Bifunctional corn protein source active peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of active peptide preparation, and particularly relates to a bifunctional corn protein source active peptide as well as a preparation method and application thereof. The invention provides a bifunctional corn protein source active peptide, and the bifunctional corn protein source active peptide is PYAEY or a composition of the PYAEY and CQDVPLL. The difunctional corn protein source active peptide is separated from corn gluten meal, is a novel difunctional corn protein source active peptide, has dual activities of antagonizing adhesion of helicobacter pylori and resisting oxidation, can be used for preparing products for inhibiting adhesion of helicobacter pylori and / or resisting oxidation, and has a wide application prospect. And a technical support is provided for prevention and treatment of helicobacter pylori infection.

Description

[0001] The present application is a divisional application of the application with the application date of August 18, 2022, the application number of 202210989989.8, and the invention title of “Bifunctional corn protein source active peptide and preparation method and application thereof”. TECHNICAL FIELD

[0002] The present application belongs to the technical field of active peptide preparation, and specifically relates to a bifunctional corn protein source active peptide and a preparation method and application thereof. BACKGROUND

[0003] Helicobacter pylori (H. pylori) can cause gastritis, peptic ulcer, indigestion, and 1% of H. pylori infected persons can develop into gastric cancer, gastric mucosa-associated tissue lymphoma and other diseases. H. pylori is mainly transmitted through mouth-to-mouth and fecal-oral transmission. At present, the commonly used treatment methods for H. pylori include triple therapy and quadruple therapy. Quadruple therapy refers to a proton pump inhibitor or a bismuth agent, plus two antibiotics; and triple therapy refers to a proton pump inhibitor plus two antibiotics; the proton pump inhibitor includes omeprazole, rabeprazole, esomeprazole and other drugs. However, due to the increasing antibiotic resistance of H. pylori year by year, the eradication rate of triple or quadruple therapy for H. pylori is also decreasing year by year. Therefore, the development of alternative therapies to antibiotics is of great significance for the prevention and treatment of H. pylori infection and maintaining the health of human gastrointestinal flora.

[0004] In recent years, some natural sources of food components that can inhibit the adhesion of H. pylori have been gradually reported in the field, including flavonoids, polysaccharides, ovomucoid peptides and wheat germ protein peptides in cranberry, but the overall types are few. Therefore, it is of great significance to develop natural, safe, low-cost and more efficient food components with the function of inhibiting the adhesion of H. pylori.

[0005] Corn gluten meal (CGM) is the largest by-product (about 60%) with the highest protein content in the wet production of corn starch, but due to its poor solubility and strong hydrophobicity, it is not easy to be effectively utilized in the food industry, resulting in that corn gluten meal is mostly used as feed, causing waste of grain resources. Therefore, if the corn protein can be modified to develop a bifunctional food with the functions of inhibiting the adhesion of H. pylori and antioxidant, and improve its added value, it is of great significance for the in-depth processing of corn protein. SUMMARY

[0006] The present application aims to provide a bifunctional corn protein source active peptide, a preparation method and application thereof, and the bifunctional corn protein source active peptide is a new bifunctional active peptide with the activities of antagonizing Helicobacter pylori adhesion and antioxidation.

[0007] The present application provides a bifunctional corn protein source active peptide, which comprises the following amino acid sequence:

[0008] PYAEY and / or CQDVPLL.

[0009] Preferably, the amino acid sequence of the bifunctional corn protein source active peptide is PYAEY and / or CQDVPLL.

[0010] The present application further provides a method for preparing the bifunctional corn protein source active peptide, comprising the following preparation steps: neutral protease is used to enzymatically hydrolyze corn gluten meal to obtain an enzymatic hydrolysate; and the corn protein source active peptide is included in the enzymatic hydrolysate.

[0011] Preferably, the method further comprises, before the enzymatic hydrolysis, preparing the corn gluten meal into a suspension, and the mass concentration of the corn gluten meal in the suspension is 15% (w / v).

[0012] Preferably, the amount of the neutral protease is 400 U / g of protein; the temperature of the enzymatic hydrolysis is 45℃, the time is 150 min, and the pH is 7.0.

[0013] Preferably, the method further comprises separating and purifying the enzymatic hydrolysate, collecting components less than 1000 Da to obtain the bifunctional corn protein source active peptide.

[0014] Preferably, the chromatography used for the separation and purification comprises gel chromatography and ion exchange chromatography.

[0015] Preferably, the pre-packed column of the gel chromatography is Superdex Peptide 10 / 300 GL; and the ion exchange chromatography comprises Q-Sepharose High Performance ion exchange chromatography and Mono Q ion exchange chromatography in sequence.

[0016] The present application further provides the application of the bifunctional corn protein source active peptide prepared by the above technical solution or the preparation method in any one or more of the following I-IV:

[0017] I: a drug for antagonizing Helicobacter pylori adhesion;

[0018] II: a food for antagonizing Helicobacter pylori adhesion;

[0019] III: medicine with antioxidant function;

[0020] IV: food with antioxidant function.

[0021] The application further provides a product for antagonizing Helicobacter pylori adhesion and / or oxidation, and active ingredients of the product include the bifunctional corn protein source active peptide or the bifunctional corn protein source active peptide prepared by the preparation method.

[0022] Beneficial effects:

[0023] The application provides a bifunctional corn protein source active peptide, which includes PYAEY and / or CQDVPLL. The bifunctional corn protein source active peptide provided by the application is isolated from corn gluten meal and is a new bifunctional corn protein source active peptide, which has the dual activities of antagonizing Helicobacter pylori adhesion and oxidation and can be used for preparing a product for inhibiting Helicobacter pylori adhesion and / or oxidation, thereby providing technical support for the prevention and treatment of Helicobacter pylori infection. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0025] Figure 1 A technical route diagram for preparing the bifunctional corn protein source active peptide in Embodiment 1;

[0026] Figure 2 A mass spectrum detection diagram of the bifunctional corn protein source active peptide (PYAEY);

[0027] Figure 3 A mass spectrum detection diagram of the bifunctional corn protein source active peptide (CQDVPLL);

[0028] Figure 4 A standard curve of colony concentration and OD 600 ; and

[0029] Figure 5 A standard curve of FITC fluorescence intensity value and OD 600 . DETAILED DESCRIPTION

[0030] The application provides a bifunctional corn protein source active peptide, which includes the following amino acid sequences: PYAEY and / or CQDVPLL.

[0031] The amino acid of the bifunctional corn protein source active peptide is preferably PYAEY (SEQ ID NO. 1) and / or CQDVPLL (SEQ ID NO. 2). The bifunctional corn protein source active peptide is separated from corn protein peptide, has good antagonistic effect on the adhesion of Helicobacter pylori, and has antioxidant activity.

[0032] The application further provides a method for preparing the bifunctional corn protein source active peptide.

[0033] Before the enzyme hydrolysis, the application preferably further comprises mixing the corn gluten meal with water to obtain a suspension. The mass concentration of the corn gluten meal in the suspension is preferably 15% (w / v). In the application, the corn gluten meal is preferably corn gluten meal after extrusion and starch removal. The application does not have special limitations on the source of the corn gluten meal, and any purchased source meeting the requirements of corn gluten meal after extrusion and starch removal can be used. The corn gluten meal after extrusion and starch removal in the application can remove starch substances closely combined with proteins, which is beneficial to the enzyme hydrolysis of proteins.

[0034] After obtaining the suspension, the application preferably uses neutral protease to hydrolyze the suspension to obtain an enzyme hydrolysate. The amount of the neutral protease is preferably 400 U / g of protein, and the protein is preferably calculated based on the protein content in the corn gluten meal. The temperature of the enzyme hydrolysis is preferably 45°C, the time is preferably 150 min, and the pH value is preferably 7.0. The application preferably further comprises, after completing the enzyme hydrolysis, performing enzyme inactivation treatment on the enzyme hydrolysate, and the temperature of the enzyme inactivation treatment is preferably 100°C, and the time of the enzyme inactivation treatment is preferably 10 min. The application preferably centrifuges the mixture obtained after the enzyme inactivation treatment to obtain supernatant, i.e., an enzyme hydrolysate. The rotation speed of the centrifugation is preferably 4000 r / min, and the time is preferably 10 min. The enzyme hydrolysate comprises the bifunctional corn protein source active peptide.

[0035] After obtaining the enzyme hydrolysate, the application preferably separates and purifies the enzyme hydrolysate, collects components with a molecular weight less than 1000 Da, and obtains a mixture containing the bifunctional corn protein source active peptide.

[0036] The present application preferably performs gel chromatography separation on the enzymatic hydrolysate, and collects components with relatively stronger antagonistic H. pylori adhesion activity and antioxidant activity and molecular weight less than 1000 Da. The pre-packed column used in the gel chromatography separation of the present application is preferably Superdex Peptide 10 / 300 GL. The conditions for performing the gel chromatography separation of the present application preferably include: sample concentration of 50 mg / L, sample volume of 1 mL; eluent is 20 mM PBS buffer containing 0.15 mol / L NaCl at pH 7.0; flow rate of the eluent is 0.25 mL / min; detection wavelength is 214 nm. The present application preferably further includes determining the antagonistic H. pylori adhesion activity and antioxidant activity of each component collected, to obtain components with relatively stronger antagonistic H. pylori adhesion activity and antioxidant activity and molecular weight less than 1000 Da. The present application preferably uses the method described in Example 1 to determine the inhibitory H. pylori adhesion activity and antioxidant activity, and the same applies here, which will not be repeated.

[0037] After obtaining the components with relatively stronger antagonistic H. pylori adhesion activity and antioxidant activity and molecular weight less than 1000 Da, the present application preferably performs ion exchange chromatography separation on the components with relatively stronger antagonistic H. pylori adhesion activity and antioxidant activity and molecular weight less than 1000 Da. The chromatography used in the ion exchange chromatography separation of the present application preferably includes Q-Sepharose High Performance ion exchange chromatography and Mono Q ion exchange chromatography. The conditions for performing separation using the Q-Sepharose High Performance ion exchange chromatography of the present application preferably include: sample volume of 50 mL; eluent A is preferably Tris-HCl buffer, the concentration of the Tris-HCl buffer is preferably 20 mM, and the pH value is preferably 7.5; eluent B is 20 mM Tris-HCl buffer containing 1 mol / L NaCl at pH 7.5; flow rate of the eluent is 2 mL / min, detection wavelength is 214 nm, gradient elution volume is 60 mL, and peak component collection volume is 6 mL / tube. The present application preferably further includes determining the antagonistic H. pylori adhesion activity and antioxidant activity of each component collected, to obtain components with relatively stronger antagonistic H. pylori adhesion activity and antioxidant activity, i.e., component A. The protein concentration in the component A of the present application is preferably 2 mg / mL.

[0038] After obtaining the component A, the present application preferably separates the component A by using Mono Q ion exchange chromatography. The conditions for separating by using the Mono Q ion exchange chromatography in the present application preferably include a sample amount of 10 mL; eluent A is preferably Tris-HCl buffer, the concentration of the Tris-HCl buffer is preferably 20 mM, and the pH value is preferably 7.0; eluent B is 20 mM Tris-HCl buffer containing 1 mol / L NaCl at pH 7.0; the flow rate of the eluent is 1 mL / min, the detection wavelength is 214 nm, the gradient elution volume is 20 mL, and the peak component collection volume is 1 mL / tube. The present application preferably further includes determining the anti-H. pylori adhesion activity and the antioxidant activity of each component collected to obtain a component with relatively stronger anti-H. pylori adhesion activity and antioxidant activity, i.e., component B.

[0039] After obtaining the component B, the present application preferably performs mass spectrometry sequencing on the component B to obtain the bifunctional corn protein source active peptide. The amino acid sequence of the bifunctional corn protein source active peptide in the present application is PYAEY and CQDVPLL. The present application preferably performs the mass spectrometry sequencing by using LC-MS / MS. The process and steps of the mass spectrometry sequencing in the present application are not particularly limited, and the conventional mass spectrometry sequencing steps in the art can be used. Before performing the mass spectrometry sequencing, the present application further includes desalting and freeze-drying the component B. The process, steps of the desalting and freeze-drying, and the state after desalting are not particularly limited in the present application, and the conventional desalting and freeze-drying process and steps in the art can be used.

[0040] The present application also provides the application of the bifunctional corn protein source active peptide as described in the above technical solution or the bifunctional corn protein source active peptide prepared by the preparation method in any one or more of the following I-IV: I: a drug for antagonizing H. pylori adhesion; II: a food for antagonizing H. pylori adhesion; III: a drug with antioxidant function; and IV: a food with antioxidant function.

[0041] The present application also provides a product for antagonizing H. pylori adhesion and / or antioxidant function, and the active ingredient of the product includes the bifunctional corn protein source active peptide as described in the above technical solution or the bifunctional corn protein source active peptide prepared by the preparation method. The product in the present application preferably includes a drug and / or a food, and the food preferably includes a functional food. The product in the present application preferably further includes an excipient and / or other active ingredients. When the product further includes other active ingredients, the type, efficacy and amount of the other active ingredients are not particularly limited in the present application, and the other active ingredients can be reasonably added according to the prepared product. The amount of the bifunctional corn protein source active peptide in the product is not particularly limited in the present application, and the bifunctional corn protein source active peptide can be added according to the conventional addition of the prepared product.

[0042] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0043] In the following examples, the methods used are all conventional experimental methods in the art unless otherwise specified; the biological materials and experimental materials used are all available through conventional channels in the art unless otherwise specified.

[0044] Example 1

[0045] The bifunctional corn protein-derived active peptide consists of the following steps (see the technical roadmap in Figure 1 ) :

[0046] 1. Preparation of corn protein enzymatic hydrolysate

[0047] A certain amount of extruded and starch-removed corn meal (purchased from Qiqihar Longjiang Fufeng Biological Technology Co., Ltd.) was taken and water was added to prepare a suspension with a substrate concentration of 15% (w / v), and then neutral protease was used for enzymolysis. The enzymolysis conditions were as follows: enzyme addition amount 400 U / g protein, enzymolysis temperature 45°C, enzymolysis time 150 min, and enzymolysis pH 7.0. After the enzymolysis was completed, the enzyme was inactivated by heating at 100°C for 10 min. The obtained supernatant was the corn protein enzymatic hydrolysate after centrifugation at 4000 r / min for 10 min and discarding the precipitate. The corn protein enzymatic hydrolysate was confirmed to be a mixture of polypeptides with high antagonistic activity against Helicobacter pylori adhesion and antioxidant activity by determining the antagonistic activity against Helicobacter pylori adhesion and the antioxidant activity. The methods for determining the antagonistic activity against Helicobacter pylori adhesion and the antioxidant activity were the same as those in Step 5, and the same applied below.

[0048] 2. Gel chromatography separation of corn protein enzymatic hydrolysate

[0049] The gel chromatography column used was a Superdex Peptide 10 / 300 GL pre-packed column. The mixture of polypeptides with high antagonistic activity against Helicobacter pylori adhesion and antioxidant activity in Step 1 was separated by gel chromatography to obtain a mixture of polypeptides with different molecular weights. The loading concentration was 50 mg / mL, the loading amount was 1 mL, the eluent was 20 mM PBS buffer solution containing 0.15 mol / L NaCl at pH 7.0, the flow rate was 0.25 mL / min, and the detection wavelength was 214 nm. The antagonistic activity against Helicobacter pylori adhesion and the antioxidant activity of each molecular weight component were determined, and the component with a molecular weight less than 1000 Da and relatively high antagonistic activity against Helicobacter pylori adhesion and antioxidant activity was collected for ion exchange chromatography separation.

[0050] 3. Ion exchange chromatography separation

[0051] 3.1 Q-Sepharose High Performance strong anion exchange chromatography separation

[0052] The component with molecular weight less than 1000 Da and relatively high anti-H. pylori adhesion activity and antioxidant activity obtained from gel chromatography was filtered through a 0.22 μm microporous filter, and the protein concentration of the obtained sample was 4 mg / mL. The sample was separated using a strong anion exchanger, Q-Sepharose High Performance. The sample loading amount was 50 mL, the eluent A of the strong anion exchange chromatography was 20 mM Tris-HCl buffer at pH 7.5, the eluent B was 20 mM Tris-HCl buffer at pH 7.5 containing 1 mol / L NaCl, the flow rate was 2 mL / min, the detection wavelength was 214 nm, the gradient elution volume was 60 mL, and the peak component was collected in 6 mL per tube. The anti-H. pylori adhesion activity and antioxidant activity of each tube were determined, and the component with relatively high anti-H. pylori adhesion activity and antioxidant activity (tube 7) was collected for Mono Q ion exchange chromatography separation.

[0053] 3.2 Mono Q ion exchange chromatography

[0054] The high activity component obtained from step 3.1 was further separated using Mono Q ion exchange chromatography. The high activity component obtained from the previous ion exchange chromatography was filtered through a 0.22 μm microporous filter, and the protein concentration of the obtained sample was 2 mg / mL. The sample loading amount was 10 mL, the eluent A of the Mono Q ion exchange chromatography was 20 mM Tris-HCl buffer at pH 7.0, the eluent B was 20 mM Tris-HCl buffer at pH 7.0 containing 1 mol / L NaCl, the flow rate was 1 mL / min, the detection wavelength was 214 nm, the gradient elution volume was 20 mL, and the peak component was collected in 1 mL per tube. The anti-H. pylori adhesion activity and antioxidant activity of each tube were determined, and the component with high anti-H. pylori adhesion activity and antioxidant activity (tube 14) was collected for standby use.

[0055] 4. LC-MS / MS mass spectrometry sequencing

[0056] The component obtained from step 3.2 was desalted and freeze-dried, and then subjected to mass spectrometry sequencing to obtain the amino acid sequences of the bifunctional active peptides derived from corn protein, PYAEY (hereinafter referred to as polypeptide I) and CQDVPLL (hereinafter referred to as polypeptide II), as shown in Figure 2 and 3 .

[0057] 5. Determination of anti-H. pylori adhesion activity and antioxidant activity of the bifunctional active peptides derived from corn protein

[0058] The bifunctional corn protein-derived active peptide obtained in step 4 was chemically synthesized (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) and its antagonistic H. pylori adhesion activity and DPPH and ABTS free radical scavenging capacity were determined.

[0059] 5.1 Determination of the antagonistic H. pylori adhesion activity of the bifunctional corn protein-derived active peptide:

[0060] 1) The H. pylori ATCC43504 strain was used as the adhesion inhibition activity test strain. The H. pylori ATCC43504 strain was thawed at 37°C, mixed with a liquid culture medium (3 g soybean peptone, 2.5 g K2HPO4, 17 g tryptone and 5 g NaCl, 1 L of deionized water was added, mixed and dissolved, and the pH value was adjusted to 7.2, sterilized at 121°C and 0.1 MPa for 1 h), then inoculated on a slant culture medium (15 g tryptone, 5 g soybean peptone, 15 g agar, 5 g NaCl and 950 mL deionized water, stirred and dissolved, the pH value was adjusted to 7.2, sterilized at 121°C and 0.1 MPa for 1 h, when the medium cooled to about 45°C, 50 mL of sterile defibrillated sheep blood was added, mixed and then poured into test tubes to prepare the slant culture medium), and cultured at 37°C under microaerobic conditions (5% O2, 85% N2 and 10% CO2) for 48-72 h. The obtained bacterial solution was used for strain passage and adhesion inhibition activity detection.

[0061] The passed H. pylori ATCC43504 bacterial solution was diluted by 10 times in gradient for four times to obtain five bacterial solutions with different concentrations. The OD value of the H. pylori bacterial solution was determined at 600 nm, and the colony concentration was calculated by the plate coating method to establish the standard curve of the colony concentration and OD value, as shown in 600 . Figure 4

[0062] 2) The frozen human gastric mucosa epithelial cells (GES-1) were thawed and transferred into a cell culture bottle. The cell culture medium was composed of 1% penicillin-streptomycin mixture, 10% fetal bovine serum and 89% DMEM medium. The cells were incubated at 37°C and 5% CO2 until a single layer of cells was formed, then passaged by trypsin-EDTA digestion, centrifuged, resuspended with the cell culture medium without antibiotics, and the cell concentration was adjusted to 3×10 5 cells / mL. The cell suspension was inoculated into a 96-well plate at 100 μL per well, and incubated in a 37°C, 5% CO2 incubator for 24 h for the determination of the antagonistic H. pylori adhesion activity.

[0063] 3) Fluorescein isothiocyanate (FITC) labeled H. pylori

[0064] ​FITC concentration of 2 mg / mL in DMSO solution, sterile filter membrane treatment. According to the volume ratio of 1:1 prepared in step 1) with the passage of H. pylori ATCC43504 bacteria liquid and its mixing, in the absence of light conditions, biochemical swing table mixing 30 min, centrifugal treatment at 4500 r / min for 3 min, remove the supernatant, washed 3 times with 1 x PBS buffer, remove the excess FITC, finally the bacteria in the liquid medium (3 g soybean peptone, 2.5 g K2HPO4, 17 g tryptone and 5 g NaCl, add 1 L deionized water, mixed and dissolved, adjust the pH value to 7.2, 121 ℃, 0.1 MPa sterilization 1 h) diluted to OD 600 value of about 0.1 (10 8 cfu / mL), ready for use.

[0065] 4) FITC fluorescence intensity value and OD 600 standard curve

[0066] The H. pylori bacteria liquid treated with FITC in the above step, according to the four times 10 times gradient dilution, under the conditions of excitation wavelength 485 nm and emission wavelength 530 nm, the fluorescence intensity value was measured, at the same time, the OD value was measured under the condition of 600 nm, the standard curve of FITC fluorescence intensity value and OD 600 was established, as shown in Figure 5 .

[0067] 5) bifunctional corn protein source active peptide antagonistic H. pylori adhesion activity test

[0068] The polypeptide I prepared in example 1 was prepared using 100% DMEM medium to prepare a polypeptide I solution with a certain protein concentration, and mixed with the bacteria liquid labeled with FITC in step 3) under the conditions of room temperature and light avoidance for 30 min, so that the final concentration of polypeptide I obtained in example 1 was 4 mg / mL, and the mixed bacteria liquid was obtained.

[0069] 100 μL of mixed bacteria liquid was added to the 96 well plate with gastric mucosal epithelial cells (GES-1), and 100 μL of 100% DMEM medium was added as a negative control group, and placed in the incubator for 90 min. Then remove the solution, wash with PBS buffer for 3 times, then add PBS buffer to each well at the amount of 100 μL, and measure the fluorescence intensity value under the conditions of emission wavelength 530 nm and excitation wavelength 485 nm. According to the steps 3) and 4) of example 1 5.1, the colony concentration of the negative control group and the test group was calculated, and the adhesion inhibition rate was calculated by the following formula:

[0070]

[0071] The result is: when the concentration of polypeptide I (PYAEY) is 4 mg / mL, its antagonistic Helicobacter pylori adhesion activity is 40.75%.

[0072] The anti-Helicobacter pylori adhesion activity of polypeptide II (CQDVPLL) is determined by the same method, and when the concentration of polypeptide II (CQDVPLL) is 4 mg / mL, its antagonistic Helicobacter pylori adhesion activity is 47.64%.

[0073] 5.2 Determination of antioxidant activity of bifunctional corn protein-derived active peptide:

[0074] (1) Determination of DPPH free radical scavenging ability of bifunctional corn protein-derived active peptide: 2 mL of polypeptide I solution of different concentrations (polypeptide I solution is prepared by dissolving polypeptide I in water) is taken, 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution is added, mixed uniformly, and reacted in the dark for 30 min, and the absorbance value (A i ) at 517 nm is measured; 2 mL of polypeptide I solution is taken in a test tube, 2 mL of anhydrous ethanol is added, and the absorbance value (A j ) at 517 nm is measured; 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution (0.1 mmol / L) and 2 mL of anhydrous ethanol are reacted as a reference, and the absorbance value (A0) at 517 nm is measured. The DPPH free radical scavenging rate K of the sample is calculated according to formula (1):

[0075]

[0076] In the formula:

[0077] K - DPPH free radical scavenging rate, %;

[0078] A0 - absorbance value at 517 nm of 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution and 2 mL of anhydrous ethanol;

[0079] A i - absorbance value at 517 nm of 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution reacted with 2 mL of sample solution;

[0080] A j - absorbance value at 517 nm of 2 mL of anhydrous ethanol and 2 mL of sample solution.

[0081] From the above DPPH free radical scavenging activity determination results, it can be concluded that the IC50 value of polypeptide I (PYAEY) for DPPH free radical scavenging ability is 1.658 mg / mL.

[0082] The DPPH free radical scavenging capacity of polypeptide II (CQDVPLL) is determined by the same method, and the IC50 value of the DPPH free radical scavenging capacity of polypeptide II (CQDVPLL) is 0.096 mg / mL.

[0083] (2) Determination of ABTS free radical scavenging capacity of bifunctional corn protein source active peptide: accurately prepare 7.0 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate aqueous solution, mix well, and stand at room temperature for 12-16 h under light-proof condition to obtain ABTS+ mother liquor. Dilute the ABTS+ mother liquor with deionized water to make the absorbance value at 734 nm be 0.70±0.023, and equilibrate at 30℃ for 30 min to obtain ABTS+ working solution. Take 2.0 mL of polypeptide I solution with different concentrations and 2.0 mL of ABTS+ working solution into test tubes, mix well, and place at room temperature for 20 min under light-proof condition, and then determine the absorbance value at 734 nm. Take deionized water instead of polypeptide I solution to prepare reaction mixture with the same volume as the control, and set three replicates to obtain the average value. The ABTS free radical scavenging rate of polypeptide I solution is calculated according to the following formula.

[0084] The scavenging rate (%) = (1-A sample / A blank) x 100, wherein A sample is the absorbance value of the solution after adding the sample; and A blank is the absorbance value of the solution without adding the sample.

[0085] From the above ABTS determination results, it can be concluded that the IC50 value of the ABTS free radical scavenging capacity of polypeptide I (PYAEY) is 0.007 mg / mL.

[0086] The ABTS free radical scavenging capacity of polypeptide II (CQDVPLL) is determined by the same method, and the IC50 value of the ABTS free radical scavenging capacity of polypeptide II (CQDVPLL) is 0.016 mg / mL.

[0087] From the above examples, it can be concluded that the bifunctional corn protein source active peptide provided by the present application has the dual activity of simultaneously antagonizing Helicobacter pylori adhesion and antioxidant.

[0088] Although the above examples have made a detailed description of the present application, it is only a part of the examples of the present application, but not all the examples, and other examples can be obtained according to the present examples without creativity, and these examples all belong to the protection scope of the present application.

Claims

1. A bifunctional zein-derived active peptide, characterized in that, The bifunctional corn protein source active peptide is PYAEY, or a combination of PYAEY and CQDVPLL.

2. A method of preparing the bifunctional zein-derived active peptide of claim 1, characterized by, The method comprises the following preparation steps: corn gluten meal is enzymatically hydrolyzed by neutral protease to obtain an enzymatic hydrolysate; and the bifunctional corn protein source active peptide is included in the enzymatic hydrolysate.

3. The method of claim 2, wherein, The method further comprises, before the enzymatic hydrolysis, preparing the corn gluten meal into a suspension with a mass concentration of 15% w / v.

4. The method of claim 2 or 3, wherein, The neutral protease is used in an amount of 400 U / g protein; the enzymatic hydrolysis is performed at a temperature of 45°C for 150 min and at a pH of 7.

0.

5. The method of claim 2, wherein, The method further comprises separating and purifying the enzymatic hydrolysate to collect components with a molecular weight of less than 1000 Da to obtain the bifunctional corn protein source active peptide.

6. The method of claim 5, wherein, The chromatography used for the separation and purification comprises gel chromatography and ion exchange chromatography.

7. The method of claim 6, wherein, The pre-packed column of the gel chromatography is Superdex Peptide 10 / 300 GL; and the ion exchange chromatography comprises Q-Sepharose High Performance ion exchange chromatography and Mono Q ion exchange chromatography in sequence.

8. The bifunctional corn protein source active peptide of claim 1 or prepared by the method of any one of claims 2-7 is used in the preparation of any one or more of the following I-IV: I: a drug for antagonizing Helicobacter pylori adhesion; II: a food for antagonizing Helicobacter pylori adhesion; III: a drug with antioxidant function; IV: a food with antioxidant function.

9. A medicament for antagonizing the adhesion of Helicobacter pylori and / or for antioxidation, characterized in that, The active ingredient of the drug comprises the bifunctional corn protein source active peptide of claim 1 or prepared by the method of any one of claims 2-7.

10. An antioxidant functional food, characterized by comprising the antioxidant composition according to claim 1. The active ingredient of the functional food comprises the bifunctional corn protein source active peptide of claim 1 or prepared by the method of any one of claims 2-7.

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