Grape seed protein peptide with dpp-iv inhibitory activity and preparation method thereof

Abstract

The application discloses grape seed protein peptides with DPP-IV inhibiting activity and a preparation method thereof, and belongs to the field of comprehensive utilization of grape wine fermentation by-products. The grape seed protein peptides are prepared from an enzymatic hydrolysate of grape seed protein prepared from grape seed protein by an ultrasonic-assisted alkali method through alkaline protease enzymolysis. The grape seed protein peptides have good DPP-IV inhibiting effect, are a natural plant source DPP-IV inhibitor, and can be applied to functional food and nutrition health care products.

Description

TECHNICAL FIELD

[0001] The present application relates to grape seed protein peptide with DPP-Ⅳ inhibitory activity and preparation method, belong to grape wine fermentation by-product comprehensive utilization field. BACKGROUND

[0002] Type Ⅱ diabetes is a chronic disease that affects the body's ability to metabolize glucose, mostly due to insulin resistance or deficiency. Among them, glucagon-like peptide-1 (GLP-1) and gut insulinotropic hormone glucose-dependent insulinotropic polypeptide (GIP) are the main regulatory factors of insulin release, which mainly reduce blood glucose levels by inhibiting the production of glucagon, stimulating insulin release and reducing gastric emptying, however, when the two gut incretins are released into the intestinal tract, they will be cleaved by a cell surface serine protease, dipeptidyl peptidase-Ⅳ, resulting in inactivation of the gut incretin, thereby causing blood glucose disorders in patients. The main mechanism of action of the dipeptidyl peptidase-Ⅳ (DPP-Ⅳ) inhibiting peptide in the present application is to inhibit DPP-Ⅳ enzyme and thus prolong the half-life of gut incretin, so as to promote insulin secretion, thereby controlling blood glucose in type Ⅱ diabetes patients.

[0003] With the continuous development of the wine industry, the increase in wine production is accompanied by a large amount of grape skin residue. According to statistics, about 1 kg of grape wine skin residue is produced for every 6 L of grape wine produced. Therefore, in order to promote green low-carbon development and sustainability of the industry, and to improve the rational resource utilization of grape wine processing waste, the present application proposes that the grape seeds produced by fermentation can be pressed for oil, and the defatted grape seeds are also a good source of DPP-Ⅳ inhibitory activity functional peptides.

[0004] Currently, the development of natural source blood glucose lowering functional peptides includes: Chinese patent application with publication number CN107141336A discloses a preparation method of yak glutelin peptide, which is prepared by using yak glutelin for multi-stage complex protease stepwise enzymolysis, then removing impurities, membrane separation, gel separation, reverse phase HPLC separation, concentration, and freeze-drying; so far, the newly published DPP-Ⅳ derived inhibitory peptides have not shown an inhibitory force comparable to that of drugs. While promoting the development of grape wine by-products, we are committed to developing new plant-derived DPP-Ⅳ inhibitory peptides that show higher DPP-Ⅳ inhibitory activity.

[0005] Meanwhile, the dipeptidyl peptidase-IV (DPP-IV) inhibitors on the market, such as sitagliptin and saxagliptin, can effectively control blood sugar, but are accompanied by side effects and high production and use costs. Natural plant-derived functional peptides are easily obtained by patients and medical researchers in the current "health" trend, and have the advantages of no side effects, high safety, easy absorption, and the like, and thus have become one of the main research directions in the field of food processing in recent years. Therefore, it is particularly important and urgent to develop a safe and efficient plant-derived functional peptide having DPP-IV inhibitory activity. SUMMARY

[0006] To solve the above technical problems and further improve the recycling method of the grape seed, a fermentation by-product of grape wine, the present application evaluates the potential of grape seed protein as a source of DPP-IV (dipeptidyl peptidase-IV) inhibitory peptide, and verifies that the grape seed protein hydrolysate has good DPP-IV inhibitory effect, and provides a grape seed protein peptide having DPP-IV inhibitory activity and a preparation method thereof.

[0007] The present application provides a grape seed protein peptide having DPP-IV inhibitory activity, 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 an embodiment of the present application, 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 an embodiment of the present application, the grape seed protein peptide is prepared from defatted grape seeds.

[0012] In an embodiment of the present application, the grape seed protein peptide can be used as a drug, health product or dietary supplement, or added to ordinary food such as beverages, dairy products, etc. as a food base; it is used for inhibiting DPP-IV, reducing blood sugar, and used as a prophylactic or adjuvant therapy for high blood sugar patients, type II diabetes patients, or chronic metabolic syndrome patients with obesity, immunodeficiency, cardiovascular and cerebrovascular diseases, etc.

[0013] In an embodiment of the present application, the preparation method of the grape seed protein peptide comprises the steps of:

[0014] In one aspect of the present application, the method for extracting protein by ultrasonic-assisted alkali dissolution and acid precipitation is used, and the extraction rate and extraction effect are improved by adding cross-linked polyvinylpyrrolidone to adsorb phenols in the protein solution; in another aspect, alkaline protease is selected for enzymolysis to release grape seed protein peptides with DPP-IV inhibitory activity.

[0015] In one embodiment of the present application, the grape seed protein peptide preparation method comprises the following steps:

[0016] 1) Extracting protein from grape seed powder by ultrasonic-assisted alkali dissolution and acid precipitation;

[0017] 2) Enzymolysis of the grape seed protein prepared in step 1) to release protein peptides;

[0018] 3) Purification of the hydrolysate after enzymolysis in step 2) by ultrafiltration and RP-HPLC, and freeze-drying after rotary evaporation to obtain the product.

[0019] In order to obtain 100% grape seed protein peptides, the above grape seed extraction process can preferably comprise the following steps:

[0020] In order to obtain 100% grape seed protein peptides, the above grape seed extraction process can preferably comprise the following steps:

[0021] In one preferred embodiment of the present application, the solid-liquid ratio of grape seed powder mixed with 50% anhydrous ethanol is 1:5.

[0022] In one preferred embodiment of the present application, 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 application, the use of 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 application, the amount of cross-linked polyvinylpyrrolidone used for adsorbing polyphenols is 4% (w / w), which can significantly reduce the content of polyphenols in grape seed protein, and the color of the obtained protein powder is also more pure due to the substantial reduction of anthocyanins.

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

[0025] The grape seed protein powder prepared in step 1) is dissolved in a 0.01M phosphate buffer (PBS) system, and the temperature is adjusted to 45°C and the pH is adjusted to 10.0. Alkaline protease is added at an enzyme / protein concentration ratio of 4000U / g for the next step of enzymolysis. The pH and temperature of the reaction system are maintained stable during the process. After the reaction, the reaction is terminated by boiling water bath. A 15% trichloroacetic acid solution is added to precipitate and remove excess protein by centrifugation. The grape seed protein hydrolysate concentrate is obtained by freeze-drying.

[0026] In a preferred embodiment of the present application, it is verified that alkaline protease has the highest DPP-IV inhibition potential, and the optimal enzymolysis time is 4h. Under this enzymolysis mode, 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 inhibition effect of the obtained grape seed protein solution, the ultrafiltration operation in step 3) is as follows:

[0028] The grape seed protein hydrolysate concentrate obtained in step 2) is purified using a Labscale TFF ultrafiltration system with a molecular weight cut-off of 5kda to obtain a grape seed protein hydrolysate with a molecular weight of <5kda. The grape seed protein hydrolysate with a molecular weight of <3kda is obtained by using an ultrafiltration centrifuge tube with a molecular weight cut-off of 3kda for purification.

[0029] In a preferred embodiment of the present application, the reverse phase HPLC in step 3) can be as follows:

[0030] The grape seed protein hydrolysate with a molecular weight of <3kda is separated by RP-HPLC reverse phase high performance liquid chromatography. The mobile phase A is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is HPLC grade acetonitrile, the detection wavelength is 214nm, and the elution peak collected at 9-12min is obtained. The solvent is removed by rotary evaporation to obtain the grape seed protein peptide crude product.

[0031] In a preferred embodiment of the present application, the grape seed protein peptide with DPP-IV inhibition 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 application, the grape seed protein peptide having DPP-IV inhibitory function prepared by the above method includes polypeptides having the amino acid sequences of 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 either 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 application also provides an expression vector or a recombinant microorganism, wherein the expression vector or the recombinant microorganism contains at least one of the above grape seed protein peptides.

[0036] In an embodiment of the present application, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector.

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

[0038] The present application also provides a pharmaceutical composition containing a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable pharmaceutical adjuvant; the active ingredient contains 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 peptide can significantly inhibit DPP-IV activity.

[0040] In an embodiment of the present application, the pharmaceutical adjuvant refers to a conventional pharmaceutical carrier in the pharmaceutical field.

[0041] In one embodiment of the present application, the adjuvant includes one or more of the following: a binder such as a cellulose derivative, an alginate, gelatin, and polyvinylpyrrolidone; a diluent such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; a filler such as starch, sucrose, etc.; a wetting agent such as glycerin; a disintegrant such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch, etc.; an absorption enhancer such as a quaternary ammonium compound; a surfactant such as polysorbate, sorbitan fatty acid, and fatty acid glyceride, etc.; a coloring agent such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red, etc.; a lubricant such as hydrogenated vegetable oil, talc, and polyethylene glycol, etc.; a coating material such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate phthalate, etc.; and other adjuvants such as a flavoring agent, a sweetening agent, etc. can be further added to the composition.

[0042] The present application also provides a food, a pharmaceutical, a health food, or a nutritional product containing an effective amount of at least one of the above-described grape seed protein peptides.

[0043] In one embodiment of the present application, 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 application, the pharmaceutical further contains a pharmaceutically acceptable pharmaceutical adjuvant; the pharmaceutical adjuvant refers to a conventional pharmaceutical carrier in the pharmaceutical field.

[0045] In one embodiment of the present application, the adjuvant includes one or more of the following: a binder such as a cellulose derivative, an alginate, gelatin, and polyvinylpyrrolidone; a diluent such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; a filler such as starch, sucrose; a wetting agent such as glycerin; a disintegrant such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; an absorption enhancer such as a quaternary ammonium compound; a surfactant such as polysorbate, sorbitan fatty acid, and fatty acid glyceride; a coloring agent such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red, etc.; a lubricant such as hydrogenated vegetable oil, talc, and polyethylene glycol; a coating material such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate phthalate; and other adjuvants such as a flavoring agent, a sweetening agent.

[0046] In one embodiment of the present application, the dosage form of the pharmaceutical includes, but is not limited to, an oral dosage form, an injection dosage form, an inhalation dosage form.

[0047] In one embodiment of the present application, the oral dosage form includes, but is not limited to, a tablet, a capsule, a granule, an oral liquid, an oral suspension.

[0048] In an embodiment of the present application, the injection forms include, but are not limited to, injection solutions, injection powders.

[0049] In an embodiment of the present application, the inhalation forms include, but are not limited to, aerosols, powder aerosols.

[0050] In an embodiment of the present application, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, beverages; the food also includes special dietary foods.

[0051] In an embodiment of the present application, the health products further contain acceptable excipients.

[0052] The present application also provides the use of at least one or more of the above grape seed protein peptides in the preparation of food, medicine, health products or nutritional products; which are used for inhibiting DPP-IV activity, lowering blood sugar, preventing or treating diabetes or diseases that benefit from DPP-IV inhibition, or preventing or treating chronic metabolic syndrome with obesity, immunodeficiency or cardiovascular and cerebrovascular diseases.

[0053] In an embodiment of the present application, 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 an embodiment of the present application, the medicine further contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional pharmaceutical carriers in the pharmaceutical field.

[0055] In an embodiment of the present application, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; diluents such as starch, pre-gelatinized starch, dextrin, sucrose, lactose, mannitol and the like; fillers such as starch, sucrose; humectants 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 and fatty acid glycerol ester; colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red and the like; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate phthalate; in addition, other auxiliary agents such as flavoring agents, sweeteners can also be added in the composition.

[0056] In an embodiment of the present application, the dosage forms of the medicine include, but are not limited to, oral dosage forms, injection dosage forms, inhalation dosage forms.

[0057] In an embodiment of the present application, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral solutions, oral suspensions.

[0058] In an embodiment of the present application, the injection forms include, but are not limited to, injection solutions and injection powders.

[0059] In an embodiment of the present application, the inhalation forms include, but are not limited to, aerosols and powder sprays.

[0060] In an embodiment of the present application, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, bean products, beverages; and the food also includes special dietary foods.

[0061] In an embodiment of the present application, the health products further contain acceptable excipients.

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

[0063] In an embodiment of the present application, 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] Advantages:

[0065] (1) The present application 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, which are novel in structure, easy to prepare, and can be obtained from grape seed proteins or artificially synthesized.

[0066] (2) The two polypeptides obtained in the present application have strong DPP-IV inhibitory activity; the half inhibitory concentration (IC 50 ) of Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF) is 231.9 ± 13.23 μmol / L; and 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 application can be used as a drug, health product or dietary supplement, or added to common food such as beverage, dairy product and the like as a food base; it is used for inhibiting DPP-IV, reducing blood sugar, as a prophylaxis or adjuvant therapy for hyperglycemia patients, type 2 diabetes patients, or chronic metabolic syndrome population with obesity, immunodeficiency, cardiovascular and cerebrovascular diseases, and has a wide application prospect in the fields of functional food, medicine, health product and the like. BRIEF DESCRIPTION OF DRAWINGS

[0068] Fig. 1 is a result graph of DPP-IV inhibition rate under different hydrolysis times;

[0069] Fig. 2 is a result graph of free radical scavenging rate under different hydrolysis times. DETAILED DESCRIPTION

[0070] The specific embodiments of the present application are described in further detail below in conjunction with examples. The following examples are used to illustrate the present application, but are not used to limit the scope of the present application.

[0071] If not specifically indicated, the technical means used in the examples are the conventional technical means well known to those skilled in the art. If not specifically indicated, the reagents used in the examples are commercially available.

[0072] The defatted grape seed powder involved in the following examples can be prepared by the following method: defatted grape seed powder is prepared by mixing grape seed powder with n-hexane at a solid-liquid ratio of 1:5, standing and then filtering;

[0073] The defatted grape seed in the by-product of grape wine fermentation can also be directly selected as the raw material.

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

[0075] pvpp (cross-linked polyvinylpyrrolidone) is purchased from Shanghai Maier Biotech Co., Ltd., and the product code is M86828-100G;

[0076] Folin phenol is purchased from Shanghai Titan Science and Technology Co., Ltd., and the product code is 4140369A;

[0077] Alkaline protease is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., and the enzyme activity is 200 U / mg, and the product code is S10154-100g;

[0078] Papain is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., and the enzyme activity is 800 U / mg, and the product code is S10011-100g;

[0079] Pepsin is purchased from China Merck Life Science, and the enzyme activity is ≥500 U / mg, and the product code is 77160-25g;

[0080] Papain was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., with an enzyme activity of 300 U / mg, and a product code of S10009-100g;

[0081] Neutral protease was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., with an enzyme activity of 100 U / mg, and a product code of S10013-250g;

[0082] BCA protein concentration determination kit (enhanced) was purchased from Biyun Tian Biotechnology Co., Ltd., with a product code of P0010S;

[0083] DPP-Ⅳ inhibitor screening kit was purchased from Taiwan Yanofa Biotechnology Co., Ltd., with a product code of KA1311.

[0084] The determination method involved in the following examples is as follows:

[0085] 1. DPP-Ⅳ (dipeptidyl peptidase-Ⅳ) inhibition activity determination method:

[0086] The method is based on the DPP-Ⅳ inhibitor screening kit instruction of Abnova Company (product code: KA1311). The kit contains DPP-Ⅳ buffer (Assay Buffer), DPP-Ⅳ enzyme, and substrate Gly-Pro-AMC. The activity determination is carried out by using 96-well plate.

[0087] Sample well: 10 μl of DPP-Ⅳ enzyme, 10 μl of sample, 30 μl of Assay Buffer, and 50 μl of substrate were sequentially added to the well.

[0088] Negative control: 10 μl of DPP-Ⅳ enzyme, 10 μl of solvent, 30 μl of Assay Buffer, and 50 μl of substrate were sequentially added to the well.

[0089] Background blank: 10 μl of solvent, 40 μl of Assay Buffer, and 50 μl of substrate were sequentially added to the well.

[0090] The above wells were placed in a 37℃ constant temperature incubator for 30 min, and the fluorescence value was detected by using an enzyme label instrument at an excitation wavelength of 355 nm and an emission wavelength of 455 nm.

[0091] Ic 50 The value is the half-inhibitory concentration, i.e. the sample concentration when the DPP-Ⅳ inhibition rate is 50%. The sample to be tested was diluted into 5 concentration gradients across 50% according to the inhibition rate data of the pre-experiment, the DPP-Ⅳ inhibition rate of each gradient concentration was determined, and the data was imported into GraghPad nonlinear fitting curve to obtain Ic 50 value.

[0092] Calculation formula: All hole fluorescence value minus background fluorescence value, so as to avoid the fluorescence interference of buffer, enzyme and substrate.

[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 of BCA protein concentration determination kit (enhanced) of Biyun Tian Biotechnology (product number: P0010S). The kit contains BCA working solution, protein standard (20mg BSA) and protein standard configuration solution. The activity determination is carried out by using 96-well plate.

[0096] Dissolve the protein standard with the protein standard configuration solution. After fully dissolving, configure the protein standard solution to 0.5mg / ml, and then dilute it with distilled water to 0.4, 0.3, 0.2, 0.1, 0.05, 0.025mg / ml. In the 96-well plate, add 20μl protein standard and sample solution, then add 200μl BCA working solution to each well, and place it at 37℃ for 25min. Measure the absorbance at A562 by using the enzyme label instrument, and make the protein standard curve. According to the standard curve, calculate the protein concentration of the sample.

[0097] (w / v) in the following examples is 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 grape wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under the protection of liquid nitrogen to prepare defatted grape seed powder;

[0101] Mix the prepared defatted grape seed powder with 50% (volume percentage) ethanol aqueous solution at a ratio of 1:5 (w / v), and place it in the dark for 1h. Remove the top phase by filtration, and repeat the above operation twice on the residue to obtain defatted and dephenolized grape seed powder.

[0102] 2) The defatted and dephenolized grape seed powder prepared in step 1) is dissolved with 0.1M sodium hydroxide at a solid-liquid ratio of 1:16 (w / v), and its pH is adjusted to 11 with 6M sodium hydroxide, and it is extracted for 40 min with stirring and ultrasonic assistance (frequency of 40 kHz), and the lower solid phase is removed by filtration. Then, pvpp (cross-linked polyvinylpyrrolidone) is added to the obtained filtrate at a solid-liquid ratio of 4% (w / v), and the mixture is reacted for 30 min, and the lower solid phase is removed to obtain an alkaline solution of grape seed protein;

[0103] 3) The pH of the alkaline solution of grape seed protein obtained in step 2) is adjusted to the isoelectric point of 3.5 with 6M hydrochloric acid, and it is placed in a refrigerator at 4°C for 2 h, and centrifuged at 12000 rpm to obtain an acidic precipitated protein; finally, distilled water is added to re-dissolve the obtained acidic precipitated protein, and its pH is adjusted to neutral, and centrifuged at 12000 rpm to obtain a precipitated grape seed protein, and freeze-dried to obtain a grape seed protein powder.

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

[0105] In order to verify the influence of the pretreatment of ethanol extraction and the removal of impurities by polyvinylpyrrolidone pvpp on the preparation efficiency of grape seed protein, and select the same four groups of Cabernet Sauvignon defatted grape seeds from wine fermentation by-products, the whole grape seeds are ground into powder under the protection of liquid nitrogen to prepare defatted grape seed powder (preparation method is the first step of step 1), and four groups of control experiments are designed (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] The prepared defatted grape seed powder is dissolved with 0.1M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH is adjusted to 11 with 6M sodium hydroxide, and it is extracted for 40 min with stirring and ultrasonic assistance (frequency of 40 kHz), and the lower solid phase is removed by filtration. Then, pvpp (cross-linked polyvinylpyrrolidone) is added to the obtained filtrate at a solid-liquid ratio of 4%, and the mixture is reacted for 30 min, and the lower solid phase is removed to obtain an alkaline solution of grape seed protein, and freeze-dried to obtain #1 grape seed protein (GSP#1).

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

[0112] The defatted grape seed powder prepared was dissolved with 0.1M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6M sodium hydroxide. The extraction was performed for 40 min with stirring and ultrasonic assistance (frequency of 40 kHz). The lower solid phase was removed by filtration to obtain a grape seed protein alkali solution, which was freeze-dried to obtain grape seed protein #2 (GSP #2).

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

[0114] The defatted grape seed powder prepared was mixed with 50% anhydrous 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, the defatted and dephenolized grape seed powder was dissolved with 0.1M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6M sodium hydroxide. The extraction was performed for 40 min with stirring and ultrasonic assistance (frequency of 40 kHz). The lower solid phase was removed by filtration. Then, 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) was added to the obtained filtrate, and the mixture was allowed to react for 30 min. The lower solid phase was removed to obtain a grape seed protein alkali solution, which was freeze-dried to obtain grape seed protein #3 (GSP #3).

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

[0116] The defatted grape seed powder prepared was mixed with 50% anhydrous 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, the defatted and dephenolized grape seed powder was dissolved with 0.1M sodium hydroxide at a solid-liquid ratio of 1:16, and the pH was adjusted to 11 with 6M sodium hydroxide. The extraction was performed for 40 min with stirring and ultrasonic assistance (frequency of 40 kHz). The lower solid phase was removed by filtration to obtain a grape seed protein alkali solution, which was freeze-dried to obtain grape seed protein #4 (GSP #4).

[0117] The protein content was determined by GB 5009.5-2016 Determination of protein in foods - Kjeldahl method for nitrogen:

[0118] A fully 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. Digestion was performed in a digestion furnace. When the temperature of the digestion furnace reached 420°C, the digestion was continued for 1 h. At this time, the liquid in the digestion tube was green and transparent. After cooling, 50 ml of water was added, and automatic liquid addition, distillation, titration, and recording of titration data were realized on an automatic Kjeldahl nitrogen apparatus. According to the acid consumption, the content of protein was calculated by multiplying the conversion factor of 6.25.

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

[0120] A standard curve was established using the standard phenolic compound catechin. 0.1 mL of sample with a concentration of 1 mg / mL was added to a 10 mL volumetric flask, 7 mL of distilled water was added and mixed, 0.5 mL of 1 M Folin phenol was added, 1 min later, 1.5 mL of 20% (v / v) sodium carbonate solution was added, mixed, and made up to 10 mL with distilled water. The reaction was carried out in the dark for 120 min, and the absorbance was measured at 765 nm. The results were expressed in terms of 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 above obtained #1 grape seed protein, #2 grape seed protein, #3 grape seed protein, and #4 grape seed protein were determined, and the determination results are shown in Table 2.

[0123] Table 2: Grape seed protein content under different extraction methods

[0124]

[0125] As can be seen from Table 2, the protein content of #3 grape seed protein (GSP#3) is the highest and the total phenol content is the lowest, and the effect of impurity removal is basically 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 grape seed protein hydrolysate concentrate

[0127] The preparation method of grape seed protein peptide with DPP-Ⅳ inhibition is as follows:

[0128] 1. Preparation of grape seed protein

[0129] 1) Selecting Cabernet Sauvignon defatted grape seeds as raw materials, the whole grape seeds were ground into powder under the protection of liquid nitrogen 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 placed in the dark for 1 h, and the top phase was removed by filtration. The residue was repeatedly operated twice, and defatted and dephenolized grape seed powder was obtained. Then 0.1 M sodium hydroxide was used to dissolve the defatted and dephenolized grape seed powder at a solid-liquid ratio of 1:16, and 6 M sodium hydroxide was used to adjust the pH to 11. The mixture was stirred and ultrasonicated (frequency of 40 kHz) for 40 min, and the lower solid phase was removed by filtration. Then 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) was added to the obtained filtrate, mixed for 30 min, and the lower solid phase was removed to obtain grape seed protein alkali solution;

[0131] 3) The pH of the grape seed protein alkaline solution obtained in step 2) is adjusted to the isoelectric point 3.5 with 6M hydrochloric acid, and the solution is stored at 4°C for 2h, and then centrifuged at 12000 rpm to obtain an acidic precipitate; finally, the acidic precipitate is dissolved in distilled water, and the pH is adjusted to neutral, and then centrifuged at 12000 rpm to obtain a grape seed protein precipitate, which is freeze-dried to obtain grape seed protein powder.

[0132] 2) Preparation of a grape seed protein hydrolysate concentrate with DPP-IV inhibitory activity

[0133] The grape seed protein powder prepared in step 1) above is dissolved in a 0.01M phosphate buffer (PBS) system, and a magnetic stirrer connected with a pH electrode is adjusted to the optimal reaction temperature 45°C, pH=10.0, and stirring speed 40 rpm. Alkaline protease is added at an enzyme / protein concentration ratio of 4000 U / g, and the next enzymatic hydrolysis is carried out. The pH and temperature of the whole reaction system are maintained stable during the process, and the reaction is terminated after 4h by boiling water bath. A 15% (v / v) 1M trichloroacetic acid solution is added to precipitate and remove the excess protein by centrifugation at 12000 rpm, and the freeze-dried product is grape seed protein hydrolysate concentrate.

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

[0135] The specific implementation is the same as in Example 2, except that the reaction time in step 2, (1) is replaced by 0.5, 1, 2, 3, 4, 5 or 6h, and the alkaline protease in step 2, (2) is replaced by 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. The DPP-IV inhibitory rate and polypeptide concentration of the grape seed protein hydrolysate concentrate prepared by hydrolysis with alkaline protease, papain, pepsin, neutral protease or bromelain for different reaction times are determined by the above-mentioned DPP-IV inhibitory activity determination method and protein concentration determination method.

[0136] The results are shown in Table 3 and Figure 1. Figs. 1-2 and Table 3.

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

[0138]

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

[0140] The DPP-IV inhibitory activity of the grape seed protein hydrolysate concentrate prepared by hydrolysis with alkaline protease, papain, pepsin, neutral protease or bromelain for 4h is determined 50Values, results are shown in Table 4:

[0141] Table 4: DPP-IV inhibition Ic of different hydrolysis enzymes of grape seed protein 4h hydrolysate 50 Values

[0142]

[0143] Results show that the hydrolysate of alkaline protease has the most significant effect on DPP-IV enzyme inhibition Ic 50 96±4.3μg soluble protein hydrolysate / mL (pepsin Ic 50 980±16.7μg soluble protein hydrolysate / mL, neutral protease Ic 50 160±7μg soluble protein hydrolysate / mL, bromelain Ic 50 369.7±29.79μg soluble protein hydrolysate / mL, papain Ic 50 1028±91.19μg 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) Selecting Cabernet Sauvignon defatted grape seeds from wine fermentation by-products as raw materials, grinding the whole grape seeds into powder under the protection of liquid nitrogen to prepare defatted grape seed powder.

[0148] 2) Mixing the prepared defatted grape seed powder with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and standing in the dark for 1h, filtering to remove the top phase, repeating the above operation twice on the residue to prepare defatted and dephenolized grape seed powder. Then, using 0.1M sodium hydroxide at a solid-liquid ratio of 1:16, adjusting the pH to 11 with 6M sodium hydroxide, extracting for 40min while stirring and assisting with ultrasonic waves (frequency of 40kHz), and filtering to remove the lower solid phase. Then, adding 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) to the obtained filtrate, mixing for 30min, and removing the lower solid phase to obtain grape seed protein alkali solution;

[0149] 3) Adjusting the pH of the grape seed protein alkali solution obtained in step 2) to the isoelectric point of 3.5 with 6M hydrochloric acid, placing it in a refrigerator at 4℃ for 2h, centrifuging at 12000rpm to obtain acid precipitated protein; finally, redissolving the obtained acid precipitated protein with distilled water, adjusting its pH to neutral, centrifuging at 12000rpm to obtain grape seed protein precipitate, and freeze-drying to obtain grape seed protein powder.

[0150] 2. Preparation of grape seed protein peptides with DPP-IV inhibitory activity

[0151] 1) The grape seed protein powder prepared in step 1 above was dissolved in 0.01 M phosphate buffer (PBS) system, and a magnetic stirrer connected with a pH electrode was adjusted to the optimal reaction condition temperature of 45°C, pH = 10.0, and stirring speed of 40 rpm. Alkaline protease was added at an enzyme / protein concentration ratio of 4000 U / g for the next enzymatic hydrolysis. The pH and temperature of the whole reaction system were maintained stable during the process, and after 4 h of reaction, the reaction was terminated by boiling water bath. A 15% (v / v) 1 M trichloroacetic acid solution was added to precipitate and remove excess protein by centrifugation at 12000 rpm. The grape seed protein hydrolysate concentrate was obtained by freeze-drying.

[0152] 2) The grape seed protein hydrolysate concentrate obtained in step 1) was subjected to ultrafiltration separation, and purified using a Labscale TFF ultrafiltration system with a molecular weight cut-off of 5 kda to obtain grape seed protein hydrolysate <5 kda. The ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kda was used again for purification to obtain grape seed protein hydrolysate 3-5 kda and <3 kda.

[0153] 3) The <3 kda grape seed protein hydrolysate obtained in step 2) was subjected to RP-HPLC reverse phase high performance liquid chromatography separation, with mobile phase A being 0.1% (v / v) trifluoroacetic acid aqueous solution and mobile phase B being HPLC grade acetonitrile, detection wavelength being 214 nm, 0-5 min, 2% B, 5-9 min, 2%-7% B, 9-28 min, 7%-30% B, 28-40 min, 30%-50% B, 40-41 min, 50%-100% B, 41-45 min, 100% B, 45-46 min, 100%-2% B, 46-60 min, 2% B. The flow rate was set to 2 mL / min; the elution peaks collected at 9-12 min were removed by rotary evaporation to obtain grape seed protein peptide crude product.

[0154] 4) The grape seed protein peptide crude product obtained in step 3) was subjected to mass spectrometry identification, and the grape seed protein peptide composition was determined by LC-MS / MS.

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

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

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

[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 grape seed protein peptide components are: a total of 10 peptides with DPP-Ⅳ inhibitory activity are identified (Table 5-Table 6), and the amino acid sequences of the two peptides with the highest DPP-Ⅳ inhibitory activity of the grape seed protein peptide are Ser-Gly-Met-Phe-Pro-Phe-Pro-Phe (SGMFPFPF) or Phe-Phe-Thr-Phe-Pro-Thr (FFTFPT).

[0161] Experimental Example 5: Determination of DPP-Ⅳ inhibitory capacity of grape seed protein peptide

[0162] The sample is the DPP-Ⅳ inhibitory peptide obtained by hydrolysis, ultrafiltration, reverse phase HPLC and mass spectrometry identification in Example 4 (Table 5-Table 6), which is synthesized by Shanghai Jil Biochemical Company, and the purity is ≥98%.

[0163] The 10 identified peptide segments (Table 5-Table 6) such as SGMFPFPF, FFTFPT were detected for DPP-Ⅳ inhibitory Ic 50 value.

[0164] Ic 50 The value determination method is as follows:

[0165] The method is based on the DPP-IV inhibitor screening kit instruction of Abnova Company (article number: KA1311). The kit contains DPP-IV buffer (Assay Buffer), DPP-IV enzyme, and substrate Gly-Pro-AMC. The activity determination is performed by using a 96-well plate.

[0166] Sample concentration preparation: 2 mg of polypeptide sample (SGMFPFPF) is dissolved in 100 μl of distilled water to obtain 20 mg / ml of peptide (SGMFPFPF), which is then diluted to obtain 10 mg / ml, 5 mg / ml, 2 mg / ml, and 1 mg / ml of peptide (SGMFPFPF).

[0167] 2 mg of polypeptide sample (FFTFPT) is dissolved in 200 μl of distilled water to obtain 10 mg / ml of peptide (FFTFPT), which is then diluted to obtain 8 mg / ml, 5 mg / ml, 2 mg / ml, and 1 mg / ml of peptide (FFTFPT).

[0168] Sample well: 10 μl of DPP-IV enzyme, 10 μl of two peptide samples with different concentrations, 30 μl of Assay Buffer, and 50 μl of substrate are sequentially added to the well.

[0169] Negative control: 10 μl of DPP-IV enzyme, 10 μl of solvent, 30 μl of Assay Buffer, and 50 μl of substrate are sequentially added to the well.

[0170] Background blank: 10 μl of solvent, 40 μl of Assay Buffer, and 50 μl of substrate are sequentially added to the well.

[0171] The above well is placed in a 37°C constant temperature incubator for dark incubation for 30 min. The fluorescence value is detected by using an enzyme label instrument at an excitation wavelength of 355 nm and an emission wavelength of 455 nm.

[0172] Ic 50 The value is the half-inhibitory concentration, i.e., the sample concentration when the DPP-IV inhibition rate is 50%. The sample to be tested is diluted to 5 concentration gradients across 50% according to the inhibition rate data of the pre-experiment. The DPP-IV inhibition rate of each gradient concentration is determined. The Ic value is obtained by importing the data into the GraphPad nonlinear fitting curve. 50 value,

[0173] Calculation formula: The fluorescence value of all wells is subtracted by the background fluorescence value to avoid the fluorescence interference of 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 values of the remaining 8 peptides were detected as above, and the results are as follows: 50

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

[0180]

[0181] Although the present application has been disclosed in the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application 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 the amino acid sequences are shown in SEQ ID NO. 1 or SEQ ID NO. 2 respectively.

2. An expression vector, characterized by, The expression vector encodes the grape seed protein peptide of claim 1.

3. The expression vector of claim 2, wherein, The expression vector is selected from a DNA vector, an RNA vector or a viral vector.

4. The expression vector of claim 2, wherein, The expression vector is selected from a plasmid, a transposon vector or a CRISPR / Cas9 vector.

5. A recombinant microorganism, characterized in that, The recombinant microorganism expresses the grape seed protein peptide of claim 1.

6. The recombinant microorganism of claim 5, wherein, The recombinant microorganism is bacteria or fungi.

7. A food, drug, nutraceutical or nutritional product, characterized in that, The food, drug, health product or nutritional product contains an effective dose of the grape seed protein peptide of claim 1.

8. The food, drug, nutraceutical or nutritional product of claim 7, wherein, The drug also contains a pharmaceutically acceptable pharmaceutical excipient; the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field.

9. The food, drug, nutraceutical or nutritional product of claim 8, wherein, The excipient includes one or more of the following: binder: cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluent: pregelatinized starch, dextrin, sucrose, lactose, mannitol; filler: starch, sucrose; humectant: glycerol; disintegrant: sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption enhancer: quaternary ammonium compound; surfactant: polysorbate, sorbitan fatty acid and fatty acid glyceride; colorant: titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red; lubricant: hydrogenated vegetable oil, talc and polyethylene glycol; coating material: acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; other excipients can also be added to the composition: flavoring agent, sweetener. The dosage form of the drug is oral dosage form, injection dosage form, inhalation dosage form. The oral dosage form is tablet, capsule, granule, oral liquid, oral suspension.

10. The food, drug, nutraceutical or nutritional product of claim 8, wherein, The injection dosage form is injection liquid, injection powder.

11. The food, drug, nutraceutical or nutritional product of claim 10, wherein, The inhalation dosage form is aerosol, powder aerosol.

12. The food, drug, nutraceutical or nutritional product of claim 10, wherein, The food is cereal product, vegetable product, fruit product, meat product, seafood product, egg product, dairy product, bean product, beverage; the food also includes special dietary food.

13. The food, drug, nutraceutical or nutritional product of claim 10, wherein, The health product also contains an acceptable excipient.

14. The food, drug, nutraceutical or nutritional product of claim 7, wherein, 16. Use of the grape seed protein peptide SGMFPFPF peptide and / or FFTFPT peptide of claim 1 in the preparation of a drug or health product for assisting in reducing blood sugar, or a drug for treating type II diabetes.

15. The food, drug, nutraceutical or nutritional product of claim 7, wherein, The drug also contains a pharmaceutically acceptable pharmaceutical excipient; the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field. The excipient includes one or more of the following: binder: cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluent: pregelatinized starch, dextrin, sucrose, lactose, mannitol; filler: starch, sucrose; humectant: glycerol; disintegrant: sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption enhancer: quaternary ammonium compound; surfactant: polysorbate, sorbitan fatty acid and fatty acid glyceride; colorant: titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red; 17. Use according to claim 16, characterized in that, ​ 18. The use according to claim 17, characterized in that, ​ ​ Lubricants: hydrogenated vegetable oil, talc and polyethylene glycol; coating materials: acrylic resin, hydroxypropyl methyl cellulose, povidone, cellulose acetate phthalate; other auxiliary agents can also be added to the composition: flavoring agents, sweeteners.

19. The use according to claim 16, characterized in that, The dosage form of the medicine is oral dosage form, injection dosage form, inhalation dosage form.

20. The use according to claim 19, characterized in that, The oral dosage form is tablet, capsule, granule, oral liquid, oral suspension.

21. The use according to claim 19, characterized in that, The injection dosage form is injection liquid, injection powder.

22. The use according to claim 19, characterized in that, The inhalation dosage form is aerosol, powder mist.

23. The use according to claim 16, characterized in that, The health care product also contains acceptable auxiliary materials.

24. An adjunct blood glucose lowering drug or nutraceutical, or a drug for the treatment of type 2 diabetes, characterized in that, It takes the grape seed protein peptide SGMFPFPF peptide or FFTFPT peptide in claim 1 as the active ingredient.

25. A method of preparing a food, pharmaceutical, nutraceutical or nutritional product according to any one of claims 7 to 15, characterised in that, The method comprises mixing the grape seed protein peptide in claim 1 with at least one acceptable auxiliary material.

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