Oat active polypeptide with activity of promoting GLP-1 secretion and reducing blood glucose and lipid as well as preparation method and application of oat active polypeptide

Abstract

The invention relates to the technical field of deep processing of oat bran, in particular to an oat active polypeptide with activity of promoting GLP-1 secretion, reducing blood glucose and reducing lipid as well as a preparation method and application thereof. After homogenization, enzymolysis and alkali extraction and acid precipitation pretreatment, alkaline protease and ficin are added for double-enzyme step-by-step hydrolysis, then filtration, ultrafiltration desalination and drying are performed, the oat active polypeptide with the activity of promoting GLP-1 secretion and reducing blood glucose and lipid can be prepared, operation is easy, the requirement for equipment is low, and the oat active polypeptide is suitable for industrial large-scale production. The prepared oat active polypeptide has the activity of promoting GLP-1 secretion, reducing blood glucose and reducing lipid, has a remarkable inhibition effect on pancreatic lipase and alpha-glucosidase, has a remarkable secretion promoting effect on pancreatic hyperglycemia-like peptide-1, and is remarkable in blood glucose reducing and lipid reducing effects. In addition, the traditional separation and purification technology is combined with bioinformatics, four non-toxic oat active peptide fragments with high hypoglycemic and lipid-lowering activity are screened out, and the hypoglycemic and lipid-lowering effects are accurate and efficient.

Description

Technical Field

[0001] The invention relates to the technical field of oat bran deep processing, and in particular to an oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid, and a preparation method and application thereof. Background Art

[0002] Oat bran is the outer part of oats. Its main components include carbohydrates, proteins, minerals and vitamins, which have a significant positive impact on human health. Oat bran also has many health benefits, such as controlling blood sugar, lowering cholesterol, controlling weight, and enhancing immunity. It can slow down the digestion and absorption of food and help keep blood sugar stable. At the same time, its high fiber content can increase satiety, reduce food intake, and help with weight management and weight loss.

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

[0004] At present, research on oat active peptides mainly focuses on the preparation method of oat active peptides. Chinese patent CN113699208A disclosed an oat peptide and its preparation method on November 26, 2021. After CO2 supercritical extraction of oat bran to remove lipids, plant composite protease was added for enzymatic hydrolysis, and then filtered through a ceramic membrane to obtain oat active peptides with high sensory acceptance; Chinese patent CN114958947A disclosed a method for the preparation and purification of oat antioxidant peptides on August 30, 2022. Oat antioxidant peptides were obtained by alkaline protease hydrolysis, and two antioxidant peptide segments HWPLPPF and GWLDRFPMF were screened out. However, there are few reports on characteristic peptide segments of oat active peptides for lowering blood sugar and lipids. Therefore, how to obtain an oat active polypeptide with blood sugar and lipid lowering activity is still a problem that technicians in this field need to explore and solve. Summary of the invention

[0005] In order to solve the problem of how to obtain an oat active polypeptide with hypoglycemic and lipid-lowering activities, the present invention provides a method for preparing an oat active polypeptide with GLP-1 secretion-promoting, hypoglycemic and lipid-lowering activities, comprising the following steps: The oat bran after the initial screening is mixed with pure water, and added into a homogenizer for homogenization to obtain an oat bran slurry; Adding amylase to the oat bran slurry for primary pretreatment, and performing enzyme inactivation treatment to obtain a primary pretreatment mixed solution; The primary pretreatment mixed solution is subjected to secondary pretreatment of alkali extraction and acid precipitation, the pH of the primary pretreatment mixed solution is adjusted to alkaline for leaching, centrifuged after leaching, the supernatant is taken, the pH is adjusted to acidic, and centrifuged to obtain oat protein precipitate; The oat protein precipitate is mixed with pure water, alkaline protease is added for primary hydrolysis, and the enzyme is inactivated after the enzymatic hydrolysis is completed to obtain a first hydrolyzate; Adding ficin to the first hydrolyzate for secondary hydrolysis, adjusting the pH to acidic after the enzymatic hydrolysis, and performing enzyme inactivation treatment to obtain a second hydrolyzate; The second hydrolyzate is centrifuged, filtered, ultrafiltered, and dried to obtain oat active polypeptides.

[0006] In one embodiment, the oat bran is initially screened with a mesh size of 40-60, and after the initial screening, the addition ratio of the oat bran to pure water is 1:(10-20) by mass percentage, and the homogenization time is 30-60 min.

[0007] In one embodiment, during the primary pretreatment, the amount of amylase added is 1%~2%, the enzymolysis temperature is 45~55°C, the pH is 7.0, the reaction time is 0.5~1.5 h, the temperature of the enzyme inactivation treatment is 85~90°C, and the treatment time is 15~20 min; during the secondary pretreatment, the alkaline extraction pH is 9.5~10.5, the extraction time is 60~120 min, the acid precipitation pH is 3.8~4.2, the precipitation time is 10~20 min, the treatment temperature is 50~55°C, the centrifugal speed is 4000~6000 rpm, and the centrifugal time is 5~10 min.

[0008] In one embodiment, during the primary hydrolysis, the ratio of the amount of the oat protein precipitate to the amount of pure water added in mass percentage is 1:(2-4), the amount of the alkaline protease added is 0.1%-0.3% of the mass of the oat bran, the enzymolysis temperature is 50-55°C, the pH is 8.0-9.0, the reaction time is 1-3 h, the temperature of the enzyme inactivation treatment is 85°C, and the treatment time is 15-20 min; during the secondary hydrolysis, the amount of the ficin added is 0.5%-0.8% of the mass of the oat bran, the enzymolysis temperature is 50-55°C, 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 for enzyme inactivation, the temperature of the enzyme inactivation treatment is 100°C, and the treatment time is 10-15 min.

[0009] Furthermore, during the primary hydrolysis, the alkaline protease is Bacillus licheniformis alkaline protease; during the secondary hydrolysis, after the enzymatic reaction is completed, an HCl solution is used to adjust the pH to acidic and then the enzyme is inactivated.

[0010] In one embodiment, the ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 1 to 2 nm.

[0011] In one embodiment, the drying is spray drying to obtain oat active polypeptide powder.

[0012] The present invention also provides an oat active polypeptide prepared according to the preparation method of an oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid as described in any one of the above items, comprising the following oat active peptide segment: its amino acid sequence is 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.

[0013] The present invention also provides a functional product, the components of which include the above-mentioned oat active polypeptides having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid.

[0014] Furthermore, the functional product includes at least one of the following functions: (1) Promote GLP-1 secretion; (2) Inhibition of PTP1B; (3) Inhibit pancreatic lipase; (4) Blood sugar lowering; (5) Reduces lipids.

[0015] The present invention provides a method for preparing an oat active polypeptide having the activity of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid. After homogenization, enzymatic hydrolysis and alkali extraction and acid precipitation pretreatment, alkaline protease and ficin are added for double enzyme step-by-step hydrolysis, and then the oat active polypeptide having the activity of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid is obtained through filtration, ultrafiltration desalination and drying. The method has simple operation, low requirements on equipment, and a yield of up to 13.33%, which is suitable for large-scale industrial production.

[0016] The oat active polypeptide provided by the present invention has the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipids, has no obvious bitterness or astringency, has a good taste and flavor, has significant blood sugar and blood lipid lowering effects, and has an inhibition rate of 45.13% on pancreatic lipase and 34.31% on α-glucosidase; Specifically, in the lipid-lowering experiment of Caenorhabditis elegans, the oat active polypeptide provided in Example 1 of the present invention has a significant promoting effect on the head swing and swallowing frequency of the nematode, which is close to the effect of the positive control orlistat capsule. Through Oil Red O staining, triglyceride content determination and lipid metabolism gene expression level test, it can be further determined that the oat active polypeptide can down-regulate the expression levels of daf-2, fat-5, fat-6 and fat-7 genes, and effectively reduce the fat synthesis ability of the nematode; In the STC-1 cell experiment, at a concentration of 0.08 mg / mL oat active polypeptide, the oat active polypeptide provided in Example 1 of the present invention can significantly increase the expression of GLP-1 in STC-1 cells, which is about 18% higher than that of the blank group, and about 45% higher at a concentration of 0.1 mg / mL. This shows that the oat active polypeptide provided by the present invention has a very significant ability to promote GLP-1 secretion and has a significant blood sugar lowering effect.

[0017] Based on the preparation method of the oat active polypeptides having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid, the present invention combines separation and purification-bioinformatics combined technology to screen out 4 oat active peptides that have not been reported and have the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid, and the amino acid sequences thereof 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), Gln-Gly-Arg-Gly-Phe-Thr-Gly-Leu (sequence abbreviation: QGRGFTGL); the 4 peptides are highly active and non-toxic, and can bind to the receptors of protein tyrosine phosphatase 1B and pancreatic lipase to play the role of lowering blood sugar and lowering blood lipid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a data analysis diagram of the number of nematode head swings within 30 seconds in Experiment 2 of the present invention; Figure 2 This is a data analysis diagram of nematode swallowing frequency in Experiment 3 of the present invention; Figure 3 This is the fat deposition in the nematode in Experiment 2 of the present invention; Figure 4 This is a data analysis diagram of triglyceride content in nematodes in Experiment 2 of the present invention; Figure 5 The expression of daf-2 gene in nematodes in Experiment 2 of the present invention; Figure 6 The expression of fat-5 gene in nematodes in Experiment 2 of the present invention; Figure 7 The expression of fat-6 gene in nematodes in Experiment 2 of the present invention; Figure 8 The expression of fat-7 gene in nematodes in Experiment 2 of the present invention; Fig. 9 is the relative expression of GLP-1 content in STC-1 cells in Experiment 3 of the present invention; Fig.10 This is a 2D diagram of the docking of the peptide SGVFTPK and the pancreatic lipase molecule in Experiment 4 of the present invention; Fig.11 This is a 2D diagram of the docking of the peptide GCRFDRL and the pancreatic lipase molecule in Experiment 4 of the present invention; Fig.12 This is a 2D diagram of the docking of the peptide NSKNFPTL and the pancreatic lipase molecule in Experiment 4 of the present invention; Fig.13 This is a 2D diagram of the docking of the peptide QGRGFTGL with the pancreatic lipase molecule in Experiment 4 of the present invention; Fig.14 It is a 2D diagram of the docking of the peptide SGVFTPK and the PTP1B molecule in Experiment 4 of the present invention; Fig.15 It is a 2D diagram of the docking of the peptide GCRFDRL and the PTP1B molecule in Experiment 4 of the present invention; Fig.16 It is a 2D diagram of the docking of the peptide NSKNFPTL and the PTP1B molecule in Experiment 4 of the present invention; Fig.17 This is a 2D diagram of the docking of the peptide QGRGFTGL and the PTP1B molecule in Experiment 4 of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and the technical features designed in different implementations 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The present invention provides the following examples 1 to 3 for preparing an oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipids: Example 1 S100, mixing 500 g of oat bran after 60 mesh primary screening with 7500 g of pure water, adding the mixture into a homogenizer and homogenizing for 60 min to obtain an oat bran slurry; S101, heating the oat bran slurry to 55°C, adjusting the pH to 7.0 with a NaOH solution, adding 5 g of amylase, performing enzymolysis for 1 h, and then heating to 85°C to inactivate the enzyme for 20 min to obtain a primary pretreated mixed solution; S102, cooling the pre-treated mixed solution to 50°C, adjusting the pH to 10.0 with NaOH solution, stirring the solution for leaching for 120 min, centrifuging at 5000 rpm for 10 min after the leaching, taking the supernatant, heating it to 50°C, adjusting the pH to 4.0 with HCl solution, precipitating for 20 min, centrifuging at 5000 rpm for 10 min, discarding the supernatant, and obtaining oat protein precipitate; S103, mixing the oat protein precipitate with pure water in a mass percentage ratio of 1:2, heating to 55°C, adjusting the pH to 8.5, adding 1 g of Bacillus licheniformis alkaline protease, performing enzymolysis for 2 h, and then heating to 85°C to inactivate the enzyme for 20 min to obtain a first hydrolyzate; S104, cooling the first hydrolyzate to 50°C, adjusting the pH to 5.7, adding 3 g of ficin, and performing enzymolysis for 2 h. After the enzymolysis, adjusting the pH to 4.0 with HCl solution, and then heating to 100°C and keeping boiling for 15 min to inactivate the enzyme, to obtain a second hydrolyzate; S105, cooling the second hydrolyzate to room temperature and filtering it, and then performing ultrafiltration desalination using an ultrafiltration membrane with a pore size of 1 nm to obtain an oat active polypeptide solution; S106, spray drying the oat active polypeptide solution to obtain oat active polypeptide powder.

[0022] Example 2 S100, mixing 500 g of oat bran after 50 mesh primary screening with 5000 g of pure water, adding the mixture into a homogenizer and homogenizing for 30 min to obtain an oat bran slurry; S101, heating the oat bran slurry to 45°C, adjusting the pH to 7.0 with a NaOH solution, adding 7.5 g of amylase, performing enzymolysis for 1.5 h, and then heating to 90°C to inactivate the enzyme for 15 min to obtain a primary pretreated mixed solution; S102, cooling the pre-treated mixed solution to 55°C, adjusting the pH to 9.5 with NaOH solution, stirring the solution for 80 min, centrifuging at 5000 rpm for 10 min after the extraction, taking the supernatant, heating it to 50°C, adjusting the pH to 3.8 with HCl solution, precipitating for 15 min, centrifuging at 5000 rpm for 10 min, discarding the supernatant, and obtaining oat protein precipitate; S103, mixing the oat protein precipitate with pure water in a mass percentage ratio of 1:3, heating to 50°C, adjusting the pH to 8.0, adding 0.5 g of Bacillus licheniformis alkaline protease, performing enzymolysis for 1 h, and then heating to 90°C to inactivate the enzyme for 15 min to obtain a first hydrolyzate; S104, cooling the first hydrolyzate to 50°C, adjusting the pH to 5.7, adding 2.5 g of ficin, and performing enzymolysis for 3 hours. After the enzymolysis is completed, adjusting the pH to 3.8 with HCl solution, and then heating to 100°C and keeping boiling for 15 minutes to inactivate the enzyme, to obtain a second hydrolyzate; S105, cooling the second hydrolyzate to room temperature and filtering it, and then performing ultrafiltration desalination using an ultrafiltration membrane with a pore size of 1 nm to obtain an oat active polypeptide solution; S106, spray drying the oat active polypeptide solution to obtain oat active polypeptide powder.

[0023] Example 3 S100, mixing 500 g of oat bran after 40 mesh primary screening with 6500 g of pure water, adding the mixture into a homogenizer and homogenizing for 45 min to obtain an oat bran slurry; S101, heating the oat bran slurry to 50°C, adjusting the pH to 7.0 with a NaOH solution, adding 10 g of amylase, performing enzymolysis for 0.5 h, and then heating to 85°C to inactivate the enzyme for 20 min to obtain a primary pretreated mixed solution; S102, cooling the pre-treated mixed solution to 55°C, adjusting the pH to 10.5 with NaOH solution, stirring the solution for 60 min, centrifuging at 6000 rpm for 5 min after the extraction, taking the supernatant, heating it to 55°C, adjusting the pH to 4.2 with HCl solution, precipitating for 10 min, centrifuging at 6000 rpm for 5 min, discarding the supernatant, and obtaining oat protein precipitate; S103, mixing the oat protein precipitate with pure water in a mass percentage ratio of 1:4, heating to 55°C, adjusting the pH to 9.0, adding 1.5 g of Bacillus licheniformis alkaline protease, performing enzymolysis for 3 h, and then heating to 95°C to inactivate the enzyme for 15 min to obtain a first hydrolyzate; S104, cooling the first hydrolyzate to 50°C, adjusting the pH to 5.7, adding 4 g of ficin, and performing enzymolysis for 1 h. After the enzymolysis, adjusting the pH to 4.2 with HCl solution, and then heating to 100°C and keeping boiling for 15 min to inactivate the enzyme, to obtain a second hydrolyzate; S105, cooling the second hydrolyzate to room temperature and filtering it, and then performing ultrafiltration desalination using an ultrafiltration membrane with a pore size of 2 nm to obtain an oat active polypeptide solution; S106, spray drying the oat active polypeptide solution to obtain oat active polypeptide powder.

[0024] In Examples 1 to 3 of the present invention, preferably, the protein content of the oat bran is 16%.

[0025] In Examples 1 to 3 of the present invention, preferably, the enzymatic activity of the amylase is 5000 U / g.

[0026] In Examples 1 to 3 of the present invention, preferably, the enzyme activity of the Bacillus licheniformis alkaline protease is 200,000 U / g.

[0027] In Examples 1 to 3 of the present invention, preferably, the enzymatic activity of the ficin is 100,000 U / g.

[0028] The present invention also provides the following comparative examples 1 to 7 for preparing an oat active polypeptide: Comparative Example 1 This comparative example prepared an oat active polypeptide, which differed from Example 1 in that: 3 g of ficin in step S104 was replaced by 0.6 g of trypsin, the enzymatic pH was 7.5, the enzymatic time was 1 h, and the enzyme was inactivated at 85°C for 20 min; and after the trypsin enzyme was inactivated, 1 g of aminopeptidase was added, the enzyme was inactivated at 50°C and pH=7.0 for 1 h, and then the enzyme was inactivated at 85°C for 20 min. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0029] 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.

[0030] It should be noted that, based on the protein content in oat bran, the total amount of Bacillus licheniformis alkaline protease, trypsin and aminopeptidase in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1, and the specific calculation process is as follows: In Example 1, the amount of oat bran added is 500 g, and its protein content is 16%. It is calculated that the amount of oat protein in 500 g of oat bran is 80 g; the amount of Bacillus licheniformis alkaline protease added is 1 g, and its enzyme activity is 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, which is converted to the enzyme activity of the added enzyme per unit gram weight of protein in oat bran, that is, 200,000 U / 80 g=2500 U / g; similarly, the amount of ficin added is 3 g, and its enzyme activity is 100,000 U / g. It is calculated that, based on the protein mass in oat bran, an enzyme with an enzyme activity of 3750 U / g is added; then in Example 1, based on the protein mass in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used is 6250 U / g.

[0031] In Comparative Example 1, the amount of Bacillus licheniformis alkaline protease added is 1 g, and the amount of Bacillus licheniformis alkaline protease used is 2500 U / g based on the protein mass in oat bran; the amount of trypsin added is 0.6 g, and its enzyme activity is 250,000 U / g, and the amount of trypsin used is 1875 U / g based on the protein mass in oat bran; the amount of aminopeptidase added is 1 g, and its enzyme activity is 150,000 U / g, and the amount of aminopeptidase used is 1875 U / g based on the protein mass in oat bran; then in Comparative Example 1, based on the protein mass in oat bran, the total amount of Bacillus licheniformis alkaline protease, trypsin and aminopeptidase used is 6250 U / g.

[0032] Therefore, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease, trypsin and aminopeptidase in Comparative Example 1 is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0033] Comparative Example 2 This comparative example prepared an oat active polypeptide, which differed 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 enzymolysis temperature was 37°C, and the enzymolysis pH was 2.5; and step S104 was deleted. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0034] In the comparative example of the present invention, the enzyme activity of the acidic protease is 200,000 U / g.

[0035] It should be noted that, based on the protein content in oat bran, the amount of acidic protease in this comparative example and the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1 can be regarded as equal, and the specific calculation process is as follows: In Example 1, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used was 6250 U / g.

[0036] In Comparative Example 2, the added amount of acidic protease is 2.5 g, and its enzyme activity is 200,000 U / g. Calculated based on the protein content in oat bran, the amount of acidic protease is 200,000 U / g×2.5 g / 80 g=6250 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0037] Comparative Example 3 This comparative example prepared an oat active polypeptide, which differed from Example 1 in that: 1 g of Bacillus licheniformis alkaline protease in step S103 was replaced by 1.25 g of alkaline composite protease, and the enzymatic hydrolysis pH was 6.5; and step S104 was deleted. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0038] In the comparative example of the present invention, the enzyme activity of the alkaline composite protease is 400,000 U / g.

[0039] It should be noted that, based on the protein content in oat bran, the amount of alkaline composite protease in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1, and the specific calculation process is as follows: In Example 1, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used was 6250 U / g.

[0040] In Comparative Example 3, the added amount of alkaline composite protease is 1.25 g, and its enzyme activity is 400,000 U / g. Calculated based on the protein mass in oat bran, the amount of alkaline composite protease is 400,000 U / g×1.25 g / 80 g=6250 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0041] Comparative Example 4 This comparative example prepared an oat active polypeptide, which differed from Example 1 in that: the Bacillus licheniformis alkaline protease in step S103 was replaced by Bacillus subtilis neutral protease, and the enzymatic hydrolysis pH was 7.0; 3 g of ficin in step S104 was replaced by 2 g of aminopeptidase, the enzymatic hydrolysis temperature was 50°C, the pH was 7.0, and the enzyme was inactivated at 85°C for 20 min. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0042] In the comparative example of the present invention, the enzyme activity of the Bacillus subtilis neutral protease is 200,000 U / g.

[0043] It should be noted that, based on the protein content in oat bran, the total amount of Bacillus subtilis neutral protease and aminopeptidase in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1, and the specific calculation process is as follows: In Example 1, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used was 6250 U / g.

[0044] In Comparative Example 4, the addition amount of Bacillus subtilis neutral protease is 1 g, and its enzyme activity is 200,000 U / g. According to the protein mass in oat bran, the dosage of Bacillus subtilis neutral protease is 200,000 U / g×1 g / 80 g=2500 U / g; the addition amount of aminopeptidase is 2 g, and its enzyme activity is 150,000 U / g. According to the protein mass in oat bran, the dosage of aminopeptidase is 150,000 U / g×2 g / 80 g=3750 U / g; then in Comparative Example 4, the total dosage of Bacillus subtilis neutral protease and aminopeptidase is 6250 U / g, which is equal to the total dosage of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0045] Comparative Example 5 This comparative example prepared an oat active polypeptide, which differed from Example 1 in that: 3 g of ficin in step S104 was replaced by 0.6 g of Bacillus subtilis neutral protease, the enzymatic hydrolysis pH was 6.5, and the enzyme was inactivated at 85°C for 20 min; and after the Bacillus subtilis neutral protease was inactivated, 1.2 g of aminopeptidase was added, the enzymatic hydrolysis pH was 7.0, the enzymatic hydrolysis time was 1 h, and then the enzyme was inactivated at 85°C for 20 min. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0046] It should be noted that, based on the protein content in oat bran, the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1, and the specific calculation process is as follows: In Example 1, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used was 6250 U / g.

[0047] In Comparative Example 5, the addition amount of Bacillus licheniformis alkaline protease is 1 g, and the amount of Bacillus licheniformis alkaline protease is 2500 U / g based on the protein mass in oat bran; the addition amount of Bacillus subtilis neutral protease is 0.6 g, and its enzyme activity is 200,000 U / g. Based on the protein mass in oat bran, the amount of Bacillus subtilis neutral protease is 200,000 U / g×0.6 g / 80 g=1500 U / g; the addition amount of aminopeptidase is 1.2 g, and its enzyme activity is 150,000 U / g. Based on the protein mass in oat bran, the amount of aminopeptidase is 150,000 U / g×1.2 g / 80 g=2250 U / g; then in Comparative Example 5, the total amount of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease and aminopeptidase is 6250 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0048] Comparative Example 6 This comparative example prepared an oat active polypeptide, which differed from Example 1 in that 1 g of Bacillus licheniformis alkaline protease in step S103 was changed to 2.5 g, and step S104 was deleted. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0049] It should be noted that, based on the protein content in oat bran, the amount of Bacillus licheniformis alkaline protease in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1, and the specific calculation process is as follows: In Example 1, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used was 6250 U / g.

[0050] In Comparative Example 6, the added amount of Bacillus licheniformis alkaline protease is 2.5 g. Calculated based on the protein content in oat bran, the amount of Bacillus licheniformis alkaline protease is 200,000 U / g×2.5 g / 80 g=6250 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0051] Comparative Example 7 This comparative example prepared an oat active polypeptide, which differed from Example 1 in that: Step S103 was deleted; and 3 g of ficin in Step S104 was changed to 5 g. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0052] It should be noted that, based on the protein content in oat bran, the amount of ficin used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and ficin used in Example 1, and the specific calculation process is as follows: In Example 1, based on the amount of protein in oat bran, the total amount of Bacillus licheniformis alkaline protease and ficin used was 6250 U / g.

[0053] In Comparative Example 7, the added amount of ficin is 5 g. Based on the protein content in oat bran, the amount of ficin is 100,000 U / g×5 g / 80 g=6250 U / g, which is equal to the total amount of Bacillus licheniformis alkaline protease and ficin in Example 1.

[0054] The yields of oat active polypeptides prepared in Examples 1 to 3 of the present invention and Comparative Examples 1 to 7 are shown in Table 1: Table 1 Yield of oat active polypeptides prepared in Examples 1 to 3 and Comparative Examples 1 to 7

[0055] According to the data in Table 1, it can be seen that under the same experimental conditions, the yield of Example 1 is significantly higher than that of Comparative Examples 6 and 7, while Comparative Examples 6 and 7 are oat active polypeptides prepared by hydrolysis of Bacillus licheniformis alkaline protease and ficin in Example 1, respectively, which indicates that the yield of double enzyme hydrolysis is better than that of single enzyme hydrolysis.

[0056] Furthermore, the present invention provides the following test for activity detection and peptide screening of the oat active polypeptides prepared by the present invention: Test 1 Determination of pancreatic lipase inhibition rate and α-glucosidase inhibition rate The oat active polypeptides obtained in Examples 1 to 3 and Comparative Examples 1 to 5 were prepared into a 20 mg / mL solution, and the pancreatic lipase inhibition rate and α-glucosidase inhibition rate were determined; wherein, the pancreatic lipase inhibition rate detection method was referred to "Inhibitory effect and mechanism of ethyl p-hydroxycinnamate on pancreatic lipase" (DO1: 10.13386 / j.issn1002-0306.2020060193), and the α-glucosidase inhibition rate detection method was referred to "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 starches" (DOI: 10.1016 / j.foodchem.2020.126847); the measurement data are summarized in Table 2: Table 2 Pancreatic lipase inhibition rate and α-glucosidase inhibition rate of oat active polypeptides obtained in Examples 1 to 3 and Comparative Examples 1 to 5

[0057] According to the data in Table 2, the oat active polypeptides prepared in Examples 1 to 3 and Comparative Examples 1 to 5 all have certain pancreatic lipase and α-glucosidase inhibitory abilities, but the pancreatic lipase and α-glucosidase inhibitory abilities of Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 5, among which the pancreatic lipase inhibition rate and α-glucosidase inhibition rate of the oat active polypeptide prepared in Example 1 are the highest, which are 45.13% and 34.31%, respectively.

[0058] Experiment 2: The lipid-lowering effect of oat active peptides on Caenorhabditis elegans In this experiment, a high-fat model of obese nematodes was established to conduct experiments in the normal group (NC group), model group, experimental group and positive control group; The method for establishing the high-fat model of obese nematodes is to prepare NGM culture medium containing 10 mmol / L glucose, transfer the synchronized L1 nematodes to a normal culture dish and culture them for 48 hours. NGM medium (Rui Chu Biotechnology): Peptone (peptone) 2.5 g; Sodium chloride (sodium chloride) 3.0 g; Calcium chloride (calcium chloride) 0.111 g; Cholesterol (cholesterol) 0.005 g; Agar (agar) 17.0 g; Instructions for use: Weigh 22.6 g of this product in 1 L of distilled water or deionized water, heat and boil for more than 1 min to completely dissolve, divide into packages, sterilize under high pressure at 121℃ for 15 min, cool to 50~5℃, add 1 bottle of T2245 for every 100 mL of culture medium, mix gently, divide into packages and set aside.

[0059] Normal group (NC group): 100 μL of E. coli OP50 solution was spread on NGM medium, and then 20 nematodes at the L4 stage were transferred to each culture dish and cultured at constant temperature for 48 h; Model group: 100 μL of E. coli OP50 solution was spread on NGM medium containing 10 mmol / L glucose, and then 20 nematodes at the L4 stage were transferred to each culture dish and cultured at a constant temperature for 48 h; Experimental group: The oat active polypeptide solutions with a concentration of 200 mg / mL obtained in Example 1 and Comparative Examples 1 to 5 were mixed with the activated Escherichia coli OP50 bacterial solution at a volume ratio of 1:9, so that the concentration of the oat active polypeptide in the mixed solution was 20 mg / mL, and 100 μL of each was applied to NGM medium containing 10 mmol / L glucose, and then 20 nematodes in the L4 stage were transferred to each culture dish and cultured at a constant temperature for 48 h; Positive control group: Take 1 orlistat capsule (0.12 g), dissolve it in 100 mL 2% DMSO solution to obtain an orlistat solution with a concentration of 1.2 mg / mL, and mix it with the activated Escherichia coli OP50 bacterial solution at a volume ratio of 1:9, so that the DMSO concentration is 0.2%, and the orlistat concentration is 120 μg / mL. Take 100 μL and spread it on NGM medium containing 10 mmol / L glucose, and then transfer 20 nematodes in the L4 stage to a culture dish and culture at a constant temperature for 48 h.

[0060] 2.1 Determination of nematode movement indicators Motor behavior is a rapid assessment indicator of whether the nervous system of C. elegans is damaged. In related studies, head shaking and swallowing frequency are often chosen to evaluate the motor ability of C. elegans.

[0061] After the nematodes were cultured on the NGM culture plates of each group for 48 h, the number of head swings of the nematodes within 30 s was observed, and the number of swallowing movements of the nematode pharynx within 1 min was observed under a microscope to evaluate the vitality of the nematodes.

[0062] Figure 1 This is a data analysis chart of the number of nematode head swings within 30 seconds. Figure 2 This is a data analysis chart of nematode swallowing frequency. Figure 1 and Figure 2 It can be seen that the sample containing the oat active polypeptide prepared in Example 1 is the closest to the positive control group in terms of the head swing and swallowing frequency of the nematodes in the experimental group, indicating that the oat active polypeptide in Example 1 has a significant promoting effect on the head swing and swallowing frequency of Caenorhabditis elegans.

[0063] 2.2 Oil Red O staining experiment The fat-soluble dye Oil Red O can specifically attach to neutral fat molecules and make them appear red, so the fat deposition in the nematode can be directly observed. Through Oil Red O staining, the fat deposition can be preliminarily judged. The darker the color, the more fat is deposited.

[0064] Figure 3 is the fat deposition in the nematode body, according to Figure 3 It can be seen that the nematodes of Example 1, Comparative Example 4, Comparative Example 5 and Comparative Example 6 are lighter in color, indicating that there is less fat deposition in their bodies.

[0065] 2.3 Determination of triglyceride content The nematodes at the L4 stage were transferred to each group of NGM culture dishes and cultured at a constant temperature for 48 h. The nematodes were washed three times with M9 buffer, allowed to settle naturally, the supernatant was discarded, and an appropriate amount of M9 buffer was added for resuspending. The supernatant was obtained by centrifugation after ultrasonic disruption and the triglyceride content was determined according to the instructions of the Nanjing Jiancheng Triglyceride Kit, with three parallel experiments in each group.

[0066] Figure 4 This is a data analysis chart of triglyceride content in nematodes. Figure 4 It can be seen that the triglyceride content (TG) in the nematodes treated with Example 1 was significantly reduced and lower than that of each comparative example. 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 fat synthesis in nematodes.

[0067] 2.4 Effects of oat active peptides on the expression level of lipid metabolism genes in nematodes Hangzhou Biori RNA extraction kit was used to extract total RNA from nematodes, and real-time fluorescence quantitative PCR was performed after reverse transcription. The primer sequences are listed in Table 3. The amplification conditions were 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s, 60°C annealing and extension for 34 s, for a total of 40 cycles; actin-1 was used as the internal reference gene, and the results were processed using 2-ΔΔCt.

[0068] Table 3 Primer sequence list

[0069] In Caenorhabditis elegans, the daf-2 gene is a key gene in the insulin signaling pathway that regulates the lipid metabolism and lifespan of Caenorhabditis elegans. The expression of the daf-2 gene can be used to effectively evaluate the lipid metabolism of Caenorhabditis elegans.

[0070] Figure 5 The expression of daf-2 gene in nematodes is shown in Figure 2. Figure 5 It can be seen that the expression of daf-2 gene in the experimental group is significantly lower than that in the model group, which shows that the presence of oat active polypeptide significantly down-regulated the expression of daf-2 gene, among which Example 1, Comparative Example 4 and Comparative Example 5 were the most obvious; it can be inferred that oat active polypeptide can prolong the lifespan and antioxidant capacity of Caenorhabditis elegans by regulating the expression level of upstream genes, thereby affecting the lipid accumulation in the nematode, thereby achieving the effect of lowering lipids.

[0071] A large number of studies have confirmed that fatty acid synthesis inhibitors inhibit the synthesis of fatty acids 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 the effect of lowering lipids; 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.

[0072] Figure 6 is the expression of fat-5 gene in nematodes. Figure 7 is the expression of fat-6 gene in nematodes. Figure 8 is the expression of fat-7 gene in nematodes. Figures 6-8 It can be seen that compared with the model group, oat active polypeptides can reduce the mRNA expression levels of fat-5, fat-6 and fat-7 genes, among which Example 1, Comparative Example 4 and Comparative Example 5 have the most obvious downregulation.

[0073] In summary, the oat active polypeptide provided by the present 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 the nematode body and achieving the effect of lowering lipids.

[0074] Experiment 3 Effect of oat active peptides on GLP-1 content in STC-1 cells Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic hormone secreted by the intestine, especially the terminal ileum, which has the advantages of improving insulin resistance, reducing weight, promoting the recovery of pancreatic islet function and restoring the physiological secretion of insulin. STC-1 is an endocrine cell line derived from the mouse intestine. It is an enteroendocrine cell that can secrete cholecystokinin (CCK) and GLP-1, and is currently recognized as a cell line for studying GLP-1 secretion.

[0075] 3.1 Determination of the optimal experimental concentration of oat active peptides The cell viability was determined by CCK8 (Cell Counting Kit-8) method. The results were analyzed by significance, and two concentrations of 0.08 mg / mL and 0.1 mg / mL that were non-toxic to cells were selected as the cell dosing concentrations.

[0076] 3.2 Experimental steps of the effect of oat active peptides on GLP-1 content in STC-1 cells Cell plating: STC-1 cells in good condition were seeded into 24-well plates with a plating density of 85%, 1 mL per well, and cultured in a cell culture incubator for 48 h.

[0077] Incubation with drug addition: Before adding drugs, prepare complete culture medium containing the optimal dosing concentration samples of oat active polypeptides prepared in Example 1 and Comparative Examples 1 to 5, shake and mix, and set aside; discard the original culture medium in the well plate, add 1 mL of complete culture medium containing drugs to each well, and culture at 37°C for 2 h.

[0078] Collect cell supernatant: After the drug addition and incubation, collect the supernatant in a sterile tube, centrifuge at 4°C and 1000 rpm for 20 min, and transfer the supernatant to a new sterile tube. It should be noted that if the experiment cannot be carried out in time, the samples need to be aliquoted and frozen at -80°C to avoid repeated freezing and thawing.

[0079] GLP-1 content detection: The determination was carried out according to the instructions of Nanjing Jiancheng GLP-1 Elisa kit.

[0080] 3.3 Results Analysis ANOVA statistical method was used to analyze the significant differences among different groups. Fig. 9 is the relative expression of GLP-1 content in STC-1 cells, according to Fig. 9 It can be seen that at a concentration of 0.08 mg / mL, the relative expression of GLP-1 in Example 1 is higher than that in the control group, and is increased by about 18% compared with the blank group, and is increased by about 12% compared with the control group 1 having the highest relative expression of GLP-1 among the control groups; at a concentration of 0.1 mg / mL, the relative expression of GLP-1 in Example 1, Control group 1, and Control groups 3 to 5 is significantly higher than that in the blank group, among which the relative expression of GLP-1 in Example 1 is increased by about 45% compared with the blank group, and is increased by about 13% compared with the control group 3 having the highest relative expression of GLP-1 among the control groups.

[0081] Experiment 4 Peptide screening of oat protein peptides In this experiment, the oat active polypeptide obtained in Example 1 was screened and analyzed for peptide segments.

[0082] 4.1 Mass spectrometry experimental method (1) Pretreatment In this experiment, the oat active peptide was pre-treated by reductive alkylation method, and the specific steps are as follows: S200, weigh 10.4 mg and 11.3 mg of the samples, respectively, add 100 μL of water to fully dissolve, and carry out subsequent experiments; S201. Use a pipette to accurately transfer 1 μL of 1M DTT solution to the sample to make the final concentration of DTT 10 mmol / L. Reduce in a water bath at 56°C for 1 h. S202. Use a pipette to accurately transfer 2 μL of 1M IAM solution to the sample to make the final IAM concentration 20 mmol / L. Incubate in dark at room temperature for 40 min. S203. Use a pipette to accurately transfer 1 μL of 1M DTT solution to the sample to make the final concentration of DTT 10 mmol / L to neutralize the unreacted IAM.

[0083] (2) C18 desalting (Stage-Tip) Desalt using a C18 stage-tip and dry under vacuum at 45°C.

[0084] 4.2 Computer Requirements (1) Liquid chromatography conditions 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; 2) Mobile phase A: 0.1% FA; 3) Mobile phase B: 0.1% FA, 80% ACN; 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min; 6) Specific chromatographic conditions are listed in Table 4: Table 4 Specific chromatographic conditions of liquid chromatography

[0085] (2) Mass spectrometry conditions The primary mass spectrometry parameters are: 1) Resolution: 70,000; 2) AGCtarget: 3e6; 3) MaximumIT: 100ms; 4) Scanrange: 100to1500m / z.

[0086] The secondary mass spectrometry parameters are: 1) Resolution: 17,500; 2) AGCtarget: 1e5; 3) MaximumIT: 50ms; 4) TopN: 20; 5) NCE / steppedNCE: 28.

[0087] The original data was obtained through mass spectrometry acquisition.

[0088] 4.3 Search conditions The mass spectrometry raw file uses software to search the target protein database, and the search parameters are as follows: 1) Fixed modifications: Carbamidomethyl (C); 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term); 3) Enzyme: Non specific; 4) Database: uniprotkb_Acipenser (genus)_2024_11_13; uniprotkb_Oreochromis (genus)_2024_12_05; 5) Peptide Mass Tolerance: 20 ppm; 6) Fragment Mass Tolerance: 0.02 Da.

[0089] 4.4 Results Analysis (1) Peptide screening The raw files of mass spectrometry were searched by the Byonic database software, and peptides with abundance greater than 10400000 were screened. The activity of the fragments was predicted (http: / / distilldeep.ucd.ie / PeptideRanker / ), and peptides with results > 0.5 were selected. At the same time, they were screened in combination with abundance ranking, and 10 peptides were obtained; after searching the peptide database BIOPEP (http: / / bis.zju.edu.cn / biopepdbr / index.php), the 10 peptides were all new peptides that had not been reported, and all were non-toxic after toxicity prediction (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php); the relevant information of the peptides is listed in Table 5: Table 5 Peptide information

[0090] Note: Peptide<ProteinMetrics Confidential> : The amino acid sequence of the identified peptide; Mass: the molecular weight of the peptide; Score: the activity prediction score; Lenth: the number of amino acids in the peptide; RT: the retention time of the peptide, in min; -10 lgP: the significance score of the peptide identified by database retrieval or de novo sequencing; Intensity: the abundance of the peptide.

[0091] (2) Molecular docking Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of the insulin signaling pathway, which can dephosphorylate tyrosine phosphorylated insulin receptors (IR) and downstream key proteins related to the signaling pathway to hinder insulin signal transduction. PTP1B is a therapeutic target for diabetes and obesity, and effective specific PTP1B inhibitors can become drugs for the treatment of diabetes.

[0092] Pancreatic lipase is the main lipase secreted by the pancreas, which can directly affect the intestinal absorption of fatty acids. Pancreatic lipase can hydrolyze dietary lipids in the digestive system and convert triglyceride substrates in ingested oils 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 lipase part of the pancreas and small intestine, change the conformation of trypsin, inhibit catalytic activity, and thus inhibit the hydrolysis of lipids in food, reduce the digestion and absorption of lipids and the accumulation of adipose tissue. Research and development of pancreatic lipase inhibitors is an important direction for the treatment of hyperlipidemia and the control of obesity.

[0093] Molecular docking software was used to dock the above 10 fragments with the above two enzymes, and the peptide-receptor docking binding energies were 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 / ).

[0094] Table 6 Docking binding energy of peptides and receptors

[0095] According to the data in Table 6, the 10 peptides screened out all have certain binding energies with pancreatic lipase and PTP1B. Based on the binding energies, the four peptides SGVFTPK, GCRFDRL, NSKNFPTL, and QGRGFTGL were screened out as characteristic peptide sequences with obvious hypoglycemic and lipid-lowering activities in oat active polypeptides.

[0096] Fig.10 It is represented as a 2D diagram of the docking of SGVFTPK and pancreatic lipase molecules; Fig.11 2D diagram showing the docking of GCRFDRL and pancreatic lipase molecules; Fig.12 2D diagram showing the docking of NSKNFPTL and pancreatic lipase molecules; Fig.13 It is represented as a 2D diagram of the docking of QGRGFTGL and pancreatic lipase molecule; Fig.14 2D diagram of the molecular docking of SGVFTPK and PTP1B; Fig.15 2D diagram of GCRFDRL and PTP1B molecular docking; Fig.16 2D image of the molecular docking of NSKNFPTL and PTP1B; Fig.17 2D image of the docking of QGRGFTGL and PTP1B molecules.

[0097] like Fig.10 As shown, SGVFTPK interacts with pancreatic lipase via carbon-hydrogen bonds, van der Waals forces, and covalent hydrogen bonds; Fig.11 As shown, GCRFDRL interacts with pancreatic lipase via carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds; Fig.12 As shown, NSKNFPTL interacts with pancreatic lipase via carbon-hydrogen bonds, van der Waals forces, attractive forces, and covalent hydrogen bonds; Fig.13 As shown, QGRGFTGL interacts with pancreatic lipase via carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds; like Fig.14 As shown, the interactions between SGVFTPK and PTP1B are mainly covalent hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces; Fig.15As shown, GCRFDRL interacts with PTP1B via carbon-hydrogen bonds, van der Waals forces, and attractive charges; Fig.16 As shown, the interactions between NSKNFPTL and PTP1B are mainly covalent hydrogen bonds, carbon-hydrogen bonds, van der Waals forces, and salt bridges; Fig.17 The interactions between QGRGFTGL and PTP1B are mainly covalent hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces and salt bridges.

[0098] It should be noted that the terms used in the present invention, such as ultrafiltration, centrifugation, spray drying, mass spectrometry experiment, molecular docking, RNA extraction, etc., are the names of processing steps commonly used by those skilled in the art, and their names can accurately describe the processing process and will not be repeated in the present invention.

[0099] The raw materials used in the present invention, such as Bacillus licheniformis alkaline protease, acid protease, ficin, Bacillus subtilis neutral protease, trypsin, aminopeptidase, alkaline composite protease and other enzymes, are all existing commercially available enzymes, and can be purchased by those skilled in the art; in addition, unless otherwise specified, other raw materials used in the present 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 the present invention without indicating the manufacturer and other information can also be purchased from the market.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing oat active polypeptides having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipids, characterized in that: The following steps are involved: The oat bran after the initial screening is mixed with pure water, and added into a homogenizer for homogenization to obtain an oat bran slurry; Adding amylase to the oat bran slurry for primary pretreatment, and performing enzyme inactivation treatment to obtain a primary pretreatment mixed solution; The primary pretreatment mixed solution is subjected to secondary pretreatment of alkali extraction and acid precipitation, the pH of the primary pretreatment mixed solution is adjusted to alkaline for leaching, centrifuged after leaching, the supernatant is taken, the pH is adjusted to acidic, and centrifuged to obtain oat protein precipitate; The oat protein precipitate is mixed with pure water, alkaline protease is added for primary hydrolysis, and the enzyme is inactivated after the enzymatic hydrolysis is completed to obtain a first hydrolyzate; Adding ficin to the first hydrolyzate for secondary hydrolysis, adjusting the pH to acidic after the enzymatic hydrolysis, and performing enzyme inactivation treatment to obtain a second hydrolyzate; The second hydrolyzate is centrifuged, filtered, ultrafiltered, and dried to obtain oat active polypeptides.

2. The method for preparing the oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid according to claim 1, characterized in that: The oat bran is initially screened at a mesh size of 40-60 meshes, and after the initial screening, the addition ratio of the oat bran to pure water is 1:(10-20) by mass percentage, and the homogenization time is 30-60 min.

3. The method for preparing the oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid according to claim 1, characterized in that: In the first pretreatment, the amount of amylase added is 1%-2%, the enzymolysis temperature is 45-55°C, the pH is 7.0, the reaction time is 0.5-1.5 h, the temperature of enzyme inactivation treatment is 85-90°C, and the treatment time is 15-20 min; During the secondary pretreatment, the alkaline extraction pH was 9.5~10.5, the extraction time was 60~120 min, the acid precipitation pH was 3.8~4.2, the precipitation time was 10~20 min, the treatment temperature was 50~55℃, the centrifugal speed was 4000~6000 rpm, and the centrifugal time was 5~10 min.

4. The method for preparing the oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid according to claim 1, characterized in that: During the primary hydrolysis, the ratio of the amount of the oat protein precipitate to the amount of pure water added is 1:(2-4) by mass percentage, the amount of the alkaline protease added is 0.1%-0.3% of the mass of the oat bran, the enzymatic hydrolysis temperature is 50-55°C, the pH is 8.0-9.0, the reaction time is 1-3 h, the temperature of the enzyme inactivation treatment is 85°C, and the treatment time is 15-20 min; During the secondary hydrolysis, the added amount of the ficin is 0.5%-0.8% of the mass of the oat bran, the enzymolysis temperature is 50-55°C, 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 temperature of the enzyme inactivation treatment is 100°C, and the treatment time is 10-15 min.

5. The method for preparing the oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid according to claim 4, characterized in that: During the primary hydrolysis, the alkaline protease is Bacillus licheniformis alkaline protease; During the secondary hydrolysis, after the enzymatic reaction is completed, HCl solution is used to adjust the pH to acidic and then the enzyme is inactivated.

6. The method for preparing the oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid according to claim 1, characterized in that: The ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 1 to 2 nm.

7. The method for preparing the oat active polypeptide having the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid according to claim 1, characterized in that: The drying is spray drying to obtain oat active polypeptide powder.

8. An oat active polypeptide obtained by the method for preparing an oat active polypeptide having GLP-1 secretion promoting, blood sugar lowering and lipid lowering activities according to any one of claims 1 to 7, characterized in that: The invention comprises the following oat active peptide segments: the amino acid sequence of which is 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.

9. A functional product, characterized in that: Its components include the oat active polypeptide with the activities of promoting GLP-1 secretion, lowering blood sugar and lowering blood lipid as described in claim 8.

10. The functional product according to claim 9, characterized in that: Include at least one of the following functions: (1) Promote GLP-1 secretion; (2) Inhibition of PTP1B; (3) Inhibit pancreatic lipase; (4) Blood sugar lowering; (5) Reduces lipids.

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