Bifunctional corn protein-derived active peptide and its preparation method and application

Abstract

The present invention belongs to the technical field of active peptide preparation, and specifically relates to bifunctional corn protein-derived active peptides, preparation methods, and applications thereof. The present invention provides bifunctional corn protein-derived active peptides, which include PYAEY and / or CQDVPLL. The bifunctional corn protein-derived active peptides of the present invention are isolated from corn yellow powder and are novel bifunctional corn protein-derived active peptides having dual activities of antagonizing Helicobacter pylori adhesion and anti-oxidation. They can be used to prepare products that inhibit Helicobacter pylori adhesion and / or anti-oxidation, providing technical support for the prevention and treatment of Helicobacter pylori infection.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptide preparation, and in particular relates to a bifunctional corn protein-derived active peptide and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori ( Helicobacter pylori , H. pylori ) can cause gastritis, peptic ulcer, indigestion, 1% of which H. pylori Infected individuals can develop diseases such as gastric cancer and gastric mucosa-associated tissue lymphoma. Helicobacter pylori is primarily transmitted through oral-oral and fecal-oral routes. Currently, commonly used treatments for Helicobacter pylori include triple therapy and quadruple therapy. Quadruple therapy involves a proton pump inhibitor or a bismuth agent plus two antibiotics, while triple therapy involves a proton pump inhibitor plus two antibiotics. Proton pump inhibitors include omeprazole, rabeprazole, and esomeprazole. However, due to the increasing resistance of Helicobacter pylori to antibiotics, the eradication rate of Helicobacter pylori with triple or quadruple therapy has been declining. Therefore, the development of alternative therapies to antibiotics is crucial for preventing and treating Helicobacter pylori infection and maintaining a healthy gastrointestinal flora.

[0003] In recent years, a number of natural food-derived ingredients, including cranberry flavonoids, polysaccharides, ovomucin peptides, and wheat germ peptides, have been reported to inhibit H. pylori adhesion. However, the overall number of such ingredients is relatively small. Therefore, the development of natural, safe, affordable, and more effective food-derived ingredients that inhibit H. pylori adhesion is of great significance.

[0004] Corn gluten meal (CGM) is the largest byproduct of corn starch production, accounting for approximately 60% of its protein content. However, due to its poor solubility and strong hydrophobicity, it is difficult to effectively utilize in the food industry, resulting in its widespread use as feed, a waste of food resources. Therefore, if corn gluten can be modified to develop dual-function foods with Helicobacter pylori adhesion inhibition and antioxidant properties, thereby increasing its added value, it would be of great significance for the deep processing of corn gluten. Summary of the Invention

[0005] The purpose of the present invention is to provide a bifunctional zein-derived active peptide and a preparation method and application thereof. The bifunctional zein-derived active peptide is a new bifunctional active peptide with antagonistic Helicobacter pylori adhesion activity and antioxidant activity.

[0006] The present invention provides a bifunctional zein-derived active peptide, which comprises the following amino acid sequence:

[0007] PYAEY and / or CQDVPLL.

[0008] Preferably, the amino acid sequence of the bifunctional zein-derived active peptide is PYAEY and / or CQDVPLL.

[0009] The present invention also provides a method for preparing the bifunctional corn protein-derived active peptide described in the above technical solution, comprising the following preparation steps: enzymatically hydrolyzing corn yellow powder with a neutral protease to obtain an enzymatic hydrolysate; the enzymatic hydrolysate includes the corn protein-derived active peptide.

[0010] Preferably, the method further comprises preparing the corn yellow powder into a suspension before performing the enzymatic hydrolysis, wherein the mass concentration of the corn yellow powder in the suspension is 15% (w / v).

[0011] Preferably, the dosage of the neutral protease is 400 U / g protein; the enzymatic hydrolysis temperature is 45° C., the time is 150 min, and the pH is 7.0.

[0012] Preferably, the method further comprises separating and purifying the enzymatic hydrolysate, collecting components smaller than 1000 Da, and obtaining the bifunctional corn protein-derived active peptide.

[0013] Preferably, the chromatography used for the separation and purification includes gel chromatography and ion exchange chromatography.

[0014] Preferably, the prepacked column of the gel chromatography is Superdex Peptide 10 / 300 GL; the ion exchange chromatography includes Q-Sepharose High Performance ion exchange chromatography and Mono Q ion exchange chromatography in sequence.

[0015] The present invention also provides the use of the bifunctional zein-derived active peptide according to the above technical solution or the bifunctional zein-derived active peptide prepared by the preparation method in the preparation of drugs that antagonize Helicobacter pylori adhesion and / or have antioxidant function.

[0016] The present invention also provides a drug for antagonizing Helicobacter pylori adhesion and / or anti-oxidation, wherein the active ingredient of the drug includes the bifunctional zein-derived active peptide as described in the above technical solution or the bifunctional zein-derived active peptide prepared by the preparation method. Beneficial effects

[0017] The present invention provides a bifunctional zein-derived active peptide comprising PYAEY and / or CQDVPLL. The bifunctional zein-derived active peptide is isolated from corn yellow powder and is a novel bifunctional zein-derived active peptide with dual activities of antagonizing Helicobacter pylori adhesion and providing antioxidant activity. The peptide can be used to prepare products that inhibit Helicobacter pylori adhesion and / or provide antioxidant activity, providing technical support for the prevention and treatment of Helicobacter pylori infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0019] Figure 1 This is the technical roadmap for the preparation of the bifunctional zein-derived active peptide in Example 1;

[0020] Figure 2 This is the mass spectrometry detection diagram of the bifunctional zein-derived active peptide (PYAEY);

[0021] Figure 3 This is the mass spectrometry detection diagram of the bifunctional corn protein-derived active peptide (CQDVPLL);

[0022] Figure 4 The colony concentration and OD 600 Standard curve of

[0023] Figure 5 FITC fluorescence intensity and OD 600 The standard curve of DETAILED DESCRIPTION

[0024] The present invention provides a bifunctional zein-derived active peptide, which comprises the following amino acid sequence: PYAEY and / or CQDVPLL.

[0025] The amino acids of the bifunctional zein-derived active peptide of the present invention are preferably PYAEY (SEQ ID NO. 1) and / or CQDVPLL (SEQ ID NO. 2). The bifunctional zein-derived active peptide of the present invention, isolated from zein peptides, has a strong antagonistic effect on the adhesion of Helicobacter pylori and also exhibits antioxidant activity.

[0026] The present invention also provides a method for preparing the bifunctional corn protein-derived active peptide described in the above technical solution, comprising the following preparation steps: enzymatically hydrolyzing corn yellow powder with a neutral protease to obtain an enzymatic hydrolysate; the enzymatic hydrolysate comprises the bifunctional corn protein-derived active peptide.

[0027] Before performing the enzymatic hydrolysis, the present invention preferably further comprises mixing corn yellow powder with water to obtain a suspension. The mass concentration of corn yellow powder in the suspension is preferably 15% (w / v). In the present invention, the corn yellow powder is preferably extruded and de-starched corn yellow powder. The source of the corn yellow powder is not particularly limited; any source that meets the requirements for de-starched corn yellow powder can be purchased. The de-starched corn yellow powder extruded and puffed can fully remove starchy substances tightly bound to proteins, facilitating enzymatic hydrolysis of proteins.

[0028] After obtaining the suspension, the present invention preferably uses a neutral protease to enzymatically hydrolyze the suspension to obtain an enzymatic hydrolysate. The amount of the neutral protease used in the present invention is preferably 400U / g protein; the protein is preferably calculated based on the protein content in the corn yellow powder. The temperature for the enzymatic hydrolysis in the present invention is preferably 45°C; the time is preferably 150 minutes, and the pH value is preferably 7.0. The present invention preferably further includes performing an enzyme inactivation treatment on the enzymatic hydrolysate after completing the enzymatic hydrolysis, and the temperature of the enzyme inactivation treatment is preferably 100°C; the time of the enzyme inactivation treatment is preferably 10 minutes. The present invention preferably centrifuges the mixture obtained by the enzyme inactivation treatment after completing the enzyme inactivation treatment to obtain a supernatant, which is an enzymatic hydrolysate. The rotation speed of the centrifugation in the present invention is preferably 4000r / min, and the time is preferably 10 minutes. The enzymatic hydrolysate of the present invention includes the bifunctional corn protein-derived active peptide.

[0029] After obtaining the enzymatic hydrolysate, the present invention preferably separates and purifies the enzymatic hydrolysate to collect components with a molecular weight less than 1000 Da to obtain a mixture containing the bifunctional zein-derived active peptides.

[0030] The present invention preferably subjects the enzymatic hydrolysate to gel chromatography separation to collect fractions exhibiting relatively stronger anti-H. pylori adhesion and antioxidant activities and a molecular weight less than 1000 Da. The prepacked column used for the gel chromatography separation is preferably Superdex Peptide 10 / 300 GL. The conditions for the gel chromatography separation preferably include: a sample concentration of 50 mg / L and a sample volume of 1 mL; an eluent of 20 mM PBS buffer containing 0.15 mol / L NaCl at pH 7.0; an eluent flow rate of 0.25 mL / min; and a detection wavelength of 214 nm. The present invention preferably further includes determining the anti-H. pylori adhesion and antioxidant activities of the collected fractions to obtain fractions exhibiting relatively stronger anti-H. pylori adhesion and antioxidant activities and a molecular weight less than 1000 Da. The present invention preferably employs the method described in Example 1 to determine the inhibitory H. pylori adhesion and antioxidant activities, which will be discussed hereinafter.

[0031] After obtaining the component with relatively stronger antagonistic Helicobacter pylori adhesion activity and antioxidant activity and a molecular weight of less than 1000Da, the present invention preferably performs ion exchange chromatography separation on the component with relatively stronger antagonistic Helicobacter pylori adhesion activity and antioxidant activity and a molecular weight of less than 1000Da. The chromatogram used for the ion exchange chromatography separation in the present invention preferably includes Q-Sepharose High Performance ion exchange chromatography and Mono Q ion exchange chromatography. The conditions for separation using the Q-Sepharose High Performance ion exchange chromatography in the present invention preferably include: a sample load of 50mL; eluent A is preferably Tris-HCl buffer, the concentration of the Tris-HCl buffer is preferably 20mM, and the pH value is preferably 7.5; eluent B is 20mM Tris-HCl buffer containing 1mol / L NaCl at pH 7.5; the flow rate of the eluent is 2mL / min, the detection wavelength is 214nm, the gradient volume is 60mL, and the volume of peak component collection is 6mL / tube. The present invention preferably further comprises measuring the anti-Helicobacter pylori adhesion activity and antioxidant activity of the collected components, and obtaining a component with relatively stronger anti-Helicobacter pylori adhesion activity and antioxidant activity, namely component A. The protein concentration in component A of the present invention is preferably 2 mg / mL.

[0032] After obtaining the component A, the present invention preferably uses Mono Q ion exchange chromatography to separate the component A. The conditions for separation using the Mono Q ion exchange chromatography in the present invention preferably include a sample load of 10 mL; eluent A is preferably a 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 a 20 mM Tris-HCl buffer at pH 7.0 containing 1 mol / L NaCl; the flow rate of the eluent is 1 mL / min, the detection wavelength is 214 nm, the gradient volume is 20 mL, and the volume of the peak fraction collected is 1 mL / tube. The present invention preferably also includes measuring the anti-Helicobacter pylori adhesion activity and antioxidant activity of each collected fraction, and obtaining a fraction with relatively stronger anti-Helicobacter pylori adhesion activity and antioxidant activity, namely, component B.

[0033] After obtaining the component B, the present invention preferably performs mass spectrometry sequencing on the component B to obtain the bifunctional corn protein-derived active peptide. The amino acid sequence of the bifunctional corn protein-derived active peptide of the present invention is PYAEY and CQDVPLL. The present invention preferably uses LC-MS / MS to perform the mass spectrometry sequencing. The present invention has no special restrictions on the process and steps of the mass spectrometry sequencing, and the conventional mass spectrometry sequencing steps in the art can be used. Before performing the mass spectrometry sequencing, the present invention also includes desalting and lyophilizing the component B. The present invention has no special restrictions on the process, steps and state after desalting of the desalting and lyophilizing, and the conventional desalting and lyophilizing process and steps in the art can be used.

[0034] The present invention also provides the use of the bifunctional zein-derived active peptide according to the above technical solution or the bifunctional zein-derived active peptide prepared by the preparation method in the preparation of drugs that antagonize Helicobacter pylori adhesion and / or have antioxidant function.

[0035] The present invention also provides a drug for antagonizing Helicobacter pylori adhesion and / or anti-oxidation, the active ingredient of the drug including the bifunctional corn protein-derived active peptide as described in the above technical solution or the bifunctional corn protein-derived active peptide prepared by the preparation method. The product of the present invention preferably also includes excipients and / or other active ingredients. When the product also includes other active ingredients, the present invention has no special restrictions on the type, efficacy and dosage of the other active ingredients, and they can be reasonably added according to the prepared product. The present invention has no special restrictions on the dosage of the bifunctional corn protein-derived active peptide in the product, and it can be routinely added according to the prepared product.

[0036] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Unless otherwise specified, the methods used in the following examples are all conventional experimental methods in the art; the biological materials and experimental materials used, unless otherwise specified, can be obtained through conventional purchasing channels in the art. Example

[0038] The bifunctional corn protein-derived active peptide is composed of the following steps (see the technical roadmap for details). Figure 1 ):

[0039] Preparation of corn protein hydrolysate

[0040] A certain amount of extruded and de-starched corn yellow powder (purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.) was prepared with water to a 15% (w / v) substrate concentration. The suspension was then enzymatically hydrolyzed with a neutral protease. The hydrolysis conditions were: 400 U / g protein, 45°C, 150 min, and a pH of 7.0. After completion, the enzyme was inactivated by heating at 100°C for 10 min. The hydrolyzate was centrifuged at 4000 rpm for 10 min, and the precipitate was discarded. The resulting supernatant, the corn protein hydrolyzate, was assayed for its anti-H. pylori adhesion and antioxidant activities, confirming that the peptide mixture possessed both high anti-H. pylori adhesion and antioxidant activities. The methods for assaying the anti-H. pylori adhesion and antioxidant activities were similar to those described in step 5 and are not further detailed here.

[0041] Gel Chromatographic Separation of Corn Protein Hydrolysate

[0042] The gel chromatography column used was a Superdex Peptide 10 / 300 GL prepacked column. The polypeptide mixture with high antagonistic Helicobacter pylori adhesion activity and antioxidant activity in step 1 was separated by gel chromatography to obtain a polypeptide mixture of different molecular weight components. The loading concentration was 50 mg / mL, the loading volume was 1 mL, the eluent was a 20 mM PBS buffer solution with pH 7.0 containing 0.15 mol / L NaCl, the flow rate was 0.25 mL / min, and the detection wavelength was 214 nm. The antagonistic Helicobacter pylori adhesion activity and antioxidant activity of each molecular weight component were determined, and the components with a molecular weight less than 1000 Da and relatively high antagonistic Helicobacter pylori adhesion activity and antioxidant activity were collected for ion exchange chromatography separation.

[0043] Ion exchange chromatography

[0044] 3.1 Q-Sepharose High Performance Strong Anion Exchange Chromatography Separation

[0045] Fractions with molecular weights less than 1000 Da and relatively high anti-H. pylori adhesion and antioxidant activities, obtained through gel chromatography, were filtered through a 0.22 μm microporous filter membrane. The resulting sample protein concentration was 4 mg / mL. Separation was performed using Q-Sepharose High Performance, a strong anion exchanger. A 50 mL sample load was used. Eluent A consisted of 20 mM Tris-HCl buffer, pH 7.5, and eluent B consisted of 20 mM Tris-HCl buffer, pH 7.5, containing 1 mol / L NaCl. The flow rate was 2 mL / min, the detection wavelength was 214 nm, the gradient volume was 60 mL, and 6 mL of peak fractions were collected per tube. The anti-H. pylori adhesion and antioxidant activities of each tube were determined. The fraction with relatively high H. pylori adhesion and antioxidant activities (tube 7) was collected for separation by Mono Q ion exchange chromatography.

[0046] 3.2 Mono Q ion exchange chromatography

[0047] The highly active fractions isolated in step 3.1 were further separated using Mono Q ion exchange chromatography. The highly active fractions from the previous ion exchange chromatography step were filtered through a 0.22 μm microporous filter membrane. The resulting sample protein concentration was 2 mg / mL, and the sample load was 10 mL. The eluent A for the Mono Q ion exchange chromatography was 20 mM Tris-HCl buffer, pH 7.0, and the eluent B was 20 mM Tris-HCl buffer, pH 7.0, containing 1 mol / L NaCl. The flow rate was 1 mL / min, the detection wavelength was 214 nm, the gradient volume was 20 mL, and 1 mL of peak fractions were collected in each tube. The anti-H. pylori adhesion and antioxidant activities of the collected fractions from each tube were assayed. The fraction with the highest H. pylori adhesion and antioxidant activities (tube 14) was collected for later use.

[0048] 4. LC-MS / MS mass spectrometry sequencing

[0049] The fraction obtained in step 3.2 was desalted and lyophilized, and then subjected to mass spectrometry sequencing to obtain a bifunctional corn protein-derived active peptide with the amino acid sequence of PYAEY (hereinafter referred to as polypeptide I) and CQDVPLL (hereinafter referred to as polypeptide II). Figure 2 and 3 shown.

[0050] 5. Determination of the Anti-Helicobacter Pylori Adhesion and Antioxidant Activities of Bifunctional Zein-Derived Active Peptides

[0051] The bifunctional corn protein-derived active peptide obtained in step 4 was chemically synthesized (commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis), and its anti-Helicobacter pylori adhesion activity and scavenging ability against DPPH and ABTS free radicals were measured.

[0052] 5.1 Determination of the Antagonistic Activity of Bifunctional Zein-Derived Active Peptides against Helicobacter Pylori Adhesion

[0053] 1) Utilize H. pylori ATCC43504 strain was used as the strain for anti-adhesion activity test. H. pylori Thaw ATCC43504 strain and mix with liquid culture medium (3 g soy peptone, 2.5 g K2HPO4, 17 g tryptone, and 5 g NaCl in 1 L of deionized water, shake well to dissolve, adjust the pH to 7.2, and sterilize at 121°C, 0.1 MPa for 1 h). Then, inoculate the mixture onto a slant culture medium (15 g tryptone, 5 g soy peptone, 15 g agar, 5 g NaCl, and 950 mL of deionized water, stir well to dissolve, adjust the pH to 7.2, and sterilize at 121°C, 0.1 MPa for 1 h. After the culture medium cools to approximately 45°C, add 50 mL of sterile defibrinated sheep blood, mix well, and pour into a test tube to prepare the slant culture medium). Culture the mixture at 37°C under microaerophilic conditions (5% O2, 85% N2, 10% CO2) for 48–72 h. The resulting bacterial suspension can be used for subculturing and anti-adhesion activity testing.

[0054] will be passed down H. pylori The bacterial solution of ATCC43504 was diluted four times by 10 times to obtain five different concentrations of bacterial solution. The OD value of the Helicobacter pylori bacterial solution was measured at 600 nm. The colony concentration was calculated by plate spreading method. The relationship between colony concentration and OD was established. 600 The standard curve of Figure 4 shown.

[0055] 2) Thaw frozen human gastric epithelial cells (GES-1) and transfer them to cell culture flasks in a culture medium composed of 1% penicillin-streptomycin, 10% fetal bovine serum, and 89% DMEM. Incubate at 37°C, 5% CO₂ until a monolayer of cells forms. Subculture cells using trypsin-EDTA digestion, centrifugation, and resuspend in antibiotic-free cell culture medium to a cell concentration of 3 × 10 cells. 5 The cell suspension was inoculated into 96-well plates, 100 μL per well, and incubated in a 37°C, 5% CO2 incubator for 24 h. H. pylori Adhesion activity assay.

[0056] 3) Fluorescein isothiocyanate (FITC) labeling of Helicobacter pylori

[0057] Prepare a 2 mg / mL DMSO solution of FITC and filter it with a sterile filter membrane. H. pylori The bacterial suspension of ATCC43504 was mixed with the suspension, mixed on a biochemical rocking table for 30 minutes under the condition of avoiding light, and then centrifuged at 4500r / min for 3 minutes. The supernatant was removed and the suspension was washed three times with 1×PBS buffer to remove excess FITC. Finally, the bacterial suspension was diluted to OD 0.05 in liquid culture medium (3g soy peptone, 2.5g K2HPO4, 17g tryptone and 5g NaCl, added to 1L deionized water, mixed and shaken to dissolve, and then the pH value was adjusted to 7.2, and sterilized at 121℃, 0.1Mpa for 1h). 600 The value is around 0.1 (10 8 cfu / mL), set aside.

[0058] 4) FITC fluorescence intensity value and OD 600 Construction of standard curve

[0059] The H. pylori solution labeled with FITC in the previous step was diluted four times in a 10-fold gradient. The fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. At the same time, the OD value was measured at 600 nm to establish the relationship between the FITC fluorescence intensity value and the OD value. 600 The standard curve of Figure 5 shown.

[0060] 5) Antagonistic activity test of Helicobacter pylori adhesion by bifunctional corn protein-derived active peptides

[0061] The polypeptide I prepared in Example 1 was used in 100% DMEM medium to prepare a polypeptide I solution having a certain protein concentration. The solution was then mixed with the FITC-labeled bacterial solution from step 3) at a ratio of 1:1 (v / v) at room temperature in the dark for 30 minutes to obtain a final concentration of 4 mg / mL of polypeptide I obtained in Example 1, thereby obtaining a mixed bacterial solution.

[0062] 100 μL of the mixed bacterial solution was added to each well of a 96-well plate containing gastric epithelial cells (GES-1). A negative control group was treated with 100 μL of 100% DMEM medium. The cells were incubated in an incubator for 90 minutes. The solution was then removed and the cells were washed three times with PBS buffer. Subsequently, 100 μL of PBS buffer was added to each well. Fluorescence intensity was measured at an emission wavelength of 530 nm and an excitation wavelength of 485 nm. The colony concentrations of the negative control and test groups were calculated according to steps 3) and 4) of 5.1 in Example 1. The adhesion inhibition rate was calculated using the following formula:

[0063]

[0064] The results showed that when the concentration of polypeptide I (PYAEY) was 4 mg / mL, its antagonistic activity against Helicobacter pylori adhesion was 40.75%.

[0065] The same method was used to determine the anti-Helicobacter pylori adhesion activity of polypeptide II (CQDVPLL). When the concentration of polypeptide II (CQDVPLL) was 4 mg / mL, its antagonistic Helicobacter pylori adhesion activity was 47.64%.

[0066] 5.2 Determination of Antioxidant Activity of Bifunctional Zein-Derived Active Peptides:

[0067] (1) Determination of the ability of bifunctional corn protein-derived active peptides to scavenge DPPH free radicals: Take 2 mL of polypeptide I solution of different concentrations (polypeptide I solution is prepared by dissolving polypeptide I in water), add 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution, mix well, react in the dark for 30 minutes, and measure its absorbance at 517 nm (A i ); Take 2mL of polypeptide I solution in a test tube, add 2mL of anhydrous ethanol, and measure its absorbance at 517nm (A j ); Take 2mL of 0.1mmol / L DPPH anhydrous ethanol solution (0.1 mmol / L) and 2mL of anhydrous ethanol to react as a reference, and measure its absorbance (A0) at 517nm. The scavenging rate K of the sample for DPPH free radicals is calculated according to formula (1):

[0068]

[0069] Where:

[0070] Scavenging rate of DPPH free radicals, %;

[0071] A0-2mL 0.1 mmol / L DPPH anhydrous ethanol solution and 2mL anhydrous ethanol absorbance at 517nm;

[0072] A i -The absorbance at 517 nm of the reaction between 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution and 2 mL of sample solution;

[0073] A j -The absorbance of 2 mL of anhydrous ethanol and 2 mL of sample solution at 517 nm.

[0074] From the above DPPH free radical scavenging activity test results, it can be concluded that the IC50 value of polypeptide I (PYAEY) for DPPH free radical scavenging ability is 1.658 mg / mL.

[0075] The same method was used to determine the DPPH free radical scavenging ability of peptide II (CQDVPLL), and the IC50 value of peptide II (CQDVPLL) on DPPH free radical scavenging ability was 0.096 mg / mL.

[0076] (2) Determination of the ability of bifunctional corn protein-derived active peptides to scavenge ABTS free radicals: Accurately prepare 7.0 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate aqueous solution, mix well, and let it stand at room temperature in the dark for 12-16 hours to obtain ABTS+ mother solution. Dilute the ABTS+ mother solution with deionized water so that its absorbance at 734 nm is 0.70±0.023, and equilibrate at 30°C for 30 minutes to obtain ABTS+ working solution. Take 2.0 mL of different concentrations of peptide I solution and 2.0 mL of ABTS+ working solution and add them to the test tube, mix well, let it stand at room temperature in the dark for 20 minutes, and measure the absorbance at 734 nm. Use deionized water instead of the same volume of peptide I solution as the control reaction mixture, set up 3 replicates, and calculate the average value. Calculate the scavenging rate of peptide I solution on ABTS free radicals according to the following formula.

[0077] Clearance rate (%) = (1-A sample / A blank) × 100, where A sample is the absorbance value of the solution after adding the sample; A blank is the absorbance value of the solution without adding the sample.

[0078] From the above ABTS determination results, it can be concluded that the IC50 value of polypeptide I (PYAEY) for ABTS free radical scavenging ability is 0.007 mg / mL.

[0079] The same method was used to determine the ABTS free radical scavenging ability of peptide II (CQDVPLL), and the IC50 value of peptide II (CQDVPLL) on ABTS free radical scavenging ability was 0.016 mg / mL.

[0080] It can be concluded from the above examples that the bifunctional zein-derived active peptide provided by the present invention has the dual activities of antagonizing Helicobacter pylori adhesion and anti-oxidation.

[0081] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A bifunctional corn protein-derived active peptide, characterized in that: The amino acid sequence of the bifunctional zein-derived active peptide is CQDVPLL.

2. A method for preparing the bifunctional zein-derived active peptide according to claim 1, characterized in that: The method comprises the following preparation steps: using neutral protease to enzymatically hydrolyze corn yellow powder to obtain enzymatic hydrolysate; the enzymatic hydrolysate comprises the bifunctional corn protein-derived active peptide.

3. The method according to claim 2, wherein The method further comprises preparing the corn yellow powder into a suspension before performing the enzymatic hydrolysis, wherein the mass concentration of the corn yellow powder in the suspension is 15% w / v.

4. The method according to claim 2 or 3, wherein: The dosage of the neutral protease is 400 U / g protein; the enzymatic hydrolysis temperature is 45° C., the time is 150 min, and the pH is 7.

0.

5. The method according to claim 2, wherein The method further comprises separating and purifying the enzymatic hydrolysate, collecting components smaller than 1000 Da, and obtaining the bifunctional corn protein-derived active peptide.

6. The method according to claim 5, wherein The chromatography used for the separation and purification includes gel chromatography and ion exchange chromatography.

7. The method according to claim 6, wherein The prepacked column of the gel chromatography is Superdex Peptide 10 / 300GL; the ion exchange chromatography includes Q-Sepharose High Performance ion exchange chromatography and MonoQ ion exchange chromatography in sequence.

8. Use of the bifunctional zein-derived active peptide according to claim 1 or the bifunctional zein-derived active peptide prepared by the preparation method according to any one of claims 2 to 7 in the preparation of a drug that antagonizes Helicobacter pylori adhesion and / or has antioxidant function.

9. A drug for antagonizing Helicobacter pylori adhesion and / or anti-oxidation, characterized in that: The active ingredient of the medicine includes the bifunctional zein-derived active peptide according to claim 1 or the bifunctional zein-derived active peptide prepared by the preparation method according to any one of claims 2 to 7.

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