A method for preparing osteopontin peptides based on in-vitro dynamic simulation of a digestive system and application thereof

Abstract

The present application relates to a kind of preparation of bone bridge protein peptide based on in-vitro dynamic simulation digestion system method and its application, belong to the technical field of infant milk product.The method of the present application comprises the following steps: S1, by esophageal device, bone bridge protein solution is injected into stomach model, and start extrusion device to contract stomach model, duodenum model and small intestine model, and start simulation digestion;S2, inject gastric simulation fluid and HCl solution into stomach model during simulation digestion, inject intestinal simulation fluid and NaHCO3 solution into duodenum model;Gastric simulation fluid is dissolved in water by NaCl, KCl and pepsin, and pH is adjusted to 2.5-3.5;Intestinal simulation fluid is dissolved in water by NaCl, KCl, CaCl2, trypsin and bile salt, and pH is adjusted to 6.2-6.8;S3, collect the digesta flowing from small intestine model, after enzyme inactivation, centrifugation, desalination, obtain bone bridge protein peptide, and the bone bridge protein peptide prepared has excellent chelating activity to calcium ion, ferrous ion and zinc ion, has potential ability to promote calcium, ferrous and zinc absorption.

Description

Technical Field

[0001] This invention belongs to the field of infant formula technology, and particularly relates to a method for preparing osteopontin peptides based on an in vitro dynamic simulation of the digestive system and its application. Background Technology

[0002] Osteopontoxin is a naturally occurring protein widely distributed in human body fluids, with the highest concentration found in breast milk. Osteopontoxin derived from the milk of various mammals, including human milk, is called milk-derived osteopontin, or simply lactopontin. In recent years, scientific research has conducted in-depth studies on the structure, function, and safety of milk-derived osteopontin, and it has achieved industrial application in some countries and regions. Scientific research has shown that after humans ingest protein, it is not primarily absorbed in the form of amino acids after digestion by enzymes, but also in the form of peptides. Some peptides not only provide the nutrients needed for human growth and development but also have various physiological functions, which are determined by the amino acid sequence. Therefore, further enzymatically hydrolyzing osteopontin into osteopontin peptides is beneficial for more in-depth research on peptide activity and absorption.

[0003] In vitro digestion simulation experiments are an important method for understanding the digestive behavior and characteristics of food, pharmaceuticals, or novel functional products. To date, the main method for studying the digestive properties of food is the use of in vitro digestion models, which simulate the physiological conditions of in vivo digestion. These models allow for the study of the structure, composition, digestive properties, stability, nutritional value, and digestibility of digestion products at different stages, offering advantages such as ease of operation and high reproducibility. Currently, research on the digestive properties of food primarily relies on in vitro digestion models, which are divided into two categories: static simulated digestion and dynamic simulated digestion.

[0004] In previous experiments, most researchers used static simulated digestion to enzymatically hydrolyze proteins. However, static digestion models have limitations in terms of food digestion and cannot fully simulate the dynamic digestive process in humans. These limitations are mainly manifested in the following ways: First, static simulated digestion does not simulate intestinal contractions and peristalsis, as well as gastric emptying, lacking experimental rigor. Second, it is difficult to control the injection rate of gastric and intestinal juices and the pH changes within the digestive tract during digestion, making it difficult to reflect the digestive characteristics of proteins. Third, in simulated digestion in infants, there is a lack of experimental parameters for the amount of digestive juice added, the injection rate, and the pH adjustment of the digestive tract. Furthermore, current technologies cannot effectively desalt digestion products, which is detrimental to subsequent separation, purification, and activity studies of osteopontin peptides. Moreover, existing desalting techniques are not suitable for large-scale processing of digestion products.

[0005] Therefore, the preparation of osteopontin peptides based on dynamic simulated digestion and enzymatic hydrolysis is of great significance. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system and its application. The method utilizes an in vitro dynamic simulated gastrointestinal digestive system, combined with parameters reflecting the digestive characteristics of infants, to simulate the digestion of osteopontin and prepare osteopontin peptides. The in vitro dynamic simulated gastrointestinal digestive system can simulate not only continuous pH changes and the sequential secretion of gastric and pancreatic juices, but also parameters such as gastric emptying or clearance of digestive products, and the peristaltic contractions of the stomach and small intestine. Furthermore, by controlling the injection rate and time of simulated gastric and intestinal fluids, as well as HCl and NaHCO3 solutions, the digestion process of osteopontin in infants is better reproduced. The final digestive products are desalted to enhance the chelation activity of the osteopontin peptides, which is beneficial for subsequent separation, purification, and activity studies.

[0007] The first objective of this invention is to provide a method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system. The method uses the in vitro dynamic simulated digestive system as the generating device. The in vitro dynamic simulated digestive system includes, in sequence, an esophageal device, a stomach model, a pyloric device, a duodenal model, and a small intestine model. The stomach model includes a gastric tilting device and a gastric peristalsis device; the small intestine model includes a small intestinal peristalsis device; and each of the stomach model, duodenal model, and small intestine model is equipped with a squeezing device. The method includes the following steps:

[0008] S1. Inject 120 mL of osteopontin solution into the stomach model through the esophageal device, and start the squeezing device to contract the stomach model, duodenal model and small intestine model, and begin to simulate digestion;

[0009] S2. During the simulated digestion process, a gastric simulation solution and an HCl solution are injected into the stomach model, and an intestinal simulation solution and a NaHCO3 solution are injected into the duodenal model. The gastric simulation solution is made by dissolving NaCl, KCl, and pepsin in water and adjusting the pH to 2.5-3.5. The intestinal simulation solution is made by dissolving NaCl, KCl, CaCl2, trypsin, and bile salts in water and adjusting the pH to 6.2-6.8.

[0010] S3. Collect the digestive material flowing out of the small intestine model, and obtain osteopontin peptides by enzyme inactivation, centrifugation, and desalting.

[0011] In one embodiment of the present invention, in S1, the mass concentration of the osteopontin solution is 4.8%-5.2%.

[0012] In one embodiment of the present invention, in S1, during the contraction process, the stomach model contracts 3 times / min, the duodenum model contracts 12 times / min, and the small intestine model contracts 6 times / min.

[0013] In one embodiment of the present invention, in S2, the concentration of NaCl in the gastric simulation solution is 94 mmol / L, the concentration of KCl is 13 mmol / L, and the activity of pepsin is 525 U / mL.

[0014] The concentration of the HCl solution is 1 mol / L;

[0015] The intestinal simulation solution contained NaCl at a concentration of 164 mmol / L, KCl at a concentration of 10 mmol / L, CaCl2 at a concentration of 3 mmol / L, trypsin activity at 20 U / mL, and bile salt concentration at 9.8 mmol / L.

[0016] The concentration of the NaHCO3 solution is 1 mol / L.

[0017] In one embodiment of the present invention, in S2, the injection process of the gastric simulated fluid is: 1 mL / min-1.1 mL / min, 0-10 min; 0.5 mL / min-1 mL / min, 10 min-180 min;

[0018] The HCl solution injection process is as follows: 0.5 mL / min - 1 mL / min, 1 min - 25 min;

[0019] The injection process for the intestinal simulation solution is: 0.5 mL / min - 1.2 mL / min, 1 - 180 min;

[0020] The injection process of the NaHCO3 solution is as follows: 0.5 mL / min-1 mL / min, 1 min-60 min; the injection rate and time of the gastric simulated fluid, intestinal simulated fluid, HCl solution, and NaHCO3 solution are controlled to better reproduce the digestive characteristics of osteopontin in infants.

[0021] In one embodiment of the present invention, in S2, the process parameters of the gastric tilt angle device are: -3° / min, 1min-6min; -0.3° / min, 6min-96min;

[0022] The process parameters of the gastric peristalsis device are as follows: the forward speed of the first stage is 500 mm / min, the return speed is 600 mm / min, and the running time is 6 min; the forward speed of the second stage is 500 mm / min, the return speed is 600 mm / min, and the running time is 175 min.

[0023] In one embodiment of the present invention, in S2, the process parameters of the small intestinal peristalsis device are: speed of 100 mm / min and running time of 181 min.

[0024] In one embodiment of the present invention, in S3, the resin used for desalination is DA201-C type macroporous adsorption resin.

[0025] In one embodiment of the present invention, in S3, the enzyme inactivation is performed at 92℃-98℃ for 8min-12min.

[0026] In one embodiment of the present invention, in S3, the centrifugation is performed at 3800 r / min-4200 r / min for 8 min-12 min.

[0027] In one embodiment of the present invention, before simulating digestion, 6 mL of gastric simulation solution without pepsin is injected into the gastric model to simulate the fasting state of the stomach.

[0028] In one embodiment of the invention, to reflect the physiological digestive status of infants (1-3 years old), several digestive parameters, including osteopontin intake, the composition of the simulated solution, the volume of the simulated solution, enzyme activity, and the pH of the gastrointestinal environment, were adjusted. In the Chinese dairy market, children's boxed / bottled liquid dairy products are commonly found in volumes of 120 mL, while adult products are typically 240 mL. Therefore, the "intake" of osteopontin solution was adjusted to 120 mL per serving. Furthermore, according to the standardized INFOGEST protocol for adult in vitro digestion, the ratio of simulated solution to food was maintained at 1:1 (by weight). Considering that infants have smaller stomach capacities, resulting in lower simulated solution secretion, the total amount of simulated solution injected in the in vitro infant digestion experiment was reduced to half the adult digestion volume. Additionally, given the difference in digestive capacity between infants and adults, the enzyme activities of pepsin and trypsin in the simulated solution were adjusted. Moreover, compared to adults, infants have a higher pH in their gastrointestinal environment; therefore, the injection volumes of HCl and NaHCO3 were adjusted to meet the pH requirements of infants' gastrointestinal environment.

[0029] A second objective of this invention is to provide an application of the osteopontin peptide prepared by the method in the preparation of products that promote the absorption of mineral elements.

[0030] In one embodiment of the present invention, the mineral element is selected from one or more of iron, calcium, and zinc.

[0031] In one embodiment of the present invention, calcium absorption is promoted, thereby promoting bone health, which is beneficial to growth and development, and can also prevent osteoporosis.

[0032] In one embodiment of the invention, iron absorption is promoted to prevent iron deficiency anemia, enhance immune system function, maintain energy metabolism and reduce fatigue, and support brain development and cognitive health.

[0033] In one embodiment of the present invention, by promoting zinc absorption, optimizing digestive function and appetite regulation, enhancing immune function and anti-infection ability, promoting vitamin A metabolism, and promoting growth and development and improving intelligence.

[0034] In one embodiment of the present invention, the osteopontin peptide has a calcium ion chelation content of 79.07±1.91μg / mg, a zinc ion chelation rate of 63.48±5.86%, and a ferrous ion chelation rate of 16.14±1.02%.

[0035] The technical solution of the present invention has the following advantages compared with the prior art:

[0036] The in vitro dynamic simulated digestive system described in this invention is based on the digestive environment and physiological processes in young children. It simulates the digestive process under similar conditions, providing not only digestive data under chemical conditions but also simulating physical processes. It can simulate dynamic gastrointestinal digestion processes, such as digestive enzyme secretion and pH changes, and through dynamic simulations of peristalsis and continuous gastric emptying of the digested sample, it can more realistically reproduce the bioavailability of osteopontin after human ingestion, resulting in more reliable experimental results. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0038] In this invention, unless otherwise stated, the DA201-C macroporous adsorption resin in the embodiments of this invention needs to be soaked in 95% anhydrous ethanol for 24 hours before use, filtered and set aside, and washed with anhydrous ethanol until A220nm<0.05 and then thoroughly rinsed with deionized water until there is no ethanol odor, to obtain the pretreated DA201-C macroporous adsorption resin.

[0039] Example 1

[0040] The present invention discloses a method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system. The method uses the in vitro dynamic simulated digestive system as the generating device. The in vitro dynamic simulated digestive system includes, in sequence, an esophageal device, a stomach model, a pyloric device, a duodenal model, and a small intestine model. The stomach model includes a gastric tilting device and a gastric peristalsis device; the small intestine model includes a small intestinal peristalsis device; and each of the stomach, duodenal, and small intestine models is equipped with a compression device. The method specifically includes the following steps:

[0041] S1. Dissolve NaCl, KCl and pepsin in 150 mL of primary water and adjust the pH to 3 to obtain a gastric simulation solution; the concentration of NaCl in the gastric simulation solution is 94 mmol / L, the concentration of KCl is 13 mmol / L, and the activity of pepsin is 525 U / mL.

[0042] NaCl, KCl, CaCl2, trypsin, and bile salts were dissolved in 210 mL of primary water, and the pH was adjusted to 6.5 to obtain an intestinal simulation solution. The concentration of NaCl in the intestinal simulation solution was 164 mmol / L, the concentration of KCl was 10 mmol / L, the concentration of CaCl2 was 3 mmol / L, the activity of trypsin was 20 U / mL, and the concentration of bile salts was 9.8 mmol / L.

[0043] Dissolve 6g of osteopontin in primary water and heat at 37°C to obtain an osteopontin solution with a mass concentration of 5% (w / w).

[0044] 40g of pretreated DA201-C macroporous adsorption resin was placed in a 500mL polyethylene bottle for later use.

[0045] S2. Before performing in vitro dynamic digestion simulation, adjust the ambient temperature of the equipment to 37°C, and use an injection pump to inject 6 mL of unenzymed gastric simulation solution into the gastric model to simulate the fasting state of the stomach.

[0046] S3. Inject 120 mL of osteopontin solution into the gastric model within 1 min through the funnel at the top of the esophageal device; the upper valve of the esophageal device opens for 0.5 min, the lower valve opens for 0.5 min, the speed is 200 rpm, and the running time is 10 min.

[0047] S4. Activate the pneumatically controlled squeezing device to generate 3 contractions / min on the stomach model, 12 contractions / min on the duodenum model, and 6 contractions / min on the small intestine model, and begin simulating digestion.

[0048] S5, gastric simulation solution and 25 mL of 1 mol / L HCl solution, and intestinal simulation solution and 50 mL of 1 mol / L NaHCO3 solution were injected into the gastric model and duodenal model respectively at a time-controlled rate;

[0049] The gastric simulated solution injection pump speeds are: 1 mL / min, 0-10 min, 0.5 mL / min, 10 min-180 min; the HCl solution injection pump speeds are: 0.5 mL / min, 1 min-25 min.

[0050] Gastric tilt angle device: -3° / min, 1min-6min; -0.3° / min, 6min-96min;

[0051] Gastric peristalsis device: The first stage has an advancing speed of 500 mm / min, a returning speed of 600 mm / min, and a running time of 6 min; the second stage has an advancing speed of 500 mm / min, a returning speed of 600 mm / min, and a running time of 175 min.

[0052] Duodenal model: 12 rpm, 120 min;

[0053] Duodenal valve: clamping time is 6 min, releasing time is 0 min, running time is 6 min; clamping time is 5 min, releasing time is 0.5 min, running time is 174 min;

[0054] Intestinal simulation solution injection pump speed: 0.8 mL / min, 1 min-180 min; NaHCO3 solution injection pump: 0.5 mL / min, 1 min-60 min;

[0055] Small intestinal peristalsis device: speed 100 mm / min, running time 181 min;

[0056] Small intestine valve: clamping time is 4.9 min, releasing time is 0.1 min, running time is 181 min;

[0057] S6. The pneumatically driven pyloric valve opens once per minute for 2 seconds, with an opening size of 0.45 mm; Pyloric device: the opening width of segment 1 is 4.5 mm, the segment speed is 100 mm / min, and the number of gastric compressions is 30; the opening width of segment 2 is 4.5 mm, the segment speed is 100 mm / min, the number of gastric compressions is 5, and the end point (clamping width) is 0.00 mm.

[0058] S7. During the in vitro dynamic digestion process, the digestive fluid flowing out of the small intestine model was collected in a beaker; after digestion for 180 min, the digestive fluid was placed at 95℃ for 10 min to inactivate enzymes, cooled to room temperature, and centrifuged at 4000 r / min for 10 min to collect the supernatant.

[0059] S8. Add 200 mL of supernatant to a polyethylene bottle containing 40 g of pretreated macroporous adsorption resin, and shake at 30 °C to allow the resin and supernatant to come into full contact. Collect the residual sample liquid in the beaker every 30 min, take the supernatant, and measure the absorbance value at 220 nm. The adsorption status can be inferred by measuring the absorbance A220 of the collected sample.

[0060] S9. Take the adsorbed resin and filter it using a vacuum filtration flask. Place the filtered resin in a polyethylene plastic bottle and use 75% ethanol solution as the eluent to elute the peptides in the macroporous resin. Place the bottle in a constant temperature shaker for room temperature desorption. Take samples every 15 minutes and measure the absorbance value at 220 nm. The desorption status can be inferred by measuring the absorbance A220 of the collected samples. After desorption, filter and collect the eluent, remove the ethanol by rotary evaporation, collect the residue and freeze-dry to obtain osteopontin peptide lyophilized powder.

[0061] Comparative Example 1

[0062] The process is basically the same as in Example 1, except that the digestion and enzymatic hydrolysis process includes the following steps:

[0063] S1. Add 200mL of water to a beaker, then add 47mL of 2mol / L NaCl solution, followed by 26mL of 0.5mol / L KCl solution and mix thoroughly. Adjust the pH of the solution to 5.3, then bring the volume to 400mL. Divide the solution into 50mL test tubes and freeze at -20℃. The resulting gastric simulated solution is 2.5 times its original volume. After thawing and dilution, it is ready for use.

[0064] Add 200 mL of water to a beaker, then add 82 mL of 2 mol / L NaCl solution, 20 mL of 0.5 mol / L KCl solution, and 85 mL of 1 mol / L NaHCO3 solution, and mix well. Adjust the pH of the solution to 6.6, then bring the volume to 400 mL. Divide the solution into 50 mL test tubes and freeze at -20°C. The resulting intestinal simulation solution is 2.5 times dilution. After thawing and dilution, it is ready for use.

[0065] S2. Simulating the gastric digestion stage of an infant: Weigh 25.2 mg of osteopontin and dissolve it in 12.6 mL of ultrapure water, add 6.9 mL of gastric simulation solution, and adjust the pH to 5.3 with 0.5 mol / L HCl. Then add 0.5 mL of pepsin solution, whose activity reaches 268 U / mL during the gastric digestion stage. Next, magnetically stir at 150 r / min in a shaker at 37 °C to simulate the gastric digestion of an infant. After incubation for 1 h, add 0.5 mol / L NaOH to adjust the pH to 7.0 to inactivate pepsin.

[0066] Simulating the small intestinal digestion stage of an infant: 10.635 mL of intestinal simulation solution was added to the obtained gastric digestion products, and the pH was adjusted to 6.6 with 0.1 mol / L HCl. Then, 1 mL of bile salts and 0.5 mL of trypsin were added, with their concentrations and activities set at 9.8 mmol / L and 16 U / mL, respectively. The mixture was then magnetically stirred at 150 r / min in a shaker at 37°C for 1 h, and finally heated in a boiling water bath for 10 min to terminate digestion.

[0067] Comparative Example 2

[0068] It is basically the same as Example 1, except that no desalination treatment is performed.

[0069] Test Example 1

[0070] The chelating activity of the osteopontin peptides prepared in Example 1 and Comparative Examples 1-2 was tested.

[0071] (1) Determination of calcium ion chelation activity: The osteopontin peptide lyophilized powders prepared in Example 1 and Comparative Examples 1-2 were dissolved in ultrapure water to prepare sample solutions with a concentration of 30 mg / mL. A CaCl2 solution with a mass concentration of 120 mg / mL was prepared according to a peptide-to-CaCl2 mass ratio of 1:1. The pH of the sample solution was adjusted with 1 mol / L NaOH and 1 mol / L HCl so that the pH of the chelation system after adding 1 mL of CaCl2 was 7.0. After chelation at 37℃ for 2 h, 6 times the volume of anhydrous ethanol was added, and the mixture was allowed to stand for 20 min. It was then centrifuged at 4000 r / min for 10 min, and the precipitate was the peptide-calcium chelate. The calcium ion content (μg / mg) in 1 mg of the peptide-calcium chelate was determined using flame atomic absorption spectrometry.

[0072] (2) Determination of zinc ion chelation activity: The osteopontin peptide lyophilized powders prepared in Example 1 and Comparative Examples 1-2 were dissolved in 40 mmol / L HEPES-KOH buffer to prepare a sample solution with a concentration of 1 mg / mL. 150 μL of the sample solution was placed in a 96-well plate and preheated at 37 °C for 10 min. After preheating, 75 μL of 8 mmol / L DTT solution and 250 μmol / L ZnSO4 solution were added, mixed evenly, and chelated at 37 °C for 30 min. After chelation, 15 μL of 2 mmol / L PAR solution was added to terminate the reaction. The absorbance was measured at 500 nm after 10 min. Deionized water was used as the blank group. Zinc ion chelation rate (%) = (Abs blank - Abs sample) / Abs blank × 100%;

[0073] (3) Ferrous ion chelating activity assay: The osteopontin peptide lyophilized powders prepared in Example 1 and Comparative Examples 1-2 were dissolved at a concentration of 1 mg / mL in sodium acetate buffer at pH 5 and a concentration of 50 mmol / L to prepare sample solutions with a concentration of 1 mg / mL. 250 μL of the sample solution was added to a 96-well plate and preheated in a constant temperature shaker at 37°C. After 10 min, the plate was removed, and 20 μL of FeSO4·7H2O with a concentration of 0.5 mmol / L was added. After shaking and mixing, the timer was started. After 50 min (37°C), 15 μL of phenanthroxazine solution with a concentration of 2.5 mmol / L was added to terminate the reaction for 10 min. The absorbance was measured at 562 nm using an ELISA reader. Deionized water was used as the control group, and each sample was tested in triplicate. Ferrous ion chelation rate (%) = (Abs blank - Abs sample) / Abs blank × 100%.

[0074] Table 1 shows the final measured parameters:

[0075] Table 1 Comparison of the chelating activities of osteopontin peptides for mineral ions under different preparation methods

[0076]

[0077] Note: Compared with Comparative Example 1, *p<0.05, **p<0.01, ***p<0.001;

[0078] Compared with Comparative Example 2, #p<0.05, ##p<0.01, ###p<0.001.

[0079] As can be seen from Table 1, the scheme in Example 1 significantly improved the chelating activity of osteopontin peptides for various mineral ions.

[0080] Comparing Example 1 and Comparative Example 1, it can be seen that the osteopontin peptide prepared in Example 1 exhibits superior chelating activity in terms of calcium ions, zinc ions, and ferrous ions. This is because Comparative Example 1 uses a static simulation of the digestion process, which cannot simulate various factors in the dynamic digestion process of the human body, such as gastrointestinal peristalsis and continuous secretion of digestive juices, as in Example 1. This results in a relatively low degree of hydrolysis of osteopontin and a relatively low yield of the final digested osteopontin peptide, thus affecting the final chelating activity.

[0081] Comparing Example 1 and Comparative Example 2, it can be seen that the osteopontin peptide prepared in Example 1 exhibits superior chelating activity against calcium ions, zinc ions, and ferrous ions. This is because the undesalted osteopontin peptide still contains many salt ions, which compete with calcium ions for chelating sites, thus reducing its chelating activity. This indicates that desalting treatment can significantly improve the chelating activity of osteopontin peptide.

[0082] Test Example 2

[0083] The process of Example 1 was repeated three times, and the volume (mL) of the supernatant before desalting and the salt content (mg / mL) of the supernatant were tested, as well as the weight (g) of the osteopontin peptide lyophilized powder after desalting and the yield (%) of osteopontin peptide. The results are shown in Table 2.

[0084] Table 2. Desalting treatment and osteopontin peptide yield analysis in repeated experiments.

[0085]

[0086] As shown in Table 2, the data for each indicator in Example 1 showed minimal fluctuation across three repeated experiments, indicating that the scheme possesses good stability and reproducibility. Both the volume and salt content of the supernatant before desalting, and the weight and yield of osteopontin peptides after desalting, remained relatively stable, which is crucial for industrial production and practical applications.

[0087] In summary, the method described in Example 1, through the use of an in vitro dynamic simulated digestive system and an optimized desalting process, not only significantly improved the chelating activity of osteopontin peptides but also demonstrated high stability and excellent efficiency during preparation. This provides strong technical support for further research and application of osteopontin peptides.

[0088] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system, wherein the in vitro dynamic simulated digestive system is used as the generating device, the in vitro dynamic simulated digestive system comprising, in sequence, an esophageal device, a stomach model, a pyloric device, a duodenal model, and a small intestine model, wherein the stomach model includes a gastric tilting device and a gastric peristalsis device; the small intestine model includes a small intestinal peristalsis device; and each of the stomach model, the duodenal model, and the small intestine model is provided with a squeezing device; characterized in that, The method includes the following steps: S1. Inject 120 mL of osteopontin solution into the stomach model through the esophageal device, and start the squeezing device to contract the stomach model, duodenal model and small intestine model, and begin to simulate digestion; S2. During the simulated digestion process, a gastric simulation solution and an HCl solution are injected into the stomach model, and an intestinal simulation solution and a NaHCO3 solution are injected into the duodenal model. The gastric simulation solution is made by dissolving NaCl, KCl, and pepsin in water and adjusting the pH to 2.5-3.

5. The intestinal simulation solution is made by dissolving NaCl, KCl, CaCl2, trypsin, and bile salts in water and adjusting the pH to 6.2-6.

8. S3. Collect the digestive material flowing out of the small intestine model, and obtain osteopontin peptides by enzyme inactivation, centrifugation, and desalting.

2. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, In S1, the mass concentration of the osteopontin solution is 4.8%-5.2%.

3. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, In S1, during the contraction process, the gastric model contracted 3 times / min, the duodenal model contracted 12 times / min, and the small intestine model contracted 6 times / min.

4. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, In S2, the concentration of NaCl in the gastric simulation solution is 94 mmol / L, the concentration of KCl is 13 mmol / L, and the activity of pepsin is 525 U / mL. The concentration of the HCl solution is 1 mol / L; The intestinal simulation solution contained NaCl at a concentration of 164 mmol / L, KCl at a concentration of 10 mmol / L, CaCl2 at a concentration of 3 mmol / L, trypsin activity at 20 U / mL, and bile salt concentration at 9.8 mmol / L. The concentration of the NaHCO3 solution is 1 mol / L.

5. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, In S2, the injection process of the gastric simulated fluid is as follows: 1 mL / min-1.1 mL / min, 0-10 min; 0.5 mL / min-1 mL / min, 10 min-180 min; The HCl solution injection process is as follows: 0.5 mL / min - 1 mL / min, 1 min - 25 min; The injection process for the intestinal simulation solution is: 0.5 mL / min - 1.2 mL / min, 1 - 180 min; The injection process for the NaHCO3 solution is as follows: 0.5 mL / min - 1 mL / min, 1 min - 60 min.

6. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, In S2, the process parameters for the gastric tilt angle device are: -3° / min, 1min-6min; -0.3° / min, 6min-96min; The process parameters of the gastric peristalsis device are as follows: the forward speed in the first stage is 500 mm / min, the return speed is 600 mm / min, and the running time is 6 min; The second stage has a forward speed of 500 mm / min, a return speed of 600 mm / min, and a running time of 175 min.

7. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, In S2, the process parameters of the small intestinal peristalsis device are: speed of 100 mm / min and running time of 181 min.

8. The method for preparing osteopontin peptides based on an in vitro dynamic simulated digestive system according to claim 1, characterized in that, Before simulating digestion, 6 mL of gastric simulation solution without pepsin was injected into the gastric model to simulate the stomach's fasting state.

9. The use of osteopontin peptide prepared by the method of any one of claims 1-8 in the preparation of products that promote the absorption of mineral elements.

10. The application according to claim 9, characterized in that, The mineral element is selected from one or more of iron, calcium, and zinc.

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