Preparation method and application of wheat oligopeptide with improved gastrointestinal function

Abstract

This invention discloses a method for preparing wheat oligopeptides that improve gastrointestinal function and their applications, relating to the technical field of preparing oligopeptides through enzymatic hydrolysis and their applications. The invention comprises wheat oligopeptides, a 5-HT4 receptor agonist, dietary fiber, prebiotics, and probiotics in a specific ratio, which can be formulated into powders, granules, or tablets for oral administration. It enhances propulsive peristalsis and secretion by activating the 5-HT4 pathway, while the oligopeptides provide a nutrient substrate to promote mucosal protein synthesis and barrier repair. The fiber / prebiotic / probiotic combination regulates the gut microbiota and produces short-chain fatty acids, thereby synergistically improving gastric emptying and small intestinal propulsion, improving defecation, and reducing the risk of recurrence.

Description

Technical Field

[0001] This invention relates to the field of preparing oligopeptides using enzymatic hydrolysis technology and the application technology of oligopeptides, specifically to a method for preparing and applying wheat oligopeptides that improve gastrointestinal function. Background Technology

[0002] Gastrointestinal dysfunction (such as functional dyspepsia and irritable bowel syndrome) is a prevalent digestive system disease worldwide, with its incidence rate showing an increasing trend year by year, seriously affecting patients' diet, sleep, and quality of life. The pathogenesis of these diseases is complex, related to multiple factors such as abnormal intestinal motility, impaired intestinal barrier function, intestinal microecological imbalance, and neurohumoral dysregulation. Currently, clinical and commercial products for improving gastrointestinal function mainly fall into categories such as prokinetic drugs, probiotic preparations, and dietary fiber supplements, but they suffer from problems such as single target of action, limited efficacy, and significant side effects for some drugs.

[0003] 5-Hydroxytryptamine (5-HT), an important neurotransmitter and regulator in the gut, has its type 4 receptor (5-HT4 receptor) widely distributed in gastrointestinal smooth muscle cells, intestinal epithelial cells, and the enteric nerve plexus. It plays a crucial role in regulating intestinal motility, promoting intestinal fluid secretion, and improving intestinal sensory function. 5-HT4 receptor agonists, by specifically binding to the 5-HT4 receptor, can effectively enhance intestinal smooth muscle contractility, accelerate intestinal transit, promote intestinal epithelial cell repair, and improve intestinal barrier integrity, thus becoming important targets for improving gastrointestinal motility disorders.

[0004] However, existing single 5-HT4 receptor agonists have significant limitations in application: on the one hand, some drugs have side effects such as diarrhea and headaches, and long-term use is poorly tolerated; on the other hand, they only target intestinal motility regulation, without considering intestinal microecological balance and nutrient supply, making it difficult to fundamentally solve the multifactorial pathogenesis of gastrointestinal dysfunction. Furthermore, while traditional probiotics and dietary fiber products can regulate intestinal flora or increase stool volume, they lack a direct effect on improving intestinal motility, resulting in poor synergistic effects.

[0005] Meanwhile, wheat oligopeptides, as small-molecule bioactive peptides, have the advantages of easy absorption, providing nutrition to intestinal cells, and enhancing intestinal barrier function. However, current technologies have not yet scientifically combined them with 5-HT4 receptor agonists, prebiotics, probiotics, etc., failing to fully leverage the synergistic effects of each component. Therefore, developing a composition with 5-HT4 receptor agonists as the core, synergistically incorporating wheat oligopeptides, prebiotics, probiotics, and other components to achieve multi-target synergistic improvement of gastrointestinal function through "dynamic regulation, microecological balance, and nutritional support," has become a key direction for overcoming the shortcomings of existing technologies and meeting clinical and market demands. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a method for preparing wheat oligopeptides that improve gastrointestinal function and their applications. This invention uses the wheat oligopeptide as the core, and combines it with a composite composition of soluble dietary fiber, prebiotics, freeze-dried probiotic powder, and a specific 5-HT4 receptor agonist to achieve a multi-dimensional synergistic effect of "intestinal motility regulation, microecological balance, nutritional support, and barrier repair," thereby enhancing the efficacy and safety of improving gastrointestinal function. Furthermore, this preparation method and composition can be widely applied in the fields of pharmaceuticals, food, health products, or food additives, meeting the application needs of different scenarios.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing wheat oligopeptides that improve gastrointestinal function includes the following steps: S1. After grinding wheat, defatting is performed to obtain defatted wheat flour. The defatted wheat flour is mixed with water, the pH is adjusted to 6.0-10.0, and the mixture is stirred and extracted, followed by solid-liquid separation to obtain an extract containing wheat protein. S2. Add protease to the extract to carry out enzymatic hydrolysis to obtain enzymatic hydrolysate; S3. The enzyme hydrolysate is heated to inactivate the enzyme and then clarified. S4. The clarified enzymatic hydrolysate is subjected to ultrafiltration separation, and the permeate with a molecular weight cutoff of less than 1 kDa is collected; S5. After desalting the permeate, spray dry or freeze dry to obtain the wheat oligopeptide product.

[0008] Furthermore, the enzyme preparation used in S2 is one or more of alkaline protease, neutral protease, and flavor protease in combination. The amount of enzyme added is 1-5% of the substrate protein mass. The enzymatic hydrolysis temperature is 45-55℃, the enzymatic hydrolysis pH is 7.5-9.0, the enzymatic hydrolysis time is 2-6 hours, and the degree of hydrolysis at the end of the enzymatic hydrolysis is 15-35%. In step S4, an ultrafiltration membrane with a molecular weight cutoff of 0.5-1.0 kDa is used to obtain oligopeptide components mainly composed of 200-1000 Da.

[0009] Furthermore, the wheat oligopeptides are used to prepare pharmaceuticals, foods, health products, or food additives that improve gastrointestinal function.

[0010] A composition containing wheat oligopeptides to improve gastrointestinal function, comprising the following components in parts by weight: 5-50 parts wheat oligopeptides, 1-40 parts soluble dietary fiber, 1-30 parts prebiotics, 0.01-10 parts lyophilized probiotic powder, 0.5-1 part 5-HT4 receptor agonist, and 10-80 parts pharmaceutically or food-grade acceptable carrier.

[0011] The 5-HT4 receptor agonist is N-(1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-yl)-5-fluoro-2,3-dihydrobenzofuran-7-carboxamide.

[0012] Furthermore, the structure of the 5-HT4 receptor agonist is as follows: .

[0013] Furthermore, the preparation method of the 5-HT4 receptor agonist is as follows: .

[0014] Furthermore, the wheat oligopeptide is a wheat oligopeptide prepared by the above-described preparation method.

[0015] Furthermore, the prebiotic is selected from one or more of inulin, fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, and mannan-oligosaccharides.

[0016] Furthermore, the probiotic freeze-dried powder contains live bacteria of the genus Lactobacillus and / or Bifidobacterium, with a live bacteria count of 1 × 10⁻⁶. 8 -1×10 11 CFU / g.

[0017] Furthermore, the soluble dietary fiber is selected from one or more of resistant dextrin, polydextrose, pectin, β-glucan, guar gum, and gum arabic.

[0018] Furthermore, the carrier is one or more of maltodextrin, dextrin, glucose, starch, sucrose, lactose, mannitol, erythritol, microcrystalline cellulose, etc.

[0019] Furthermore, the method for preparing the composition containing wheat oligopeptides to improve gastrointestinal function is as follows: A1. The 5-HT4 receptor agonist and 1 / 3 of the carrier are mixed evenly using an equal-volume incremental method, and then passed through a 60-80 mesh sieve to obtain a drug premix. A2. Mix the wheat oligopeptides, soluble dietary fiber, prebiotics and 2 / 3 of the carrier evenly, then add the drug premix and mix again; A3. After the temperature of the material obtained in S3 drops below 25°C, add the probiotic freeze-dried powder, mix at low speed until uniform in an environment with a relative humidity of less than 45%, discharge, and package to obtain the composition containing wheat oligopeptides that improves gastrointestinal function.

[0020] Application of a composition containing wheat oligopeptides that improves gastrointestinal function in the preparation of pharmaceuticals and health products that improve gastrointestinal function.

[0021] A composition containing wheat oligopeptides that improves gastrointestinal function can be used in the manufacture of biopharmaceuticals.

[0022] A composition containing wheat oligopeptides to improve gastrointestinal function can also be added to products made from seafood such as astaxanthin and lutein.

[0023] The formula described in this invention systematically addresses the coexisting technical problems of "insufficient motility, damaged barrier, dysbiosis, and insufficient nutrient supply" in existing gastrointestinal dysfunction through a multi-target, hierarchical synergistic mechanism. Specifically, wheat oligopeptides, in their small molecular form of 200-1000 Da, are easily absorbed by the intestinal epithelium, directly providing nitrogen and functional amino acids to intestinal mucosal cells and enteric nerve cells, promoting intestinal epithelial repair and enhancing tight junction protein expression, thereby improving intestinal barrier function. Soluble dietary fiber, through water absorption, swelling, and fermentation to produce short-chain fatty acids, enhances the volume of intestinal contents and the mechanical stimulation of the intestinal wall. Simultaneously, it provides metabolic substrates for beneficial bacteria; the prebiotic and probiotic freeze-dried powder form a "substrate-strain" synergistic system, rapidly rebuilding the beneficial bacteria structure centered on Lactobacillus and Bifidobacterium, inhibiting the growth of pathogenic bacteria and regulating the local immune microenvironment; on this basis, a low-dose 5-HT4 receptor agonist specifically activates 5-HT4 receptors on the enteric nerve plexus and smooth muscle cells, significantly enhancing intestinal propulsive peristalsis and secretory function. Its dosage is controlled within the synergistic effective range, thus exerting a prokinetic effect under the aforementioned nutritional support and microecological stability, significantly reducing the risk of side effects such as diarrhea caused by single-drug use. The above components are mutually matched and amplified within the mass fraction range, ultimately forming a synergistic closed loop of "motor regulation - microecological balance - barrier repair - nutritional support," comprehensively solving the multifactorial technical challenges of gastrointestinal dysfunction from a mechanistic perspective.

[0024] Compared with the prior art, the beneficial effects of the present invention are: 1. Compared with existing technologies that tend to rely on "single prokinetic drugs" or "single dietary fiber / probiotic conditioning", this invention directly enhances gastrointestinal propulsion through the 5-HT4 pathway, showing a more significant improvement trend in gastric emptying and small intestinal propulsion.

[0025] 2. The introduction of nutrients / functional components such as wheat oligopeptides not only improves motility but also promotes colon tissue protein synthesis and barrier repair, demonstrating the trend advantage of "improving symptoms + improving baseline condition" rather than just short-term stimulation.

[0026] 3. Fiber, prebiotics, probiotics, and oligopeptides provide a foundation for the regulation of the microecology and inflammation, complementing and synergizing with 5-HT4 prokinetic agents to achieve a more balanced overall effect. This approach is expected to reduce dependence on high-dose single prokinetic agents, thereby improving safety and compliance. Attached Figure Description

[0027] Figure 1This is a photograph of the N-(1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-yl)-5-fluoro-2,3-dihydrobenzofuran-7-carboxamide described in this invention.

[0028] Figure 2 This is a Western blot diagram showing the effects of the 5-HT4 receptor agonist and wheat oligopeptide described in this invention on 5-HT4 receptor expression.

[0029] Figure 3 This is a statistical analysis of the relative protein expression levels of the effects of the 5-HT4 receptor agonist and wheat oligopeptide described in this invention on 5-HT4 receptor expression.

[0030] Figure 4 This is the 1H NMR spectrum of compound 1-A described in this invention.

[0031] Figure 5 This is the 1H NMR spectrum of compound 2-A described in this invention.

[0032] Figure 6 The image shows the 1H NMR spectrum of the 5-HT4 receptor agonist described in this invention. Detailed Implementation

[0033] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Preparation Example 1 Preparation of 5-HT4 receptor agonists: ; Add 60 ml of acetonitrile, 5 g of compound 1-1, and 8.63 g of potassium carbonate to a three-necked flask equipped with a mechanical stirrer, reflux condenser, and nitrogen protection. Stir until homogeneous, then purge the air three times with nitrogen. Add 4.39 g of compound 1-2 and 0.41 g of potassium iodide to one of the purgings. Under a continuous nitrogen flow, heat to 80.0°C and reflux for 12 hours. Cool to room temperature, filter to remove inorganic salts, wash the filter cake with a small amount of acetonitrile, concentrate the filtrate under reduced pressure until dry, and recrystallize from ethyl acetate / petroleum ether (1:1). Filter and dry to obtain 6.46 g of compound 1-A. Compound 1-1 is: tert-butylpiperidine-4-ylcarbamate; Compounds 1-2 are: 1-(3-chloropropyl)-1H-tetrazole; Compound 1-A is: tert-butyl(1-(3-(1H-1,2,3,4-tetrazol-1-yl)propyl)piperidin-4-yl)carbamate; Mass spectrometry of compound 1-A (MS+1): 311.

[0035] Add 80 ml of dichloromethane and 6.46 g of compound 1-A to a three-necked flask equipped with a mechanical stirrer, reflux condenser, and nitrogen protection. Cool to 0°C and slowly add 23.8 ml of compound 2-2 dropwise, controlling the internal temperature to not exceed 10°C. After the addition is complete, allow the mixture to rise naturally to room temperature and react for 4 hours. Remove most of the dichloromethane and trifluoroacetic acid by rotary evaporation under reduced pressure. Add 50 ml of dichloromethane to redissolve the compound and evaporate to dryness again. Repeat this process twice to remove residual TFA, yielding 4.31 g of a brown, viscous, oily compound 2-A, which can be used directly in the next step.

[0036] Compound 2-2 is trifluoroacetic acid; Compound 2-A is: 1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-amine; Mass spectrometry of compound 2-A (MS+1): 211.

[0037] 50 mL of DMF and 3.73 g of compound 3-1 were added to a three-necked flask equipped with a mechanical stirrer, reflux condenser, and nitrogen protection. After stirring until homogeneous, 10.60 g of DIPEA was added, followed by stirring until homogeneous again. Then, 9.35 g of HATU was added, and the mixture was activated at room temperature for 15 minutes. 4.31 g of compound 2-A was slowly added, and the reaction was stirred at room temperature for 16 hours. The reaction mixture was slowly poured into ice water (250 mL), and stirred vigorously for 30 minutes. The pH was adjusted to between 8 and 9 with saturated NaHCO3 solution. The mixture was extracted with ethyl acetate (EA, 100 mL × 3). The organic phases were combined and washed successively with water (100 mL × 2) and saturated brine (100 mL) (to thoroughly remove DMF). The mixture was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography using a gradient elution of DCM:MeOH = 40:1 to 20:1. The product fraction was collected, concentrated, and dried under vacuum to obtain 5.94 g of a 5-HT4 receptor agonist. Compound 3-1 is: 5-fluoro-2,3-dihydrobenzofuran-7-carboxylic acid; The 5-HT4 receptor agonist is: N-(1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-yl)-5-fluoro-2,3-dihydrobenzofuran-7-carboxamide; Mass spectrometry (MS+1:375) of 5-HT4 receptor agonists.

[0038] See the physical image of N-(1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-yl)-5-fluoro-2,3-dihydrobenzofuran-7-carboxamide. Figure 1.

[0039] The 1H NMR spectrum of compound 1-A is shown below. Figure 4 As shown; The 1H NMR spectrum of compound 2-A is shown below. Figure 5 As shown; The 1H NMR spectrum of 5-HT4 receptor agonists, as shown below. Figure 6 As shown.

[0040] Preparation Example 2 Preparation of a wheat oligopeptide that improves gastrointestinal function: S1: High-quality wheat grains are cleaned and impurities are removed. Wheat coarse flour is prepared using an ultrafine pulverizer and passed through an 80-mesh sieve to obtain uniformly sized wheat flour. The wheat flour is placed in a Soxhlet extractor, and defatted with n-hexane as the defatting solvent at a material-to-liquid ratio of 1:8 (mass-to-volume ratio, g / mL). Reflux at 60℃ for 3 hours. After defatting, residual solvent is removed by rotary evaporation, and the mixture is vacuum dried (50℃, -0.09MPa) to constant weight to obtain defatted wheat flour. 100g of the defatted wheat flour is added to 800mL of deionized water and stirred until evenly dispersed. The pH of the system is adjusted to 7.5 using 1mol / L NaOH solution. The mixture is placed in a constant-temperature water bath and extracted at 50℃ with stirring for 2 hours, stirring once every 20 minutes for 5 minutes each time. After extraction, the mixture is placed in a high-speed centrifuge and centrifuged at 8000r / min for 20 minutes. The supernatant is taken as the extract containing wheat protein. S2: Add a complex protease (alkaline protease to flavor protease in a mass ratio of 1:1) to the above wheat protein extract. The amount of enzyme added is 3% of the substrate protein in the extract. Adjust the pH of the system to 8.0, place it in a constant temperature water bath shaker, and enzymatically hydrolyze it at 50℃ and 150r / min for 4 hours. Take samples every 1 hour during the enzymatic hydrolysis process and determine the degree of hydrolysis using the ninhydrin colorimetric method. When the degree of hydrolysis reaches 25%, terminate the enzymatic hydrolysis reaction to obtain the enzymatic hydrolysate. S3 rapidly heats the above enzymatic hydrolysate to 90°C and holds it at that temperature for 15 minutes to inactivate the enzyme. After inactivation, the hydrolysate is allowed to cool naturally to room temperature. 0.2% (by mass / volume) of diatomaceous earth is added to the system, stirred evenly, and allowed to stand for 30 minutes. Then, the hydrolysate is filtered using a plate and frame filter to remove the diatomaceous earth and insoluble impurities, and the clear hydrolysate is collected. S4: The clarified enzymatic hydrolysate is passed into an ultrafiltration system. A ceramic ultrafiltration membrane with a molecular weight cutoff of 0.8 kDa is selected, and ultrafiltration separation is performed under the conditions of operating pressure of 0.3 MPa and temperature of 40℃. The ultrafiltration permeate is collected, and the molecular weight distribution of peptides in the permeate is detected by gel permeation chromatography. More than 90% of the peptides have a molecular weight in the range of 200-1000 Da. S5: The ultrafiltration permeate is passed through an ion exchange resin column (a 732-type cation exchange resin and a 717-type anion exchange resin connected in series) for desalination. The flow rate is controlled at 1 BV / h, and the desalinated liquid is collected. The conductivity of the desalinated liquid is measured using a conductivity meter. When the conductivity is ≤50 μS / cm, the desalination is considered qualified. The qualified desalinated permeate is dried using a spray dryer with an inlet air temperature of 180℃, an outlet air temperature of 80℃, and a feed flow rate of 15 mL / min. After drying, the powdered product is collected. Alternatively, it can be dried using a freeze dryer with a freezing temperature of -40℃ and a vacuum of 10 Pa until constant weight is obtained to obtain the wheat oligopeptide product. The wheat oligopeptide product is a white to light yellow uniform powder with no odor and good water solubility. The protein content is ≥85%, the proportion of oligopeptides with a molecular weight of 200-1000 Da is ≥92%, the moisture content is ≤5%, and the ash content is ≤3%.

[0041] Preparation Example Performance Testing: Human colonic smooth muscle cells (HCSMCs) in the logarithmic growth phase were randomly divided into 5 groups: 5-HT4 receptor agonist group (N-(1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-yl)-5-fluoro-2,3-dihydrobenzofuran-7-carboxamide), wheat oligopeptide group, 5-HT4 receptor agonist + wheat oligopeptide group, positive control group 1 (mosapride), positive control group 2 (cisapride), and a blank control group (cell culture medium only). The blank control group used DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-drug antibodies; the 5-HT4 receptor agonist group, wheat oligopeptide group, combination drug group (10 μg 5-HT4 receptor agonist + 90 μg wheat oligopeptide group), positive drug control group 1 (mosapride), and positive drug control group 2 (cisapride) used DMEM / F12 medium containing 100 μg / ml 10% fetal bovine serum and 1% penicillin-drug antibodies. Cells in each group were incubated at 37℃ in a 5% CO2 incubator for 24 h. Cells were then lysed on ice for 30 min using RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors), centrifuged at 12000 r / min for 15 min, and the supernatant was collected. Protein concentration was determined and standardized using the BCA method. Equal amounts of protein samples were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4℃ with rabbit anti-human 5-HT4 receptor primary antibody (1:1000 dilution) and internal control protein GAPDH primary antibody (1:5000 dilution). After washing the membrane three times with TBST, HRP-labeled goat anti-rabbit secondary antibody (1:2000 dilution) was added, and the membrane was incubated at room temperature for 1 h. The membrane was washed three more times with TBST. The membranes were developed using an ECL chemiluminescence kit, and the gray ratio of the 5-HT4 receptor protein band to the internal control GAPDH band was quantitatively analyzed using ImageJ software. The relative expression level of 5-HT4 receptor in each group was calculated using the blank control group as a baseline.

[0042] Western blot quantitative results are shown below Figure 2 and Figure 3 Compared with the blank control, both 5-HT4 receptor agonists and wheat oligopeptides can upregulate 5-HT4R protein expression, with the combined treatment showing the greatest upregulation. The two positive control drugs, mosapride and cisapride, can also significantly increase 5-HT4R expression, with the combined use of these drugs having the strongest promoting effect on 5-HT4R expression.

[0043] Example 1 Preparation of a composition containing wheat oligopeptides to improve gastrointestinal function: 1. Raw material composition by weight: 25 parts of wheat oligopeptides (prepared by the method described in Preparation Example 2); 15 portions of soluble dietary fiber (resistant dextrin to polydextrose mass ratio 2:1); 10 servings of prebiotics (fructooligosaccharides to xylooligosaccharides in a 1:1 mass ratio). Two portions of freeze-dried probiotic powder (containing live Bifidobacterium and Lactobacillus strains, with a live count ≥ 5 × 10¹) 0 CFU / g); 0.5 parts of 5-HT4 receptor agonist (prepared by the method described in Preparation Example 1); 47.2 parts of carrier (maltodextrin to microcrystalline cellulose mass ratio 3:1).

[0044] 2. Preparation method: A1. Take 0.8 parts of the 5-HT4 receptor agonist according to the formula and mix it with 1 / 3 parts by weight of the carrier (15.73 parts, 11.8 parts of maltodextrin + 3.93 parts of microcrystalline cellulose) in an equal increment method until uniform. Pass the mixture through a 70-mesh sieve to ensure that the material is uniformly dispersed and there is no obvious particle agglomeration, and obtain the drug premix. A2. Take 25 parts of wheat oligopeptides, 15 parts of soluble dietary fiber, and 10 parts of prebiotics according to the formula, and add them to the remaining 2 / 3 of the carrier (31.47 parts, 23.6 parts of maltodextrin + 7.87 parts of microcrystalline cellulose) in a three-dimensional mixer. Set the speed to 25 r / min and mix for 15 min. Then add the drug premix prepared in step A1 and continue mixing for 20 min to ensure that all components are fully mixed. A3. Transfer the mixture obtained in step A2 to a low-temperature dryer and cool it to below 22°C; in a clean environment with a relative humidity of 35%, add 2 parts of the prescribed amount of probiotic freeze-dried powder and put it into a V-type mixer. Set the speed to a low speed of 15 r / min and mix for 8 minutes to avoid inactivation of probiotics due to shear force or high temperature; after mixing, immediately perform aseptic packaging to obtain the finished product of the composition containing wheat oligopeptides to improve gastrointestinal function.

[0045] Example 2 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function is carried out according to the preparation method of Example 1, except that the mass fraction of wheat oligopeptides is replaced with 45 parts, and the rest remains the same as in Example 1.

[0046] Example 3 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function is carried out according to the preparation method of Example 1, except that the mass fraction of the 5-HT4 receptor agonist is replaced with 1 part, and the rest is the same as in Example 1.

[0047] Comparative Example 1 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function is carried out by referring to the preparation method of Example 1, except that the mass fraction of wheat oligopeptides is replaced with 4 parts, and the rest remains the same as in Example 1.

[0048] Comparative Example 2 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function is carried out according to the preparation method of Example 1, except that the mass fraction of the 5-HT4 receptor agonist is replaced with 0.3 parts, and the rest remains the same as in Example 1.

[0049] Comparative Example 3 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function was carried out according to the preparation method of Example 1, except that the 5-HT4 receptor agonist was replaced with mosapride, and the rest remained the same as in Example 1.

[0050] Comparative Example 4 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function was carried out according to the preparation method of Example 1, except that the 5-HT4 receptor agonist was not added, and the rest remained the same as in Example 1.

[0051] Comparative Example 5 The preparation of a composition containing wheat oligopeptides to improve gastrointestinal function is carried out according to the preparation method of Example 1, except that the wheat oligopeptides are not added, and the rest is the same as in Example 1.

[0052] Performance tests of the examples and comparative examples: Examples 1-3 and Comparative Examples 1-5 were all ground into a fine powder and prepared into a suspension with a concentration of 100 mg / mL using physiological saline. Forty-eight SPF-grade SD rats (half male, half female, weighing 200-220 g) were selected and, after one week of acclimatization, randomly divided into eight groups (Example 1-3 groups and Comparative Examples 1-5 groups), with six rats in each group. A rat model of gastrointestinal dysfunction (simulating functional dyspepsia / irritable bowel syndrome) was established using a "complex stress method." Restraint stress: Rats were restrained in a cage for 2 hours every morning for 14 days. Dietary intervention: Free access to drinking water containing 0.1% sodium deoxycholate, while feeding a high-fat diet (30% fat content) for 14 days; Model validation: After modeling, the gastric emptying rate (phenol red method) and small intestinal propulsion rate (charcoal powder method) of rats were measured to confirm the successful construction of the model (both gastric emptying rate and small intestinal propulsion rate were significantly lower than those of normal rats).

[0053] 1. Intestinal motility test: After successful modeling, rats in each group were administered the drug by gavage at a dose of 10 mL / kg body weight, while the blank control group was administered an equal volume of physiological saline by gavage. The administration was once daily for 14 consecutive days. Two hours after the last administration, each group of rats was administered 1 mL of a nutritional paste containing 0.05% phenol red (10% gum arabic + 10% starch + 0.05% phenol red) by gavage. Thirty minutes after gavage, the rats were euthanized by cervical dislocation, the stomach was quickly separated, the outer wall of the stomach was rinsed with physiological saline, the stomach cavity was cut open, the stomach contents were collected, 5 mL of 0.1 mol / L NaOH solution was added and soaked for 30 minutes, then centrifuged (3000 r / min, 10 min) after thorough shaking. The supernatant was collected, and the absorbance (A value) was measured at 560 nm using a spectrophotometer. At the same time, the absorbance (A0 value) of the standard phenol red solution (same concentration) was measured. The gastric emptying rate was calculated as follows: Gastric emptying rate (%) = (1 - A value of phenol red in stomach contents / A0 value of standard phenol red) × 100%. The data are shown in Table 1.

[0054] Small intestinal propulsion rate determination (charcoal method): 2 hours after the last administration, each group of rats was administered 1 mL of physiological saline suspension containing 10% activated charcoal by gavage; 20 minutes after gavage, the rats were euthanized by cervical dislocation, and the small intestine (from the pylorus to the ileocecal junction) was quickly separated, laid flat on filter paper, and the total length of the small intestine (L1) and the length of the frontal propulsion of the activated charcoal (L2) were measured; the small intestinal propulsion rate was calculated as follows: small intestinal propulsion rate (%) = (L2 / L1) × 100%, and the data are shown in Table 1.

[0055] 2. Intestinal protein concentration test: After the intestinal motility test, colon tissue was quickly separated, fixed in 4% paraformaldehyde, and partially frozen in liquid nitrogen. The frozen colon tissue was taken, RIPA lysis buffer (containing protease inhibitor) was added, and the tissue was ground and lysed on ice for 30 min. The supernatant was collected by centrifugation (12000 r / min, 15 min), and the protein concentration was determined by BCA method. The data are shown in Table 1.

[0056] Table 1

[0057] Table 1 shows the synergistic effect of the three indicators: "motility enhancement, repair / nutrition, and microecology." 5-HT4 receptor agonists directly enhance the release of cholinergic neurotransmitters and propulsive peristalsis in the intestinal wall, thereby simultaneously increasing gastric emptying rate and small intestinal propulsion rate. Therefore, Example 3 with increased dosage showed the highest levels of both motility indicators, while the control example 4 lacking 5-HT4 showed the lowest motility. Wheat oligopeptides mainly provide absorbable nitrogen sources and small molecule peptide signals, promoting mucosal cell protein synthesis / barrier repair. Therefore, Example 2 with increased oligopeptides showed the highest BCA protein concentration in colonic tissue, while significantly reduced or absent oligopeptides... The comparative example 1 / 5 showed the lowest protein concentration and limited improvement in motility. Meanwhile, dietary fiber, prebiotics, and probiotics improved the gut microbiota structure and short-chain fatty acid production, reduced inflammation, and enhanced the mucus layer, creating a "background gain" for motility and repair. This allowed Example 1 to achieve a more balanced performance across the three indicators without extreme dosage increases. In contrast, Comparative Example 3, using mosapride, achieved a near-improvement in motility, but its effect on tissue protein recovery was not as good as the combination of "oligopeptides + specific 5-HT4 agonists + microecological base," demonstrating the synergistic advantage of this formula in integrating "propulsion" and "repair."

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composition comprising wheat oligopeptides to improve gastrointestinal function, characterized in that, It contains the following components by weight: 5-50 parts wheat oligopeptides, 1-40 parts soluble dietary fiber, 1-30 parts prebiotics, 0.01-10 parts freeze-dried probiotic powder, 0.5-1 part 5-HT4 receptor agonist, and 10-80 parts pharmaceutically or food-grade acceptable carrier. The 5-HT4 receptor agonist is N-(1-(3-(1H-tetrazol-1-yl)propyl)piperidin-4-yl)-5-fluoro-2,3-dihydrobenzofuran-7-carboxamide.

2. The composition containing wheat oligopeptides to improve gastrointestinal function according to claim 1, characterized in that, The preparation method of the wheat oligopeptides includes the following steps: S1. After grinding wheat, defatting is performed to obtain defatted wheat flour. The defatted wheat flour is mixed with water, the pH is adjusted to 6.0-10.0, and the mixture is stirred and extracted, followed by solid-liquid separation to obtain an extract containing wheat protein. S2. Add protease to the extract to carry out enzymatic hydrolysis to obtain enzymatic hydrolysate; S3. The enzyme hydrolysate is heated to inactivate the enzyme and then clarified. S4. The clarified enzymatic hydrolysate is subjected to ultrafiltration separation, and the permeate with a molecular weight cutoff of less than 1 kDa is collected; S5. After desalting the permeate, spray dry or freeze dry to obtain the wheat oligopeptide product.

3. The composition comprising wheat oligopeptides for improving gastrointestinal function according to claim 2, characterized in that, The enzyme preparation used in S2 is one or more of alkaline protease, neutral protease, and flavor protease in combination. The amount of enzyme added is 1-5% of the substrate protein mass. The enzymatic hydrolysis temperature is 45-55℃, the enzymatic hydrolysis pH is 7.5-9.0, the enzymatic hydrolysis time is 2-6 hours, and the degree of hydrolysis at the end of the enzymatic hydrolysis is 15-35%. In step S4, an ultrafiltration membrane with a molecular weight cutoff of 0.5-1.0 kDa is used to obtain oligopeptide components mainly composed of 200-1000 Da.

4. The composition comprising wheat oligopeptides for improving gastrointestinal function according to claim 1, characterized in that, The wheat oligopeptide is a wheat oligopeptide prepared by the preparation method described in claim 1.

5. A composition comprising wheat oligopeptides to improve gastrointestinal function according to claim 1, characterized in that, The prebiotics are selected from one or more of inulin, fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, and mannan-oligosaccharides.

6. A composition comprising wheat oligopeptides for improving gastrointestinal function according to claim 1, characterized in that, The probiotic freeze-dried powder contains live bacteria of the genus Lactobacillus and / or the genus Bifidobacterium, with a live bacteria count of 1 × 10⁻⁶. 8 -1×10 11 CFU / g.

7. A composition comprising wheat oligopeptides for improving gastrointestinal function according to claim 1, characterized in that, The soluble dietary fiber is selected from one or more of resistant dextrin, polydextrose, pectin, β-glucan, guar gum, and gum arabic.

8. A composition comprising wheat oligopeptides for improving gastrointestinal function according to claim 1, characterized in that, The carrier is one or more of maltodextrin, dextrin, glucose, starch, sucrose, lactose, mannitol, erythritol, and microcrystalline cellulose.

9. The use of a composition comprising wheat oligopeptides for improving gastrointestinal function as described in any one of claims 1-8 in the field of preparing a pharmaceutical product for improving gastrointestinal function.

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