Small molecule active peptide with antioxidant and blood sugar lowering effects, composition and use

Abstract

The present invention discloses small molecule active peptides, compositions and uses with antioxidant and hypoglycemic effects, and belongs to the technical field of bioactive peptides. The amino acid sequence of the small molecule active peptide with antioxidant and hypoglycemic effects of the present invention is at least one of FSNAPRL, QFGRLY and SVPGHF. The present invention extracts and obtains multiple small molecule active peptides with antioxidant and hypoglycemic effects from walnut meal, performs molecular docking and chemical synthesis on the obtained peptide sequences, and verifies the antioxidant activity and hypoglycemic activity of the single peptide segment and the composite peptide segment of the synthetic peptide segment. The active peptide and its composite peptide segment have good DPPH and ABTS clearance rates and α-glucosidase and α-amylase inhibition capabilities. Therefore, they have good application prospects in the preparation of products with antioxidant and hypoglycemic effects, and lay a foundation for the comprehensive utilization of walnut meal by-products and the promotion of the research and development and application of functional active peptides.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and relates to small molecule active peptides with antioxidant and hypoglycemic effects, compositions and uses. Background Art

[0002] Walnut is one of the main economic tree species in China, and its planting area and annual output are both the highest in the world. Walnut is mainly used for oil extraction. The walnut meal after oil extraction is rich in nutritional value, including rich protein (more than 40%), dietary fiber and phenolic substances. However, the walnut meal after oil extraction is not fully utilized, resulting in a large amount of resource waste.

[0003] Antioxidants are substances that can effectively inhibit free radical oxidation reactions. At present, most of the antioxidants mainly used are chemically synthesized, such as L-ascorbic acid and its sodium salt, isoascorbic acid and its sodium salt. Hypoglycemia mainly relies on drug treatment, such as glimepiride, metformin, acarbose, sitagliptin, repaglinide, etc. In addition, blood sugar can also be improved through diet. However, chemical synthetic reagents are less safe for the human body, and the method of regulating self-function through diet is slow to take effect. Therefore, it is urgent to develop green, safe, and highly active antioxidant hypoglycemic products. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a small molecule active peptide with antioxidant and blood sugar lowering effects, a composition and a use thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A small molecule active peptide with antioxidant and blood sugar lowering effects, wherein the amino acid sequence of the small molecule active peptide is at least one of FSNAPRL, QFGRLY, and SVPGHF.

[0007] The use of the above-mentioned small molecule active peptide with antioxidant and blood sugar lowering effects in the preparation of drugs with antioxidant and blood sugar lowering functions.

[0008] A small molecule active peptide composition with antioxidant and blood sugar lowering effects, the small molecule active peptide composition comprises at least one of the above-mentioned active peptides with antioxidant and blood sugar lowering effects.

[0009] Based on the above scheme, the small molecule active peptide composition is any two of FSNAPRL, QFGRLY and SVPGHF.

[0010] Based on the above scheme, the mass ratio of the two active peptides in the small molecule active peptide composition is 1:1.

[0011] Based on the above scheme, the antioxidant has the ability to scavenge free radicals.

[0012] Based on the above scheme, the free radical is at least one of DPPH and ABTS.

[0013] On the basis of the above scheme, the blood sugar lowering effect is to inhibit α-glucosidase and / or α-amylase.

[0014] The use of the above-mentioned small molecule active peptide composition with antioxidant and hypoglycemic effects in the preparation of drugs with antioxidant and hypoglycemic effects.

[0015] Based on the above scheme, the medicine further comprises pharmaceutically acceptable excipients.

[0016] The method for extracting small molecule active peptides with antioxidant and hypoglycemic effects from walnut meal is as follows:

[0017] 1. First, crush the walnut meal, add distilled water to mix, stir evenly and adjust its pH value to 10, then shake at 45℃, 150 r / min for 30 min; take out the solution and centrifuge, discard the oil layer and filter the supernatant; collect the supernatant and adjust its pH value to 4.5 for acid precipitation, wash it with distilled water 3 times during the precipitation process, finally centrifuge, discard the supernatant and retain the precipitate, and freeze-dry to obtain walnut protein powder;

[0018] 2. Prepare walnut protein solution, heat at 90℃ for 10 min for pre-denaturation, then add protease for enzymatic hydrolysis. During the process, stir the protein solution continuously and use 0.1 M NaOH solution to maintain a constant pH of the system; after the enzymatic hydrolysis is completed, inactivate the enzyme, cool and centrifuge, and filter the supernatant;

[0019] 3. Dilute the walnut protein solution after enzymatic hydrolysis, first pass it through a 0.45 μm filter membrane, and then through a 0.22 μm filter membrane; use membrane ultrafiltration with a molecular weight of 10 KDa and 3 KDa, the ultrafiltration centrifugal force is 8000 r / min, the operating temperature is 25°C, and the time is 15 min and 40 min respectively; after the ultrafiltration is completed, the filtrate is freeze-dried to obtain walnut active peptide powders with molecular weights >10 KDa, 3-10 KDa and <3 KDa, and 2 mg / mL sample peptide solution is prepared to determine the free radical scavenging ability and blood sugar inhibition effect of active peptides with molecular weights >10 KDa, 3-10 KDa and <3 KDa; select active peptides with a molecular weight of <3 KDa for further identification;

[0020] 4. The active peptides with the most antioxidant and hypoglycemic activity and the molecular weight less than 3 KDa separated above were screened and analyzed by liquid chromatography-mass spectrometry technology to identify the structure of the active peptides. The Easy-nLC high performance liquid chromatography system and RSLC C18 column were used to separate the walnut polypeptide peak with the most antioxidant and hypoglycemic activity and the molecular weight less than 3 KDa;

[0021] 5. Use the Peptide Ranker program to predict the potential biological activity of the peptides in the walnut meal protein hydrolysate obtained in step 4 (0-1), select peptides with a score > 0.5, and use the ToxinPred3.0 website to select peptides with a toxicity threshold prediction of < 0.4, and obtain the small molecule active peptides FSNAPRL, QFGRLY, and SVPGHF.

[0022] Based on the above scheme, the protease is at least one of neutral protease, alkaline protease, papain and trypsin; preferably, it is alkaline protease.

[0023] On the basis of the above scheme, the enzymatic hydrolysis conditions are: enzyme addition amount 0.5%-2%, enzymatic hydrolysis time 1h-4h, solid-liquid ratio 0.1-0.5; preferably, enzyme addition amount 1.25%, enzymatic hydrolysis time 2.5h, solid-liquid ratio 0.3.

[0024] Advantages of the technical solution of the present invention:

[0025] The present invention extracts protein from walnut meal and separates and purifies multiple small molecule active peptides with antioxidant and hypoglycemic effects through enzymatic hydrolysis, ultrafiltration and other technologies, and preliminarily identifies its components and sequences through liquid chromatography-mass spectrometry, peptide Ranker and molecular docking technology. Chemical synthesis is performed according to the identified peptide sequence, and the antioxidant activity and hypoglycemic activity of the single peptide segment and the composite peptide segment of the synthetic peptide segment are detected. The small molecule active peptide and its composite peptide segment have good free radical scavenging and α-glucosidase and α-amylase inhibitory effects. In addition, the small molecule active peptide of the present invention is a specific protein fragment, which has high activity and small molecular weight compared to protein, and is therefore more easily absorbed and utilized by the human body. Therefore, it has a good application prospect in the preparation of products with antioxidant and hypoglycemic effects, and lays a foundation for the high added value utilization of walnut meal and the promotion of the research and development and application of functional active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of free radical scavenging ability test of different molecular weight products obtained by alkaline protease hydrolysis of walnut meal protein;

[0027] Figure 2The results of the determination of the inhibitory capacity of α-glucosidase and α-amylase of the products with different molecular weights obtained by alkaline protease hydrolysis of walnut meal protein;

[0028] Figure 3 Secondary spectrum results of walnut meal protein peptide FSNAPRL;

[0029] Figure 4 Secondary spectrum results of walnut meal protein peptide QFGRLY;

[0030] Figure 5 The secondary spectrum results of walnut meal protein peptide SVPGHF;

[0031] Figure 6 The secondary structure prediction diagram of walnut meal protein peptide FSNAPRL;

[0032] Figure 7 The secondary structure prediction diagram of walnut meal protein peptide QFGRLY;

[0033] Figure 8 The secondary structure prediction diagram of walnut meal protein peptide SVPGHF;

[0034] Fig. 9 Molecular docking results of small molecule active peptide sequences FSNAPRL, QFGRLY, SVPGHF and DPPH,

[0035] Among them, (a) the molecular docking result diagram of the small molecule active peptide sequence FSNAPRL and DPPH, (b) the molecular docking result diagram of the small molecule active peptide sequence QFGRLY and DPPH, (c) the molecular docking result diagram of the small molecule active peptide sequence SVPGHF and DPPH;

[0036] Fig.10 Molecular docking results of small molecule active peptide sequence FSNAPRL and α-amylase;

[0037] Fig.11 Molecular docking results of small molecule active peptide sequence QFGRLY and α-amylase;

[0038] Fig.12 Molecular docking results of small molecule active peptide sequence SVPGHF and α-amylase;

[0039] Fig.13 Molecular weight plot of mass spectrum of small molecule active peptide sequence FSNAPRL;

[0040] Fig.14 Molecular weight plot of mass spectrum of small molecule active peptide sequence QFGRLY;

[0041] Fig.15Molecular weight diagram of the mass spectrum of the small molecule active peptide sequence SVPGHF;

[0042] Fig.16 The results of the free radical scavenging ability determination of chemically synthesized FSNAPRL, QFGRLY, and SVPGHF peptides;

[0043] Fig.17 The results of the determination of the α-glucosidase and α-amylase inhibitory abilities of the chemically synthesized FSNAPRL, QFGRLY, and SVPGHF peptides;

[0044] Fig.18 Degree of hydrolysis of different walnut meal protein hydrolysates;

[0045] Fig.19 The results of the determination of DPPH inhibition rate of different walnut meal protein hydrolysates;

[0046] Fig. 20 The results of the determination of α-glucosidase inhibition rate of different walnut meal protein hydrolysates;

[0047] Fig.21 Three-dimensional surface diagram of hydrolysis degree of walnut meal protein under different hydrolysis conditions;

[0048] Fig. 22 Three-dimensional surface diagram of DPPH, ABTS, α-amylase and α-glucosidase inhibition rates under different walnut meal proteolysis conditions.

[0049] Among them, (a) the three-dimensional surface graph of DPPH inhibition rate under different walnut meal protein hydrolysis conditions; (b) the three-dimensional surface graph of ABTS inhibition rate under different walnut meal protein hydrolysis conditions; (c) the three-dimensional surface graph of α-amylase inhibition rate under different walnut meal protein hydrolysis conditions; (d) the three-dimensional surface graph of α-glucosidase inhibition rate under different walnut meal protein hydrolysis conditions;

[0050] Fig.23 Results of free radical scavenging ability of the composite peptides FSNAPRL+QFGRLY, FSNAPRL+SVPGHF, and QFGRLY+SVPGHF;

[0051] Fig.24 The results of the α-glucosidase and α-amylase inhibitory abilities of the complex peptides FSNAPRL+QFGRLY, FSNAPRL+SVPGHF, and QFGRLY+SVPGHF. DETAILED DESCRIPTION

[0052] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0053] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0054] In the following embodiments,

[0055] 1. Determination of hydrolysis degree

[0056] During the protein hydrolysis process, as protons are released, the pH value of the solution will drop sharply. At this time, 0.01M NaOH solution is added to maintain the pH. The degree of hydrolysis (DH) can be calculated by the volume of alkali solution added. Calculation formula:

[0057] DH=(h / h tot )×100%=[(C×V) / (a×m×h tot )]×100%

[0058] C——Concentration of NaOH solution, moL / L; V——Volume of NaOH solution consumed, mL;

[0059] a——dissociation of amino group; a=10 pH-pKa / (1+10 pH-pKa ); M——the mass of walnut protein in the solution, g;

[0060] h tot ——Total number of peptide bonds in substrate protein, mmol / g, for walnut protein h tot =7.13 (calculated based on the amino acid composition of walnut protein).

[0061] Wherein, pH is the pH value of the hydrolysis system, pKa is the pKa of the amino group, pKa=7.8+[(298-T)×2400] / (298×T), where T is the Kelvin temperature.

[0062] 2. Protein concentration calculation

[0063] The standard curve for determining the peptide concentration. The ordinate is the absorbance at 280 nm, and the abscissa is the bovine serum albumin (BSA) concentration. The standard curve equation is: y=0.4217x-0.0003, R 2=0.98888. The protein content in the hydrolysate was finally determined based on the standard curve.

[0064] 3. Determination of DPPH free radical scavenging rate

[0065] Weigh a certain amount of hydrolyzate and dissolve it in deionized water to prepare a sample solution with a concentration of 0.2~2.0 mg / mL. Mix 100 μL of the sample solution with 100 μL of 0.1 mM DPPH 95% ethanol solution, react at room temperature in the dark for 30 min, and measure the absorbance value A at 517 nm. S An equal volume of ethanol solution was used to replace DPPH as the blank group A0, an equal volume of water was used to replace the sample solution as the control group A1, and an equal volume of water and ethanol mixture was used to adjust the zero. Three parallels were used for each group.

[0066] The calculation formula of DPPH free radical scavenging activity is as follows:

[0067] DPPH free radical scavenging rate (%) = [(A1 – A s +A0) / A1]×100

[0068] Where A1 is the absorbance value of the control group, A S is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0069] 4. ABTS free radical scavenging rate determination

[0070] Preparation of ABTS free radical solution: Mix 7 mM ABTS solution and 2.45 mM potassium persulfate in equal volumes, place in a dark place, and react for 12-16 hours away from light. Then dilute with anhydrous ethanol to make its absorbance at 734 nm wavelength 0.700±0.02, and let stand at room temperature for 30 minutes before use. Use anhydrous ethanol as the reference solution.

[0071] Weigh a certain amount of hydrolyzate and dissolve it in deionized water to prepare sample solutions of different concentrations (2 mg / mL). Take 30 μL of sample and mix it evenly with 570 μL of LABTS free radical solution. After reacting at room temperature in the dark for 10 min, measure the absorbance value of the sample at 734 nm, A1. Use 30 μL of distilled water instead of the sample solution as a blank group, and measure the absorbance value of the blank group as A0. Repeat the measurement 3 times for each group.

[0072] The calculation formula of ABTS free radical scavenging rate is as follows:

[0073] ABTS free radical scavenging rate (%) = [(A0-A1) / A0] × 100

[0074] Where A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0075] 5. Determination of α-glucosidase inhibitory activity

[0076] α-Glucosidase and p-nitrophenyl-α-D-glucoside (pNPG) were prepared into 0.2 U / mL and 1 mg / mL solutions respectively using 0.2 M sodium phosphate buffer at pH 6.8. 100 μL of buffer, 100 μL of pNPG, and 100 μL of sample peptide solution were added to the experimental groups respectively, and then preheated in a constant temperature water bath at 37°C for 5 min. After being taken out, 20 μL of α-glucosidase solution was immediately added to start the reaction. After mixing, the mixture was incubated in a water bath at 37°C for 30 min. After the reaction was terminated, 160 μL of 1M Na2CO3 solution was immediately added.

[0077] The experiment also set up a blank group, a control group and a sample background group. The blank group only added 100 μL of substrate pNPG, and the rest was replaced by an equal amount of buffer, eliminating the interference of the substrate pNPG spontaneously converted into the color-developing substance 4-nitrophenol (PNP) during the preheating process; the control group was the same as the experimental group except that the sample peptide solution was not added. The peptide solution was replaced by an equal amount of buffer, and the highest content of the color-developing substance PNP generated after the reaction of α-glucosidase and substrate pNPG was obtained under the same conditions; the sample background group only added the sample peptide solution, and the rest was replaced by an equal amount of distilled water, thus eliminating the influence of the absorbance value of the sample peptide solution at the corresponding wavelength. After the reaction was terminated, the content of the reactant PNP was detected at a wavelength of 405 nm by an ultraviolet spectrophotometer and recorded. The specific scheme is shown in Table 1.

[0078] The calculation formula of α-glucosidase activity inhibition rate is as follows:

[0079] α-glucosidase activity inhibition rate (%) = [OD c -(OD s -OD d )] / (OD c -OD b )×100

[0080] OD b : The absorbance of the blank group, the absorbance of the solution in which the walnut peptide solution and α-glucosidase solution were replaced by an equal amount of buffer;

[0081] OD c : Absorbance value of control group, absorbance value of solution in which walnut peptide solution was replaced by an equal amount of buffer solution;

[0082] OD s : absorbance value of experimental group;

[0083] OD d : Absorbance value of sample background group, absorbance value of solution containing only walnut peptide solution system.

[0084] Table 1 α-glucosidase inhibitory activity assay

[0085]

[0086] 6. Determination of α-amylase inhibitory activity

[0087] The DNS colorimetric method was used to determine the α-amylase inhibitory activity of the sample. Soluble starch can be hydrolyzed to produce maltose under the catalysis of α-amylase. Maltose, as a reducing sugar, can react with the DNS reagent to produce an orange-red product with maximum ultraviolet absorption at 540 nm.

[0088] PBS was used to prepare α-amylase into a 0.5 U / mL solution, and the concentration of starch solution was 1%. The experimental group added 50 μL of walnut peptide solution and α-amylase solution (0.5 U / mL, 50 μL) in turn, preheated at 37°C for 10 min, and added substrate soluble starch solution (1%, 50 μL) to start the reaction, and then incubated at 37°C for 15 min. After the reaction, 100 μL of DNS reagent was immediately added to the boiling water bath for 5 min to terminate the reaction. After cooling to room temperature, 250 μL of double distilled water was added, and the absorbance was measured at 540 nm. The experiment also set up a control group and a sample background group. The control group was to replace the 50 μL walnut peptide solution of the experimental group with 50 μL buffer; the sample background group was to replace the 50 μL starch solution of the experimental group with 50 μL buffer, and the rest of the operations were the same. The specific scheme is shown in Table 2.

[0089] The calculation formula of α-amylase activity inhibition rate is as follows:

[0090] α-Amylase activity inhibition rate (%) = [OD A0 -(OD A1 -OD A2 ) / OD A0 ]×100

[0091] Where, OD A0 : absorbance value of control group;

[0092] OD A1 : absorbance value of experimental group;

[0093] OD A2 : Absorbance value of sample background group.

[0094] Table 2 α-amylase inhibitory activity assay

[0095]

[0096] Example 1

[0097] A small molecule active peptide with antioxidant and blood sugar lowering effects, wherein the active peptide is a peptide having an amino acid sequence shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 3:

[0098] SEQ ID NO: 1: FSNAPRL;

[0099] SEQ ID NO:2: QFGRLY;

[0100] SEQ ID NO:3: SVPGHF.

[0101] Example 2

[0102] The method for extracting small molecule active peptides with antioxidant and hypoglycemic effects from walnut meal is as follows:

[0103] 1. Crude protein extraction by alkali dissolution and acid precipitation: First, crush the walnut meal with a pulper, mix the walnut pulp with distilled water in a ratio of 1:6 (w:v), stir evenly, adjust its pH value to 10, and then oscillate at 150 r / min in a 45°C constant temperature water bath oscillator for 30 min; take out the solution and centrifuge it at 4000 r / min for 15 min in a low-speed centrifuge, discard the oil layer and filter the supernatant; collect the supernatant and adjust its pH value to 4.5 for acid precipitation, wash it with distilled water 3 times during the precipitation process, and finally centrifuge it at 4000 r / min for 10 min, discard the supernatant and retain the precipitate, and freeze-dry to obtain walnut protein powder.

[0104] 2. Enzyme hydrolyzes walnut protein to obtain walnut peptides

[0105] Prepare a 30% walnut protein solution (for example, weigh 15 g of protein powder and dissolve it in 50 mL of water), heat it at 90°C for 10 min for pre-denaturation (passivation). Then add alkaline protease (1.25% of the protein powder weight), temperature 55°C, pH 10, and enzymolysis for 2.5 hours. During this period, the protein solution is constantly stirred and the pH of the system is maintained constant (always maintained at the optimal pH) with 0.1 M NaOH solution.

[0106] After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is placed in a 90°C water bath for 15 min to inactivate the enzyme. After cooling, it is centrifuged in a high-speed refrigerated centrifuge at a speed of 4000 r / min for 20 min. The temperature is controlled at 4°C. The supernatant is filtered and freeze-dried to obtain walnut peptide.

[0107] 3. Isolation and activity detection of active peptides

[0108] The walnut peptide freeze-dried powder after enzymatic hydrolysis was diluted, first passed through a 0.45 μm filter membrane, then through a 0.22 μm filter membrane, and poured into a liquid cylinder. The membrane ultrafiltration with a molecular weight of 10 KDa and 3 KDa was used, the centrifugal force of the ultrafiltration was 8000 r / min, the operating temperature was 25°C, and the time was 15 min and 40 min, respectively. After the ultrafiltration was completed, the filtrate was freeze-dried to obtain walnut active peptide powders with molecular weights >10 KDa, 3-10 KDa and <3 KDa. A 2 mg / mL sample peptide solution was prepared to determine the free radical scavenging ability and blood sugar inhibition effect of walnut active peptides with molecular weights >10KDa, 3-10 KDa and <3 KDa.

[0109] The results are as follows Figure 1 and Figure 2 As shown, the DPPH and ABTS free radical scavenging abilities, α-glucosidase and α-amylase inhibitory activities of active peptides with a molecular weight of <3 KDa were significantly better than those of walnut peptides with a molecular weight of >10 KDa and 3-10 KDa. Therefore, active peptides with a molecular weight of <3 KDa were selected for further identification.

[0110] 4. Amino acid sequence identification

[0111] The active peptides with a molecular weight of less than 3 KDa and the most antioxidant and hypoglycemic activity obtained by the above separation were screened and analyzed by liquid chromatography-mass spectrometry technology, and the structures of the active peptides were identified. The Easy-nLC high performance liquid chromatography system was used, and the RSLCC18 chromatographic column was used to separate the walnut polypeptide peak with a molecular weight of less than 3 KDa and the most antioxidant and hypoglycemic activity; gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; the mass spectrometry scanning method was in the information-dependent acquisition working mode, the primary mass spectrometry scanning resolution was 70000, and a maximum of 20 secondary spectra with charges of 2+ to 5+ were collected in each working cycle.

[0112] Through liquid chromatography-mass spectrometry technology, the results showed that a total of 2,618 peptides were obtained in the walnut peptide sample.

[0113] 5. Peptide screening and functional verification

[0114] The potential biological activity of the peptides in the walnut meal protein hydrolysate obtained in step 4 was predicted using the Peptide Ranker program (0-1), and peptides with a score > 0.5 were selected. The ToxinPred3.0 website was used to select peptides with a toxicity threshold prediction of < 0.4. Finally, three peptides were obtained. The secondary mass spectrometry analysis showed that the peptides had a molecular weight of 803.429 and a sequence of FSNAPRL ( Figure 3 ); a peptide with a molecular weight of 782.4075 and a sequence of QFGRLY ( Figure 4); a peptide with a molecular weight of 642.3126 and a sequence of SVPGHF ( Figure 5 ), the specific information is shown in Table 3. The secondary structures of walnut meal polypeptides FSNAPRL, QFGRLY, and SVPGHF predicted by PepDraw software are as follows Figure 6 , Figure 7 and Figure 8 shown.

[0115] Table 3 Walnut meal polypeptide FSNAPRL, QFGRLY, SVPGHF information list

[0116]

[0117] The 3D structures of DPPH (PubChem CID: 2735032) and α-amylase (PDB ID: 1UA7) were obtained from the PubChem database. The AutodockTools software was used to perform molecular docking of the walnut meal polypeptide sequence with DPPH and α-amylase to study the binding energy, hydrogen bonds and intermolecular forces between DPPH and α-amylase (receptors) and polypeptides (ligand molecules). The Pymol software was used to visualize the molecular docking results of walnut meal polypeptide molecules FSNAPRL, QFGRLY, and SVPGHF with DPPH and α-amylase ( Figure 9-12 ), and three sequences were chemically synthesized ( Figure 13-Figure 15 ).

[0118] The free radical scavenging ability and α-glucosidase and α-amylase inhibition of the synthesized peptides were verified by single peptides (2 mg / mL). The effects of free radical scavenging ability and α-glucosidase and α-amylase inhibition were: SVPGHF>FSNAPRL>QFGRLY( Fig.16 , Fig.17 SVPGHF, a single peptide, had the best antioxidant and hypoglycemic activities, among which the DPPH free radical scavenging ability was 89.57%, the ABTS free radical scavenging ability was 84.29%, the α-glucosidase activity inhibition rate was 82.34%, and the α-amylase activity inhibition rate was 65.47%.

[0119] Effects of different proteases on enzymatic hydrolysis of walnut meal and product activity

[0120] Prepare a 10% walnut protein solution and heat it at 90℃ for 10 min for pre-denaturation (passivation). Then add 1% of the protein powder mass of trypsin, alkaline protease, neutral protease or papain, and use the optimal conditions of the protease reaction (as shown in Table 4) as the temperature and pH of the hydrolysis system during the enzymatic hydrolysis process. Stir the protein solution continuously and use 0.1 M NaOH solution to maintain the pH of the system constant (always maintain it at the optimal pH). After hydrolysis for 4 h, the enzymatic solution was placed in a 90℃ water bath for enzyme inactivation for 15 min. After cooling, it was centrifuged in a high-speed refrigerated centrifuge at a speed of 4000 r / min for 20 min. The temperature was controlled at 4℃. The supernatant was filtered and freeze-dried to obtain walnut peptides.

[0121] Table 4 Optimum temperature and pH of different proteases

[0122]

[0123] After enzymatic hydrolysis, the effects of different proteases on the degree of hydrolysis (DH, %), DPPH inhibition ability related to antioxidant activity, and α-glucosidase inhibition related to hypoglycemic activity of walnut protein hydrolysates were determined.

[0124] The results showed that alkaline protease had better hydrolysis ability ( Fig.18 ), and the enzymatic hydrolysis products have significant antioxidant and hypoglycemic effects ( Fig.19 , Fig. 20 ).

[0125] Effects of enzymatic hydrolysis conditions on enzymatic hydrolysis of walnut meal and product activity

[0126] Using Design-expert software, 17 groups of response surface experiments were designed according to three factors and three levels by adjusting the amount of enzyme added, enzymatic hydrolysis time, and material-liquid ratio. The specific enzymatic hydrolysis conditions are shown in Table 5. Alkaline protease was used as the hydrolase, and the enzymatic hydrolysis steps were the same as above. Different hydrolysis degrees were obtained according to different enzymatic hydrolysis conditions, and the DPPH and ABTS inhibition abilities related to the antioxidant activity and the α-glucosidase and α-amylase inhibition effects related to the hypoglycemic activity were determined respectively ( Fig.21 , Fig. 22 ), select a set of enzymatic hydrolysis conditions with the best effect.

[0127] Effects of different enzyme addition amounts and enzymatic hydrolysis time on the degree of hydrolysis (DH) of walnut meal protein Fig.21 As shown in the figure, the effects of different enzyme addition amounts and material-liquid ratios on the free radical scavenging ability of walnut meal protein (DPPH, ABTS activity inhibition rate), and the effects of different enzyme addition amounts and enzymolysis time on the blood sugar inhibition effect of walnut meal protein (α-amylase, α-glucosidase activity inhibition rate) are shown in the figure. Fig. 22 shown.

[0128] The results showed that among the 17 groups of enzymatic hydrolysis conditions, the inhibition rates of DPPH and ABTS free radicals, α-glucosidase and α-amylase activities were the highest when the enzyme addition amount was 1.25%, the enzymatic hydrolysis time was 2.5h, and the solid-liquid ratio was 0.3, so it was taken as the optimal enzymatic hydrolysis condition.

[0129] Table 5 Enzymatic hydrolysis parameters of response surface experiment

[0130]

[0131] Note: The sample concentration for determining the inhibition rate of DPPH and ABTS free radicals, α-glucosidase and α-amylase activities was 2 mg / mL.

[0132] Example 3

[0133] A small molecule active peptide composition with antioxidant and blood sugar lowering effects comprises FSNAPRL and QFGRLY, wherein the mass ratio of FSNAPRL to QFGRLY is 1:1.

[0134] Example 4

[0135] A small molecule active peptide composition with antioxidant and blood sugar lowering effects comprises FSNAPRL and SVPGHF, wherein the mass ratio of FSNAPRL to SVPGHF is 1:1.

[0136] Example 5

[0137] A small molecule active peptide composition with antioxidant and blood sugar lowering effects comprises QFGRLY and SVPGHF, wherein the mass ratio of QFGRLY to SVPGHF is 1:1.

[0138] The FSNAPRL, QFGRLY, and SVPGHF peptides were compounded in pairs to obtain three compositions of FSNAPRL+QFGRLY, FSNAPRL+SVPGHF, and QFGRLY+SVPGHF. The mass ratio of the two peptides in the compositions was 1:1. Ultrapure water was used as the solvent to dissolve the peptides, and the concentration of each peptide was 2 mg / mL.

[0139] The free radical scavenging ability and α-glucosidase and α-amylase inhibitory effects of the above composite peptides were determined. Fig.23 and Fig.24As shown in the figure, the peptides of FSNAPRL and SVPGHF have the best free radical scavenging ability and α-glucosidase and α-amylase inhibition effects after compounding, among which, the DPPH free radical scavenging ability is 92.1%, the ABTS free radical scavenging ability is 95.37%, the α-glucosidase activity inhibition rate is 86.48%, and the α-amylase activity inhibition rate is 77.01%, which is significantly better than the effect of FSNAPRL or SVPGHF alone.

[0140] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A small molecule active peptide with antioxidant and hypoglycemic effects, characterized in that: The amino acid sequence of the small molecule active peptide is any one of FSNAPRL, QFGRLY and SVPGHF.

2. Use of the small molecule active peptide with antioxidant and hypoglycemic efficacy as claimed in claim 1 in the preparation of a drug with antioxidant and hypoglycemic function.

3. A small molecule active peptide composition with antioxidant and hypoglycemic effects, characterized in that: The small molecule active peptide composition comprises at least one of the small molecule active peptides with antioxidant and hypoglycemic effects as described in claim 1.

4. The small molecule active peptide composition with antioxidant and hypoglycemic effects according to claim 3, characterized in that: The small molecule active peptide composition is any two of FSNAPRL, QFGRLY and SVPGHF.

5. The small molecule active peptide composition with antioxidant and hypoglycemic effects according to claim 4, characterized in that: The mass ratio of the two active peptides in the small molecule active peptide composition is 1:

1.

6. The small molecule active peptide composition with antioxidant and hypoglycemic effects according to claim 5, characterized in that: The antioxidant has the ability to remove free radicals, and the free radicals are at least one of DPPH and ABTS.

7. The small molecule active peptide composition with antioxidant and hypoglycemic effects according to claim 5, characterized in that: The blood sugar lowering effect is to inhibit α-glucosidase and / or α-amylase.

8. Use of the small molecule active peptide composition with antioxidant and hypoglycemic effects according to any one of claims 3 to 7 in the preparation of a drug with antioxidant and hypoglycemic effects.

9. Use of the small molecule active peptide composition with antioxidant and hypoglycemic efficacy according to claim 8 in the preparation of a drug with antioxidant and hypoglycemic efficacy, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

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