Lipid in vitro digestion simulation method based on digestive physiological characteristics of infants and young children

Abstract

The present application relates to the field of bionics, and in particular to a liposome in-vitro digestion simulation method based on the digestive physiological characteristics of infants and young children. Based on the digestive physiological characteristics of infants and young children in the early life, BSSL and PLRP2 are used in the simulation process, so that the simulation environment is more realistic, and the simulation effect is improved. The fitting degree of the method of the present application to the in-vivo lipid digestion conditions of infants and young children is improved by 30-50% compared with the prior art. By dynamically regulating the simulated gastric juice secretion rate, the simulated intestinal juice secretion rate, the acid liquid secretion rate, the alkaline liquid secretion rate, the mechanical parameters and the enzyme kinetic parameters, high-precision fitting of the digestive environment characteristics of infants and young children after feeding is achieved. The present application first realizes the system integration of special digestive enzymes and dynamic physiological parameters of infants and young children, and provides a high-bionic standardized scheme for the evaluation of the lipid digestion characteristics of infant formula food.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bionics, in particular to a lipids in-vitro digestion simulation method based on the physiological characteristics of infants. BACKGROUND

[0002] In-vitro digestion is a method for studying the process of food digestion. By simulating the pH and enzyme activity of the human digestive system, using buffer solutions to simulate gastric juice and intestinal juice, and creating an in-vitro environment similar to in-vivo digestion, in-vitro digestion simulation is achieved. This method is time-saving, labor-saving, low-cost, short experimental period, and strong repeatability of results, and avoids ethical issues, so it has become an important tool for studying the process of food digestion.

[0003] In-vitro digestion models are mainly divided into static, semi-dynamic and dynamic. These models increase in complexity, consisting of a single or multiple bioreactors, simulating the environment of different regions of the digestive tract. Dynamic models are particularly complex, as they can simulate multiple parameters such as digestion fluid flow rate, digestion enzyme secretion, pH changes, duration, peristalsis, and emptying, thus more closely simulating the in-vivo physiological environment and fully simulating the physiological processes of each digestion stage.

[0004] Currently, there is international consensus on adult in-vitro digestion, but the particularity of the infant digestive system presents significant challenges to the construction of in-vitro models. The main challenge is that the gastrointestinal physiology of infants presents typical immature characteristics, specifically insufficient gastric acid secretion (high pH), low and slow intestinal motility, and limited secretion of digestive fluids. The inventors of the present application have found that the expression levels of key enzymes for lipid digestion in infants, including bile salts stimulate lipase (BSSL) and pancreatic lipase related protein 2 (PLRP2), are fundamentally different from those of adults. These physiological characteristics make it difficult for existing models based on adult physiological parameters to accurately assess the lipid digestion process in infants. SUMMARY

[0005] Therefore, the present application provides a lipids in-vitro digestion simulation method based on the physiological characteristics of infants, which at least solves one problem in the prior art.

[0006] The lipids in-vitro digestion simulation method based on the physiological characteristics of infants provided by the present application comprises the following steps:

[0007] Using a gastrointestinal in-vitro digestion simulation system, secreting simulated gastric fluid (SGF) according to a first segmented gradient infusion strategy, and secreting simulated gastric fluid containing gastric lipase according to a second segmented gradient infusion strategy;

[0008] The simulated intestinal fluid (SIF) is secreted according to the third segmented gradient infusion strategy, and the simulated intestinal fluid containing bile salt-stimulated lipase (BSSL) is secreted according to the fourth segmented gradient infusion strategy, and the simulated intestinal fluid containing pancreatic lipase-related protein 2 (PLRP2) is secreted according to the fifth segmented gradient infusion strategy.

[0009] It should be noted that the gastrointestinal in vitro digestion simulation system is prior art, for example, the gastrointestinal in vitro digestion simulation system can be DIS-II gastrointestinal in vitro digestion simulation system (The Dynamic In Vitro Infant Stomach-Duodenum System II) of Xiaodong Yijian (Suzhou) Instrument and Equipment Co., Ltd. Both BSSL and PLRP2 are key lipid digestion enzymes in the early life of infants, which improves the lipid in vitro digestion simulation effect for infants.

[0010] In some optional embodiments, the concentration of gastric lipase in the simulated gastric juice 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, the gastric lipase, the bile salt-stimulated lipase, and the pancreatic lipase-related protein 2 can well simulate the infant gastrointestinal juice, thereby improving the lipid in vitro digestion simulation effect for infants.

[0011] In some optional embodiments, the first segmented gradient infusion strategy described above is that the first stage flow rate is 10-13 mL / min, the second stage flow rate decreases exponentially to 0.4-0.6 mL / min, and the third stage flow rate is 0.1-0.3 mL / min. This segmented gradient infusion strategy accurately simulates the environment of infant gastric juice secretion. Among them, the first stage can be 0-1 min, the second stage can be 1-60 min, and the third stage can be 60-120 min.

[0012] In some optional embodiments, the second segmented gradient infusion strategy described above is that the first stage flow rate is 10-13 mL / min, the second stage flow rate decreases exponentially to 0.4-0.6 mL / min, and the third stage flow rate is 0.1-0.3 mL / min. This segmented gradient infusion strategy accurately simulates the environment of infant gastric lipase secretion. Among them, the first stage can be 0-1 min, the second stage can be 1-60 min, and the third stage can be 60-120 min.

[0013] In some alternative embodiments, the third segmented gradient infusion strategy described above is: the first stage flow rate is 0.8-1.2 mL / min, the second stage flow rate is 1.2-1.6 mL / min, the third stage flow rate is 0.8-1.2 mL / min, and the fourth stage flow rate is 0.8-1.1 mL / min. This segmented gradient infusion strategy accurately simulates the environment of infantile intestinal fluid secretion. Among them, the first stage can be 0-30 min, the second stage can be 30-60 min, the third stage can be 60-90 min, and the fourth stage can be 90-120 min.

[0014] In some alternative embodiments, the fourth segmented gradient infusion strategy described above is: the first stage flow rate is 0.60-0.88 mL / min, the second stage flow rate is 0.90-1.20 mL / min, the third stage flow rate is 0.60-0.90 mL / min, and the fourth stage flow rate is 0.50-0.70 mL / min. This segmented gradient infusion strategy accurately simulates the environment of infantile BSSL secretion. Among them, the first stage can be 0-30 min, the second stage can be 30-60 min, the third stage can be 60-90 min, and the fourth stage can be 90-120 min. That is, the SIF containing 26-30 U / mL BSSL is infused at a constant speed of 0.60-0.88 mL / min for the initial 30 minutes, the infusion speed is adjusted to 0.90-1.20 mL / min for 30-60 minutes, it is restored to 0.60-0.90 mL / min for 60-90 minutes, and it is further reduced to 0.50-0.70 mL / min for 90-120 minutes.

[0015] In some alternative embodiments, the fifth segmented gradient infusion strategy described above is: the first stage flow rate is 0.80-1.10 mL / min, the second stage flow rate is 1.00-1.40 mL / min, the third stage flow rate is 0.80-1.20 mL / min, and the fourth stage flow rate is 0.70-0.90 mL / min. This segmented gradient infusion strategy accurately simulates the environment of infantile PLRP2 secretion. Among them, the first stage can be 0-30 min, the second stage can be 30-60 min, the third stage can be 60-90 min, and the fourth stage can be 90-120 min. That is, the SIF containing 60-65 U / mL PLRP2 is infused at a constant speed of 0.80-1.10 mL / min for the initial 30 minutes, the infusion speed is adjusted to 1.00-1.40 mL / min for 30-60 minutes, it is restored to 0.80-1.20 mL / min for 60-90 minutes, and it is further reduced to 0.70-0.90 mL / min for 90-120 minutes.

[0016] In some optional embodiments, the method further comprises a step of simulating gastric emptying. For example, gastric emptying can be simulated by adjusting the squeezing peristalsis parameters and the body position inclination angle of the simulated stomach. The squeezing peristalsis parameters can be a frequency of 420-460 mm / min (preferably 450 mm / min), and the body position inclination angle can be 30°-45°.

[0017] In some optional embodiments, the method further comprises a step of simulating gastric acid secretion. For example, gastric acid secretion can be simulated by controlling the flow rate of the acid solution to control the pH. The acid solution can be a 0.1 M hydrochloric acid solution, and the gastric pH change can be 3.0-3.6→4.8-5.2→6.6-6.9.

[0018] In some optional embodiments, the method further comprises a step of simulating intestinal alkali secretion. For example, intestinal alkali secretion can be simulated by controlling the flow rate of the alkali solution to control the pH. The alkali solution can be a 0.1 M sodium hydroxide solution, and the intestinal pH change can be 6.4-6.6→7.4-7.6.

[0019] In some optional embodiments, the method further comprises a step of controlling the temperature of the gastrointestinal in vitro digestion simulation system to be 37±0.5℃.

[0020] Thanks to the above technical solutions, the embodiments of the present application have at least the following advantages: based on the physiological characteristics of early life of infants and young children, BSSL and PLRP2 are used in the simulation process, which makes the simulation environment more realistic and improves the simulation effect. The fitting degree of the method of the embodiments of the present application to the lipid digestion conditions in infants and young children is improved by 30-50% compared with the prior art. By dynamically adjusting the simulated gastric juice secretion rate, the simulated intestinal juice secretion rate, the acid secretion rate, the alkali secretion rate, the mechanical parameters and the enzyme kinetic parameters, the high-precision fitting of the digestion environment characteristics after the infants and young children eat is achieved. The system integration of the special digestive enzyme system of infants and young children and dynamic physiological parameters is realized for the first time, and a high-bionic standardized scheme for evaluating the lipid digestion characteristics of infant formula food is provided. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 FIG. 1 is a gastric juice flow rate diagram of dynamic in vitro digestion of early life of infants and young children in Example 1 of the present application.

[0022] Figure 2 FIG. 2 is a gastric lipase flow rate diagram of dynamic in vitro digestion of early life of infants and young children in Example 1 of the present application.

[0023] Figure 3 FIG. 3 is an intestinal juice flow rate diagram of dynamic in vitro digestion of early life of infants and young children in Example 1 of the present application.

[0024] Figure 4 Gastric phase acid flow rate profile for dynamic in-vitro digestion of infant formula in Example 1 of the present application.

[0025] Figure 5 Intestinal phase base flow rate profile for dynamic in-vitro digestion of IF / HM in Example 1 of the present application.

[0026] Figure 6 Gastric phase acid flow rate profile for dynamic in-vitro digestion of infant formula in Example 1 of the present application.

[0027] Figure 7 Intestinal phase base flow rate profile for dynamic in-vitro digestion of IF / HM in Example 1 of the present application.

[0028] Figure 8 Gastric phase acid flow rate profile for dynamic in-vitro digestion of breast milk in Example 2 of the present application. DETAILED DESCRIPTION

[0029] The concept and technical effects of the present application will be described clearly and completely below to fully explain the purpose, scheme and effects of the present application.

[0030] The existing related researches involving human body digestion simulation still have certain limitations: (1) the current in-vitro digestion method does not involve the digestion in early infancy; (2) the digestive enzymes used in the current in-vitro digestion system are all animal-derived exogenous enzymes, which have certain differences with the fat digestion enzymes in the body of infants; (3) the gastrointestinal characteristics of infants are not considered, especially the dynamic changes of the pH of the stomach of infants, gastric emptying and intestinal transit and other parameter changes.

[0031] To solve at least one of the above technical problems, the embodiment of the present application provides an in-vitro digestion simulation method based on the physiological characteristics of lipid digestion in early infancy. Based on the key lipases (BSSL and PLRP2) and the physiological characteristics of digestion in early infancy, an in-vitro digestion model of lipids in early infancy is constructed, which provides a more accurate evaluation model for the nutritional evaluation of infant food.

[0032] According to the embodiment of the present application, first, the infant-specific lipid digestion enzyme system is constructed, and BSSL and PLRP2 can be prepared by recombinant expression technology. For example, the BSSL and PLRP2 gene sequences are synthesized by using gene recombination technology, and after codon optimization, they are integrated into the Pichia pastoris expression vector pPICZαA. The X-33 Pichia pastoris host strain is transformed by using electroporation, and the extracellular secretion expression of the key enzymes of lipid digestion is realized by the methanol induction characteristics of the Pichia pastoris expression system (the preparation method of BSSL and PLRP2 is prior art, and can refer 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 Lipase in Pichia pastoris [J]. Protein Expression & Purification, 2001, 23 (2): 282-288. DOI: 10.1006 / prep.2001.1509).

[0033] Secondly, a dynamic biomimetic digestion environment is constructed, and a multi-parameter coupled regulation model is established based on the physiological parameters of the infant digestive system. The model includes a mechanical dynamics module, an enzyme kinetics module and a fluid control module, and realizes the spatiotemporal synchronous regulation of gastric emptying rate, gastrointestinal pH gradient and dynamic secretion of digestive enzymes. For example, the DIS-II gastrointestinal in vitro digestion simulation system can be used to construct the dynamic biomimetic digestion environment. The temperature of the digestion solution and the environment is stably maintained at 37±0.5℃ by using a digestion solution heater and an environment heater.

[0034] According to the embodiment of the present application, the lipid in vitro digestion simulation method based on the physiological characteristics of infants includes the following contents:

[0035] (1) Gastric digestion simulation

[0036] Simulated gastric fluid (SGF, artificial gastric fluid): SGF flow rate presents a significant peak (12.5 mL / min) in the initial digestion stage (0-1 min), followed by a regulation period (1-60 min), the flow rate decreases exponentially to the basic level (0.5 mL / min), and finally maintains a stable secretion state (60-120 min, 0.2 mL / min).

[0037] Intragastric acid solution: To simulate the dynamic change of intragastric pH value, the concentration of 0.1 M hydrochloric acid solution is used to simulate the dynamic change of intragastric pH value after feeding in infants and young children.

[0038] Gastric lipase: Gastric lipase is added to SGF (19 U / mL), and the lipase is secreted with SGF, presenting a significant peak (12.5±0.3 mL / min) in the initial stage (0-1 min); followed by a regulation period (1-60 min), the flow rate decreases exponentially to the basic level (0.5 mL / min); finally maintains a stable secretion state (60-120 min, 0.2 mL / min).

[0039] Gastric half-emptying: By changing the peristalsis frequency (420-460 mm / min), the inclination angle (± 90°) and the time parameter (t=0-120 min) of the DIS-II gastrointestinal in vitro digestion simulation system, a controllable gastric emptying kinetics model is established. According to the gastric emptying kinetics characteristics of infants and young children, it is found that the gastric half-emptying time (t 1 / 2 ) of infants and young children after intake of human milk (HM) and infant formula (IF) is 48±15 minutes and 78±14 minutes, respectively. This difference reflects the different gastric emptying characteristics of HM and IF in the digestion process, which can be used as the basis for designing key parameters. Specifically, by optimizing the inclination angle and time gradient and other key parameters (see Table 1), the precise simulation of the gastric emptying process is achieved.

[0040] Table 1 Inclination angle changes of DIS-II gastrointestinal in vitro digestion simulation system

[0041]

[0042] (2) Intestinal digestion simulation

[0043] Simulated intestinal fluid (SIF, artificial intestinal fluid): SIF has different flow rates at different digestion time periods, with a secretion rate of 1.0 mL / min for 0-30 minutes; the secretion rate increases slightly to 1.4 mL / min for 30-60 minutes; the secretion rate decreases to 1.0 mL / min for 60-90 minutes; and the secretion rate further decreases to 0.9 mL / min for 90-120 minutes.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 1

[0048] Infant formula (IF) was digested according to the following experimental parameters:

[0049] 100 mL of infant formula (IF) was added to the simulated stomach of the DIS-II gastrointestinal in vitro digestion simulation system;

[0050] 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;

[0051] Infuse artificial gastric fluid containing 19U / mL gastric lipase to secrete lipase together with artificial gastric fluid. Figure 2 As shown;

[0052] 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;

[0053] Infusion of artificial intestinal fluid containing 28 U / mL BSSL, BSSL is secreted together with artificial intestinal fluid, the flow rate is as shown in Figure 4 ;

[0054] Infusion of artificial intestinal fluid containing 62 U / mL PLRP2, PLRP2 is secreted together with artificial intestinal fluid, the flow rate is as shown in Figure 5 ;

[0055] According to Table 1, simulate gastric emptying;

[0056] Infusion of 0.1 M hydrochloric acid solution, the flow rate is as shown in Figure 6 ;

[0057] Infusion of 0.1 M sodium hydroxide solution, the flow rate is as shown in Figure 7 .

[0058] Example 2

[0059] This example is basically the same as Example 1, only the infant formula is replaced by human milk, and the flow rate of the 0.1 M hydrochloric acid solution is as shown in Figure 8 .

[0060] Comparative Example 1

[0061] According to the following experimental parameters, the infant formula (IF) is digested:

[0062] 100 mL of infant formula (IF) is added to the simulated stomach of the DIS-II gastrointestinal in vitro digestion simulation system;

[0063] Infusion of artificial gastric juice, wherein the composition of the artificial gastric juice is NaCl 94 mmol / L, KCl 13 mmol / L, and the solvent is water;

[0064] Infusion of artificial gastric juice containing 19 U / mL gastric lipase, so that the lipase is secreted together with the artificial gastric juice;

[0065] Infusion of 0.1 M hydrochloric acid solution, the flow rate is 0.1~0.5 mL / min, to simulate the pH change of the stomach after the baby eats;

[0066] Infusion of artificial intestinal fluid, wherein the composition of the artificial intestinal fluid is NaCl 164 mmol / L, KCl 10 mmol / L, NaHCO385 mmol / L, CaCl2119 mmol / L, and the solvent is water;

[0067] Infusion of artificial intestinal fluid containing 90 U / mL pancreatic lipase.

[0068] Comparative Example 2

[0069] This example is basically the same as Comparative Example 1, except that the infant formula is replaced by human milk.

[0070] The in vivo digestion and in vitro digestion were evaluated according to the gastric pH change, gastric half emptying time, and intestinal pH change in Example 1, Example 2, Comparative Example 1 and Comparative Example 2. The fitting degree of in vivo digestion and in vitro digestion was evaluated according to the following formula:

[0071]

[0072] wherein n represents the number of in vitro-in vivo data pairs used for MAPE and Bias calculation in the in vitro-in vivo correlation (IVIVR) study. MAPE (mean absolute percentage error) and Bias (systematic bias) were selected as the evaluation indexes of the accuracy of the in vitro method, in which the in vivo data were used as the "true value" for reference. These two indexes have a wide application in the quantitative analysis of modeling and prediction data accuracy.

[0073] As shown in Table 2, the in vitro simulation of Example 1 or Example 2 of the present application showed a high consistency with the in vivo measured gastric pH kinetic curve during the digestion of HM and IF. The Pearson correlation coefficient r = 0.971, p < 0.01 for the in vitro simulation of the digestion of HM, and r = 0.997, p < 0.01 for the in vitro simulation of the digestion of IF. The Bias and MAPE of the in vitro simulation were 1.72% and 2.22%, respectively.

[0074] Table 2 In vivo-in vitro gastric pH fitting degree in early life of infants

[0075]

[0076] As shown in Table 3, the in vitro simulation of Example 1 or Example 2 of the present application showed a high consistency with the in vivo measured gastric emptying time during the digestion of HM and IF. The Bias and MAPE of the in vitro simulation were 6.97% and 6.97%, respectively.

[0077] Table 3 In vivo-in vitro gastric emptying fitting degree in early life of infants

[0078]

[0079] As shown in Table 4, the in vitro simulation of Example 1 or Example 2 of the present application showed a high consistency with the in vivo measured intestinal pH kinetic curve during the digestion of HM and IF. The Pearson correlation coefficient r = 0.971, p < 0.01 for the in vitro simulation of the digestion of HM, and r = 0.997, p < 0.01 for the in vitro simulation of the digestion of IF. The Bias and MAPE of the in vitro simulation were 1.03% and 1.89%, respectively.

[0080] Table 4 Fitting of in-vivo-in-vitro intestinal pH in early life of infants

[0081]

[0082] In summary, the constructed infant early life in vitro lipid digestion model method can provide a more accurate evaluation model for the nutritional evaluation of lipids in infant food.

[0083] The above merely describes preferred embodiments of the present application, and the present application is not limited to the above-described embodiments. As long as the same or equivalent means achieve the technical effects of the present application, they should all fall within the protection scope of the present application. Within the protection scope of the present application, the technical solutions and / or embodiments can have various modifications and changes.

Claims

1. A method for in-vitro digestion simulation of lipids based on the physiological characteristics of the infant's digestion, characterized in that, The method comprises the following steps: The simulated gastric juice is secreted according to a first segmented gradient infusion strategy, and the simulated gastric juice containing gastric lipase is secreted according to a second segmented gradient infusion strategy; The simulated intestinal juice is secreted according to a third segmented gradient infusion strategy, and the simulated intestinal juice containing bile salt-stimulated lipase is secreted according to a fourth segmented gradient infusion strategy, and the simulated intestinal juice containing pancreatic lipase-related protein 2 is secreted according to a fifth segmented gradient infusion strategy; The first segmented gradient infusion strategy is that the first stage flow rate is 10-13 mL / min, the second stage flow rate is exponentially decreased to 0.4-0.6 mL / min, and the third stage flow rate is 0.1-0.3 mL / min; The second segmented gradient infusion strategy is that the first stage flow rate is 10-13 mL / min, the second stage flow rate is exponentially decreased to 0.4-0.6 mL / min, and the third stage flow rate is 0.1-0.3 mL / min; The third segmented gradient infusion strategy is that the first stage flow rate is 0.8-1.2 mL / min, the second stage flow rate is 1.2-1.6 mL / min, the third stage flow rate is 0.8-1.2 mL / min, and the fourth stage flow rate is 0.8-1.1 mL / min; The fourth segmented gradient infusion strategy is that the first stage flow rate is 0.60-0.88 mL / min, the second stage flow rate is 0.90-1.20 mL / min, the third stage flow rate is 0.60-0.90 mL / min, and the fourth stage flow rate is 0.50-0.70 mL / min; The fifth segmented gradient infusion strategy is that the first stage flow rate is 0.80-1.10 mL / min, the second stage flow rate is 1.00-1.40 mL / min, the third stage flow rate is 0.80-1.20 mL / min, and the fourth stage flow rate is 0.70-0.90 mL / min.

2. The method of claim 1, wherein, The concentration of the gastric lipase in the simulated gastric juice containing gastric lipase is 18-20 U / mL, the concentration of the bile salt-stimulated lipase in the simulated intestinal juice containing bile salt-stimulated lipase is 26-30 U / mL, and the concentration of the pancreatic lipase-related protein 2 in the simulated intestinal juice containing pancreatic lipase-related protein 2 is 60-65 U / mL.

3. The method of claim 1, wherein, The method further comprises a step of simulating gastric emptying.

4. The method of claim 1, wherein, The method further comprises a step of simulating gastric acid secretion and a step of simulating intestinal alkali secretion.

5. The method of claim 1, wherein, The method further comprises a step of controlling the temperature of the gastrointestinal in-vitro digestion simulation system to be 37±0.5 ℃.

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