CN112029663A - Integrated human gastrointestinal tract digestion and absorption in-vitro substitution model - Google Patents

Integrated human gastrointestinal tract digestion and absorption in-vitro substitution model Download PDF

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CN112029663A
CN112029663A CN202010698350.5A CN202010698350A CN112029663A CN 112029663 A CN112029663 A CN 112029663A CN 202010698350 A CN202010698350 A CN 202010698350A CN 112029663 A CN112029663 A CN 112029663A
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simulation system
absorption
digestion
cells
control system
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CN112029663B (en
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杨杏芬
吴炜亮
李跃麒
范维林
李长林
徐飞飞
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Southern Medical University
Tianjin Hope Industry and Trade Co Ltd
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Southern Medical University
Tianjin Hope Industry and Trade Co Ltd
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Abstract

The invention provides an integrated human gastrointestinal tract digestion and absorption in-vitro substitution model, which is an integrated in-vitro simulation digestion model integrated by 12 systems and comprises a stomach digestion simulation system, a small intestine absorption simulation system, a large intestine decomposition simulation system, a central intelligent control system, a pH control system, a temperature control system, a stirring rate control system, a conveying system, a central purification system, an automatic cleaning and disinfection system and an anaerobic system. Through the synergistic effect of each system, the in vitro substitution model not only simulates the digestive absorption process of the gastrointestinal tract in vitro, but also simulates the fermentation and decomposition process of the intestinal microflora. The model is suitable for accurately quantifying the metabolic process and toxic effect of nutrients or pollutants in a food matrix in vivo, obtaining relevant data such as biological accessibility, biological effectiveness, metabolic tendency and interaction with microorganisms of the nutrients or the pollutants and realizing accurate risk assessment.

Description

Integrated human gastrointestinal tract digestion and absorption in-vitro substitution model
Technical Field
The invention belongs to the technical fields of food safety, in-vitro substitution toxicology, microbiology, nutriology, organic chemistry and the like, and particularly relates to an integrated in-vitro substitution model for human gastrointestinal tract digestion and absorption.
Background
After food enters the gastrointestinal tract of a human body through the oral cavity, nutrients or pollutants combined with a food matrix are released into gastrointestinal fluid under the digestive action of the gastrointestinal tract, absorbed through the gastrointestinal tract and finally reach target organs through blood or lymph tissue and other human body internal circulation to form a health effect, and the series of processes are complex processes of digestive fluid secretion, enzyme reaction, gastrointestinal motility, intestinal microflora and the like. However, the binding form, structure, physicochemical properties, etc. of nutrients or contaminants to the food matrix can affect the rate of transport through the gastrointestinal wall into the blood circulation to the target organ, e.g., a highly lipid-soluble chemical contaminant cannot be completely absorbed through the gastrointestinal tract due to its inability to form a molecular solution in the gastrointestinal tract. However, currently, in evaluating the health effects of nutrients or contaminants in food products, factors that affect gastrointestinal digestive absorption are not considered, which may result in overestimation or underestimation of the associated health effects in humans.
In order to quantify and evaluate the influence of absorption and metabolism processes, toxic effects and decomposition and transformation of nutrients or pollutants in vivo on human Health more precisely, researchers have attempted to construct in vitro gastrointestinal tract models by simulating the digestive and absorption processes of the human body, such as the more widely used physiological principles Extraction model (PBET), the RIVM model established by the National Institute of Public Health and Environment in the netherlands (National Institute of Public Health and the Environment), the DIN model recognized by the german standards Institute (Deutsches Institute of north norm), the european standard Method developed by the european bioavailability research group (UBM model, Unified bar Method) and the infogenst model. Although these models can better simulate the physiological and biochemical processes of gastrointestinal tract digestion, they fail to truly reflect the different characteristics of microorganisms in the colon reactor and the metabolic conditions of nutrients or pollutants under the action of human intestinal flora. Therefore, in vitro substitution models such as a Simulator of Human Intestinal Microbial Ecosystem (Simulator of Human Intestinal Microbial Ecosystem of Infants, SHIME), and a TIM model (TNO gastroenterological model) developed by Nutrition and food research institute of the Netherlands applied scientific research institute have introduced Intestinal microbes to simulate the absorption and metabolism of the colon part of the Human body. Although the model has more completely simulated the digestive situation of the whole digestive tract, the model fails to simulate the absorption mechanism, the immune mechanism, the microbial metabolism mechanism and the like of the digestive tract compared with the in vivo condition.
Based on the principle of the model, partial scientific research institutions in China also set forth the establishment of in vitro simulation systems and corresponding methodology research. For example, the invention patent CN102533543B discloses a device for intestinal microorganism culture, which can simulate the human intestinal environment; the invention patent with publication number CN101665758B discloses a human gastrointestinal tract digestive system based on SHIME model principle and aiming at improving living habits of Asian people; the invention patent with publication number CN103740589B discloses a bionic system for simulating human gastrointestinal tract digestion and intestinal microecology and a simulation test method based on the system; the invention patent with publication number CN108318625B discloses a visual bionic digestive system of a human intestinal tract model. The invention patent establishes a relevant in-vitro digestion model for simulating human intestinal microorganisms and human gastrointestinal tract digestion and intestinal tract microecology, but also fails to realize the simulation of the aspects of the absorption mechanism, the immune mechanism, the microbial metabolism mechanism and the like of the simulated digestive tract.
Disclosure of Invention
Aiming at the limitation of the existing human gastrointestinal tract digestion in-vitro simulation system, the invention aims to provide an integrated human gastrointestinal tract digestion and absorption in-vitro substitution model which can simulate a digestion mechanism, an absorption mechanism, an immunization mechanism and intestinal microecology, is used for truly simulating the digestion and absorption process of nutrients or pollutants in food in the human gastrointestinal tract, and provides data support for food safety risk assessment.
To achieve the above objects, the present invention provides an integrated in vitro substitution model for digestive absorption in the gastrointestinal tract of a human body, comprising:
the gastric digestion simulation system comprises a spherical polytetrafluoroethylene reactor, wherein a digestive juice inlet of the spherical polytetrafluoroethylene reactor is connected with a gastric juice storage bottle through a silicone tube;
the small intestine digestion simulation system comprises a cylindrical polytetrafluoroethylene reactor, wherein a gas inlet of the reactor is connected with a gas outlet of the stomach digestion simulation system through a silicone tube, and a digestive juice inlet of the reactor is connected with a digestive juice outlet of the stomach digestion simulation system through a silicone tube; the small intestinal juice inlet is connected with a small intestinal juice storage bottle through a silicone tube;
the small intestine absorption simulation system is a simulation system which co-cultures 1 human colon adenocarcinoma cell, 1 cup-shaped cell and 1 human liver precursor cell in a Transwell small-scale simulation digestion device; the digestive juice is filtered and transferred to an upper chamber of a Transwell small-sized analog simulation digestion device through a polyether sulfone filter membrane through a sampling port of a small intestine digestion analog system to simulate an absorption metabolic process;
the large intestine decomposition simulation system comprises three cylindrical polytetrafluoroethylene reactors connected through silicone tubes; the gas inlet of the large intestine decomposition simulation system is connected with the gas outlet of the small intestine digestion simulation system through a silicone tube; a digestive juice inlet of the large intestine decomposition simulation system is connected with a digestive juice outlet of the small intestine digestion simulation system through a silicone tube;
the central intelligent control system comprises a touch screen intelligent controller which is connected with other systems through communication lines;
the pH control system comprises five pH electrodes, a pH controller, an acid liquid bottle, an alkali liquid bottle, an acid liquid peristaltic pump and an alkali liquid peristaltic pump; the pH electrode is connected with a pH controller through a communication line; the pH controller is connected with the central intelligent controller through a communication line so as to transmit the pH value of each simulation system to the central intelligent controller in real time and control the acid liquid and alkali liquid peristaltic pumps to start the transfer of the acid liquid and the alkali liquid; the acid liquor bottle and the alkali liquor bottle are respectively connected with acid liquor and alkali liquor inlets of the stomach digestion simulation system, the small intestine digestion simulation system and the large intestine decomposition simulation system through silicone tubes;
the temperature control system is connected with the central intelligent control system through a communication line and comprises a movable upper cover, an annular water bath vessel, a water bath pump, a plurality of heaters and a temperature sensor; the heaters are connected with the temperature sensors through communication lines, and the real-time temperature of the water bath is fed back to the central intelligent control system through the communication lines by the temperature sensors to form records and control the heaters to be turned on or turned off;
the stirring speed control system comprises five magnetic stirring devices which are connected with the central intelligent control system through a communication line;
the conveying system comprises eight peristaltic pumps which are connected with the central intelligent controller through communication lines;
the central purification system comprises a sealing cover, three groups of deodorization devices, an air draft device and an electric switch; two ends of the electric switch are respectively connected with the central intelligent control system and the air draft device through communication lines; the air draft device is connected with the sealing cover and the deodorization device through an ABS plastic pipe;
the automatic cleaning and disinfecting system comprises three cleaning and disinfecting bottles and three delivery pumps, wherein the three cleaning and disinfecting bottles are respectively connected with the three delivery pumps through silicone tubes, the three delivery pumps are all connected with the gastric digestion simulation system through the silicone tubes, and the three delivery pumps are all connected with the central intelligent control system through communication lines;
the anaerobic system is connected with the gastric digestion simulation system through a silicone tube and comprises a nitrogen generating device, an electric switch and a waste gas bottle; the electric switch is connected with the central intelligent control system through a communication line; the nitrogen generating device is connected with a gas inlet of the gastric digestion simulation system through a silicone tube.
Compared with the prior art, the invention integrates a plurality of systems for simulating the digestive absorption of the gastrointestinal tract of the human body, realizes the full-automatic control of the whole process of the digestive absorption of the gastrointestinal tract of the human body by the central intelligent control system, and has the characteristics of low cost and high efficiency for simulating the digestive absorption of the gastrointestinal tract in vitro. The small intestine absorption simulation system can simulate the absorption mechanism and the immune mechanism of the small intestine, is closer to the real absorption process of the small intestine to related substances, and is better than the mode in the existing intestinal simulation model. The large intestine decomposition simulation system can realize real-time detection of microbial metabolites, intestinal microbial communities, intestinal microbial-host interaction and the like, and obtain the catabolic fate of nutrients and pollutants in the intestinal microbial ecosystem and the interaction mechanism with the intestinal microbial communities and the host. The model has good stability and repeatability, meets the requirements of standardized scientific research and application, is suitable for simulating the convergence of nutrients and pollutants in a food matrix in the digestion and absorption of human intestinal tracts and the catabolism of the nutrients and the pollutants in an intestinal microorganism ecosystem, accurately quantifies the metabolic process and the toxic action of the nutrients or the pollutants in the food matrix in vivo, and obtains relevant data such as the biological accessibility, the biological effectiveness and the like of the nutrients or the pollutants.
Further, the small intestine absorption simulation system is a simulation system formed by co-culturing human colon adenocarcinoma cells Caco-2, goblet cells HT29-MTX and human liver precursor cells HepaRG in a Transwell small simulation digestion device by using DMEM medium and William's E medium. The Caco-2 cells, the HT29-MTX cells and the HepaRG cells are co-cultured in a Transwell small simulation digestion device, so that the absorption mechanism and the immune mechanism of the simulated small intestine can be realized, the real absorption process of the small intestine to related substances is more approximate, and the model is better than that in the existing intestinal simulation model.
Further, the small intestine absorption simulation system is constructed according to the following steps:
(1) cell culture: the Caco-2 cells and HT29-MTX cells were cultured in DMEM medium, and the culture medium was added thereto at 37 ℃ with 5% CO2And culturing in an environment with 90% humidity; when the cells grow to 80%, digesting and passaging the cells by using 0.25% of pancreatin-EDTA at a passage ratio of 1: 2-1: 4; HepRG cells were cultured in William's E medium, added with culture medium, and incubated at 37 ℃ with 5% CO2Culturing under the condition of 90% humidity, and replacing a DMEM culture medium and a William's E culture medium every 2-3 days; cells were passaged every 2 weeks at a density of 2.7X 104Cells/cm2；
(2) Establishing a co-culture model: caco-2 cells and HT29-MTX cells were inoculated into 6-well transwells at inoculation concentration ratios of 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50, respectively, the cell concentration when the final plate was adjusted to about 5X 104 cells/mL using complete medium, the total amount of suspension added to the upper chamber was 1.5mL, and 2.5mL of complete medium was added to the lower chamber, and the plate was placed in 5% CO2Culturing at 37 deg.C in cell culture box, and changing every 2dCulturing in complete culture medium at conventional time to 21d, and making 3 parallel wells at each ratio; during the period, the transmembrane resistance value and the AKP enzyme activity are monitored, and a fluorescent yellow permeability experiment and morphological observation are carried out after 21d to determine the optimal culture proportion;
(3) constructing a small intestine absorption simulation system: firstly, inverting the Transwell nested chamber in a 6-hole culture plate, and then tightly attaching another Transwell nested bracket without a polyester carbonate film to the first nested chamber to avoid the leakage of culture medium and cells; then approximately 5 x 104 HepaRG cells were resuspended in 100 μ L of medium, seeded on the bottom of the inverted nested chamber, and cultured with the corresponding complete medium for 3-4 h until the cells adhered; then the nested chamber with the HepaRG cells on the bottom surface is turned over and placed in a 6-well plate; then Caco-2 cells and HT29-MTX cells were seeded at the top side of the Transwell nest in the optimal ratio selected in step (2) to give a final cell concentration of 5X 104 cells/mL; adding 1.5mL of complete culture medium into the upper chamber, adding 2.5mL of complete culture medium into the lower chamber, and continuing culturing, and completing the construction of the small intestine absorption simulation system after stabilization.
Further, in the small intestine absorption simulation system construction step, the culture solution of Caco-2 cells is 1% of non-essential amino acid, 1% of penicillin-streptomycin double-antibody solution, 2mmol/L L-glutamine and 20% of fetal bovine serum; the culture solution of HT29-MTX cell is 1% non-essential amino acid, 1% penicillin-streptomycin double-antibody solution, 2mmol/L L-glutamine and 10% fetal calf serum; the culture solution of HepRG cells is 1% non-essential amino acid, 1% penicillin-streptomycin double antibody solution, 2mmol/L L-glutamine, 10% fetal calf serum and 5. mu.g/mL insulin and 50. mu.M hydrocortisone hemisuccinate.
Furthermore, the large intestine decomposition simulation system is formed by connecting three cylindrical polytetrafluoroethylene reactors which respectively simulate ascending colon, transverse colon and descending colon through silicone tubes.
Further, the large intestine decomposition simulation system is used for constructing intestinal microbial ecology by inoculating excrement of healthy people in an SHIME culture medium for 24h anaerobic culture.
Further, each set of deodorizing device comprises a calcium chloride dehumidifying device and an activated carbon adsorbing device. Through the deodorizing device, the purifying system can adsorb peculiar smell generated by intestinal microorganism fermentation during the running of the in-vitro substitution model of the digestion and absorption of the gastrointestinal tract of a human body.
Further, the automatic cleaning and disinfecting system is a CIP clean-in-place system. The CIP in-situ cleaning system can realize the disinfection and cleaning of the in-vitro surrogate model through the central intelligent controller under the condition that the in-vitro surrogate model is digested and absorbed by the gastrointestinal tract of a human body is not disassembled or moved, effectively removes residues of fat, protein, microorganisms and the like, and ensures the accuracy of a next batch of simulation experiments.
Further, the integrated human gastrointestinal tract digestion and absorption in-vitro substitution model further comprises a functional rack, and the stomach digestion simulation system, the small intestine absorption simulation system, the large intestine decomposition simulation system, the central intelligent control system, the pH control system, the temperature control system, the stirring rate control system, the conveying system, the central purification system, the automatic cleaning and disinfection system and the anaerobic system are integrated in the functional rack. All systems can be integrated into an integrated model through a functional bench.
Furthermore, the functional rack is of a three-layer structure and sequentially comprises a rack bottom plate, a rack guard plate and a support frame plate from bottom to top; the bottom of the function rack is also provided with a shock absorption foot pad. The functional rack has simple and practical structure, and can conveniently erect various simulation systems, thereby forming an integrated model structure.
Drawings
FIG. 1 is a schematic diagram of an integrated in-vitro substitution model for digestive absorption in the human gastrointestinal tract according to the present invention;
FIG. 2 is a schematic diagram of a GI tract digestion simulation system of the present invention;
FIG. 3 is a schematic view of a small intestine absorption simulation system of the present invention;
FIG. 4 is a schematic diagram of a large intestine breakdown simulation system of the present invention;
FIG. 5 is a schematic diagram of a temperature control system of the present invention;
FIG. 6 is a schematic view of a central purification system of the present invention;
FIG. 7 is a schematic view of the automatic cleaning and disinfecting system of the present invention;
FIG. 8 is a schematic view of an anaerobic system of the present invention;
FIG. 9 is a schematic view of the structure of the utility stand of the present invention;
FIG. 10 is a graph showing the effectiveness of the model of the present invention in combination with a mouse model and a conventional in vitro digestion and absorption model.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described in detail and completely with reference to the accompanying drawings. The following description refers to the accompanying drawings, in which like reference numerals refer to the same or similar elements throughout the different views, unless otherwise specified. It is to be understood that the described embodiments are merely examples of some, but not all, of the disclosed embodiments of the invention, which are in keeping with certain aspects of the invention, as detailed in the appended claims. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The use of "first," "second," and similar terms in the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item appearing before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect. The singular forms "a", "an", and "the" may include plural referents unless the context clearly dictates otherwise. "and/or" means any and all possible combinations of one or more of the associated listed items.
The integrated human gastrointestinal tract digestion and absorption in-vitro substitution model provided by the embodiment of the invention is explained in the following with the accompanying drawings.
As shown in fig. 1, the integrated in-vitro human gastrointestinal tract digestion and absorption model provided by the embodiment of the present invention is an integrated in-vitro simulated digestion model formed by integrating 12 systems, including a stomach digestion simulation system 1, a small intestine digestion simulation system 2, a small intestine absorption simulation system 3, a large intestine decomposition simulation system 4, a central intelligent control system 5, a pH control system 6, a temperature control system 7, a stirring rate control system 8, a conveying system 9, a central purification system 10, an automatic cleaning and disinfecting system 11, and an anaerobic system 12. Through the synergistic effect of each system, the in vitro substitution model not only simulates the digestive absorption process of the gastrointestinal tract in vitro, but also simulates the fermentation and decomposition process of the intestinal microflora.
Referring also to fig. 2, the gastric digestion simulation system 1, the small intestine digestion simulation system 2, the pH control system 6, the delivery system 9, and the anaerobic system 12 form a gastrointestinal digestion simulation system. Wherein, the gastric digestion simulation system 1 comprises a spherical polytetrafluoroethylene reactor 101, the small intestine digestion simulation system 2 comprises a cylindrical polytetrafluoroethylene reactor 201, and the pH control system comprises five pH electrodes 601-605, a pH controller 611-615, an acid bottle 621-625, an alkali bottle 631-635, an acid peristaltic pump 641-645 and an alkali peristaltic pump 651-655; the conveying system 9 comprises peristaltic pumps 921-928; the anaerobic system 12 includes a nitrogen generating device 1201, an electrical switch 1202, and an exhaust bottle 1203.
Specifically, in the gastric digestion simulation system 1, the gas inlet 102 of the spherical polytetrafluoroethylene reactor 101 is connected with the nitrogen generation device 1201 of the anaerobic system 12 through a silicone tube; the gas outlet 103 is connected with a gas inlet 202 of the small intestine digestion simulation system 2 through a silicone tube; the food liquid inlet 104 is connected with the food liquid storage bottle 901 through a silicone tube; the digestive juice inlet 105 is connected with a gastric juice storage bottle 902 through a silicone tube; the digestive juice outlet 106 is connected with a digestive juice inlet 204 of the small intestine digestion simulation system 2 through a silicone tube; the acid liquid inlet 107 is connected with the acid liquid peristaltic pump 641 through a silicone tube, and the alkali liquid inlet 108 is connected with the alkali liquid peristaltic pump 651 through a silicone tube; the sample is collected at the sampling port 109.
In the small intestine digestion simulation system 2, a gas inlet 202 of a cylindrical polytetrafluoroethylene reactor 201 is connected with a gas outlet 103 of the stomach digestion simulation system 1 through a silicone tube; the gas outlet 203 is connected with a gas inlet 402 of the large intestine decomposition simulation system 4 through a silicone tube; the digestive juice inlet 204 is connected with the digestive juice outlet 106 of the gastric digestion simulation system 1 through a silicone tube; the small intestinal juice inlet 205 is connected with a small intestinal juice storage bottle 903 through a silicone tube; the digestive juice outlet 206 is connected with the digestive juice inlet of the large intestine decomposition simulation system 4 through a silicone tube; the acid liquid inlet 207 is connected with the acid liquid peristaltic pump 642 through a silicone tube, and the alkali liquid inlet 208 is connected with the alkali liquid peristaltic pump 652 through a silicone tube; the sample is collected at the sampling port 209.
Referring to FIG. 3, in the present embodiment, the intestinal absorption simulation system 3 is formed by co-culturing Caco-2 cells, HT29-MTX cells and HepaRG cells in a Transwell mini-simulation digestion unit 301. The sample taken from the sampling port 209 was filtered through a Polyethersulfone (PES) filter and transferred to a Transwell mini-simulated digestion unit 301.
The small intestine absorption simulation system 3 is constructed according to the following steps:
(1) cell culture: caco-2 cells and HT29-MTX cells were cultured in DMEM medium (high sugar), added with a culture medium, and incubated at 37 ℃ with 5% CO2And culturing in an environment with 90% humidity; when the cells grow to 80%, digesting and passaging the cells by using 0.25% of pancreatin-EDTA at a passage ratio of 1: 2-1: 4; HepRG cells were cultured in William's E medium, added with culture medium, and incubated at 37 ℃ with 5% CO2Culturing under the condition of 90% humidity, and replacing a DMEM culture medium and a William's E culture medium every 2-3 days; the cells were passaged every 2 weeks at a density of 2.7X 104 cells/cm2(ii) a Wherein, the culture solution of Caco-2 cells is 1% of non-essential amino acid, 1% of penicillin-streptomycin double-antibody solution, 2mmol/L L-glutamine and 20% of fetal calf serum; the culture solution of HT29-MTX cell is 1% non-essential amino acid, 1% penicillin-streptomycin double-antibody solution, 2mmol/L L-glutamine and 10% fetal calf serum; HepRG cell culture solution1% of non-essential amino acid, 1% of penicillin-streptomycin double-resistant liquid, 2mmol/L L-glutamine, 10% of fetal bovine serum, 5 mu g/mL of insulin and 50 mu M of hydrocortisone hemisuccinate.
(2) Establishing a co-culture model: caco-2 cells and HT29-MTX cells were inoculated into 6-well transwells at inoculation concentration ratios of 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50, respectively, the cell concentration when the final plate was adjusted to about 5X 104 cells/mL using complete medium, the total amount of suspension added to the upper chamber (AP) was 1.5mL, the total amount of complete medium added to the lower chamber (BL) was 2.5mL, and the plate was placed in 5% CO2Culturing at 37 deg.C in cell culture box, replacing complete culture medium every 2d, culturing to 21d conventionally, and making 3 holes in parallel at each ratio; during the period, the transmembrane resistance value and the AKP enzyme activity are monitored, and a fluorescent yellow permeability experiment and morphological observation are carried out after 21d to determine the optimal culture proportion;
(3) constructing a small intestine absorption simulation system: firstly, inverting the Transwell nested chamber in a 6-hole culture plate, and then tightly attaching another Transwell nested bracket without a polyester carbonate film to the first nested chamber to avoid the leakage of culture medium and cells; then approximately 5 x 104 HepaRG cells were resuspended in 100 μ L of medium, seeded on the bottom of the inverted nested chamber, and cultured with the corresponding complete medium for 3-4 h until the cells adhered; then the nested chamber with the HepaRG cells on the bottom surface is turned over and placed in a 6-well plate; then Caco-2 cells and HT29-MTX cells were seeded at the top side of the Transwell nest in the optimal ratio selected in step (2) to give a final cell concentration of 5X 104 cells/mL; adding 1.5mL of complete culture medium into the upper chamber, adding 2.5mL of complete culture medium into the lower chamber, and continuing culturing, and completing the construction of the small intestine absorption simulation system after stabilization.
Referring also to fig. 4, the simulated large intestine dissociation system 4 comprises three cylindrical ptfe reactors 401, 411, and 421 connected by silicone tubing. Wherein, the gas inlet of the cylindrical polytetrafluoroethylene reactor 401 is connected with the gas outlet 203 of the small intestine digestion simulation system 2 through a silicone tube; the gas outlet is connected with the gas inlet of the cylindrical polytetrafluoroethylene reactor 411 through a silicone tube; the gas outlet of the cylindrical polytetrafluoroethylene reactor 411 is connected with the gas inlet of the cylindrical polytetrafluoroethylene reactor 421 through a silicone tube; the gas outlet of the cylindrical polytetrafluoroethylene reactor 421 is connected with the waste gas bottle 1203 by a silicone tube; a digestive juice inlet of the cylindrical polytetrafluoroethylene reactor 401 is connected with a digestive juice outlet 206 of the small intestine digestion simulation system 2 through a silicone tube; the digestive juice outlet is connected with the digestive juice inlet of the cylindrical polytetrafluoroethylene reactor 411 through a silicone tube; a digestive juice outlet of the cylindrical polytetrafluoroethylene reactor 411 is connected with a digestive juice inlet of the cylindrical polytetrafluoroethylene reactor 421 through a silicone tube; a digestive juice outlet of the cylindrical polytetrafluoroethylene reactor 421 is connected with the waste liquid bottle 904 through a silica gel tube; acid liquor inlets of the cylindrical polytetrafluoroethylene reactors 401, 411 and 421 are respectively connected with acid liquor peristaltic pumps 643, 644 and 645 through silicone tubes; the alkali liquor inlets 407, 417 and 427 of the cylindrical polytetrafluoroethylene reactors 401, 411 and 421 are respectively connected with alkali liquor peristaltic pumps 653, 654 and 655 through silicone tubes; the sample is collected at the sampling port. The large intestine decomposition simulation system 4 constructs intestinal microbial ecology by inoculating feces of healthy people in SHIME culture medium for 24h for anaerobic culture.
The central intelligent control system 5 is a touch screen intelligent controller, and is connected with other systems through communication lines, and the starting and action setting of each system are realized in the touch intelligent controller.
Specifically, in the pH control system 6, each pH electrode is connected to each pH controller through a communication line; each pH controller is respectively connected with the central intelligent control system 5 through a communication line, so as to transmit the pH values of the gastric digestion simulation system 1, the small intestine digestion simulation system 2 and the large intestine decomposition simulation system 4 to the central intelligent control system 5 in real time. Each acid liquor peristaltic pump and each alkali liquor peristaltic pump are connected with the central intelligent control system 5 through communication lines and are controlled to be turned on or turned off by the central intelligent control system 5. Each acid liquid bottle is connected with each acid liquid peristaltic pump through a silica gel tube, and each alkali liquid bottle is connected with each alkali liquid peristaltic pump through a silica gel tube.
Referring to fig. 5, the temperature control system 7 includes a movable cover 701, an annular water bath 702, heaters 703-705, a water bath pump 706, and a temperature sensor 707. The heaters 703-705 are respectively connected with the temperature sensor 707 through a communication line, the temperature sensor 707 is connected with the central intelligent control system 5 through a communication line, the real-time temperature of the water bath is fed back to the central intelligent control system 5 through the communication line by the temperature sensor 707 to form a record, and the heaters 703-705 are controlled to be turned on or turned off.
Specifically, the stirring speed control system 8 comprises five magnetic stirring devices 801-805. The stirring speed control system 8 is connected with the central intelligent control system 5 through a communication line, and the rotating speeds of the five magnetic stirring devices 801-805 are set through the central intelligent control system 5 and range from 0rpm to 300 rpm.
The conveying system 9 is connected with the central intelligent control system 5 through a communication line, the opening and closing of the peristaltic pumps 921-928 and the conveying speed are controlled by the central intelligent control system 5, and the conveying speed range is 0-500 mL/min. Peristaltic pumps 921, 922, 924 and 928 are respectively connected with the food liquid storage bottle 901, the gastric juice storage bottle 902, the small intestinal juice storage bottle 903 and the waste liquid bottle 904 through silicone tubes; the peristaltic pumps 923, 925, 926 and 927 are respectively connected with the gastric digestion simulation system 1, the small intestine digestion simulation system 2 and the large intestine decomposition simulation system 4 through silicone tubes.
Referring to fig. 6, the central purification system 10 includes a sealing cover 1001, deodorizing devices 1002 to 1004, an air draft device 1005, and an electric switch 1006. Wherein, central purification system 10 passes through the communication line with central intelligent control system 5 and is connected, and electric switch 1006 passes through the communication line with updraft ventilator 1005 and is connected, and updraft ventilator 1005 passes through the ABS plastic tubing with sealed cowling 1001, deodorizing device 1002 ~ 1004 and is connected. The deodorizing devices 1002-1004 can be used for adsorbing peculiar smell generated by intestinal microorganism fermentation during the running period of the in-vitro substitution model for the digestion and absorption of the gastrointestinal tract of a human body.
Referring to fig. 7, the automatic cleaning and disinfecting system 11 includes cleaning and disinfecting bottles 1101-1103 and delivery pumps 1111-1113. Wherein, the cleaning and disinfecting reagent bottles 1101-1103 are respectively connected with the delivery pumps 1111-1113 through silicone tubes; the delivery pumps 1111-1113 are respectively connected with the gastric digestion simulation system 1 through silicone tubes; the delivery pumps 1111-1113 are respectively connected with the central intelligent control system 5 through communication lines. Preferably, the automatic cleaning and disinfecting system 11 is a CIP in-situ cleaning system, which can realize the disinfection and cleaning of the in-vitro surrogate model through the central intelligent control system 5 without disassembling or moving the device, effectively remove the residues of fat, protein, microorganisms and the like, and ensure the accuracy of the next batch of simulation experiments.
Referring also to fig. 8, the anaerobic system 12 includes a nitrogen generator 1201, an electrical switch 1202, and an exhaust bottle 1203. Wherein, the electric switch 1202 is connected with the central intelligent control system 5 through a communication line, so as to realize the timing on and off of the anaerobic system every day; the anaerobic system 12 is connected with the gastric digestion simulation system 1 through a silicone tube, and because the gastric digestion simulation system 1 is communicated with the small intestine digestion simulation system 2 and the large intestine decomposition simulation system 4 through the silicone tube, the communication of the three systems and the anaerobic system 12 can simulate the anaerobic environment similar to the gastrointestinal tract.
In order to integrate all the systems into an integrated model, in one embodiment, the integrated human gastrointestinal tract digestive absorption in vitro surrogate model of the present invention further comprises a functional rack into which each of the above-described systems is integrated. As shown in fig. 9, the functional rack has a three-layer structure, which includes a rack bottom plate 1310, a rack guard plate 1320, and a support frame plate 1330 from bottom to top, which are arranged in parallel to each other to form a three-layer rack structure for accommodating various simulation systems. The bottom of the function rack is also provided with a plurality of shock absorption foot pads 1340, and the shock absorption foot pads 1340 can be provided with rotating wheels to facilitate the movement of the function rack.
The following will further describe the use of the integrated in vitro substitution model for digestive absorption of gastrointestinal tract of human body, which is proposed by the present invention, by taking the digestive absorption process of vomitoxin as an example.
The integrated human gastrointestinal tract digestion and absorption in-vitro substitution model is started, relevant in-vitro digestion and absorption model parameters are set in a central intelligent control system 5, wherein the temperature of an annular water bath 702 is 37 ℃, the rotating speed of a magnetic stirring device is 30rpm, the pH range of a stomach digestion simulation system 1 is 1.6-2.2, the pH range of a small intestine digestion simulation system 2 is 5.5-6.0, and the pH ranges of cylindrical polytetrafluoroethylene reactors 401, 411 and 421 of a large intestine decomposition simulation system 4 are 5.6-6.0, 6.0-6.4 and 6.4-6.9 respectively.
Collecting 3-5 adult feces without antibiotic intake for 6 months, sampling 50g of feces, uniformly mixing, adding phosphate buffer solution with pH 7, adding 1g of sodium thioacetate as reducing agent, homogenizing the sample by using a homogenizer, centrifuging the sample at 3000rpm for 5min, inoculating 50mL, 80mL and 50mL of intestinal microbial solution in three cylindrical polytetrafluoroethylene reactors 401, 411 and 421 in a large intestine decomposition simulation system 4 respectively after centrifugation, and then adding SHIME culture medium (1.0g/L arabinogalactan, 2.0g/L pectin, 3.0g/L starch, 1.0g/L xylan, 0.5g/L cysteine, 0.4g/L glucose, 3.0g/L yeast extract, 4.0g/L mucin and 1.0g/L peptone) 500mL, adding, 800mL and 500mL, stirring at 37 ℃ for 24h at 30rpm, and introducing nitrogen for 10min at intervals of 8 h. After stabilization, the pH values of the three reactors are controlled to be 5.6-6.0, 6.0-6.4 and 6.4-6.9 respectively by adding 0.05mol/L HCl or 0.05mol/L NaOH respectively.
After inoculation of microorganisms in the intestinal tract of the human body of the model, the model and the central purification system are started, and the flow rate and the working time of each peristaltic pump, the rotating speed of the magnetic stirrer and the working sequence are set through the central intelligent control system 5. After the set-up was completed, the peristaltic pump was turned on to transfer 200mL of SHIME medium in the food solution storage bottle to the gastric digestion simulation system 1 at a flow rate of 20mL/min and 60mL of gastric fluid to the gastric digestion simulation system 1 at a flow rate of 10 mL/min. After the transfer was completed, the magnetic stirring apparatus was turned on at a rotation speed of 30 rpm. The food solution was digested at 37 ℃ for 2 h. After digestion, 250mL of the liquid was transferred from the gastric digestion simulation system 1 to the small intestine digestion simulation system 2 at a flow rate of 25mL/min, and 60mL of the small intestine digestion liquid (12.5g/L sodium bicarbonate, 6.0g/L bile salts, and 0.9g/L pancreatin) was transferred from the small intestine liquid storage bottle 903 to the small intestine digestion simulation system 2 at a flow rate of 12 mL/min. After the transfer was completed, the magnetic stirring apparatus was turned on at 30rpm and digested in the small intestine digestion simulation system at 37 ℃ for 2 h. After digestion is finished, digestive juice is transferred from the small intestine digestion simulation system 2 to the large intestine decomposition simulation system 4 at the flow rate of 25mL/min, the liquid volumes in the three cylindrical polytetrafluoroethylene reactors 401, 411 and 421 in the large intestine decomposition simulation system 4 are kept constant at 500mL, 800mL and 500mL in the transfer process, and the residual waste liquid is transferred from the digestive juice outlet of the third cylindrical polytetrafluoroethylene reactor in the large intestine decomposition simulation system to a waste liquid bottle by a peristaltic pump. After the transfer was completed, the magnetic stirring apparatus was started at 30rpm, and digestion and decomposition were carried out at 37 ℃ in a large intestine decomposition simulation system. The model is operated according to the steps at 8:00, 12:00 and 18:00 every day, 10mL of samples are collected at sampling ports of three cylindrical polytetrafluoroethylene reactors of a large intestine decomposition simulation system every day, the microbial plate technology method and 16S rRNA high-throughput sequencing are used for detecting the number and the structure of flora, and gas phase and liquid phase are used for measuring the yield of the intestinal microbial short-chain fatty acid to obtain baseline data.
After the model continued to run for two weeks until it stabilized, the 25mg/L vomitoxin-containing food solution in the food solution storage bottle 901 was transferred to the gastric digestion simulation system 1 at a flow rate of 20mL/min for 200mL, and the gastric juice was transferred to the gastric digestion simulation system 1 at a flow rate of 10mL/min for 60 mL. After the transfer was completed, the magnetic stirring apparatus was turned on at a rotation speed of 30 rpm. The food solution was digested at 37 ℃ for 2 h. After digestion, 250mL of the liquid was transferred from the gastric digestion simulation system to the small intestine digestion simulation system at a flow rate of 25mL/min, and 60mL of the small intestine digestion liquid was transferred from the small intestine liquid storage bottle 903 to the small intestine digestion simulation system 2 at a flow rate of 12 mL/min. After the transfer was completed, the magnetic stirring apparatus was turned on at 30rpm and digested in the small intestine digestion simulation system 2 at 37 ℃ for 2 h. After digestion is finished, transferring 10mL of digestive juice to the small intestine absorption simulation system 2 from a sampling port of the small intestine digestion simulation system 2, transferring the digestive juice from the small intestine digestion simulation system 2 to the large intestine decomposition simulation system 4 at a flow rate of 25mL/min, keeping the liquid volumes in three cylindrical polytetrafluoroethylene reactors 401, 411 and 421 in the large intestine decomposition simulation system 4 to be 500mL, 800mL and 500mL constant in the transferring process, and transferring the residual waste liquid into a waste liquid bottle from a digestive juice outlet of a third cylindrical polytetrafluoroethylene reactor 401, 411 and 421 in the large intestine decomposition simulation system 4 by a peristaltic pump. After the transfer was completed, the magnetic stirring apparatus was started at 30rpm, and digestion and decomposition were carried out at 37 ℃ in a large intestine decomposition simulation system. The model was run at 8:00, 12:00 and 18:00 per day according to the above procedure, and 10mL of samples were taken at the sampling ports of the three cylindrical teflon reactors 401, 411 and 421 of the large intestine decomposition simulation system 4 per day.
After passing through PES filter membrane, 10mL of digestive juice transferred from 2 sampling ports of small intestine digestion simulation system was subjected to liquid phase-mass spectrometry to determine the content of vomitoxin, and simultaneously transferred to 5X 10 final cell concentration4The biological accessibility, biological effectiveness and other related data of the small intestine absorption simulation system 3 co-cultured by Caco-2, HT29-MTX and HepaRG triple cells of each/mL are obtained, and FIG. 10 is a comparison between the biological effectiveness of the vomitoxin obtained by the in vitro model and the animal model. As can be seen from the figure, the simulated biological effectiveness of the in vitro substitution model for the digestive absorption of the human gastrointestinal tract is close to that obtained by a mouse model and is far higher than that of the traditional in vitro digestion and absorption model.
10mL of samples collected at a sampling port of a large intestine decomposition simulation system are subjected to microbial plate technology method and 16S rRNA high-throughput sequencing to detect the number and structure of flora, and gas phase, liquid phase and liquid phase-mass spectrometry are used for measuring the content of intestinal microbial metabolites and the like and the content of vomitoxin metabolites, and metabolism tends to interact with microbes and other related data.
After the in vitro digestion and absorption experiment is finished, an automatic cleaning and disinfection system 11 is started to clean a stomach digestion simulation system 1, a small intestine digestion simulation system 2, a large intestine decomposition simulation system 4, a food liquid storage bottle 901 and a waste liquid bottle 904, hot water washing is carried out at 60 ℃ for 3-5 min, 2% NaOH solution is used for washing for 10-20 min, hot water washing is carried out at 60 ℃ for 3-5 min, 2% HCl solution is used for washing for 10-20 min, hot water washing is carried out at 60 ℃ for 3-5 min, 0.05% sodium hypochlorite disinfectant is used for disinfection for 5min, and clear water washing is carried out for 20 min.
Compared with the prior art, the invention integrates a plurality of systems for simulating the digestive absorption of the gastrointestinal tract of the human body, realizes the full-automatic control of the whole process of the digestive absorption of the gastrointestinal tract of the human body by the central intelligent control system 5, and has the characteristics of low cost and high efficiency for simulating the digestive absorption of the gastrointestinal tract in vitro. The small intestine absorption simulation system 3 can simulate the absorption mechanism and the immune mechanism of the small intestine, is closer to the real absorption process of the small intestine to related substances, and is better than the mode in the existing intestinal simulation model. The large intestine decomposition simulation system 4 can realize real-time detection of microbial metabolites, intestinal microbial communities, intestinal microbial-host interaction and the like, and obtain catabolic fates of nutrients and pollutants in the intestinal microbial ecosystem and interaction mechanisms with the intestinal microbial communities and hosts. The model has good stability and repeatability, meets the requirements of standardized scientific research and application, is suitable for simulating the convergence of nutrients and pollutants in a food matrix in the digestion and absorption of human intestinal tracts and the catabolism of the nutrients and the pollutants in an intestinal microorganism ecosystem, accurately quantifies the metabolic process and the toxic action of the nutrients or the pollutants in the food matrix in vivo, and obtains relevant data such as the biological accessibility, the biological effectiveness and the like of the nutrients or the pollutants.
It should be noted that, according to actual requirements, two or more than two of all embodiments provided by the present invention may be combined to solve two or more corresponding technical problems; moreover, various technical features in the above embodiments may be arbitrarily combined as long as there is no conflict or contradiction between the combinations of features, but the combination is not described one by one at the time of space.
The present invention is not limited to the above-described embodiments, and various modifications and variations of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.

Claims (10)

1. An integrated in vitro substitution model for digestive absorption in the gastrointestinal tract of a human being, comprising:
the gastric digestion simulation system comprises a spherical polytetrafluoroethylene reactor, wherein a digestive juice inlet of the spherical polytetrafluoroethylene reactor is connected with a gastric juice storage bottle through a silicone tube;
the small intestine digestion simulation system comprises a cylindrical polytetrafluoroethylene reactor, wherein a gas inlet of the reactor is connected with a gas outlet of the stomach digestion simulation system through a silicone tube, and a digestive juice inlet of the reactor is connected with a digestive juice outlet of the stomach digestion simulation system through a silicone tube; the small intestinal juice inlet is connected with a small intestinal juice storage bottle through a silicone tube;
the small intestine absorption simulation system is a simulation system which co-cultures 1 human colon adenocarcinoma cell, 1 cup-shaped cell and 1 human liver precursor cell in a Transwell small-scale simulation digestion device; the digestive juice is filtered and transferred to an upper chamber of a Transwell small-sized analog simulation digestion device through a polyether sulfone filter membrane through a sampling port of a small intestine digestion analog system to simulate an absorption metabolic process;
the large intestine decomposition simulation system comprises three cylindrical polytetrafluoroethylene reactors connected through silicone tubes; the gas inlet of the large intestine decomposition simulation system is connected with the gas outlet of the small intestine digestion simulation system through a silicone tube; a digestive juice inlet of the large intestine decomposition simulation system is connected with a digestive juice outlet of the small intestine digestion simulation system through a silicone tube;
the central intelligent control system comprises a touch screen intelligent controller which is connected with other systems through communication lines;
the pH control system comprises five pH electrodes, a pH controller, an acid liquid bottle, an alkali liquid bottle, an acid liquid peristaltic pump and an alkali liquid peristaltic pump; the pH electrode is connected with a pH controller through a communication line; the pH controller is connected with the central intelligent controller through a communication line so as to transmit the pH value of each simulation system to the central intelligent controller in real time and control the acid liquid and alkali liquid peristaltic pumps to start the transfer of the acid liquid and the alkali liquid; the acid liquor bottle and the alkali liquor bottle are respectively connected with acid liquor and alkali liquor inlets of the stomach digestion simulation system, the small intestine digestion simulation system and the large intestine decomposition simulation system through silicone tubes;
the temperature control system is connected with the central intelligent control system through a communication line and comprises a movable upper cover, an annular water bath vessel, a water bath pump, a plurality of heaters and a temperature sensor; the heaters are connected with the temperature sensors through communication lines, and the real-time temperature of the water bath is fed back to the central intelligent control system through the communication lines by the temperature sensors to form records and control the heaters to be turned on or turned off;
the stirring speed control system comprises five magnetic stirring devices which are connected with the central intelligent control system through a communication line;
the conveying system comprises eight peristaltic pumps which are connected with the central intelligent controller through communication lines;
the central purification system comprises a sealing cover, three groups of deodorization devices, an air draft device and an electric switch; two ends of the electric switch are respectively connected with the central intelligent control system and the air draft device through communication lines; the air draft device is connected with the sealing cover and the deodorization device through an ABS plastic pipe;
the automatic cleaning and disinfecting system comprises three cleaning and disinfecting bottles and three delivery pumps, wherein the three cleaning and disinfecting bottles are respectively connected with the three delivery pumps through silicone tubes, the three delivery pumps are all connected with the gastric digestion simulation system through the silicone tubes, and the three delivery pumps are all connected with the central intelligent control system through communication lines;
the anaerobic system is connected with the gastric digestion simulation system through a silicone tube and comprises a nitrogen generating device, an electric switch and a waste gas bottle; the electric switch is connected with the central intelligent control system through a communication line; the nitrogen generating device is connected with a gas inlet of the gastric digestion simulation system through a silicone tube.
2. The in vitro substitution model for digestive absorption in human gastrointestinal tract according to claim 1, wherein the simulated system for intestinal absorption is a simulated system formed by co-culturing human colon adenocarcinoma cells Caco-2, goblet cells HT29-MTX and human liver precursor cells Heparg in a Transwell small simulated digestion device by using DMEM medium and William's E medium.
3. The in vitro substitution model for digestive absorption of human gastrointestinal tract according to claim 2, wherein the small intestine absorption simulation system is constructed according to the following steps:
(1) cell culture: the Caco-2 cells and HT29-MTX cells were cultured in DMEM medium, and the culture medium was added thereto at 37 ℃ with 5% CO2And culturing in an environment with 90% humidity; in cellsWhen the growth rate reaches 80%, digesting and passaging the seeds by using 0.25% pancreatin-EDTA (ethylene diamine tetraacetic acid) in a passage ratio of 1: 2-1: 4; HepRG cells were cultured in William's E medium, added with culture medium, and incubated at 37 ℃ with 5% CO2Culturing under the condition of 90% humidity, and replacing a DMEM culture medium and a William's E culture medium every 2-3 days; the cells were passaged every 2 weeks at a density of 2.7X 104 cells/cm2；
(2) Establishing a co-culture model: caco-2 cells and HT29-MTX cells were inoculated into 6-well transwells at inoculation concentration ratios of 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50, respectively, the cell concentration when the final plate was adjusted to about 5X 104 cells/mL using complete medium, the total amount of suspension added to the upper chamber was 1.5mL, and 2.5mL of complete medium was added to the lower chamber, and the plate was placed in 5% CO2Culturing at 37 deg.C in cell culture box, replacing complete culture medium every 2d, culturing to 21d conventionally, and making 3 holes in parallel at each ratio; during the period, the transmembrane resistance value and the AKP enzyme activity are monitored, and a fluorescent yellow permeability experiment and morphological observation are carried out after 21d to determine the optimal culture proportion;
(3) constructing a small intestine absorption simulation system: firstly, inverting the Transwell nested chamber in a 6-hole culture plate, and then tightly attaching another Transwell nested bracket without a polyester carbonate film to the first nested chamber to avoid the leakage of culture medium and cells; then approximately 5 x 104 HepaRG cells were resuspended in 100 μ L of medium, seeded on the bottom of the inverted nested chamber, and cultured with the corresponding complete medium for 3-4 h until the cells adhered; then the nested chamber with the HepaRG cells on the bottom surface is turned over and placed in a 6-well plate; then Caco-2 cells and HT29-MTX cells were seeded at the top side of the Transwell nest in the optimal ratio selected in step (2) to give a final cell concentration of 5X 104 cells/mL; adding 1.5mL of complete culture medium into the upper chamber, adding 2.5mL of complete culture medium into the lower chamber, and continuing culturing, and completing the construction of the small intestine absorption simulation system after stabilization.
4. The in vitro substitution model for digestive absorption in human gastrointestinal tract of claim 3, wherein the culture solution of Caco-2 cells is 1% non-essential amino acid, 1% penicillin-streptomycin double antibody, 2mmol/L L-glutamine and 20% fetal bovine serum; the culture solution of HT29-MTX cell is 1% non-essential amino acid, 1% penicillin-streptomycin double-antibody solution, 2mmol/L L-glutamine and 10% fetal calf serum; the culture solution of HepRG cells is 1% non-essential amino acid, 1% penicillin-streptomycin double antibody solution, 2mmol/L L-glutamine, 10% fetal calf serum and 5. mu.g/mL insulin and 50. mu.M hydrocortisone hemisuccinate.
5. The in vitro substitution model for digestive absorption of human gastrointestinal tract according to claim 1, wherein the large intestine decomposition simulation system is formed by connecting three cylindrical polytetrafluoroethylene reactors for respectively simulating ascending colon, transverse colon and descending colon through silicone tubes.
6. The in vitro substitution model for digestive absorption in human gastrointestinal tract according to claim 1, wherein the large intestine decomposition simulation system constructs intestinal microbial ecology by inoculating feces of healthy people in SHIME culture medium for 24h anaerobic culture.
7. The in vitro substitution model for digestive absorption in human gastrointestinal tract according to claim 1, wherein each set of deodorizing means comprises a calcium chloride dehumidifying means and an activated carbon adsorbing means.
8. The in vitro substitution model for digestive absorption in human gastrointestinal tract according to claim 1, wherein the automatic cleaning and disinfecting system is a CIP clean-in-place system.
9. The in vitro substitution model for digestive absorption of human gastrointestinal tract according to claim 1, further comprising a functional rack in which the gastric digestion simulation system, the small intestine absorption simulation system, the large intestine decomposition simulation system, the central intelligent control system, the pH control system, the temperature control system, the stirring rate control system, the delivery system, the central purification system, the automatic cleaning and disinfecting system and the anaerobic system are integrated.
10. The in vitro substitution model for digestive absorption of human gastrointestinal tract of claim 1, wherein the functional rack is a three-layer structure comprising a rack bottom plate, a rack guard plate and a support rack plate from bottom to top; the bottom of the function rack is also provided with a shock absorption foot pad.
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CN112837590A (en) * 2020-12-29 2021-05-25 浙江工商大学 Method for simulating dynamic digestion of liquid food of infant based on artificial stomach system
CN112837590B (en) * 2020-12-29 2023-03-31 浙江工商大学 Method for simulating dynamic digestion of liquid food of infant based on artificial stomach system
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CN114525226A (en) * 2022-03-15 2022-05-24 陈晓东 Method for preparing culture medium of fecal transplantation bacteria by simulating in-vivo digestion process
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