LU501261B1 - Method for evaluating dynamic food allergen digestion model and in-vitro simulation - Google Patents
Method for evaluating dynamic food allergen digestion model and in-vitro simulation Download PDFInfo
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- LU501261B1 LU501261B1 LU501261A LU501261A LU501261B1 LU 501261 B1 LU501261 B1 LU 501261B1 LU 501261 A LU501261 A LU 501261A LU 501261 A LU501261 A LU 501261A LU 501261 B1 LU501261 B1 LU 501261B1
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Abstract
The present disclosure discloses a method for evaluating a dynamical food allergen digestion model and in-vitro simulation, which mainly includes establishment of a method for evaluating a dynamical allergen digestion model and in-vitro simulation. A digestion system simulating device simulates human gastroduodenal "biochemical reaction" conditions. The method for evaluating simulation proposes an experimental method for establishing an in-vitro simulation of dynamic allergen digestion, an experimental method for establishing an intestinal epithelial cell model using Caco-2 cell (human colon carcinoma cell line) to simulate absorption and transport actions of the intestine, and an experimental method for establishing a KU812 (human peripheral blood basophilic leukemia cell) cell model to evaluate a mast cell degranulation inducing ability of digestion and transport products of food allergens. The present disclosure is applicable to evaluation on digestion and sensitization of purified food allergens or an allergen extract containing complicated food matrix.
Description
METHOD FOR EVALUATING DYNAMIC FOOD ALLERGEN DIGESTION 10501261 MODEL AND IN-VITRO SIMULATION
[01] The present disclosure relates to the field of evaluation on sensitization of food allergens, and particularly to a method for evaluating a dynamical food allergen digestion model and in-vitro simulation.
[02] Food allergies are food safety issues drawn great attention at home and abroad. How to effectively and quickly evaluate the sensitization of food allergens is a problem which needs to be urgently solved in the current food safety study field. Existing evaluation methods all face certain limitations in use and fail to truly and effectively evaluate the sensitization of food allergens.
[03] An objective of the present disclosure is to provide a method for evaluating a dynamic food allergen digestion model and in-vitro simulation, including:
[04] (1) establishing a dynamic allergen digestion model;
[05] (2) establishing an experimental method for in-vitro simulation of dynamic allergen digestion, and analyzing structural changes and digestion stability of a food allergen;
[06] (3) establishing an intestinal epithelial cell model using Caco-2 cells (human colon carcinoma cell line) to simulate digestion, transport and absorption processes of digestion products, and evaluating cytotoxicity and absorption transmittance of an allergen digestion product; and
[07] (4) establishing a KU812 (human peripheral blood basophilic leukemia cell) cell model, and evaluating the mast cell degranulation inducing ability of digestion and transport products of the food allergen.
[08] Further, changes in a structure and sensitization of the food allergen in a digestion process are studied using a simulation device.
[09] Further, the simulation device includes a simulating gastric digestion system, a simulating duodenal digestion system, an automatic pH adjustment system, a constant-temperature heating plate and a magnetic stirrer, wherein secretion of digestive juice and bile salt solution, emptying of gastrointestinal contents, and pH adjustment in the simulating gastric digestion system and the simulating duodenal digestion system are all integrally controlled using stepper motorized peristaltic pumps, all to-be-implemented operations and reaction conditions (running time segments and flow rates of all peristaltic pumps, and target pH curves of a simulating gastric cavity and an enteric cavity) are capable of being preset on a touch-screen control panel before digestion begins, reaction conditions are changed without stopping a digestion process halfway, automatic digestion is achieved in the whole process, and after quick enzyme deactivation, micro-samples are capable of being used for further analyses of digestibility, allergy, etc. 1
[10] Further, digestion products at different time nodes of the continuous digestion LUS01261 process of the stomach and the intestine are collected, a dynamic digestion result of an allergen sample 1s determined through an SDS-polyacrylamide gel experiment, and an IgG/IgE binding ability of the digestion products is evaluated by Westen-blot.
[11] Further, when the intestinal epithelial cell model is established using Caco-2 cells (human colon carcinoma cell line), the Caco-2 cell 1s inoculated into a substrate such as a polycarbonate porous membrane; the Caco-2 cell is cultured under proper culture conditions to spontaneously form monolayers which have a polarity, microvilli and tight junctions, are morphologically similar to human intestinal epithelia, and are used to simulate digestion, transport and absorption of brush border peptidases of allergens by the intestinal epithelial cells.
[12] Further, when the KU812 cell model is established, a mast cell in-vitro degranulation model is established through in-vitro culture of KU812 cell lines and by use of a serum of a patient with an allergic reaction to determine the content of a cellular activity medium in a cell culture fluid after antigen challenge and to evaluate the sensitization of the food allergen after digestion and transport.
[13] Further, the simulating gastric digestion system includes a simulating gastric juice tank, a sample storage tank, and a simulating gastric cavity, wherein the simulating gastric juice tank and the sample storage tank are respectively connected and communicated with the simulating gastric cavity via a peristaltic pump, and the simulating gastric cavity is internally provided with a pH sensor and a temperature sensor;
[14] the simulating duodenal digestion system includes a simulating enteric cavity, a simulating pancreatic juice tank, a bile salt solution tank and a waste liquid tank, wherein the simulating pancreatic juice tank, the bile salt solution tank and the waste liquid tank are respectively connected and communicated with the simulating enteric cavity via a peristaltic pump, and the simulating enteric cavity is internally provided with a pH sensor and a temperature sensor;
[15] the automatic pH adjustment device includes a gastric cavity pH adjustment tank and an enteric cavity pH adjustment tank, wherein the gastric cavity pH adjustment tank and the enteric cavity pH adjustment tank are respectively connected and communicated with the stimulating gastric cavity and the stimulating enteric cavity via a peristaltic pump; and,
[16] a constant-temperature heating plate for preheating and heat insulating and a magnetic stirrer which plays a shearing and mixing role are arranged at a bottom portion of each of the simulating gastric cavity and the simulating enteric cavity.
[17] All peristaltic pumps, pH sensors, temperature sensors, constant-temperature heating plates and magnetic stirrers are respectively electrically connected to a circuit board, and the circuit board is electrically connected to the control panel.
[18] Beneficial effects:
[19] 1. The dynamic digestion model can be preset with digestion conditions at all stages, does not need to stop in the digestion process, and automatically performs continuous digestion and regulates the pH value of a reaction system to a target pH value in real time in the entire process, thereby reducing experimental errors caused by 2 inconsistent operation methods or by stopping to change reaction conditions halfway; LUS01261
[20] 2. The running of the dynamic allergen digestion model is controlled using the touch-screen control panel, and electromagnetic heating is adopted to replace the conventional water-bath heating, thereby reducing space occupied by devices;
[21] 3. The micro-samples can be taken at any time in the sample digestion process, are not required to be extracted for the second time after sampling, and can be used for relevant analyses such as determination of absorption transmittance and sensitization evaluation after termination of digestion, thereby maintaining the primitiveness of the digestion product to the maximum extent;
[22] 4. The present disclosure uses the intestinal epithelial cell model to simulate the digestion, transport and absorption of the brush border peptidases of the gastrointestinal digestion product of allergens by the intestine;
[23] 5. The present disclosure can systematically and effectively reflect an IgE-mediated allergic reaction induced by food allergens, and can be used to evaluate influences of food substrates and processing methods on the digestion stability and sensitization of the allergens.
[24] 6. The present disclosure establishes a human mast cell detecting method, and can be used in combination with the serum of the patient with an allergic reaction to evaluate the sensitization of the food allergens.
[25] FIG. 1 is a schematic structural diagram of a simulation device of a dynamic allergen digestion model according to the present disclosure;
[26] Description of reference numerals: O1: gastric cavity pH adjustment tank; 02- simulating gastric juice tank; 03: sample storage tank; 04: simulating gastric cavity; 05: constant-temperature heating plate; 06: simulating enteric cavity, 07: magnetic stirrer; 08: enteric cavity pH adjustment tank; 09: simulating pancreatic juice tank; 10: bile salt solution tank; 11: waste liquid tank; 12: first peristaltic pump; 13: second peristaltic pump; 14: third peristaltic pump; 15: pH sensor a; 16: temperature sensor a; 17: fourth peristaltic pump; 18: pH sensor b; 19: temperature sensor b; 20: fifth peristaltic pump; 21: sixth peristaltic pump; 22: seventh peristaltic pump: 23: eighth peristaltic pump; 24: circuit board; and 25: touch-screen control panel.
[27] FIG. 2 is a schematic diagram of a Caco-2 cell model.
[28] Changes in a structure and sensitization of a food allergen in a digestion process are studied using a simulation device as shown in FIG. 1. The simulation device includes a simulating gastric digestion system, a simulating duodenal digestion system, an automatic pH adjustment system, a constant-temperature heating plate 05 and a magnetic stirrer 07, wherein secretion of digestive juice and bile salt solution, emptying of the gastrointestinal contents, and pH adjustment in the simulating gastric digestion system and the simulating duodenal digestion system are all integrally controlled using stepper motorized peristaltic pumps, all to-be-implemented operations and reaction conditions are capable of being preset on a touch-screen control panel before digestion 3 begins, reaction conditions are changed without stopping a digestion process halfway, LUS01261 automatic digestion is achieved in the whole process, and after quick enzyme deactivation, micro-samples are capable of being used for further analyses of digestibility, allergy, etc.
[29] The simulating gastric digestion system includes a simulating gastric juice tank 02, a sample storage tank 03 and a simulating gastric cavity 04, wherein the simulating gastric juice tank 02 and the sample storage tank 03 are respectively connected and communicated with the simulating gastric cavity 04 via a second peristaltic pump 13 and a third peristaltic pump 14, and the simulating gastric cavity 04 is internally provided with a pH sensor al5 and a temperature sensor al6; the simulating duodenal digestion system includes a simulating enteric cavity 06, a simulating pancreatic juice tank 09, a bile salt solution 10 and a waste liquid tank 11, wherein the simulating pancreatic juice tank 09, the bile salt solution 10 and the waste liquid tank 11 are respectively connected and communicated with the simulating enteric cavity 06 via a sixth peristaltic pump 21, a seventh peristaltic pump 22 and an eight peristaltic pump 23, and the simulating enteric cavity 06 is internally provided with a pH sensor b18 and a temperature sensor b10; the automatic pH adjustment device includes a gastric cavity pH adjustment tank 01 and an enteric cavity pH adjustment tank 08, wherein the gastric cavity pH adjustment tank O1 and the enteric cavity pH adjustment tank 08 are respectively connected and communicated with the simulating gastric cavity 04 and the simulating enteric cavity 06 via a first peristaltic pump 12 and a fifth peristaltic pump 20; a constant-temperature heating plate 05 for preheating and heat insulating is arranged at a bottom portion of each of the simulating gastric cavity 04 and the simulating enteric cavity 06, while a magnetic stirrer 07 is arranged at an inner bottom portion of each of the simulating gastric cavity 04 and the simulating enteric cavity 06; all peristaltic pumps, pH sensors, temperature sensors, constant-temperature heating plates and magnetic stirrers are respectively electrically connected with a circuit board 24 and a touch-screen control panel 25.
[30] Embodiment 1: Experiment on in-vitro simulation of dynamic allergen digestion
[31] (1) Filling of liquid materials: a digestive juice, acid-alkali adjustment reagents (HCL, NaHCO3) and a bile salt solution required in a gastro-intestinal digestion are prepared; and the digestive juice is stored in a 4°C refrigerator before use to maintain enzyme activity.
[32] (2) Startup and preheating of software: an instrument is powered on and then starts initiating; after it is confirmed that the stepper motorized pumps, pH sensors and constant-temperature heating plates (electromagnetic heating plates) are in good connection, a “Set” key on a homepage is tapped; a “temperature control” mode is selected; a temperature for the heating plate is set, and to-be-digested samples, a simulating gastric cavity and a simulating enteric cavity are placed at the constant-temperature heating plate and then preheated for 10 min.
[33] (3) pH calibration: the “Set” key on the homepage is tapped, then “pH calibration “is selected; next, pH sensors 1 and 2 are respectively calibrated at pH values of 6.86, 4.01 and 9.18 in sequence, and “Record” and “Save” keys are tapped in time.
4
[34] (4) Full filling of pipelines: all liquid material tanks and reacting cavities are LU501261 placed at corresponding positions; a “Manual control” key on the homepage is tapped, then, a “Fully fill the pipelines” mode is selected on the homepage; and a “Stop” key is tapped after the pipelines are full. It is predicted that this process lasts 60 seconds, and consumes 1.0 ml liquid material per pump.
[35] (5) Edition of digestion procedures: an “Edit process” on the homepage is tapped, then, starting and ending time, a flow rate and target pH value of each of pumps in the simulating gastric and duodenal digestion systems are respectively set according to experimental conditions to simulate the continuous secretion of the gastro-intestinal digestive juice and emptying of the gastrointestinal contents, to make sure that pH changes in the simulating gastric cavity conform to pH change laws during digestion in the empty stomach of a person: pH=1.68+3.82 (-t/42), and to maintain the pH of the simulating enteric cavity at about 6.5.
[36] (6) Digestion simulation: an “Operating process” key is tapped; preset conditions are selected and checked; a preset operating process is switched on; a “Stir” key and a “Run” key are tapped in turn; then, digestion simulation begins. During digestion, the pH value and temperature of a reacting system and running conditions of all pumps can be observed in real time, and a target temperature can be controlled using “+” and “-” keys according to the real-time temperature. Trace samples can be taken at any time during sample digestion. After digestion is terminated, relative analyses such as determination of absorption transmittance and sensitization evaluation can be carried out.
[37] (7) Pipeline cleaning and maintenance: After digestion is completed, “X” is tapped to exit, a “Manual control” key on the homepage is tapped; the liquid in the pipeline is forced to flow back reversely; the pipeline is cleaned and emptied with hyperpure water and 75% ethanol in turn; and after all the previous operations are completed, a “Stop” key is tapped.
[38] Embodiment 2: SDS-polyacrylamide gel experiment
[39] Simulated gastric digestion products at different time nodes (0, 1, 2, 5, 10, 20, 30, 60 and 90) and simulated duodenal digestion products at different time modes (0, 1, 5, 10, 15, 30, 60, 90 and 120) are collected; and structural changes and digestion stability of an allergen sample in an in-vitro dynamic digestion process are determined through an SDS-polyacrylamide gel experiment.
[40] Embodiment 3: In-vitro evaluation on the IgG/IgE binding ability of the allergen digestion product.
[41] Changes of an IgG/IgE binding ability of the allergen digestion product in the dynamic digestion process is analyzed through Westen-blot.
[42] Embodiment 4: Construction of a Caco-2 intestinal epithelial cell model
[43] Embodiment 5: Evaluation on the cytotoxicity of the allergen digestion product
[44] Cytotoxicity activity (8) = re x 100%
[45] Notes: A (positive): absorbency of pores with cells, a cck-8 solution and a protein solution:
[46] A (blank): absorbency of pores with culture medium and the cck-8 solution and LUS01261 without cells;
[47] A (negative): absorbency of pores with cells and the cck-8 Solution and without the protein solution.
[48] Embodiment 6: Experiment of simulating digestion, transport and absorption of the allergen digestion product on the intestine
[49] FIG. 2 is a schematic diagram of a Caco-2 cell model. According to experiment results obtained in Embodiment 5, the allergen digestion product is diluted until reaching a proper concentration by using a Hank’s balanced salt solution which does not contain calcium ions and magnesium ions; the model successfully established in Embodiment 4 is washed using a D-Hank’s balanced salt solution; then, 0.5 mL of sterilized D-Hank’s balanced salt solution which contains the allergen product is added to an AP side, while 1.5 ml of D-Hank’s balanced salt solution without drugs is added to a BL side (substrate side); a culture plate is placed in a 37°C water bath oscillator and is slowly oscillated; 2 hours later, the sample solution on the BL side is taken out and transferred in an EP tube; the sample solution is filtered using a microporous filter membrane; and the allergen content is determined through enzyme linked immunosorbent assay; then the absorption transparency of the allergen digestion product is evaluated, and the allergen digestion product is transferred into an in-vitro mast cell model in Embodiment 8 to evaluate the sensitization thereof.
[50] Embodiment 7: Serums of at least 10 patents with an allergic reaction are correspondingly selected from a serum bank; the serums are blended by an equal volume to prepare a serum pool, and a serum information form of each of the patents is established. [S1] Embodiment 8: Establishment of a method for evaluating the sensitization of in-vitro cells
[52] KU812 cells in good conditions are centrifuged (1000 rpm, 5 min) at room temperature; supernant is removed; Human MyelomalgE (human myeloma IgE) is dissolved in a 1640 complete medium such that the final concentration is 0.5 pg/mL; and, the KU812 cells are incubated for one week in a 37°C incubator to promote expression of an Fc ¢ RI receptor on surfaces of the KU812 cells. The cells are washed with the Hank’s balanced salt solution, then added into the perfect medium which contains the serum pool of patents with an allergic reaction, and next incubated for 12 h (serum: culture medium is 1: 30). On the next day, the cells are added into a 48-pore cell culture plate, 1 mL in each pore; a to-be-tested sample with a proper concentration is added into each of the pores to motivate the KU812 cells; 4 hours later, the content of the mast cell active mediums (B-hexosaminidase, tryptases, histamine, etc.) 1s detected; and then, the sensitization of purified allergen extract which contains food allergens or complex food matrix 1s evaluated with reference to experiment results of embodiments 2, 3 and 6.
6
Claims (3)
1. A method for evaluating a dynamic food allergen digestion model and in-vitro simulation, characterized by comprising: (1) establishing a dynamic allergen digestion model; (2) establishing an experimental method for in-vitro simulation of dynamic allergen digestion, and analyzing structural changes and digestion stability of a food allergen; (3) establishing an intestinal epithelial cell model using Caco-2 cells to simulate digestion, transport and absorption processes of digestion products, and evaluating cytotoxicity and absorption transmittance of an allergen digestion product; and (4) establishing a KU812 cell model, and evaluating a mast cell degranulation inducing ability of digestion and transport products of the food allergen.
2. The method for evaluating a dynamic food allergen digestion model and in-vitro simulation according to claim 1, characterized in that changes to a structure and sensitization of the food allergen in a digestion process are studied using a simulation device.
3. The method for evaluating a dynamic food allergen digestion model and in-vitro simulation according to claim 2, characterized in that the simulation device comprises a simulating gastric digestion system, a simulating duodenal digestion system, an automatic pH adjustment system, a constant-temperature heating plate and a magnetic stirrer, wherein secretion of digestive juice and bile, emptying of gastrointestinal contents, and pH adjustment in the simulating gastric digestion system and the simulating duodenal digestion simulation system are all integrally controlled using stepper motorized peristaltic pumps, all operations are capable of being preset on a touch-screen control panel before digestion begins, reaction conditions are changed without stopping a digestion process halfway, and after being subjected to quick enzyme deactivation treatment, micro-samples obtained are capable of being used for further analyses of digestibility, allergy, etc.
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