AU2021100067A4 - Active peptide capable of inhibiting helicobacter pylori (h. pylori), and preparation method and use thereof - Google Patents

Abstract

The present disclosure provides to an active peptide capable of inhibiting Helicobacter pylori (H. pylori), and a preparation method and use thereof, and belongs to the technical field of active peptides. A wheat peptide mixture capable of inhibiting H. pylori is obtained by grinding and homogenizing a vital wheat gluten solution, pretreating a resulting homogenate, subjecting a pretreated homogenate to stepwise enzymolysis with papain and neutral protease successively, and filtering through a ceramic membrane and an organic membrane. The wheat peptide mixture is subjected to high-performance liquid chromatography (HPLC) to obtain a wheat peptide M, and the wheat peptide M is further subjected to chromatographic analysis by a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain a wheat peptide Ml with a sequence shown in SEQ ID NO: 1.In vivo and in vitro H. pylori-inhibiting experiments have verified that the wheat peptide mixture, wheat peptide M, and wheat peptide Ml all have the activity of inhibiting H. pylori and enable the repair of gastrointestinal mucosa, thus achieving the therapeutic effect of gastrointestinal diseases. DRAWINGS - Q 4G P G -PG- - 2 3 4 44 4 44 4 4410 10 10 12 14 140 Y. FIG.

Description

DRAWINGS - Q PG 4G -PG- -

2 3 4 44 4 10 44 4 4410 10 12 14 140 Y.

FIG. ACTIVE PEPTIDE CAPABLE OF INHIBITING HELICOBACTER PYLORI(H. PYLORI), AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD The present disclosure belongs to the technical field of active peptides, and particularly relates to an active peptide capable of inhibiting Helicobacter pylori (H. pylori), and a preparation method and use thereof.

BACKGROUND Helicobacter pylori (H. pylori) is a gram-negative bacterium that grows on the gastric mucous layer, which is a pathogenic factor for common diseases of the digestive system such as gastritis and gastric ulcer. The World Health Organization (WHO) has listed H. pylori as a Class I carcinogen. Diseases of the digestive system caused by H. pylori have become a major global public health problem. At present, two of metronidazole, clarithromycin, amoxicillin, and tetracycline are commonly used to eradicate H. pylori. Currently, the antibiotic treatment can achieve prominent therapeutic effects in a short term, but exhibits side effects. Moreover, long-term use of antibiotics will not only cause drug resistance, but also result in physical discomfort such as nausea and vomit, which will further stimulate the stomach.

SUMMARY In view of this, the present disclosure aims provide a novel active peptide capable of inhibiting H. pylori, and a preparation method and use thereof. The active peptide is isolated from wheat, which is safe and can effectively inhibit H. pylori, without side effects. The present disclosure provides a wheat peptide mixture capable of inhibiting Helicobacterpylori (H. pylori), where, a preparation method of the wheat peptide mixture includes the following steps: 1) grinding and homogenizing a vital wheat gluten solution to obtain a stable homogenate; 2) stirring the homogenate at 50°C to 60°C for 1 h to 2 h to obtain a pretreated homogenate; 3) subjecting the pretreated homogenate to stepwise enzymolysis with papain and neutral protease successively and to enzymatic inactivation to obtain an enzymatic hydrolysate; and 4) filtrating the enzymatic hydrolysate through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and collecting a resulting filtrate to obtain the wheat peptide mixture.

Preferably, in step 3), the papain has a mass concentration of 1.0% to 2.0% in the pretreated homogenate, and enzymolysis with the papain is conducted for 5 h to 7 h; before the papain is added, pH of the pretreated homogenate is adjusted to neutral; the neutral protease has a mass concentration of 0.3% to 1.0% in the pretreated homogenate, and enzymolysis with the neutral protease is conducted for 3 h to 5 h; before the enzymolysis with the neutral protease is conducted, pH of a resulting system is adjusted to 6.9 to 7.2; and in step 1), particles in the homogenate have a particle size of 50 m to 150 [m. The present disclosure further provides a wheat peptide M capable of inhibiting H. pylori, where, the wheat peptide M is obtained by subjecting the above wheat peptide mixture to high performance liquid chromatography (HPLC) and collecting a component with peak time of 8 min to 9 min. Preferably, parameters for the HPLC are as follows: a mixed solution of mobile phase A and mobile phase B used for the following gradient elution: from 0 min to 5 min, a volume percentage of mobile phase B gradiently increasing from % to 5%; from 5 min to 31 min, the volume percentage of mobile phase B gradiently increasing from 5% to 7 8 %; from 31 min to 33 min, the volume percentage of mobile phase B remaining at 78%; and from 33 min to 36 min, the volume percentage of mobile phase B gradiently decreasing from 78% to 5%; where, the mobile phase A is a mixture of trifluoroacetic acid (TFA) and ultrapure water (UPW), with a TFA-UPW volume ratio of 0.1:99.9; and the mobile phase B is a mixture of TFA and acetonitrile, with a TFA-acetonitrile volume ratio of 0.1:99.9; detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21°C to 0 C. The present disclosure further provides an active peptide capable of inhibiting H. pylori, including a wheat peptide M1 or a salt form of the wheat peptide M1; and the wheat peptide M1 has an amino acid sequence shown in SEQ ID No: 1. The present disclosure further provides a method for preparing the above active peptide capable of inhibiting H. pylori, where, the active peptide is obtained by subjecting the above wheat peptide M to chromatographic analysis and identification using a liquid chromatography-Q EXACTIVE mass spectrometry system. Preferably, conditions of the chromatographic analysis are as follows: mobile phase: phase A: a mixture of formic acid and purified water, with a purified water- formic acid volume ratio of 100:0.1; phase B: a mixture of acetonitrile and formic acid, with an acetonitrile-formic acid volume ratio of 100:0.1; flow rate of the mobile phase: 300 nl/min; injection volume: IpL supernatant; gradient procedure of the mobile phase as follows:

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0 Volume percentage of phase A 97% 97% 63% 10% 10% 97% 97% Volume percentage of phase B 3% 3% 37% 90% 90% 3% 3%

The present disclosure further provides a drug capable of inhibiting H. pylori, including the above active peptide, the above wheat peptide mixture, or the above wheat peptide M. The present disclosure further provides use of the above active peptide, the above wheat peptide mixture, or the above wheat peptide M in the preparation of a drug for inhibiting H. pylori. The present disclosure further provides use of the above active peptide, the above wheat peptide mixture, or the above wheat peptide M in the preparation of a drug for treating gastrointestinal diseases. The active peptide capable of inhibiting H. pylori provided in the present disclosure includes a wheat peptide M1 or a salt form of the wheat peptide M1; and the wheat peptide M1 has an amino acid sequence shown in SEQ ID No: 1. The wheat peptide MI exhibits an excellent effect on repairing damaged gastric mucosa. The present disclosure screens the wheat peptide M1 with the effect of inhibiting H. pylori through in vitro experiments, and further verifies the inhibitory effect of the wheat peptide M1 on H. pylori through H. pylori-infected zebrafish models. The inhibitory effect of the wheat peptide M1 and wheat peptide mixture on H. pylori and the repair effect of the wheat peptide M1 and wheat peptide mixture on gastric mucosa are evaluated, and results show that, compared with the H. pylori-infected model, the wheat peptide M1 and wheat peptide mixture show a significant inhibitory effect on H.pylori infection; and compared with the H. pylori-infected zebrafish model group, the wheat peptide M1 significantly promotes the regression of colitis inflammation. It can be seen that the wheat peptide M1 can not only inhibit H. pylori, but also eliminate gastrointestinal inflammation caused by H. pylori infection, thereby achieving the purpose of treating gastrointestinal diseases caused by H. pylori infection. The wheat peptide M capable of inhibiting H. pylori provided in the present disclosure is obtained by subjecting the wheat peptide mixture to high-performance liquid chromatography (HPLC) and collecting a component with peak time of 8 min to 9 min. In vitro H. pylori inhibiting experiments show that, compared with other components isolated from the wheat peptide mixture, the wheat peptide M shows the strongest inhibitory effect on H. pylori, with an inhibitory zone diameter (IZD) reaching 36 0.56 mm. It indicates that the wheat peptide M has a strong anti-H. pylori activity.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a mass spectrum of the wheat peptide M1 provided in the present disclosure; FIG. 2 is a phenotype diagram of the H. pylori intestinal infection in zebrafish; FIG. 3 shows the inhibitory effect of wheat peptide M1 at different concentrations on H. pylori (H. pylori fluorescence intensity), where, compared with the model control group, *p < 0.05, ***p < 0.001; FIG. 4 shows the inhibitory effect of wheat peptide M1 at different concentrations on H. pylori, where, compared with the model control group, **p < 0.01, ***p < 0.001; FIG. 5 is a phenotype diagram of the promoting effect of wheat peptide M1 on the regression of colitis inflammation; FIG. 6 shows the promoting effect of wheat peptide M1 at different concentrations on the regression of colitis inflammation (the number of neutrophils), where, compared with the model control group, *p < 0.05, ***p < 0.001; FIG. 7 shows the promoting effect of wheat peptide MI at different concentrations on the regression of colitis inflammation, where, compared with the model control group, *p < 0.05, ***p < 0 .0 0 1 .

DETAILED DESCRIPTION The present disclosure provides an active peptide capable of inhibiting H. pylori, including a wheat peptide M1; and the wheat peptide M1 has an amino acid sequence shown in SEQ ID No: 1 (GQQPGQGQQPGQGQPGYYPT), which is composed of 20 amino acid residues and has a molecular weight of 2071.93 Da. The active peptide may also include a salt form of the wheat peptide M1, such as an acetate form and a hydrochloride form. The present disclosure has no specific limitation on a source of the active peptide, which can be obtained by artificial synthesis or a separation and purification method. In the present disclosure, the wheat peptide Ml used in the animal experiment is entrusted to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis.

The present disclosure provides a method for preparing the active peptide capable of inhibiting H. pylori, including the following steps: 1) grinding and homogenizing a vital wheat gluten solution to obtain a stable homogenate; 2) stirring the homogenate at 50°C to 60°C for 1 h to 2 h to obtain a pretreated homogenate; 3) subjecting the pretreated homogenate to enzymolysis with papain and neutral protease successively and to enzymatic inactivation to obtain an enzymatic hydrolysate; 4) filtrating the enzymatic hydrolysate through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and collecting a resulting filtrate to obtain the wheat peptide mixture; 5) subjecting the wheat peptide mixture to HPLC and collecting a component capable of inhibiting H. pylori with peak time of 8 min to 9 min; and 6) subjecting the component capable of inhibiting H. pylori to chromatographic analysis using a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain a wheat peptide M1 or a salt form of the wheat peptide M1. In the present disclosure, a vital wheat gluten solution is ground and homogenized to obtain a stable homogenate. In the present disclosure, the vital wheat gluten solution may have a concentration preferably of 10% to 30%, more preferably of 15% to 25%, and most preferably of 20%. The present disclosure has no specific limitation on a preparation method of the vital wheat gluten solution, and a preparation method for a protein solution well known in the art may be adopted, such as heating a solution at a temperature preferably of 70°C to 80°C and more preferably of 0 C. In the present disclosure, the grinding may preferably be conducted with a colloidal mill. The grinding with a colloidal mill may be conducted preferably for 2 h to 3 h and more preferably for 2.5 h. The homogenization may preferably be conducted at a pressure of 25 MPa for 2 h. After the homogenization, particles in the homogenate may have a particle size preferably of 50 m to 150 m, more preferably of 80 m to 120 [m, and most preferably of 100 [m. The prepared homogenate has a fine texture and is not separated into layers, which is beneficial to the stability of the homogenate, thereby increasing the content of wheat peptide M1. In the present disclosure, after the homogenate is obtained, the homogenate is stirred at 0C to 60 0C for 1 h to 1.5 h to obtain a pretreated homogenate. In the present disclosure, the temperature may preferably be kept at 550 C. In the present disclosure, the temperature may preferably be kept for 1.2 h. A too-high temperature may cause denaturation of protein particles in the homogenate, and a too-low temperature is not conducive to the full dissolution of materials. The temperature-keeping treatment of the homogenate is beneficial to improve the solubility of materials and facilitate the enzymolysis process. In the present disclosure, after a pretreated homogenate is obtained, the pretreated homogenate is subjected to enzymolysis with papain and neutral protease successively and then to enzymatic inactivation to obtain an enzymatic hydrolysate. In the present disclosure, the papain in the pretreated homogenate may have a mass concentration preferably of 1% to 2% and more preferably of 1.2%.The enzymolysis with the papain may be conducted preferably for 3 h to 4 h and more preferably for 3.5 h. With strong enzyme cleavage characteristics, the papain can enzymatically destroy the spatial structure of the protein in vital wheat gluten to expose peptide chains and thus enzymatically cleave the chains into large fragment polypeptides at a specific site. Before the papain is added, pH of the pretreated homogenate may be adjusted preferably to neutral and more preferably to 7.2.A solution for adjusting the pH of the pretreated homogenate is not specifically limited, and a reagent for adjusting pH well known in the art may be used, such as sodium hydroxide and hydrochloric acid. After the enzymolysis with papain is conducted, enzymolysis with neutral protease is conducted. Before the enzymolysis with neutral protease is conducted, pH of a resulting system may be adjusted preferably to 6.9 to 7.2 and more preferably to 7.1. The neutral protease in the pretreated homogenate may have a mass concentration preferably of 0.3% to 1% and more preferably of 0.5%; and the enzymolysis with neutral protease may be conducted preferably for 3 h to 5 h and more preferably for 4 h. On the basis of the enzymolysis with papain, the enzymolysis with neutral protease is conducted to enzymatically cleave obtained large fragment polypeptides at a specific site, where, an amino acid sequence of the active peptide will not be cut during this enzymatic cleavage process, and the active peptide is obtained from small fragments resulting from the enzymatic cleavage with neutral protease. During the enzymolysis process, the present disclosure tries to use other kinds of proteases alone or in combination to enzymatically cleave vital wheat gluten particles, but a wheat peptide M1 cannot be obtained or an obtained wheat peptide M1 shows a poor effect. The present disclosure has no specific limitation on a method for the enzymatic inactivation, and a scheme for enzymatic inactivation well known in the art may be adopted, such as high temperature enzymatic inactivation. In the present disclosure, after an enzymatic hydrolysate is obtained, the enzymatic hydrolysate is filtered through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and a resulting filtrate is collected to obtain the wheat peptide mixture. In the present disclosure, after the enzymatic hydrolysate is filtered through the 30,000 Da ceramic membrane, a resulting filtrate is collected. The filtrate is filtered through the1 KDa organic membrane. The present disclosure has no specific limitation on sources of the ceramic membrane and the organic membrane, and sources of the ceramic membrane and the organic membrane well known in the art may be adopted. In an example of the present disclosure, the ceramic membrane and the organic membrane are purchased from Jiangsu Jiuwu Hi-Tech Co., Ltd. The filtration with the ceramic membrane and organic membrane is conducted to remove large fragment polypeptides in the enzymatic hydrolysate and collect polypeptides with a small molecular weight below 1 KDa. Pigments may preferably be removed before the enzymatic hydrolysate is filtered through the 30,000 Da ceramic membrane. The method for removing pigments may preferably be conducted using activated carbon. The activated carbon may be added at a mass preferably accounting for 4% of the total mass of the total system. After a filtrate is collected, the filtrate may preferably be dried. The present disclosure has no specific limitation on a method for the drying, and a drying method for protein or polypeptide well known in the art may be adopted, such as lyophilizing or spray drying. Evaluation experiments of the inhibitory effect of the wheat peptide mixture on H. pylori and the repair effect of the wheat peptide mixture on gastric mucosa show that, compared with the H. pylori-infected model, the wheat peptide mixture shows a significant inhibitory effect on H. pylori infection. In the present disclosure, after a wheat peptide mixture is obtained, the wheat peptide mixture is subjected to HPLC, and a component with peak time of 8 min to 9 min is collected. In the present disclosure, parameters for the HPLC are as follows: a mixed solution of mobile phase A and mobile phase B used for the following gradient elution: from 0 min to 5 min, a volume percentage of mobile phase B gradiently increasing from % to 5%; from 5 min to 31 min, the volume percentage of mobile phase B gradiently increasing from 5% to 7 8 %; from 31 min to 33 min, the volume percentage of mobile phase B remaining at 78%; and from 33 min to 36 min, the volume percentage of mobile phase B gradiently decreasing from 78% to 5%; where, the mobile phase A is a mixture of trifluoroacetic acid (TFA) and ultrapure water (UPW), with a TFA-UPW volume ratio of 0.1:99.9; and the mobile phase B is a mixture of TFA and acetonitrile, with a TFA-acetonitrile volume ratio of 0.1:99.9; detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21°C to 25°C. The HPLC can exclude polypeptides of less than 1 KDa collected in the present disclosure in the order of time, which is beneficial to the fine separation of polypeptides of various molecular weights. In the separation of wheat peptides by HPLC, a collection tube is changed every 1 min to obtain 36 components. In vitro H. pylori-inhibiting experiments show that, compared with other 35 components isolated from the wheat peptide mixture, the wheat peptide M shows the strongest inhibitory effect on H. pylori, indicating an excellent H. pylori-inhibiting activity. In the present disclosure, after a component with peak time of 8 min to 9 min, the component with peak time of 8 min to 9 min is subjected to chromatographic analysis using a liquid chromatography-Q EXACTIVE mass spectrometry system to obtain an active peptide, namely, a wheat peptide M1. In the present disclosure, the liquid chromatography-Q EXACTIVE mass spectrometry system may preferably be a nano-liquid chromatography-Q EXACTIVE mass spectrometry system. Conditions of the chromatographic analysis may preferably as follows: mobile phase: phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100:0.1; phase B: a mixture of acetonitrile and formic acid, with an acetonitrile-formic acid volume ratio of 100:0.1; flow rate of the mobile phase: 300 nl/min; injection volume: 1IpL supernatant; gradient procedure of the mobile phase:

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

A (volume percentage) 97% 97% 63% 10% 10% 97% 97%

B (volume percentage) 3% 3% 37% 90% 90% 3% 3%

It is determined by the liquid chromatography-mass spectrometry technique that the wheat peptide M1 accounts for 52% of the total mass of component 9, and it is determined by the liquid chromatography-mass spectrometry technique that a mass percentage of the single-chain polypeptide in the wheat peptide mixture is 0.92%. The present disclosure provides a drug capable of inhibiting H. pylori, including the active peptide, the wheat peptide mixture, or the wheat peptide M. Given that the active peptide, the wheat peptide mixture, or the wheat peptide M has an effect of inhibiting H. pylori in vitro, the present disclosure provides use of the active peptide, the wheat peptide mixture, or the wheat peptide M in the preparation of a drug for inhibiting H. pylori. Since H. pylori infection is likely to cause diseases of the digestive system such as the gastrointestinal tract, the present disclosure provides use of the active peptide, the wheat peptide mixture, or the wheat peptide M in the preparation of a drug for treating gastrointestinal diseases. The gastrointestinal diseases include gastritis, enteritis, gastric ulcer, and other gastrointestinal diseases caused by H. pylori. The present disclosure has no specific limitation on a dosage form and a preparation method of the drug, and a dosage form and a preparation method of drugs well known in the art may be adopted. The drug may be administered at a dosage preferably making a concentration of the wheat peptide M1 not less than 33.3 [g/mL. The active peptide capable of inhibiting H. pylori, and a preparation method and use thereof provided in the present disclosure will be described in detail below with reference to examples, but these examples cannot be understood as limiting the claimed scope of the present disclosure.

Example 1 Preparation methods of a wheat peptide mixture and a wheat peptide M1 Step 1. 1,000 kg of vital wheat gluten was taken for preparing a slurry, 5,000 L of deionized water was added, and a resulting mixture was heated to 80°C and stirred for fully dissolving the vital wheat gluten to obtain a vital wheat gluten solution with a concentration of 20%. Step 2. The vital wheat gluten solution in step 1 was thoroughly stirred, treated with a colloidal mill for 2.5 h, and then treated with a high-pressure homogenizer at a pressure of 25 MPa for 2 h to obtain a homogenate with a particle size of 100 m, ensuring that the homogenate had a fine texture and was not separated into layers, thus improving the stability of the homogenate. Step 3. The homogenate obtained from high-pressure homogenization was fed, heated to °C under stirring, and kept at the temperature for 1 h. Step 4. pH of the vital wheat gluten solution in step 3 was adjusted to 7.5 with a 0.5 M NaOH solution, and the temperature was kept constant. Step 5. 1.2% papain (12.0 Kg) was added to the pH-adjusted vital wheat gluten solution in step 4, and enzymolysis was conducted for 6 h; the pH was adjusted to about 7.0 with a 0.1 M HCl solution; and 0.5% neutral protease (5.0 kg) was then added, and enzymolysis was further conducted for 4 h. Step 6. An enzymatic hydrolysate in step 5 was heated to 110°C and kept at the temperature for 5 min to inactivate the proteases. Step 7. An enzyme-inactivated solution in step 6 was cooled to 55°C, 4% of activated carbon (40 Kg) was added, and a resulting mixture was kept at the temperature for 1 h; the mixture was filtered through a 30,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was collected and spray dried (inlet temperature: 170°C; and outlet temperature: 100°C) to obtain a wheat peptide mixture. Determination indexes of the basic physical and chemical properties of the wheat peptide mixture: protein content determination (GB5009.9), moisture content determination (GB5009.3), and ash content determination (GB5009.4). Results are shown in Table 1. Table 1 Basic physical and chemical properties of the wheat peptide mixture

Item Protein content () Moisture content ()Ash content (%)

Casein peptide mixture 95 1.5 3.5

Example 2 A separation and purification method of a wheat peptide The preparative HPLC was used to separate and purify a wheat peptide from the wheat peptide mixture: (1) Mobile phase A: 0.1% TFA + 99.9% UPW; mobile phase B: 0.1% TFA + 99.9% acetonitrile; (2) Detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21°C to 25°C. (3) Equilibration was conducted using 6 column volumes of mobile phase A at a speed of mL/min until a baseline was stable. (4) The obtained wheat peptide mixture was dissolved with mobile phase A to obtain a wheat peptide mixture solution with a concentration of 10 mg/mL; the wheat peptide mixture solution was centrifuged at 10,000 r/min for 5 min; and a resulting supernatant was filtered with a 0.22 m filter membrane and injected at a volume of 15 mL. (5) A mixed solution of mobile phase A and mobile phase B was used for the following gradient elution: from 0 min to 5 min, a concentration of mobile phase B gradiently increasing from 0% to %; from 5 min to 31 min, the concentration of mobile phase B gradiently increasing from 5% to 78%; from 31 min to 33 min, the concentration of mobile phase B remaining at 78%; and from 33 min to 36 min, the concentration of mobile phase B gradiently decreasing from 78% to 5%. (6) A component was collected every 1 min for activity assay.

36 components were collected. The peak time of the 36 components is shown in Table 2. Table 2 Peak time of 36 components

Component Peak time Component Peak time Component Peak time

1 0 min to 1 min 13 12 min to 13 min 25 24 min to 25 min

2 1 min to 2 min 14 13 min to 14 min 26 25 min to 26 min

3 2 min to 3 min 15 14 min to 15 min 27 26 min to 27 min

4 3 min to 4 min 16 15 min to 16 min 28 27 min to 28 min

5 4 min to 5 min 17 16 min to 17 min 29 28 min to 29 min

6 5 min to 6 min 18 17 min to 18 min 30 29 min to 30 min

7 6 min to 7 min 19 18 min to 19 min 31 30 min to 31 min

8 7 min to 8 min 20 19 min to 20 min 32 31 min to 32 min

9 8 min to 9 min 21 20 min to 21 min 33 32 min to 33 min

10 9 min to 10 min 22 21 min to 22 min 34 33 min to 34 min

11 10 min to 11 min 23 22 min to 23 min 35 34 min to 35 min

12 11 min to 12 min 24 23 min to 24 min 36 35 min to 36 min

Example 3 In vitro H. pylori-inhibiting experiments of the 36 components At present, there is still a lack of effective drugs for treating this disease. Therefore, screening of candidate compounds capable of inhibiting H. pylori is of great significance for developing drugs for treating gastrointestinal diseases such as gastritis. With the advantages of high efficiency, non-toxicity, high temperature resistance, no residue, no drug resistance, etc., polypeptides are a kind of materials with great potential to resist H. pylori. So far, there has been no report on the isolation of peptides with anti-H. pylori activity from wheat peptides. Step 1. Cultivation of H. pylori: A brain heart infusion (BHI) medium was used for H. pylori cultivation. An H. pylori solution was picked using an L-shaped glass rod (after sterilization) and intensively streaked on a plate. The medium inoculated with H. pylori was continuously cultivated in a microaerobic incubator for 72 h. In the incubator, nitrogen, carbon dioxide, and oxygen were filled in a volume percentage ratio of 8 5 %:10 % :5% , a petri dish filled with sterile water was placed to ensure a humidity required for the growth of H. pylori (above 90%), and a temperature was controlled at 37°C. The bacterial solution could be used for passage. Step 2. Preparation of an indicator bacterium plate: A loopful of H. pylori cultivated in step 1 was picked up with an inoculation loop and inoculated in 10 mL of sterile water, and a resulting mixture was shaken with a shaker to obtain a 1 x 106 bacterial suspension. 1 mL of the bacterial suspension was inoculated in an H. pylori-selective medium added with defiberized sheep blood and a bacteriostatic agent (sodium azide) (the medium had a temperature of about 37°C), a resulting medium with indicator bacteria was thoroughly shaken and then quickly poured into a sterile petri dish with a diameter of 9 cm, and an indicator bacterium plate was obtained after the medium was solidified. Step 3. Antibacterial activity of different separated wheat peptide components: Different separated components were prepared into 10 mg/mL solutions, separately. The agar diffusion method was used to measure the activity of different separated wheat peptide components. A pipette tip (after sterilization) was used to punch holes in the center of the indicator plate, and a pipette was used to pipette different wheat peptide component solutions, separately. The above 10 mg/mL sample solution was added to a sample hole on the indicator bacterium plate, with 20 1 per hole. This plate was placed in an anaerobic tank, then a microaerobic gas generating bag (5% 02, 10% C02, and 85% N 2 ) was placed in the anaerobic tank, and finally a small petri dish with sterile water was placed in the anaerobic tank to ensure a humidity required for the growth of H. pylori. The anaerobic tank was closed and incubated in a constant temperature incubator at 37°C for 2 d to 3 d. The antibacterial activity was determined by observing the size of inhibitory zones. Each set of data was repeated 3 times. Results are shown in Table 3 and Table 4.

Table 3 Inhibitory effect of different wheat peptide components on H. pylori

Component Antibacterial Component Antibacterial Component Antibacterial effect effect effect 1 -- 13 ++ 25 ++

2 -- 14 ++ 26 -

3 -- 15 27

4 -- 16 28 -

5 -- 17 ++ 29

6 18 ++ 30 --

7 19 ++ 31

8 ++ 20 32

9 ++ 21 ++ 33 -

10 ++ 22 34 -

11 23 ++ 35 -

12 ++ 24 36 -

Notes: ++ means that the IZD is greater than 20 mm, - means that the IZD is 12 mm to mm, and -- means that the IZD is less than 12 mm.

Table 4 Determination of anti-H. pylori activity of different wheat peptide components

Component 8 9 10 12 13 14

IZD (mm) 25 0.36 36 0.56 23 0.21 26 0.46 26 0.20 22 0.12

Component 17 18 19 21 23 25

IZD (mm) 20 0.13 21 0.12 24 0.31 27 0.39 23 0.32 22 0.12

Results showed that the component 9 had the strongest antibacterial activity. The component 9 was defined as a wheat peptide M, and the purity and structure were determined for the wheat peptide M.

Example 4 Identification of the purity and structure of the wheat peptide M The purity and structure of the wheat peptide M obtained in Example 3 were identified by the nano-liquid chromatography-Q EXACTIVE mass spectrometry system. 1. Chromatographic conditions: (1) mobile phase: phase A: 100% purified water + 0.1% formic acid; phase B: 100% acetonitrile + 0.1% formic acid; (2) flow rate of the mobile phase: 300 nL/min; (3) injection volume: 1IpL supernatant; and (4) gradient procedure of the mobile phase as shown in Table 5.

Table 5 Gradient procedure of the mobile phase

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

A (%) 97 97 63 10 10 97 97

B (%) 3 3 37 90 90 3 3

The nano-liquid chromatography-Q EXACTIVE mass spectrometry system was used to purify a polypeptide from the wheat peptide M, and a structure thereof was identified as GQQPGQGQQPGQGQPGYYPT (Gly--Gln--Gln-Pro-Gly--Gln-Gly--Gln--Gln-Pro-Gly- GlnGly--Gln-Pro-Gly-Tyr-Tyr-Pro-Thr), which was a single-chain polypeptide of 2071.93 Da, as shown in FIG. 1. It was determined by the liquid chromatography-mass spectrometry technique that the single-chain polypeptide accounted for 52% of the total mass of the wheat peptide M, and it was determined by the liquid chromatography-mass spectrometry technique that a mass percentage of the single-chain polypeptide in the wheat peptide mixture was 0.92%. Therefore, the single-chain polypeptide was named wheat peptide M1.

Example 5 Evaluation of the inhibitory effect of the wheat peptide M1 on H. pylori and the repair of the wheat peptide M1 on gastric mucosa 1. Concentration groups Experimental group 1 normal control group (no treatment) Experimental group 2 model control group Experimental group 3 positive control drug of metronidazole 15 pg/mL Experimental group 4 wheat peptide mixture 55 pg/mL Experimental group 5 wheat peptide M1 11.1 pg/mL Experimental group 6 wheat peptide M1 33.3 pg/mL Experimental group 7 wheat peptide M1 100 pg/mL 2. Model establishment Fluorescently-labeled H. pylori (prepared by Hunter Biotechnology Co., Ltd.) with a concentration of 106 CFU/mL was added into fish farming water, and zebrafish were cultivated in the water for 48 h to 72 h. The H. pylori, after swallowed orally by the zebrafish, was colonized in the gastrointestinal tract of the zebrafish. A test sample was administered by water soluble administration. The antibacterial effect of a test sample was determined by observing the fluorescence intensity of intestinal H. pylori. 3. Basis for concentration determination According to results of the concentration exploration experiment in experiment 4, the maximum tolerated concentration (MTC)of the wheat peptide M1 was 100 pg/mL, and pharmacodynamic evaluation concentrations were set to 11.1 pg/mL, 33.3 pg/mL, and 100 pg/mL. 4. Experimental method (1) Basis for concentration determination 360 zebrafish (3 dpf wild-type AB strain) were randomly selected and added to a 6-well plate, with 30 per well. Each well had a volume of 3 mL. Fluorescently-labeled H. pylori was added into the fish farming water to induce H. pylori-infected zebrafish models. The wheat peptide M1 was administered by water-soluble administration at concentrations of 10 g/mL, pg/mL, 100 [g/mL, 200 [g/mL, and 400 [g/mL; the wheat peptide mixture was administered by water-soluble administration at concentrations of 10 g/mL, 50 [g/mL, 100

[tg/mL, 200 [g/mL, and 400 [g/mL; and a normal control group (zebrafish were treated with the fish farming water) and a model control group were set. The zebrafish were observed and dead zebrafish were removed every day. The death conditions and the death number of zebrafish were counted, and the MTC of zebrafish was determined based on the toxicity and death of zebrafish. (2) Index detection Zebrafish (3 dpf wild-type AB strain) were randomly selected and added to a 6-well plate, with 30 per well. Fluorescently-labeled H. pylori was added into the fish farming water to induce H. pylori-infected zebrafish models. The wheat peptide M1 (hereinafter referred to as "1#") was administered by water-soluble administration at concentrations 11.1 [g/mL, 33.3

[tg/mL, and 100 [g/mL; the wheat peptide mixture was administered by water-soluble administration at a concentration of 55 pg/mL, the positive control drug of metronidazole was administered by water-soluble administration at a concentration of 15 pg/mL; and a normal control group and a model control group were set. Each well (concentration group) had 30 zebrafish and a volume of 3 mL. After the zebrafish were incubated in an incubator for 120 h at 35°C, the fluorescence intensity of gastrointestinal H. pylori was acquired under a fluorescence microscope for the zebrafish of each group, and the statistical analysis of fluorescence intensity was adopted to evaluate the inhibitory effect of a sample on H. pylori infection in zebrafish. The inhibitory effects of the wheat peptide mixture, wheat peptide M1, and metronidazole on H. pylori infection in zebrafish were calculated according to the following formula I:

Inhibitory effect(%) = S(model control group) - S(test sample group) X 100% S(model control group) formula I.

Since H. pylori infection in zebrafish would cause gastrointestinal inflammation, after the cultivation was completed, pictures were taken and data were acquired to analyze and count the number of neutrophils (N) in the intestine of zebrafish. The statistical significance of the number of neutrophils (N) in the intestine was used to evaluate the regression effect of a sample on the gastrointestinal inflammation in zebrafish, which was calculated according to the following formula II:

Inflammationregressioneffect(0%)= (N(model control group) - N(test sample group) N(model control group) 100% formula II. Statistical analysis was conducted using analysis of variance (ANOVA) and Dunnett's T test. p < 0.05 indicates a significant difference. 5. Experimental results Results showed that the wheat peptide M1 caused no adverse reactions to H. pylori induced colitis zebrafish models at a concentration of 100 pg/mL, but resulted in zebrafish death at a concentration higher than 100 pg/mL, with a mortality of 100% at a concentration of 400 pg/mL (30/30) and a mortality of 40% at a concentration of 200 pg/mL. Therefore, the MTC of the wheat peptide M1 for H. pylori-induced colitis zebrafish models was 100 pg /mL. Zebrafish death occurred when the wheat peptide mixture was at a concentration higher than 100 pg/mL. Therefore, a concentration higher than 50 pg/mL but lower than 100 pg/mL was selected as a drug concentration for H. pylori-induced colitis zebrafish models. Details can be seen in Table 6.

Table 6 Exploration results of MTC

Concentration (pg/mL) Death count Mortality (%) 10 0 0 50 0 0 WheatpeptideMl 100 0 0 200 12 40 400 30 100 Concentration ( g/mL) Death count Mortality (%) 10 0 0 50 0 0 Wheat peptide 100 5 16.7 mixture 200 18 60 400 30 100

The results showed that the gastrointestinal fluorescence intensity of zebrafish in the H. pylori-infected model control group (977,955 pixels) was significantly higher than that of the normal control group (132,327 pixels), suggesting that the H. pylori-infected zebrafish models were successfully constructed (p < 0.001). The gastrointestinal fluorescence intensity in zebrafish administered with the positive control drug of metronidazole (413,117 pixels) was significantly weaker than that of the model control group (977,955 pixels) (p < 0.05), with an inhibitory effect on H. pylori of 57.8%, indicating that metronidazole exhibited a significant inhibitory effect on H. pylori infection. The gastrointestinal fluorescence intensities in zebrafish administered with the wheat peptide M1 at concentrations of 11.1 [g/mL, 33.3 [g/mL, and 100 g/mL were 729,757 pixels, 533,368 pixels, and 433,256 pixels, respectively, all withp < 0.01 as compared with the model control group; and the inhibitory effects on H. pylori infection were 25.4%, 45.5%, and 55. 7

% respectively. The gastrointestinal fluorescence intensity in zebrafish administered with the wheat peptide mixture at a concentration of 55 g/mL was 732,654 pixels, with p < 0.01 as compared with the model control group; and the inhibitory effect on H. pylori infection was 45.5%. It showed that the wheat peptide M1 and the wheat peptide mixture both exhibited a significant inhibitory effect on H. pylori infection. Details can be seen in Table 7, FIG. 2, FIG. 3, and FIG. 4.

Table 7 Evaluation of the inhibitory effect of test samples on H. pylori (n = 30)

Concentration Fluorescence intensity of H. Inhibitory Group ([g/mL) pylori (pixel, mean SE) effect (%) Normal control 132327 6305 group Model control - 977955 16581 group Metronidazole 15 413117 15297*** 57.8*** Wheat peptide 55 732654 14363* 25.1* mixture 11.1 729757 12655* 25.4*** Wheat peptide 33.3 533368 11361*** 45.5*** Ml 100 433256 10133*** 55.7***

Note: as compared with the model control group, *p < 0.05, and ***p < 0.001.

The number of neutrophils in the intestine of zebrafish in the model control group (9) showed p < 0.001 as compared with that of the normal control group (3), indicating that the model was successfully established. The number of neutrophils in the group administered with the positive control drug of metronidazole (5) showed p < 0.001 as compared with that of the model control group, with an inflammation regression effect of 44.4%. The number of neutrophils (6) in the intestine of zebrafish administered with the wheat peptide mixture at a concentration of 55 pg/mL showed p < 0.05 as compared with the model control group, with an inflammation regression effect of 33.3%. The numbers of neutrophils in the intestine of zebrafish administered with the wheat peptide M1 at concentrations of 11.1 pg/mL, 33.3 pg/mL, and 100 pg/mL were 5, 4, and 3, respectively, showing p < 0.001 as compared with the model control group, with inflammation regression effects of 44.4%, 55.6%, and 66.7%, respectively. It was suggested that "1#" exhibited a significant inflammation regression effect at the experimental concentration. Details can be seen in Table 8, FIG. 5, FIG. 6, and FIG. 7.

Table 8 Quantitative results of the promotion of test samples on the regression of colitis inflammation (n = 30)

Concentration Number of Inflammation regression Group (pg/mL) neutrophils(mean effect (%) SE) Normal control 3 ±0.4 group Model control - 9 0.5 group Metronidazole 15 5 0.3*** 44.4*** Wheat peptide 55 6 0.5* 33.3* mixture 11.1 5 0.5*** 44.4*** Wheat peptide M1 33.3 4 0.6*** 55.6*** 100 3 0.3*** 66.7*** Note: as compared with the model control group, *p < 0.05, and ***p < 0.001.

Comparative Example 1 1. Optimization of single-enzyme hydrolysis conditions 1,000 kg of vital wheat gluten was taken for preparing a slurry, and 5,000 L of deionized water was added to obtain a vital wheat gluten solution with a concentration of 20%, which was heated to 80°C for fully dissolving the vital wheat gluten. The vital wheat gluten solution was thoroughly stirred, treated with a colloidal mill for 2.5 h, and then treated with a high pressure homogenizer at a pressure of 25 MPa for 2 h, ensuring that the homogenate had a fine texture and was not separated into layers, thus improving the stability of the homogenate. The homogenate obtained from high-pressure homogenization was fed, stirred, heated to 55°C, and kept at the temperature for 1 h. Then a weighed protease (see Table 9) was added, and enzymolysis was conducted for 6 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis, see Table 9); and a resulting enzymatic hydrolysate was heated to 110°C and kept at the temperature for 5 min to inactivate the protease. An enzyme-inactivated solution was cooled to 55°C, 4% of activated carbon (40 kg) was added, and a resulting mixture was kept at the temperature for 1 h; the mixture was filtered through a 30,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined.

Table 9 List of enzymolysis conditions for different types of proteases

Optimal Optimal Protein Added Protease enzymolysis enzymolysis concentration amount pH type temperature time (%) (%)

(0C) (h)

Alkaline 20 1.2 8.5 55 6 protease

Papain 20 1.2 7.5 55 6

Neutral 20 1.2 7.0 55 6 protease

Trypsin 20 1.2 6.9 37 6

1.1 Enzymolysis results of alkaline protease A 20% vital wheat gluten solution was prepared and heated to 550 C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 8.5 (the optimal pH of the corresponding protease); a resulting solution was incubated for 1 h; then an alkaline protease was added, and enzymolysis was conducted for 6 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); an enzyme-inactivated solution was cooled to 55°C, and 4% of activated carbon (40 kg) was added; a resulting solution was kept at the temperature for 1 h, and then filtered through a 30,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected as 16 mm; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined as 0.16%. 1.2 Enzymolysis results of papain A 20% vital wheat gluten solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.5 (the optimal pH of the corresponding protease); a resulting solution was incubated for 1 h; then a papain was added, and enzymolysis was conducted for 6 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); an enzyme-inactivated solution was cooled to 55°C, and 4% of activated carbon (40 kg) was added; a resulting solution was kept at the temperature for 1 h, and then filtered through a ,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected as 29 mm; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined as 0.6%. 1.3 Enzymolysis results of neutral protease A 20 % vital wheat gluten solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.0 (the optimal pH of the corresponding protease); a resulting solution was incubated for 1 h; then a neutral protease was added, and enzymolysis was conducted for 6 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); an enzyme-inactivated solution was cooled to 55°C, and 4% of activated carbon (40 kg) was added; a resulting solution was kept at the temperature for 1 h, and then filtered through a 30,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected as 23 mm; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined as 0.52%. 1.4 Enzymolysis results of trypsin A 20% vital wheat gluten solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 6.9 (the optimal pH of the corresponding protease); a resulting solution was incubated for 1 h; then a trypsin was added, and enzymolysis was conducted for 6 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); an enzyme-inactivated solution was cooled to 55°C, and 4% of activated carbon (40 kg) was added; a resulting solution was kept at the temperature for 1 h, and then filtered through a ,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected as 9 mm; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined as 0.09%. 2. Stepwise enzymolysis of vital wheat gluten using a combination of two enzymes A single one of alkaline protease, papain, neutral protease, and trypsin was first used to conduct hydrolysis under the optimal enzymolysis conditions of the single enzyme. According to the detection results of the proliferation activity of macrophages and the content of albumin peptide B1, it was intended to use the papain and neutral protease for enzymolysis. 2.1 Stepwise enzymolysis results of a combination of neutral protease and papain A 20% vital wheat gluten solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.0 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; then a neutral protease was added with a final concentration of 0.5%, and enzymolysis was conducted for 4 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to 7.5; a papain was added with a final concentration of 1.2%, and enzymolysis was further conducted for 6 h; an enzyme-inactivated solution was cooled to 55°C, and 4% of activated carbon (40 kg) was added; a resulting solution was kept at the temperature for 1 h, and then filtered through a ,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected as 30 mm; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined as 0.69%. 2.2 Stepwise enzymolysis results of a combination of papain and neutral protease A 20% vital wheat gluten solution was prepared and heated to 55°C (the optimal enzymolysis temperature of the protease) using a constant temperature magnetic stirrer; pH of the solution was measured using a pH meter (Leici) in real time and adjusted to 7.5 (the optimal pH of the corresponding protease); a resulting solution was incubated for 30 min; a papain was added with a final concentration of 1.2%, and enzymolysis was conducted for 6 h under the corresponding enzymolysis conditions (the optimal pH and temperature were maintained during the enzymolysis); pH of a resulting solution was adjusted to 7.0; then a neutral protease was added with a final concentration of 0.5%, and enzymolysis was further conducted for 4 h; an enzyme-inactivated solution was cooled to 55°C, and 4% of activated carbon (40 kg) was added; a resulting solution was kept at the temperature for 1 h, and then filtered through a ,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray dried to obtain a wheat peptide mixture. According to the detection method of in vitro inhibition on H. pylori described in Example 3, the inhibitory effect of the wheat peptide mixture on H. pylori was detected as 32 mm; and according to the chromatographic analysis method described in Example 4, the mass percentage of the wheat peptide M1 in the wheat peptide mixture was determined as 0.92%. The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims (5)

What is claimed is:

1. A wheat peptide mixture capable of inhibiting Helicobacterpylori(H. pylori), wherein, a preparation method of the wheat peptide mixture comprises the following steps: 1) grinding and homogenizing a vital wheat gluten solution to obtain a stable homogenate; 2) stirring the homogenate at 50°C to 60°C for 1 h to 2 h to obtain a pretreated homogenate; 3) subjecting the pretreated homogenate to stepwise enzymolysis with papain and neutral protease successively and to enzymatic inactivation to obtain an enzymatic hydrolysate; and 4) filtrating the enzymatic hydrolysate through a 30,000 Da ceramic membrane and a 1 KDa organic membrane successively, and collecting a resulting filtrate to obtain the wheat peptide mixture; wherein, in step 3), the papain has a mass concentration of 1.0% to 2.0% in the pretreated homogenate, and enzymolysis with the papain is conducted for 5 h to 7 h; before the papain is added, pH of the pretreated homogenate is adjusted to neutral; the neutral protease has a mass concentration of 0.3% to 1.0% in the pretreated homogenate, and enzymolysis with the neutral protease is conducted for 3 h to 5 h; before the enzymolysis with the neutral protease is conducted, pH of a resulting system is adjusted to 6.9 to 7.2; and in step 1), particles in the homogenate have a particle size of 50 pm to 150 pm.

2. A wheat peptide M capable of inhibiting H. pylori, wherein, the wheat peptide M is obtained by subjecting the wheat peptide mixture according to claim 1 to high-performance liquid chromatography (HPLC) and collecting a component with peak time of 8 min to 9 min; wherein, parameters for the HPLC are as follows: a mixed solution of mobile phase A and mobile phase B used for the following gradient elution: from 0 min to 5 min, a volume percentage of mobile phase B gradiently increasing from % to 5%; from 5 min to 31 min, the volume percentage of mobile phase B gradiently increasing from 5% to 78%; from 31 min to 33 min, the volume percentage of mobile phase B remaining at 78%; and from 33 min to 36 min, the volume percentage of mobile phase B gradiently decreasing from 78% to 5%; wherein, the mobile phase A is a mixture of trifluoroacetic acid (TFA) and ultrapure water (UPW), with a TFA-UPW volume ratio of 0.1:99.9; and the mobile phase B is a mixture of TFA and acetonitrile, with a TFA-acetonitrile volume ratio of 0.1:99.9; detection wavelength: 214 nm; detection time: 36 min; and column temperature: 21C to 0 C.

3. An active peptide capable of inhibiting H. pylori, comprising a wheat peptide M1 or a salt form of the wheat peptide M1; and the wheat peptide M1 has an amino acid sequence shown in SEQ ID No: 1.

4. A method for preparing the active peptide capable of inhibiting H. pylori according to claim 3, wherein, the active peptide is obtained by subjecting the wheat peptide M according to claim 2 to chromatographic analysis and identification using a liquid chromatography-Q EXACTIVE mass spectrometry system; wherein, conditions of the chromatographic analysis are as follows: mobile phase: phase A: a mixture of formic acid and purified water, with a purified water formic acid volume ratio of 100:0.1; phase B: a mixture of acetonitrile and formic acid, with an acetonitrile-formic acid volume ratio of 100:0.1; flow rate of the mobile phase: 300 nl/min; injection volume: 1IpL supernatant; gradient procedure of the mobile phase as follows:

Time (min) 0 2.0 36.0 38.0 41.0 42.0 45.0

Volume percentage of phase A 97% 97% 63% 10% 10% 97% 97%

Volume percentage of phase B 3% 3% 37% 90% 90% 3% 3%

5. A drug capable of inhibiting H. pylori, comprising the active peptide according to claim 3, the wheat peptide mixture according to claim 1, or the wheat peptide M according to claim 2.