A hypoglycemic antioxidant oat peptide composition, preparation method and application thereof

Abstract

This invention discloses an oat peptide composition with hypoglycemic and antioxidant properties, its preparation method, and its applications, relating to the field of bioactive peptide technology. The oat peptide composition is prepared from oat bran and comprises: a first active peptide component with a molecular weight of 300-1000 Da, comprising 45%-60% of the total mass of the oat peptide composition; a second active peptide component with a molecular weight of 1000-3000 Da, comprising 30%-40% of the total mass of the oat peptide composition; the first active peptide component exhibiting a half-maximal inhibitory concentration (IC50) of not more than 0.5 mg / mL against α-glucosidase; and the second active peptide component exhibiting a half-maximal inhibitory concentration (IC50) of not more than 1.0 mg / mL against DPPH free radical scavenging. This invention achieves a balance and synergy of dual activities, and the correlation between molecular weight distribution and activity parameters makes the product characteristics clearer.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to an oat peptide composition with hypoglycemic and antioxidant properties, its preparation method, and its application. Background Technology

[0002] Oats, a common grain, are rich in nutrients such as protein, dietary fiber, and beta-glucan. Studies have shown that oat peptides, produced by enzymatic hydrolysis of oat protein, possess various biological activities, such as antioxidant, blood sugar-lowering, and blood pressure-lowering effects, and have broad application prospects in the field of functional foods and health products.

[0003] Currently, there are technologies available regarding oat bioactive peptides and their preparation methods. However, the following technical problems still need to be solved: Dual activities are difficult to achieve simultaneously: Current methods for preparing oat peptides mostly focus on a single activity (antioxidant or hypoglycemic), making it difficult to obtain products with both high hypoglycemic and antioxidant activities. Studies have shown that peptides in different molecular weight ranges often have different bioactivity preferences; peptides in the 300-1000 Da range are more likely to exhibit α-glucosidase inhibitory activity, while peptides in the 1000-3000 Da range tend to exhibit antioxidant activity. However, current technologies have not been able to target this pattern for specific preparation.

[0004] Severe enzyme activity loss and membrane fouling: Oat bran contains a large amount of β-glucan and starch, which are released into the solution during enzymatic hydrolysis, forming a high-viscosity system. In the continuous preparation process of the enzyme membrane reactor, the shear force of the peristaltic pump and the adsorption of the ultrafiltration membrane lead to significant loss of enzyme activity. At the same time, the viscous polysaccharides easily cause membrane fouling, affecting production efficiency and product quality.

[0005] Difficulty in controlling molecular weight distribution: Current technology struggles to precisely control the proportion of peptides with different molecular weights in the product, leading to poor batch-to-batch consistency and unstable activity. In particular, it is difficult to simultaneously obtain high proportions of 300-1000 Da and 1000-3000 Da peptides.

[0006] Low raw material utilization: The large molecular peptides generated during enzymatic hydrolysis are usually treated as waste, resulting in raw material waste and increased production costs.

[0007] To address the above problems, this invention provides an oat peptide composition and its preparation method that can simultaneously achieve both hypoglycemic and antioxidant activities, have a high yield, and are suitable for industrial production. Summary of the Invention

[0008] The purpose of this invention is to provide an oat peptide composition with hypoglycemic and antioxidant properties, its preparation method, and its application, in order to solve the problems of difficulty in achieving dual activities, large enzyme activity loss, serious membrane fouling, and low raw material utilization in the current technology.

[0009] To achieve the above objectives, the present invention provides an oat peptide composition with hypoglycemic and antioxidant properties, prepared from oat bran, comprising: The first active peptide component has a molecular weight of 300-1000 Da; the content of the first active peptide component accounts for 45%-60% of the total mass of the oat peptide composition; A second active peptide component with a molecular weight of 1000-3000 Da; the content of the second active peptide component accounts for 30%-40% of the total mass of the oat peptide composition; The half-maximal inhibitory concentration (IC50) of the first active peptide component against α-glucosidase 50 The concentration of the second active peptide component is not higher than 0.5 mg / mL; the half-maximal inhibitory concentration (IC50) of the second active peptide component for scavenging DPPH free radicals is not higher than 0.5 mg / mL. 50 (Not higher than 1.0 mg / mL)

[0010] Preferably, the half-maximal inhibitory concentration (IC50) of the α-glucosidase is determined using p-nitrophenyl-α-D-glucopyranoside as a substrate at a temperature of 37°C and a pH of 6.8; the DPPH free radical scavenging rate is determined using the DPPH working solution method under light-protected reaction conditions for 30 min.

[0011] Preferably, the first active peptide component contains a peptide derived from oat bran and possessing α-glucosidase inhibitory activity, identified by liquid chromatography-tandem mass spectrometry; the second active peptide component contains a peptide derived from oat bran and possessing DPPH free radical scavenging activity, identified by liquid chromatography-tandem mass spectrometry.

[0012] This invention provides a method for preparing the above-mentioned oat peptide composition with hypoglycemic and antioxidant properties, comprising the following steps: S1. Mix defatted oat bran with water, adjust the pH to 8.5-9.5, stir and separate the solid and liquid at 45-55℃, collect the supernatant after solid-liquid separation, adjust the pH of the supernatant to 4.0-5.0 and then perform isoelectric point precipitation, collect the precipitate by centrifugation, and reconstitute to obtain oat protein solution. S2. Adjust the temperature of the oat protein solution to 50-60℃ and the pH value to 8.0-9.0. Add alkaline protease and hydrolyze for 2-4 hours. Then adjust the pH value to 6.0-7.0 and add flavor protease to continue hydrolysis for 1-3 hours to obtain the hydrolysate. S3. Within 0.5-1.5 hours after the start of flavor protease hydrolysis, β-glucanase and fungal amylase are added simultaneously, and the enzymatic hydrolysis reaction is carried out at 45-55℃ for 1-2 hours to obtain purified enzymatic hydrolysate. S4. Separate the purified enzyme hydrolysate sequentially through a microfiltration membrane and an ultrafiltration membrane system: A. The purified enzymatic hydrolysate is passed through a microfiltration membrane with a pore size of 0.1-0.5 μm, and the permeate is collected. B. Pass the microfiltration permeate through a first ultrafiltration membrane with a molecular weight cutoff of 3000 Da, collect the first permeate, and simultaneously concentrate the first retentate online and recycle it to step S2 for deep enzymatic hydrolysis. C. Pass the first permeate through a second ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and collect the second retentate and the second permeate respectively; the second retentate is the second active peptide component with a molecular weight of 1000-3000 Da; the second permeate is the crude peptide solution with a molecular weight of less than 1000 Da. S5. Dry the second retentate to obtain the second active peptide component; pass the second permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da for desalting and concentration, collect the nanofiltration retentate, and dry it to obtain the first active peptide component.

[0013] This invention employs a stepwise enzymatic hydrolysis strategy, first using alkaline protease followed by flavor protease. This allows the alkaline protease to preferentially cleave hydrophobic amino acid sites, generating a large number of hydrophobic peptides with antioxidant potential. Then, the flavor protease directionally modifies amide-containing amino acids, improving bitterness while releasing hypoglycemic peptides. Furthermore, the stepwise hydrolysis strategy allows for a natural pH transition, eliminating the need for additional acid-base adjustments, thus aligning with green chemical engineering principles. This invention simultaneously adds β-glucanase and fungal amylase within 0.5-1.5 hours after the start of flavor protease hydrolysis. This avoids both the competitive inhibition of protease activity caused by premature addition of glycosylases and the viscosity increase problem caused by polysaccharide accumulation due to delayed addition.

[0014] This invention constructs a four-stage membrane separation system that concentrates the retentate with a protein concentration greater than 3000 Da to 5%-10% online and then recycles it to the enzymatic hydrolysis process. This allows large molecular peptides, which were originally waste, to have multiple opportunities to be cleaved and transformed into target small molecular peptides. This not only improves the utilization rate of raw materials but also enables precise control of the molecular weight range of the products.

[0015] Preferably, the amount of alkaline protease added in S2 is 1.5%-3.0% of the mass of the oat protein solution; the amount of flavor protease added is 1.0%-2.5% of the mass of the oat protein solution.

[0016] Preferably, the operating pressure of the microfiltration membrane in S4 is 0.1-0.3 MPa; the operating pressure of the first ultrafiltration membrane and the second ultrafiltration membrane is 0.3-0.6 MPa; and the protein concentration of the first retentate after online concentration is 5%-10%.

[0017] Preferably, the amount of β-glucanase added in S3 is 0.1%-0.5% of the mass of the enzymatic hydrolysate; the amount of fungal amylase added is 0.2%-0.6% of the mass of the enzymatic hydrolysate, and the mass ratio of β-glucanase to fungal amylase is 1:1.2-2.

[0018] Preferably, the pH value of the enzymatic hydrolysis reaction in S3 is 6.0-7.0; the viscosity of the reaction system is continuously monitored during the enzymatic hydrolysis reaction, and the reaction is terminated when the relative viscosity of the reaction system decreases to 30%-50% of the initial viscosity. This invention introduces the relative viscosity of the system as a criterion for determining the reaction endpoint, precisely controlling the degree of enzymatic hydrolysis by real-time monitoring of viscosity changes, ensuring the consistency and activity stability of the product, and achieving precise process control. Simultaneously, viscosity control also creates favorable conditions for subsequent membrane separation, significantly reducing the risk of membrane fouling.

[0019] This invention provides the application of the above-mentioned oat peptide composition with hypoglycemic and antioxidant properties in the preparation of in vitro reagents for inhibiting α-glucosidase.

[0020] The present invention also provides the application of the above-mentioned oat peptide composition with hypoglycemic and antioxidant properties in the preparation of an antioxidant in vitro reagent for scavenging DPPH free radicals.

[0021] In summary, the oat peptide composition, preparation method, and application provided by this invention, which have hypoglycemic and antioxidant properties, offer the following advantages compared to traditional technologies: (1) This invention employs a specific molecular weight distribution design, in which the first active peptide component with a molecular weight of 300-1000 Da accounts for 45%-60% of the total mass of the oat peptide composition, and the second active peptide component with a molecular weight of 1000-3000 Da accounts for 30%-40% of the total mass of the oat peptide composition. This design allows the first peptide component to focus on hypoglycemic activity, while the second peptide component focuses on antioxidant activity, achieving a balance and synergy of dual activities. This correlation between molecular weight distribution and activity parameters makes the product characteristics clearer.

[0022] (2) The oat peptide composition of the present invention, which has the functions of lowering blood sugar and anti-oxidation, contains 75%-100% target peptides, has a clear active peptide sequence, and has small batch-to-batch differences, making it suitable for industrial production. Furthermore, activity verification shows that the half-maximal inhibitory concentration (IC50) of the first peptide component against α-glucosidase can reach 0.32 mg / mL, and the half-maximal inhibitory concentration (IC50) of the second peptide component against DPPH free radical scavenging can reach 0.76 mg / mL.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1This is a flowchart of the preparation method of the oat peptide composition with hypoglycemic and antioxidant properties in this invention; Figure 2 This is a molecular weight distribution diagram of the oat peptide composition with hypoglycemic and antioxidant properties in this invention. Figure 3 Figure showing the results of an experiment on the use of zebrafish to assist in lowering blood sugar. Figure 4 A graph showing the changes in fluorescence intensity in the yolk sac of zebrafish; Figure 5 The image shows the results of fluorescence intensity changes in the yolk sac of zebrafish. Detailed Implementation

[0025] The technical method of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the scope of this application and its application or use.

[0027] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. All materials and reagents used in this invention are commercially available products.

[0028] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0029] This invention provides a method for preparing an oat peptide composition with hypoglycemic and antioxidant properties, such as... Figure 1 As shown, the process includes: raw material pretreatment, stepwise enzymatic hydrolysis with two enzymes, synergistic enzymatic hydrolysis-impurity removal coupled treatment, multi-stage coupled membrane separation and recycling, and post-fractionation treatment. Specific embodiments are as follows: Example 1 A method for preparing an oat peptide composition with hypoglycemic and antioxidant properties includes the following steps: (1) Raw material pretreatment: Take 1 kg of defatted oat bran, add 10 L of water (solid-to-material ratio 1:10), adjust the pH to 9.0 with 1 mol / L NaOH solution, extract by stirring at 50℃ for 2 h, centrifuge at 4000 rpm for 15 min and collect the supernatant. Then adjust the pH of the supernatant to 4.5 with 1 mol / L HCl solution, let stand for 1 h for isoelectric point precipitation, centrifuge at 4000 rpm for 15 min and collect the precipitate, redissolve in 5 L of water to obtain oat protein solution. The protein concentration of the oat protein solution was determined by Kjeldahl method to be approximately 2.5%.

[0030] (2) Stepwise enzymatic hydrolysis with two enzymes: Adjust the temperature of the oat protein solution to 55°C and the pH value to 8.5, add 25g of alkaline protease (enzyme addition amount is 2.0%, based on the mass of oat protein solution), and enzymatically hydrolyze for 3h; then adjust the pH value of the system to 6.5, add 20g of flavor protease (enzyme addition amount is 1.6%, based on the mass of oat protein solution), and continue enzymatic hydrolysis for 2h to obtain the hydrolysate.

[0031] (3) Synergistic enzymatic hydrolysis-impurity removal coupling treatment: 1.0 h after the start of flavor protease enzymatic hydrolysis, 3 g β-glucanase and 5 g fungal amylase (mass ratio of β-glucanase to fungal amylase is 1:1.67) were added to the enzymatic hydrolysis system simultaneously. The enzymatic hydrolysis reaction was continued at 50 °C. The viscosity of the system was continuously monitored during the process. When the relative viscosity decreased to 40% of the initial viscosity (about 1.5 h), the reaction was terminated to obtain the purified enzymatic hydrolysate.

[0032] (4) Multi-stage coupling membrane separation and recycling: The purified enzyme hydrolysate is passed through a 0.2 μm microfiltration membrane (operating pressure of 0.2 MPa), and the microfiltration permeate is collected.

[0033] The microfiltration permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da (operating pressure of 0.4 MPa) to collect the first permeate. At the same time, the first retentate is concentrated online to a protein concentration of 8% and then recycled to the two-enzyme stepwise enzymatic hydrolysis process in step (2).

[0034] The first permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da (operating pressure of 0.4 MPa), and the second retentate and the second permeate are collected separately.

[0035] (5) Post-fractionation: The second retentate was freeze-dried at -50℃ for 24h to obtain 32.5g of the second active peptide component with a molecular weight of 1000-3000Da, with a yield of 3.25% (based on the dry weight of oat bran).

[0036] The second permeate was desalted and concentrated by passing it through a nanofiltration membrane with a molecular weight cutoff of 300 Da (operating pressure of 1.0 MPa). The nanofiltration retentate was collected and freeze-dried under vacuum at -50°C for 24 h to obtain 51.2 g of the first active peptide component with a molecular weight of 300-1000 Da, with a yield of 5.12% (based on the dry weight of oat bran).

[0037] High-performance gel filtration chromatography (HPGPC) analysis revealed that peptides with a molecular weight of 300-1000 Da accounted for 92% of the total mass of the first active peptide component, while peptides with a molecular weight of 1000-3000 Da accounted for 88% of the total mass of the second active peptide component. Based on the total mass of the composition, the first active peptide component accounted for 61.2%, and the second active peptide component accounted for 38.8%.

[0038] The sequences of the first and second active peptide components were identified using liquid chromatography-tandem mass spectrometry. The results are as follows: Figure 2 The first active peptide component contains multiple peptides with molecular weights ranging from 300 to 1000 Da. Some of these peptides, after being searched in a database, match peptides derived from oat storage proteins and exhibit α-glucosidase inhibitory activity. The second active peptide component contains multiple peptides with molecular weights ranging from 1000 to 3000 Da. Some of these peptides exhibit DPPH free radical scavenging activity.

[0039] Example 2 The difference between this embodiment and Example 1 is that the mass ratio of β-glucanase to fungal amylase in step (3) is 1:1.2. The viscosity of the system is continuously monitored during the enzymatic hydrolysis process, and the reaction is terminated when the relative viscosity decreases to 30% of the initial viscosity.

[0040] The yield of the first active peptide component was 4.85%, the yield of the second active peptide component was 3.12%, and the total yield was 7.97%.

[0041] Example 3 The difference between this embodiment and embodiment 1 is that the mass ratio of β-glucanase to fungal amylase in step (3) is 1:2. The viscosity of the system is continuously monitored during the enzymatic hydrolysis process, and the reaction is terminated when the relative viscosity decreases to 50% of the initial viscosity.

[0042] The yield of the first active peptide component was 4.62%, the yield of the second active peptide component was 2.95%, and the total yield was 7.57%.

[0043] Comparative Example 1 The difference between this comparative example and Example 1 is that the synergistic enzymatic hydrolysis-impurity removal coupling treatment in step (3) is not performed, that is, the membrane separation in step (4) is performed directly after the flavor protease hydrolysis for 2 hours.

[0044] Results: Both the microfiltration and ultrafiltration membranes showed significant clogging after 30 minutes of operation, with membrane flux decreasing to 35% of the initial value. The final yield of the first active peptide component was only 2.86%, the yield of the second active peptide component was 1.92%, and the total yield was 4.78%. Furthermore, molecular weight distribution analysis showed that the final product contained a large amount of polysaccharide impurities larger than 3000 Da.

[0045] The yields of the first and second active peptide components, the total yield, and the half-maximal inhibitory concentration (IC50) of the first active peptide component against α-glucosidase in the examples and comparative examples are as follows: 50 The half-maximal inhibitory concentration (IC50) of the second active peptide component in terms of its ability to scavenge DPPH free radicals. 50 The membrane flux retention rate and the membrane flux retention rate are shown in Table 1.

[0046] Table 1. Yields of the first active peptide component, the second active peptide component, the total yield, and the half-maximal inhibitory concentration (IC50) of the first active peptide component against α-glucosidase in the examples and comparative examples. 50 The half-maximal inhibitory concentration (IC50) of the second active peptide component in terms of its ability to scavenge DPPH free radicals. 50 ) and membrane flux retention data table

[0047] As shown in Table 1, compared with Comparative Example 1 (without synergistic enzymatic hydrolysis-impurity removal coupling treatment), the total yield of Example 1 increased from 4.78% to 8.37%, an improvement of 75.1%; the IC50 of the first active peptide component against α-glucosidase... 50 The concentration decreased from 0.68 mg / mL to 0.32 mg / mL, resulting in a 2.1-fold increase in activity; the IC50 of the second active peptide component's ability to scavenge DPPH free radicals... 50 The concentration was reduced from 1.35 mg / mL to 0.76 mg / mL, resulting in a 1.8-fold increase in activity; membrane flux retention increased from 35% to 92%. This demonstrates that the absence of synergistic enzymatic hydrolysis-decontamination coupling treatment leads to severe membrane fouling and decreased activity, proving the necessity of the synergistic enzymatic hydrolysis-decontamination coupling treatment in this invention. Comparison of Examples 1-3 shows that a mass ratio of β-glucanase to fungal amylase within the range of 1:1.2-2 yields good results, with total yields between 7.57% and 8.37% and membrane flux retention rates between 85% and 92%, with Example 1 showing the best performance.

[0048] Following conventional methods, the oat peptide composition prepared in Example 1 was formulated into in vitro reagents for inhibiting α-glucosidase at concentrations of 0.003125%, 0.00625%, and 0.0125% (oat peptide powder with a concentration of 0.003125%, oat peptide powder with a concentration of 0.00625%, and oat peptide powder with a concentration of 0.0125%). These reagents, along with a 0.04% metformin solution, were used as the experimental group in a zebrafish-assisted hypoglycemic assay. The results are as follows... Figure 3 As shown in Table 2, the normal control group had normal blood glucose levels, while the model control group and the experimental group were both hyperglycemic groups with the same blood glucose levels. Figure 3 It can be seen that the in vitro reagents for inhibiting α-glucosidase at concentrations of 0.003125%, 0.00625%, and 0.0125% significantly reduced blood glucose levels compared with the model control group, indicating that the in vitro reagents for inhibiting α-glucosidase at concentrations of 0.003125%, 0.00625%, and 0.0125% have an auxiliary hypoglycemic effect.

[0049] Table 2. Blood Glucose Level Detection Results

[0050] Note: A p-value < 0.05 is considered statistically significant.

[0051] Following conventional methods, the oat peptide composition prepared in Example 1 was formulated into in vitro antioxidant reagents for scavenging DPPH free radicals at concentrations of 0.000078125%, 0.00015625%, and 0.0003125% (oat peptide powder with a concentration of 0.000078125%, oat peptide powder with a concentration of 0.00015625%, and oat peptide powder with a concentration of 0.0003125%). These reagents, along with a reagent containing 0.00625% N-acetyl-L-cysteine ​​(NAC), were used as the experimental group to verify the antioxidant activity by analyzing the changes in fluorescence intensity in the yolk sac of zebrafish. The fluorescence images are shown below. Figure 4 As shown in Table 3, the normal control group used normal zebrafish yolk sacs, while the model control group and experimental group both used zebrafish yolk sacs treated with menadione. Figure 5 It can be seen that the fluorescence intensity of the in vitro antioxidant reagents for scavenging DPPH free radicals at concentrations of 0.003125%, 0.00625%, and 0.0125% was significantly reduced compared with the model control group, indicating that the in vitro antioxidant reagents for scavenging DPPH free radicals at concentrations of 0.003125%, 0.00625%, and 0.0125% have antioxidant properties.

[0052] Table 3. Results of fluorescence intensity detection in zebrafish yolk sacs

[0053] Note: A p-value < 0.05 is considered statistically significant.

[0054] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An oat peptide composition with hypoglycemic and antioxidant properties, characterized in that, The oat peptide composition is prepared from oat bran and includes the following components: The first active peptide component has a molecular weight of 300-1000 Da; the content of the first active peptide component accounts for 45%-60% of the total mass of the oat peptide composition; A second active peptide component with a molecular weight of 1000-3000 Da; the content of the second active peptide component accounts for 30%-40% of the total mass of the oat peptide composition; The half-maximal inhibitory concentration (IC50) of the first active peptide component against α-glucosidase is not higher than 0.5 mg / mL; the half-maximal inhibitory concentration (IC50) of the second active peptide component in scavenging DPPH free radicals is not higher than 1.0 mg / mL.

2. The oat peptide composition with hypoglycemic and antioxidant properties according to claim 1, characterized in that, The half-maximal inhibitory concentration (IC50) of the α-glucosidase was determined using p-nitrophenyl-α-D-glucopyranoside as a substrate at 37°C and pH 6.

8. The DPPH free radical scavenging rate was determined using the DPPH working solution method under light-protected reaction conditions for 30 min.

3. The oat peptide composition with hypoglycemic and antioxidant properties according to claim 1, characterized in that, The first active peptide component contains a peptide derived from oat bran and possessing α-glucosidase inhibitory activity, identified by liquid chromatography-tandem mass spectrometry; the second active peptide component contains a peptide derived from oat bran and possessing DPPH free radical scavenging activity, identified by liquid chromatography-tandem mass spectrometry.

4. A method for preparing an oat peptide composition with hypoglycemic and antioxidant properties as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Mix defatted oat bran with water, adjust the pH to 8.5-9.5, stir and separate the solid and liquid at 45-55℃, collect the supernatant after solid-liquid separation, adjust the pH of the supernatant to 4.0-5.0 and then perform isoelectric point precipitation, collect the precipitate by centrifugation, and reconstitute to obtain oat protein solution. S2. Adjust the temperature of the oat protein solution to 50-60℃ and the pH value to 8.0-9.

0. Add alkaline protease and hydrolyze for 2-4 hours. Then adjust the pH value to 6.0-7.0 and add flavor protease to continue hydrolysis for 1-3 hours to obtain the hydrolysate. S3. Within 0.5-1.5 hours after the start of flavor protease hydrolysis, β-glucanase and fungal amylase are added simultaneously, and the enzymatic hydrolysis reaction is carried out at 45-55℃ for 1-2 hours to obtain purified enzymatic hydrolysate. S4. Separate the purified enzyme hydrolysate sequentially through a microfiltration membrane and an ultrafiltration membrane system: A. The purified enzymatic hydrolysate is passed through a microfiltration membrane with a pore size of 0.1-0.5 μm, and the permeate is collected. B. Pass the microfiltration permeate through a first ultrafiltration membrane with a molecular weight cutoff of 3000 Da, collect the first permeate, and simultaneously concentrate the first retentate online and recycle it to step S2 for deep enzymatic hydrolysis. C. Pass the first permeate through a second ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and collect the second retentate and the second permeate respectively; the second retentate is the second active peptide component with a molecular weight of 1000-3000 Da; the second permeate is the crude peptide solution with a molecular weight of less than 1000 Da. S5. Dry the second retentate to obtain the second active peptide component; pass the second permeate through a nanofiltration membrane with a molecular weight cutoff of 300 Da for desalting and concentration, collect the nanofiltration retentate, and dry it to obtain the first active peptide component.

5. The method for preparing the oat peptide composition with hypoglycemic and antioxidant properties according to claim 4, characterized in that, The amount of alkaline protease added in S2 is 1.5%-3.0% of the mass of the oat protein solution; the amount of flavor protease added is 1.0%-2.5% of the mass of the oat protein solution.

6. The method for preparing the oat peptide composition with hypoglycemic and antioxidant properties according to claim 4, characterized in that, The operating pressure of the microfiltration membrane in S4 is 0.1-0.3 MPa; the operating pressure of the first ultrafiltration membrane and the second ultrafiltration membrane is 0.3-0.6 MPa; and the protein concentration of the first retentate after online concentration is 5%-10%.

7. The method for preparing the oat peptide composition with hypoglycemic and antioxidant properties according to claim 4, characterized in that, The amount of β-glucanase added in S3 is 0.1%-0.5% of the mass of the enzymatic hydrolysate; the amount of fungal amylase added is 0.2%-0.6% of the mass of the enzymatic hydrolysate, and the mass ratio of β-glucanase to fungal amylase is 1:1.2-2.

8. The method for preparing the oat peptide composition with hypoglycemic and antioxidant properties according to claim 4, characterized in that, The pH value of the enzymatic hydrolysis reaction described in S3 is 6.0-7.0; the viscosity of the reaction system is continuously monitored during the enzymatic hydrolysis reaction, and the reaction is terminated when the relative viscosity of the reaction system decreases to 30%-50% of the initial viscosity.

9. The use of an oat peptide composition having hypoglycemic and antioxidant properties as described in any one of claims 1-3 in the preparation of an in vitro reagent for inhibiting α-glucosidase.

10. The use of an oat peptide composition having hypoglycemic and antioxidant properties as described in any one of claims 1-3 in the preparation of an antioxidant in vitro reagent for scavenging DPPH free radicals.

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