Bifunctional corn peptides with activities of antagonizing Helicobacter pylori adhesion and antioxidant activity, and preparation method and application thereof

Abstract

The present invention belongs to the technical field of protein peptide preparation, and particularly relates to a bifunctional corn peptide with activities of antagonizing Helicobacter pylori adhesion and antioxidation, and a preparation method and application thereof. The present invention provides a bifunctional corn peptide with activities of antagonizing Helicobacter pylori adhesion and antioxidation, comprising polypeptide I and / or polypeptide II; the polypeptide I comprises the amino acid sequence shown in SEQ ID NO. 1; the polypeptide II comprises the amino acid sequence shown in SEQ ID NO. 2. The bifunctional corn peptide of the present invention is isolated from corn protein powder, has dual activities of antagonizing Helicobacter pylori adhesion and antioxidation, and particularly has a strong inhibitory effect on the adhesion of Helicobacter pylori, and can be used for preparing products with functions of antagonizing Helicobacter pylori adhesion and / or antioxidation.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein peptide preparation, and specifically relates to a bifunctional corn peptide with antagonistic Helicobacter pylori adhesion activity and antioxidant activity, and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori ( 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. Helicobacter pylori grows under microaerophilic conditions, and the optimal growth environment is 5% O2, 10% CO2, 85% N2, 37 °C and high humidity. Although Helicobacter pylori can colonize the gastric mucosa, it can only grow within the pH range of 5.5-8.0.

[0003] Helicobacter pylori is a special pathogen. Once the body is infected with Helicobacter pylori, it is generally difficult to clear itself. Without eradication treatment, it will be carried for life. Helicobacter pylori can specifically adhere to gastric mucosal epithelial cells, triggering gastric oxidative stress and inflammatory responses. It is one of the recognized important pathogenic factors for chronic gastritis and ulcerative diseases, and 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 with a commonly used proton pump inhibitor and two antibiotics, and the quadruple therapy with bismuth, a proton pump inhibitor and two antibiotics combined. However, as the antibiotic resistance of Helicobacter pylori increases 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.

[0004] In recent years, some food-derived components of natural origin have been reported to inhibit the adhesion activity of Helicobacter pylori, 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 Helicobacter pylori adhesion activity.

[0005] 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 adverse 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

[0006] The purpose 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.

[0007] 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;

[0008] 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.

[0009] The present invention also provides a preparation method of the bifunctional corn peptide described in the above technical solution, including the following steps:

[0010] Using neutral protease to enzymatically hydrolyze corn gluten meal, and the enzymatic hydrolysate includes the bifunctional corn peptide.

[0011] 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).

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

[0013] Preferably, after 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.

[0014] Preferably, the separation and purification include gel chromatography separation and ion exchange chromatography separation.

[0015] Preferably, the pre-packed column for gel chromatography separation is Superdex Peptide 10 / 300 GL; 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.

[0016] The present invention also provides an application of the bifunctional corn peptide described in the above technical solution or the bifunctional corn peptide prepared by the preparation method in the preparation of drugs and / or foods with the functions of antagonizing Helicobacter pylori adhesion and / or antioxidation.

[0017] 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 preparation method.

[0018] Preferably, the product includes drugs and / or foods. Beneficial effects

[0019] 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, has the dual activities of antagonizing Helicobacter pylori adhesion and antioxidation, and particularly has a strong inhibitory effect on the adhesion of Helicobacter pylori, and can be used for preparing products with the activities of antagonizing Helicobacter pylori adhesion and / or antioxidation. Description of the drawings

[0020] 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 to be used in the embodiments.

[0021] Figure 1 is the technical route diagram for the preparation of the bifunctional corn peptide in Example 1;

[0022] Figure 2 is the mass spectrometry detection chart of polypeptide I (IIPQCS);

[0023] Figure 3 is the mass spectrometry detection chart of polypeptide II (VCENPIL);

[0024] Figure 4 is the standard curve of the colony concentration and OD 600 of;

[0025] Figure 5 is the standard curve of FITC fluorescence intensity value and OD 600 . Specific embodiments

[0026] The present invention provides a bifunctional corn peptide with antagonistic activity against Helicobacter pylori adhesion 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 400U / g protein. The temperature of the enzymatic hydrolysis of the present invention is preferably 45°C, the time is preferably 150min, 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 10min. 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 4000r / min, and the centrifugation time is preferably 10min. The enzymatic hydrolysate of the present invention includes the bifunctional corn peptide.

[0031] After obtaining the enzymatic hydrolysate, the present invention preferably further includes separating and purifying the enzymatic hydrolysate, collecting the fraction with a molecular weight < 1000Da, and obtaining the bifunctional corn peptide.

[0032] The separation and purification in the present invention preferably includes the following steps: separating the enzymolysis solution by gel chromatography, and collecting the fraction with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity and a molecular weight < 1000 Da; after obtaining the fraction, separating the fraction by ion exchange chromatography, and collecting the fraction with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity to obtain the bifunctional corn peptide.

[0033] The present invention preferably separates the enzymolysis solution by gel chromatography, and collects the fraction 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 in the present invention is preferably Superdex Peptide 10 / 300 GL. The conditions for gel chromatography separation in 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 at pH 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 fraction to obtain the fraction 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 hereinafter, and will not be repeated. The protein concentration in the fraction with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity and a molecular weight < 1000 Da in the present invention is preferably 4 mg / mL.

[0034] After obtaining the fraction with relatively stronger antagonistic activity against Helicobacter pylori adhesion 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 antagonistic activity against Helicobacter pylori adhesion 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 with a pH of 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 antagonistic activity against Helicobacter pylori adhesion and antioxidant activity of the collected peak fractions to obtain the fraction with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity, which is Fraction I. The protein concentration in the Fraction I of the present invention is preferably 2 mg / mL.

[0035] 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 with a pH of 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 antagonistic activity against Helicobacter pylori adhesion and antioxidant activity of the collected peak fractions to obtain the fraction with relatively stronger antagonistic activity against Helicobacter pylori adhesion and antioxidant activity, which is Fraction II.

[0036] 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, and the bifunctional corn peptide 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 steps of desalting, freeze-drying and mass spectrometry sequencing in the art can be used.

[0037] 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.

[0038] 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 specific prepared product.

[0039] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0040] In the methods used in the following embodiments, 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 purchase channels in the art. Example

[0041] 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:

[0042] Preparation of corn protein hydrolysate

[0043] Take a certain amount of corn protein powder (purchased from Qiqihar Longjiang Fufeng Biotechnology 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, heat to inactivate the enzyme at 100°C for 10 min, centrifuge the enzymatic hydrolysate at 4000 r / min for 10 min and discard the precipitate. 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 all carried out according to step 5 below, and the same applies hereinafter, and will not be elaborated.

[0044] Gel chromatography separation of corn protein hydrolysate

[0045] The gel chromatography column used was a Superdex Peptide 10 / 300 GL 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 determined, 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.

[0046] Ion exchange chromatography separation

[0047] 3.1 Q-Sepharose High Performance strong anion exchange chromatography separation

[0048] 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 separation was performed using the strong anion exchanger Q-Sepharose High Performance. 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 determined, 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.

[0049] 3.2 Mono Q ion exchange chromatography

[0050] The highly active component separated in Step 3.1 is further separated by Mono Q ion exchange chromatography. The highly active component obtained from the previous ion exchange chromatography is passed through a 0.22 μm microporous filter membrane. The protein concentration of the obtained sample is 2 mg / mL, and the sample loading volume is 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 is 1 mL / min, the detection wavelength is 214 nm, the gradient elution volume is 20 mL, and 1 mL is collected for each tube of the peak component; the anti-Helicobacter pylori adhesion activity and antioxidant activity of each tube of the collected solution are measured, and the component with relatively high anti-Helicobacter pylori adhesion activity and antioxidant activity (the 16th tube) is collected for standby.

[0051] 4. LC-MS / MS Mass Spectrometry Sequencing

[0052] The component obtained in Step 3.2 is desalted and freeze-dried and then subjected to mass spectrometry sequencing to obtain a bifunctional corn peptide with amino acid sequences of IIPQCS (peptide I) and VCENPIL (peptide II). The results are as Figure 2 and 3 shown.

[0053] 5. Determination of Anti-Helicobacter pylori Adhesion Activity and Antioxidant Activity of Bifunctional Corn Peptide

[0054] After the bifunctional corn peptide obtained in Step 4 is chemically synthesized (commissioned by Shanghai Qiangyao Biotechnology Co., Ltd.), its anti-Helicobacter pylori adhesion activity and the scavenging ability against DPPH and ABTS free radicals are measured.

[0055] 5.1 Determination of Anti-Helicobacter pylori Adhesion Activity of Bifunctional Corn Peptide:

[0056] 1) Use H.pylori ATCC43504 strain as the strain for the anti-adhesion activity test. At 37 °C, the H.pyloriThe strain ATCC43504 was thawed and first mixed with a liquid medium (3 g of soy peptone, 2.5 g of K2HPO4, 17 g of tryptone, and 5 g of NaCl, added to 1 L of deionized water, mixed and shaken well to dissolve, then the pH value was adjusted to 7.2, sterilized at 121 °C and 0.1 Mpa for 1 h). Subsequently, it was inoculated onto a slant medium (15 g of tryptone, 5 g of soy peptone, 15 g of agar, 5 g of NaCl, and 950 mL of 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 defibrinated sheep blood was added, mixed well, and then poured into test tubes to make slant medium). Under microaerophilic conditions (5% O2, 85% N2, 10% CO2) at 37 °C, it was cultured for 48 - 72 h, and the obtained bacterial liquid could be used for bacterial strain passage and anti - adhesion activity detection.

[0057] The passaged H.pylori bacterial liquid of ATCC43504 was subjected to four - fold 10 - fold serial dilutions to obtain five different concentrations of bacterial liquid. The OD value of Helicobacter pylori bacterial liquid was measured at 600 nm. At the same time, the colony concentration was calculated by the plate coating method, and a standard curve of colony concentration versus OD 600 was established, as Figure 4 shown.

[0058] 2) The cryopreserved human gastric mucosal epithelial cells (GES - 1) were thawed and transferred into a cell culture flask. The cell culture medium consisted of 1% penicillin - streptomycin mixture, 10% fetal bovine serum, and 89% DMEM medium. Incubated at 37 °C and 5% CO2 until a monolayer of cells formed, digested and passaged with trypsin - EDTA, centrifuged, resuspended with 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, 100 μL per well, and cultured and incubated in a 37 °C, 5% CO2 incubator for 24 h for the determination experiment of anti - H. pylori adhesion activity.

[0059] 3) Fluorescein isothiocyanate (FITC) - labeled Helicobacter pylori

[0060] A DMSO solution with a FITC concentration of 2 mg / mL was prepared and filtered through a sterile filter membrane. According to a volume ratio of 1:1, the passaged H. pyloriMix the bacterial solution of ATCC43504 with it. Under the condition of avoiding light, mix it on a biochemical shaker for 30 min, then centrifuge it 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 solution in a liquid medium (3 g soy peptone, 2.5 g K2HPO4, 17 g tryptone and 5 g NaCl, add 1 L 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) until the OD 600 value is about 0.1 (10 8 cfu / mL), and set aside.

[0061] 4) Construction of the standard curve of FITC fluorescence intensity value and OD 600 Standard curve construction

[0062] Dilute the Helicobacter pylori bacterial solution labeled with FITC in the previous step by 10-fold gradient four times. Measure its fluorescence intensity value under the conditions of excitation wavelength 485 nm and emission wavelength 530 nm. At the same time, measure the OD value under the condition of 600 nm, and establish the standard curve of FITC fluorescence intensity value and OD 600 as shown in Figure 5 the figure.

[0063] 5) Bifunctional zein peptide antagonistic Helicobacter pylori adhesion activity assay

[0064] 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 bacterial solution labeled with FITC in step 3) at a ratio of 1:1 (v / v) under the condition of room temperature and avoiding light for 30 min, so that the final concentration of polypeptide Ⅰ obtained in Example 1 is 4 mg / mL to obtain the mixed bacterial solution.

[0065] 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 conditions of emission wavelength 530 nm and excitation wavelength 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:

[0066]

[0067] The result was as follows: when the concentration of polypeptide I (IIPQCS) was 4 mg / mL, its antagonistic activity against Helicobacter pylori adhesion was 21.96%.

[0068] The anti-Helicobacter pylori adhesion activity of polypeptide II (VCENPIL) was determined by the same method. When the concentration of polypeptide II (VCENPIL) was 4 mg / mL, its antagonistic activity against Helicobacter pylori adhesion was 36.86%.

[0069] 5.2 Determination of the antioxidant activity of bifunctional corn peptides:

[0070] (1) Determination of the ability of bifunctional corn peptides to scavenge DPPH free radicals: Take 2 mL of polypeptide I solutions with different concentrations (polypeptide I solutions were 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 min, and measure its absorbance value (A i ) at 517 nm; Take 2 mL of polypeptide I solution in a test tube, add 2 mL of anhydrous ethanol, and measure its absorbance value (A j ) at 517 nm; Take 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution (0.1 mmol / L) and 2 mL of anhydrous ethanol to react as a reference, and measure its absorbance value (A0) at 517 nm. The scavenging rate K of the sample on DPPH free radicals was calculated according to formula (1):

[0071]

[0072] In the formula:

[0073] K - Scavenging rate of DPPH free radicals, %;

[0074] A0 - Absorbance value of 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution and 2 mL of anhydrous ethanol at 517 nm;

[0075] A i - Absorbance value of 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution and 2 mL of sample solution at 517 nm;

[0076] A j - Absorbance value of 2 mL of anhydrous ethanol and 2 mL of sample solution at 517 nm.

[0077] From the above determination results of DPPH free radical scavenging activity, it can be concluded that the IC50 value of polypeptide I (IIPQCS) for DPPH free radical scavenging ability was 0.090 mg / mL.

[0078] The same method was used to determine the DPPH radical scavenging ability of polypeptide II (VCENPIL). The IC50 value of the DPPH radical scavenging ability of polypeptide II (VCENPIL) was 0.082 mg / mL.

[0079] (2) Determination of the ABTS radical scavenging ability of bifunctional corn peptides: Prepare 7.0 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate aqueous solution accurately, 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 for 30 min at 30 °C to obtain the ABTS+ working solution. 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 deionized water to replace the same volume of the reaction mixture of 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 radicals according to the following formula.

[0080] 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.

[0081] From the above ABTS determination results, it can be obtained that the IC50 value of the ABTS radical scavenging ability of polypeptide I (IIPQCS) is 0.011 mg / mL.

[0082] The same method was used to determine the ABTS radical scavenging ability of polypeptide II (VCENPIL). The IC50 value of the ABTS radical scavenging ability of polypeptide II (VCENPIL) was 0.019 mg / mL.

[0083] From the above examples, it can be obtained that the bifunctional corn peptide provided by the present invention has dual activities of simultaneously antagonizing Helicobacter pylori adhesion and antioxidation.

[0084] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not 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 antagonistic activity against Helicobacter pylori adhesion and antioxidant activity, characterized in that, The amino acid sequence of the bifunctional corn peptide is shown in SEQ ID NO.

1.

2. The preparation method of the bifunctional corn peptide according to claim 1, characterized in that, It includes the following steps: Hydrolyze corn protein powder with neutral protease, and the hydrolysate contains the bifunctional corn peptide; The corn protein powder is the corn protein powder after extrusion and expansion and de-starching; before 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; The dosage of the neutral protease is 400U / g protein; The temperature of the hydrolysis is 45°C, the time is 150 min, and the pH is 7.0; After the hydrolysis, it also includes: separating and purifying the hydrolysate, collecting the fraction with a molecular weight <1000 Da to obtain the bifunctional corn peptide; The separation and purification include gel chromatography separation and ion exchange chromatography separation; The pre-packed chromatography column for the gel chromatography separation is Superdex Peptide 10 / 300 GL; 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.

3. Use of the bifunctional corn peptide described in claim 1 or the bifunctional corn peptide prepared by the preparation method described in claim 2 in the preparation of a drug for antagonizing Helicobacter pylori adhesion and / or a food with antioxidant function.

4. A product, characterized in that, The active ingredient of the product includes the bifunctional corn peptide described in claim 1 or the bifunctional corn peptide prepared by the preparation method described in claim 2.

5. The product according to claim 4, characterized in that, The product is a drug for antagonizing Helicobacter pylori adhesion and / or a food with antioxidant function.

Images