Difunctional corn peptide with helicobacter pylori adhesion antagonism activity and antioxidant activity as well as preparation method and application of difunctional corn peptide

Abstract

The invention belongs to the technical field of protein peptide preparation, and particularly relates to a bifunctional corn peptide with helicobacter pylori adhesion antagonism activity and antioxidant activity and a preparation method and application thereof. The invention provides a bifunctional corn peptide with helicobacter pylori adhesion antagonism activity and antioxidant activity. The bifunctional corn peptide is polypeptide II or a composition of polypeptide I and polypeptide II. The amino acid sequence of the polypeptide I is as shown in SEQ ID NO. 1; the amino acid sequence of the polypeptide II is as shown in SEQ ID NO. 2. The bifunctional corn peptide is separated from corn protein powder, has dual activities of antagonizing adhesion of helicobacter pylori and resisting oxidation, particularly has a relatively strong inhibition effect on adhesion of helicobacter pylori, and can be used for preparing products with functions of antagonizing adhesion of helicobacter pylori and / or resisting oxidation.

Description

[0001] This application is a divisional application of the application with the application date of August 18, 2022, application number 202210989940.2, and invention title "Bifunctional Corn Peptide with Antagonistic Activity against Helicobacter pylori Adhesion and Antioxidant Activity, Its Preparation Method and Application". Technical Field

[0002] The present invention belongs to the technical field of protein peptide preparation, and specifically relates to a bifunctional corn peptide with antagonistic activity against Helicobacter pylori adhesion and antioxidant activity, its preparation method and application. Background Art

[0003] Helicobacter pylori is a Gram-negative bacterium, 2-4 μm in length and 0.5-1 μm in width, usually in a spiral shape. The bacterium has 2-6 flagella, which endow the bacterium with motility, enabling it to move rapidly in the mucus layer of gastric epithelial cells. The growth of Helicobacter pylori has the characteristic of microaerophilia, and the conditions of 5% O 2 , 10% CO 2 , 85% N 2 , 37 °C and high humidity are its optimal growth environment. Although Helicobacter pylori can colonize on the gastric mucosa, it can only grow within the range of pH 5.5-8.0.

[0004] Helicobacter pylori is a special pathogen. After the body is infected with Helicobacter pylori, it is generally difficult to clear itself. If eradication treatment is not taken, it will be carried for life. Helicobacter pylori can specifically adhere to gastric mucosal epithelial cells, triggering gastric oxidative stress and inflammatory reactions, and is one of the recognized important pathogenic factors for chronic gastritis and ulcerative diseases. It may also be closely related to the incidence of gastric cancer and gastric mucosa-associated lymphoma. At present, the World Health Organization has classified Helicobacter pylori as a class I carcinogen. At present, the most common method for eradicating Helicobacter pylori is antibiotic treatment, such as the triple therapy of commonly used proton pump inhibitors and two antibiotics, and the quadruple therapy of bismuth, proton pump inhibitors and two antibiotics combined. However, with the increasing antibiotic resistance of Helicobacter pylori year by year, its eradication rate is also decreasing, and taking antibiotics has many side effects, such as intestinal discomfort, allergies, etc. Therefore, developing alternative therapies to antibiotics is of great significance for preventing and treating Helicobacter pylori infection and maintaining the health of the human gastrointestinal flora.

[0005] In recent years, some food-derived components of natural origin that inhibit the adhesion activity of Helicobacter pylori have been reported, such as flavonoids, polysaccharides, ovomucin peptides and wheat germ peptides in cranberries, etc., but the overall types are few. Therefore, it is still very necessary to further develop natural, safe, low-cost and more efficient food-derived components with antagonistic activity against Helicobacter pylori adhesion.

[0006] Corn gluten meal (CGM) is the by-product with the largest output and the highest protein content (about 60%) in the wet production of corn starch. However, there are many unfavorable factors restricting its application in the food industry, such as poor solubility and strong hydrophobicity. Therefore, using corn gluten meal directly as feed causes a great waste of grain resources. Therefore, if corn protein can be modified to develop functional foods with the activity of antagonizing Helicobacter pylori adhesion and improve its added value, it is of great significance for the intensive processing of corn protein. Summary of the Invention

[0007] The object of the present invention is to provide a bifunctional corn peptide with the activities of antagonizing Helicobacter pylori adhesion and antioxidant activity, and its preparation method and application. The corn peptide is a newly discovered bifunctional peptide with the dual activities of antagonizing Helicobacter pylori adhesion and antioxidant activity.

[0008] The present invention provides a bifunctional corn peptide with the activities of antagonizing Helicobacter pylori adhesion and antioxidant activity, including polypeptide I and / or polypeptide II;

[0009] The polypeptide I includes the amino acid sequence shown in SEQ ID NO.1; the polypeptide II includes the amino acid sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a preparation method of the bifunctional corn peptide described in the above technical solution, including the following steps:

[0011] Using neutral protease to enzymatically hydrolyze corn gluten meal, and the enzymatic hydrolysate includes the bifunctional corn peptide.

[0012] Preferably, the corn gluten meal is the corn gluten meal after extrusion and expansion and starch removal; before enzymatic hydrolysis, it also includes preparing the corn gluten meal into a suspension, and the mass concentration of corn gluten meal in the suspension is 15% (w / v).

[0013] Preferably, the dosage of the neutral protease is 400 U / g protein; the temperature of enzymatic hydrolysis is 45 °C, the time is 150 min, and the pH is 7.0.

[0014] Preferably, after enzymatic hydrolysis, it also includes: separating and purifying the enzymatic hydrolysate, collecting the fraction with a molecular weight < 1000 Da to obtain the bifunctional corn peptide.

[0015] Preferably, the separation and purification include gel chromatography separation and ion exchange chromatography separation.

[0016] Preferably, the pre-packed column for gel chromatography separation is Superdex Peptide 10 / 300GL; the ion exchange chromatography separation includes two-step ion exchange chromatography separation. The ion exchanger used in the first-step ion exchange chromatography separation is Q-Sepharose High Performance, and the ion exchanger used in the second-step ion exchange chromatography separation is Mono Q.

[0017] The present invention also provides the use of the bifunctional corn peptide described in the above technical solution or the bifunctional corn peptide prepared by the preparation method described above in the preparation of drugs and / or foods with the functions of antagonizing Helicobacter pylori adhesion and / or antioxidation.

[0018] The present invention also provides a product with the activity of antagonizing Helicobacter pylori adhesion and / or antioxidation. The active ingredient of the product includes the bifunctional corn peptide described in the above technical solution or the bifunctional corn peptide prepared by the preparation method described above.

[0019] Preferably, the product includes drugs and / or foods.

[0020] Beneficial effects:

[0021] The present invention provides a bifunctional corn peptide with the activities of antagonizing Helicobacter pylori adhesion and antioxidation, including polypeptide I and / or polypeptide II; the polypeptide I includes the amino acid sequence shown in SEQ ID NO.1; the polypeptide II includes the amino acid sequence shown in SEQ ID NO.2. The bifunctional corn peptide of the present invention is separated from corn protein powder and has dual activities of antagonizing Helicobacter pylori adhesion and antioxidation. In particular, it has a strong inhibitory effect on the adhesion of Helicobacter pylori and can be used to prepare products with the activities of antagonizing Helicobacter pylori adhesion and / or antioxidation. In particular, products with both the functions of antagonizing Helicobacter pylori adhesion and antioxidation can prevent Helicobacter pylori infection and at the same time relieve the oxidative stress response and inflammatory response generated after the body is infected with Helicobacter pylori. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 It is the technical route diagram for the preparation of the bifunctional corn peptide in Example 1;

[0024] Figure 2 It is the mass spectrometry detection diagram of polypeptide I (IIPQCS);

[0025] Figure 3 It is the mass spectrometry detection diagram of polypeptide II (VCENPIL);

[0026] Figure 4 is the standard curve of colony concentration and OD600;

[0027] Figure 5 is the standard curve of FITC fluorescence intensity value and OD600. Specific embodiments

[0028] The present invention provides a bifunctional corn peptide having anti-Helicobacter pylori adhesion activity and antioxidant activity, including polypeptide I and / or polypeptide II; the polypeptide I includes the amino acid sequence shown in SEQ ID NO.1; the polypeptide II includes the amino acid sequence shown in SEQ ID NO.2.

[0029] The amino acid sequence shown in SEQ ID NO.1 of the present invention is IIPQCS, and the amino acid sequence shown in SEQ ID NO.2 is VCENPIL. The bifunctional corn peptide of the present invention is isolated from corn protein powder and has dual activities of antagonizing Helicobacter pylori adhesion and antioxidant, especially having a strong inhibitory effect on the adhesion of Helicobacter pylori.

[0030] The present invention also provides a preparation method of the bifunctional corn peptide described in the above technical solution, including the following steps: enzymatically hydrolyzing corn protein powder with neutral protease, and the enzymatic hydrolysate includes the bifunctional corn peptide.

[0031] Before the enzymatic hydrolysis of the present invention, it is preferably further included to mix the corn protein powder with water to prepare a suspension, and the mass concentration of the suspension is preferably 15% (w / v). The corn protein powder of the present invention is preferably the corn protein powder after extrusion and expansion and de-starching. The present invention has no special limitation on the source of the corn protein powder after extrusion and expansion and de-starching, and the corn protein powder after extrusion and expansion and de-starching purchased conventionally in the art can be used.

[0032] After obtaining the suspension, the present invention enzymatically hydrolyzes the suspension with neutral protease to obtain an enzymatic hydrolysis mixture. Calculated based on the protein content in the corn protein powder, the dosage of the neutral protease of the present invention is preferably 400 U / g protein. The temperature of the enzymatic hydrolysis of the present invention is preferably 45 °C, the time is preferably 150 min, and the pH value is preferably 7.0. After completing the enzymatic hydrolysis, the present invention preferably further includes inactivating the enzyme in the enzymatic hydrolysis mixture, and the temperature of the enzyme inactivation treatment is preferably 100 °C; the time of the enzyme inactivation treatment is preferably 10 min. After completing the enzyme inactivation treatment, the present invention preferably centrifuges the mixture after enzyme inactivation treatment, takes the supernatant, and obtains an enzymatic hydrolysate. The rotation speed of the centrifugation of the present invention is preferably 4000 r / min, and the centrifugation time is preferably 10 min. The enzymatic hydrolysate of the present invention includes the bifunctional corn peptide.

[0033] After obtaining the enzymolysis solution, the present invention preferably further includes separating and purifying the enzymolysis solution, collecting the components with a molecular weight < 1000 Da, and obtaining the bifunctional corn peptide.

[0034] The separation and purification of the present invention preferably include the following steps: separating the enzymolysis solution by gel chromatography, and collecting the components with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity and a molecular weight < 1000 Da; after obtaining the components, separating the components by ion exchange chromatography, and collecting the components with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity to obtain the bifunctional corn peptide.

[0035] The present invention preferably separates the enzymolysis solution by gel chromatography, and collects the components with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity and a molecular weight < 1000 Da. The pre-packed column for separation of the gel chromatography of the present invention is preferably Superdex Peptide 10 / 300GL. The conditions for gel chromatography separation of the present invention preferably include: the sample loading concentration is 50 mg / L, and the sample loading volume is 1 mL; the eluent is 20 mM PBS buffer containing 0.15 mol / L NaCl with a pH of 7.0; the flow rate of the eluent is 0.25 mL / min; the detection wavelength is 214 nm. The present invention preferably further includes measuring the antagonistic activity against Helicobacter pylori adhesion and antioxidant activity of each collected component, and obtaining the components with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity and a molecular weight < 1000 Da. The present invention preferably uses the method in Example 1 to measure the antagonistic activity against Helicobacter pylori adhesion and antioxidant activity, and the same applies hereinafter and will not be elaborated. The protein concentration in the components with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity and a molecular weight < 1000 Da is preferably 4 mg / mL.

[0036] After obtaining the fraction with relatively stronger anti-Helicobacter pylori adhesion activity and antioxidant activity and a molecular weight < 1000 Da, the present invention preferably performs the first ion exchange chromatography separation on the fraction with relatively stronger anti-Helicobacter pylori adhesion activity and antioxidant activity and a molecular weight < 1000 Da to obtain Fraction I. The ion exchanger used in the first ion exchange chromatography separation of the present invention is preferably Q-Sepharose High Performance. The conditions for performing the first ion exchange chromatography separation of the present invention preferably include: the sample loading volume is 50 mL; the 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; the eluent B is 20 mM Tris-HCl buffer containing 1 mol / L NaCl at pH 7.5; the flow rate of the eluent is 2 mL / min, the detection wavelength is 214 nm, the gradient elution volume is 60 mL, and the volume of the peak fraction collected is 6 mL / tube. The present invention preferably further includes measuring the anti-Helicobacter pylori adhesion activity and antioxidant activity of the collected peak fractions to obtain the fraction with relatively stronger anti-Helicobacter pylori adhesion activity and antioxidant activity, which is Fraction I. The protein concentration in the Fraction I of the present invention is preferably 2 mg / mL.

[0037] After obtaining the Fraction I, the present invention preferably performs the second ion exchange chromatography separation on the Fraction I to obtain Fraction II. The ion exchanger used in the second ion exchange chromatography separation of the present invention is preferably Mone Q. The conditions for performing the second ion exchange chromatography separation of the present invention preferably include: the sample loading volume is 10 mL; the 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; the 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 volume of the peak fraction collected is 1 mL / tube. The present invention preferably further includes measuring the anti-Helicobacter pylori adhesion activity and antioxidant activity of the collected peak fractions to obtain the fraction with relatively stronger anti-Helicobacter pylori adhesion activity and antioxidant activity, which is Fraction II.

[0038] After obtaining the Fraction II, the present invention preferably performs desalting, freeze-drying and mass spectrometry sequencing on the Fraction II to obtain the bifunctional corn peptide, which is Polypeptide I with the amino acid sequence IIPQCS and Polypeptide II with the amino acid sequence VCENPIL. The present invention preferably uses LC-MS / MS for the mass spectrometry sequencing. The present invention has no special limitations on the process and steps of the desalting, freeze-drying and mass spectrometry sequencing, and the conventional desalting, freeze-drying and mass spectrometry sequencing steps in the art can be used.

[0039] The present invention also provides the use of the bifunctional corn peptide described in the above technical solution or the bifunctional corn peptide prepared by the described preparation method in the preparation of drugs and / or foods with the functions of antagonizing Helicobacter pylori adhesion and / or antioxidation.

[0040] The present invention also provides a product with the activities of antagonizing Helicobacter pylori adhesion and / or antioxidation. The active ingredient of the product includes the bifunctional corn peptide described in the above technical solution or the bifunctional corn peptide prepared by the described preparation method. The product of the present invention preferably includes drugs and / or foods. The product of the present invention preferably further includes excipients and / or other active ingredients. The present invention has no special limitation on the excipients and other active ingredients, and they can be reasonably added according to the prepared product. The present invention has no special limitation on the dosage of the bifunctional corn peptide in the product, and it can be reasonably added according to the specifically prepared product.

[0041] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0042] In the methods used in the following examples, unless otherwise specified, conventional test methods in the art are adopted; the biological materials and test materials used, unless otherwise specified, can be obtained through conventional purchasing channels in the art.

[0043] Example 1

[0044] The bifunctional corn peptide with the activities of antagonizing Helicobacter pylori adhesion and antioxidation consists of the following steps, and the technical route is as Figure 1 shown:

[0045] 1. Preparation of corn protein hydrolysate

[0046] Take a certain amount of corn protein powder (purchased from Qiqihar Longjiang Fufeng Biotech Co., Ltd.) that has been extruded and expanded and had its starch removed, add water to prepare a suspension with a substrate concentration of 15% (w / v), and then carry out enzymatic hydrolysis with neutral protease. The enzymatic hydrolysis conditions are: enzyme dosage 400 U / g protein, enzymatic hydrolysis temperature 45 °C, enzymatic hydrolysis time 150 min, enzymatic hydrolysis pH 7.0. After the enzymatic hydrolysis is completed, heat to inactivate the enzyme at 100 °C for 10 min. The hydrolysate is centrifuged at 4000 r / min for 10 min and the precipitate is discarded. The obtained supernatant is the corn protein hydrolysate. By measuring its activities of antagonizing Helicobacter pylori adhesion and antioxidation, it is confirmed that it is a polypeptide mixture with high activities of antagonizing Helicobacter pylori adhesion and antioxidation. The methods for measuring the activities of antagonizing Helicobacter pylori adhesion and antioxidation are both carried out according to step 5 below, and the same applies hereinafter, and will not be elaborated further.

[0047] 2. Gel chromatography separation of corn protein hydrolysate

[0048] The gel chromatography column used was a Superdex Peptide 10 / 300GL pre-packed column. The polypeptide mixture with high anti-Helicobacter pylori adhesion activity and antioxidant activity in Step 1 was separated by gel chromatography to obtain polypeptide mixtures with different molecular weight components. The sample loading concentration was 50 mg / mL, the sample 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 anti-Helicobacter pylori adhesion activity and antioxidant activity of each molecular weight component were measured, and the fraction with a molecular weight < 1000 Da and relatively high anti-Helicobacter pylori adhesion activity and antioxidant activity was collected for ion exchange chromatography separation.

[0049] 3. Ion exchange chromatography separation

[0050] 3.1 Q-Sepharose High Performance strong anion exchange chromatography separation

[0051] The fraction with a molecular weight < 1000 Da and relatively high anti-Helicobacter pylori adhesion activity and antioxidant activity obtained by gel chromatography was passed through a 0.22 μm microporous filter membrane. The protein concentration of the obtained sample was 4 mg / mL, and a strong anion exchanger Q-Sepharose High Performance was used for separation. The sample loading volume was 50 mL. The eluent A for the strong anion exchange chromatography: 20 mM Tris-HCl buffer solution with pH 7.5, eluent B: 20 mM Tris-HCl buffer solution with 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 6 mL was collected for each tube of the peak fraction. The anti-Helicobacter pylori adhesion activity and antioxidant activity of each tube of the collected solution were measured, and the fraction with relatively high anti-Helicobacter pylori adhesion activity and antioxidant activity (the 6th tube) was collected for Mono Q ion exchange chromatography separation.

[0052] 3.2 Mono Q ion exchange chromatography

[0053] The highly active component separated in Step 3.1 was further separated using Mono Q ion exchange chromatography. The highly active component obtained from the previous ion exchange chromatography was passed through a 0.22 μm microporous filter membrane. The protein concentration of the obtained sample was 2 mg / mL, and the sample loading volume was 10 mL. The eluent A of the Mono Q ion exchange chromatography: 20 mM Tris-HCl buffer with pH 7.0, eluent B: 20 mM Tris-HCl buffer containing 1 mol / L NaCl with pH 7.0, the flow rate was 1 mL / min, the detection wavelength was 214 nm, the gradient elution volume was 20 mL, and 1 mL was collected for each tube of the peak component; the anti-Helicobacter pylori adhesion activity and antioxidant activity of each tube of the collected solution were measured, and the component with relatively high anti-Helicobacter pylori adhesion activity and antioxidant activity (the 16th tube) was collected for standby.

[0054] 4. LC-MS / MS Mass Spectrometry Sequencing

[0055] The component obtained in Step 3.2 was desalted and freeze-dried and then subjected to mass spectrometry sequencing to obtain bifunctional corn peptides with amino acid sequences of IIPQCS (peptide I) and VCENPIL (peptide II). The results are as Figure 2 and 3 shown.

[0056] 5. Determination of the Anti-Helicobacter pylori Adhesion Activity and Antioxidant Activity of Bifunctional Corn Peptides

[0057] After the bifunctional corn peptides obtained in Step 4 were chemically synthesized (commissioned Shanghai Qiangyao Biotechnology Co., Ltd. to synthesize), their anti-Helicobacter pylori adhesion activity and the scavenging ability against DPPH and ABTS free radicals were measured.

[0058] 5.1 Determination of the Anti-Helicobacter pylori Adhesion Activity of Bifunctional Corn Peptides:

[0059] 1) The H. pylori ATCC43504 strain was used as the strain for the anti-adhesion activity test. The cryopreserved H. pylori ATCC43504 strain was thawed at 37 °C, first mixed with a liquid medium (3 g soy peptone, 2.5 g K 2 HPO 4 、17 g tryptone and 5 g NaCl, add 1 L of deionized water, mix well and dissolve, then adjust the pH value to 7.2, sterilize at 121 °C and 0.1 Mpa for 1 h), and then inoculate it on a slant medium (15 g tryptone, 5 g soy peptone, 15 g agar, 5 g NaCl and 950 mL of deionized water, stir and dissolve, adjust the pH value to 7.2, sterilize at 121 °C and 0.1 Mpa for 1 h. When the medium cools to about 45 °C, add 50 mL of sterile defibrinated sheep blood, mix well, and pour it into a test tube to make a slant medium), and incubate it microaerophilically at 37 °C (5% O2 , 85% N 2 , 10% CO 2 ) Under these conditions, culture for 48 - 72 h, and the obtained bacterial liquid can be used for bacterial strain passage and anti-adhesion activity detection.

[0060] Dilute the bacterial liquid of the passaged H. pylori ATCC43504 four times by a 10-fold gradient to obtain five different concentrations of bacterial liquid. Measure the OD value of the Helicobacter pylori bacterial liquid under the condition of 600 nm. At the same time, calculate the colony concentration by the plate coating method, and establish the standard curve of the colony concentration and OD 600 as Figure 4 shown.

[0061] 2) After thawing, transfer the cryopreserved human gastric mucosal epithelial cells (GES-1) into a cell culture flask. The cell culture medium consists of 1% penicillin-streptomycin mixture, 10% fetal bovine serum, and 89% DMEM medium. Incubate at 37°C, 5% CO 2 until a monolayer of cells is formed. Digest and passage with trypsin-EDTA, centrifuge, resuspend with cell culture medium without antibiotics, and adjust the cell concentration to 3×10 5 cells / mL. Inoculate the cell suspension into a 96-well plate, 100 μL per well, and culture and incubate in an incubator at 37°C, 5% CO 2 for 24 h for the determination experiment of the antagonistic activity of H. pylori adhesion.

[0062] 3) Label Helicobacter pylori with fluorescein isothiocyanate (FITC)

[0063] Prepare a DMSO solution with a FITC concentration of 2 mg / mL and filter it through a sterile filter membrane. Mix it evenly with the bacterial liquid of the passaged H. pylori ATCC43504 prepared in step 1) according to a volume ratio of 1:1. Under the condition of avoiding light, mix on a biochemical shaker for 30 min, then centrifuge at a speed of 4500 r / min for 3 min, remove the supernatant, wash it 3 times with 1×PBS buffer to remove the excess FITC. Finally, dilute the bacterial liquid in a liquid medium (3 g soy peptone, 2.5 g K 2 HPO 4 、17 g tryptone and 5 g NaCl, add 1 L of deionized water, mix well and dissolve, then adjust the pH value to 7.2, sterilize at 121°C, 0.1 Mpa for 1 h) until the OD 600 value is about 0.1 (10 8 cfu / mL) for standby.

[0064] 4) Construction of the standard curve of the FITC fluorescence intensity value and OD 600

[0065] Dilute the Helicobacter pylori bacterial solution treated with FITC labeling in the previous step by a factor of 10 four times. Measure its fluorescence intensity value under the excitation wavelength of 485 nm and the emission wavelength of 530 nm. At the same time, measure the OD value under the condition of 600 nm, and establish the standard curve of the FITC fluorescence intensity value and OD 600 as shown in Figure 5 the figure

[0066] 5) Bifunctional zein peptide antagonistic activity test against Helicobacter pylori adhesion

[0067] Use 100% DMEM medium to prepare a solution of polypeptide I with a certain protein concentration from the polypeptide I prepared in Example 1. Mix it with the FITC-labeled bacterial solution in step 3) at a ratio of 1:1 (v / v) for 30 min in the dark at room temperature, so that the final concentration of polypeptide I obtained in Example 1 is 4 mg / mL, and obtain the mixed bacterial solution

[0068] Add 100 μL of the mixed bacterial solution to a 96-well plate with gastric mucosal epithelial cells (GES-1). Use 100 μL of 100% DMEM medium as the negative control group and place it in an incubator for 90 min. Then remove the solution and wash it 3 times with PBS buffer. Immediately, add 100 μL of PBS buffer to each well and measure the fluorescence intensity value under the emission wavelength of 530 nm and the excitation wavelength of 485 nm. According to steps 3) and 4) of 5.1 in Example 1, calculate the colony concentration of the negative control group and the test group, and calculate the adhesion inhibition rate using the following formula

[0069]

[0070] Result: When the concentration of polypeptide I (IIPQCS) is 4 mg / mL, its antagonistic activity against Helicobacter pylori adhesion is 21.96%

[0071] Use the same method to measure the anti-Helicobacter pylori adhesion activity of polypeptide II (VCENPIL). When the concentration of polypeptide II (VCENPIL) is 4 mg / mL, its antagonistic activity against Helicobacter pylori adhesion is 36.86%

[0072] 5.2 Determination of the antioxidant activity of bifunctional zein peptides

[0073] (1) Determination of the ability of bifunctional zein peptides to scavenge DPPH free radicals: Take 2 mL of polypeptide I solutions with different concentrations (prepare polypeptide I solutions by dissolving polypeptide I in water), add 2 mL of 0.1 mmol / L DPPH absolute ethanol solution, mix well, react in the dark for 30 min, and measure its absorbance value (A i) Take 2 mL of polypeptide I solution in a test tube, add 2 mL of absolute ethanol, and measure its absorbance (A) at 517 nm j ) Take 2 mL of 0.1 mmol / L DPPH absolute ethanol solution (0.1 mmol / L) and 2 mL of absolute ethanol for reaction as a reference, and measure its absorbance (A) at 517 nm 0 ). The scavenging rate K of the sample for DPPH radicals is calculated according to formula (1):

[0074]

[0075] In the formula:

[0076] K - Scavenging rate for DPPH radicals, %;

[0077] A 0 - Absorbance of 2 mL of 0.1 mmol / L DPPH absolute ethanol solution and 2 mL of absolute ethanol at 517 nm;

[0078] A i - Absorbance of the reaction of 2 mL of 0.1 mmol / L DPPH absolute ethanol solution and 2 mL of sample solution at 517 nm;

[0079] A j - Absorbance of 2 mL of absolute ethanol and 2 mL of sample solution at 517 nm.

[0080] From the above determination results of DPPH radical scavenging activity, it can be obtained that the IC50 value of polypeptide I (IIPQCS) for the ability to scavenge DPPH radicals is 0.090 mg / mL.

[0081] Using the same method to determine the ability of polypeptide II (VCENPIL) to scavenge DPPH radicals, the IC50 value of polypeptide II (VCENPIL) for the ability to scavenge DPPH radicals is 0.082 mg / mL.

[0082] (2) Determination of the ability of bifunctional corn 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 for 12 - 16 h under dark conditions at room temperature to obtain the ABTS⁺ mother liquor. Dilute the ABTS⁺ mother liquor with deionized water to make its absorbance value at 734 nm be 0.70 ± 0.023, and equilibrate it at 30 °C for 30 min to obtain the ABTS⁺ working solution. Respectively take 2.0 mL of polypeptide I solutions with different concentrations and 2.0 mL of the ABTS⁺ working solution and add them into test tubes, mix well, place them in the dark at room temperature for 20 min, and measure the absorbance value at 734 nm. Use the reaction mixture with the same volume of deionized water replacing the polypeptide I solution as the control, set 3 replicates, and calculate the average value. Calculate the scavenging rate of the polypeptide I solution on ABTS free radicals according to the following formula.

[0083] Scavenging 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.

[0084] From the above ABTS determination results, it can be obtained that the IC50 value of polypeptide I (IIPQCS) for the ability to scavenge ABTS free radicals is 0.011 mg / mL.

[0085] Use the same method to determine the ability of polypeptide II (VCENPIL) to scavenge ABTS free radicals. The IC50 value of polypeptide II (VCENPIL) for the ability to scavenge ABTS free radicals is 0.019 mg / mL.

[0086] From the above examples, it can be obtained that the bifunctional corn peptides provided by the present invention have dual activities of simultaneously antagonizing Helicobacter pylori adhesion and antioxidation.

[0087] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A bifunctional corn peptide with activities of antagonizing Helicobacter pylori adhesion and antioxidant activity, characterized in that, the bifunctional corn peptide is polypeptide II, or a composition of polypeptide I and polypeptide II; the amino acid sequence of the polypeptide I is shown as SEQ ID NO.1; the amino acid sequence of the polypeptide II is shown as SEQ ID NO.

2.

2. A preparation method of the bifunctional corn peptide according to claim 1, characterized in that, it comprises the following steps: enzymatically hydrolyzing corn protein powder with neutral protease, and the enzymatic hydrolysate contains the bifunctional corn peptide.

3. According to the preparation method of claim 2, characterized in that, the corn protein powder is the corn protein powder after extrusion puffing and starch removal; before the enzymatic hydrolysis, it also includes preparing the corn protein powder into a suspension, and the mass concentration of the corn protein powder in the suspension is 15% w / v.

4. According to the preparation method of claim 2 or 3, characterized in that, the dosage of the neutral protease is 400U / g protein; the temperature of the enzymatic hydrolysis is 45°C, the time is 150min, and the pH is 7.

0.

5. According to the preparation method of claim 2, characterized in that, after the enzymatic hydrolysis, it also includes: separating and purifying the enzymatic hydrolysate, collecting the fraction with a molecular weight < 1000Da, to obtain the bifunctional corn peptide.

6. According to the preparation method of claim 5, characterized in that, the separation and purification include gel chromatography separation and ion exchange chromatography separation.

7. According to the preparation method of claim 6, characterized in that, the chromatography pre-packed column for the gel chromatography separation is Superdex Peptide 10 / 300GL; the ion exchange chromatography separation includes two-step ion exchange chromatography separation. The ion exchanger used in the first-step ion exchange chromatography separation is Q-Sepharose High Performance, and the ion exchanger used in the second-step ion exchange chromatography separation is Mone Q.

8. Use of the bifunctional corn peptide according to claim 1 or the bifunctional corn peptide prepared by the preparation method according to any one of claims 2 to 7 in the preparation of drugs and / or foods with functions of antagonizing Helicobacter pylori adhesion and / or antioxidant activity.

9. A product with functions of antagonizing Helicobacter pylori adhesion and / or antioxidant activity, characterized in that, the active ingredient of the product includes the bifunctional corn peptide according to claim 1 or the bifunctional corn peptide prepared by the preparation method according to any one of claims 2 to 7.

10. According to the product of claim 9, characterized in that, the product includes drugs and / or foods.

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