A wheat peptide with the function of promoting intestinal peristalsis, its preparation method and application

Abstract

The present invention discloses a wheat peptide with the function of promoting intestinal peristalsis, its preparation method and application, belonging to the technical field of biomedicine. The wheat peptide is obtained by directional enzymatic hydrolysis of wheat protein, and contains a hexapeptide TR6 with the amino acid sequence of Thr-Ser-Val-Pro-Phe-Arg. Function verification shows that the hexapeptide TR6 and the wheat peptide containing this peptide segment have the function of promoting intestinal peristalsis, which is mainly reflected in shortening the time interval of intestinal peristalsis peaks and increasing the promotion rate of intestinal peristalsis, and the food-derived bioactive peptide has high biological safety. Therefore, it can be applied to the preparation of drugs for improving intestinal peristalsis disorders. The present invention provides a new solution for improving intestinal peristalsis disorders, and has good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a wheat peptide having the function of promoting intestinal peristalsis, a preparation method thereof, and an application thereof. Background Art

[0002] Intestinal peristalsis refers to the movement similar to that of a worm, especially the wave-like contraction of the intestine, which is crucial for the normal digestion of food. The main functions of peristalsis are to mix and break down food and transport it to the posterior segment of the digestive tract, as well as to stimulate the absorption of nutrients, water, and electrolytes. Intestinal motility disorder refers to the insufficient or excessive slow peristalsis of the intestine, resulting in the dysfunction of the digestive system. Long-term slow peristalsis will not only lead to the occurrence of gastrointestinal diseases such as intestinal obstruction and constipation, but also cause cardiovascular and cerebrovascular diseases.

[0003] Due to being affected by various factors, the exact pathogenesis of motility disorder has not been clarified. At present, laxatives are mostly relied on to improve this symptom. However, laxatives have different degrees of side effects on the human body. For example, long-term use of anthraquinone laxatives can lead to melanosis coli and has the risk of suffering from colorectal cancer (Tack J, et al . Diagnosis and treatment of chronicconstipation - a European perspective. Neurogastroenterol Motil , 2011, 23(8):697-710.). Therefore, it is particularly important to find safe and effective natural substances to promote intestinal peristalsis.

[0004] Food-derived bioactive peptides refer to protein fragments generated by enzymatic hydrolysis or fermentation in edible animal and plant proteins. In addition to providing essential amino acids, they also have various biological activities such as blood pressure lowering, blood sugar lowering, antioxidant, and immune regulation. As natural functional factors, food-derived bioactive peptides have good health promotion effects, no toxic side effects, low cost, high tissue permeability, easy synthesis and modification, etc., and have excellent potential for development as health foods. For example, Zhang Ting et al. reported that walnut oligopeptides have the function of moistening the intestine and relieving constipation. The walnut oligopeptides are extracted from walnuts by using biological enzymatic hydrolysis technology, and the main component is that the content of small molecule oligopeptides with a relative molecular weight < 1000 Da is > 95% (Function of walnut oligopeptides in moistening the intestine and relieving constipation. Chinese Journal of Public Health, 2019, 35(9): 1225-1228.). The above research objects are mixtures of bioactive peptides. At present, the research on small molecule peptides of bioactive peptides promoting intestinal peristalsis in this field is still insufficient.

[0005] Wheat peptides are the enzymatic hydrolysis products of wheat gluten. Previous studies by the research group found that wheat peptides have the function of promoting and alleviating constipation. Therefore, by analyzing the peptide spectrum of wheat peptides, screening out bioactive small peptides related to the function of promoting intestinal peristalsis will provide a new solution for improving intestinal peristalsis dysfunction. Summary of the Invention

[0006] The purpose of the present invention is to provide a natural small molecule bioactive peptide with the function of promoting intestinal peristalsis and apply it to the development of drugs for improving intestinal peristalsis dysfunction.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] The present invention uses LC-MS / MS peptide spectrum analysis technology to analyze the polypeptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between the peptide segment and the acetylcholine receptor. A candidate peptide segment is screened out. After mass spectrometry identification, its amino acid sequence is Thr-Ser-Val-Pro-Phe-Arg (TSVPFR), and the molecular weight is 705.38 Da. It is named TR6. Further, the hexapeptide TR6 is synthesized artificially, and functional verification finds that this hexapeptide has a significant function of promoting intestinal peristalsis.

[0009] Therefore, the present invention provides a bioactive hexapeptide TR6, and the amino acid sequence of the hexapeptide TR6 is Thr-Ser-Val-Pro-Phe-Arg.

[0010] The present invention also provides a method for preparing the hexapeptide TR6. The hexapeptide TR6 can be prepared by solid-phase synthesis method. The specific method includes: adopting the Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as the solid-phase carrier, and introducing arginine, phenylalanine, proline, valine, serine, and threonine residues in turn to extend the peptide chain from the C-terminus to the N-terminus to solid-phase synthesize the hexapeptide TR6.

[0011] The hexapeptide TR6 can also be obtained by enzymatic hydrolysis of wheat gluten. Specifically, using wheat protein (gluten powder) as the raw material, successively undergoing enzymatic hydrolysis by alkaline protease, neutral protease, and flavor protease, taking the supernatant and filtering it through a filter membrane with a molecular weight cut-off of 5 kDa. The obtained filtrate contains the hexapeptide TR6.

[0012] The present invention also provides the application of the hexapeptide TR6 in the preparation of drugs for intestinal peristalsis disorders and / or alleviating constipation. The research of the present invention shows that the hexapeptide TR6 has the function of promoting intestinal peristalsis. Administering the hexapeptide TR6 to an animal model of intestinal peristalsis disorder can significantly promote intestinal peristalsis and improve the condition of intestinal peristalsis disorder. Therefore, it can be applied to the development of related products for improving intestinal peristalsis disorder.

[0013] Furthermore, the promotion of intestinal peristalsis includes at least one of shortening the time interval of intestinal peristalsis peaks and increasing the intestinal peristalsis promotion rate.

[0014] Another object of the present invention is to provide a wheat peptide having the function of promoting intestinal peristalsis. Its preparation method includes: mixing wheat gluten powder and water at a mass ratio of 1:10 - 20, adjusting the pH to 8.0 ± 0.2, adding alkaline protease accounting for 0.5% of the weight of gluten powder, enzymolyzing for 30 min, and shearing the enzymolysis solution at a rotation speed of 10000 - 15000 r / min for 15 - 30 min; then successively adding alkaline protease accounting for 0.5 - 2.0% of the weight of gluten powder for enzymolysis for 30 - 60 min, adding neutral protease accounting for 0.5 - 1.0% of the weight of gluten powder for enzymolysis for 15 - 45 min, and adding flavor protease accounting for 0.1 - 1.0% of the weight of gluten powder for 30 min; after the enzymolysis is completed, centrifuging at a rotation speed of 4000 - 6000 r / min, taking the supernatant and filtering it through a filter membrane with a cut-off molecular weight of 5 kDa, and sterilizing and drying the filtrate to obtain the wheat peptide.

[0015] The wheat peptide contains the hexapeptide TR6. Animal experiments show that administering the wheat peptide in a constipation model can significantly shorten the time for the first black stool to be excreted, significantly increase the number of fecal pellets and the small intestine propulsion rate, indicating that the wheat peptide has the function of promoting intestinal peristalsis. The promotion of intestinal peristalsis includes increasing the small intestine propulsion rate. Therefore, it can be applied to the development of related products for improving intestinal peristalsis disorders.

[0016] The present invention also provides the application of the wheat peptide in the preparation of drugs for intestinal peristalsis disorders and / or relieving constipation.

[0017] The present invention provides a pharmaceutical composition for treating diseases related to intestinal peristalsis disorders. The active ingredient of the pharmaceutical composition includes the hexapeptide TR6 with the amino acid sequence Thr - Ser - Val - Pro - Phe - Arg or the wheat peptide. The diseases related to intestinal peristalsis disorders can be, but are not limited to, intestinal obstruction, constipation.

[0018] In the pharmaceutical composition provided by the present invention, the hexapeptide TR6 or the wheat peptide can be used as the only active ingredient that plays the role of promoting intestinal peristalsis, or can be used in combination with other active ingredients having the function of promoting intestinal peristalsis. The active components with the function of promoting intestinal peristalsis can be, but are not limited to, dietary fiber, probiotics or other protein peptides.

[0019] The pharmaceutical composition also includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver the active substance of the effective dose of the present invention, does not interfere with the biological activity of the active substance and has no toxic or side effects on the host or subject.

[0020] The present invention uses the hexapeptide TR6 or the wheat peptide as the main active ingredient, adds a pharmaceutically acceptable carrier, and is made into a preparation according to the preparation method of the preparation recorded in pharmacy.

[0021] Furthermore, the pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.

[0022] Furthermore, the dosage form of the pharmaceutical composition can be, but is not limited to, an oral preparation. Specifically, the dosage form can be, but is not limited to, an oral liquid, a capsule, a microcapsule powder, a tablet, a granule, or an emulsion.

[0023] The present invention also provides a health food for promoting digestion or promoting defecation, which includes the hexapeptide TR6 or the wheat peptide as an active ingredient, and food-grade excipients acceptable in food science. The amino acid sequence of the hexapeptide TR6 is Thr-Ser-Val-Pro-Phe-Arg. The food-grade excipients acceptable in food science are food-grade excipients that can deliver the active substance in an effective dose of the present invention and do not interfere with the biological activity of the active substance.

[0024] Furthermore, the dosage form of the health food is a beverage, an oral liquid, a capsule, a microcapsule powder, a tablet, a granule, or an emulsion.

[0025] The beneficial effects of the present invention:

[0026] The present invention provides a hexapeptide TSVPFR having a function of promoting intestinal peristalsis and a wheat peptide containing this peptide segment. The peptide segment can be obtained by artificial synthesis or by directional enzymatic hydrolysis of wheat gluten. Functional verification of the animal model shows that the hexapeptide TSVPFR and the wheat peptide containing this peptide segment have the function of promoting intestinal peristalsis, which is mainly reflected in shortening the time interval of intestinal peristalsis peaks and increasing the promotion rate of intestinal peristalsis. Moreover, the food-derived bioactive peptide has high biological safety. Therefore, it can be applied to the preparation of drugs for improving intestinal peristalsis disorders. The present invention provides a new solution for improving intestinal peristalsis disorders and has good market prospects and application potential. Description of the Drawings

[0027] Figure 1 Effects of wheat peptide on the defecation function of constipated mice, where A is the experimental design of the mice, B is the weight gain, C is the energy intake, D is the time of the first black feces excretion, E is the number of fecal pellets within 5 h, F is the dry fecal weight, G is the wet fecal weight, H is the fecal water content, I is the small intestine propulsion rate, and J is the small intestine image; the letters a, b, and c in the figure represent the difference comparison between groups. The same letter indicates no significant difference between groups, and different letters indicate significant differences between groups. P <0.05.

[0028] Figure 2 It is the secondary mass spectrometry diagram of the hexapeptide TSVPFR. In the figure, #1:1 represents the serial number; T / S / V / P / F / R represents the hexapeptide sequence; the vertical coordinate Intensity(%) represents the relative abundance; y1 represents the first fragment ion generated by the cleavage at the C-terminus of the peptide segment; b2 represents the second fragment ion generated by the cleavage at the N-terminus of the peptide segment; b3-H2O represents the third dehydrated fragment ion generated by the cleavage at the N-terminus of the peptide segment; y2 represents the second fragment ion generated by the cleavage at the C-terminus of the peptide segment; y3-NH3 represents the third deaminated fragment ion generated by the cleavage at the C-terminus of the peptide segment; y3 represents the third fragment ion generated by the cleavage at the C-terminus of the peptide segment; y4-NH3 represents the fourth deaminated fragment ion generated by the cleavage at the C-terminus of the peptide segment; y4 represents the fourth fragment ion generated by the cleavage at the C-terminus of the peptide segment; y5-H2O represents the fifth dehydrated fragment ion generated by the cleavage at the C-terminus of the peptide segment; y5 represents the fifth fragment ion generated by the cleavage at the C-terminus of the peptide segment.

[0029] Figure 3 It is a schematic diagram of the binding of the hexapeptide TR6 to CHRM1.

[0030] Figure 4 It is a picture of intestinal peristalsis recorded by a stereomicroscope after the hexapeptide TR6 acts on the zebrafish model.

[0031] Figure 5 It is the influence of the hexapeptide TR6 on the time interval of zebrafish intestinal peristalsis. Among them, the * sign represents the significant difference compared with the blank group (NC), and * represents P <0.05; the # sign represents the significant difference compared with the constipation model group (MC), and ## represents P <0.01.

[0032] Figure 6 It is a picture under a stereofluorescence microscope after the hexapeptide TR6 acts on the zebrafish model.

[0033] Figure 7 It is a statistical chart of the fluorescence intensity after the hexapeptide TR6 is used in the zebrafish model. Among them, the * sign represents the significant difference compared with the blank group (NC), and ** represents P <0.01, and *** represents P <0.001.

[0034] Figure 8 It is the influence of the hexapeptide TR6 on the promotion rate of zebrafish intestinal peristalsis. Among them, the * sign represents the significant difference compared with the blank group (NC), and ** represents P <0.01, and *** represents P <0.001. Specific implementation mode

[0035] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the method, steps or conditions of the present invention shall fall within the scope of the present invention.

[0036] Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0037] Wheat protein (gluten) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.

[0038] Example 1: Preparation and analysis of wheat peptides

[0039] (1) Preparation of wheat peptides (glutamine peptides)

[0040] Put wheat protein (gluten) and water into the enzymatic hydrolysis reaction tank at a ratio of 1:15, adjust the pH of the material liquid to 8.0 ± 0.2, add 0.5% of alkaline protease (derived from Bacillus licheniformis) based on the total weight of gluten and carry out enzymatic hydrolysis for 30 min, then turn on the shearing machine, with a shearing speed of 15000 r / min and shear for 30 min.

[0041] After the shearing is completed, adjust the pH of the material liquid to 8.0 ± 0.2, add 1.5% of alkaline protease (derived from Bacillus licheniformis) based on the total weight of gluten and continue enzymatic hydrolysis for 30 min. After completion, add 0.5% of neutral protease (derived from Bacillus subtilis) based on the total weight of gluten and continue enzymatic hydrolysis for 45 min. After completion, use 0.5% of flavor protease based on the total weight of gluten and act for 30 min.

[0042] After the enzymatic hydrolysis is completed, separate by a disc centrifuge (rotating speed 6000 r / min), take the supernatant and filter it through a membrane with a molecular weight cut-off of 5 kDa. After filtration, take the supernatant, sterilize it and spray-dry it to obtain wheat peptide (glutamine peptide) powder.

[0043] (2) Analysis of wheat peptides (glutamine peptides)

[0044] The main components, amino acid composition and molecular weight distribution range of the wheat peptides (glutamine peptides) prepared in step (1) were measured, and the results are shown in Table 1 - Table 3:

[0045] Table 1. Content of main components of glutamine peptides

[0046] Component Content (%) Moisture 4.02 Ash 4.15 Protein content (on dry basis) 78.12 Degree of hydrolysis 7.26 Peptide content (on dry basis) 30.25

[0047] Table 2. Relative molecular weight distribution of glutamine peptides

[0048] Relative molecular weight range, Da Wheat peptide (glutamine peptide), % >10000 2.14 5000-10000 7.95 5000-3000 7.78 3000-2000 8.38 2000-1000 12.51 1000-500 24.49 500-180 26.98 <180 9.77

[0049] Table 3. Amino acid composition of glutamine peptides

[0050] Sequence Amino acid species Absolute content, mg / g 1 Aspartic acid 21.06 2 Glutamic acid 290.11 3 Serine 33.26 4 Glycine 21.57 5 Histidine 5.21 6 Arginine 18.76 7 Threonine 23.5 8 Alanine 15.23 9 Proline 100.76 10 Tyrosine 25.42 11 Valine 31.06 12 Methionine 7.18 13 Cystine 3.02 14 Isoleucine 25.1 15 Leucine 54.12 16 Phenylalanine 35.33 17 Lysine 13.12 Total 723.81

[0051] Example 2: Effect of wheat peptides (prepared in Example 1) on intestinal peristalsis in constipated mice

[0052] (1) Grouping and intervention of mouse experiments

[0053] SPF-grade male BALB / C mice (weighing about 20 g) were selected for the experiment. All procedures were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of Zhejiang Chinese Medical University and approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (No. 20220425-28). Thirty-six mice were housed in a breeding cage at a temperature of 25 ± 1 °C, a humidity of 50-55%, and a light cycle of 12 h / 12 h, and the mice were allowed to eat and drink freely. The experiment started after 1 week of environmental adaptation. The mice were randomly divided into 3 groups according to body weight (n = 12). The specific grouping was as follows: ① Normal (CON) group: gavaged with an equal volume of sterile normal saline every day, and gavaged with an equal volume of sterile normal saline half an hour later; ② Modeling (LOP) group: gavaged with loperamide hydrochloride (10 mg / kg.bw) every day, and gavaged with an equal volume of sterile normal saline half an hour later; ③ Wheat peptide (WP) group: gavaged with loperamide hydrochloride (10 mg / kg.bw) every day, and gavaged with wheat peptides (0.5 mg / g.bw) half an hour later. The body weight and food intake of the mice were recorded at fixed times every week. A 5-h defecation experiment was carried out on the 14th day, and a small intestine propulsion experiment was carried out on the 17th day.

[0054] (2) Mouse defecation experiment

[0055] On the 13th day of the experiment, the mice were fasted but allowed to drink water for 16 h. On the 14th day of the experiment, except for the CON group, the other groups were gavaged with loperamide hydrochloride (10 mg / kg.bw), and the CON group was gavaged with the same volume of normal saline. Half an hour later, the CON group and the LOP group were gavaged with activated carbon solution, and the WP group was gavaged with activated carbon solution containing wheat peptides, and the timing started immediately. The mice were placed in a metabolic cage and immediately resumed normal diet. Observe and record the time of the first black stool excretion of each mouse, and use the time of the first black stool of the last mouse in the LOP group as the termination time. The wheat peptide group that exceeded the first black stool time of the LOP group was considered ineffective. Collect the feces of the mice within 5 h and analyze the number of defecation particles, the total weight of feces within 5 h, and the water content of feces.

[0056] (3) Small intestine propulsion experiment

[0057] On the evening of the 16th day of the experiment, the mice were fasted but given water for 16 h to empty the intestine. After 16 h of fasting, except for the CON group, the remaining groups were intragastrically administered loperamide hydrochloride (7.5 mg / kg.bw), and the CON group was intragastrically administered the same volume of normal saline. Half an hour later, the CON group and the LOP group intragastrically administered activated carbon solution to the mice, and the WP group intragastrically administered activated carbon solution containing wheat peptides, and the timing started. Half an hour later, the mice were dissected, the eyeballs were removed to collect blood, and the serum was separated. When dissecting the mice, open the abdominal cavity of the mice, cut the complete gastrointestinal tract (from the pylorus at the upper end to the ileocecal junction at the lower end), and cut the mesentery of the small intestine to slowly straighten the small intestine. On the plate sprinkled with normal saline, straighten the small intestine. After natural retraction, use a 1m ruler to measure the distance from the pylorus to the activated carbon juice (the propulsion length of the activated carbon solution) and the distance from the pylorus to the starting point of the ileocecal junction (the total length of the small intestine), and calculate the small intestine propulsion rate (the ratio of the propulsion length of the activated carbon solution to the total length of the small intestine).

[0058] (4) Data processing

[0059] The experimental data were statistically analyzed using GraphPad Prism 6.0 and SPSS 22.0 software, and the results were expressed as mean ± standard error. The Tukey test in One-way ANOVA was used to compare the differences between multiple groups, and different letters represented significant differences ( P <0.05).

[0060] (5) Result analysis

[0061] As Figure 1 shown, compared with the CON group, the weight gain in the LOP group decreased ( P <0.001), while the WP group reversed this trend. However, there was no significant difference in energy intake among the groups ( P <0.05). Compared with the CON group, the time to the first black stool excretion in the LOP group increased significantly ( P <0.001), and at the same time, the number of fecal pellets, fecal wet weight, water content, and gastrointestinal transit in the LOP group decreased ( P <0.05). However, compared with the LOP group, the time to the first black stool excretion in the WP group was significantly shortened by 22.10% ( P <0.01), and the number of fecal pellets and small intestine propulsion rate increased significantly by 20.10% ( P <0.05) and 37.50% ( P <0.01).

[0062] Based on the above results, it is shown that wheat peptides can effectively relieve constipation induced by loperamide hydrochloride in mice.

[0063] Example 3: Screening of bioactive peptide segments

[0064] (1)Identification of wheat peptide sequence

[0065] Dissolve the wheat peptide sample prepared in Example 1 in NH4HCO3 solution, add dithiothreitol solution, and place it in a water bath at 56 °C for 1 h for reduction. Subsequently, add iodoacetamide solution and react in the dark for 40 min. After desalting, evaporate the solvent to dryness, and then dissolve the sample in 10 μL of mobile phase A (0.1% formic acid) into a liquid phase injection vial. Subsequently, LC-MS / MS analysis is carried out.

[0066] Chromatographic conditions: Analytical column (Acclaim PepMap RPLC C18, 150×150 mm, 3 μm); mobile phase A (0.1% formic acid); mobile phase B (0.1% formic acid and 80% acetonitrile); flow rate (600 nL / min). Gradient elution program: 0 - 2 min, 4% B - 8% B; 2 - 45 min, 8% B - 40% B; 45 - 55 min, 40% B - 60% B; 55 - 56 min, 60% B - 95% B; 56 - 66 min, 95% B.

[0067] Mass spectrometry conditions: Full scan MS is performed by Orbitrap for the first - level scan, scanning range (100~1500 m / z), resolution (70,000), maximum ion injection time (100 ms), automatic gain control (3×10 6 ); The top 20 precursor ions with the highest intensities that meet the tandem (MS / MS) fragmentation conditions are fragmented using high - energy collision dissociation and scanned by Orbitrap, resolution (17,500), maximum ion injection time (50 ms), automatic gain control (1×10 5 ). The raw data obtained by mass spectrometry is analyzed for polypeptide sequence using the De novo software of PEAKSStudio.

[0068] (2)Screening of bioactive peptides with potential function of promoting intestinal peristalsis

[0069] The enteric nervous system is the largest component of the peripheral nervous system and can autonomously control intestinal function independent of the central nervous system. Patients with severe constipation, especially those who have long-term used stimulant laxatives, often exhibit dysfunctions of the enteric nervous system, including a reduction in the number of colonic neurons and damage to cell populations. In intestinal neurons, the activation of muscarinic acetylcholine receptors (mAChRs) can lead to calcium ion influx, triggering a series of intracellular signal transduction, thereby participating in the regulation of intestinal motility, secretion, and other physiological processes. CHRM1 is one of the mAChR subtypes. Therefore, molecular docking of wheat peptide segments with CHRM1 was performed to screen for small peptide segments that may promote intestinal peristalsis.

[0070] Peptide segments meeting the criteria were screened according to the conditions that the average local confidence (ALC) was greater than 95%, the peak area was greater than 2×10 6 , and the PeptideRanker score was greater than 0.8. Subsequently, the peptide segments were subjected to molecular docking with the M1-muscarinic acetylcholine receptor (CHRM1).

[0071] First, the crystal structure (5CXV) of CHRM1 was downloaded from the PDB protein database. After removing water molecules and adding hydrogen atoms to the receptor target through the Discovery Studio software, its active center was defined. The structures of the selected wheat peptide segments were constructed by Discovery Studio, and their energies were minimized by the CHARMm force field. These peptides were defined as ligands. The constructed peptides were docked with CHRM1 using CDOCKER to simulate the binding mode, site, and amino acid residues involved with the lowest binding energy and the highest binding degree, and screening was performed according to the binding energy and the number of hydrogen bonds. Finally, the hexapeptide TSVPFR (TR6) was determined, as shown in Table 4.

[0072] Table 4. Peptide segments in wheat peptides that promote intestinal peristalsis

[0073] Peptide sequence ALC score (%) Mass-to-charge ratio (m / z) Charge number (z) Relative abundance Molecular weight (Da) PeptideRanker score Docking score (kcal / mol) TSVPFR 96.6 353.70 2 2.34E+06 705.38 0.532 -90.17

[0074] Specifically, the secondary mass spectrometry diagram of the hexapeptide TR6 is as Figure 2 shown. The cleavage fragment ions of peptides include: N-terminal fragment ions (types a, b, c) and C-terminal fragment ions (types x, y, z). The side-chain cleavage of a, y, and z-type ions forms d, v, and w-type ions respectively. In addition, there are internal ions formed by cleavage at both ends. The b and y series ions are the most common. The primary structure of the peptide can be deduced and analyzed based on the b or y series fragment ions of the peptide. 353.70 m / z is the [M+H] of the hexapeptide +The ion signal has a charge number (z) of 2 and a molecular weight of 705.38 Da, which is consistent with the relative molecular weight of the hexapeptide TSVPFR. Further, the hexapeptide was analyzed by in-source collision-induced dissociation technology for its secondary mass spectrometry to determine the primary structure of the hexapeptide as Thr-Ser-Val-Pro-Phe-Arg.

[0075] The 2D and 3D maps of the molecular docking of TR6 with CHRM1 are as Figure 3 shown. Analysis of the chemical bonds revealed that TR6 binds to CHRM1 mainly through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and electrostatic interactions (π-salt bridges and Attractivecharge). TR6 forms 12 van der Waals forces with amino acid residues HIS1030, LEU1031, TYR1023, MET1105, THR1025, GLN1140, TRP1137, ASP1019, TYR1017, THR1141, ASN1143, and ARG1147, 12 hydrogen bonds with PHE1103, GLY1029, GLU1021, LYS1034, GLN1104, GLU1010, THR1020, ARG1144, and ASP1009, and 2 electrostatic interactions with LYS1034 and GLU1010.

[0076] From the molecular docking results, it can be seen that the hexapeptide TR6 can bind to CHRM1, thereby activating CHRM1, and then regulating the intestinal nerves, thus exerting the effect of promoting intestinal peristalsis.

[0077] The peptide segment TSVPFR was synthesized by Shenzhen Borun Sida Biotechnology Co., Ltd. with a purity of ≥98% for subsequent functional verification.

[0078] Example 4: Effect of hexapeptide TSVPFR on intestinal peristalsis in a zebrafish constipation model

[0079] (1) Effect of hexapeptide TSVPFR on the time interval of intestinal peristalsis in zebrafish

[0080] In this example, the prokinetic effect of the sample was evaluated by measuring the time interval between two intestinal peristaltic peaks in zebrafish.

[0081] Wild-type AB strain zebrafish at 5 dpf were selected and placed in six-well plates, divided into 8 groups, with 20 fish in each well. The specific grouping was a blank group (NC), a constipation model group (MC), a low-dose wheat peptide group (10 μg / mL, WP-10) and a high-dose wheat peptide group (100 μg / mL, WP-100), a lower-dose TR6 group (0.5 μg / mL, TR6-0.5), a low-dose TR6 group (1 μg / mL, TR6-1), a medium-dose TR6 group (5 μg / mL, TR6-5) and a high-dose TR6 group (10 μg / mL, TR6-10). The blank group was added with the same volume of system water, the model group was added with 10 g / mL ropivacaine hydrochloride, and the sample groups were added with the corresponding doses of samples and ropivacaine hydrochloride at the same time. After culturing in a constant temperature incubator at 28 ± 0.5 °C for 24 h, a stereomicroscope was used to record the intestinal peristalsis videos of zebrafish in each group. After the experiment, by repeatedly watching the videos, the time interval between two intestinal peristalsis wave peaks of zebrafish was recorded.

[0082] In this study, Graphpad Prism 8.0 software was used for statistical difference analysis and drawing, and all data were expressed as mean ± standard error. One-way analysis of variance was used to compare the differences between groups (* P <0.05, ** P <0.01, *** P <0.001; compared with the NC group, # P <0.05, ## P <0.01, P <0.001).

[0083] The results are as Figure 4 and Figure 5 shown. Compared with the NC group, the intestinal peristalsis time interval of the MC group increased significantly ( P <0.05), indicating that the intestinal peristalsis disorder model was successfully established. Compared with the MC group, although 10 μg / mL wheat peptide shortened the intestinal peristalsis time interval, it did not reach significance ( P >0.05); when the concentration of wheat peptide was 100 μg / mL, it could significantly shorten the intestinal peristalsis time interval ( P <0.01). Compared with the MC group, when the concentration of hexapeptide TR6 was 0.5 μg / mL and 1 μg / mL, although the intestinal peristalsis time interval was shortened, it did not reach significance ( P >0.05); when the concentration of hexapeptide TR6 was 5 μg / mL and 10 μg / mL, both could significantly shorten the intestinal peristalsis time interval ( P <0.01).

[0084] Based on the above data, it can be seen that from the perspective of the intestinal peristalsis time interval, the promoting effect of hexapeptide TR6 on intestinal peristalsis is about 20 times that of wheat peptide.

[0085] (2)Effect of hexapeptide TSVPFR on the promotion rate of intestinal peristalsis in zebrafish

[0086] The fluorescent dye Nile red is not interfered by other tissues in zebrafish and will not be absorbed. In this example, the promotion effect of intestinal peristalsis can be evaluated by quantifying Nile red in the gastrointestinal tract of zebrafish.

[0087] Select zebrafish at 5 dpf and stain them with Nile red dye. After culturing the zebrafish in the dark for 16 h, then wash away the dye with system water, transfer the zebrafish to a 6-well plate, and divide them into 7 groups with 20 tails in each well. The specific grouping is the blank group (NC), the low-dose (10 μg / mL, WP-10) and high-dose (100 μg / mL, WP-100) groups of wheat peptide, the lower-dose (0.5 μg / mL, TR6-0.5), low-dose (1 μg / mL, TR6-1), medium-dose (5 μg / mL, TR6-5) and high-dose (10 μg / mL, TR6-10) groups of TR6. The blank group is added with the same volume of system water, and the sample groups are added with the corresponding doses of samples. After culturing in the dark for 24 h, select the same magnification, fluorescence intensity, exposure time and fluorescence gain, take pictures under a stereomicroscope, and use the NIS-Elements D 3.10 advanced image processing software to analyze the total fluorescence signal of the zebrafish intestine. The promotion effect of the sample on the intestinal peristalsis of zebrafish is judged by the amplitude of the decrease in fluorescence intensity before and after. The calculation method of the promotion rate of intestinal peristalsis is the ratio of (fluorescence intensity of the blank group - fluorescence intensity of the sample group) to the fluorescence intensity of the blank group.

[0088] The results are as Figures 6 - 8 shown. Compared with the NC group, although the wheat peptide at 10 μg / mL increased the promotion rate of intestinal peristalsis, it did not reach significance ( P >0.05); when the concentration of wheat peptide was 100 μg / mL, it could significantly increase the promotion rate of intestinal peristalsis ( P <0.001). Compared with the NC group, when the concentration of hexapeptide TR6 was 0.5 μg / mL, although it increased the promotion rate of intestinal peristalsis, it did not reach significance ( P >0.05); when the concentration of hexapeptide TR6 was 1 μg / mL, 5 μg / mL and 10 μg / mL, it could significantly increase the promotion rate of intestinal peristalsis ( P <0.01, P <0.001, P <0.001).

[0089] Based on the above data, it can be seen that from the perspective of the promotion rate of intestinal peristalsis, the promotion effect of hexapeptide TR6 on intestinal peristalsis is about 100 times that of wheat peptide.

[0090] In summary, through mass spectrometry identification of peptide segments and molecular docking, the hexapeptide TSVPFR was screened from wheat peptides. This peptide segment has the effect of promoting intestinal peristalsis, mainly reflected in the intestinal peristalsis disorder model. Compared with the model group, the time interval of intestinal peristalsis peaks can be significantly shortened after treatment with the hexapeptide TSVPFR; in the zebrafish model, compared with the blank group, the intestinal peristalsis promotion rate can be significantly increased after treatment with the hexapeptide TSVPFR. The present invention provides a theoretical basis for the development of novel active substances for promoting intestinal peristalsis and can be applied to drugs for improving intestinal peristalsis disorders. The hexapeptide TSVPFR can be used alone in the preparation of drugs for improving intestinal peristalsis disorders or can be used in combination with other active ingredients having the function of promoting intestinal peristalsis. The active components with the function of promoting intestinal peristalsis can be, but are not limited to, dietary fiber, probiotics or other protein peptides. The drug can be prepared into microcapsules to improve the gastrointestinal digestion stability, bioavailability and shelf life of the polypeptide, so that it can be better applied to the food industry and the health care field.

Claims

1. A biologically active hexapeptide TR6, characterized in that: The amino acid sequence of the hexapeptide TR6 is Thr-Ser-Val-Pro-Phe-Arg.

2. The method for preparing hexapeptide TR6 according to claim 1, characterized in that: The hexapeptide TR6 is prepared by solid phase synthesis; or obtained by enzymatic hydrolysis of wheat gluten, wherein the enzymatic hydrolysis conditions are as follows: wheat gluten powder and water are mixed at a mass ratio of 1:10-20, the pH is adjusted to 8.0±0.2, 0.5% alkaline protease by weight of the gluten powder is added, the enzymatic hydrolysis is carried out for 30 minutes, and the enzymatic hydrolyzate is sheared for 15-30 minutes at a speed of 10000-15000 r / min; then 0.5-2.0% alkaline protease by weight of the gluten powder is added for enzymatic hydrolysis for 30-60 minutes, 0.5-1.0% neutral protease by weight of the gluten powder is added for enzymatic hydrolysis for 15-45 minutes, and 0.1-1.0% flavor protease by weight of the gluten powder is added for 30 minutes; after the enzymatic hydrolysis is completed, centrifugation is carried out at a speed of 4000-6000 r / min, and the supernatant is taken and purified by a 5% molecular weight cut-off method. kDa filter membrane, and the filtrate was sterilized and dried to obtain wheat peptides, which contained the hexapeptide TR6.

3. Use of the hexapeptide TR6 according to claim 1 in the preparation of a drug for treating intestinal peristalsis disorders and / or relieving constipation, characterized in that: The hexapeptide TR6 has the function of promoting intestinal peristalsis.

4. The use according to claim 3, characterized in that The promoting of intestinal peristalsis includes at least one of shortening the intestinal peristalsis peak time interval and increasing the intestinal peristalsis promotion rate.

5. A wheat peptide having the function of promoting intestinal peristalsis, characterized in that: The wheat peptide comprises the hexapeptide TR6 as claimed in claim 1; the preparation method of the wheat peptide comprises: mixing wheat gluten powder and water at a mass ratio of 1:10-20, adjusting the pH to 8.0±0.2, adding 0.5% alkaline protease by weight of the gluten powder, enzymolysis for 30 minutes, and shearing the enzymolysis solution at a speed of 10000-15000 r / min for 15-30 minutes; then successively adding 0.5-2.0% alkaline protease by weight of the gluten powder for enzymolysis for 30-60 minutes, adding 0.5-1.0% neutral protease by weight of the gluten powder for enzymolysis for 15-45 minutes, and adding 0.1-1.0% flavor protease by weight of the gluten powder for 30 minutes; after the enzymolysis is completed, centrifuging at a speed of 4000-6000 r / min, filtering the supernatant through a filter membrane with a molecular weight cutoff of 5 kDa, and sterilizing and drying the filtrate to obtain the wheat peptide.

6. Use of the wheat peptide according to claim 5 in the preparation of a medicament for treating intestinal peristalsis disorders and / or relieving constipation.

7. A pharmaceutical composition for treating diseases related to intestinal peristalsis disorders, characterized in that: The pharmaceutical composition comprises hexapeptide TR6 with an amino acid sequence of Thr-Ser-Val-Pro-Phe-Arg or a wheat peptide containing the hexapeptide TR6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is in the form of an oral preparation.

9. A health food for promoting digestion or laxative, characterized in that: The invention comprises hexapeptide TR6 with the amino acid sequence of Thr-Ser-Val-Pro-Phe-Arg or wheat peptide containing the hexapeptide TR6, and excipients acceptable in food science.

10. The health food according to claim 9, characterized in that The dosage form of the health food is beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

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