阅读说明：本技术 一种利用核桃粕制备二肽基肽酶ⅳ抑制活性肽的方法及其应用 (Method for preparing dipeptidyl peptidase IV inhibiting active peptide by using walnut meal and application thereof ) 是由 孔祥珍 张丽娜 华欲飞 宋伟光 张彩猛 陈业明 李兴飞 于 2020-05-18 设计创作，主要内容包括：一种利用核桃粕制备二肽基肽酶Ⅳ抑制活性肽的方法及其应用,属于植物蛋白质深加工技术领域。本发明以脱脂核桃粕为原料,首先对其进行醇洗、酸洗和高速剪切处理,加入碱性蛋白酶水解得到酶解液；经脱盐和干燥得到二肽基肽酶Ⅳ抑制活性肽。本发明方法制备的核桃肽具有良好的降血糖和抗氧化功能活性,产品得率和蛋白质含量高,实现了核桃粕的深加工和综合利用,对于提高其附加值具有重要意义。(A method for preparing dipeptidyl peptidase IV inhibiting active peptide by using walnut meal and application thereof belong to the technical field of plant protein deep processing. The invention takes the degreased walnut dregs as the raw material, firstly carries out alcohol washing, acid washing and high-speed shearing treatment on the degreased walnut dregs, and adds alkaline protease for hydrolysis to obtain enzymatic hydrolysate; desalting and drying to obtain the dipeptidyl peptidase IV inhibiting active peptide. The walnut peptide prepared by the method has good activity of reducing blood sugar and resisting oxidation, high product yield and high protein content, realizes deep processing and comprehensive utilization of walnut meal, and has important significance for improving the added value of the walnut meal.)

1. A method for preparing dipeptidyl peptidase IV inhibiting active peptide by using walnut meal is characterized by comprising the following steps:

(1) alcohol washing: carrying out alcohol washing treatment on the degreased walnut dregs to obtain alcohol-washed walnut dregs;

(2) acid washing: carrying out acid washing treatment on the alcohol-washed walnut pulp to obtain walnut protein curd;

(3) shearing: mixing the walnut protein curd with deionized water, and carrying out high-speed shearing treatment;

(4) hydrolysis: adding alkaline protease to carry out enzymatic hydrolysis under certain conditions to obtain a crude enzymatic hydrolysate;

(5) and (3) post-treatment: desalting and drying the crude enzymolysis liquid to obtain powdery dipeptidyl peptidase IV inhibiting active peptide.

2. The method for preparing the dipeptidyl peptidase IV inhibiting active peptide by using the walnut meal according to claim 1, which is characterized in that: adding degreased walnut cake powder into an ethanol solution with the volume concentration of 85% and the volume of 4-6 times that of the degreased walnut cake powder, stirring and washing for 0.5-2 hours at the temperature of 40-50 ℃, and then performing suction filtration; and taking out the filter cake, and drying at 30-40 ℃ to remove residual ethanol to obtain the alcohol-washed walnut meal.

3. The method for preparing the dipeptidyl peptidase IV inhibiting active peptide by using the walnut meal according to claim 1, which is characterized in that: in the step (2), the alcohol-washed walnut pulp prepared in the step (1) is mixed with deionized water to prepare a suspension with the mass concentration of 10% -15%, the pH value is adjusted to be 4.5-6.5, the suspension is stirred for 1 hour at normal temperature, the suspension is centrifuged for 15-30 min at 3000-5000 r/min, and precipitates are collected; and repeating the steps for 2-4 times, and collecting the precipitate to obtain the walnut protein curd.

4. The method for preparing the dipeptidyl peptidase IV inhibiting active peptide by using the walnut meal according to claim 1, which is characterized in that: in the step (3), the walnut protein curd prepared in the step (2) is mixed with deionized water to prepare a suspension with the mass concentration of 8-12%, the pH is adjusted to 8.0-9.0, the temperature is 50-65 ℃, the shearing speed is 8000-12000 r/min, and the shearing time is 4-10 min.

5. The method for preparing the dipeptidyl peptidase IV inhibiting active peptide by using the walnut meal according to claim 1, which is characterized in that: in the step (4), alkaline protease is added into the walnut protein suspension obtained in the step (3) after high-speed shearing treatment, the enzyme addition amount E/S is 1.5% -3%, the temperature is kept at 50-65 ℃ and the pH is kept constant at 8.0-9.0 in the enzymolysis process, after enzymolysis is carried out for 4-6 hours, the pH is adjusted to be neutral, enzyme is inactivated in a boiling water bath for 10min, and centrifugation is carried out for 20min at 3000-5000 r/min, so that the obtained supernatant is crude enzymolysis liquid.

6. The method for preparing the dipeptidyl peptidase IV inhibiting active peptide by using the walnut meal according to claim 1, which is characterized in that: and (5) during desalting, circulating for 3-6 times, wherein the mass concentration of the material is 2-5%, the pH is 6.0-8.0, the cut-off Molecular Weight (MWCO) of the nanofiltration membrane is 150-300 Da, the operation temperature is 20-30 ℃, the pressure is 0.4-0.7 MPa, and the cut-off liquid is collected.

7. The method for preparing the dipeptidyl peptidase IV inhibiting active peptide by using the walnut meal according to claim 1, which is characterized in that: and (5) during drying, performing spray drying on the filtrate collected after nanofiltration to obtain powdery dipeptidyl peptidase IV inhibiting active peptide.

8. Use of a dipeptidyl peptidase iv inhibiting active peptide prepared according to the method of any one of claims 1 to 7, characterized in that: it can be used for preparing products for reducing blood sugar or resisting oxidation.

Technical Field

The invention relates to a method for preparing dipeptidyl peptidase IV inhibiting active peptide by using walnut meal and application thereof, belonging to the technical field of plant protein deep processing.

Background

Diabetes Mellitus (DM) is a metabolic disease associated with hyperglycemia, a common disease worldwide. There are two types of diabetes, insulin-dependent diabetes (type I diabetes) and non-insulin-dependent diabetes (type II diabetes). Type I diabetes (T1 DM) is caused by the inability of the pancreas to secrete insulin due to destruction of the islet beta cells by an autoimmune response. Type II diabetes (T2 DM) is a complex disease caused by insufficient production of insulin or the inability of the human body to utilize the produced insulin. The incidence rate of type II diabetes mellitus is gradually increased, and accounts for about 90-95% of diagnosis cases, so that a heavy burden is caused to the society, health and economy. No medicine capable of curing T2DM is found at present. The existing drugs mainly play a role by controlling the rise of blood sugar level, increasing the sensitivity of the body to insulin and reducing the damage of hyperglycemia to the body.

Dipeptidyl peptidase IV (DPP-IV) is a serine protease that is widely distributed in many tissues of the human body and is responsible for the degradation and inactivation of the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). DPP-IV inhibitors can prolong the action of GLP-1 and GIP, thereby stimulating insulin secretion to regulate blood glucose levels, and thus are the latest means for treating T2 DM. Some chemically synthesized DPP-IV inhibitors such as sitagliptin, vildagliptin and saxagliptin are effective drugs for clinically treating diabetes, but the drugs are expensive, can generate side effects when treating diabetes, and have no established long-term safety. Therefore, it is of great significance to extract active ingredients with DPP-IV inhibitory effect from natural food proteins. At present, food-derived vegetable protein peptides have shown inhibitory effects on DPP-IV.

The walnut has the beneficial effects that the cultivation area and the yield of the walnut are in the front of the world, and the walnut has the effects of strengthening the brain and improving the intelligence due to the rich physiological active components. The walnut protein contains a large amount of arginine, glutamic acid and aspartic acid, the arginine is used as a precursor for synthesizing Nitric Oxide (NO), plays an important role in regulating and controlling the physiological activities of a human body, and the generated NO is favorable for promoting vasodilatation and enhancing the immune activity of an organism; glutamic acid has the function of improving nerve function, and is involved in the synthesis of gamma-aminobutyric acid (GABA) in vivo, so that the energy metabolism of brain is promoted, and the oxygen supply amount is increased. The walnut protein has unique nutritive value, contains rich bioactive peptide sequences and is a good source for developing active polypeptides.

In recent years, the market demand of walnut oil is increasing, and the protein content of defatted walnut meal as a walnut oil processing byproduct is up to more than 50%, but due to the limitation of a protein deep processing technology, the walnut meal is generally used as feed or fertilizer, so that the waste of walnut resources is caused. Therefore, the walnut polypeptide with the antioxidant activity and the DPP-IV inhibitory activity is prepared by an enzymolysis technology, which is not only beneficial to widening the application range of the walnut protein, but also promoting the development of natural antioxidants and hypoglycemic foods.

Disclosure of Invention

The invention aims to overcome the defects and provide a method for preparing dipeptidyl peptidase IV inhibiting active peptide by using walnut meal and application thereof.

The technical scheme of the invention is a method for preparing dipeptidyl peptidase IV inhibiting active peptide by using walnut meal, which is characterized by comprising the following steps:

(1) alcohol washing: carrying out alcohol washing treatment on the degreased walnut dregs to obtain alcohol-washed walnut dregs;

(2) acid washing: carrying out acid washing treatment on the alcohol-washed walnut pulp to obtain walnut protein curd;

(3) shearing: mixing the walnut protein curd with deionized water, and carrying out high-speed shearing treatment;

(4) hydrolysis: adding alkaline protease to carry out enzymatic hydrolysis under certain conditions to obtain a crude enzymatic hydrolysate;

(5) and (3) post-treatment: desalting and drying the crude enzymolysis liquid to obtain powdery dipeptidyl peptidase IV inhibiting active peptide.

Further, in the step (1), adding 4-6 times volume of ethanol solution with volume concentration of 85%, stirring and washing for 0.5-2 h at 40-50 ℃, and then carrying out suction filtration; and taking out the filter cake, and drying at 30-40 ℃ to remove residual ethanol to obtain the alcohol-washed walnut meal.

Further, in the step (2), mixing the alcohol-washed walnut pulp prepared in the step (1) with deionized water to prepare a suspension with the mass concentration of 10% -15%, adjusting the pH value to be 4.5-6.5, stirring for 1h at normal temperature, centrifuging for 15-30 min at 3000-5000 r/min, and collecting precipitates; and repeating the steps for 2-4 times, and collecting the precipitate to obtain the walnut protein curd.

Further, in the step (3), the walnut protein curd prepared in the step (2) is mixed with deionized water to prepare a suspension with the mass concentration of 8-12%, the pH is adjusted to 8.0-9.0, the temperature is 50-65 ℃, the shearing time is 4-10 min, and the shearing speed is 8000-12000 r/min.

Further, in the step (4), adding alkaline protease into the walnut protein suspension obtained in the step (3) after high-speed shearing treatment, wherein the enzyme adding amount E/S is 1.5% -3%, the temperature is kept at 50-65 ℃ and the pH is kept constant at 8.0-9.0 in the enzymolysis process, after 4-6 hours of enzymolysis, adjusting the pH to be neutral, inactivating the enzyme in a boiling water bath for 10min, and centrifuging for 20min at 3000-5000 r/min to obtain a supernatant, namely a crude enzymolysis liquid.

Further, in the desalting in the step (5), the mass concentration of the material is 2-5%, the pH value is 6.0-8.0, the cut-off molecular weight MWCO of the nanofiltration membrane is 150-300 Da, the operation temperature is 20-30 ℃, the pressure is 0.4-0.7 MPa, the circulation is performed for 3-6 times, and the cut-off liquid is collected.

Further, in the step (5), during drying, the filtrate collected after nanofiltration is spray-dried to obtain powdery dipeptidyl peptidase IV inhibiting active peptide.

The application of the dipeptidyl peptidase IV inhibiting active peptide prepared by the method is applied to preparing a product for reducing blood sugar or resisting oxidation.

The walnut dipeptidyl peptidase IV inhibiting active peptide prepared by the method has good color and luster, the yield is more than 65%, the protein content is more than 80%, and the peptide segment with the molecular weight less than 2000Da accounts for more than 80%.

The walnut dipeptidyl peptidase IV inhibiting active peptide prepared by the method has good dipeptidyl peptidase IV inhibiting activity (IC)₅₀Not higher than 1.0 mg/mL) and antioxidant activity including: in vitro DPPH free radical scavenging Activity (IC)₅₀Not higher than 0.20 mg/mL), ABTS free radical scavenging activity (IC)₅₀Not higher than 50. mu.g/mL) and hydroxyl radical scavenging activity (IC)₅₀Not higher than 1.5 mg/mL).

The analysis method comprises the following steps:

1. determination of protein content: a semi-micro Kjeldahl method (GB 5009.5-2016);

2. determination of peptide yield: the total yield of the peptide powder (%) = the protein mass of the walnut peptide powder is multiplied by 100/the total protein mass in the walnut meal;

3. molecular weight determination of peptides: the prepared peptide product was subjected to molecular weight distribution measurement by gel chromatography. Preparing a sample to be detected into a concentration of 5-10 mg/mL, centrifuging for 10min at a rotating speed of 10000 r/min, filtering with a 0.22 mu m cellulose acetate microporous filter membrane, taking 20 mu L of the sample, and introducing the sample into a chromatographic column TSK-gel G2000 SW_XL(7.8 mm × 300 mm) the column was previously equilibrated with mobile phase (acetonitrile, water, TFA at 45:55:0.1 by volume), detection wavelength and column temperature were 214 nm, 30 ℃ respectively, mobile phase flow rate was 0.5 mL/min, time per sample was 45 min and molecular weight distribution standards were used cytochrome C (12500 Da), cecropin (6500 Da), bacitracin (1450 Da), GGAT (450 Da), GGG (189 Da).

4. Measurement of dipeptidyl peptidase iv inhibitory Activity: mu.L of the sample (dissolved in Tris-HCl buffer, pH 8.0, 100 mmol/L) was mixed with 25. mu.L of Gly-Pro-PNA (1.6 mmol/L) in a 96-well microplate, incubated at 37 ℃ for 10min, the reaction was started by adding 50. mu.L of DPP-IV (8U/L) solution, the reaction was started at 37 ℃ for 60 min, then the reaction was stopped by adding 100. mu.L of sodium acetate buffer (1 mol/L, pH 4.0), and the absorbance was measured at 405 nm. Tris-HCl buffer solution is used as a blank group instead of DPP-IV solution, and Tris-HCl buffer solution is used as a control group instead of sample. The DPP-IV inhibition rate calculation formula is as follows:

in the formula:

A_s: absorbance of the sample reaction system at 405 nm;

A_b: absorbance of blank at 405 nm;

A_c: absorbance at 405 nm for the control.

5. Determination of antioxidant Activity:

the DPPH free radical scavenging activity determination method comprises the following steps: in a 96-well microplate, 100. mu.L of walnut peptide solution was mixed with an equal volume of 0.2 mmol/L DPPH solution, reacted at room temperature in the dark for 30min, and the absorbance was read at 517 nm using a microplate reader (Bio-Rad). Taking polypeptide concentration and DPPH free radical scavenging activity as regression curve, establishing regression equation, and calculating IC₅₀The value, i.e. the peptide concentration corresponding to 50% scavenging of DPPH radicals.

The determination method of ABTS free radical scavenging activity comprises the following steps: mixing 7 mmol/L ABTS solution and 2.45 mmol/L potassium persulfate solution (final concentration), and then reacting for 12-16 h at room temperature in a dark place to generate ABTS free radical cation (ABTS)^•+). Before use, ABTS^•+The solution was diluted with 10 mmol/L phosphate buffer (pH 7.4) to an absorbance at 734 nm of 0.70. + -. 0.02. 50 μ L of the sample was mixed with 150 μ L of ABTS^•+The solutions were combined, reacted at 30 ℃ for 30min in the absence of light, and then the absorbance was measured at 734 nm. Establishing regression equation by using polypeptide concentration and ABTS free radical scavenging activity as regression curve, and calculating IC₅₀Value, i.e. the corresponding peptide concentration at which 50% of the ABTS radicals are scavenged.

The method for measuring the hydroxyl free radical scavenging activity comprises the following steps: 1.0 mL of sample was mixed with 1.0 mL of 3 mmol/L FeSO₄Solutions and1.0 mL of a 3 mmol/L salicylic acid-ethanol solution. Then, 1.0 mL of 6 mmol/L H was added₂O₂Reacting the solution at 37 deg.C in dark for 30min, measuring absorbance at 510 nm, making regression curve with polypeptide concentration and hydroxyl radical scavenging activity, establishing regression equation, and calculating IC₅₀The value, i.e. the corresponding peptide concentration at which 50% of the hydroxyl radicals are scavenged.

The invention has the beneficial effects that: the dipeptidyl peptidase IV inhibiting active peptide prepared by the invention has strong antioxidant and dipeptidyl peptidase IV inhibiting activities, and the product yield and the protein content are higher. The invention is suitable for industrial production, and the prepared active polypeptide has good functional characteristics and certain physiological function, and can be applied to various fields of food, feed, health products, medicine, daily chemical industry and the like.

Detailed Description

The invention is further illustrated below with reference to specific examples.