, resulting in loss of GLP-1's insulinotropic activity. DPP-IV is a novel adipokine that is highly secreted during adipogenesis / obesity and is associated with the low-grade inflammation observed. It plays a crucial role in glucose and insulin metabolism and immune regulation. Therefore, DPP-IV inhibitors are a novel therapeutic agent for type 2 diabetes.

[0004] In addition, GLP-1 receptor agonists are a class of drugs used to treat type 2 diabetes by activating the GLP-1 receptor and exerting an incretin effect. GLP-1 receptor agonists have similar effects to GLP-1, and due to structural differences from natural GLP-1, their duration of action can be prolonged. GLP-1 receptor agonists promote insulin release primarily by activating the GLP-1 receptor. After binding to the GLP-1 receptor, GLP-1 receptor agonists increase calcium influx and endoplasmic reticulum calcium release through the cAMP / PKA pathway, activating calmodulin and ultimately enhancing insulin exocytosis. The use of GLP-1 receptor agonists is beneficial for patients with type 2 diabetes to achieve stable blood sugar reduction. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation and application of a whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] On the one hand, the present invention provides a blood sugar lowering peptide derived from bovine whey protein that is stable in gastrointestinal digestion and absorption, characterized in that the amino acid sequence of the peptide is shown in any one of SEQ ID NOs: 1-5. It has been verified that the obtained peptide segment has gastrointestinal digestion and absorption stability, DPP-IV inhibitory activity and GLP-1 receptor agonist activity in the body, can improve insulin resistance and lipid accumulation induced by a high-fat diet, and reduce body weight. The blood sugar lowering peptide segment can be used to reduce DPP-IV activity in samples, increase GLP-1 content, and activate GLP-1 receptors. The auxiliary blood sugar lowering and blood lipid lowering products prepared using the peptide segment can be used for the prevention, health care and auxiliary treatment of hyperglycemia, hyperlipidemia, obesity and other related diseases, and have broad application prospects in the fields of health care and medical biology.

[0008] On the other hand, the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes a peptide as shown in any one of SEQ ID NOs: 1-5, and the nucleic acid molecule is DNA or RNA.

[0009] On the other hand, the present invention provides an expression vector, characterized in that the nucleotide sequence of the expression vector is the above-mentioned nucleic acid molecule.

[0010] In another aspect, the present invention provides a recombinant cell, characterized in that the recombinant cell is obtained by introducing the above-mentioned expression vector into a host cell.

[0011] On the other hand, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a peptide as shown in any one of SEQ ID NOs: 1-5, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned recombinant cell.

[0012] In another aspect, the present invention provides a method for preparing a whey protein hydrolysate having hypoglycemic activity, characterized in that the method comprises the following steps:

[0013] S1. A composite enzymatic hydrolysis of bovine whey protein using alkaline protease and pepsin at a certain temperature to obtain a bovine whey protein hydrolysate;

[0014] S2. Simulated gastric digestion and intestinal digestion using bovine whey protein hydrolysate;

[0015] S3. Absorption was simulated using a monolayer of fully differentiated human colon adenocarcinoma cells Caco-2 in a Transwell plate. The bottom permeate was collected and freeze-dried to obtain a stable enzymatic hydrolysate of bovine whey protein for gastrointestinal digestion and absorption.

[0016] On the other hand, the present invention demonstrates at the cellular level that peptides such as those shown in any one of SEQ ID NOs: 1-5 can enhance the uptake and glycogen synthesis of insulin-resistant HepG2 hepatocytes, promote GLP-1 secretion in enteroendocrine cells NCI-H716, and that the combined use of multiple peptides can further promote GLP-1 secretion and activate the GLP-1 receptor. According to an embodiment of the present invention, the peptides having DPP-IV inhibitory activity and GLP-1 receptor agonist activity have the above-mentioned amino acid sequence or an amino acid sequence of a conservatively modified form thereof.

[0017] On the other hand, the present invention demonstrates in vivo that the peptide segments shown in any one of SEQ ID NOs: 1-3 can reduce the body weight and blood lipid levels of mice fed a high-fat diet, activate the insulin signaling pathway to improve insulin resistance, increase serum GLP-1 levels, reduce lipid accumulation in white adipose tissue, and regulate intestinal flora. According to an embodiment of the present invention, the peptides with DPP-IV inhibitory activity and GLP-1 receptor agonist activity have the above-mentioned amino acid sequence or an amino acid sequence of a conservatively modified form thereof.

[0018] On the other hand, the present invention provides the use of the above-mentioned peptide, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned recombinant cell, or the above-mentioned pharmaceutical composition, or the above-mentioned method in preparing a drug for preventing and / or treating diabetes, hyperlipidemia or obesity.

[0019] On the other hand, the present invention provides the use of the above-mentioned peptide, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned recombinant cell, or the above-mentioned pharmaceutical composition, or the above-mentioned method in preparing a health care product or functional food that assists in improving insulin resistance or lowering blood lipid levels or losing weight.

[0020] The beneficial effects of the present invention compared to the prior art are:

[0021] 1. The present invention obtains whey protein hypoglycemic peptides with stable gastrointestinal digestion and absorption through enzymatic hydrolysis, simulated gastrointestinal digestion, and Caco-2 cell absorption. The whey protein peptide sequence is identified by LC-MS / MS, and the obtained multiple peptide fragments have gastrointestinal digestion stability, DPP-IV inhibitory activity, and glucagon-like peptide-1 (GLP-1) receptor agonist activity.

[0022] 2. The glucose-lowering peptide provided by the present invention has good effects in in vitro cell experiments. The peptide segment can increase the uptake of insulin-resistant HepG2 liver cells and utilize glucose to synthesize glycogen, and can promote the secretion of GLP-1 by enteroendocrine cells NCI-H716. The combined use of multiple peptide segments can further promote GLP-1 secretion and activate GLP-1 receptors.

[0023] 3. The glucose-lowering peptide provided by the present invention also has excellent effects in the insulin resistance mouse model constructed by high-fat diet. The peptide segment has obvious effects on weight loss, reducing lipid accumulation and assisting in lowering blood lipids, and can also improve symptoms related to insulin resistance.

[0024] 4. The milk-based hypoglycemic peptide provided by the present invention has gastrointestinal digestion stability and blood sugar-lowering activity, and can be used as a functional milk-based material to develop foods, medicines or health products with blood sugar-regulating effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The in vitro DPP-IV inhibition rate of the peptides is shown.

[0026] Figure 2 The effect of the peptide on glucose utilization in insulin-resistant HepG2 cells is shown.

[0027] Figure 3 Shown is the effect of the peptide on the relative glycogen content in insulin-resistant HepG2 cells.

[0028] Figure 4 Shown are the effects of the peptides on GLP-1 secretion in NCI-H716 cells.

[0029] Figure 5 Shown are the effects of the peptides on cAMP in NCI-H716 cells.

[0030] Figure 6 The results show that the peptides can exert synergistic effects on GLP-1 secretion in NCI-H716 cells.

[0031] Figure 7 The mass spectrum of the peptide LPMHIR is shown.

[0032] Figure 8 Shown is the mass spectrum of the peptide KFDK.

[0033] Figure 9 Shown is the mass spectrum of the peptide IPAVFKID.

[0034] Figure 10 This figure shows the effects of peptides on glucose tolerance in mice with insulin resistance induced by a high-fat diet. A shows blood glucose monitoring results at different times after oral gavage; B shows the effects of different peptides on the area under the curve during the OGTT test.

[0035] Figure 11 This figure shows the effects of peptides on insulin tolerance in mice induced by a high-fat diet. A shows blood glucose monitoring results at different times after intraperitoneal insulin injection; B shows the effects of different peptides on the area under the blood glucose curve in mice.

[0036] Figure 12 The effect of the peptide fragment on DPP-IV enzyme activity in the serum of mice with insulin resistance induced by a high-fat diet was shown.

[0037] Figure 13 The effect of the peptide on the serum GLP-1 content in high-fat diet-induced insulin-resistant mice is shown.

[0038] Figure 14 The effects of the peptide on GLP-1R mRNA expression in the liver of high-fat diet-induced insulin-resistant mice are shown.

[0039] Figure 15 Shown is the effect of the peptide on body weight in mice fed a high-fat diet.

[0040] Figure 16 The effects of the peptides on hepatic lipid accumulation in high-fat diet-induced insulin-resistant mice are shown.

[0041] Figure 17 The effects of peptides on the diversity of gut microbiota in mice with insulin resistance induced by a high-fat diet are shown. A shows the effect of different peptides on the Ace index of gut microbiota; B shows the effect of different peptides on the Chao index of gut microbiota; and C shows the effect of different peptides on the Shannon index of gut microbiota.

[0042] Figure 18 The effects of the peptides on the composition of the intestinal microbiota in mice with insulin resistance induced by a high-fat diet are shown. DETAILED DESCRIPTION

[0043] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] The present invention will be described in detail below through embodiments in conjunction with the accompanying drawings, but this does not limit the present invention and is only used as an example.

[0047] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Therefore, features specified as "first" or "second" may explicitly or implicitly include one or more of such features.

[0048] The quality inspection index data in the examples are all average values.

[0049] As used herein, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the biological activity of a polypeptide comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the xanthine oxidase inhibitory peptides of the present invention using standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

[0050] Example 1. Preparation of Gastrointestinal Digestion-Stable Hypoglycemic Peptides

[0051] In this example, a bovine whey protein hydrolysate having DPP-IV inhibitory activity was prepared by the following method:

[0052] (1) Enzymatic hydrolysis: bovine whey protein was dissolved in distilled water at a ratio of 10%, and the bovine whey protein was subjected to a composite enzymatic hydrolysis at 37°C using alkaline protease and pepsin (alkaline protease: pepsin = 1:1) at a ratio of enzyme to substrate of 5% (w / w); samples were taken after enzymatic hydrolysis for 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, and the enzymes were inactivated by heating at 85°C for 20 min; the pH value of the enzymatic hydrolyzate was adjusted to 7.0, and the composite enzymatic hydrolyzate (WPCH) was obtained by freeze-drying.

[0053] (2) Determine the degree of hydrolysis and DPP-IV inhibition rate of the hydrolysates obtained at different hydrolysis times. The study found that the bovine whey protein hydrolysates under different hydrolysis time conditions all had DPP-IV inhibitory activity. As the hydrolysis time increased, the degree of hydrolysis increased and the DPP-IV inhibition rate gradually increased. When the hydrolysis time reached 4 hours, the degree of hydrolysis and DPP-IV inhibition rate were not significantly different from those of the hydrolysate obtained at a hydrolysis time of 5 hours. On the other hand, the camel milk casein protein hydrolysate hydrolyzed for 4 hours had the greatest DPP-IV inhibitory activity, which can be used for further experiments.

[0054] (3) Simulated gastrointestinal digestion: The composite enzymatic hydrolysate was dissolved in distilled water at a ratio of 5% and incubated in an 85°C water bath for 15 min. Preheat simulated gastric fluid at 37°C and add the simulated gastric fluid to the sample solution to a final ratio of 1:1 (vol / vol). Add 1M HCl to adjust the pH to 3.0. Add pepsin to a final activity of 2000 U / mL in the digestion mixture. Maintain the pH at 3.0 and stir in a 37°C water bath for 2 h. Preheat simulated intestinal fluid in a 37°C water bath. Add simulated intestinal fluid to the simulated gastric digestion product to a final ratio of 1:1 (vol / vol). Add 1M NaOH to adjust the pH to 7.0. Add bile salts to a final concentration of 10 mM. Place the solution in a 37°C water bath and stir for at least 30 min to completely dissolve the bile salts. Add CaCl2(H2O)2 solution to a final concentration of 0.6 mM in the simulated intestinal fluid. Pancreatin was added to a final mixture with a pancreatic enzyme activity of 100 U / mL. The pH was maintained at 7.0 and the mixture was stirred in a 37°C water bath for 2 h. The enzyme was inactivated in a boiling water bath for 5 min and freeze-dried to obtain a bovine whey protein simulated gastrointestinal digest (GD-WPCH).

[0055] (4) Caco-2 cell absorption: The fully differentiated Caco-2 cell monolayer was washed twice with preheated Hank's balanced salt solution (HBSS) (pH 7.2). 1.5 mL of HBSS was added to the upper chamber of the Transwell plate, and 2 mL of HBSS was added to the bottom chamber, and the plates were equilibrated at 37°C for 30 min. Subsequently, the HBSS in the upper chamber was replaced with bovine whey protein simulated gastrointestinal digest (concentration of 25 mg / mL, dissolved in HBSS buffer). After incubation at 37°C for 2 h, the bottom permeate was collected and freeze-dried to obtain bovine whey protein gastrointestinal digestion and absorption stable enzymatic hydrolysate (CA-WPCH).

[0056] (5) Peptide Identification: Bovine whey protein gastrointestinal digestion and absorption stable enzymatic hydrolysate (CA-WPCH) was dissolved in 0.1% formic acid solution. The high performance liquid chromatography system was connected to a mass spectrometer equipped with an electrospray ionization source. The first order spectral detection scanning range was 50-1500 m / z, the scanning resolution was 60000, and the second order spectral scanning range was 50-1400 m / z, the scanning resolution was 15000. The mobile phase A was 0.1% (v / v) formic acid, and the mobile phase B was 80% acetonitrile (containing 0.1% formic acid). During the elution process, solvent B was increased from 8% to 50% (v / v) at a flow rate of 200 nL / min.

[0057] (6) Peptide screening: Molecular docking of peptides with DPP-IV enzyme and GLP-1 receptor was performed. The crystal structures of DPP-IV (PDB code: 4PNZ) and GLP-1 receptor (PDB code: 6X18) were obtained from the RCSB protein database; the peptide structures were generated in ChemDraw 20.0 and converted into stereostructures in Chem3D 20.0; the DPP-IV and GLP-1 receptors were deliganded and hydrogenated in Discovery Studio, and their active sites were determined; the interactions between DPP-IV, GLP-1 receptor, and peptides were determined using the LibDock module.

[0058] Five polypeptides were screened from the identified peptides (Table 1), namely LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), EVFR (SEQ ID NO: 4), and ILDKVGINY (SEQ ID NO: 5).

[0059] Table 1 Amino acid sequence obtained by identifying the bovine whey protein hydrolysate in Example 1

[0060]

[0061] *α-La: bovine α-lactalbumin; β-Lg: bovine β-lactoglobulin.

[0062] The active peptides provided herein can be derived from a complex enzymatic hydrolysate of bovine whey protein or obtained through solid-phase synthesis. The peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), EVFR (SEQ ID NO: 4), and ILDKVGINY (SEQ ID NO: 5) used in the following examples were obtained through solid-phase synthesis. Shanghai Nuoyou Biotechnology Co., Ltd. was commissioned to perform the solid-phase synthesis of these hypoglycemic peptides for subsequent experiments.

[0063] Example 2. Determination of DPP-IV inhibitory activity of peptide fragments

[0064] To a 96-well plate, 100 μL of Gly-Pro-PNA HCl solution (1 mM), 50 μL of Tris-HCl buffer (100 mM, pH 8.0), and 20 μL of sample (10 mg / mL) were added and incubated at 37°C for 10 min. Then, 30 μL of DPP-IV solution in Tris-HCl (0.02 μg / μL) was added for a 30-min enzyme reaction. The absorbance at 405 nm was measured in triplicate for each sample. The DPP-IV inhibition rate was calculated according to the following formula. The sample control group used the same volume of Tris-HCl buffer (100 mM, pH 8.0) instead of DPP-IV. The negative control group used the same volume of Tris-HCl buffer (100 mM, pH 8.0) instead of sample. The blank control group used the same volume of Tris-HCl buffer (100 mM, pH 8.0) instead of sample and DPP-IV. The DPP-IV inhibition rate was calculated using the following formula.

[0065]

[0066] DPP-IV, also known as CD26, is a transmembrane serine protease and a member of the prolyl oligopeptidase family. It can specifically cleave the N-terminal dipeptide residue of GLP-1: AA-Pro or AA-Ala (AA is any amino acid), and is one of the key enzymes that promote the degradation and inactivation of GLP-1 in vivo and in vitro. DPP-IV is widely distributed in the body, not only in plasma, but also in tissues and organs such as the kidneys, small intestine, bile ducts and pancreatic epithelial cells, and endothelial cells of blood vessels. Therefore, selective inhibition of DPP-4 can increase the concentration of GLP-1 in the body and prolong its duration of action. It can also inhibit the production of glucagon and prolong the duration of GLP-1 stimulation of insulin secretion. Therefore, the DPP-IV enzyme plays a vital role in glucose and insulin metabolism. The DPP-IV inhibition rate of peptides is as follows: Figure 1 As shown in Table 2, in the positive control group using sitagliptin, the peptides LPMHIR (LR), KFDK (KK), and IPAVFKID (ID) all exhibited significant DPP-IV inhibitory activity (>70%). Among them, at the same concentration, the peptide KFDK exhibited the greatest DPP-IV inhibition rate, reaching 78.28±4.29%. Furthermore, the peptides EVFR (ER) and ILDKVGINY (IY) exhibited in vitro DPP-IV inhibition rates <50%.

[0067] Table 2 DPP-Ⅳ inhibition rate of peptides

[0068]

[0069]

[0070] half maximal inhibitory concentration (IC 50 ) refers to the concentration at which a substance inhibits another substance, such as an enzyme, by 50%, and is used to measure sensitivity. There are many studies on milk-derived DPP-IV inhibitory peptides, and the IC of the peptide VLGP derived from bovine β-casein is 50 =580.0 μM, IC of bovine β-lactoglobulin VLVLDTDYK 50 =424.0 μM, IC of the peptide WLAHKAL derived from bovine α-lactalbumin 50 =286.0μM. IC 50 The smaller the value, the stronger the inhibitory effect of the peptide on DPP-Ⅳ. 50 The results of the value determination are shown in Table 3.

[0071] Table 3 Half inhibitory concentration of glucose-lowering peptides

[0072]

[0073] Example 3. Effect of peptides on improving glucose utilization in insulin-resistant HepG2 cells

[0074] By constructing insulin-resistant HepG2 cells, the effects of the peptide on glucose utilization and glycogen synthesis in insulin-resistant liver cells were evaluated.

[0075] Construction of a HepG2 cell model of insulin resistance: HepG2 hepatocytes were seeded in a 96-well plate and allowed to adhere for 24 hours. A 600mM glucose PBS solution was prepared and filtered through a 0.22μm aqueous filter. 10μL was aspirated to achieve a glucose concentration of 30mM in the wells. A blank control group was supplemented with 10μL of PBS. The cells were incubated at 37°C, 5% CO2 for 24 hours, and the glucose consumption rate and relative glycogen content were measured. The results are shown in Figure 2. Figure 2 As shown, the glucose consumption rate and relative glycogen content of HepG2 cells stimulated by high glucose were significantly lower than those of normal HepG2 cells, and the insulin-resistant HepG2 cell model was successfully constructed.

[0076] Effects on Glucose Consumption Rate and Relative Glycogen Content: HepG2 hepatocytes were seeded in 96-well plates. After 24 hours of adherence, 10 μL of glucose (final concentration 30 mM) was added, along with 10 μL of a 100 μM glucose-lowering peptide solution in PBS. The glucose and sample solutions were replaced with the same volume of PBS in blank wells, and the sample solution was replaced with the same volume of PBS in standard wells. After 24 hours of incubation, the culture medium was aspirated and assayed using a glucose oxidase assay kit (Nanjing Jiancheng Bioengineering Institute, A154-1-1). Cells were seeded into 6-cm culture dishes and allowed to adhere for 12 hours before stimulation with 30 mM glucose for 24 hours. Cells were digested with trypsin to prepare a cell suspension, centrifuged at 1000 rpm for 10 minutes, and the supernatant discarded, retaining the cell pellet. 1 mL of PBS buffer was added to the cell pellet, gently mixed, and centrifuged at 1000 rpm for 10 minutes. The supernatant discarded, retaining the cell pellet. 0.2 mL of PBS buffer was added, and the cells were sonicated in an ice-water bath without centrifugation for direct measurement. The glycogen content of cells was determined using a glycogen kit.

[0077] The glucose consumption rate and relative glycogen content in cells of each group were as follows Figure 2 and Figure 3 As shown in the results, compared with the control group, the intervention of peptides LPMHIR (LR), KFDK (KK), and IPAVFKID (ID) significantly increased the glucose consumption rate and intracellular glycogen content of insulin-resistant HepG2 cells, indicating that the peptides improved the glucose uptake and utilization ability of insulin-resistant liver cells, and to a certain extent played a role in lowering blood sugar and improving insulin resistance. The peptides EVFR (ER) and ILDKVGINY (IY) had no obvious effect on improving the glucose uptake and utilization ability of insulin-resistant liver cells.

[0078] Example 4. Effect of peptide fragments on GLP-1 secretion by enteroendocrine cells

[0079] Using enteroendocrine cells NCI-H716 cells as a model, we studied the promoting effect of peptides on the secretion of GLP-1 by enteroendocrine cells and whether there is a synergistic effect between peptides in promoting GLP-1 secretion.

[0080] Stably growing NCI-H716 cells were cultured at a rate of 1.5×10 6 The cells were seeded at a density of 100 μg / mL in a 12-well culture plate coated with matrix gel and cultured for 48 hours. NCI-H716 cells were incubated with the five peptides for 24 hours. Sitagliptin was used as the positive control group. The GLP-1 content in the cell supernatant was detected using an enzyme-linked immunosorbent assay kit. NCI-H716 is a human colorectal adenocarcinoma cell that can secrete hormones such as GLP-1. This study was used to study the effect of active peptide intervention on the secretion of GLP-1 by intestinal epithelial cells. Figure 4As shown, with the control group as the benchmark, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) all promoted the secretion of GLP-1 by NCI-H716, indicating that glucose-lowering peptides can promote the secretion of GLP-1 at the cellular level, and exert the effects of lowering blood sugar and improving insulin resistance through GLP-1. The synthesized and secreted GLP-1 exerts its beneficial effects by binding to the GLP-1 receptor on the cell surface. After binding to the GLP-1 receptor, the GLP-1 receptor agonist increases calcium influx and endoplasmic reticulum calcium ion release through the cAMP / PKA pathway to activate calmodulin and promote insulin release. In order to further determine whether the peptide has the activity of a GLP-1 receptor agonist, the cAMP level in the culture medium supernatant was measured. As shown Figure 5 As shown, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), and EVFR (SEQ ID NO: 4) all increased the cAMP level to a certain extent, indicating that the glucose-lowering peptides can activate the GLP-1 receptor by increasing the cAMP level.

[0081] Combined with Examples 2 and 3, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) have good GLP-1 receptor agonist activity. On this basis, further study was conducted to determine whether there is a synergistic effect between the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) in promoting GLP-1 secretion. Figure 6 As shown, simultaneous peptide combination interventions resulted in higher GLP-1 secretion levels in the cell culture medium than either peptide alone. The combination of LPMHIR and KFDK, and the combination of KFDK and IPAVFKID, resulted in GLP-1 secretion effects greater than the sum of the effects of either peptide alone, indicating that KFDK exhibits a synergistic effect with LPMHIR and IPAVFKID, respectively, and that their combined use can enhance their ability to promote GLP-1 secretion and activate the GLP-1 receptor. The peptides LPMHI and IPAVFKID did not exhibit significant synergistic effects. These results suggest that the combined use of KFDK and LPMHIR, or KFDK and IPAVFKID, can further enhance the glucose-lowering peptide's ability to promote GLP-1 secretion and activate the GLP-1 receptor.

[0082] Example 5. Effect of peptides on improving insulin resistance in mice fed a high-fat diet

[0083] According to Examples 2, 3, and 4, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) have good hypoglycemic effects and can simultaneously play the dual roles of DPP-IV inhibitors and GLP-1 agonists in vitro. Whey protein source to lower blood sugar The peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) were used in animal experiments to study their hypoglycemic effects in vivo. The mass spectra of the three peptides are shown in Figure 2. Figure 7-9 shown.

[0084] Male C57 / 6J mice, 3-4 weeks old, were purchased from Beijing Weitonglihua Experimental Technology Co., Ltd. and housed according to SPF animal husbandry standards. Animal experiments were reviewed and approved by the Animal Experimentation Committee of China Agricultural University. Mice had free access to food and water and were acclimated for one week in an environment with a 12:12 day / night cycle at 22±1°C. After one week of acclimation, the mice were randomly divided into a normal diet (ND) group (n=6) and a high-fat diet (HFD) group (n=30). The ND group was fed a normal diet, while the HFD group was fed a high-fat diet (energy supply ratio: 20% protein, 20% carbohydrates, 60% fat) for 10 weeks to establish high-fat diet-induced insulin resistance in mice. After 10 weeks, fasting blood glucose and fasting insulin levels were measured, and the insulin resistance index (HOMA-IR) was calculated. The insulin resistance index of mice in the HFD group was greater than 2.69, indicating that the high-fat diet-induced insulin resistance model was successfully established. Mice fed a high-fat diet were randomly divided into a HFD group, a LPMHIR group (LR), a KFDK group (KK group), an IPAVFKID group (ID group), and a sitagliptin group (Sitagliptin group), with six mice in each group. The mice were given the corresponding intervention substances for ten weeks, during which time their food intake and body weight were monitored weekly.

[0085] After nine weeks of intervention, the mice were fasted but not watered for 8 h and gavaged with glucose at a dose of 2 g / kg BW. Blood was collected from the tip of the tail of the mice at 0, 30, 60, 90, and 120 min after gavage. The blood glucose levels were measured with a glucometer, and the curve was drawn to calculate the area under the curve (AUC).

[0086] like Figure 10As shown, after oral administration of glucose, the blood glucose levels of mice rose, then decreased, and essentially leveled off after 90 minutes. Compared with the ND group, the blood glucose levels of mice in the HFD group remained consistently higher. The area under the curve (AUC) during the OGTT test was 1.39 times that of the ND group, significantly higher than that of the control group. This indicates that the high-fat diet impairs glucose tolerance in mice and reduces their ability to regulate blood glucose. Intervention with glucose-lowering peptides and sitagliptin improved the glucose regulation ability of HFD mice and significantly improved the high-fat diet-induced glucose tolerance impairment. Sitagliptin treatment reduced the AUC of HFD mice by 27.2%. Three glucose-lowering peptides, LPMHIR (SEQ ID NO:1), KFDK (SEQ ID NO:2), and IPAVFKID (SEQ ID NO:3), reduced the AUC of HFD mice by 18.8%, 23.0%, and 21.3%, respectively. The above results indicate that glucose-lowering peptides can improve impaired glucose tolerance induced by a high-fat diet and enhance the blood glucose regulation ability of mice fed a high-fat diet.

[0087] Mice were fasted for 6 h but not water, and insulin was injected intraperitoneally at a dose of 0.75 U / kg BW. Blood was collected from the tip of the tail at 0, 30, 60, 90, and 120 min after injection. Blood glucose levels were measured with a glucometer, and the curve was drawn to calculate the area under the curve (AUC).

[0088] like Figure 11 As shown, compared with the ND group, fasting blood glucose levels in mice in the HFD group were significantly elevated. Furthermore, after insulin injection, blood glucose levels decreased more slowly and by a smaller amount. The area under the curve (AUC) was 1.63 times that of the ND group, significantly higher than that of the control group. This indicates that a high-fat diet leads to elevated fasting blood glucose and impaired insulin tolerance in mice. Intervention with glucose-lowering peptides and sitagliptin increased the rate of blood glucose reduction in HFD mice, while also increasing the magnitude of the reduction, with a significant decrease in the AUC. Sitagliptin treatment decreased the AUC by 42.3% in HFD mice. Three glucose-lowering peptides, LPMHIR (SEQ ID NO:1), KFDK (SEQ ID NO:2), and IPAVFKID (SEQ ID NO:3), improved high-fat diet-induced impaired glucose tolerance, decreasing the AUC by 26.1%, 33.3%, and 30.8%, respectively, in HFD mice. This suggests that glucose-lowering peptides improve insulin tolerance and alleviate insulin resistance in HFD mice.

[0089] After ten weeks of intervention, the mice were killed, serum was collected, and the DPP-IV enzyme activity in the serum was measured to explore the effect of the application example of the present invention on the DPP-IV enzyme activity in the serum of mice with insulin resistance induced by a high-fat diet. Figure 12Compared with the ND group, the DPP-IV enzyme activity in the serum of HFD mice was significantly increased. Intervention with different glucose-lowering peptides reduced DPP-IV activity in the serum of HFD mice, indicating that glucose-lowering peptide intervention can inhibit DPP-IV activity in mice fed a high-fat diet, thereby inhibiting the degradation and inactivation of GLP-1.

[0090] GLP-1 activity in serum was measured to explore the effect of the application examples of the present invention on the GLP-1 content in the serum of mice with insulin resistance induced by a high-fat diet. GLP-1 not only stimulates insulin secretion, but also has a protective effect on pancreatic β cells and the cardiovascular system. It can inhibit β cell apoptosis, promote β cell growth and proliferation, upregulate insulin secretion, lower blood sugar, and is associated with reducing food intake, promoting gastric emptying and weight loss. It plays an important role in alleviating diabetes. As shown in Figure 13, the GLP-1 content in the serum of HFD mice was significantly reduced compared to the ND group, indicating that in mice fed a high-fat diet, GLP-1 has a lower effect on improving insulin resistance. The intervention of different glucose-lowering peptides significantly increased the GLP-1 content in the serum of mice fed a high-fat diet. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) increased the GLP-1 content in the serum of HFD mice by 46.4%, 57.8%, and 61.8%, respectively, indicating that the glucose-lowering peptides in this application example are beneficial for increasing the GLP-1 content in serum and exerting the effect of GLP-1 on improving insulin resistance. On the other hand, GLP-1 exerts its beneficial effects by binding to GLP-1 receptors on the surface of different tissue cells. In order to study the effect of glucose-lowering peptides on the expression of GLP-1R in the liver of mice with insulin resistance induced by a high-fat diet, the expression of GLP-1R in the liver of mice was detected by PCR, as shown in Figure 3. Figure 14 Compared with the ND group, the HFD diet significantly reduced GLP-1R mRNA expression, indicating that while the high-fat diet induced a decrease in serum GLP-1 levels, it also reduced GLP-1R expression, hindering the binding of GLP-1 to tissue cell receptors and thus improving blood sugar. Intervention with the glucose-lowering peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) increased GLP-1R mRNA expression in the liver tissue of HFD mice and activated the GLP-1 receptor.

[0091] During the intervention period, the weight changes of mice were monitored to explore the improvement effect of the application example of the present invention on high-fat diet-induced obesity. Figure 15As shown in Figure 3, compared with the ND group, the body weight of mice in the HFD group increased significantly, reaching 1.52 times that of the ND group. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) significantly reduced high-fat diet-induced obesity and reduced the body weight of mice.

[0092] Mouse epididymal fat was taken for H&E staining to explore the effect of the application example of the present invention on lipid accumulation induced by high-fat diet. Figure 16 As shown, compared with the ND group, the cell diameter of epididymal fat in HFD mice increased and the fat index increased, indicating that the high-fat diet induced lipid accumulation in mice and increased white adipocytes. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) significantly reduced the diameter of white fat, indicating that the peptides have an improvement effect on reducing lipid accumulation.

[0093] Total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in mouse serum were measured to characterize the mouse lipid levels. As shown in Table 4, high-fat diet intervention significantly increased TC, TG, and LDL-c in mice, inducing hyperlipidemia. Administering LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) intervention reduced TC, TG, and LDL-c levels in the serum of HFD mice, while increasing HDL-c levels. This indicates that peptide intervention significantly improved the lipid levels of mice fed a high-fat diet, improved hyperlipidemia, and had an auxiliary lipid-lowering effect.

[0094] Table 4 Effects of hypoglycemic peptide intervention on blood lipid levels in HFD mice

[0095]

[0096]

[0097] The fasting insulin and glycated hemoglobin of mice were measured to explore the effects of the application examples of the present invention on the indicators in the serum of mice with insulin resistance induced by a high-fat diet. Glycated hemoglobin (HbA1c) is the product of the combination of hemoglobin in red blood cells and glucose in the blood. It can directly and effectively reflect the patient's blood sugar level in the past 3-6 months, and is positively correlated with the level of insulin resistance, directly correlated with cholesterol, triglycerides and low-density lipoprotein cholesterol, and negatively correlated with high-density lipoprotein cholesterol. As shown in Table 5, compared with the ND group, the glycated hemoglobin level of HFD mice was significantly increased, indicating that the high-fat diet induced diabetes and insulin resistance in mice. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) significantly reduced the glycated hemoglobin level of mice on a high-fat diet.

[0098] Table 5 Effects of hypoglycemic peptide intervention on serum parameters of HFD mice

[0099]

[0100] Alpha diversity analysis reflects the diversity and differences of microbial communities. The most commonly used indices include Ace index, Chao index and Shannon index. The intestinal flora of mice was measured to explore the effect of the application example of the present invention on the diversity of intestinal flora of mice with insulin resistance induced by high-fat diet. Figure 17 As shown in the figure, compared with the ND group, the Ace index and Chao index of HFD mice were significantly decreased, indicating that HFD significantly reduced the richness of intestinal microorganisms in mice. Intervention with peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) increased the diversity of the intestinal flora in mice fed a high-fat diet and restored it to a certain extent.

[0101] Gut microbes are thought to be factors associated with fat storage, weight gain, and insulin resistance. When weight gain is in an obese state, the abundance of Bacteroidetes decreases and the abundance of Firmicutes increases. Figure 18 As shown in the figure, compared with the ND group, the relative abundance ratio of Bacteroidetes / Firmicutes in the intestinal flora of mice in the HFD group was significantly decreased, indicating that a high-fat diet can cause an imbalance in the intestinal flora of mice at the phylum level. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) significantly increased the ratio of Bacteroidetes / Firmicutes, indicating that the three peptides regulate the intestinal flora composition of HFD mice at the phylum level, which is beneficial to the role of intestinal flora in improving obesity, insulin resistance and other related symptoms.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A blood sugar lowering peptide derived from bovine whey protein with stable gastrointestinal digestion and absorption, characterized in that: The amino acid sequence of the peptide is shown in either SEQ ID NO: 2 or SEQ ID NO:

4.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the peptide according to claim 1, and the nucleic acid molecule is DNA or RNA.

3. An expression vector, characterized in that The nucleotide sequence of the expression vector comprises the nucleic acid molecule according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 3 into a host cell.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the peptide according to claim 1, or the nucleic acid molecule according to claim 2, or the expression vector according to claim 3, or the recombinant cell according to claim 4.

6. A method for preparing an enzymatic hydrolysate having hypoglycemic activity, characterized in that: The method comprises the following steps: S1. A composite enzymatic hydrolysis of bovine whey protein using alkaline protease and pepsin at a certain temperature to obtain a bovine whey protein hydrolysate; S2. Simulated gastric digestion and intestinal digestion using bovine whey protein hydrolysate; S3. Absorption was simulated using a monolayer of fully differentiated human colon adenocarcinoma cells Caco-2 in a Transwell plate. The bottom permeate was collected and freeze-dried to obtain a stable enzymatic hydrolysate of bovine whey protein for gastrointestinal digestion and absorption.

7. Use of a peptide as shown in any one of SEQ ID NOs: 1 to 5, or a nucleic acid molecule as described in claim 2, or an expression vector as described in claim 3, or a recombinant cell as described in claim 4, or a pharmaceutical composition as described in claim 5, or a method as described in claim 6 in the preparation of a drug for preventing and / or treating diabetes.

8. Use of a peptide as shown in any one of SEQ ID NOs: 1 to 5, or a nucleic acid molecule as described in claim 2, or an expression vector as described in claim 3, or a recombinant cell as described in claim 4, or a pharmaceutical composition as described in claim 5, or a method as described in claim 6 in the preparation of a medicament for preventing and / or treating hyperlipidemia.

9. Use of a peptide as shown in any one of SEQ ID NOs: 1 to 5, or a nucleic acid molecule as described in claim 2, or an expression vector as described in claim 3, or a recombinant cell as described in claim 4, or a pharmaceutical composition as described in claim 5, or a method as described in claim 6 in the preparation of a drug for preventing and / or treating obesity.

10. Use of a peptide as shown in any one of SEQ ID NOs: 1 to 5, or a nucleic acid molecule as described in claim 2, or an expression vector as described in claim 3, or a recombinant cell as described in claim 4, or a pharmaceutical composition as described in claim 5, or a method as described in claim 6 in the preparation of a health product or functional food that assists in improving insulin resistance, lowering blood lipid levels, or losing weight.

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