Grape seed protein peptide with DPP-IV inhibitory activity and preparation method thereof

Abstract

The invention discloses a grape seed protein peptide with DPP-IV inhibitory activity and a preparation method thereof, and belongs to the field of comprehensive utilization of wine fermentation byproducts. The grape seed protein peptide disclosed by the invention is prepared from a zymolyte obtained by carrying out alkaline protease enzymolysis on grape seed protein prepared from grape seeds by an ultrasonic-assisted alkaline method. The grape seed protein peptide disclosed by the invention has a good DPP-IV inhibition effect, is a DPP-IV inhibitor from a natural plant source, and can be applied to functional foods and nutritional health-care products.

Description

Technical Field

[0001] The present invention relates to grape seed protein peptides with DPP-IV inhibitory activity and a preparation method thereof, and belongs to the field of comprehensive utilization of wine fermentation by-products. Background Art

[0002] Type 2 diabetes is a chronic disease that affects the body's ability to metabolize glucose, mostly caused by insulin resistance or deficiency. Among them, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone, are the main regulators of insulin release, mainly reducing blood glucose levels by inhibiting the production of glucagon, stimulating insulin release, and reducing gastric emptying. However, when these two incretins are released into the intestine, they will be cleaved by a serine protease on the cell surface - dipeptidyl peptidase-IV (DPP-IV), resulting in the inactivation of incretins, thus causing blood glucose disorders in patients. The main mechanism of action of the DPP-IV inhibitory peptide in the present invention is to inhibit the DPP-IV enzyme, thereby prolonging the half-life of incretins and achieving the effect of promoting insulin secretion, so as to control the blood glucose in patients with type 2 diabetes.

[0003] With the continuous development of the wine industry, while the wine production increases, a large amount of grape pomace is also generated. According to statistics, about 1 kg of wine pomace is produced for every 6 L of wine produced. Therefore, in order to promote green and low-carbon development and the sustainability of the industry, and improve the reasonable resource utilization of wine processing waste, the present invention proposes that in addition to being able to extract oil by pressing, the grape seeds produced by fermentation are also a high-quality source for exploring DPP-IV inhibitory active functional peptides after defatting.

[0004] At present, the development of hypoglycemic functional peptides from natural sources includes: the Chinese patent application text with the publication number CN107141336A discloses a preparation method of yak gluten peptides, that is, using yak gluten to be stepwise enzymolyzed by a multi-stage composite protease, and then the enzymolysis product is decontaminated, membrane-separated, gel-separated, reverse-phase HPLC-separated, concentrated, and freeze-dried; so far, the newly developed DPP-IV-derived inhibitory peptides that have been publicly reported have never shown inhibitory power equivalent to that of the drug. While promoting the research and development of wine by-products, we are committed to exploring new plant-derived DPP-IV inhibitory peptides with higher DPP-IV inhibitory power.

[0005] Meanwhile, among the dipeptidyl peptidase-IV (DPP-IV) inhibitors currently on the market, such as sitagliptin and saxagliptin, although they can effectively control blood sugar, they are also accompanied by side effects and high production and use costs. Under the current trend of "big health", plant-derived functional peptides of natural origin are extremely likely to attract the attention of patients and medical researchers. Their advantages of having no side effects, high safety, and easy absorption have also become one of the main research directions in the food processing field in recent years. Therefore, it is particularly important and urgent to develop a plant-derived functional peptide with both safety and high efficiency and having DPP-IV inhibitory activity. Summary of the Invention

[0006] To solve the above technical problems and further improve the reuse method of wine fermentation by-products - grape seeds, the present invention evaluates the potential of grape seed protein as a source of DPP-IV (dipeptidyl peptidase-IV) inhibitory peptides, and verifies that the grape seed protein hydrolysate has good DPP-IV inhibitory effects, and provides a grape seed protein peptide with DPP-IV inhibitory activity and its preparation method.

[0007] The present invention provides a grape seed protein peptide with DPP-IV inhibitory activity, and the amino acid sequence of the grape seed protein peptide is Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF, SEQ ID NO.1) or Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT, SEQ ID NO.2).

[0008] SEQ ID NO.1: SMFFPFPF;

[0009] SEQ ID NO.2: FFTFPT.

[0010] In one embodiment of the present invention, the grape seed protein peptide has good DPP-IV inhibitory activity, and the DPP-IV inhibitory activity (IC 50 value) is less than 0.5 mg / mL.

[0011] In one embodiment of the present invention, the grape seed protein peptide is prepared from defatted grape seeds.

[0012] In one embodiment of the present invention, the grape seed protein peptide can be used as a medicine, a health product or a dietary supplement, or added as a food base material to common foods such as beverages and dairy products; it is used to inhibit DPP-IV and lower blood sugar, and is used for the prevention or adjuvant treatment of hyperglycemic patients, type II diabetic patients, or people with chronic metabolic syndrome such as obesity, immune deficiency, cardiovascular and cerebrovascular diseases.

[0013] In one embodiment of the present invention, the preparation method of the grape seed protein peptide includes the steps:

[0014] On the one hand, the present invention uses a method of ultrasonic-assisted alkali dissolution and acid precipitation to extract proteins, and improves the extraction rate and extraction effect by adding cross-linked polyvinylpyrrolidone to adsorb phenols in the protein solution; on the other hand, alkaline protease is selected for enzymatic hydrolysis to release grape seed protein peptides with DPP-IV inhibitory activity.

[0015] In one embodiment of the present invention, the method for preparing the grape seed protein peptide includes the steps of:

[0016] 1) Grape seed powder is used to extract proteins by ultrasonic-assisted alkali dissolution and acid precipitation;

[0017] 2) The grape seed protein obtained in step 1) is enzymatically hydrolyzed to release protein peptides;

[0018] 3) The hydrolyzate after enzymatic hydrolysis in step 2) is purified by ultrafiltration and RP-HPLC, and then obtained after rotary evaporation and freeze-drying.

[0019] In order to obtain 100% grape seed protein peptide, the above grape seed extraction process preferably may include the following steps:

[0020] Select defatted grape seeds in wine fermentation by-products as raw materials, grind the grape seeds into powder to obtain defatted grape seed powder, mix it with 50% absolute ethanol and let it stand in the dark for 1 h, filter to remove the top phase to obtain defatted and dephenolized grape seed powder. Dissolve the defatted grape seed powder with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, adjust its pH to 11 with 6 M sodium hydroxide, assist with ultrasonic extraction while stirring, filter to remove the lower solid phase, then add pvpp (cross-linked polyvinylpyrrolidone) to the obtained filtrate, mix and react for 30 min, remove the lower solid phase to obtain grape seed protein alkaline solution; then adjust the pH of the obtained alkaline solution to the isoelectric point of 3.5 with hydrochloric acid, refrigerate and place for 2 h, centrifuge to obtain acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0021] In a preferred embodiment of the present invention, the solid-liquid ratio of the mixture of grape seed powder and 50% absolute ethanol is 1:5.

[0022] In a preferred embodiment of the present invention, the ultrasonic wave for assisting extraction is 40 kHz, the power is 200 W, and the reaction time is 40 min. In the embodiment of the present invention, using the specific frequency ultrasonic wave technology for extraction can significantly improve the extraction efficiency and reduce the time cost.

[0023] In a preferred embodiment of the present invention, the amount of cross-linked polyvinylpyrrolidone adsorbing polyphenols is 4% (w / w), which can significantly reduce the polyphenol content in grape seed protein. Due to the significant reduction of anthocyanins, the color of the obtained protein powder is also purer.

[0024] In a preferred embodiment of the present invention, the specific steps of enzymatic hydrolysis in step 2) are as follows:

[0025] Dissolve the grape seed protein powder prepared in step 1) in a 0.01M phosphate buffer (PBS) system, adjust to the optimal reaction conditions of temperature 45°C and pH = 10.0, and add alkaline protease at an enzyme / protein concentration ratio of 4000U / g for the next enzymatic hydrolysis; maintain the pH and temperature of the entire reaction system stable during the process, terminate the reaction by boiling water bath after the reaction, add trichloroacetic acid solution with a final concentration of 15% to precipitate and centrifuge to remove excess protein, and freeze-dry to obtain a concentrate of grape seed protein hydrolysate.

[0026] In a preferred embodiment of the present invention, it has been verified that alkaline protease is the enzyme with the most potential for DPP-IV inhibition, and the enzymatic hydrolysis for 4 hours is the optimal enzymatic hydrolysis time. Under this enzymatic hydrolysis method, the obtained grape seed protein hydrolysate has the highest degree of hydrolysis and the strongest DPP-IV inhibition potential.

[0027] In order to obtain the best inhibitory effect of the obtained grape seed protein solution, the ultrafiltration operation in step 3) is specifically as follows:

[0028] Purify the concentrate of grape seed protein hydrolysate obtained in step 2) using a Labscale TFF ultrafiltration system with a molecular weight cut-off of 5 kDa to obtain grape seed protein hydrolysate with <5 kDa, and then purify it again using an ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kDa to obtain grape seed protein hydrolysates with 3 - 5 kDa and <3 kDa;

[0029] In a preferred embodiment of the present invention, the RP-HPLC in step 3) can be:

[0030] Perform RP-HPLC reverse-phase high-performance liquid chromatography separation on the grape seed protein hydrolysate with <3 kDa. Mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is HPLC-grade acetonitrile, the detection wavelength is 214 nm, collect the elution peaks at 9 - 12 minutes, and rotary evaporate to remove the solvent to obtain a crude product of grape seed protein peptide.

[0031] In a preferred embodiment of the present invention, the grape seed protein peptide with DPP-IV inhibitory function prepared by the above method includes polypeptides with the following amino acid sequences:

[0032] Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF, SEQ ID NO.1) or Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT, SEQ ID NO.2);

[0033] In a preferred embodiment of the present invention, the grape seed protein peptide with DPP-IV inhibitory function prepared by the above method comprises a polypeptide with the following amino acid sequence: Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF, SEQ ID NO.1) or Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT, SEQ ID NO.2).

[0034] The grape seed protein peptide containing any one of the above two polypeptides has good DPP-IV inhibitory activity, and the DPP-IV inhibitory activity (Ic 50 value) is less than 0.5 mg / mL.

[0035] The present invention also provides an expression vector or a recombinant microorganism, which contains at least one of the above grape seed protein peptides.

[0036] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0037] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0038] The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable pharmaceutical excipient; the active ingredient comprises any one or more of the above grape seed protein peptides: Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe or Phe-Phe-Thr-Phe-Pro-Thr;

[0039] The above grape seed protein peptides can significantly inhibit DPP-IV activity.

[0040] In one embodiment of the present invention, the pharmaceutical excipient refers to a conventional pharmaceutical carrier in the pharmaceutical field.

[0041] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, sorbitan fatty acids, and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hypromellose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents, sweetening agents, etc. can also be added to the composition.

[0042] The present invention also provides a food, drug, health product, or nutritional product, which contains at least one of the above-mentioned grape seed protein peptides in an effective dose.

[0043] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe or Phe-Phe-Thr-Phe-Pro-Thr.

[0044] In one embodiment of the present invention, the drug also contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0045] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, sorbitan fatty acids, and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hypromellose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents, sweetening agents can also be added to the composition.

[0046] In one embodiment of the present invention, the dosage forms of the drug include, but are not limited to, oral dosage forms, injection dosage forms, inhalation dosage forms.

[0047] In one embodiment of the present invention, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, oral suspensions.

[0048] In one embodiment of the present invention, the injection dosage form includes, but is not limited to, injection solutions and injection powder injections.

[0049] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder aerosols.

[0050] In one embodiment of the present invention, the food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, beverages; the food also includes special dietary foods.

[0051] In one embodiment of the present invention, the health product also contains acceptable excipients.

[0052] The present invention also provides the use of at least one or more of the above-mentioned grape seed protein peptides in the preparation of foods, drugs, health products or nutritional products; it is used to inhibit DPP-IV activity, lower blood sugar, prevent or treat diabetes or diseases benefited from DPP-IV inhibition, or prevent or treat chronic metabolic syndrome with conditions such as obesity, immune deficiency or cardiovascular and cerebrovascular diseases.

[0053] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe or Phe-Phe-Thr-Phe-Pro.

[0054] In one embodiment of the present invention, the drug also contains pharmaceutically acceptable medicinal excipients; the medicinal excipients refer to conventional drug carriers in the pharmaceutical field.

[0055] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc powder and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can be added to the composition.

[0056] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage forms, injection dosage forms, inhalation dosage forms.

[0057] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, oral suspensions.

[0058] In one embodiment of the present invention, the injection dosage form includes, but is not limited to, injection solutions and injection powder for injection.

[0059] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder aerosols.

[0060] In one embodiment of the present invention, the food includes, but is not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0061] In one embodiment of the present invention, the health care product also contains acceptable excipients.

[0062] The present invention also provides a method for preparing the above-mentioned food, medicine, health care product or nutritional product, the method comprising mixing at least one of the above-mentioned grape seed protein peptides with at least one acceptable excipient.

[0063] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF) or Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT).

[0064] Beneficial effects:

[0065] (1) The present invention obtains grape seed protein peptides with DPP-IV inhibitory activity, and the specific sequences are Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe or Phe-Phe-Thr-Phe-Pro. The structure is novel and easy to prepare. It can be obtained from grape seed protein or artificially synthesized.

[0066] (2) The two polypeptides obtained in the present invention have strong DPP-IV inhibitory activity; among them, the half inhibitory concentration (IC 50 ) of Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF) is 231.9 ± 13.23 μmol / L; the half inhibitory concentration (IC 50 ) of Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT) is 308.8 ± 21.19 μmol / L.

[0067] (3) The grape seed protein peptide of the present invention can be used as a medicine, a health product or a dietary supplement, or added to common foods such as beverages and dairy products as a food base material; it is used to inhibit DPP-IV and lower blood sugar, and is used for the prevention or adjuvant treatment of hyperglycemic patients, type 2 diabetic patients, or people with chronic metabolic syndrome such as obesity, immune deficiency, cardiovascular and cerebrovascular diseases, etc., and has broad application prospects in the fields of functional foods, medicines, health products, etc. Brief Description of the Drawings

[0068] Figure 1 It is a graph of the DPP-IV inhibition rate results at different hydrolysis times;

[0069] Figure 2 It is a graph of the free radical scavenging rate results at different hydrolysis times. Detailed Embodiments

[0070] The following combines examples to further describe in detail the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0071] Unless otherwise specified, the technical means used in the examples are conventional technical means well-known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are commercially available.

[0072] The defatted grape seed powder involved in the following examples can be prepared with reference to the following method: Mix grape seed powder with n-hexane at a solid-liquid ratio of 1:5, let it stand and then filter to obtain defatted grape seed powder;

[0073] Defatted grape seeds in the by-products of wine fermentation can also be directly selected as raw materials.

[0074] The sources of the raw materials involved in the following examples are as follows:

[0075] pvpp (crosslinked polyvinylpyrrolidone) was purchased from Shanghai Merck Biochemical Technology Co., Ltd., with the product number M86828-100G;

[0076] Folin-Ciocalteu reagent was purchased from Shanghai Titan Technology Co., Ltd., with the product number 4140369A;

[0077] Alkaline protease was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 200 U / mg and the product number S10154-100g;

[0078] Papain was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 800 U / mg and the product number S10011-100g;

[0079] Pepsin was purchased from Merck Life Science China, with an enzyme activity of ≥500 U / mg and the product number 77160-25g;

[0080] Bromelain was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 300 U / mg and a product number of S10009-100g;

[0081] Neutral protease was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 100 U / mg and a product number of S10013-250g;

[0082] BCA Protein Concentration Assay Kit (Enhanced) was purchased from Beyotime Biotechnology Co., Ltd., with a product number of P0010S;

[0083] DPP-Ⅳ Inhibitor Screening Kit was purchased from GeneScript Biotech Corporation, with a product number of KA1311.

[0084] The measurement methods involved in the following examples are as follows:

[0085] 1. DPP-Ⅳ (Dipeptidyl Peptidase-IV) Inhibitory Activity Measurement Method:

[0086] The method is based on the instruction manual of the DPP-Ⅳ Inhibitor Screening Kit from Abnova Corporation (product number: KA1311). The kit contains DPP-Ⅳ buffer (Assay Buffer), DPP-Ⅳ enzyme, and the substrate is Glycyl-Proline-7-amino-4-methylcoumarin hydrobromide (Gly-Pro-AMC). The activity measurement is carried out using a 96-well plate.

[0087] Sample wells: Add 10 μl of DPP-Ⅳ enzyme, 10 μl of sample, 30 μl of Asaay Buffer, and 50 μl of substrate into the wells in sequence;

[0088] Negative control: Add 10 μl of DPP-Ⅳ enzyme, 10 μl of solvent, 30 μl of Asaay Buffer, and 50 μl of substrate into the wells in sequence;

[0089] Background blank: Add 10 μl of solvent, 40 μl of Asaay Buffer, and 50 μl of substrate into the wells in sequence;

[0090] Place the above wells in a 37 °C constant temperature incubator and let them stand in the dark for 30 min. Use a microplate reader to detect the fluorescence value at an excitation wavelength of 355 nm and an emission wavelength of 455 nm.

[0091] Ic 50 Value, the half-inhibitory concentration, that is, the sample concentration when the DPP-Ⅳ inhibition rate is 50%. Dilute the sample to be tested into 5 concentration gradients spanning 50% according to the inhibition rate data of the preliminary experiment, measure the DPP-Ⅳ inhibition rate of each gradient concentration, and import the data into GraghPad non-linear fitting curve to obtain the Ic 50 Value.

[0092] Calculation formula: Subtract the background fluorescence value from the fluorescence value of all pores to avoid fluorescence interference from buffers, enzymes, and substrates.

[0093] DPP-IV inhibition rate (%) = F 阴性对照 -F 样品 / F 阴性对照 -F 背景空白

[0094] 2. BCA protein concentration determination method:

[0095] The method is based on the instruction manual of the BCA Protein Concentration Assay Kit (Enhanced) from Beyotime Biotechnology (Cat. No.: P0010S). The kit contains BCA working solution, protein standard (20 mg BSA), and protein standard preparation solution. The activity is measured using a 96-well plate.

[0096] Dissolve the protein standard with the protein standard preparation solution. After complete dissolution, prepare a 0.5 mg / ml protein standard solution, and then dilute it with distilled water to 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 mg / ml. In a 96-well plate, add 20 μl of protein standard and sample solution, then add 200 μl of BCA working solution to each well. Incubate at 37 °C for 25 min, measure the absorbance at A562 with an enzyme-linked immunosorbent assay (ELISA) reader, and make a protein standard curve. Calculate the protein concentration of the sample according to the standard curve.

[0097] In the following examples, (w / v) refers to g / ml.

[0098] Experimental Example 1: Grape seed protein extraction process

[0099] 1. Preparation of grape seed protein

[0100] 1) Select Cabernet Sauvignon defatted grape seeds from wine fermentation by-products as raw materials. Under liquid nitrogen protection, grind the whole grape seeds into powder to obtain defatted grape seed powder.

[0101] Mix the prepared defatted grape seed powder with 50% ethanol aqueous solution at a solid-liquid ratio of 1:5 (w / v), and let it stand in the dark for 1 h. Filter to remove the top phase, and repeat the above operation twice for the residue to obtain defatted and de-phenolized grape seed powder.

[0102] 2) Dissolve the defatted and dephenolized grape seed powder prepared in step 1) with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16 (w / v), adjust its pH to 11 with 6 M sodium hydroxide, extract for 40 min with stirring assisted by ultrasound (frequency 40 kHz), and filter to remove the lower solid phase. Then add pvpp (crosslinked polyvinylpyrrolidone) at a solid-liquid ratio of 4% (w / v) to the obtained filtrate, mix and react for 30 min, and remove the lower solid phase to obtain the grape seed protein alkaline solution;

[0103] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point of 3.5 with 6 M hydrochloric acid, refrigerate at 4 °C for 2 h, and centrifuge at 12000 rpm to obtain the acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain the grape seed protein precipitate, and freeze-dry to obtain the grape seed protein powder.

[0104] 2. Optimization of the grape seed protein extraction process

[0105] In order to verify the effects of ethanol extraction pretreatment and polyvinylpyrrolidone pvpp impurity removal on the preparation efficiency of grape seed protein, and select Cabernet Sauvignon defatted grape seeds from the same four groups of wine fermentation by-products, grind the whole grape seeds into powder under liquid nitrogen protection to obtain defatted grape seed powder (the preparation method is the same as the first step of step 1), and the present invention designs four groups of control experiments (Table 1):

[0106] Table 1: Four groups of control experiments

[0107]

[0108] The specific method is as follows:

[0109] 1) Preparation of #1 grape seed protein:

[0110] Dissolve the prepared defatted grape seed powder with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, adjust its pH to 11 with 6 M sodium hydroxide, extract for 40 min with stirring assisted by ultrasound (frequency 40 kHz), and filter to remove the lower solid phase. Then add pvpp (crosslinked polyvinylpyrrolidone) at a solid-liquid ratio of 4% to the obtained filtrate, mix and react for 30 min, and remove the lower solid phase to obtain the grape seed protein alkaline solution, and freeze-dry to obtain #1 grape seed protein (GSP#1).

[0111] 2) Preparation of #2 grape seed protein:

[0112] The obtained defatted grape seed powder was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted for 40 min with stirring assisted by ultrasound (frequency 40 kHz). The lower solid phase was removed by filtration to obtain grape seed protein alkaline solution, which was freeze-dried to obtain grape seed protein #2 (GSP#2).

[0113] 3) Preparation of grape seed protein #3:

[0114] The obtained defatted grape seed powder was mixed with 50% absolute ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 h. The top phase was removed by filtration, and the residue was subjected to the above operation twice to obtain defatted and dephenolized grape seed powder. Then it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted for 40 min with stirring assisted by ultrasound (frequency 40 kHz). The lower solid phase was removed by filtration. Then 4% (w / w) pvpp (crosslinked polyvinylpyrrolidone) was added to the obtained filtrate, and the mixture was reacted for 30 min. The lower solid phase was removed to obtain grape seed protein alkaline solution, which was freeze-dried to obtain grape seed protein #3 (GSP#3).

[0115] 4) Preparation of grape seed protein #4:

[0116] The obtained defatted grape seed powder was mixed with 50% absolute ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 h. The top phase was removed by filtration, and the residue was subjected to the above operation twice to obtain defatted and dephenolized grape seed powder. Then it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted for 40 min with stirring assisted by ultrasound (frequency 40 kHz). The lower solid phase was removed by filtration to obtain grape seed protein alkaline solution, which was freeze-dried to obtain grape seed protein #4 (GSP#4).

[0117] The protein content was determined by the Kjeldahl method in accordance with GB5009.5-2016 Determination of Protein in Foods:

[0118] A well-mixed solid grape seed protein powder sample was weighed into a digestion tube, and then 0.4 g of copper sulfate, 6 g of potassium sulfate and 20 ml of sulfuric acid were added, and digestion was carried out in a digestion furnace. After the temperature of the digestion furnace reached 420 °C, digestion was continued for 1 h. At this time, the liquid in the digestion tube was green and transparent. After taking it out and cooling, 50 ml of water was added, and automatic liquid addition, distillation, titration and recording of titration data were realized on an automatic Kjeldahl distiller. The protein content was obtained by multiplying the acid consumption by the conversion factor 6.25.

[0119] The polyphenol content was determined by the Folin-phenol method:

[0120] First, a standard curve was established using the standard phenolic substance catechin. 0.1 mL of a sample with a concentration of 1 mg / ml was added to a 10 mL volumetric flask, 7 mL of distilled water was added and shaken well, then 0.5 mL of 1 M Folin-Ciocalteu reagent was added. After 1 minute, 1.5 mL of 20% (v / v) sodium carbonate solution was added, mixed well, and the volume was made up to 10 mL with distilled water. The reaction was carried out in the dark for 120 minutes, and the absorbance was measured at 765 nm. The results were expressed as the equivalent value of the catechin standard curve.

[0121] 5) Detection of experimental results

[0122] The protein content and polyphenol content of defatted grape seed powder and the obtained Grape Seed Protein #1, Grape Seed Protein #2, Grape Seed Protein #3, and Grape Seed Protein #4 were measured, and the measurement results are shown in Table 2.

[0123] Table 2: Protein content of grape seed protein under different extraction methods

[0124]

[0125] As can be seen from Table 2, the protein content of Grape Seed Protein #3 (GSP#3) is the highest and the total phenol content is the lowest. Basically, it can be seen that the effect of impurity removal is good, the operation is simple, and the cost is low. It is an effective method for extracting grape seed protein.

[0126] Example 2: Preparation of concentrated grape seed protein hydrolysate

[0127] The preparation method of grape seed protein peptides with DPP-IV inhibitory activity is as follows:

[0128] 1. Preparation of grape seed protein

[0129] 1) Cabernet Sauvignon defatted grape seeds in wine fermentation by-products were selected as raw materials, and the whole grape seeds were ground into powder under liquid nitrogen protection to obtain defatted grape seed powder.

[0130] 2) The prepared defatted grape seed powder was mixed with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and left to stand in the dark for 1 h. The top phase was filtered off, and the residue was repeated the above operation twice to obtain defatted and de-phenolized grape seed powder. Then it was dissolved with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted with ultrasound (frequency 40 kHz) for 40 min while stirring, and the lower solid phase was filtered off. Then 4% (w / w) pvpp (crosslinked polyvinylpyrrolidone) was added to the obtained filtrate, and the mixture was reacted for 30 min to remove the lower solid phase to obtain grape seed protein alkaline solution;

[0131] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point of 3.5 with 6M hydrochloric acid, refrigerate at 4°C for 2 h, and centrifuge at 12,000 rpm to obtain acidic precipitated protein; finally, redissolve the obtained acidic precipitated protein with distilled water, adjust its pH to neutral, centrifuge at 12,000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0132] 2. Preparation of grape seed protein hydrolysate concentrate with DPP-IV inhibition

[0133] Dissolve the grape seed protein powder prepared in step 1 above in a 0.01M phosphate buffer (PBS) system, adjust the magnetic stirrer connected to the pH electrode to the optimal reaction conditions of temperature 45°C, pH = 10.0, and stirring speed 40 rpm, add alkaline protease at an enzyme / protein concentration ratio of 4000 U / g, and carry out the next enzymatic hydrolysis; maintain the pH and temperature of the entire reaction system stable during the process, terminate the reaction by boiling water bath after 4 h, add 15% (v / v) of 1M trichloroacetic acid solution to precipitate and centrifuge at 12,000 rpm to remove excess protein, and freeze-dry to obtain grape seed protein hydrolysate concentrate.

[0134] Experimental Example 3: Optimization of the preparation method of grape seed protein hydrolysate concentrate

[0135] The specific implementation method is the same as that in Example 2, the difference is that the reaction for 4 h in step 2, (1) is replaced with 0.5, 1, 2, 3, 4, 5 or 6 h, and the alkaline protease in step 2, (2) is replaced with papain, pepsin or neutral protease, bromelain (Table 3). The remaining steps are the same as in Example 2 to obtain grape seed protein hydrolysate concentrate. For the grape seed protein hydrolysate concentrates prepared by hydrolyzing with alkaline protease, papain, pepsin, neutral protease or bromelain for different reaction times, the inhibition rate of DPP-IV and the polypeptide concentration are measured by using the above DPP-IV inhibition activity measurement method and protein concentration measurement method.

[0136] The results are as Figures 1-2 shown in Table 3.

[0137] Table 3: Different enzymatic hydrolysis reactions and corresponding results

[0138]

[0139] It was found that the hydrolysate by alkaline protease had the strongest biological activity and the highest polypeptide content at 3.5 - 4 h.

[0140] Detect the DPP-IV inhibition Ic of the grape seed protein hydrolysate concentrates prepared by hydrolyzing with alkaline protease, papain, pepsin, neutral protease or bromelain for 4 h respectively 50Values, and the results are shown in Table 4:

[0141] Table 4: DPP-IV inhibition Ic of hydrolysates of grape seed protein by different hydrolases for 4 h 50 value

[0142]

[0143] The results showed that the hydrolysate of alkaline protease had the most significant inhibitory effect on DPP-IV enzyme, with an Ic 50 of 96 ± 4.3 μg soluble protein hydrolysate / mL (pepsin Ic 50 = 980 ± 16.7 μg soluble protein hydrolysate / mL, neutral protease Ic 50 = 160 ± 7 μg soluble protein hydrolysate / mL, bromelain Ic 50 = 369.7 ± 29.79 μg soluble protein hydrolysate / mL, papain Ic 50 = 1028 ± 91.19 μg soluble protein hydrolysate / mL).

[0144] Example 4: Preparation of grape seed protein peptides with DPP-IV inhibition

[0145] The preparation method of grape seed protein peptides with DPP-IV inhibition is as follows:

[0146] 1. Preparation of grape seed protein

[0147] 1) Select Cabernet Sauvignon defatted grape seeds from wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection to obtain defatted grape seed powder.

[0148] 2) Mix the obtained defatted grape seed powder with 50% absolute ethanol at a solid-liquid ratio of 1:5 and let it stand in the dark for 1 h. Filter to remove the top phase, and repeat the above operation twice for the residue to obtain defatted and de-phenolized grape seed powder. Then dissolve it with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, adjust its pH to 11 with 6 M sodium hydroxide, extract for 40 min with stirring and assisted by ultrasound (frequency 40 kHz), and filter to remove the lower solid phase. Then add 4% (w / w) pvpp (crosslinked polyvinylpyrrolidone) to the obtained filtrate, mix and react for 30 min, and remove the lower solid phase to obtain grape seed protein alkaline solution;

[0149] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point of 3.5 with 6 M hydrochloric acid, refrigerate at 4 °C for 2 h, centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0150] 2. Preparation of Grape Seed Protein Peptide with DPP-IV Inhibition

[0151] 1) Dissolve the grape seed protein powder prepared in step 1 above in a 0.01M phosphate buffer (PBS) system. Adjust the magnetic stirrer connected to the pH electrode to the optimal reaction conditions of temperature 45°C, pH = 10.0, and stirring speed 40 rpm. Add alkaline protease at an enzyme / protein concentration ratio of 4000 U / g for the next enzymatic hydrolysis. During the process, maintain the pH and temperature of the entire reaction system stable. After reacting for 4 h, terminate the reaction by boiling water bath. Add 15% (v / v) of 1M trichloroacetic acid solution to precipitate and centrifuge at 12000 rpm to remove excess protein, and then freeze-dry to obtain the concentrated grape seed protein hydrolysate.

[0152] 2) Ultrafiltration separation is carried out on the concentrated grape seed protein hydrolysate obtained in step 1). Use a Labscale TFF ultrafiltration system with a molecular weight cut-off of 5 kDa for purification to obtain grape seed protein hydrolysate with <5 kDa. Then, use an ultrafiltration centrifugal tube with a molecular weight cut-off of 3 kDa for purification again to obtain grape seed protein hydrolysates of 3 - 5 kDa and <3 kDa.

[0153] 3) Carry out RP-HPLC reverse-phase high-performance liquid chromatography separation on the <3 kDa grape seed protein hydrolysate obtained in step 2). Mobile phase A is 0.1% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B is HPLC-grade acetonitrile. The detection wavelength is 214 nm. From 0 to 5 min, 2% B; from 5 to 9 min, 2% - 7% B; from 9 to 28 min, 7% - 30% B; from 28 to 40 min, 30% - 50% B; from 40 - 41 min, 50% - 100% B; from 41 to 45 min, 100% B; from 45 to 46 min, 100% - 2% B; from 46 to 60 min, 2% B. Set the flow rate at 2 mL / min; collect the elution peak from 9 - 12 min, and rotary evaporate to remove the solvent to obtain the crude grape seed protein peptide.

[0154] 4) Carry out mass spectrometry identification on the crude grape seed protein peptide obtained in step 3), and determine the components of the grape seed protein peptide by LC-MS / MS.

[0155] The conditions of LC-MS / MS are as follows:

[0156] The model of the high-performance liquid chromatograph is Dionex U3000; the chromatographic column is C18, 3μm, 75μm x 15cm; Mobile phase A: 0.1% Formic acid in water; Mobile phase B: 0.1% Formic acid in Acetonitrile; Flow rate is 600nL / min, 8% B from 0 - 8min, 13% B from 8 - 16min, 28% B from 16 - 39min, 40% B from 39 - 50min, 95% B from 50 - 51min, 95% B from 51 - 55min, 6% B from 55 - 56min, 6% B from 56 - 60min.

[0157] The model of the mass spectrometer is Thermo Scientific Q Exactive; Resolution settings: 70,000@m / z 200 for the first stage, 17,500@m / z 200 for the second stage; Parent ion scan range: m / z 300 - 1400; Daughter ion scan range: m / z 100; MS1 AGC: 3e6, Ion injection time: 60ms; MS2 AGC: 5e4, Ion injection time: 80ms; Ion screening window: 3.0m / z; Fragmentation mode: HCD, Energy NCE 27; Data-dependent MS / MS: Top 20; Dynamic exclusion time: 15s.

[0158] 5) Screening of grape seed protein peptides with DPP-IV inhibitory activity

[0159] Through computer simulation and in vitro determination experiments, a total of 10 peptides with DPP-IV inhibitory activity were screened (Table 6).

[0160] The determination results of the grape seed protein peptide components were as follows: A total of 10 peptides with DPP-IV inhibitory activity were identified (Tables 5 - 6). The amino acid sequences of the two peptides with the highest DPP-IV inhibitory activity in the grape seed protein peptides were Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF) or Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT).

[0161] Experimental Example 5: Experiment for determining the DPP-IV inhibitory ability of grape seed protein peptides

[0162] The samples were DPP-IV inhibitory peptides (Tables 5 - 6) obtained by hydrolysis, ultrafiltration, reverse-phase HPLC, and mass spectrometry identification in Example 4, and were synthesized by Shanghai Gil Biochemical Co., Ltd., with a purity ≥ 98%.

[0163] Detection of the IC50 values of 10 identified peptide segments such as SGMFPFPF and FFTFPT (Tables 5 - 6) for DPP-IV inhibition. 50 value.

[0164] Ic 50 The method for measuring the value is as follows:

[0165] The method is based on the instruction manual of the DPP-IV inhibitor screening kit from Abnova Corporation (product number: KA1311). The kit contains DPP-IV buffer (Assay Buffer), DPP-IV enzyme, and the substrate is Glycyl-Prolyl-7-amino-4-methylcoumarin hydrobromide (Gly-Pro-AMC). The activity is measured using a 96-well plate.

[0166] Preparation of sample concentration: Dissolve 2 mg of the polypeptide sample (SGMFPFPF) in 100 μl of distilled water to obtain a peptide (SGMFPFPF) with a concentration of 20 mg / ml, and then dilute it to obtain peptides (SGMFPFPF) with concentrations of 10 mg / ml, 5 mg / ml, 2 mg / ml, and 1 mg / ml;

[0167] Dissolve 2 mg of the polypeptide sample (FFTFPT) in 200 μl of distilled water to obtain a peptide (FFTFPT) with a concentration of 10 mg / ml, and then dilute it to obtain peptides (FFTFPT) with concentrations of 8 mg / ml, 5 mg / ml, 2 mg / ml, and 1 mg / ml.

[0168] Sample wells: Sequentially add 10 μl of DPP-IV enzyme, 10 μl of two peptide samples with different concentrations, 30 μl of Asaay Buffer, and 50 μl of substrate into the wells;

[0169] Negative control: Sequentially add 10 μl of DPP-IV enzyme, 10 μl of solvent, 30 μl of Asaay Buffer, and 50 μl of substrate into the wells;

[0170] Background blank: Sequentially add 10 μl of solvent, 40 μl of Asaay Buffer, and 50 μl of substrate into the wells;

[0171] Place the above wells in a constant temperature incubator at 37 °C and let them stand in the dark for 30 min. Use an enzyme-linked immunosorbent assay reader to detect the fluorescence value at an excitation wavelength of 355 nm and an emission wavelength of 455 nm.

[0172] Ic 50 The half-inhibitory concentration value, that is, the sample concentration when the DPP-IV inhibition rate is 50%. Dilute the sample to be tested into 5 concentration gradients spanning 50% according to the inhibition rate data from the preliminary experiment, measure the DPP-IV inhibition rate of each gradient concentration, and import the data into GraghPad for non-linear fitting curve to obtain the Ic 50 value,

[0173] Calculation formula: Subtract the background fluorescence value from the fluorescence value of all wells to avoid fluorescence interference from the buffer, enzyme, and substrate

[0174] DPP-IV inhibition rate (%) = F 阴性对照 - F 样品 / F 阴性对照 - F 背景空白

[0175] The results are as follows:

[0176] Table 5 The strongest DPP-IV inhibition ability of grape seed protein peptides

[0177]

[0178] The DPP-IV inhibition Ic of the remaining 8 peptides 50 The detection of the value is the same as above, and the results are as follows:

[0179] Table 6 The DPP-IV inhibition ability of the remaining grape seed protein peptides

[0180]

[0181] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A grape seed protein peptide, characterized in that: The grape seed protein peptide is SGMFPFPF peptide or FFTFPT peptide, and its amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A method for preparing grape seed protein peptide, characterized in that: The method comprises: 1) Defatted grape seeds from wine fermentation byproducts are selected as raw materials, and the grape seeds are ground into powder to obtain defatted grape seed powder; the defatted grape seed powder is mixed with 50% ethanol water at a solid-liquid ratio of 1:4-6, and filtered after standing to obtain defatted and dephenolized grape seed powder; the defatted and dephenolized grape seed powder is dissolved in sodium hydroxide at a solid-liquid ratio of 1:15-18, the pH is adjusted to 10-12, and ultrasonic extraction is performed while stirring at a frequency of 30-50kHz for 15-30 minutes, and the extraction is filtered, and cross-linked polyvinyl pyrrolidone is added to the obtained filtrate at a mass volume ratio of 4-6% (w / v), mixed and reacted for 20-30 minutes, and the lower solid phase is removed to obtain grape seed protein alkali solution; 2) adjusting the pH of the grape seed protein alkali solution obtained in step 1) to an isoelectric point of 3.5 to 4, and centrifuging to obtain an acidic precipitated protein; finally, adding distilled water to redissolve the acidic precipitated protein, and adjusting the pH to neutral, centrifuging to obtain a grape seed protein precipitate, and freeze-drying to obtain a grape seed protein powder; 3) The grape seed protein powder obtained in the above step 2) is dissolved in a phosphate buffer system, and alkaline protease is added at 40-45° C., pH=9.5-10.5, stirring speed 100-150 r / min, with an enzyme / protein concentration ratio of 3800-4200 U / g for the next enzymatic hydrolysis; the reaction is terminated in a boiling water bath after 3.5-4 hours, trichloroacetic acid solution is added for precipitation, and excess protein is removed by centrifugation, and freeze-dried to obtain a grape seed protein peptide mixture.

3. An expression vector or a recombinant microorganism, characterized in that: The grape seed protein peptide according to claim 1 is expressed in the expression vector or recombinant microorganism; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; Preferably, the recombinant microorganism is a bacterium or a fungus.

4. A food, medicine, health product or nutritional product, characterized in that: The food, medicine, health product or nutritional product contains an effective dose of the grape seed protein peptide according to claim 1; preferably, the food, medicine, health product or nutritional product may also contain the grape seed protein peptide according to claim 1; the grape seed protein peptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino end or carbonyl end of the grape seed protein peptide; Preferably, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

5. The food, medicine, health product or nutritional product according to claim 4, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

6. Use of SGMFPFPF peptide and / or FFTFPT peptide in the preparation of DPP-IV inhibitors, or hypoglycemic drugs or health products, or drugs or health products for treating type II diabetes.

7. The use according to claim 6, characterized in that: The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

8. The use according to claim 7, characterized in that: The dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays; The health care product also contains acceptable auxiliary materials.

9. A DPP-IV inhibitor, or a hypoglycemic drug or health product, or a drug or health product for treating type II diabetes, characterized in that: The active ingredient is the grape seed protein peptide SGMFPFPF peptide or FFTFPT peptide described in claim 1.

10. A method for preparing the food, medicine, health product or nutritional product according to claim 4 or 5, characterized in that: The method comprises mixing the grape seed protein peptide or the grape seed protein peptide derivative described in claim 1 with at least one acceptable excipient; preferably, the grape seed protein peptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminal or carbonyl terminal of the polypeptide.

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