A method for evaluating the lipid-lowering effect of diglycerides and a biomimetic digestive machine device

Abstract

This invention relates to a method for evaluating the lipid-lowering effect of diglycerides and a biomimetic digestive apparatus, belonging to the field of in vitro biomimetic technology. By constructing an integrated, interconnected device including a circulating air chamber, a three-stage biomimetic digestive module, and a Caco-2 cell culture unit, triglycerides or 1,3-diglycerides are hydrolyzed by sn-1,3-specific pancreatic lipase to generate digestion products with different 1-MG / 2-MG ratios under conditions of 37℃, pH 7.0-7.4, and a peristaltic frequency of 80-120 times / min. The pathways for triglyceride resynthesis and chylomicron formation are then reconstructed in a Transwell system. By detecting the levels of intracellular triglycerides, chylomicron triglycerides, and apolipoprotein B48, and comparing with a positive control group, the lipid-lowering effect of 1,3-diglycerides is verified. The mechanism by which the resynthesis efficiency decreases due to the difficulty of 1-MG being recognized by MAGAT is elucidated, providing a highly realistic and reproducible in vitro evaluation scheme for the development of functional lipids. The lipid-lowering effect of 1,3-diglyceride-type diglycerides was verified at three levels: mechanism, metabolic intermediates, and end effects.

Description

Technical Field

[0001] This invention relates to a method for evaluating the lipid-lowering effect of diglycerides and a biomimetic digestive machine device, belonging to the field of biomimetic biotechnology. Background Technology

[0002] After the body ingests triacylglycerol (TAG) or diacylglycerol (DAG), both can be hydrolyzed by lipases to produce monoacylglycerol (MG) and free fatty acids. The fatty acids in different glycerols are positioned differently on the glycerol backbone, resulting in different monoacylglycerol isomers formed during hydrolysis, thus leading to fundamental differences in subsequent metabolic pathways and efficiency.

[0003] 1,3-DAG (1,3-diglyceride) is hydrolyzed by intestinal lipases primarily to form 1-monoglyceride (1-MG) or 3-monoglyceride (3-MG), while 1,2-DAG and conventional TAGs are hydrolyzed primarily to form 2-monoglyceride (2-MG). The TAG resynthesis pathway in the human small intestine includes the phosphatidic acid bypass and the 2-MG bypass, with approximately 80% of TAGs synthesized via the 2-MG bypass. This pathway relies on the sequential catalysis of MAGAT and DAGAT: 2-MG is converted to DAG by MAGAT, and DAG is converted to TAG by DAGAT. Subsequently, TAGs are assembled with phospholipids, cholesterol, and apolipoprotein B48 to form chylomicrons (CM), which then enter the bloodstream via the lymphatic system.

[0004] Existing research indicates that 2-MG is a high-affinity substrate for MAGAT / DAGAT, while 1-MG (or 3-MG) has a significantly lower affinity for MAGAT and is less likely to rapidly enter the "2-MG bypass." Therefore, after digestion, lipids with 1,3-diglycerides as their main component show a reduced rate of TAG resynthesis, decreased chylomicron production, and decreased postprandial and fasting serum TAG levels. This is a key mechanism by which 1,3-diglycerides lower blood lipids and reduce body fat accumulation.

[0005] Currently, the validation of the lipid-lowering effect of diglycerides mainly relies on in vivo and in vitro validation techniques. In vivo validation techniques include animal experiments and human clinical trials. Animal experiments typically last 4–8 weeks, are costly, and exhibit species-specific differences in intestinal lipase activity and MAGAT / DAGAT expression levels between animals and humans, leading to uncertainties in extrapolation. Human clinical trials require ethical review, last 2–3 months or more, are costly, and are significantly affected by subjects' diets, exercise levels, and individual metabolic differences, resulting in poor reproducibility. Furthermore, in vivo studies primarily rely on end-point indicators such as serum TAG, making it difficult to directly observe the intermediate metabolic processes of "1-MG / 2-MG formation → MAGAT / DAGAT catalytic efficiency → TAG resynthesis → chylomicron generation," thus hindering the mechanistic quantitative evaluation of the lipid-lowering effect of 1,3-diglyceride-type diglycerides. Existing in vitro validation techniques often use static digestive systems or only simple cellular absorption modules, without incorporating MAGAT / DAGAT enzyme systems to simulate 2-MG bypass TAG resynthesis. Typical models, such as the INFOOGSEST static digestion system, primarily simulate lipolysis through single-batch enzyme addition reactions with fixed reaction conditions (pH, stirring, enzyme concentration). They lack the ability to simulate continuous intestinal peristalsis and the dynamic generation and transport of digestive products, and also lack the ability to reconstruct the MAGAT / DAGAT-mediated TAG resynthesis process. They cannot dynamically simulate the human digestive process, making it difficult to quantitatively evaluate the lipid-lowering mechanism of 1,3-diglyceride-type diglycerides along the continuous chain of "1-MG / 2-MG formation—MAGAT / DAGAT recognition—TAG resynthesis—chylomicron generation."

[0006] Therefore, there is an urgent need for a method and device based on in vitro dynamic digestion biomimetic equipment that can replicate the 2-MG bypass TAG resynthesis process in vitro and quantitatively evaluate the lipid-lowering mechanism and effect of 1,3-diglyceride type diglycerides. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for evaluating the lipid-lowering effect of diglycerides and a biomimetic digestive apparatus. This overcomes the shortcomings of existing in vitro and in vivo verification techniques, which fail to construct a MAGAT / DAGAT enzyme system, thus failing to directly simulate the 2-MG bypass TAG resynthesis; the digestion process is mostly static and cannot reflect intestinal peristalsis and dynamic pH regulation; and the inability to systematically correlate 1-MG / 2-MG generation, MAGAT / DAGAT catalytic efficiency, and chylomicron generation.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for evaluating the lipid-lowering effect of diglycerides, using an evaluation device comprising, in series with the following components: a biomimetic digestion module, a selective permeation membrane, an absorption chamber, a diversion unit, a constant flow micropump, a sterilization filter, a cell culture unit, and an online detection interface, and controlled by a controller; the evaluation method includes the following steps: S1. Sample pretreatment and grouping: Set up a positive control group, an experimental group, and a negative control group, and ensure that the final substrate concentrations of the three groups are the same; prepare solutions of specific concentrations for each of the three groups of samples for later use; adjust the evaluation device through the controller, stabilize the temperature of the biomimetic digestion module and the cell culture unit within the range of 37℃±0.5℃, and preset the split ratio; and seed detection cells in the cell culture unit. S2. Dynamic biomimetic digestion: Three substrate solutions were added to the digestion chambers of the biomimetic digestion module, and sodium taurocholate was added to each chamber. Active sn-1,3-specific pancreatic lipase was added to the positive control group and the experimental group to keep the enzyme activity of the system stable at 10 U / mL. Inactivated pancreatic lipase was added to the negative control group. The biomimetic digestion module was started by the controller to obtain three digestion products containing 1-monoglyceride and 2-monoglyceride. S3, Dual Intestinal Absorption Simulation: The digestive products of the biomimetic digestion module are passed into the selective permeable membrane and the absorption chamber respectively. After separation by the selective permeable membrane, small molecules enter the absorption chamber to complete the initial absorption and separation. S4. Digestion fluid diversion and aseptic delivery: The digestion fluid after selective membrane separation is diverted according to a preset ratio, and the constant flow micropump is started to deliver the digestion fluid to be tested to the sterilization filter at a stable rate for filtration, and then delivered to the cell culture unit. S5. Evaluation of secretion linkage: Enzyme preparations were added to the three systems of the cell culture unit respectively: MAGAT and DAGAT enzyme preparations were added to the positive control group and the experimental group, and the same amount of inactivated MAGAT and DAGAT solution was added to the negative control group; the cell culture unit was incubated for a certain period of time to allow MAGAT / DAGAT to catalyze the resynthesis of monoglycerides into TAG and induce the secretion of chylomicrons, thereby simulating the lipid metabolism process of human intestinal epithelial cells; S6. Detection of lipid-lowering indicators: Offline sampling is performed through the online detection interface of the cell culture unit to sequentially complete chylomicron capture, intracellular TAG content detection, and chylomicron-related indicator detection, while mechanistic indicator analysis of digestion products is performed. S7. Result Judgment: Taking the corresponding indicators of the positive control group as 100%, if all of the following conditions are met, then 1,3-diglyceride type diglycerides are judged to have a lipid-lowering effect: the intracellular TAG content in the experimental group is 30% to 50% of that in the positive control group; the TAG content in chylomicrons in the experimental group is 30% to 50% of that in the positive control group; the apolipoprotein B48 content in chylomicrons in the experimental group is 40% to 60% of that in the positive control group; no TAG or chylomicrons are generated in the negative control group; and the above indicators of the experimental group and the positive control group are compared by t test with P < 0.05.

[0009] In one embodiment of the present invention, the pH value of the biomimetic digestion module is stabilized between 7.0 and 7.4, and the peristalsis frequency is 80 to 120 times / min.

[0010] In one embodiment of the present invention, the shunting ratio of the shunting unit is determined according to the calculation method of: concentration × shunting ratio = constant load, wherein the concentration is the digestive fluid concentration and the constant load is the cell metabolic load.

[0011] In one embodiment of the present invention, the method for capturing chylomicrons and detecting related indicators is as follows: Culture medium from each group of cell culture units is collected, 2 mL from each group is taken, and the density is adjusted to 1.006 g / mL with 0.9% NaCl solution filtered through a 0.22 μm membrane. This solution is then added to an ultracentrifuge tube, and a sucrose solution with a density of 1.063 g / mL is slowly added. The tube is centrifuged at 4°C and 40,000 rpm for 18 hours. The top 0.2 mL of liquid from the centrifuge tube is then collected as the chylomicron content. Chylomicron enrichment solution; 20 μL of chylomicron enrichment solution was taken, and the content of apolipoprotein B48 in the chylomicron enrichment solution was determined using a BCA quantitative kit with specific anti-human apolipoprotein B48 antibody. The content of TAG in the chylomicron enrichment solution was determined using high performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: C18 column, mobile phase acetonitrile:isopropanol = 65:35, flow rate 1.0 mL / min, column temperature 30℃, detection wavelength 210 nm, and injection volume 10 μL. The method for detecting intracellular TAG content is as follows: 1 mL of RIPA lysis buffer containing protease inhibitor is added to the cell culture unit, and the cells are lysed on ice for 30 min. After centrifugation at 12000 rpm and 4℃ for 15 min, 50 μL of the supernatant is taken as the cell lysis buffer. The TAG content in the cell lysis buffer is determined using an enzymatic triglyceride detection kit.

[0012] In one embodiment of the present invention, the enzyme activity of MAGAT is 10-30 U / mL and the enzyme activity of DAGAT is 40-60 U / mL.

[0013] Secondly, the present invention also provides a biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides, which is applied to the aforementioned method for evaluating the lipid-lowering effect of diglycerides. The evaluation device includes a circulating air box and a biomimetic digestive module, and the biomimetic digestive module is connected to the circulating air box. A selectively permeable membrane is connected to the outlet of the biomimetic digestion module; An absorption chamber is located on one side of the selectively permeable membrane and is connected to the outlet of the biomimetic digestion module; The diversion unit is connected to the absorption chamber and diverts the substances in the absorption chamber. A constant flow micropump and a sterilizing filter are provided. The constant flow micropump is connected to the flow splitting unit and the constant flow micropump is connected to the cell culture unit through the sterilizing filter. The cell culture unit is equipped with an online detection interface. A waste liquid collection unit, which is connected to the diversion unit and the cell culture unit; The controller is electrically connected to each unit and adjusts the operating parameters of each unit.

[0014] In one embodiment of the present invention, the pore size of the selective permeation membrane is in the range of 0.1~10kDa.

[0015] In one embodiment of the present invention, the delivery rate of the constant flow micropump is 0.5~2.0 mL / h.

[0016] In one embodiment of the present invention, the cell culture unit is a closed culture chamber, inoculated with Caco-2 cells at a density of 1.8~2.2×10⁻⁶. 5 pcs / cm 2 .

[0017] In one embodiment of the present invention, the pore size of the sterilization filter is 0.22 μm.

[0018] The beneficial effects of this invention are: (1) The present invention introduces the MAGAT / DAGAT enzyme system in vitro to simulate the main pathway of 2-MG bypass TAG resynthesis: by adding MAGAT and DAGAT to the cell culture unit, the 1-MG / 2-MG is directly linked to the TAG resynthesis process, realizing the full simulation of "monoglyceride formation → MAGAT / DAGAT recognition → TAG resynthesis → chylomicron formation". Compared with the existing technology that only detects terminal TAG indicators, the present invention can better reflect the mechanism characteristics of 1,3-diglyceride type diglycerides in lowering blood lipids by detecting intracellular TAG content, TAG content in chylomicrons, and apolipoprotein B48 content in chylomicrons.

[0019] (2) This invention achieves continuous verification from the mechanism layer to the effect layer: in the same experimental system, the following can be obtained simultaneously: the composition of monoglycerides (1-MG / 2-MG ratio); the effect of the presence or absence of MAGAT / DAGAT on the TAG resynthesis efficiency; the changes in the content of chylomicrons TAG and ApoB48; thereby verifying the lipid-lowering effect of 1,3-diglyceride type diglycerides at the three levels of mechanism, metabolic intermediate and final effect.

[0020] (3) This invention has a short experimental cycle, low cost, and good reproducibility: the single experimental cycle is about 8 hours, which is much shorter than animal experiments and clinical trials; no animal modeling or subject recruitment is required, the requirements for experimental sites and ethics are low, and it is easy to conduct batch parallel testing; key parameters in the system (peristalsis frequency, pH, enzyme activity, etc.) can be precisely controlled, so that the intra-batch coefficient of variation is ≤5%, and the reproducibility is better than that of in vivo experiments that are significantly affected by individual differences. In addition to 1,3-diglycerides, this method can be extended to the verification of various lipid components with the potential to lower blood lipids by adjusting the substrate, and is suitable for the needs of functional oil research and development, quality control, efficacy declaration and other scenarios in the field of food science. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the structure of the biomimetic digestive machine device provided by the present invention.

[0023] In the diagram: 1. Circulating air box; 2. Bionic digestion module; 3. Selective permeable membrane; 4. Absorption chamber; 5. Diversion unit; 6. Constant flow micropump; 7. Waste liquid collection unit; 8. Sterilization filter; 9. Cell culture unit; 10. Online detection interface; 11. Controller. Detailed Implementation

[0024] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. The terms "first position" and "second position" refer to two different positions.

[0026] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to fixed connections or detachable connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal connections between two components. Those skilled in the art can understand the specific meaning of these terms in this invention based on the specific circumstances.

[0027] like Figure 1 As shown, this invention provides a biomimetic digestive machine for evaluating the lipid-lowering effect of diglycerides. The device includes a circulating air chamber 1, a biomimetic digestion module 2, a selective permeable membrane 3, an absorption chamber 4, a diversion unit 5, a constant flow micropump 6, a sterilization filter 8, and a cell culture unit 9. These units are connected in series. A waste liquid collection unit 7 is also provided on one side of the diversion unit 5, and an online detection interface 10 is installed on the cell culture unit 9. The device also includes a controller 11 and a detection unit. The controller 11 is electrically connected to each unit, and it coordinates the operation of each unit. The operating parameters of each unit are adjusted based on the detection data fed back from the detection unit. This allows for programmed and precise control of digestion time, pH value, peristalsis frequency, diversion ratio, delivery rate, and cell culture conditions. Simultaneously, it automatically collects, stores, and analyzes the operating data and detection indicators of each unit, achieving automated and standardized operation of the device. The detection unit includes temperature sensors, pH sensors, and other detection devices.

[0028] In some embodiments, the circulating gas chamber 1 is connected to the flow path of the entire device, providing a constant temperature and stable gas environment for the entire device, ensuring the digestive enzyme activity of the biomimetic digestion module 2 and the cell survival and metabolic needs of the cell culture unit, and maintaining the balance of gas components within the device. One side of the biomimetic digestion module 2 is connected to the circulating gas chamber 1, and a selective permeation membrane 3 is installed at its internal outlet, through which the substance to be detected enters the absorption chamber 4. The biomimetic digestion module 2 includes three independent digestive chambers—duodenum, jejunum, and ileum—connected in series. Each chamber is independently equipped with a temperature control unit, a pH adjustment unit, and a peristalsis simulation unit. The temperature control unit stabilizes the chamber temperature at 37℃±0.5℃ through water temperature regulation. The pH control unit connects to 0.1mol / L hydrochloric acid solution and 0.1mol / L sodium hydroxide solution through two peristaltic pumps, respectively, to monitor the pH value in the biomimetic digestion module 2 in real time and adjust it accordingly, ensuring that the pH value fluctuation does not exceed ±0.1. The peristalsis simulation unit has an adjustable peristalsis frequency of 80 to 120 times / min, which simulates the dynamic digestive environment of different segments of the human gastrointestinal tract to achieve in vitro biomimetic digestion of diglycerides.

[0029] In some embodiments, the selective permeable membrane 3 is connected to the outlet of the biomimetic digestion module 2. The membrane pore size ranges from 0.1 to 10 kDa, selectively retaining large molecules in the digestive products while allowing only small-molecule monoglycerides, fatty acids, and other absorbable active ingredients to pass through, thus achieving precise separation of digestive products. The absorption chamber 4 is located on one side of the selective permeable membrane 3 and is connected to the outlet of the biomimetic digestion module 2. Working in conjunction with the selective permeable membrane 3, it performs preliminary separation and absorption simulation of the digestive products from the biomimetic digestion module 2, comprehensively simulating the physiological processes of different absorption stages in the human intestine, and enhancing the realism of digestion and absorption simulation.

[0030] In some embodiments, the diversion unit 5 is connected to the absorption chamber 4 to divert the digestion solution in the absorption chamber 4 according to a preset ratio. The diversion ratio is determined based on the cell metabolic load, combined with cell density, digestion solution concentration, and detection sensitivity, and is positively correlated with cell state and negatively correlated with digestion solution concentration. When changing cells or substrates, the ratio is adjusted according to the principle of "concentration × diversion ratio = constant load," and repeatability is verified through preliminary experiments. The side of the diversion unit 5 facing away from the absorption chamber 4 is connected to the waste collection unit 7 and the constant flow micropump 6, respectively. A portion of the diverted digestion solution is delivered to the cell culture unit 9, and excess digestion solution is discharged to the waste collection unit 7, achieving controllable distribution of the digestion solution and avoiding excessive inflow of digestion solution that could cause a large metabolic load on the cells. The constant flow micropump 6 is a power delivery unit, and its side facing away from the diversion unit 5 is connected to the sterilization filter 8. The constant flow micropump 6 can precisely control the delivery rate of the digestion solution, ensuring a stable dose and uniform flow rate of the digestion solution delivered to the cell culture unit, thereby improving experimental repeatability. The sterilization filter 8 is connected to the cell culture unit 9 on the side opposite to the constant flow micropump 6. The sterilization filter 8 is used to sterile filter the digestion solution delivered by the constant flow micropump 6, thoroughly remove microbial impurities in the digestion solution, avoid contaminating the cell culture system, and ensure the effectiveness of cell experiments.

[0031] In some embodiments, the cell culture unit 9 is a closed culture chamber that can stably maintain a cell culture environment of 37°C and 5% CO2; Caco-2 cells are seeded inside the chamber at a seeding density of 1.8~2.2×10⁻⁶ cells / cm². 5 pcs / cm 2 The cells are cultured for at least 14 days, with the culture medium changed every 24 hours during the process. Trypan blue staining is used to determine cell viability, which is ≥90%, thus constructing a mature intestinal epithelial metabolic secretion model. An online detection interface 10 is installed within the chamber, enabling real-time monitoring and offline sampling of intracellular indicators and secreted products. The waste collection unit 7 is also connected to the cell culture unit 9, collecting excess digestive fluids and cell culture waste during the experiment to maintain the overall cleanliness and orderly operation of the device.

[0032] Furthermore, this invention also provides a method for evaluating the lipid-lowering effect of diglycerides. This method uses the aforementioned biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides, and the specific steps are as follows: Preparation of experimental materials Substrates: Triglycerides (TAG) with a purity ≥98% and 1,3-DAG (1,3-DAG) with a purity ≥95%. Enzymes and reagents: sn-1,3 specific pancreatic lipase (activity 1000 U / mg), MAGAT enzyme preparation, DAGAT enzyme preparation, sodium taurocholate (purity 98%), RIPA lysis buffer (containing protease inhibitor), triglyceride assay kit (enzymatic method, detection range 0.1-10 mmol / L), anti-human apolipoprotein B48 antibody BCA quantitative kit (detection wavelength 562 nm, linear range 0.1-10 μg / mL). Cells and equipment: Caco-2 cells, horizontal rotor ultracentrifuge, high performance liquid chromatography, cell culture chamber.

[0033] S1. Sample Pretreatment and Grouping A positive control group, an experimental group, and a negative control group were set up, with a final substrate concentration of 5 mmol / L for all three groups. The substrate for the positive control group was triglycerides (TAG) with a purity ≥98%; the substrate for the experimental group was 1,3-diglyceride-type diglycerides with a purity ≥95%; and the substrate for the negative control group was 99% pure TAG dissolved and diluted to 5 mmol / L in serum-free DMEM medium. Simultaneously, the sn-1,3-specific pancreatic lipase from the precise enzyme donation unit was boiled in a 100℃ water bath for 15 min, cooled, and diluted to 10 U / mL as the enzyme donation for that group. Solutions of appropriate concentrations were prepared for each of the three groups of samples for later use.

[0034] Device parameter debugging: The controller 11 is used to programmatically debug each unit of the device: the temperature of each chamber of the biomimetic digestion module 2 and the cell culture unit 9 is stabilized at 37℃±0.5℃, with fluctuations not exceeding ±0.1℃; the pH electrode is calibrated by connecting 0.1mol / L hydrochloric acid solution and 0.1mol / L sodium hydroxide solution through two peristaltic pumps to stabilize the pH value of the biomimetic digestion module at 7.0~7.4, with fluctuations not exceeding ±0.1; the peristaltic frequency of the biomimetic digestion module is adjusted to 80~120 times / min (100 times / min is preferred under standard conditions); the diversion ratio is set to ensure that the digestive solution is distributed to the cell culture unit 9 and the waste liquid collection unit 7 as needed; the digestive solution delivery rate is adjusted by adjusting the constant flow micropump 6 to ensure stable and uniform delivery of the digestive solution.

[0035] Constructing a secretion model: Caco-2 cells were seeded onto the Transwell chamber membrane within cell culture unit 9 at a seeding density of 2 × 10⁶ cells / year. 5 pcs / cm 2The environmental conditions for cell culture unit 9 were set at 37℃ and 5% CO2. Serum-free DMEM culture medium containing 5 mmol / L glucose + 1% penicillin-streptomycin was added and replaced every 24 hours. After 14 days of culture, cell viability was detected by trypan blue staining and found to be ≥90%. Western blotting was used to verify the expression of MGAT1, MGAT2, DGAT1, DGAT2 and apolipoprotein B48, confirming cell differentiation and maturation.

[0036] S2, Dynamic Bionic Digestion The three substrate solutions were added to the digestive chambers of the biomimetic digestion module 2, respectively. Sodium taurocholate was added to each chamber to bring the final concentration to 10 mmol / L. Active sn-1,3-specific pancreatic lipase was added to the positive control group and the experimental group to keep the enzyme activity of the system stable at 10 U / mL. Inactivated pancreatic lipase was added to the negative control group. The biomimetic digestion module was started by the controller 11, and the peristalsis frequency was set to 80-120 times / min, pH 7.0-7.4, and temperature 37℃±0.5℃. The reaction was carried out continuously for 2 hours to obtain three digestion products containing 1-monoglyceride and 2-monoglyceride.

[0037] S3, Intestinal Dual Absorption Simulation The digestion products of the biomimetic digestion module 2 are passed into the selective permeation membrane 3 and the absorption chamber 4, respectively. The absorption chamber 4 completes the initial absorption and separation, while the selective permeation membrane 3 achieves the precise separation of small molecule absorbable active ingredients through a 0.1~10kDa selective permeation membrane, allowing small molecule monoglycerides and fatty acids to pass through, resulting in a digestive solution that can be used for subsequent cell metabolism evaluation.

[0038] S4. Digestive fluid diversion and aseptic delivery The controller 11 regulates the diversion unit 5 to divert the digested liquid separated by the selective permeation membrane 3 according to a preset ratio. The constant flow micropump 6 is started to deliver the digested liquid to be tested to the sterilization filter 8 at a stable rate of 0.5~2.0 mL / h. The sterilization filter 8 uses a microporous filter membrane with a pore size of 0.22μm to remove bacteria and particles in the digested liquid through physical interception and deliver it to the cell culture unit 9.

[0039] S5. Evaluation of the metabolic-secretion linkage of intestinal epithelial cells Enzyme preparations were added to the three systems in cell culture unit 9: MAGAT and DAGAT enzyme preparations were added to the positive control group and the experimental group, so that the enzyme activities in the system were 10-30 U / mL for MAGAT and 40-60 U / mL for DAGAT, respectively. The negative control group was added with the same amount of inactivated MAGAT and DAGAT solution. The cell culture unit was incubated at 37°C and 5% CO2 for 6 h to allow MAGAT / DAGAT to catalyze the resynthesis of monoglycerides into TAG and induce the secretion of chylomicrons, simulating the lipid metabolism process of human intestinal epithelial cells.

[0040] S6, Lipid-lowering related indicator testing Offline sampling was performed using the online detection interface 10 of the cell culture unit to sequentially complete chylomicron capture, intracellular TAG content detection, and chylomicron-related index detection. Simultaneously, mechanistic index analysis of the digestion products was conducted. The specific operations are as follows: Chylomicron capture: 2 mL of culture medium from each cell culture unit 9 was collected and the density was adjusted to 1.006 g / mL with 0.9% NaCl solution filtered through a 0.22 μm filter membrane. The medium was then added to an ultracentrifuge tube and slowly covered with sucrose solution with a density of 1.063 g / mL. The tube was centrifuged at 4 °C and 40,000 rpm for 18 h. 0.2 mL of the liquid from the top of the centrifuge tube was taken as the chylomicron enrichment solution. Intracellular TAG content detection: 1 mL of RIPA lysis buffer containing protease inhibitor was added to the cell culture unit. After lysing on ice for 30 min, the cells were centrifuged at 12000 rpm and 4℃ for 15 min. 50 μL of the supernatant was taken as the cell lysis buffer. The TAG content in the cell lysis buffer was determined using an enzymatic triglyceride detection kit. Detection of chylomicron-related indicators: 20 μL of chylomicron enrichment solution was used, and the content of apolipoprotein B48 in the chylomicron enrichment solution was determined using a BCA quantitative kit (detection wavelength 562 nm) with a specific anti-human apolipoprotein B48 antibody. The content of TAG in the chylomicron enrichment solution was determined using high performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: C18 column (250 mm × 4.6 mm, particle size 5 μm), mobile phase acetonitrile:isopropanol = 65:35 (v / v), flow rate 1.0 mL / min, column temperature 30 ℃, detection wavelength 210 nm, injection volume 10 μL. Metabolic mechanism index analysis: The molar ratio of 1-monoglyceride and 2-monoglyceride in the digestion products obtained in step S3 was quantitatively analyzed by high performance liquid chromatography. The composition of monoglycerides was used as an intermediate mechanism index to determine the metabolic differences between 1,3-diglyceride type diglycerides and triglycerides.

[0041] S7. Result Determination Using the positive control group as a baseline of 100%, if all of the following conditions are met, then 1,3-diglyceride type diglycerides are considered to have a lipid-lowering effect: The intracellular TAG content in the experimental group was 30%–50% of that in the positive control group; the TAG content in chylomicrons in the experimental group was 30%–50% of that in the positive control group; the apolipoprotein B48 content in chylomicrons in the experimental group was 40%–60% of that in the positive control group; no TAG or chylomicrons were generated in the negative control group. The results of the above indicators between the experimental group and the positive control group were compared using a t-test, with P < 0.05. Here, t represents the t-test, used to compare the differences in the means of measurement data between the experimental group and the positive control group; P represents the p-value, and P < 0.05 indicates that the differences between the two groups are statistically significant, proving that the lipid-lowering effect of 1,3-DAG is not due to random error and the results are reliable.

[0042] Example 1: Comparison of 1,3-DAG and TAG under standard conditions S1. Experimental Grouping and Substrate Preparation A positive control group, an experimental group, and a negative control group were set up, with a final substrate concentration of 5 mmol / L for all three groups. The substrate for the positive control group was triglyceride (TAG) with a purity ≥98%, the substrate for the experimental group was 1,3-diglyceride type diglyceride with a purity ≥95%, and the substrate for the negative control group was TAG with a purity of 99%, dissolved and diluted to 5 mmol / L in serum-free DMEM culture medium. At the same time, sn-1,3 specific pancreatic lipase from the precise enzyme supply unit was boiled in a 100℃ water bath for 15 min, cooled, and diluted to 10 U / mL as the enzyme supply for this group.

[0043] Device parameter debugging: The controller 11 was used to programmatically debug each unit of the device: the temperature of each chamber of the biomimetic digestion module 2 and the cell culture unit 9 was stabilized at 37℃±0.5℃, with fluctuations not exceeding ±0.1℃; the pH electrode was calibrated by connecting 0.1mol / L hydrochloric acid solution and 0.1mol / L sodium hydroxide solution through two peristaltic pumps, so that the pH value of the biomimetic digestion module was stabilized at 7.2±0.1, with fluctuations not exceeding ±0.1; the peristaltic frequency of the biomimetic digestion module was adjusted to 100 times / min; the shunt ratio was set to 25%; and the digestive fluid delivery rate was adjusted to 0.5 mL / h by adjusting the constant flow micropump 6.

[0044] Constructing a secretion model: Caco-2 cells were seeded onto the Transwell chamber membrane within cell culture unit 9 at a seeding density of 2 × 10⁶ cells / year. 5 pcs / cm 2The environmental conditions for cell culture unit 9 were set at 37℃ and 5% CO2. Serum-free DMEM culture medium containing 5 mmol / L glucose + 1% penicillin-streptomycin was added and replaced every 24 hours. After 14 days of culture, cell viability was detected by trypan blue staining and found to be ≥90%. Western blotting was used to verify the expression of MGAT1, MGAT2, DGAT1, DGAT2 and apolipoprotein B48, confirming cell differentiation and maturation.

[0045] S2, Dynamic Digestion Stage Three groups of 50 mL substrate solutions were added to the biomimetic digestion module 2, along with sodium taurocholate to achieve a final concentration of 10 mmol / L. For the positive control and experimental groups, active sn-1,3-specific pancreatic lipase was added to achieve an enzyme activity of 10 U / mL. For the negative control group, pancreatic lipase solution that had been boiled at 100℃ for 15 min and then cooled was added, resulting in the same nominal enzyme concentration as the other groups but with no actual enzyme activity. The peristalsis frequency was set to 100 times / min, the pH was controlled at 7.2±0.1, and the reaction was carried out continuously at 37℃ for 2 h. After the reaction, samples were taken and the MAG composition was analyzed by HPLC: the positive control group mainly produced 2-MG, accounting for approximately 75% of the total MAG; the experimental group mainly produced 1-MG, accounting for approximately 72% of the total MAG; and no obvious MG peak was detected in the negative control group.

[0046] S3, Intestinal Dual Absorption Simulation The digestion products of the biomimetic digestion module 2 are passed into the selective permeation membrane 3 and the absorption chamber 4, respectively. The absorption chamber 4 completes the initial absorption and separation, and the selective permeation membrane 3 achieves the precise separation of small molecule absorbable active ingredients through a 10kDa selective permeation membrane, allowing small molecule monoglycerides and fatty acids to pass through, resulting in a digestive solution that can be used for subsequent cell metabolism evaluation.

[0047] S4. Digestive fluid diversion and aseptic delivery The controller 11 regulates the diversion unit 5 to divert the digested liquid separated by the selective permeation membrane 3 according to a preset ratio of 25%. The constant flow micropump 6 is started to deliver the digested liquid to be tested to the sterilization filter 8 at a stable rate of 0.5 mL / h. The sterilization filter 8 uses a microporous membrane with a pore size of 0.22 μm to remove bacteria and particles in the digested liquid through physical interception and deliver it to the cell culture unit 9.

[0048] S5, MAGAT / DAGAT catalysis and metabolism-secretion stage The digestion products from the three groups were transferred to cell culture unit 9. MAGAT and DAGAT enzyme preparations were added to the positive control and experimental groups to achieve enzyme activities of 20 U / mL (MAGAT) and 50 U / mL (DAGAT), respectively. An inactivated MAGAT and DAGAT solution, treated at 100℃ for 15 min, was added to the negative control group. The cells were incubated at 37℃ in a 5% CO2 incubator for 6 h.

[0049] S6, Chylomicron Capture Offline sampling was performed via the online detection interface 10 of the cell culture unit to sequentially complete chylomicron capture, intracellular TAG content detection, and chylomicron-related index detection, while mechanistic index analysis of the digestion products was conducted. Two mL of culture medium from each cell culture unit 9 was collected, and the density was adjusted to 1.006 g / mL with 0.9% NaCl solution. This was added to an ultracentrifuge tube, slowly covered with a sucrose solution of 1.063 g / mL, and centrifuged in a horizontal rotor ultracentrifuge at 4℃ and 40,000 rpm for 18 h. After centrifugation, approximately 0.2 mL of the top layer of chylomicron-enriched solution was collected for later use.

[0050] Intracellular TAG content detection: Add 1 mL of RIPA lysis buffer containing protease inhibitor, lyse on ice for 30 min, centrifuge at 12000 rpm and 4℃ for 15 min, take 50 μL of supernatant, and determine the intracellular TAG content according to the instructions of the triglyceride detection kit.

[0051] Detection of ApoB48 chylomicron index: Take 20 μL of chylomicron enrichment solution and use the BCA quantitative kit for anti-human ApoB48 antibody to determine the protein content at a wavelength of 562 nm. Chylomicrons (TAG): Take 50 μL of chylomicron enrichment solution and detect the TAG content by HPLC: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase acetonitrile:isopropanol = 65:35 (volume ratio), flow rate 1.0 mL / min, column temperature 30℃, detection wavelength 210 nm.

[0052] S7. Result Determination Three parallel experiments were conducted. The results showed that (with the positive control group = 100%): the intracellular TAG in the experimental group was approximately 38% of that in the positive control group; the TAG content of chylomicrons in the experimental group was approximately 35% of that in the positive control group; and the ApoB48 content of chylomicrons in the experimental group was approximately 49% of that in the positive control group. In the negative control group, all three indicators were close to 0, and no obvious chylomicron formation was detected. A t-test showed a significant difference between the experimental group and the positive control group (P < 0.01), meeting the criteria of this invention.

[0053] Example 2: Reduced peristalsis frequency (80 times / min) Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the dynamic digestion phase, the peristalsis frequency was set to 80 times / min, with other conditions remaining unchanged. After 2 hours of digestion, HPLC results showed that 2-MG accounted for approximately 70% of the total MAG in the positive control group, while 1-MG accounted for approximately 68% of the total MAG in the experimental group. The MAGAT / DAGAT catalysis and subsequent detection steps were the same as in Example 1.

[0054] The results showed (based on a positive control group of 100%): the intracellular TAG content in the experimental group was approximately 41%; the TAG content in chylomicrons in the experimental group was approximately 40%; the ApoB48 content in the experimental group was approximately 52%; and no TAG or chylomicrons were detected in the negative control group.

[0055] Example 3: Increased peristalsis frequency (120 times / min) Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the dynamic digestion phase, the peristalsis frequency was set to 120 times / min, with other conditions remaining unchanged. After 2 hours of digestion, HPLC results showed that 2-MG accounted for approximately 78% of the total MAG in the positive control group, while 1-MG accounted for approximately 76% of the total MAG in the experimental group. MAGAT / DAGAT catalysis and detection were the same as in Example 1.

[0056] The results showed (based on a positive control group of 100%): the intracellular TAG content in the experimental group was approximately 36%; the TAG content in chylomicrons in the experimental group was approximately 34%; the ApoB48 content in the experimental group was approximately 46%; and no TAG or chylomicrons were detected in the negative control group.

[0057] Example 4: pH slightly decreased (pH 7.0) Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the dynamic digestion phase, the pH range was adjusted to 7.0 ± 0.1, and the peristalsis frequency was 100 times / min. After 2 hours of digestion, 2-MG accounted for approximately 72% of the total MAG in the positive group, and 1-MG accounted for approximately 70% in the experimental group. MAGAT / DAGAT catalysis and detection were the same as in Example 1.

[0058] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 33%; chylomicron TAG in the experimental group was approximately 32%; and ApoB48 in the experimental group was approximately 43%.

[0059] Example 5: Reduced MAGAT dosage Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the MAGAT / DAGAT catalytic phase, the MAGAT enzyme activity was adjusted from 20 U / mL to 15 U / mL, while the DAGAT activity was maintained at 50 U / mL. Other operating procedures were the same as in Example 1.

[0060] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 35%; chylomicron TAG in the experimental group was approximately 34%; and ApoB48 in the experimental group was approximately 48%.

[0061] Example 6: Reduced DAGAT dosage Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: The DAGAT enzyme activity was adjusted from 50 U / mL to 40 U / mL, while the MAGAT activity was maintained at 20 U / mL. Other steps were the same as in Example 1.

[0062] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 37%; chylomicron TAG in the experimental group was approximately 36%; and ApoB48 in the experimental group was approximately 50%.

[0063] Example 7: Simultaneous reduction of MAGAT and DAGAT Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the catalytic phase, the MAGAT enzyme activity was adjusted to 15 U / mL, and the DAGAT enzyme activity was adjusted to 40 U / mL. Other procedures were the same as in Example 1.

[0064] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 39%; chylomicron TAG in the experimental group was approximately 38%; and ApoB48 in the experimental group was approximately 54%.

[0065] Example 8: Increased MAGAT / DAGAT dosage and increased peristalsis frequency Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: The peristalsis frequency during the dynamic digestion phase was set to 120 times / min; during the MAGAT / DAGAT catalytic phase, the MAGAT enzyme activity was adjusted to 25 U / mL and the DAGAT enzyme activity was adjusted to 60 U / mL.

[0066] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 34%; chylomicron TAG in the experimental group was approximately 33%; and ApoB48 in the experimental group was approximately 45%.

[0067] The above eight examples show that under different peristaltic frequencies, pH and enzyme activity conditions, 1,3-DAG significantly reduces TAG resynthesis and chylomicron generation, and all indicators fall within the preset judgment range, indicating that the method has good parameter adaptability and stability.

[0068] Example 9: The diversion ratio is adjusted to 20%. Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the diversion and delivery phase, the diversion unit ratio is set to 20%, and the constant flow micropump rate is adjusted synchronously to ensure a stable dose of digestive fluid delivered to the cell culture unit. Other conditions such as aseptic filtration and cell culture are the same as in Example 1.

[0069] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 31%; chylomicron TAG in the experimental group was approximately 32%; and chylomicron ApoB48 in the experimental group was approximately 41%.

[0070] Example 10: The diversion ratio is adjusted to 30%. Except for the following differences, the remaining steps in this embodiment are the same as in Embodiment 1: During the diversion and delivery phase, the diversion unit ratio is set to 30%, and the constant flow micropump rate is adjusted synchronously to ensure a stable dose of digestive fluid delivered to the cell culture unit. Other conditions such as aseptic filtration and cell culture are the same as in Example 1.

[0071] The results showed (based on a positive control group of 100%): intracellular TAG in the experimental group was approximately 49%; chylomicron TAG in the experimental group was approximately 48%; and chylomicron ApoB48 in the experimental group was approximately 59%.

[0072] The above ten examples show that under different peristaltic frequencies, pH and enzyme activity conditions, 1,3-DAG significantly reduces TAG resynthesis and chylomicron generation, and all indicators fall within the preset judgment range, indicating that the method has good parameter adaptability and stability.

[0073] Comparative Example 1: Pancreatic lipase and MAGAT / DAGAT were both inactivated. Except for the following differences, the steps in this comparative example are the same as in Example 1: Dynamic digestion stage: The substrate was TAG 5 mmol / L; a pancreatic lipase solution boiled at 100°C for 15 min and then cooled was added to achieve the same nominal enzyme concentration as in Example 1, but with actual enzyme activity lost; peristalsis frequency 100 times / min, pH 7.2±0.1, 37°C, reaction time 2 h. MAGAT / DAGAT catalytic stage: MAGAT and DAGAT solutions that had been inactivated in the same manner were added to the system to ensure that the system contained only inactivated enzyme proteins with no substantial catalytic activity. The remaining steps were the same as in Example 1.

[0074] The results showed that no significant MAG generation was detected; intracellular TAG was close to background levels; and neither chylomicron TAG nor ApoB48 was detected.

[0075] This indicates that in the absence of effective enzyme activity, no substantial TAG resynthesis or chylomicron formation occurs, eliminating the risk of false positives caused by non-enzymatic reactions in the system.

[0076] Comparative Example 2: Diversion ratio 50% Except for the following differences, the steps in this comparative example are the same as in Example 1: During the diversion and delivery phase, the diversion unit ratio is set to 50%, and the constant flow micropump rate is adjusted synchronously to ensure a stable dose of digestive fluid delivered to the cell culture unit. Other conditions such as aseptic filtration and cell culture are the same as in Example 1.

[0077] The results showed (based on a positive control group = 100%): intracellular TAG in the experimental group was approximately 51%; chylomicron TAG in the experimental group was approximately 50%; and chylomicron ApoB48 in the experimental group was approximately 61%. Table 1. Summary of key parameters and results for different embodiments and comparative examples (with TAG positive control group = 100%)

[0078] Comparative Example 1: When pancreatic lipase and MAGAT / DAGAT were both inactivated, all indicators were close to 0, excluding interference from non-enzymatic reactions.

[0079] Comparative Example 2: When the shunt ratio is large, the cellular metabolic load increases, and TAG, chylomicrons (TAG), and ApoB48 approach the critical value for judgment, resulting in decreased method stability and cell tolerance.

[0080] The results above show that, under different peristaltic frequencies, pH values, and MAGAT / DAGAT enzyme concentrations, the intracellular TAG and chylomicron TAG levels in the examples were consistently maintained within the range of 30%–50% of the positive control group, while the chylomicron ApoB48 levels were maintained within the range of 40%–60%, meeting the preset judgment conditions of this invention. The comparative examples verified the necessity of the MAGAT / DAGAT enzyme system and pancreatic lipase activity, indicating that this method has good specificity and reliability.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can modify or make equivalent substitutions to the technical features without departing from the spirit and substance of the present invention; all modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for evaluating the lipid-lowering effect of diglycerides, characterized in that, An evaluation device is used, comprising a biomimetic digestion module (2), a selective permeation membrane (3), an absorption chamber (4), a diversion unit (5), a constant flow micropump (6), a sterilization filter (8), a cell culture unit (9), and an online detection interface (10) connected in series, and controlled by a controller (11); the evaluation method includes the following steps: S1. Sample pretreatment and grouping: Set up a positive control group, an experimental group and a negative control group, and make the final concentration of the substrate in the three groups the same; prepare solutions of specific concentrations for the three groups of samples respectively; debug the evaluation device through the controller (11), stabilize the temperature of the biomimetic digestion module (2) and the cell culture unit (9) within the range of 37℃±0.5℃, and preset the split ratio; and seed the detection cells in the cell culture unit (9); S2, Dynamic biomimetic digestion: The three substrate solutions were added to the digestion chambers of the biomimetic digestion module (2), and sodium taurocholate was added to each chamber. Active sn-1,3-specific pancreatic lipase was added to the positive control group and the experimental group to keep the enzyme activity of the system stable at 10 U / mL. Inactivated pancreatic lipase was added to the negative control group. The biomimetic digestion module (2) was started by the controller (11) to obtain three digestion products containing 1-monoglyceride and 2-monoglyceride. S3, Dual Intestinal Absorption Simulation: The digestive products of the biomimetic digestion module (2) are passed into the selective permeable membrane (3) and the absorption chamber (4) respectively. After separation by the selective permeable membrane (3), small molecules enter the absorption chamber (4) to complete the initial absorption and separation. S4. Digestive fluid diversion and aseptic delivery: The digestive fluid separated by the selective permeable membrane (3) is diverted according to a preset ratio. The constant flow micropump (6) is started to deliver the digestive fluid to be tested to the sterile filter (8) at a stable rate for filtration, and then delivered to the cell culture unit (9). S5. Evaluation of secretion linkage: Add enzyme preparations to the three systems of the cell culture unit (9): add MAGAT and DAGAT enzyme preparations to the positive control group and the experimental group, and add the same amount of inactivated MAGAT and DAGAT solution to the negative control group; incubate the cell culture unit (9) for a certain time to allow MAGAT / DAGAT to catalyze the resynthesis of monoglycerides to TAG and induce the secretion of chylomicrons, thereby simulating the lipid metabolism process of human intestinal epithelial cells; S6. Detection of lipid-lowering indicators: Offline sampling is performed through the online detection interface (10) of the cell culture unit (9) to sequentially complete the capture of chylomicrons, detection of intracellular TAG content, detection of chylomicron-related indicators, and mechanistic index analysis of digestion products. S7. Result Judgment: Taking the corresponding indicators of the positive control group as 100%, if all of the following conditions are met, 1,3-diglyceride type diglycerides are judged to have a lipid-lowering effect: the intracellular TAG content in the experimental group is 30% to 50% of that in the positive control group; the TAG content in chylomicrons in the experimental group is 30% to 50% of that in the positive control group; the apolipoprotein B48 content in chylomicrons in the experimental group is 40% to 60% of that in the positive control group; no TAG or chylomicrons are generated in the negative control group; and the above indicators of the experimental group and the positive control group are compared by t test with P < 0.

05.

2. The method for evaluating the lipid-lowering effect of diglycerides according to claim 1, characterized in that, The pH value of the biomimetic digestion module (2) is stable between 7.0 and 7.4, and the peristalsis frequency is 80 to 120 times / min.

3. The method for evaluating the lipid-lowering effect of diglycerides according to claim 1, characterized in that, The diversion ratio of the diversion unit (5) is determined according to the calculation method of: concentration × diversion ratio = constant load, where the concentration is the concentration of digestive fluid and the constant load is the cell metabolic load.

4. The method for evaluating the lipid-lowering effect of diglycerides according to claim 1, characterized in that, The method for capturing chylomicrons and detecting related indicators is as follows: collect the culture medium of each group of cell culture units (9), take 2 mL of each group, adjust the density to 1.006 g / mL with 0.9% NaCl solution filtered through a 0.22 μm filter membrane, add it to an ultracentrifuge tube, slowly cover it with sucrose solution with a density of 1.063 g / mL, centrifuge for 18 h at a temperature of 4℃ and a rotation speed of 40000 rpm, and take 0.2 mL of liquid from the top of the centrifuge tube as chylomicron enrichment solution; Take 20 μL of chylomicron enrichment solution and determine the content of apolipoprotein B48 in the chylomicron enrichment solution using a BCA quantitative kit with specific anti-human apolipoprotein B48 antibody. Determine the TAG content in the chylomicron enrichment solution using high performance liquid chromatography (HPLC). The HPLC detection conditions are as follows: C18 column, mobile phase acetonitrile:isopropanol = 65:35, flow rate 1.0 mL / min, column temperature 30℃, detection wavelength 210 nm, and injection volume 10 μL. The method for detecting intracellular TAG content is as follows: 1 mL of RIPA lysis buffer containing protease inhibitor is added to the cell culture unit (9), the cells are lysed on ice for 30 min, and then centrifuged at 12000 rpm and 4℃ for 15 min. 50 μL of the supernatant is taken as the cell lysis buffer, and the TAG content in the cell lysis buffer is determined using an enzymatic triglyceride detection kit.

5. The method for evaluating the lipid-lowering effect of diglycerides according to claim 1, characterized in that, The enzyme activity of MAGAT is 10-30 U / mL, and the enzyme activity of DAGAT is 40-60 U / mL.

6. A biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides, applied to the lipid-lowering effect evaluation method of diglycerides according to any one of claims 1-5, characterized in that, include: A circulating air box (1) and a biomimetic digestion module (2), wherein the biomimetic digestion module (2) is connected to the circulating air box (1); A selective permeable membrane (3) is connected to the outlet of the biomimetic digestion module (2); Absorption chamber (4), which is located on one side of the selective permeable membrane (3) and connected to the outlet of the biomimetic digestion module (2); Diversion unit (5), which is connected to the absorption chamber (4) and diverts the substance in the absorption chamber (4); A constant flow micropump (6) and a sterile filter (8) are provided. The constant flow micropump (6) is connected to the flow splitting unit (5). The constant flow micropump (6) is connected to the cell culture unit (9) through the sterile filter (8). An online detection interface (10) is installed on the cell culture unit (9). Waste liquid collection unit (7), which is connected to the diversion unit (5) and the cell culture unit (9); The controller (11) is electrically connected to each unit and adjusts the operating parameters of each unit.

7. The biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides according to claim 6, characterized in that, The selective permeable membrane (3) has a pore size range of 0.1~10kDa.

8. The biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides according to claim 6, characterized in that, The delivery rate of the constant flow micropump (6) is 0.5~2.0 mL / h.

9. The biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides according to claim 6, characterized in that, The cell culture unit (9) is a closed culture chamber, inoculated with Caco-2 cells at a density of 1.8~2.2×10⁻⁶ cells / year. 5 pcs / cm 2 .

10. The biomimetic digestive machine device for evaluating the lipid-lowering effect of diglycerides according to claim 6, characterized in that, The pore size of the sterilization filter (8) is 0.22 μm.

Images