Wheat peptide with function of promoting peristalsis as well as preparation method and application of wheat peptide

Abstract

The invention discloses a wheat peptide with a function of promoting peristalsis as well as a preparation method and application of the wheat peptide, and belongs to the technical field of biological medicines. The wheat peptide is obtained by directional enzymolysis of wheat protein and contains hexapeptide TR6 with the amino acid sequence of Thr-Ser-Val-Pro-Phe-Arg, functional verification shows that the hexapeptide TR6 and the wheat peptide containing the peptide fragment have the function of promoting peristalsis, the function is mainly reflected in shortening the time interval of peristalsis peaks and improving the peristalsis promoting rate, and the function of promoting the peristalsis is achieved. And the food-borne bioactive peptide is high in biological safety. Therefore, the compound can be applied to the preparation of food or medicines for improving the peristalsis disorder. The invention provides a new solution for improving the peristalsis disorder, and has good market prospect and application potential.

Description

Technical Field

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

[0002] Intestinal peristalsis refers to the worm-like movement, especially the wave-like contraction of the intestine, which is essential for the normal digestion of food. The main function of peristalsis is to mix and break down food and transport it to the later part of the digestive tract, as well as stimulate the absorption of nutrients, water and electrolytes. Intestinal peristalsis dysfunction means that the peristalsis of the intestine is not strong enough or is too slow, resulting in 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 the influence of many factors, the exact pathogenesis of peristaltic dysfunction has not yet been clarified. Currently, laxatives are mostly used 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 the risk of colorectal cancer (Tack J, et al . Diagnosis and treatment of chronic constipation - 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 produced by enzymatic hydrolysis or fermentation in edible animal and plant proteins. In addition to providing essential amino acids, they also have multiple biological activities such as lowering blood pressure, lowering blood sugar, anti-oxidation and immunomodulation. As natural functional factors, food-derived bioactive peptides have good health-promoting effects, no toxic side effects, low cost, high tissue permeability, easy synthesis and modification, and have excellent potential for development as health foods. For example, Zhang Ting et al. reported that walnut oligopeptides have a laxative effect. The walnut oligopeptides are extracted from walnuts using biological enzymatic hydrolysis technology, and the main component is a small molecule oligopeptide with a relative molecular weight of <1000 Da and a content of >95% (Walnut oligopeptides have laxative function. Chinese Public Health, 2019, 35(9): 1225-1228.). The above research objects are mixtures of bioactive peptides. At present, there is insufficient research in this field on small molecule peptides that promote intestinal peristalsis.

[0005] Wheat peptides are enzymatic hydrolysis products of wheat gluten protein. Previous studies by the research team found that wheat peptides have the function of promoting and relieving constipation. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out bioactive small molecule peptides related to the function of promoting intestinal motility, it will provide a new solution for improving intestinal motility 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 to apply the peptide to the development of medicines or health foods for improving intestinal peristalsis dysfunction.

[0007] To achieve the above object, the present invention adopts the following technical solution: The present invention uses LC-MS / MS peptide analysis technology to analyze the polypeptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between peptides and acetylcholine receptors, and screens out a candidate peptide. After mass spectrometry identification, its amino acid sequence is Thr-Ser-Val-Pro-Phe-Arg (TSVPFR), and its molecular weight is 705.38Da. It is named TR6. Further, through artificial synthesis of hexapeptide TR6, functional verification found that the hexapeptide has a significant function of promoting intestinal peristalsis.

[0008] Therefore, the present invention provides a biologically active hexapeptide TR6, wherein the amino acid sequence of the hexapeptide TR6 is Thr-Ser-Val-Pro-Phe-Arg.

[0009] The present invention also provides a method for preparing the hexapeptide TR6. The hexapeptide TR6 can be prepared by solid phase synthesis. The specific method includes: adopting Fmoc solid phase synthesis strategy, using Fmoc protected amino acids as raw materials, selecting Wang resin as a solid phase carrier, sequentially introducing arginine, phenylalanine, proline, valine, serine, and threonine residues, extending the peptide chain from the C-terminus to the N-terminus, and solid phase synthesizing the hexapeptide TR6.

[0010] The hexapeptide TR6 can also be obtained by enzymatic hydrolysis of wheat gluten protein. Specifically, wheat protein (gluten powder) is used as a raw material, and is enzymatically hydrolyzed by alkaline protease, neutral protease, and flavor protease in sequence. The supernatant is filtered through a filter membrane with a molecular weight cutoff of 5 kDa, and the resulting filtrate contains the hexapeptide TR6.

[0011] The present invention also provides the use of the hexapeptide TR6 in the preparation of drugs or foods for intestinal peristalsis and / or constipation relief. The present invention has shown that the hexapeptide TR6 has the function of promoting intestinal peristalsis, and the treatment of the hexapeptide TR6 in an intestinal peristalsis animal model can significantly promote intestinal peristalsis and improve the intestinal peristalsis condition, so it can be applied to the development of related products for improving intestinal peristalsis.

[0012] Furthermore, the promoting of intestinal motility includes at least one of shortening the intestinal motility peak time interval and increasing the intestinal motility promotion rate.

[0013] Another object of the present invention is to provide a wheat peptide with the function of promoting intestinal peristalsis. The preparation method thereof comprises: mixing wheat gluten powder and water in 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.

[0014] The wheat peptide contains the hexapeptide TR6. Animal experiments show that administration of the wheat peptide in a constipation model can significantly shorten the time of first black stool discharge, significantly increase the number of fecal particles and small intestinal propulsion rate, indicating that the wheat peptide has the function of promoting intestinal peristalsis. The promotion of intestinal peristalsis includes increasing the small intestinal propulsion rate. Therefore, it can be applied to the development of related products for improving intestinal peristalsis disorders.

[0015] The present invention also provides the use of the wheat peptide in preparing medicines or foods for intestinal peristalsis disorders and / or constipation relief.

[0016] The present invention provides a pharmaceutical composition for treating diseases related to intestinal peristalsis, wherein the active ingredient of the pharmaceutical composition comprises hexapeptide TR6 with an amino acid sequence of Thr-Ser-Val-Pro-Phe-Arg or the wheat peptide. The diseases related to intestinal peristalsis may be, but are not limited to, intestinal obstruction and constipation.

[0017] The hexapeptide TR6 or the wheat peptide in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that promotes intestinal peristalsis, or can be used in combination with other active ingredients that have the function of promoting intestinal peristalsis. The active components that promote intestinal peristalsis function can be but are not limited to dietary fiber, probiotics or other protein peptides.

[0018] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject.

[0019] The present invention uses the hexapeptide TR6 or the wheat peptide as a main active ingredient, adds a pharmaceutically acceptable carrier, and prepares the preparation according to the preparation preparation method recorded in pharmacy.

[0020] Furthermore, the pharmaceutically acceptable carrier includes: one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0021] Furthermore, the pharmaceutical composition may be in the form of, but not limited to, an oral preparation. Specifically, the pharmaceutical composition may be in the form of, but not limited to, an oral liquid, a capsule, a microcapsule powder, a tablet, a granule, or an emulsion.

[0022] The present invention also provides a health food for promoting digestion or laxative, comprising hexapeptide TR6 or the wheat peptide as an active ingredient, and a food-based acceptable auxiliary material, wherein the amino acid sequence of the hexapeptide TR6 is Thr-Ser-Val-Pro-Phe-Arg. The food-based acceptable auxiliary material is a food-grade auxiliary material that can deliver an effective dose of the active substance of the present invention and does not interfere with the biological activity of the active substance.

[0023] Furthermore, the dosage form of the health food is beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0024] The present invention has the following beneficial effects: The present invention provides a hexapeptide TSVPFR having the function of promoting intestinal peristalsis and a wheat peptide containing the peptide segment, wherein the peptide segment can be obtained by artificial synthesis or by directed enzymatic hydrolysis of wheat gluten. Functional verification of animal models shows that the hexapeptide TSVPFR and the wheat peptide containing the peptide segment have the function of promoting intestinal peristalsis, which is mainly reflected in shortening the intestinal peristalsis peak time interval and improving the intestinal peristalsis promotion rate. Moreover, the food-borne bioactive peptides have high biological safety. Therefore, they can be used to prepare foods or medicines 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2 is the effect of wheat peptide on the defecation function of constipated mice, where A is the experimental design of mice, B is the weight gain, C is the energy intake, D is the time of first black stool discharge, E is the number of stool particles within 5 hours, F is the weight of dry stool, G is the weight of wet stool, H is the water content of stool, 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 the groups, the same letters indicate that there is no significant difference between the groups, and different letters indicate that there is a significant difference between the groups. P <0.05.

[0026] Figure 2 This is the secondary mass spectrum of the hexapeptide TSVPFR, in which #1:1 represents the number; T / S / V / P / F / R represents the hexapeptide sequence; the ordinate Intensity (%) represents the relative abundance; y1 represents the first fragment ion produced by the cleavage of the C-terminus of the peptide; b2 represents the second fragment ion produced by the cleavage of the N-terminus of the peptide; b3-H2O represents the third dehydrated fragment ion produced by the cleavage of the N-terminus of the peptide; y2 represents the second fragment ion produced by the cleavage of the C-terminus of the peptide; y3-NH3 represents the third deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; y3 represents the third fragment ion produced by the cleavage of the C-terminus of the peptide; y4-NH3 represents the fourth deaminated fragment ion produced by the cleavage of the C-terminus of the peptide; y4 represents the fourth fragment ion produced by the cleavage of the C-terminus of the peptide; y5-H2O represents the fifth dehydrated fragment ion produced by the cleavage of the C-terminus of the peptide; y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide.

[0027] Figure 3 Schematic diagram of the binding interaction between hexapeptide TR6 and CHRM1.

[0028] Figure 4 This is a picture of intestinal peristalsis recorded by in vivo microscopy after the hexapeptide TR6 acted on the zebrafish model.

[0029] Figure 5 The effect of hexapeptide TR6 on the time interval of intestinal peristalsis in zebrafish, where * indicates significant difference compared with the blank group (NC), P <0.05; # indicates significant difference compared with the constipation model group (MC), ## indicates P <0.01.

[0030] Figure 6 This is a picture taken under fluorescence microscopy after the hexapeptide TR6 acted on the zebrafish model.

[0031] Figure 7 The fluorescence intensity statistics of hexapeptide TR6 after being used in zebrafish model, where * indicates significant difference compared with blank group (NC), ** indicates P <0.01, *** indicates P <0.001.

[0032] Figure 8 The effect of hexapeptide TR6 on the intestinal motility promotion rate of zebrafish, where * indicates a significant difference compared with the blank group (NC), and ** indicates P <0.01, *** indicates P <0.001. DETAILED DESCRIPTION

[0033] 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 intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0035] Wheat protein (gluten powder) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme (Yichang) Co., Ltd.

[0036] Example 1: Preparation and analysis of wheat peptides (1) Preparation of wheat peptide (glutamine peptide) Wheat protein (gluten) and water were put into the enzymatic reaction tank in a ratio of 1:15, the pH of the solution was adjusted to 8.0±0.2, and 0.5% alkaline protease (derived from Bacillus licheniformis) of the total weight of gluten was added for enzymatic hydrolysis for 30 min, and then the shearing machine was turned on with a shearing speed of 15000 r / min for 30 min.

[0037] After shearing, the pH of the feed solution was adjusted to 8.0±0.2, and 1.5% of the total weight of gluten (derived from Bacillus licheniformis) alkaline protease was added to continue enzymatic hydrolysis for 30 min. After that, 0.5% of the total weight of gluten (derived from Bacillus subtilis) neutral protease was added to continue enzymatic hydrolysis for 45 min. After that, 0.5% of the total weight of gluten flavor protease was used for 30 min.

[0038] After the enzymatic hydrolysis, the mixture was separated by a disc centrifuge (speed 6000 r / min), and the supernatant was filtered through a membrane with a molecular weight cutoff of 5 kDa. After filtration, the supernatant was sterilized and spray-dried to obtain wheat peptide (glutamine peptide) powder.

[0039] (2) Analysis of wheat peptides (glutamine peptides) The main components, amino acid composition and molecular weight distribution range of the wheat peptide (glutamine peptide) prepared in step (1) were measured, and the results are shown in Tables 1 to 3: Table 1. Content of main components of glutamine peptide Element content(%) Moisture 4.02 Ash 4.15 Protein content (dry basis) 78.12 Degree of hydrolysis 7.26 Peptide content (dry basis) 30.25 Table 2. Relative molecular weight distribution of glutamine peptides Relative molecular weight range, Da Wheat peptides (glutamine peptides), % >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 Table 3. Amino acid composition of glutamine peptide sequence Amino Acid Type Absolute content, mg / g 1 Aspartic acid 21.06 2 Glutamate 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 sum 723.81 Example 2: Effect of wheat peptide (prepared in Example 1) on intestinal peristalsis in constipated mice (1) Mouse experimental grouping and intervention SPF male BALB / C mice (weighing about 20 g) were used in the experiment. All procedures were carried out in accordance with the Guide 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). 36 mice were housed in cages with a temperature of 25 ± 1 ℃, humidity of 50-55%, and a light cycle of 12 h / 12 ​​h. The mice were allowed to eat and drink freely. The experiment began after 1 week of adaptation to the environment. The mice were randomly divided into 3 groups (n = 12) according to their body weight. The specific groups were as follows: ① Normal (CON) group: gavage with an equal amount of sterile saline every day, and gavage with an equal amount of sterile saline half an hour later; ② Modeling (LOP) group: gavage with loperamide hydrochloride (10 mg / kg.bw) every day, and gavage with an equal amount of sterile saline half an hour later; ③ Wheat peptide (WP) group: gavage with loperamide hydrochloride (10 mg / kg.bw) every day, and gavage with wheat peptide (0.5 mg / g.bw) half an hour later. The body weight and food intake of mice were recorded at fixed times every week. A 5-h defecation experiment was performed on the 14th day, and a small intestinal propulsion experiment was performed on the 17th day.

[0040] (2) Mouse defecation experiment On the 13th day of the experiment, mice were fasted but not watered for 16 h. On the 14th day of the experiment, loperamide hydrochloride (10 mg / kg.bw) was gavaged in all groups except the CON group, and the CON group was gavaged with an equal volume of 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 was started immediately. The mice were placed in metabolic cages and immediately resumed their normal diet. The time of the first black stool of each mouse was observed and recorded, and the time of the first black stool of the last mouse in the LOP group was taken as the termination time. The wheat peptide group that exceeded the time of the first black stool of the LOP group was invalid. The feces of mice within 5 h were collected and the number of fecal particles, the total weight of feces within 5 h, and the water content of feces were analyzed.

[0041] (3) Small Intestinal Propulsion Experiment On the evening of the 16th day of the experiment, mice were fasted for 16 h but not water to empty the intestine. After fasting for 16 h, loperamide hydrochloride (7.5 mg / kg.bw) was gavaged in all groups except the CON group, which was gavaged with an equal volume of saline. Half an hour later, mice in the CON and LOP groups were gavaged with activated carbon solution, and mice in the WP group were gavaged with activated carbon solution containing wheat peptides, and the timing was 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, the abdominal cavity of the mice was opened, the complete gastrointestinal tract (the upper end was from the pylorus, the lower end was to the ileocecum) was cut, and the small intestinal mesentery was cut to slowly straighten the small intestine. The small intestine was straightened on a plate sprinkled with saline. After natural retraction, 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 beginning of the ileocecum (the total length of the small intestine) were measured with a 1m ruler, and the small intestinal propulsion rate (the ratio of the propulsion length of the activated carbon solution to the total length of the small intestine) was calculated.

[0042] (4) Data processing 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. Different letters represent significant differences ( P <0.05).

[0043] (5) Results analysis like Figure 1 As shown, the LOP group had a reduced weight gain compared with the CON group ( P <0.001), while the WP group reversed this trend. However, there was no significant difference in energy intake between the groups ( P <0.05). Compared with the CON group, the time to first black stool in the LOP group was significantly increased ( P <0.001, while the number of fecal particles, fecal wet weight, water content, and gastrointestinal transit were reduced in the LOP group ( P <0.05). However, compared with the LOP group, the time to first black stool in the WP group was significantly shortened by 22.10% ( P <0.01), the number of fecal particles and small intestinal propulsion rate increased significantly by 20.10% ( P <0.05) and 37.50% ( P <0.01).

[0044] The above results show that wheat peptide can effectively relieve loperamide hydrochloride-induced constipation in mice.

[0045] Example 3: Screening of active peptides (1) Wheat peptide sequence identification The wheat peptide sample prepared in Example 1 was dissolved in NH4HCO3 solution, dithiothreitol solution was added, and the mixture was reduced in a 56°C water bath for 1 h. Then, iodoacetamide solution was added and the mixture was reacted for 40 min in the dark. After desalting, the solvent was evaporated, and then 10 μL of mobile phase A (0.1% formic acid) was used to dissolve the sample into a liquid injection bottle. LC-MS / MS analysis was then performed.

[0046] 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.

[0047] Mass spectrometry conditions: Full scan MS using Orbitrap for primary scanning, scan range (100-1500 m / z), resolution (70,000), maximum ion introduction time (100 ms), automatic gain control (3×10 6 ); The top 20 parent ions that met the MS / MS fragmentation conditions were fragmented using high-energy collision dissociation and scanned with Orbitrap, with a resolution of 17,500, a maximum ion introduction time of 50 ms, and automatic gain control (1×10 5 The raw data obtained by mass spectrometry were analyzed for peptide sequence using PEAKSStudio's De novo (de novo sequencing) software.

[0048] (2) Screening of active peptides with potential to promote intestinal motility The enteric nervous system is the largest component of the peripheral nervous system and can autonomously control intestinal function independently of the central nervous system. Patients with severe constipation, especially those who take stimulant laxatives for a long time, often show enteric nervous system dysfunction including a decrease in the number of colonic neurons and damaged cell populations. In intestinal neurons, the activation of muscarinic acetylcholine receptors (mAChR) can lead to calcium ion influx and trigger 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, so the wheat peptide segment was molecularly docked with CHRM1 to screen small molecule peptides that may promote intestinal peristalsis.

[0049] According to the average local confidence (ALC) greater than 95%, the peak area greater than 2×10 6, the peptides with a PeptideRanker score greater than 0.8 were screened to obtain qualified peptides. The peptides were then docked with the M1-muscarinic acetylcholine receptor (CHRM1).

[0050] First, the crystal structure of CHRM1 (5CXV) was downloaded from the PDB protein database. The active center was defined by removing water molecules and adding hydrogen atoms from the receptor target using Discovery Studio software. The structure of the peptide segment of the selected wheat peptide was constructed by Discovery Studio, and its energy was minimized by the CHARMm force field, and these peptides were defined as ligands. The constructed peptide was docked with CHRM1 by CDOCKER to simulate the binding mode, site and amino acid residues with the lowest binding energy and the highest binding degree, and screened according to the binding energy and the number of hydrogen bonds. Finally, the hexapeptide TSVPFR (TR6) was determined, as shown in Table 4.

[0051] Table 4. Peptides in wheat peptides that promote intestinal motility 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 Specifically, the secondary mass spectrum of hexapeptide TR6 is as follows: Figure 2 As shown in the figure, the fragmentation ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z-type ions break to form d, v, and w-type ions, respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. 353.70m / z is the [M+H] + 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. The hexapeptide was further analyzed by secondary mass spectrometry using in-source collision-induced dissociation technology, and the primary structure of the hexapeptide was determined to be Thr-Ser-Val-Pro-Phe-Arg.

[0052] Molecular docking 2D and 3D images of TR6 and CHRM1 are shown in Figure 3As shown. Analysis of chemical bonds revealed that TR6 is mainly bound to CHRM1 through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and electrostatic interactions (π salt bridges and attractive charge). 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.

[0053] 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, thereby promoting intestinal peristalsis.

[0054] Shenzhen Borunsida Biotechnology Co., Ltd. was commissioned to synthesize the peptide TSVPFR with a purity of ≥98% for subsequent functional verification.

[0055] Example 4: Effect of hexapeptide TSVPFR on intestinal peristalsis in zebrafish constipation model (1) Effect of hexapeptide TSVPFR on the interval of intestinal peristalsis in zebrafish In this example, the time interval between two intestinal peristalsis peaks of zebrafish is measured to evaluate the intestinal peristalsis-promoting effect of the sample.

[0056] Wild-type AB zebrafish of 5 dpf were selected and placed in a six-well plate and divided into 8 groups, with 20 fish in each well. The specific groups were blank group (NC), constipation model group (MC), low-dose (10 μg / mL, WP-10) and high-dose (100 μg / mL, WP-100) groups of wheat peptide, and low-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 was added with the same volume of system water, the model group was added with 10 g / mL ropiramide hydrochloride, and the sample group was added with the corresponding dose of sample and ropiramide hydrochloride. After culturing in a constant temperature incubator at 28 ± 0.5 ℃ for 24 h, the intestinal peristalsis video of each group of zebrafish was recorded by stereomicroscope. After the experiment, the time interval between two intestinal peristalsis peaks of zebrafish was recorded by repeatedly watching the video.

[0057] This study used Graphpad Prism 8.0 software for statistical analysis and plotting, and all data were expressed as mean ± standard error. One-way analysis of variance was used to compare differences between groups (compared with the NC group, * P <0.05,** P <0.01,*** P <0.001; compared with the MC group, # P <0.05,## P <0.01,### P <0.001).

[0058] The results are as follows Figure 4 and Figure 5 As shown, the bowel movement interval in the MC group was significantly increased compared with the NC group ( P <0.05), indicating that the intestinal peristalsis disorder model was successfully established. Compared with the MC group, 10 μg / mL wheat peptide shortened the intestinal peristalsis interval, but did not reach a significant level ( P >0.05); when the concentration of wheat peptide was 100 μg / mL, it could significantly shorten the intestinal peristalsis interval ( P <0.01). Compared with the MC group, when the concentration of hexapeptide TR6 was 0.5 μg / mL and 1 μg / mL, the intestinal peristalsis interval was shortened, but it did not reach a significant level ( P >0.05); when the concentration of hexapeptide TR6 was 5 μg / mL and 10 μg / mL, it could significantly shorten the intestinal peristalsis interval ( P <0.01).

[0059] Based on the above data, it can be seen that from the perspective of intestinal peristalsis time interval, the effect of hexapeptide TR6 in promoting intestinal peristalsis is about 20 times that of wheat peptide.

[0060] (2) Effect of hexapeptide TSVPFR on the intestinal motility promotion rate of zebrafish The fluorescent dye Nile red is not interfered by other tissues in the zebrafish and is not absorbed. In this example, the intestinal motility-promoting effect can be evaluated by quantifying the amount of Nile red in the gastrointestinal tract of the zebrafish.

[0061] 5 dpf zebrafish were selected for Nile red dyeing, and the zebrafish were cultured in the dark for 16 h. The dye was then washed off with system water, and the zebrafish were transferred to a 6-well plate and divided into 7 groups, with 20 fish per well. The specific groups were blank group (NC), low-dose (10 μg / mL, WP-10) and high-dose (100 μg / mL, WP-100) groups of wheat peptide, and 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 was added with the same volume of system water, and the sample group was added with samples of the corresponding dose. After culturing in the dark for 24 h, the same magnification, fluorescence intensity, exposure time and fluorescence gain were selected, and the images were taken under a stereo fluorescence microscope. The total fluorescence signal of the zebrafish intestine was analyzed using NIS-ElementsD 3.10 advanced image processing software, and the effect of the sample on the intestinal peristalsis of zebrafish was judged by the amplitude of the fluorescence intensity reduction before and after. The intestinal peristalsis promotion rate was calculated as the ratio of (blank group fluorescence intensity - sample group fluorescence intensity) to the blank group fluorescence intensity.

[0062] The results are as follows Figure 6-Figure 8 As shown in the figure, compared with the NC group, 10 μg / mL wheat peptide increased the intestinal motility promotion rate, but it did not reach a significant level ( P >0.05); when the concentration of wheat peptide was 100 μg / mL, it could significantly increase the intestinal motility promotion rate ( P <0.001). Compared with the NC group, when the concentration of hexapeptide TR6 was 0.5 μg / mL, the intestinal motility promotion rate was increased, but it did not reach a significant level ( 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 intestinal motility promotion rate ( P <0.01, P <0.001, P <0.001).

[0063] Based on the above data, it can be seen that from the perspective of intestinal motility promotion rate, the intestinal motility promoting effect of hexapeptide TR6 is about 100 times that of wheat peptide.

[0064] In summary, the present invention screened out the hexapeptide TSVPFR from wheat peptides through mass spectrometry identification and molecular docking of peptide segments. The peptide segment has the effect of promoting intestinal peristalsis, which is mainly reflected in the intestinal peristalsis disorder model. Compared with the model group, the hexapeptide TSVPFR treatment can significantly shorten the intestinal peristalsis peak time interval; in the zebrafish model, compared with the blank group, the hexapeptide TSVPFR treatment can significantly improve the intestinal peristalsis promotion rate. The present invention provides a theoretical basis for the development of new active substances that promote intestinal peristalsis, and can be applied to foods or drugs that improve intestinal peristalsis. The hexapeptide TSVPFR can be used alone to prepare foods or drugs that improve intestinal peristalsis, or it can be compounded with other active ingredients that have the function of promoting intestinal peristalsis. The active components that promote the intestinal peristalsis function can be but are not limited to dietary fiber, probiotics or other protein peptides. The food or 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 used in the food industry and health care fields.

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 protein.

3. Use of the hexapeptide TR6 according to claim 1 in the preparation of a medicine or food 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 medicines or foods 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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