An oat active polypeptide with GLP-1 secretion promoting, hypoglycemic and lipid-lowering activities, and preparation method and application thereof

Abstract

The present application relates to the technical field of oat bran deep processing, and particularly relates to an oat active polypeptide with GLP-1 secretion promotion, blood sugar reduction and lipid reduction activity, and a preparation method and application thereof.The present application is prepared by adding alkaline protease and ficin for double-enzyme step-by-step hydrolysis after homogenate, enzymolysis and alkali extraction and acid precipitation pretreatment, and then filtering, ultrafiltration desalination and drying, so that the oat active polypeptide with GLP-1 secretion promotion, blood sugar reduction and lipid reduction activity is obtained.The present application is simple to operate, low in equipment requirement, and suitable for industrial large-scale production.The prepared oat active polypeptide has GLP-1 secretion promotion, blood sugar reduction and lipid reduction activity, has a significant inhibitory effect on pancreatic lipase and alpha-glucosidase, has an obvious GLP-1 secretion promotion effect, and has a significant blood sugar reduction and lipid reduction effect.

Description

Technical Field

[0001] This invention relates to the field of oat bran deep processing technology, and in particular to an oat active polypeptide with GLP-1-promoting, blood sugar-lowering and lipid-lowering activities, its preparation method and application. Background Technology

[0002] Oat bran is the outer layer of oats, and its main components include carbohydrates, protein, minerals, and vitamins, all of which have significant positive effects on human health. Oat bran also offers numerous health benefits, such as controlling blood sugar, lowering cholesterol, managing weight, and boosting immunity. It slows down the digestion and absorption of food, helping to maintain stable blood sugar levels. Simultaneously, its high fiber content increases satiety, reducing food intake and aiding in weight management and weight loss.

[0003] In addition, oat bran contains about half of the total protein content of oat grains, and its price is much lower than that of oats. Therefore, oat bran is a suitable and economical raw material for preparing oat bioactive peptides.

[0004] Currently, research on oat bioactive peptides mainly focuses on their preparation methods. Chinese patent CN113699208A, published on November 26, 2021, discloses an oat peptide and its preparation method. This method involves removing lipids from oat bran using supercritical CO2 extraction, followed by enzymatic hydrolysis with a plant complex protease, and then filtration through a ceramic membrane to obtain oat bioactive peptides with high sensory acceptance. Chinese patent CN114958947A, published on August 30, 2022, discloses a method for preparing, purifying, and identifying oat antioxidant peptides. This method uses alkaline protease hydrolysis to obtain oat antioxidant peptides and screens out two antioxidant peptides: HWPLPPF and GWLDRFPMF. However, there are few reports on characteristic peptides of oat bioactive peptides that lower blood sugar and lipids. Therefore, how to obtain an oat bioactive peptide with hypoglycemic and lipid-lowering activities remains a problem that needs to be explored and solved by those skilled in the art. Summary of the Invention

[0005] To address the problem of how to obtain an oat-based bioactive polypeptide with hypoglycemic and lipid-lowering activities, this invention provides a method for preparing an oat-based bioactive polypeptide with GLP-1-promoting, hypoglycemic, and lipid-lowering activities, comprising the following steps:

[0006] The initial sieved oat bran is mixed with pure water and then homogenized in a homogenizer to obtain oat bran slurry.

[0007] Add amylase to oat bran slurry for a first pretreatment, and then inactivate the enzyme to obtain a pretreated mixture.

[0008] The pretreated mixture was subjected to a second pretreatment process involving alkaline extraction and acid precipitation. The pH of the pretreated mixture was adjusted to alkaline for extraction. After extraction, the mixture was centrifuged, and the supernatant was collected. The pH was adjusted to acidic, and the mixture was centrifuged again to obtain oat protein precipitate.

[0009] Oat protein precipitate was mixed with pure water, and alkaline protease was added for a first hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated to obtain the first hydrolysate.

[0010] Fig protease was added to the first hydrolysate for a second hydrolysis. After the enzymatic hydrolysis was completed, the pH was adjusted to acidic and the enzyme was inactivated to obtain the second hydrolysate.

[0011] The second hydrolysate was centrifuged, filtered, ultrafiltered, and dried to obtain oat active peptides.

[0012] In one embodiment, the initial sieve mesh size of the oat bran is 40-60 mesh, and the ratio of oat bran to pure water after initial sieve addition is 1:(10-20) by mass percentage, and the homogenization time is 30-60 min.

[0013] In one embodiment, during the first pretreatment, the amount of amylase added is 1%~2%, the enzymatic hydrolysis temperature is 45~55℃, the pH is 7.0, the reaction time is 0.5~1.5 h, the enzyme inactivation temperature is 85~90℃, and the treatment time is 15~20 min; during the second pretreatment, the alkaline extraction pH is 9.5~10.5, the extraction time is 60~120 min, the acidic precipitation pH is 3.8~4.2, the precipitation time is 10~20 min, the treatment temperature is 50~55℃, the centrifugation speed is 4000~6000 rpm, and the centrifugation time is 5~10 min.

[0014] In one embodiment, during the first hydrolysis, the ratio of the added oat protein precipitate to pure water is 1:(2~4) by mass percentage. The amount of alkaline protease added is 0.1%~0.3% of the mass of oat bran. The enzymatic hydrolysis temperature is 50~55℃, the pH is 8.0~9.0, the reaction time is 1~3 h, and the enzyme inactivation treatment temperature is 85℃ for 15~20 min. During the second hydrolysis, the amount of fig protease added is 0.5%~0.8% of the mass of oat bran. The enzymatic hydrolysis temperature is 50~55℃, the pH is 5.5~6.0, the reaction time is 1~3 h, and after the reaction, the pH is adjusted to 3.8~4.2 for enzyme inactivation. The enzyme inactivation treatment temperature is 100℃ for 10~15 min.

[0015] Furthermore, during the first hydrolysis, the alkaline protease is Bacillus licheniformis alkaline protease; during the second hydrolysis, after the enzymatic hydrolysis reaction is completed, the pH is adjusted to acidic using HCl solution before enzyme inactivation treatment.

[0016] In one embodiment, the ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 1 to 2 nm.

[0017] In one embodiment, the drying process is spray drying to obtain oat active polypeptide powder.

[0018] The present invention also provides an oat active polypeptide prepared according to the method for preparing oat active polypeptides with GLP-1 secretion-promoting, hypoglycemic and lipid-lowering activities as described in any of the preceding claims, comprising the following oat active peptide segments: whose amino acid sequences are Ser-Gly-Val-Phe-Thr-Pro-Lys or Gly-Cys-Arg-Phe-Asp-Arg-Leu or Asn-Ser-Lys-Asn-Phe-Pro-Thr-Le or Gln-Gly-Arg-Gly-Phe-Thr-Gly-Leu.

[0019] The present invention also provides a functional product, the components of which include oat active polypeptides as described above that have the activities of promoting GLP-1 secretion, lowering blood sugar and lowering lipids.

[0020] Furthermore, the functional product includes at least one of the following functions:

[0021] (1) Promotes GLP-1 secretion;

[0022] (2) Inhibit PTP1B;

[0023] (3) Inhibits pancreatic lipase;

[0024] (4) Lowering blood sugar;

[0025] (5) Lowering lipids.

[0026] The present invention provides a method for preparing oat active peptides with GLP-1-promoting, hypoglycemic, and lipid-lowering activities. After homogenization, enzymatic hydrolysis, and alkaline extraction and acid precipitation pretreatment, alkaline protease and fig protease are added for stepwise hydrolysis by two enzymes. After filtration, ultrafiltration desalting, and drying, oat active peptides with GLP-1-promoting, hypoglycemic, and lipid-lowering activities can be obtained. The operation is simple, the equipment requirements are low, and the yield can reach 13.33%, which is suitable for large-scale industrial production.

[0027] The oat-based active polypeptide provided by this invention has the effects of promoting GLP-1 secretion, lowering blood sugar and lipids. It has no obvious bitterness or astringency, has a good taste and flavor, and has significant effects in lowering blood sugar and lipids. It inhibits pancreatic lipase by 45.13% and α-glucosidase by 34.31%.

[0028] Specifically, in the lipid-lowering experiment of Caenorhabditis elegans, the oat active polypeptide provided in Example 1 of this invention significantly promoted the head swaying and swallowing frequency of the nematode, and its effect was similar to that of the positive control orlistat capsule. Oil Red O staining, triglyceride content determination and lipid metabolism gene expression level test can further confirm that the oat active polypeptide can downregulate the expression levels of daf-2, fat-5, fat-6 and fat-7 genes, effectively reducing the fat synthesis capacity of the nematode.

[0029] In the STC-1 cell experiment, at a concentration of 0.08 mg / mL oat active peptides, the oat active peptides provided in Example 1 of this invention significantly increased the expression level of GLP-1 in STC-1 cells, by about 18% compared to the blank group, and by about 45% at a concentration of 0.1 mg / mL. This indicates that the oat active peptides provided in this invention have a very significant ability to promote GLP-1 secretion and have a significant hypoglycemic effect.

[0030] This invention is based on the above-mentioned method for preparing oat bioactive peptides with GLP-1-promoting, hypoglycemic, and lipid-lowering activities. Combined with separation and purification-bioinformatics technology, four previously unreported oat bioactive peptides with GLP-1-promoting, hypoglycemic, and lipid-lowering activities were screened. Their amino acid sequences are: Ser-Gly-Val-Phe-Thr-Pro-Lys (sequence abbreviation: SGVFTPK), Gly-Cys-Arg-Phe-Asp-Arg-Leu (sequence abbreviation: GCRFDRL), Asn-Ser-Lys-Asn-Phe-Pro-Thr-Leu (sequence abbreviation: NSKNFPTL), and Gln-Gly-Arg-Gly-Phe-Thr-Gly-Leu (sequence abbreviation: QGRGFTGL). These four peptides exhibit high activity, are non-toxic, and can bind to receptors such as protein tyrosine phosphokinase 1B and pancreatic lipase, thus exerting hypoglycemic and lipid-lowering effects. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a graph showing the data analysis of the number of times the nematode's head swayed within 30 seconds in Experiment 2 of this invention;

[0033] Figure 2 This is a graph showing the data analysis of nematode swallowing frequency in Experiment 3 of this invention;

[0034] Figure 3 This describes the fat deposition within the nematodes during Experiment 2 of this invention.

[0035] Figure 4 This is a graph showing the data analysis of triglyceride content in nematodes during Experiment 2 of this invention;

[0036] Figure 5 This describes the expression of the daf-2 gene in nematodes during Experiment 2 of this invention.

[0037] Figure 6 This describes the expression of the fat-5 gene in nematodes during Experiment 2 of this invention.

[0038] Figure 7 This describes the expression of the fat-6 gene in nematodes during Experiment 2 of this invention.

[0039] Figure 8 This describes the expression of the fat-7 gene in nematodes during Experiment 2 of this invention.

[0040] Figure 9 The relative expression of GLP-1 content in STC-1 cells during Experiment 3 of this invention;

[0041] Figure 10 This is a 2D diagram of the docking of the peptide SGVFTPK with the pancreatic lipase molecule in Experiment 4 of this invention;

[0042] Figure 11 This is a 2D diagram of the docking of the peptide GCRFDRL with the pancreatic lipase molecule in Experiment 4 of this invention;

[0043] Figure 12 This is a 2D diagram of the docking of the peptide NSKNFPTL with the pancreatic lipase molecule in Experiment 4 of this invention;

[0044] Figure 13 This is a 2D diagram of the docking of peptide QGRGFTGL with pancreatic lipase molecules in Experiment 4 of this invention;

[0045] Figure 14 This is a 2D diagram of the docking of the peptide SGVFTPK with the PTP1B molecule in Experiment 4 of this invention;

[0046] Figure 15 This is a 2D diagram of the docking of the peptide GCRFDRL with the PTP1B molecule in Experiment 4 of this invention.

[0047] Figure 16 This is a 2D diagram of the docking of the peptide NSKNFPTL with the PTP1B molecule in Experiment 4 of this invention;

[0048] Figure 17This is a 2D diagram of the docking of peptide QGRGFTGL with PTP1B molecules in Experiment 4 of this invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and the technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] This invention provides the following Examples 1-3 for preparing an oat-based bioactive polypeptide with GLP-1-promoting, blood sugar-lowering, and lipid-lowering activities:

[0051] Example 1

[0052] S100. Mix 500 g of oat bran after sieving through a 60-mesh sieve with 7500 g of pure water, add it to a homogenizer and homogenize for 60 minutes to obtain oat bran slurry.

[0053] S101. Heat the oat bran slurry to 55℃, adjust the pH to 7.0 with NaOH solution, add 5 g of amylase, enzymatically hydrolyze for 1 h, and then heat to 85℃ to inactivate the enzyme for 20 min to obtain a pre-treated mixture.

[0054] S102. Cool the pretreated mixture to 50°C, adjust the pH to 10.0 with NaOH solution, stir the solution and extract for 120 min. After extraction, centrifuge at 5000 rpm for 10 min, take the supernatant, heat to 50°C, adjust the pH to 4.0 with HCl solution, precipitate for 20 min, centrifuge at 5000 rpm for 10 min, discard the supernatant, and obtain oat protein precipitate.

[0055] S103. Mix oat protein precipitate with pure water at a mass percentage ratio of 1:2, heat to 55℃, adjust pH to 8.5, add 1 g of Bacillus licheniformis alkaline protease, enzymatically hydrolyze for 2 h, then heat to 85℃ to inactivate enzyme for 20 min to obtain the first hydrolysate.

[0056] S104. Cool the first hydrolysate to 50°C, adjust the pH to 5.7, add 3 g of fig protease, and hydrolyze for 2 h. After hydrolysis, adjust the pH to 4.0 with HCl solution, then heat to 100°C and keep boiling to inactivate the enzyme for 15 min to obtain the second hydrolysate.

[0057] S105. After cooling the second hydrolysate to room temperature, filter it and then use an ultrafiltration membrane with a pore size of 1 nm for ultrafiltration desalting to obtain an oat active polypeptide solution.

[0058] S106. Spray-dry the oat active polypeptide solution to obtain oat active polypeptide powder.

[0059] Example 2

[0060] S100. Mix 500 g of oat bran after 50 mesh initial screening with 5000 g of pure water, add it to a homogenizer and homogenize for 30 minutes to obtain oat bran slurry.

[0061] S101. Heat the oat bran slurry to 45℃, adjust the pH to 7.0 with NaOH solution, add 7.5 g of amylase, hydrolyze for 1.5 h, then heat to 90℃ to inactivate the enzyme for 15 min to obtain a pre-treated mixture.

[0062] S102. Cool the pretreated mixture to 55°C, adjust the pH to 9.5 with NaOH solution, stir the solution and extract for 80 min. After extraction, centrifuge at 5000 rpm for 10 min, take the supernatant, heat to 50°C, adjust the pH to 3.8 with HCl solution, precipitate for 15 min, centrifuge at 5000 rpm for 10 min, discard the supernatant, and obtain oat protein precipitate.

[0063] S103. Mix oat protein precipitate with pure water at a mass percentage ratio of 1:3, heat to 50℃, adjust pH to 8.0, add 0.5 g of Bacillus licheniformis alkaline protease, enzymatically hydrolyze for 1 h, and then heat to 90℃ to inactivate the enzyme for 15 min to obtain the first hydrolysate.

[0064] S104. Cool the first hydrolysate to 50°C, adjust the pH to 5.7, add 2.5 g of fig protease, and hydrolyze for 3 h. After hydrolysis, adjust the pH to 3.8 with HCl solution, then heat to 100°C and keep boiling to inactivate the enzyme for 15 min to obtain the second hydrolysate.

[0065] S105. After cooling the second hydrolysate to room temperature, filter it and then use an ultrafiltration membrane with a pore size of 1 nm for ultrafiltration desalting to obtain an oat active polypeptide solution.

[0066] S106. Spray-dry the oat active polypeptide solution to obtain oat active polypeptide powder.

[0067] Example 3

[0068] S100. Mix 500 g of oat bran after 40 mesh initial sieve with 6500 g of pure water, add it to a homogenizer and homogenize for 45 min to obtain oat bran slurry.

[0069] S101. Heat the oat bran slurry to 50℃, adjust the pH to 7.0 with NaOH solution, add 10 g of amylase, hydrolyze for 0.5 h, then heat to 85℃ to inactivate the enzyme for 20 min to obtain a pretreated mixture.

[0070] S102. Cool the pretreated mixture to 55°C, adjust the pH to 10.5 with NaOH solution, stir the solution and extract for 60 min. After extraction, centrifuge at 6000 rpm for 5 min, take the supernatant, heat to 55°C, adjust the pH to 4.2 with HCl solution, precipitate for 10 min, centrifuge at 6000 rpm for 5 min, discard the supernatant, and obtain oat protein precipitate.

[0071] S103. Mix oat protein precipitate with pure water at a mass percentage ratio of 1:4, heat to 55℃, adjust pH to 9.0, add 1.5 g of Bacillus licheniformis alkaline protease, enzymatically hydrolyze for 3 h, then heat to 95℃ to inactivate enzyme for 15 min to obtain the first hydrolysate.

[0072] S104. Cool the first hydrolysate to 50°C, adjust the pH to 5.7, add 4 g of fig protease, and hydrolyze for 1 h. After hydrolysis, adjust the pH to 4.2 with HCl solution, then heat to 100°C and keep boiling to inactivate the enzyme for 15 min to obtain the second hydrolysate.

[0073] S105. After cooling the second hydrolysate to room temperature, filter it and then use an ultrafiltration membrane with a pore size of 2 nm for ultrafiltration desalting to obtain an oat active peptide solution.

[0074] S106. Spray-dry the oat active polypeptide solution to obtain oat active polypeptide powder.

[0075] In embodiments 1 to 3 of the present invention, preferably, the protein content of the oat bran is 16%.

[0076] In Examples 1-3 of the present invention, preferably, the amylase activity is 5000 U / g.

[0077] In embodiments 1-3 of the present invention, preferably, the enzyme activity of the Bacillus licheniformis alkaline protease is 200,000 U / g.

[0078] In Examples 1-3 of the present invention, preferably, the enzyme activity of the fig protease is 100,000 U / g.

[0079] This invention also provides the following comparative examples 1-7 for the preparation of an oat bioactive polypeptide:

[0080] Comparative Example 1

[0081] This comparative example prepared an oat-based active polypeptide, differing from Example 1 in that: 3 g of fig protease in step S104 was replaced with 0.6 g of trypsin; the enzymatic hydrolysis pH was 7.5; the hydrolysis time was 1 h; and the enzyme was inactivated by treatment at 85°C for 20 min. Furthermore, after trypsin inactivation, 1 g of aminopeptidase was added, and the enzyme was hydrolyzed at 50°C and pH=7.0 for 1 h, followed by treatment at 85°C for 20 min to inactivate the enzyme. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0082] In the comparative example of the present invention, the enzyme activity of the trypsin is 250,000 U / g, and the enzyme activity of the aminopeptidase is 150,000 U / g.

[0083] It should be noted that, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease, trypsin, and aminopeptidase used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0084] In Example 1, 500 g of oat bran was added, with a protein content of 16%. Calculations show that 500 g of oat bran contains 80 g of oat protein. 1 g of Bacillus licheniformis alkaline protease was added, with an enzyme activity of 200,000 U / g. The enzyme activity of 1 g of Bacillus licheniformis alkaline protease is 200,000 U / g × 1 g = 200,000 U. Converted to the enzyme activity per unit weight of protein in oat bran, this is 200,000 U / 80 g = 2500 U / g. Similarly, 3 g of fig protease was added, with an enzyme activity of 100,000 U / g. Calculations show that, based on the protein content in oat bran, an enzyme with an activity of 3750 U / g was added. Therefore, in Example 1, based on the protein content in oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used is 6250 U / g.

[0085] In Comparative Example 1, the amount of Bacillus licheniformis alkaline protease added was 1 g, which, based on the protein content of oat bran, represents 2500 U / g; the amount of trypsin added was 0.6 g, with an enzyme activity of 250,000 U / g, which, based on the protein content of oat bran, represents 1875 U / g; and the amount of aminopeptidase added was 1 g, with an enzyme activity of 150,000 U / g, which, based on the protein content of oat bran, represents 1875 U / g. Therefore, in Comparative Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease, trypsin, and aminopeptidase was 6250 U / g.

[0086] Therefore, based on the protein content in oat bran, the total amount of Bacillus licheniformis alkaline protease, trypsin, and aminopeptidase used in Comparative Example 1 is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0087] Comparative Example 2

[0088] This comparative example prepared an oat-based active polypeptide, which differs from Example 1 in that: 1 g of Bacillus licheniformis alkaline protease in step S103 was replaced with 2.5 g of acidic protease, the enzymatic hydrolysis temperature was 37℃, and the enzymatic hydrolysis pH was 2.5; and step S104 was omitted. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0089] In the comparative example of the present invention, the enzyme activity of the acidic protease is 200,000 U / g.

[0090] It should be noted that, based on the protein content of oat bran, the amount of acidic protease used in this comparative example can be considered equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0091] In Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used was 6250 U / g.

[0092] In Comparative Example 2, the amount of acidic protease added was 2.5 g, and its enzyme activity was 200,000 U / g. Based on the protein content in oat bran, the amount of acidic protease used was 200,000 U / g × 2.5 g / 80 g = 6250 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0093] Comparative Example 3

[0094] This comparative example prepared an oat-based active polypeptide, differing from Example 1 in that: the 1 g Bacillus licheniformis alkaline protease in step S103 was replaced with 1.25 g alkaline complex protease, and the enzymatic hydrolysis pH was 6.5; and step S104 was omitted. All other components, proportions, operating steps, and process parameters remained consistent with Example 1.

[0095] In the comparative example of the present invention, the enzyme activity of the alkaline complex protease is 400,000 U / g.

[0096] It should be noted that, based on the protein content of oat bran, the amount of alkaline complex protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0097] In Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used was 6250 U / g.

[0098] In Comparative Example 3, the amount of alkaline complex protease added was 1.25 g, and its enzyme activity was 400,000 U / g. Based on the protein content in oat bran, the amount of alkaline complex protease used was 400,000 U / g × 1.25 g / 80 g = 6250 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0099] Comparative Example 4

[0100] This comparative example prepared an oat-based active polypeptide, differing from Example 1 in that: the Bacillus licheniformis alkaline protease in step S103 was replaced with Bacillus subtilis neutral protease, and the enzymatic hydrolysis pH was 7.0; the 3 g fig protease in step S104 was replaced with 2 g aminopeptidase, the enzymatic hydrolysis temperature was 50℃, the pH was 7.0, and the enzyme was inactivated by treatment at 85℃ for 20 min. All other components, proportions, operating steps, and process parameters remained consistent with Example 1.

[0101] In the comparative example of the present invention, the enzyme activity of the Bacillus subtilis neutral protease was 200,000 U / g.

[0102] It should be noted that, based on the protein content of oat bran, the total amount of Bacillus subtilis neutral protease and aminopeptidase used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0103] In Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used was 6250 U / g.

[0104] In Comparative Example 4, the amount of Bacillus subtilis neutral protease added was 1 g, with an enzyme activity of 200,000 U / g. Based on the protein content of oat bran, the amount of Bacillus subtilis neutral protease used was 200,000 U / g × 1 g / 80 g = 2500 U / g. The amount of aminopeptidase added was 2 g, with an enzyme activity of 150,000 U / g. Based on the protein content of oat bran, the amount of aminopeptidase used was 150,000 U / g × 2 g / 80 g = 3750 U / g. Therefore, in Comparative Example 4, the total amount of Bacillus subtilis neutral protease and aminopeptidase used was 6250 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0105] Comparative Example 5

[0106] This comparative example prepared an oat-based active polypeptide, differing from Example 1 in that: the 3 g fig protease in step S104 was replaced with 0.6 g Bacillus subtilis neutral protease, the enzymatic hydrolysis pH was 6.5, and the enzyme was inactivated at 85°C for 20 min; furthermore, after inactivation by Bacillus subtilis neutral protease, 1.2 g aminopeptidase was added, the enzymatic hydrolysis pH was 7.0, the hydrolysis time was 1 h, and the enzyme was then inactivated again at 85°C for 20 min. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0107] It should be noted that, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0108] In Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used was 6250 U / g.

[0109] In Comparative Example 5, the amount of Bacillus licheniformis alkaline protease added was 1 g, which, based on the protein content of oat bran, corresponds to 2500 U / g; the amount of Bacillus subtilis neutral protease added was 0.6 g, with an enzyme activity of 200,000 U / g, which, based on the protein content of oat bran, corresponds to 200,000 U / g × 0.6 g / 80 g = 1500 U / g; and the amount of aminopeptidase added was 1.2 g, with an enzyme activity of 150,000 U / g, which, based on the protein content of oat bran, corresponds to 150,000 U / g × 1.2 g / 80 g = 2250 U / g. Therefore, in Comparative Example 5, the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and aminopeptidase was 6250 U / g. U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0110] Comparative Example 6

[0111] This comparative example prepared an oat-based active polypeptide, which differs from Example 1 in that: the 1 g of Bacillus licheniformis alkaline protease in step S103 was replaced with 2.5 g; and step S104 was omitted. All other components, proportions, operating steps, and process parameters remained consistent with Example 1.

[0112] It should be noted that, based on the protein content of oat bran, the amount of Bacillus licheniformis alkaline protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0113] In Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used was 6250 U / g.

[0114] In Comparative Example 6, the amount of Bacillus licheniformis alkaline protease added was 2.5 g. Based on the protein content in oat bran, the amount of Bacillus licheniformis alkaline protease was 200,000 U / g × 2.5 g / 80 g = 6250 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0115] Comparative Example 7

[0116] This comparative example prepared an oat-based active polypeptide, which differs from Example 1 in that step S103 was omitted; and the 3 g of fig protease in step S104 was replaced with 5 g. All other components, ratios, operating steps, and process parameters remained the same as in Example 1.

[0117] It should be noted that, based on the protein content of oat bran, the amount of fig protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1. The specific calculation process is as follows:

[0118] In Example 1, based on the protein content of oat bran, the total amount of Bacillus licheniformis alkaline protease and fig protease used was 6250 U / g.

[0119] In Comparative Example 7, the amount of fig protease added was 5 g. Based on the protein content in oat bran, the amount of fig protease used was 100,000 U / g × 5 g / 80 g = 6250 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and fig protease used in Example 1.

[0120] The yields of oat bioactive peptides prepared in Examples 1-3 and Comparative Examples 1-7 of this invention are shown in Table 1.

[0121] Table 1. Yields of oat bioactive peptides prepared in Examples 1-3 and Comparative Examples 1-7

[0122]

[0123] According to the data in Table 1, under the same experimental conditions, the yield of Example 1 was significantly higher than that of Comparative Examples 6 and 7. Comparative Examples 6 and 7 were prepared by hydrolyzing Bacillus licheniformis alkaline protease and fig protease in Example 1, respectively, to produce oat active peptides. This shows that the yield of dual-enzyme hydrolysis is better than that of single-enzyme hydrolysis.

[0124] Furthermore, the present invention provides the following experiments to detect the activity and screen the peptides of the oat active polypeptides obtained by the present invention:

[0125] Experiment 1: Determination of pancreatic lipase inhibition rate and α-glucosidase inhibition rate

[0126] The oat active peptides obtained in Examples 1-3 and Comparative Examples 1-5 were prepared into 20 mg / mL solutions for the determination of pancreatic lipase inhibition rate and α-glucosidase inhibition rate. The pancreatic lipase inhibition rate was determined according to the method described in "Inhibitory Effect and Mechanism of Ethyl p-hydroxycinnamate on Pancreatic Lipase" (DOI: 10.13386 / j.issn1002-0306.2020060193), and the α-glucosidase inhibition rate was determined according to the method described in "Inhibitory effect of chestnut (Castanea mollissima Blume) inner skin extract on the activity of α-amylase, α-glucosidase, dipeptidyl peptidase IV and in vitro digestibility of stars" (DOI: 10.1016 / j.foodchem.2020.126847). The measured data are summarized in Table 2.

[0127] Table 2. Pancreatic lipase inhibition rate and α-glucosidase inhibition rate of the oat bioactive peptides prepared in Examples 1-3 and Comparative Examples 1-5

[0128]

[0129] According to the data in Table 2, the oat active polypeptides prepared in Examples 1-3 and Comparative Examples 1-5 all have certain inhibitory abilities against pancreatic lipase and α-glucosidase. However, the inhibitory abilities against pancreatic lipase and α-glucosidase in Examples 1-3 are significantly higher than those in Comparative Examples 1-5. Among them, the oat active polypeptide prepared in Example 1 has the highest inhibition rates against pancreatic lipase and α-glucosidase, which are 45.13% and 34.31%, respectively.

[0130] Experiment 2: Lipid-lowering effect of oat bioactive peptides on Caenorhabditis elegans

[0131] In this experiment, an obese nematode hyperlipidemia model was established and tested in a normal group (NC group), a model group, an experimental group, and a positive control group.

[0132] The method for establishing the high-fat model of obese nematodes was to prepare NGM medium containing 10 mmol / L glucose, and transfer the synchronized L1 stage nematodes to normal culture dishes for 48 h.

[0133] NGM medium (Ruichu Biotechnology): Peptone 2.5 g; Sodium chloride 3.0 g; Calcium Chloride 0.111 g; Cholesterol 0.005 g; Agar 17.0 g;

[0134] Instructions for use: Weigh 22.6 g of this product into 1 L of distilled or deionized water, heat and boil for at least 1 min to dissolve completely, dispense into containers, autoclave at 121°C for 15 min, cool to 50°C, add 1 vial of T2245 per 100 mL of culture medium, mix gently, dispense into containers, and use for later use.

[0135] Normal group (NC group): 100 μL of Escherichia coli OP50 bacterial suspension was spread onto NGM medium, and 20 nematodes in the L4 stage were transferred to each culture dish and incubated at constant temperature for 48 h.

[0136] Model group: 100 μL of Escherichia coli OP50 bacterial suspension was spread onto NGM medium containing 10 mmol / L glucose, and 20 nematodes in the L4 stage were transferred to each culture dish and incubated at constant temperature for 48 h.

[0137] Experimental group: The oat active peptide solution with a concentration of 200 mg / mL obtained in Example 1 and Comparative Examples 1-5 was mixed with the activated Escherichia coli OP50 bacterial solution at a volume ratio of 1:9. The concentration of oat active peptide in the mixture was 20 mg / mL. 100 μL of each solution was spread onto NGM medium containing 10 mmol / L glucose. Twenty nematodes in the L4 stage were then transferred to each culture dish and incubated at a constant temperature for 48 h.

[0138] Positive control group: One orlistat capsule (0.12 g) was dissolved in 100 mL of 2% DMSO solution to obtain an orlistat solution with a concentration of 1.2 mg / mL. This solution was then mixed with activated Escherichia coli OP50 bacterial suspension at a volume ratio of 1:9, resulting in a DMSO concentration of 0.2% and an orlistat concentration of 120 μg / mL. 100 μL of this solution was spread onto NGM medium containing 10 mmol / L glucose. Twenty nematodes in the L4 stage were then transferred to a culture dish and incubated at a constant temperature for 48 h.

[0139] 2.1 Determination of Nematode Motility Indicators

[0140] Motor behavior is a rapid indicator of whether the nervous system of *C. elegans* is damaged. In related studies, head swaying and swallowing frequency are often selected to assess the motor ability of *C. elegans*.

[0141] After the nematodes were cultured on NGM culture plates for 48 h, the number of head movements of the nematodes within 30 s was observed, and the number of swallowing movements of the nematodes' pharynx within 1 min was observed under a microscope to evaluate the viability of the nematodes.

[0142] Figure 1 This is a graph analyzing the number of times the nematode's head twitches within 30 seconds. Figure 2 A graph showing the swallowing frequency data of nematodes, based on Figure 1 and Figure 2 It can be seen that the sample containing the oat active polypeptide prepared in Example 1 was the closest to the positive control group in terms of head swaying and swallowing frequency of nematodes in the experimental group, indicating that the oat active polypeptide of Example 1 has a significant promoting effect on the head swaying and swallowing frequency of Caenorhabditis elegans.

[0143] 2.2 Oil Red O Staining Experiment

[0144] The fat-soluble dye Oil Red O can specifically adhere to neutral fat molecules, turning them red. Therefore, the extent of fat deposition in nematodes can be directly observed. Oil Red O staining can provide a preliminary assessment of fat deposition; the darker the color, the greater the fat deposition.

[0145] Figure 3 To determine the deposition of fat within nematodes, based on Figure 3 It can be seen that the nematodes in Example 1, Comparative Example 4, Comparative Example 5 and Comparative Example 6 are lighter in color, indicating that they have less fat deposition in their bodies.

[0146] 2.3 Determination of Triglyceride Content

[0147] Nematodes in the L4 stage were transferred to NGM culture dishes of each group and incubated at a constant temperature for 48 h. The nematodes were washed three times with M9 buffer, allowed to settle naturally, and the supernatant was discarded. The supernatant was then resuspended in an appropriate amount of M9 buffer, sonicated, and centrifuged to collect the supernatant. The triglyceride content was determined according to the instructions of the Nanjing Jiancheng Triglyceride Reagent Kit. Three parallel experiments were conducted in each group.

[0148] Figure 4 This is a graph analyzing the triglyceride content in nematodes, based on... Figure 4 It can be seen that the triglyceride (TG) content in the nematodes treated in Example 1 was significantly reduced and lower than that in each pair of ratios. Compared with the model group, the triglyceride content in Example 1 was reduced by about 50%, indicating that the oat active polypeptide in Example 1 has a good ability to reduce lipid synthesis in nematodes.

[0149] 2.4 Effects of oat bioactive peptides on the expression levels of lipid metabolism genes in nematodes

[0150] Total RNA was extracted from nematodes using the Hangzhou Bori RNA Extraction Kit, and then reverse transcribed and subjected to real-time quantitative PCR. The primer sequences are listed in Table 3. The amplification conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s; 60℃ annealing and extension for 34 s, for a total of 40 cycles. Actin-1 was used as an internal reference gene, and the results were processed using 2-ΔΔCt.

[0151] Table 3 Primer sequence listing

[0152]

[0153] In *C. elegans*, the daf-2 gene is a key gene in the insulin signaling pathway that regulates lipid metabolism and lifespan. The expression of the daf-2 gene can effectively assess the lipid metabolism of *C. elegans*.

[0154] Figure 5 To determine the expression of the daf-2 gene in nematodes, based on Figure 5 It can be seen that the expression of the daf-2 gene in the experimental group was significantly lower than that in the model group, indicating that the presence of oat active peptides significantly downregulated the expression of the daf-2 gene. Among them, the downregulation was most obvious in Example 1, Comparative Example 4 and Comparative Example 5. It can be inferred that oat active peptides can prolong the lifespan and antioxidant capacity of Caenorhabditis elegans by regulating the expression level of upstream genes, thereby affecting lipid accumulation in the nematode and achieving the effect of lipid reduction.

[0155] Numerous studies have confirmed that fatty acid synthesis inhibitors suppress fatty acid synthesis and reduce fatty acid unsaturation by regulating the cholesterol regulatory element binding protein (SREBP) pathway and its downstream genes (fat-5, fat-6, fat-7), thereby reducing lipid accumulation and achieving a lipid-lowering effect. In addition, the three target genes (fat-5, fat-6, fat-7) can participate in the regulation of desaturases and are three important genes encoding stearoyl-CoA desaturase (Δ-9 desaturase), which is a key enzyme in fat synthesis and maintaining energy balance.

[0156] Figure 6 This shows the expression of the fat-5 gene in nematodes. Figure 7 This describes the expression of the fat-6 gene in nematodes. Figure 8 To determine the expression of the fat-7 gene in nematodes, based on Figures 6-8 It can be seen that, compared with the model group, oat active peptides can reduce the mRNA expression levels of fat-5, fat-6 and fat-7 genes, among which the downregulation is most obvious in Example 1, Comparative Example 4 and Comparative Example 5.

[0157] In summary, the oat active polypeptides provided by this invention can reduce the expression levels of daf-2, fat-5, fat-6 and fat-7 genes, thereby comprehensively inhibiting the synthesis of fat in nematodes and achieving the effect of lowering lipids.

[0158] Experiment 3: Effect of oat bioactive peptides on GLP-1 content in STC-1 cells

[0159] Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic hormone secreted by the intestine, especially the terminal ileum. It has advantages such as improving insulin resistance, weight loss, promoting the recovery of pancreatic function, and restoring physiological insulin secretion. STC-1 is an endocrine cell line derived from the mouse intestine that secretes cholecystokinin (CCK), GLP-1, and other enteroendocrine cells, and is currently the recognized cell line for studying GLP-1 secretion.

[0160] 3.1 Determination of the optimal experimental concentration of oat bioactive peptides

[0161] Cell viability was determined using the CCK8 (Cell Counting Kit-8) method. After significance analysis, two concentrations of 0.08 mg / mL and 0.1 mg / mL, which were non-toxic to cells, were selected as the cell drug concentrations.

[0162] 3.2 Experimental Procedure for Influence of Oat Bioactive Peptides on GLP-1 Content in STC-1 Cells

[0163] Cell seeding: Seed healthy STC-1 cells into 24-well plates at a density of 85%, 1 mL per well, and incubate in a cell culture incubator for 48 h.

[0164] Drug addition and incubation: Before adding the drug, prepare complete culture media containing the optimal dosing concentration of oat active peptides obtained in Example 1 and Comparative Examples 1-5, respectively, shake and mix well, and set aside; discard the original culture medium in the well plate, add 1 mL of complete culture medium containing the drug to each well, and incubate at 37°C for 2 h.

[0165] Collect cell supernatant: After drug incubation, collect the supernatant in a sterile tube, centrifuge at 4℃ and 1000rpm for 20 min, and transfer the supernatant to a new sterile tube. It should be noted that if the experiment cannot be conducted in time, the sample should be aliquoted and frozen at -80℃ to avoid repeated freeze-thaw cycles.

[0166] GLP-1 content detection: The determination was performed according to the instructions of the Nanjing Jiancheng GLP-1 Elisa kit.

[0167] 3.3 Results Analysis

[0168] The ANOVA statistical method was used to analyze the significant differences between different groups. Figure 9 The relative expression of GLP-1 in STC-1 cells, based on Figure 9 It can be seen that at a concentration of 0.08 mg / mL, the relative expression of GLP-1 in Example 1 was higher than that in the comparative examples, and increased by about 18% compared with the blank group, and by about 12% compared with the comparative example 1, which had the highest relative expression of GLP-1 among the comparative examples; at a concentration of 0.1 mg / mL, the relative expression of GLP-1 in Example 1, comparative example 1 and comparative examples 3-5 was significantly higher than that in the blank group, and the relative expression of GLP-1 in Example 1 was increased by about 45% compared with the blank group, and by about 13% compared with the comparative example 3, which had the highest relative expression of GLP-1 among the comparative examples.

[0169] Experiment 4: Peptide screening of oat protein peptides

[0170] In this experiment, the oat active polypeptide obtained in Example 1 was screened and analyzed for peptide fragments.

[0171] 4.1 Mass Spectrometry Experimental Methods

[0172] (1) Preprocessing

[0173] In this experiment, oat bioactive peptides were pretreated using a reductive alkylation method. The specific steps are as follows:

[0174] S200: Weigh 10.4 mg and 11.3 mg of the samples respectively, add 100 μL of water to dissolve them completely, and then carry out subsequent experiments;

[0175] S201. Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, and reduce it in a water bath at 56℃ for 1 h.

[0176] S202. Accurately transfer 2 μL of 1M IAM solution into the sample using a pipette to make the final IAM concentration 20 mmol / L. React in the dark at room temperature for 40 min.

[0177] S203. Accurately pipette 1 μL of 1M DTT solution and add it to the sample to make the final DTT concentration 10 mmol / L, neutralizing the unreacted IAM.

[0178] (2) C18 desalination (Stage-Tip)

[0179] Desalting was performed using C18 stage-tip, followed by vacuum drying at 45°C.

[0180] 4.2 Computer Usage Requirements

[0181] (1) Liquid chromatography conditions

[0182] 1) Pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm;

[0183] 2) Mobile phase A: 0.1% FA;

[0184] 3) Mobile phase B: 0.1% FA, 80% ACN;

[0185] 4) Flow rate: 600 nL / min;

[0186] 5) Analysis time for each component: 66 min;

[0187] 6) Specific chromatographic conditions are listed in Table 4:

[0188] Table 4 Specific chromatographic conditions for liquid chromatography

[0189]

[0190] (2) Mass spectrometry conditions

[0191] The primary mass spectrometry parameters are: 1) Resolution: 70,000; 2) AGC target: 3e6; 3) Maximum IT: 100 ms; 4) Scan range: 100 to 1500 m / z.

[0192] The secondary mass spectrometry parameters were: 1) Resolution: 17,500; 2) AGC target: 1e5; 3) Maximum IT: 50ms; 4) Top N: 20; 5) NCE / stepped NCE: 28.

[0193] Raw data was obtained through mass spectrometry.

[0194] 4.3 Search Conditions

[0195] The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows:

[0196] 1) Fixed modifications: Carbamidomethyl (C);

[0197] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term);

[0198] 3) Enzyme: Non specific;

[0199] 4) Database: uniprotkb_Acipenser (genus)_2024_11_13; uniprotkb_Oreochromis (genus)_2024_12_05;

[0200] 5) Peptide Mass Tolerance: 20 ppm;

[0201] 6) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da.

[0202] 4.4 Results Analysis

[0203] (1) Peptide screening

[0204] Raw files acquired by mass spectrometry were searched using the Byonic database software to screen for peptides with abundance greater than 10,400,000. Activity prediction was performed on these fragments (http: / / distilldeep.ucd.ie / PeptideRanker / ), and peptides with a result >0.5 were selected. This was further refined using abundance ranking, resulting in 10 peptides. A search of the BIOPEP peptide database (http: / / bis.zju.edu.cn / biopepdbr / index.php) revealed that all 10 peptides were novel and previously unreported. Toxicity prediction showed that all 10 peptides were non-toxic (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). Peptide-related information is listed in Table 5.

[0205] Table 5 Peptide Information

[0206]

[0207] Note: Peptide<ProteinMetrics Confidential> : Amino acid sequence of the identified peptide; Mass: molecular weight of the peptide; Score: activity prediction score; Lenth: number of amino acids in the peptide; RT: retention time of the peptide, in min; -10 lgP: significance score of the peptide identified by database search or de novo sequencing; Intensity: peptide abundance.

[0208] (2) Molecular docking

[0209] Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of the insulin signaling pathway. It dephosphorylates tyrosine-phosphorylated insulin receptors (IRs) and downstream key proteins involved in the signaling pathway, thereby inhibiting insulin signal transduction. PTP1B is a therapeutic target for diabetes and obesity, and effective, specific PTP1B inhibitors could become drugs for treating diabetes.

[0210] Pancreatic lipase is the main lipase secreted by the pancreas, directly affecting the absorption of fatty acids in the intestine. Pancreatic lipase hydrolyzes dietary lipids in the digestive system, converting triglyceride substrates from ingested fats into monoglycerides and free fatty acids. These hydrolysis products are absorbed by the epithelial cells of the small intestine. Pancreatic lipase inhibitors can bind to the active lipases in the pancreas and small intestine, altering the conformation of trypsin and inhibiting its catalytic activity. This inhibits the hydrolysis of dietary lipids, reducing lipid digestion and absorption and the accumulation of adipose tissue. Researching and developing pancreatic lipase inhibitors is an important direction for treating hyperlipidemia and controlling obesity.

[0211] Molecular docking software was used to dock the above 10 fragments with the above two enzymes. The binding energies of peptides to receptors are summarized in Table 6. Among them, the three-dimensional structures of pancreatic lipase (PDB code: 1LPB) and PTP1B (PDB code: 1T49) were downloaded from the PDB database (http: / / www.rcsb.org / ).

[0212] Table 6. Binding energy between peptides and receptors

[0213]

[0214] According to the data in Table 6, all 10 selected peptides have a certain binding energy with pancreatic lipase and PTP1B. Based on the binding energy, SGVFTPK, GCRFDRL, NSKNFPTL, and QGRGFTGL were selected as characteristic peptide sequences with significant hypoglycemic and lipid-lowering activities in oat bioactive polypeptides.

[0215] Figure 10This is a 2D diagram showing the docking of SGVFTPK with pancreatic lipase molecules. Figure 11 A 2D diagram showing the docking of GCRFDRL with pancreatic lipase molecules; Figure 12 A 2D diagram showing the docking of NSKNFPTL with pancreatic lipase molecules; Figure 13 This is represented as a 2D diagram showing the docking of QGRGFTGL with pancreatic lipase molecules; Figure 14 A 2D diagram showing the docking of SGVFTPK with PTP1B molecules; Figure 15 A 2D diagram showing the docking of GCRFDRL with PTP1B molecules; Figure 16 A 2D diagram showing the docking of NSKNFPTL and PTP1B molecules; Figure 17 This is a 2D diagram showing the docking of QGRGFTGL with PTP1B molecules.

[0216] like Figure 10 As shown, SGVFTPK interacts with pancreatic lipase through carbon-hydrogen bonds, van der Waals forces, and covalent hydrogen bonds; Figure 11 As shown, GCRFDRL interacts with pancreatic lipase through carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds; as Figure 12 As shown, NSKNFPTL interacts with pancreatic lipase through carbon-hydrogen bonds, van der Waals forces, attractive forces, and covalent hydrogen bonds; Figure 13 As shown, QGRGFTGL interacts with pancreatic lipase through carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds.

[0217] like Figure 14 As shown, the interactions between SGVFTPK and PTP1B are mainly covalent hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces; for example... Figure 15 As shown, GCRFDRL and PTP1B interact via carbon-hydrogen bonds, van der Waals forces, and attractive charge; as Figure 16 As shown, the interactions between NSKNFPTL and PTP1B are mainly covalent hydrogen bonds, C-H bonds, van der Waals forces, and salt bridges; Figure 17 The interaction between QGRGFTGL and PTP1B is mainly characterized by covalent hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces, and salt bridges.

[0218] It should be noted that the terms used in this invention, such as ultrafiltration, centrifugation, spray drying, mass spectrometry, molecular docking, and RNA extraction, are names of processing steps commonly used by those skilled in the art, and their names can accurately describe the processing procedures, so they will not be repeated in this invention.

[0219] The raw materials used in this invention, such as Bacillus licheniformis alkaline protease, acidic protease, fig protease, Bacillus subtilis neutral protease, trypsin, aminopeptidase, and alkaline complex protease, are all commercially available enzymes that can be purchased and obtained by those skilled in the art. In addition, unless otherwise specified, other raw materials used in this invention can also be conventional commercial products in the field or prepared by conventional methods in the field. That is, the reagents, instruments, and consumables used in this invention, unless otherwise specified, can also be purchased from the market.

[0220] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an oat bioactive polypeptide with GLP-1-promoting, hypoglycemic, and lipid-lowering activities, characterized in that, Includes the following steps: The initial sieved oat bran is mixed with pure water and then homogenized in a homogenizer to obtain oat bran slurry. Add amylase to oat bran slurry for a first pretreatment, and then inactivate the enzyme to obtain a pretreated mixture. The pretreated mixture was subjected to a second pretreatment process involving alkaline extraction and acid precipitation. The pH of the pretreated mixture was adjusted to alkaline for extraction. After extraction, the mixture was centrifuged, and the supernatant was collected. The pH was adjusted to acidic, and the mixture was centrifuged again to obtain oat protein precipitate. Oat protein precipitate was mixed with pure water, and alkaline protease was added for a first hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated to obtain the first hydrolysate. Fig protease was added to the first hydrolysate for a second hydrolysis. After the enzymatic hydrolysis was completed, the pH was adjusted to acidic and the enzyme was inactivated to obtain the second hydrolysate. The second hydrolysate was centrifuged, filtered, ultrafiltered, and dried to obtain oat active peptides; The initial sieve mesh size of the oat bran is 40-60 mesh, and the ratio of oat bran to pure water after initial sieve addition is 1:(10-20) by mass percentage, and the homogenization time is 30-60 min; During the first pretreatment, the amount of amylase added is 1%~2%, the enzymatic hydrolysis temperature is 45~55℃, the pH is 7.0, the reaction time is 0.5~1.5 h, the enzyme inactivation temperature is 85~90℃, and the treatment time is 15~20 min. During the secondary pretreatment, the alkaline extraction pH was 9.5~10.5 and the extraction time was 60~120 min; the acidic precipitation pH was 3.8~4.2 and the precipitation time was 10~20 min; the treatment temperature was 50~55℃; the centrifugation speed was 4000~6000 rpm and the centrifugation time was 5~10 min. During the first hydrolysis, the ratio of the oat protein precipitate to pure water added is 1:(2~4) by mass percentage. The amount of alkaline protease added is 0.1%~0.3% of the mass of oat bran. The enzymatic hydrolysis temperature is 50~55℃, the pH is 8.0~9.0, the reaction time is 1~3 h, the enzyme inactivation treatment temperature is 85℃, and the treatment time is 15~20 min. During the secondary hydrolysis, the amount of fig protease added is 0.5%~0.8% of the oat bran mass, the enzymatic hydrolysis temperature is 50~55℃, the pH is 5.5~6.0, the reaction time is 1~3 h, and after the reaction is completed, the pH is adjusted to 3.8~4.2 to inactivate the enzyme. The enzyme inactivation treatment temperature is 100℃ and the treatment time is 10~15 min. During the first hydrolysis, the alkaline protease is Bacillus licheniformis alkaline protease; During the secondary hydrolysis, after the enzymatic hydrolysis reaction is completed, the pH is adjusted to acidic using HCl solution before enzyme inactivation treatment.

2. The method for preparing oat bioactive polypeptides with GLP-1-promoting, hypoglycemic, and lipid-lowering activities according to claim 1, characterized in that: The ultrafiltration process is performed using an ultrafiltration membrane with a pore size of 1-2 nm.

3. The method for preparing oat bioactive polypeptides with GLP-1-promoting, hypoglycemic, and lipid-lowering activities according to claim 1, characterized in that: The drying process is spray drying, which yields oat active polypeptide powder.

4. An oat-based bioactive polypeptide, characterized in that: The oat bioactive polypeptide with GLP-1-promoting, hypoglycemic, and lipid-lowering activities as described in any one of claims 1 to 3 is prepared by the method of preparation.

5. A functional product, characterized in that: Its components include the oat active polypeptide described in claim 4.

6. The functional product according to claim 5, characterized in that, Includes at least one of the following functions: (1) Promotes GLP-1 secretion; (2) Inhibit PTP1B; (3) Inhibits pancreatic lipase; (4) Lowering blood sugar; (5) Lowering lipids.

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