Alpha-glucosidase inhibitory peptide based on fructus cannabis as well as preparation method and application of alpha-glucosidase inhibitory peptide

Abstract

The invention discloses an alpha-glucosidase inhibitory peptide based on fructus cannabis. The alpha-glucosidase inhibitory peptide comprises polypeptides of which the amino acid sequences are shown as follows: Ser-Leu-Phe-Lys, Tyr-Phe-Lys, Lys-Phe-Tyr, Lys-Phe-Leu and Ser-Trp-Gly respectively. The preparation method of the alpha-glucosidase inhibitory peptide comprises the following steps: carrying out enzymolysis on fructus cannabis protein by bromelain, sequentially carrying out microfiltration and ultrafiltration on supernate of obtained enzymolysis liquid, taking ultrafiltrate with the molecular weight of less than 10kDa, concentrating to obtain concentrated ultrafiltrate, diluting, and separating by a restricted affinity medium to obtain the alpha-glucosidase inhibitory peptide. The alpha-glucosidase inhibitory peptide provided by the invention has very high activity and inhibition rate. In the preparation method, the alpha-glucosidase inhibitory peptide with high inhibitory activity is quickly adsorbed and separated from the fructus cannabis protein enzymatic hydrolysate by utilizing a restricted access affinity medium, the content of the obtained active component is greatly increased, the separation time is short, and the operation process is simple.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an alpha-glucosidase inhibitory peptide based on hemp seed and a preparation method thereof. Background Art

[0002] Hemp seed (Cannabis sativa L.) is the dried mature fruit of the hemp plant of the Moraceae family. Its aliases are hemp seed, hemp seed, hemp seed and hemp seed. Hemp seed contains 20%-25% protein, 25%-30% oil, 20%-30% dietary fiber, and a series of rich vitamins and minerals. Hemp seed protein mainly contains globulin and albumin, which meet the various essential amino acids required by the human body, and is particularly rich in arginine, lysine and glutamic acid. These two proteins are easy to digest and are a very excellent new resource of plant protein. It has been found that hemp seed protein (HSP) has the effects of lowering blood sugar, anti-oxidation and lowering blood pressure. However, the proteases used for enzymatic hydrolysis are mostly alkaline protease, neutral protease, trypsin, etc., and there are few studies on the use of bromelain to enzymatically hydrolyze HSP.

[0003] The number of diabetic patients is expected to increase to 783 million by 2045. The number of patients is large and the complications are more, while most of the existing hypoglycemic drugs have side effects. Therefore, natural plant hypoglycemic drugs that are easy to absorb, have no side effects, are non-toxic, and have good biological activity have become a hot topic.

[0004] The existing separation and purification methods are relatively complicated and cumbersome, and require multiple and long purification and separation by chromatography and liquid phase to obtain small molecule peptides with specific high inhibition rate. The affinity chromatography method can achieve rapid and accurate screening. Therefore, the use of affinity chromatography to screen hemp seed hypoglycemic peptides is more efficient and less costly, and has broad application prospects and research significance. Summary of the invention

[0005] One of the purposes of the present invention is to provide an α-glucosidase inhibitory peptide based on hemp seed.

[0006] The present invention achieves the above-mentioned purpose through the following technical scheme: an alpha-glucosidase inhibitory peptide based on hemp seed, which comprises amino acid sequences of Ser-Leu-Phe-Lys, Tyr-Phe-Lys, Lys-Phe-Tyr, Lys-Phe-Leu, and Ser-Trp-Gly polypeptides.

[0007] The second object of the present invention is to provide a method for preparing an α-glucosidase inhibitory peptide based on hemp seed.

[0008] Specifically, the preparation method of the α-glucosidase inhibitory peptide based on hemp seed comprises the following steps: hydrolyzing hemp seed protein with bromelain, subjecting the supernatant of the obtained hydrolyzate to microfiltration and ultrafiltration, taking the ultrafiltrate with a molecular weight of less than 10 kDa, concentrating the concentrated ultrafiltrate, diluting it, and separating it with a limited-access affinity medium to obtain the α-glucosidase inhibitory peptide.

[0009] In the present invention, a hemp seed protein solution with a mass concentration of 15-25 g / L is prepared, bromelain is added to carry out enzymolysis according to the enzyme amount of bromelain in the hemp seed protein solution being 4000-7000 U / g, the enzymolysis conditions being pH 6-10, the temperature being 30-50 DEG C, the enzymolysis time being 0.5-4 h, and finally the solution is heated to 95 DEG C to inactivate the enzyme for 10-15 min to obtain an enzymolysis solution.

[0010] Furthermore, bromelain should be added in liquid form and dissolved in advance with PBS of corresponding pH, because a large amount of solid enzyme added to solution A requires time to dissolve, which will affect the enzymatic hydrolysis effect.

[0011] The pH should be monitored throughout the enzymatic hydrolysis process and maintained by adding 0.05M-0.2M sodium hydroxide solution.

[0012] The pore size of the obtained microfiltration membrane is 0.45 μm.

[0013] The limited access affinity media separates:

[0014] The concentrated ultrafiltrate is diluted with a 0.1-1M PBS solution to make its polypeptide concentration 0.25-2mg / ml to obtain a polypeptide solution; the restricted access affinity medium is dispersed in the polypeptide solution at a ratio of 5:1-3:1 (mg / ml), and adsorbed at 20-30°C for 30-60min by shaking, and then the restricted access affinity medium is separated from the solution by centrifugation. After washing the restricted access affinity medium three times, the washed restricted access affinity medium is eluted with a 1M-3M sodium chloride solution for 30min-120min, and centrifuged. The obtained supernatant is the eluate after separation and purification, which is concentrated to obtain a concentrated eluate.

[0015] The mass volume ratio of the restricted affinity medium and the 1M to 3M sodium chloride solution is 5:1-3:1 (mg / ml).

[0016] The preparation of the limited-access affinity medium in the present invention is as follows:

[0017] (1) α-glucosidase solution was mixed with 3-MPBA solution, and zinc nitrate hexahydrate solution and Bim were added to react at 25°C with stirring to obtain a white precipitate, which was separated by centrifugation to obtain 3-MPBA / AG@aZIF-7;

[0018] (2) 3-MPBA / AG@aZIF-7 was mixed with Tris-HCl containing ammonium persulfate (APS) and dopamine hydrochloride (DA) and stirred at 25°C and 150 rpm for 2 h. The collected mixture precipitate was washed with deionized water and freeze-dried to obtain 3-MPBA / AG@aZIF-7 / PDA.

[0019] In step (1), the α-glucosidase solution is α-glucosidase dissolved in 50 mM phosphate buffer solution, with a concentration of 8 mg / mL and a pH value of 6.8. The amount of the α-glucosidase solution is 1 mL.

[0020] In step (1), the 3-MPBA solution is a 3-MPBA ethanol solution, which is prepared by dissolving 5.2 mg 3-MPBA in 20 uL ethanol.

[0021] In step (1), the amount of zinc nitrate hexahydrate solution used is 0.5 mL, and the concentration is 56 mM; the amount of Bim used is 5 mL, and the concentration is 50 mM.

[0022] In step (1), the reaction was stirred at 25°C for 50 min.

[0023] In step (2), the amount of Tris-HCl containing a mixture of ammonium persulfate (APS) and dopamine hydrochloride (DA) is 10 mL, wherein the volume ratio of ammonium persulfate (APS) to 1 mg / mL dopamine hydrochloride (DA) is 2:1, the concentration of Tris-HCl is 10 mM, and the pH is 8.5.

[0024] The third object of the present invention is to provide the application of α-glucosidase inhibitory peptide based on hemp seed.

[0025] Specifically, the present invention provides an application of the hemp seed-based α-glucosidase inhibitory peptide in the preparation of functional foods. Further, the hemp seed-based α-glucosidase inhibitory peptide is used in the preparation of functional foods that alleviate hyperglycemia symptoms.

[0026] The present invention has the following beneficial effects:

[0027] 1. The method uses hemp seed protein as a raw material, hydrolyzes the protein with bromelain, and then rapidly separates components with high inhibitory activity using a limited-access affinity medium, then uses liquid phase mass spectrometry to detect amino acid sequences of the separated components, and detects the alpha-glucosidase inhibitory activity of the components separated in each step, and finally obtains five polypeptides with amino acid sequences of Ser-Leu-Phe-Lys, Tyr-Phe-Lys, Lys-Phe-Tyr, Lys-Phe-Leu, Ser-Trp-Gly, and abbreviations of SLFK, YFK, KFY, KFL, and SWG, respectively.

[0028] 2. The enzymolysis solution obtained by enzymolysis of hemp seed protein in the present invention is microfiltered and ultrafiltered using a 0.45 μm filter membrane and an ultrafiltration membrane with a molecular weight cutoff of 10 kDa in sequence, so as to effectively remove impurities and improve the efficiency of subsequent material separation.

[0029] 3. The limited-access affinity medium prepared by the present invention resists the adsorption of large molecular weight proteins while specifically adsorbing small molecule polypeptides, and compared with the original traditional separation technology, the content of the active component prepared by the present invention is greatly improved, the separation time is short and the operation process is simple. The limited-access affinity medium prepared by the present invention can quickly adsorb and separate α-glucosidase inhibitory peptides with high inhibitory activity from hemp seed protein hydrolysate. Moreover, the limited-access affinity medium prepared by the present invention is very suitable for quickly and effectively separating small molecule active substances from complex biological matrix samples, thereby improving separation efficiency.

[0030] 4. The raw materials for preparing the α-glucosidase inhibitory peptide of the present invention are abundant, the preparation process is reasonable, and the operability is strong. The new α-glucosidase inhibitory peptide can be used to prepare functional foods for alleviating symptoms of hyperglycemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the mass spectrum of the amino acid fragment KFY.

[0032] Figure 2 This is the mass spectrum of the amino acid fragment YFK.

[0033] Figure 3 This is the mass spectrum of the amino acid fragment SLFK.

[0034] Figure 4 This is the mass spectrum of the amino acid fragment KFL.

[0035] Figure 5 It is the mass spectrum of the amino acid fragment SWG. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The present invention is further described by the following examples, but is not intended to be limiting of the present invention. Specific experimental conditions and methods not specified in the following examples are conventional methods commonly known to those skilled in the art.

[0042] Example 1

[0043] Preparation of S1 Limited Access Affinity Matrix:

[0044] (1) 1 mL of α-glucosidase (8 mg / mL) dissolved in phosphate buffer solution (pH 6.8, 50 mM) and 5.2 mg of 3-MPBA (dissolved in 20 uL of ethanol) were added continuously to a 25 mL wedge-shaped flask, and then 0.5 mL of zinc nitrate hexahydrate solution (56 mM) and 5 mL of Bim (50 mM) were added and stirred at 25 °C for 50 min; the product was obtained as a white precipitate, which was then centrifuged at 10,000 rpm for 3 times, each time for 10 min, to obtain 3-MPBA / AG@aZIF-7.

[0045] (2) 3-MPBA / AG@aZIF-7 (1 g) prepared in step (1) was added to 10 mL of Tris-HCl (pH = 8.5, 10 mM) mixed with ammonium persulfate (APS) and dopamine hydrochloride (DA, 1 mg / mL) (2:1), and stirred at 25 ° C, 150 rpm for 2 h before being taken out. The collected mixture precipitate was washed with deionized water 3 times, 10 minutes each time, to obtain 3-MPBA / AG@aZIF-7 / PDA, and freeze-dried for 24 h to obtain 3-MPBA / AG@aZIF-7 / PDA.

[0046] Preparation of S2 hemp seed protein hydrolysis ultrafiltrate:

[0047] Weigh a solution A with a mass concentration of 20 g / L of hemp seed protein, add bromelain according to the enzyme amount of 5000 U / g of bromelain in the solution A for enzymolysis, the enzymolysis conditions are pH 7, temperature 35° C., enzymolysis for 1 h, and finally heat to 95° C. to inactivate the enzyme for 10-15 min to obtain an enzymolysis solution; after microfiltration through a 0.45 μm filter membrane, the supernatant of the obtained enzymolysis solution is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain an ultrafiltrate.

[0048] S3 affinity medium separation:

[0049] The ultrafiltrate obtained in S2 was dispersed in the 3-MPBA / AG@aZIF-7 / PDA obtained in S1, and the protein concentration in the system was 1 mg / ml, and the mass volume ratio of 3-MPBA / AG@aZIF-7 / PDA to the ultrafiltrate was 5:1 mg / ml. The adsorption was shaken at 25°C for 45 minutes, followed by centrifugation. After washing 3-MPBA / AG@aZIF-7 / PDA three times, the washed material was eluted with 2M sodium chloride solution for 60 minutes, and the mass volume ratio of 3-MPBA / AG@aZIF-7 / PDA to 2M sodium chloride solution was 5:1 mg / ml. The supernatant after centrifugation is the eluate after separation and purification.

[0050] S4 liquid chromatography-mass spectrometry to detect amino acid sequences:

[0051] The concentrated eluate obtained in S3 was subjected to structural identification on a Q Exactive HF-X mass spectrometer (Thermo), and the following compounds were obtained through screening: Figure 1-5 The short peptides SLFK, YFK, KFY, KFL and SWG are α-glucosidase inhibitory peptides.

[0052] Example 2

[0053] Preparation of S1 Limited Access Affinity Matrix:

[0054] (1) 1 mL of α-glucosidase (8 mg / mL) dissolved in phosphate buffer solution (pH 6.8, 50 mM) and 5.2 mg of 3-MPBA (dissolved in 20 uL of ethanol) were added continuously to a 25 mL wedge-shaped flask, and then 0.5 mL of zinc nitrate hexahydrate solution (56 mM) and 5 mL of Bim (50 mM) were added and stirred at 25 °C for 50 min; the product was obtained as a white precipitate, which was then centrifuged at 10,000 rpm for 3 times, each time for 10 min, to obtain 3-MPBA / AG@aZIF-7.

[0055] (2) 3-MPBA / AG@aZIF-7 (1 g) prepared in step (1) was added to 10 mL of Tris-HCl (pH = 8.5, 10 mM) mixed with ammonium persulfate (APS) and dopamine hydrochloride (DA, 1 mg / mL) (2:1), and stirred at 25°C, 150 rpm for 2 h before being taken out. The collected mixture precipitate was washed with deionized water 3 times for 10 minutes each time to obtain 3-MPBA / AG@aZIF-7 / PDA, which was freeze-dried for 24 h to obtain 3-MPBA / AG@aZIF-7 / PDA.

[0056] Preparation of S2 hemp seed protein hydrolysis ultrafiltrate:

[0057] Weigh a solution A with a mass concentration of 20 g / L of hemp seed protein, add bromelain according to the enzyme amount of 5000 U / g of bromelain in the solution A for enzymolysis, the enzymolysis conditions are pH 8, temperature 45° C., enzymolysis 0.5 h, and finally heat to 95° C. to inactivate the enzyme for 10-15 min to obtain an enzymolysis solution; after microfiltration through a 0.45 μm filter membrane, the supernatant of the obtained enzymolysis solution is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain an ultrafiltrate.

[0058] S3 affinity medium separation:

[0059] The ultrafiltrate obtained in S2 was dispersed in the 3-MPBA / AG@aZIF-7 / PDA obtained in S1 to obtain a protein concentration of 0.5 mg / ml in the system, that is, the mass volume ratio of 3-MPBA / AG@aZIF-7 / PDA to the ultrafiltrate was 5:1 mg / ml. The adsorption was shaken at 30°C for 30 minutes, followed by centrifugation. After washing 3-MPBA / AG@aZIF-7 / PDA three times, the washed material was eluted with a 2M sodium chloride solution for 60 minutes. The mass volume ratio of 3-MPBA / AG@aZIF-7 / PDA to the 2M sodium chloride solution was 5:1 mg / ml. The supernatant after centrifugation is the eluate after separation and purification.

[0060] S4 liquid chromatography-mass spectrometry to detect amino acid sequences:

[0061] The concentrated eluate obtained in S3 was subjected to structural identification on a Q Exactive HF-X mass spectrometer (Thermo). Figure 1-5 The short peptides SLFK, YFK, KFY, KFL and SWG are α-glucosidase inhibitory peptides.

[0062] Example 3

[0063] Preparation of S1 Limited Access Affinity Matrix:

[0064] (1) 1 mL of α-glucosidase (8 mg / mL) dissolved in phosphate buffer solution (pH 6.8, 50 mM) and 5.2 mg of 3-MPBA (dissolved in 20 uL of ethanol) were added continuously to a 25 mL wedge-shaped flask, and then 0.5 mL of zinc nitrate hexahydrate solution (56 mM) and 5 mL of Bim (50 mM) were added and stirred at 25 °C for 50 min; the product was obtained as a white precipitate, which was then centrifuged at 10,000 rpm for 3 times, each time for 10 min, to obtain 3-MPBA / AG@aZIF-7.

[0065] (2) 3-MPBA / AG@aZIF-7 (1 g) prepared in step (1) was added to 10 mL of Tris-HCl (pH = 8.5, 10 mM) mixed with ammonium persulfate (APS) and dopamine hydrochloride (DA, 1 mg / mL) (2:1), and stirred at 25°C, 150 rpm for 2 h before being taken out. The collected mixture precipitate was washed with deionized water 3 times for 10 minutes each time to obtain 3-MPBA / AG@aZIF-7 / PDA, which was freeze-dried for 24 h to obtain 3-MPBA / AG@aZIF-7 / PDA.

[0066] Preparation of S2 hemp seed protein hydrolysis ultrafiltrate:

[0067] Weigh a solution A with a mass concentration of 20 g / L of hemp seed protein, add bromelain according to the enzyme amount of bromelain in the solution A being 6000 U / g for enzymolysis, the enzymolysis conditions being pH 9, temperature 50° C., enzymolysis for 1 h, and finally heating to 95° C. to inactivate the enzyme for 10 to 15 min to obtain an enzymolysis solution; microfilter the supernatant of the obtained enzymolysis solution through a 0.45 μm filter membrane, and then ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain an ultrafiltrate.

[0068] S3 affinity medium separation:

[0069] The ultrafiltrate obtained in S2 was dispersed in the 3-MPBA / AG@aZIF-7 / PDA obtained in S1, and the protein concentration in the system was 1 mg / ml, and the mass volume ratio of 3-MPBA / AG@aZIF-7 / PDA to the ultrafiltrate was 5:1 mg / ml. The adsorption was shaken at 28°C for 60 minutes, followed by centrifugation. After washing 3-MPBA / AG@aZIF-7 / PDA three times, the washed material was eluted with 2M sodium chloride solution for 60 minutes, and the mass volume ratio of 3-MPBA / AG@aZIF-7 / PDA: 2M sodium chloride solution was 5:1 mg / ml. The supernatant after centrifugation is the eluate after separation and purification.

[0070] S4 liquid chromatography-mass spectrometry to detect amino acid sequences:

[0071] The concentrated eluate obtained in S3 was subjected to structural identification on a Q Exactive HF-X mass spectrometer (Thermo). Figure 1-5 The short peptides SLFK, YFK, KFY, KFL and SWG are α-glucosidase inhibitory peptides.

[0072] α-glucosidase inhibitory activity test of α-glucosidase inhibitory peptides:

[0073] The α-glucosidase inhibitory activity test was performed on the enzymatic hydrolysates, <10wD ultrafiltrate and eluate in the above Examples 1 to 3.

[0074] Preparation method of main reagents:

[0075] α-Glucosidase solution: Dissolve 100 U α-glucosidase in 0.5 mL of cold 0.1 mol / L pH 6.8 phosphate buffer (PBS) to prepare 200 U / mL α-glucosidase solution, divide into portions, and store at -30°C for later use.

[0076] Substrate p-nitrophenyl-α-D-glucopyranoside (PNPG) solution: Take an appropriate amount of PNPG and prepare a 0.4 mol / L PNPG solution with 0.1 mol / L pH 6.8 phosphate buffer (PBS) for use.

[0077] The reaction system environment is pH 6.8, 0.1mol / L phosphate buffer (PBS). In a 1.5mL centrifuge tube, take 100μL, 0.4mol / L pnpg, add a certain concentration of sample solution, mix evenly, preheat in a 37℃±0.5℃ constant temperature water bath for 10min, then add 20μL, 1U / mL α-glucosidase, and mix thoroughly. After keeping warm in a 37℃ constant temperature water bath for 15min, add 400μL, 0.4mol / L sodium carbonate solution to stop the reaction. After the reaction is completed, measure the absorbance (OA) at 405nm on an ELISA reader.

[0078] The α-glucosidase inhibition rate (IR) was calculated according to the following formula. The results are shown in Table 1:

[0079]

[0080] OD A The absorbance of the supernatant of the sample to be tested is replaced by an equal amount of buffer; OD B OD is the absorbance of the supernatant of the sample being tested; c The absorbance of the supernatant of the sample and PNPG solution was replaced by an equal amount of buffer.

[0081] The half-inhibitory concentration of the α-glucosidase inhibitory peptide obtained after separation and concentration using the preparation method of the α-glucosidase inhibitory peptide in Examples 1 to 3 was determined:

[0082] The peptide concentration of the concentrated eluate in S3 was determined using the microbiuret method. The concentrated eluate was diluted using 0.1M phosphate buffer (PBS) at pH 6.8. Different amounts of α-glucosidase inhibitory peptide sample solutions were taken to calculate the inhibition rate according to the method of the α-glucosidase inhibitory activity test of the α-glucosidase inhibitory peptide described above, and then the natural logarithmic concentration of the α-glucosidase inhibitory peptide sample was used as the horizontal axis, and the inhibition rate was used as the vertical axis to draw a graph to calculate the inhibition rate of the α-glucosidase inhibitory peptide sample on α-glucosidase, and the results are shown in Table 1.

[0083] Among them, trace amount of biuret reagent: weigh 86.5gNa 3 C 6 H 5 O 7 ·2H 2 0, 50.0gNa 2 CO 3 ; Dissolve in 250ml deionized water; weigh 8.65gCuSO 4 ·5H 2 0 is dissolved in 100ml of deionized water; the above two solutions are mixed and the volume is adjusted to 500mL. This reagent can be stored for a long time, and it needs to be re-prepared if precipitation occurs.

[0084] 6% NaOH solution: Weigh 30.0 g of NaOH and prepare a 6% solution with deionized water.

[0085] Take 0.5 mL of sample, 0.5 mL of NaOH solution, and 0.050 mL of biuret reagent and mix them evenly. After leaving them at room temperature for 15 min, detect the absorbance at a wavelength of 310 nm.

[0086] Table 1 α-glucosidase IC50 of α-glucosidase inhibitory peptides obtained in Examples 1 to 3

[0087]

[0088] It can be seen from Table 1 that the α-glucosidase inhibitory peptide eluate prepared by the method of the present invention has higher activity and better inhibitory effect.

[0089] The eluate in Example 1-3 was subjected to liquid chromatography-mass spectrometry to identify active peptides, and five new active peptides, SLFK, YFK, KFY, KFL and SWG, were obtained. The secondary mass spectra of SLFK, YFK, KFY, KFL and SWG were as follows: Figure 1-5 shown.

[0090] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An α-glucosidase inhibitory peptide based on hemp seed, characterized in that: The polypeptides comprise the amino acid sequences shown below: Ser-Leu-Phe–Lys, Tyr-Phe–Lys, Lys-Phe–Tyr, Lys-Phe–Leu and Ser-Trp–Gly, respectively.

2. A method for preparing an α-glucosidase inhibitory peptide based on hemp seed, characterized in that: Bromelain is used to enzymolyze hemp seed protein, and the supernatant of the obtained enzymolysis solution is successively subjected to microfiltration and ultrafiltration, and the ultrafiltrate with a molecular weight less than 10kDa is concentrated to obtain a concentrated ultrafiltrate, which is diluted and separated by a limited access affinity medium to obtain an alpha-glucosidase inhibitory peptide.

3. The method for preparing an α-glucosidase inhibitory peptide based on hemp seed according to claim 2, characterized in that: A hemp seed protein solution with a mass concentration of 15-25 g / L is prepared, bromelain is added according to the enzyme amount of bromelain in the hemp seed protein solution being 4000-7000 U / g for enzymolysis, the enzymolysis conditions are pH 6-10, the temperature is 30-50 DEG C, the enzymolysis time is 0.5-4 h, and finally the solution is heated to 95 DEG C to inactivate the enzyme for 10-15 min to obtain an enzymolysis solution.

4. The method for preparing an α-glucosidase inhibitory peptide based on hemp seed according to claim 2, characterized in that: The pore size of the obtained microfiltration membrane is 0.45 μm.

5. The method for preparing an α-glucosidase inhibitory peptide based on hemp seed according to claim 2, characterized in that: The limited access affinity media separates: The concentrated ultrafiltrate is diluted with a 0.1-1M PBS solution to make its polypeptide concentration 0.25-2mg / ml to obtain a polypeptide solution; the restricted access affinity medium is dispersed in the polypeptide solution at a mass volume ratio of 5:1-3:1, and adsorbed at 20-30°C for 30-60min by shaking, and then the restricted access affinity medium and the solution are separated by centrifugation. After washing the restricted access affinity medium three times, the washed restricted access affinity medium is eluted with a 1M-3M sodium chloride solution for 30min-120min, and centrifuged. The obtained supernatant is the eluate after separation and purification, which is concentrated to obtain a concentrated eluate.

6. The method for preparing an α-glucosidase inhibitory peptide based on hemp seed according to claim 2, wherein: The limited-access affinity matrix was prepared as follows: (1) α-glucosidase solution was mixed with 3-MPBA solution, and zinc nitrate hexahydrate solution and Bim were added to react at 25°C with stirring to obtain a white precipitate, which was separated by centrifugation to obtain 3-MPBA / AG@aZIF-7; (2) 3-MPBA / AG@aZIF-7 was mixed with Tris-HCl containing ammonium persulfate (APS) and dopamine hydrochloride (DA) and stirred at 25°C and 150 rpm for 2 h. The collected mixture precipitate was washed with deionized water and freeze-dried to obtain 3-MPBA / AG@aZIF-7 / PDA.

7. The method for preparing an α-glucosidase inhibitory peptide based on hemp seed according to claim 6, characterized in that: In step (1), the α-glucosidase solution is α-glucosidase dissolved in 50 mM phosphate buffer solution, with a concentration of 8 mg / mL, and the pH value of the phosphate buffer solution is 6.8; the amount of the α-glucosidase solution is 1 mL; The 3-MPBA solution is a 3-MPBA ethanol solution, which is prepared by dissolving 5.2 mg 3-MPBA in 20 μL ethanol; The zinc nitrate hexahydrate solution is used in an amount of 0.5 mL, with a concentration of 56 mM; the Bim is used in an amount of 5 mL, with a concentration of 50 mM; The reaction was stirred at 25°C for 50 min.

8. The method for preparing an α-glucosidase inhibitory peptide based on hemp seed according to claim 6, characterized in that: In the step (2), the amount of Tris-HCl containing a mixture of ammonium persulfate and dopamine hydrochloride is 10 mL, wherein the volume ratio of the ammonium persulfate to 1 mg / mL dopamine hydrochloride is 2:1, the concentration of the Tris-HCl is 10 mM, and the pH is 8.

5.

9. Use of the hemp seed-based α-glucosidase inhibitory peptide according to claim 1 in the preparation of functional foods.

10. The hemp seed-based α-glucosidase inhibitory peptide of claim 1 is used in the preparation of functional food for alleviating hyperglycemia symptoms.

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