Lipid in-vitro digestion simulation method based on infant digestion physiological characteristics

Abstract

The invention relates to the technical field of bionics, in particular to a lipid in-vitro digestion simulation method based on infant digestion physiological characteristics. According to the method, BSSL and PLRP2 are used in the simulation process on the basis of the early-stage digestion physiological characteristics of the life of the infant, so that the simulation environment is more real, the simulation effect is improved, and the fitting degree of the method and the lipid digestion condition in the body of the infant is improved by 30-50% compared with the prior art; by dynamically regulating and controlling the simulated gastric juice secretion rate, the simulated intestinal juice secretion rate, the simulated acid liquid secretion rate, the simulated alkali liquid secretion rate, mechanical parameters and enzyme kinetic parameters, high-precision fitting of digestive environment characteristics after infant ingestion is realized; system integration of a special digestive enzyme system and dynamic physiological parameters of infants is achieved for the first time, and a high-bionic-degree standardized scheme is provided for lipid digestion characteristic evaluation of infant formula food.

Description

Technical Field

[0001] The present invention relates to the field of bionic technology, and in particular to a method for simulating in vitro lipid digestion based on the digestive physiological characteristics of infants and young children. Background Art

[0002] In vitro digestion is a method for studying the digestion process of food. By simulating the pH and enzyme activity of the human digestive system and using buffers to simulate digestive fluids such as gastric and intestinal fluids, this creates an in vitro environment similar to in vivo digestion, thereby simulating in vitro digestion. This method is time-saving, labor-saving, low-cost, has a short experimental cycle, highly reproducible results, and avoids ethical issues, making it an important tool for studying the digestive process.

[0003] In vitro digestion models are primarily categorized as static, semi-dynamic, and dynamic. These models, with increasing complexity, consist of single or multiple bioreactors, simulating the environments of different regions of the digestive tract. Dynamic models are particularly complex, simulating a variety of parameters, including digestive fluid flow rate, digestive enzyme secretion, pH changes, duration, motility, and emptying. These models more closely resemble the in vivo physiological environment and comprehensively simulate the physiological processes at each digestive stage.

[0004] While there is currently an international consensus on in vitro digestion in adults, the unique characteristics of the infant digestive system present significant challenges in developing in vitro models. This is primarily due to the typical immature gastrointestinal physiology of infants and young children, manifested by insufficient gastric acid secretion (high pH), slow intestinal motility, and limited secretion of digestive juices. The inventors of this application discovered that the expression levels of key lipid digestion enzymes in infants and young children, including bile salts stimulated lipase (BSSL) and pancreatic lipase-related protein 2 (PLRP2), differ fundamentally from those in adults. These physiological characteristics make it difficult to accurately assess the lipid digestion process in infants and young children using existing models based on adult physiological parameters. Summary of the Invention

[0005] Based on this, the present invention provides a method for simulating in vitro lipolysis digestion based on the physiological characteristics of infant digestion, which at least solves one problem in the prior art.

[0006] The present invention provides a method for simulating in vitro lipid digestion based on the physiological characteristics of infant digestion, comprising the following steps: Using a gastrointestinal in vitro digestion simulation system, simulated gastric fluid (SGF) was secreted according to the first segmented gradient infusion strategy, and simulated gastric fluid containing gastric lipase was secreted according to the second segmented gradient infusion strategy; According to the third segmented gradient infusion strategy, simulated intestinal fluid (SIF) was secreted, according to the fourth segmented gradient infusion strategy, simulated intestinal fluid containing bile salt-stimulated lipase (BSSL) was secreted, and according to the fifth segmented gradient infusion strategy, simulated intestinal fluid containing pancreatic lipase-related protein 2 (PLRP2) was secreted.

[0007] It should be noted that the in vitro gastrointestinal digestion simulation system is an existing technology. For example, the Dynamic In Vitro Infant Stomach-Duodenum System II, manufactured by Xiaodong Yijian (Suzhou) Instruments and Equipment Co., Ltd., can be used. BSSL and PLRP2 are both key lipid-digesting enzymes in early life in infants and young children, enhancing the effectiveness of in vitro lipid digestion simulation for infants and young children.

[0008] In some optional embodiments, the concentration of gastric lipase in the simulated gastric fluid containing gastric lipase is 18-20 U / mL, the concentration of bile salt-stimulated lipase in the simulated intestinal fluid containing bile salt-stimulated lipase is 26-30 U / mL, and the concentration of pancreatic lipase-related protein 2 in the simulated intestinal fluid containing pancreatic lipase-related protein 2 is 60-65 U / mL. Under these concentration conditions, gastric lipase, bile salt-stimulated lipase, and pancreatic lipase-related protein 2 can well simulate the gastrointestinal digestive fluid of infants and young children, thereby improving the in vitro lipid digestion simulation effect for infants and young children.

[0009] In some optional embodiments, the first segmented gradient infusion strategy is as follows: the flow rate in the first phase is 10-13 mL / min, the flow rate in the second phase decreases exponentially to 0.4-0.6 mL / min, and the flow rate in the third phase is 0.1-0.3 mL / min. This segmented gradient infusion strategy accurately simulates the environment in which infants secrete gastric juice. The first phase can be 0-1 min, the second phase can be 1-60 min, and the third phase can be 60-120 min.

[0010] In some optional embodiments, the second segmented gradient infusion strategy is as follows: the flow rate in the first phase is 10-13 mL / min, the flow rate in the second phase decreases exponentially to 0.4-0.6 mL / min, and the flow rate in the third phase is 0.1-0.3 mL / min. This segmented gradient infusion strategy accurately simulates the environment in which infant gastric lipase is secreted. The first phase can be 0-1 min, the second phase can be 1-60 min, and the third phase can be 60-120 min.

[0011] In some optional embodiments, the third segmented gradient infusion strategy is as follows: the flow rate in the first stage is 0.8-1.2 mL / min, the flow rate in the second stage is 1.2-1.6 mL / min, the flow rate in the third stage is 0.8-1.2 mL / min, and the flow rate in the fourth stage is 0.8-1.1 mL / min. This segmented gradient infusion strategy accurately simulates the environment of intestinal fluid secretion in infants and young children. The first stage can be 0-30 minutes, the second stage can be 30-60 minutes, the third stage can be 60-90 minutes, and the fourth stage can be 90-120 minutes.

[0012] In some optional embodiments, the fourth segmented gradient infusion strategy includes a first-stage flow rate of 0.60-0.88 mL / min, a second-stage flow rate of 0.90-1.20 mL / min, a third-stage flow rate of 0.60-0.90 mL / min, and a fourth-stage flow rate of 0.50-0.70 mL / min. This segmented gradient infusion strategy accurately simulates the environment in which BSSL is secreted in infants and young children. The first stage can be 0-30 minutes, the second stage can be 30-60 minutes, the third stage can be 60-90 minutes, and the fourth stage can be 90-120 minutes. That is, SIF containing 26-30 U / mL BSSL was infused at a constant rate of 0.60-0.88 mL / min for the first 30 minutes, the infusion rate was adjusted to 0.90-1.20 mL / min from 30 to 60 minutes, restored to 0.60-0.90 mL / min from 60 to 90 minutes, and further reduced to 0.50-0.70 mL / min from 90 to 120 minutes.

[0013] In some optional embodiments, the fifth segmented gradient infusion strategy is as follows: a first-stage flow rate of 0.80-1.10 mL / min, a second-stage flow rate of 1.00-1.40 mL / min, a third-stage flow rate of 0.80-1.20 mL / min, and a fourth-stage flow rate of 0.70-0.90 mL / min. This segmented gradient infusion strategy accurately simulates the environment in which PLRP2 is secreted in infants and young children. The first stage can be 0-30 minutes, the second stage can be 30-60 minutes, the third stage can be 60-90 minutes, and the fourth stage can be 90-120 minutes. That is, SIF containing 60-65 U / mL PLRP2 was infused at a constant rate of 0.80-1.10 mL / min for the initial 30 minutes, the infusion rate was adjusted to 1.00-1.40 mL / min for 30-60 minutes, restored to 0.80-1.20 mL / min for 60-90 minutes, and further reduced to 0.70-0.90 mL / min for 90-120 minutes.

[0014] In some optional embodiments, the method further includes the step of simulating gastric emptying. For example, simulated gastric emptying can be achieved by regulating the squeezing and peristaltic parameters of the simulated stomach and the body tilt angle; the squeezing and peristaltic parameters may be a frequency of 420-460 mm / min (preferably 450 mm / min), and the body tilt angle may be 30° to 45°.

[0015] In some optional embodiments, the method further includes the step of simulating gastric acid secretion. For example, the pH is controlled by controlling the flow rate of the acid solution, thereby simulating gastric acid secretion. The acid solution may be a 0.1 M hydrochloric acid solution, and the gastric pH may vary from 3.0-3.6 to 4.8-5.2 to 6.6-6.9.

[0016] In some optional embodiments, the method further comprises the step of simulating intestinal alkali secretion. For example, the pH is controlled by controlling the flow rate of the alkali solution, thereby simulating gastric acid secretion; wherein the alkali solution can be a 0.1 M sodium hydroxide solution, and the intestinal pH can change from 6.4-6.6 to 7.4-7.6.

[0017] In some optional embodiments, the above method further comprises the step of controlling the temperature of the gastrointestinal in vitro digestion simulation system to 37±0.5°C.

[0018] Due to the adoption of the above technical scheme, the embodiments of the present invention have at least the following beneficial effects: based on the digestive physiological characteristics of infants in early life, BSSL and PLRP2 are used in the simulation process, making the simulation environment more realistic and improving the simulation effect. The method of the embodiment of the present invention is 30% to 50% more compatible with the lipid digestion conditions in infants than the existing technology; by dynamically regulating the simulated gastric juice secretion rate, simulated intestinal juice secretion rate, acid secretion rate, alkali secretion rate, mechanical parameters and enzyme kinetic parameters, high-precision fitting of the digestive environment characteristics of infants after feeding is achieved; for the first time, the systematic integration of infants' special digestive enzyme system and dynamic physiological parameters is achieved, providing a highly biomimetic standardized scheme for the evaluation of lipid digestion characteristics of infant formula. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of gastric juice flow rate during dynamic in vitro digestion of infants in the early stages of life in Example 1 of the present invention.

[0020] Figure 2 This is a flow rate diagram of gastric lipase in dynamic in vitro digestion of infants and young children in early life in Example 1 of the present invention.

[0021] Figure 3 This is a diagram of the dynamic in vitro digestion intestinal fluid flow rate of infants in the early stages of life in Example 1 of the present invention.

[0022] Figure 4 This is a flow rate diagram of dynamic in vitro digestion of intestinal lipase (BSSL) in early life of infants in Example 1 of the present invention.

[0023] Figure 5 This is a flow rate diagram of the dynamic in vitro digestion of intestinal lipase (PLRP2) in early life of infants in Example 1 of the present invention.

[0024] Figure 6 This is a graph of acid flow rates in the gastric stage during the dynamic in vitro digestion of infant formula in the early stages of life of an infant in Example 1 of the present invention.

[0025] Figure 7 This is a diagram of the alkali solution flow rate in the intestinal stage during the dynamic in vitro digestion of IF / HM in infants and young children in early life in Example 1 of the present invention.

[0026] Figure 8 This is a graph of the acid flow rate in the gastric stage during the dynamic in vitro digestion of breast milk in the early life of infants in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effects of the present invention.

[0028] Existing research on human digestion simulation still has certain limitations: (1) Current in vitro digestion methods do not involve digestion in the early stages of infant life; (2) The digestive enzymes used in current in vitro digestion systems are all exogenous enzymes of animal origin, which are somewhat different from the fat digestion enzymes in infants; (3) The gastrointestinal characteristics of infants are not taken into account, especially the dynamic changes in gastric pH, gastric emptying, intestinal motility and other parameters of infants.

[0029] To address at least one of the above technical issues, embodiments of the present invention provide an in vitro digestion simulation method based on the physiological characteristics of lipid digestion in early life in infants and young children. Based on the key lipases for lipid digestion (BSSL and PLRP2) and digestive physiological characteristics of early life infants and young children, an in vitro lipid digestion model for early life infants and young children was constructed, providing a more accurate nutritional evaluation model for infant and young children's food.

[0030] According to an embodiment of the present invention, an infant-specific lipid digestion enzyme system is first constructed, and BSSL and PLRP2 can be prepared by recombinant expression technology. For example, BSSL and PLRP2 gene sequences were synthesized by gene recombination technology, and after codon optimization, they were integrated into the Pichia pastoris expression vector pPICZαA; they were transformed into the X-33 Pichia pastoris host strain by electroporation, and the extracellular secretion expression of key lipid digestion enzymes was achieved through the methanol-inducible characteristics of the Pichia pastoris expression system (the preparation method of BSSL and PLRP2 is an existing technology, and reference can be made to the literature Xunjun, Xiao, Mukherjee, et al. Pancreatic Lipase-related Protein-2 (PLRP2) Can Contribute to Dietary Fat Digestion in Human Newborns[J]. Journal of Biological Chemistry, 2011, 286(30):26353-26363.DOI:10.1074 / jbc.M111.249813 or the literature Anjali V. Sahasrabudhe, Suresh M. Solapure, Rajeev Khurana. Production of Recombinant Human Bile Salt-Stimulated Lipasein Pichia pastoris[J]. Protein Expression&Purification, 2001, 23(2):282-288.DOI:10.1006 / prep.2001.1509).

[0031] Secondly, a dynamic bionic digestive environment is constructed. A multi-parameter coupling control model is established based on the physiological parameters of the infant digestive system. The model includes a mechanical dynamics module, an enzyme dynamics module, and a fluid control module to achieve spatiotemporal synchronous control of gastric emptying rate, gastrointestinal pH gradient, and digestive enzyme secretion dynamics. For example, the DIS-II gastrointestinal in vitro digestion simulation system can be used to realize the construction of a dynamic bionic digestive environment. Among them, the digestive fluid temperature and the ambient temperature are stably maintained at 37±0.5℃ by a digestive fluid heater and an environmental heater.

[0032] According to an embodiment of the present invention, a method for simulating in vitro lipid digestion based on the digestive physiological characteristics of infants and young children includes the following: (1) Gastric digestion simulation Simulated gastric fluid (SGF): The SGF flow rate shows a significant peak (12.5 mL / min) in the initial digestion stage (0-1 min), then enters the regulation period (1-60 min), and the flow rate decreases exponentially to the basal level (0.5 mL / min), and finally maintains a stable secretion state (60-120 min, 0.2 mL / min).

[0033] Gastric acid: To simulate the dynamic changes of gastric pH, a 0.1 M hydrochloric acid solution was used to simulate the dynamic changes of gastric pH in infants and young children after feeding.

[0034] Gastric lipase: Gastric lipase was added to SGF (19 U / mL), causing the lipase to be secreted together with SGF, with a significant peak (12.5±0.3 mL / min) in the initial stage (0-1 min). This was followed by a regulation period (1-60 min), during which the flow rate decreased exponentially to the basal level (0.5 mL / min). Finally, a stable secretion state was maintained (60-120 min, 0.2 mL / min).

[0035] Gastric half-emptying: A controllable gastric emptying kinetic model was established by dynamically changing the peristaltic frequency (420-460 mm / min), tilt angle (± 90°), and time parameters (t=0-120 min) of the DIS-II gastrointestinal in vitro digestion simulation system. Based on the gastric emptying kinetic characteristics of infants and young children, it was found that the gastric half-emptying time (t) of infants and young children after consuming breast milk (HM) and infant formula (IF) was significantly different. 1 / 2 ) were 48 ± 15 minutes and 78 ± 14 minutes, respectively. This difference reflects the different gastric emptying characteristics of HM and IF during digestion, which can be used to design key parameters. Specifically, by systematically optimizing key parameters such as tilt angle and time gradient (see Table 1 for details), accurate simulation of the gastric emptying process was achieved.

[0036] Table 1 Changes in the tilt angle of the DIS-II gastrointestinal in vitro digestion simulation system (2) Intestinal digestion simulation Simulated intestinal fluid (SIF): SIF has different flow rates in different digestion time periods. The secretion rate is 1.0 mL / min from 0 to 30 minutes; the secretion rate increases slightly to 1.4 mL / min from 30 to 60 minutes; the secretion rate decreases again to 1.0 mL / min from 60 to 90 minutes; and the secretion rate further decreases to 0.9 mL / min from 90 to 120 minutes.

[0037] Intestinal alkali solution: To simulate the dynamic changes in intestinal pH, 0.1 M sodium hydroxide solution was added dropwise at a flow rate of 0.1 mL / min between 60 and 90 minutes to stabilize the intestinal pH at around 6.5. During the later stages of digestion (90 to 120 minutes), the pH of the gastric chyme further decreased, and the sodium hydroxide solution addition rate was increased to 0.2 mL / min to maintain the intestinal pH within the physiological range for infants and young children.

[0038] SIF containing BSSL: BSSL was added to SIF at a concentration of 28 U / mL, and the SGF containing BSSL was infused at a constant rate of 0.77 mL / min for the first 30 minutes. The infusion rate was adjusted to 1.01 mL / min from 30 to 60 minutes, restored to 0.77 mL / min from 60 to 90 minutes, and further reduced to 0.69 mL / min from 90 to 120 minutes.

[0039] PLRP2-containing SIF: PLRP2 was added to the simulated SIF at a concentration of 62 U / mL, and the PLRP2-containing SIF was infused at a constant rate of 0.98 mL / min for the first 30 minutes. The infusion rate was adjusted to 1.37 mL / min from 30 to 60 minutes, restored to 0.98 mL / min from 60 to 90 minutes, and further reduced to 0.88 mL / min from 90 to 120 minutes.

[0040] Example 1 Infant formula (IF) was digested according to the following experimental parameters: Add 100 mL of infant formula (IF) into the simulated stomach of the DIS-II gastrointestinal in vitro digestion simulation system; Infuse artificial gastric fluid at a flow rate of Figure 1 As shown; wherein, the composition of artificial gastric juice is NaCl 94 mmol / L, KCl 13mmol / L, and the solvent is water; Infuse artificial gastric fluid containing 19U / mL gastric lipase to secrete lipase together with artificial gastric fluid. Figure 2 As shown; Infuse artificial intestinal fluid at a flow rate of Figure 3 As shown; the composition of artificial intestinal fluid is NaCl 164 mmol / L, KCl10 mmol / L, NaHCO385 mmol / L, CaCl2119 mmol / L, and the solvent is water; Infuse artificial intestinal fluid containing 28 U / mL BSSL to secrete BSSL together with the artificial intestinal fluid. Figure 4 As shown; Infuse artificial intestinal fluid containing 62 U / mL PLRP2 to secrete PLRP2 together with the artificial intestinal fluid. Figure 5As shown; Simulate gastric emptying according to Table 1 ; Infuse 0.1 M hydrochloric acid solution at a flow rate of Figure 6 As shown; Infuse 0.1 M sodium hydroxide solution at a flow rate of Figure 7 shown.

[0041] Example 2 This embodiment is basically the same as embodiment 1, except that the infant formula milk powder is replaced with human milk, and the flow rate of the infusion of 0.1 M hydrochloric acid solution is as follows: Figure 8 shown.

[0042] Comparative Example 1 Infant formula (IF) was digested according to the following experimental parameters: Add 100 mL of infant formula (IF) into the simulated stomach of the DIS-II gastrointestinal in vitro digestion simulation system; Infuse artificial gastric juice, wherein the artificial gastric juice composition is NaCl 94 mmol / L, KCl 13 mmol / L, and the solvent is water; Artificial gastric fluid containing 19 U / mL gastric lipase was infused to allow the lipase to be secreted together with the artificial gastric fluid; Infuse 0.1 M hydrochloric acid solution at a flow rate of 0.1-0.5 mL / min to simulate the pH change in the stomach after feeding. Infuse artificial intestinal fluid, wherein the composition of artificial intestinal fluid is NaCl 164 mmol / L, KCl 10 mmol / L, NaHCO3 85 mmol / L, CaCl2 119 mmol / L, and the solvent is water; Artificial intestinal fluid containing 90 U / mL pancreatic lipase was infused.

[0043] Comparative Example 2 This embodiment is substantially the same as Comparative Example 1, except that the infant formula milk powder is replaced with human milk.

[0044] The degree of fit between the in vivo digestion conditions and the in vitro digestion conditions was evaluated based on the changes in gastric pH, gastric half-emptying time, and intestinal pH during digestion in Example 1, Example 2, Comparative Example 1, and Comparative Example 2. The formula for determining the degree of fit with the in vivo digestion conditions in infants and young children is as follows: Here, n represents the number of in vitro-in vivo data pairs used in the in vitro-in vivo goodness of fit (IVIVR) study to calculate MAPE and Bias. MAPE (mean absolute percentage error) and Bias (systematic bias) were selected as metrics for evaluating the accuracy of in vitro methods, with in vivo data serving as the "true value" reference. These two metrics are widely used in the quantitative analysis of modeling and prediction data accuracy.

[0045] As shown in Table 2, the gastric pH dynamics profiles simulated in vitro and in vivo for digestion of HM and IF were highly consistent with those measured in Example 1 or Example 2 of the present invention. The Pearson correlation coefficient for the in vitro simulated digestion of HM was r=0.971, p<0.01; the Pearson correlation coefficient for the in vitro simulated digestion of IF was r=0.997, p<0.01. The in vitro simulated Bias and MAPE were 1.72% and 2.22%, respectively.

[0046] Table 2 Goodness of fit between in vivo and in vitro gastric pH in early life of infants and young children As shown in Table 3, the in vitro simulated and in vivo measured gastric emptying times for digestion of HM and IF were highly consistent with the clinically measured values ​​for in vitro simulated and in vivo gastric emptying times according to Examples 1 and 2 of the present invention. The in vitro simulated Bias and MAPE were 6.97% and 6.97%, respectively.

[0047] Table 3 Goodness of fit of in vivo and in vitro gastric emptying in early life of infants and young children As shown in Table 4, the intestinal pH dynamics profiles simulated in vitro and in vivo for digestion of HM and IF were highly consistent with those measured in vivo. The Pearson correlation coefficient for the in vitro simulated digestion of HM was r=0.971, p<0.01; the Pearson correlation coefficient for the in vitro simulated digestion of IF was r=0.997, p<0.01. The in vitro simulated Bias and MAPE were 1.03% and 1.89%, respectively.

[0048] Table 4 In vivo-in vitro intestinal pH fit in early life of infants and young children In summary, the constructed in vitro lipid digestion model method for infants and young children in early life can provide a more accurate evaluation model for the nutritional evaluation of lipids in infant and young children's food.

[0049] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any method that achieves the technical effects of the present invention by the same or equivalent means shall fall within the scope of protection of the present invention. Within the scope of protection of the present invention, various modifications and variations of the technical solutions and / or implementation methods are possible.

Claims

1. A method for simulating in vitro lipid digestion based on the digestive physiological characteristics of infants and young children, characterized in that: The following steps are involved: Using a gastrointestinal in vitro digestion simulation system, the simulated gastric fluid is secreted according to a first segmented gradient infusion strategy, and the simulated gastric fluid containing gastric lipase is secreted according to a second segmented gradient infusion strategy; According to the third segmented gradient infusion strategy, simulated intestinal fluid is secreted, according to the fourth segmented gradient infusion strategy, simulated intestinal fluid containing bile salt-stimulated lipase is secreted, and according to the fifth segmented gradient infusion strategy, simulated intestinal fluid containing pancreatic lipase-related protein 2 is secreted.

2. The method according to claim 1, characterized in that The concentration of gastric lipase in the simulated gastric fluid containing gastric lipase was 18-20 U / mL, the concentration of bile salt-stimulated lipase in the simulated intestinal fluid containing bile salt-stimulated lipase was 26-30 U / mL, and the concentration of pancreatic lipase-related protein 2 in the simulated intestinal fluid containing pancreatic lipase-related protein 2 was 60-65 U / mL.

3. The method according to claim 1, characterized in that The above-mentioned first segmented gradient infusion strategy is: the flow rate in the first stage is 10-13 mL / min, the flow rate in the second stage decreases exponentially to 0.4-0.6 mL / min, and the flow rate in the third stage is 0.1-0.3 mL / min.

4. The method according to claim 1, wherein The second segmented gradient infusion strategy is as follows: the flow rate in the first stage is 10-13 mL / min, the flow rate in the second stage decreases exponentially to 0.4-0.6 mL / min, and the flow rate in the third stage is 0.1-0.3 mL / min.

5. The method according to claim 1, wherein The above-mentioned third segmented gradient infusion strategy is: the flow rate of the first stage is 0.8-1.2 mL / min, the flow rate of the second stage is 1.2-1.6 mL / min, the flow rate of the third stage is 0.8-1.2 mL / min, and the flow rate of the fourth stage is 0.8-1.1 mL / min.

6. The method according to claim 1, characterized in that The fourth segmented gradient infusion strategy is as follows: the flow rate in the first stage is 0.60-0.88 mL / min, the flow rate in the second stage is 0.90-1.20 mL / min, the flow rate in the third stage is 0.60-0.90 mL / min, and the flow rate in the fourth stage is 0.50-0.70 mL / min.

7. The method according to claim 1, characterized in that The fifth segmented gradient infusion strategy is as follows: the flow rate in the first stage is 0.80-1.10 mL / min, the flow rate in the second stage is 1.00-1.40 mL / min, the flow rate in the third stage is 0.80-1.20 mL / min, and the flow rate in the fourth stage is 0.70-0.90 mL / min.

8. The method according to claim 1, characterized in that The above method further comprises the step of simulating gastric emptying.

9. The method according to claim 1, characterized in that The method further comprises the steps of simulating the secretion of acid in the stomach and simulating the secretion of alkaline solution in the intestine.

10. The method according to claim 1, characterized in that The above method also includes the step of controlling the temperature of the gastrointestinal in vitro digestion simulation system to 37±0.5°C.

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