A method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model

Abstract

This invention discloses a method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model. Soybean oil is emulsified using WPI emulsifier and then digested sequentially through simulated gastric and intestinal fluids to obtain the gastric digestion products of soybean oil. Fatty acids are extracted from the intestinal digestion products of soybean oil, dissolved in dimethyl sulfoxide, and linked to fatty acid-free bovine serum albumin to obtain in vitro digestion products of soybean oil that can be absorbed by the intestines. These are then added to a cell co-culture model for cell incubation and transport. HepG2 cells from the cell co-culture model are collected, and Oil Red O staining and total triglyceride content are measured to obtain lipid deposition, thereby evaluating the bioavailability of soybean oil. The in vitro digestion / cell co-culture model of this invention is specifically designed for soybean oil digestion. This method objectively and scientifically evaluates the bioavailability of soybean oil at different metabolic stages, providing a standard basis for the scientific and healthy consumption of soybean oil and possessing significant practical value.

Description

Technical Field

[0001] This invention relates to a method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model. Background Technology

[0002] Soybean oil is one of the most commonly used edible oils in the world, ranking among the top producers in the global oilseed market. It is a staple oil for Chinese consumers, especially those in northern China. Soybean oil is rich in various valuable nutrients, with a high content of polyunsaturated fatty acids, a moderate level of monounsaturated fatty acids compared to other vegetable oils, and a relatively low content of saturated fatty acids. Furthermore, soybean oil is rich in linoleic acid, an essential fatty acid for the human body. Therefore, soybean oil is a cost-effective edible oil.

[0003] In vitro simulated digestion technology utilizes biomimetic principles, selecting appropriate reagents to highly simulate the in vivo digestive environment. It simulates the digestion and absorption process of food in the body to the greatest extent possible, and is a detection method for measuring changes in nutrient content, digestibility, and release rate of target components during food digestion and absorption. In vitro simulated digestion requires establishing a digestive model in an in vitro environment based on the body's digestive physiology, realizing processes including oral, gastric, and intestinal digestion. The principle of in vitro simulated digestion is to create a digestive environment similar to that in vivo by selecting biological pH, temperature conditions, and digestive enzyme systems in an in vitro environment, mimicking the enzymatic breakdown process of food in the oral cavity, stomach, and intestines; examining the digestion and absorption of ingested substances in vivo, and evaluating the digestion and absorption of intermediate or final digestive products.

[0004] In vitro simulated digestion / cell co-culture combines in vitro digestion simulation with intestinal epithelial cell membrane simulation to create a digestive environment and digestive enzyme system similar to that in humans or animals, enabling in vitro studies of the digestive physiological processes of ingested food. Caco-2 cells, derived from human colon cancer cells, are cultured on cellulose acetate membranes and can be used to construct intestinal transit models. They are widely used due to their high throughput, low cost, and close resemblance to human responses. For example, Aziz et al. used an in vitro simulated digestion / Caco-2 cell model to study the bioavailability and bioavailability of rice with excessive cadmium levels and used the HL-7702 cell model to study its toxicity to humans. Fu Jin et al. used an in vitro digestion / Caco-2 cell model to determine the effects of cooking conditions and additives on the bioavailability of lead and cadmium in leafy greens and spinach, assessing the potential health risks of lead and cadmium in vegetables.

[0005] However, these studies on in vitro simulated digestion / cell co-culture mainly focus on exploring the metabolism of harmful elements in grains or vegetables and assessing their harm to human health, without addressing bioavailability research. Furthermore, the establishment of existing in vitro digestion models also faces significant challenges: ① In vitro digestion models highly simulate in vivo digestion processes, making it difficult to guarantee the consistency and accuracy between in vitro simulated digestion and in vivo digestion. ② Existing in vitro digestion models lack universality and adaptability; different digestion models are established for different samples. Further exploration is needed to refine the types and amounts of reagents used in each digestion stage of the model to more closely resemble the in vivo digestion process.

[0006] There are currently no research reports on the in vitro digestion of soybean oil, and no in vitro digestive environment or digestive enzyme system has been established specifically for soybean oil digestion. Therefore, how to establish an in vitro digestion system for soybean oil, and how to objectively and scientifically evaluate the bioavailability of soybean oil at different metabolic stages (enzyme digestion, small intestinal absorption, nutrient transport, etc.), in order to provide a basis for formulating scientific and healthy intake standards for edible oils, remain urgent problems to be solved. Summary of the Invention

[0007] To address the aforementioned problems and achieve the above-mentioned technical objectives, this invention provides a method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model. This method establishes an in vitro digestion environment and digestive enzyme system for soybean oil digestion, and establishes a specific cell co-culture model for soybean oil absorption to scientifically evaluate the bioavailability of soybean oil. The specific technical solution is as follows:

[0008] This invention provides a method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model, comprising the following steps:

[0009] S1: Soybean oil emulsification: Soybean oil is emulsified and homogenized using WPI emulsifier to obtain a homogeneous soybean oil emulsion;

[0010] S2: Gastric digestion: The emulsified soybean oil emulsion is digested in simulated gastric juice to obtain the soybean oil gastric digestion products;

[0011] S3: Intestinal digestion: The stomach digestion products of soybean oil are digested in simulated intestinal fluid to obtain the intestinal digestion products of soybean oil.

[0012] S4: Preparation of intestinal absorbable products: Extract fatty acids from the intestinal digestion products of soybean oil, dissolve them in dimethyl sulfoxide, and link them with fatty acid-free bovine serum albumin to obtain intestinal absorbable in vitro digestion products of soybean oil.

[0013] S5: Cell co-culture: Discard the original culture medium of the cell co-culture model, add the in vitro digestion product of soybean oil that can be absorbed by the intestine into the small chamber of the Transwell plate, add fresh cell culture medium to the basal side, and incubate and transport the cells.

[0014] S6: Determining physiological activity: HepG2 cells from the cell co-culture model were collected, and Oil Red O staining and total triglyceride content were measured to obtain lipid deposition, thereby evaluating the bioavailability of soybean oil.

[0015] The aforementioned method for evaluating the bioavailability of soybean oil, wherein the soybean oil emulsification in step S1 is as follows: WPI is dissolved in phosphate buffer to obtain a WPI solution, which is placed in a refrigerator at 4°C overnight to allow it to fully hydrate; the next day, the pH of the WPI solution is readjusted to 7.0, soybean oil is added, and the mixture is stirred and homogenized until no oil droplets float on the surface of the liquid to obtain a soybean oil reserve emulsion; the mass-volume concentration of the WPI solution is 1%; and the mass fraction concentration of the soybean oil reserve emulsion is 10%.

[0016] The aforementioned method for evaluating the bioavailability of soybean oil, wherein the gastric digestion in step S2 is as follows: soybean oil reserve emulsion is added to simulated gastric juice and diluted to a mass fraction concentration of 1%, and then shaken at 37°C for 2 hours in a constant temperature water bath to obtain the gastric digestion product; the simulated gastric juice has a pH of 2.0 and its components, by weight, include: 3.2 parts pepsin, 0.5 parts potassium chloride, 0.12 parts potassium dihydrogen phosphate, 2.1 parts sodium bicarbonate, 2.76 parts sodium chloride, 0.02 parts magnesium chloride hexahydrate, 0.05 parts ammonium carbonate, 1.3 parts concentrated hydrochloric acid, and 0.02 parts calcium chloride dihydrate.

[0017] The aforementioned method for evaluating the bioavailability of soybean oil, in step S3, involves incubating the gastric digestion products in a 37°C constant temperature water bath for 10 minutes to allow the temperature to reach equilibrium. After adding simulated intestinal fluid, the mixture is shaken at 37°C for 2 hours in a constant temperature water bath to obtain the intestinal digestion products. The simulated intestinal fluid has a pH of 7.0 and its components, by weight, include: 6.79 parts of dipotassium hydrogen phosphate, 8.76 parts of sodium chloride, 5 parts of ox bile salts, and 1.6 parts of pancreatic lipase.

[0018] The aforementioned method for evaluating the bioavailability of soybean oil, in step S4, involves extracting fatty acids as follows: Take the intestinal digestion product, add 5 times the volume of extraction reagent, shake and mix for 3 minutes, centrifuge to remove the lower chloroform layer, dry it with nitrogen, dissolve it in dimethyl sulfoxide, add BSA solution, shake and mix well to obtain the in vitro digestion product of soybean oil; the extraction reagent is a chloroform-methanol-deionized water solution, and the volume ratio of each component is 2:2:1.

[0019] The method for evaluating the bioavailability of soybean oil, specifically the method for establishing the cell co-culture model described in step S5, includes the following steps:

[0020] S5-1: Culture and grow Caco-2 cells and HT29 cells until they reach more than 80% confluence with the bottom of the culture dish;

[0021] S5-2: Digest the two cell lines from step S5-1 separately with 0.25% trypsin digestion solution and count them, then adjust the density ratio of the two cell suspensions.

[0022] S5-3: Seed the cell suspension from step S5-2 into Transwell plates and culture for 20 consecutive days to form intestinal epithelial cell membranes;

[0023] S5-4: Digest HepG2 cells with 0.25% trypsin digestion solution and count them. Adjust the cell suspension density and then seed them on the basal side of the Transwell plate in step S5-3. Culture for 24 hours to obtain a cell co-culture model.

[0024] Preferably, in step S5-2, the ratio of the density of the two cell suspensions is adjusted to: Caco-2 cell concentration: HT29 cell concentration = 7:3; in step S5-3, the transmembrane resistance of the intestinal epithelial cell membrane reaches 424.42±3.88Ω / cm2, and the ratio of alkaline phosphatase activity (AP / BL) is 2.34±0.22; in step S5-4, the HepG2 cells in the cell co-culture model are completely adherent and grow with the required cell morphology, and the cell co-culture model is considered to have been successfully established.

[0025] The aforementioned method for evaluating the bioavailability of soybean oil, specifically the Oil Red O staining in step S6, involves: aspirating the original culture medium from the cell co-culture model, rinsing twice with PBS, adding 4% paraformaldehyde solution, fixing the cells at room temperature for 30 minutes, aspirating the formaldehyde solution, rinsing three times with PBS solution, adding Oil Red O staining solution, staining at room temperature for 1 hour, washing away the excess stain with PBS, and then taking pictures and observing under an inverted microscope.

[0026] The aforementioned method for evaluating the bioavailability of soybean oil, specifically the determination of total triglyceride content in step S6, is as follows: The original culture medium of the cell co-culture model is discarded, the cells are washed twice with PBS, and then digested with 0.25% trypsin digestion solution. After the cells are completely detached, the cell suspension is aspirated into an EP tube, centrifuged, and the supernatant is discarded. The cells are then washed once with PBS and resuspended in 100 μL of PBS, and agitated to form a homogeneous cell suspension. Under ice-water bath conditions, the cells are homogenized in an ultrasonic homogenizer for 3–5 seconds every 30 seconds, repeated 5 times, to obtain a cell homogenate. The homogenate is then measured using a cell triglyceride assay kit.

[0027] The beneficial effects of this invention are:

[0028] 1) This invention establishes for the first time an in vitro digestion simulation system for soybean oil, and creatively uses Caco-2 cells and HT29 cells to simulate small intestinal epithelial cells and HepG2 cells to simulate liver cells. It objectively and scientifically simulates different metabolic stages of soybean oil digestion, small intestinal absorption, and nutrient transport. The bioavailability of soybean oil is evaluated by measuring the lipids accumulated in HepG2 cells. This invention successfully achieves an objective and scientific evaluation of the bioavailability of soybean oil through in vitro experiments, providing a basis for formulating scientific and healthy intake standards for edible oils.

[0029] 2) The in vitro simulated digestion / cell co-culture model of this invention is established based on the digestive characteristics of soybean oil: First, soybean oil is emulsified using WPI to approximate real oral digestion; second, various salts such as potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, magnesium chloride hexahydrate, ammonium carbonate, and calcium chloride dihydrate are combined with pepsin and concentrated hydrochloric acid to simulate gastric juice, ensuring that soybean oil is in the pH environment of simulated gastric digestion and preventing the reaction between soybean oil and salts in gastric juice under acidic conditions, which would affect the experimental results; third, the soybean oil is digested in the intestines... The product is linked to fatty acid-free bovine serum albumin, enabling its absorption by the small intestine. Fourth, the simulated intestinal epithelial cell membrane transmembrane resistance is limited to 424.42±3.88 Ω / cm², and the alkaline phosphatase activity (AP / BL) ratio is 2.34±0.22, ensuring the transmembrane conditions for lipids after intestinal digestion. Finally, HepG2 cells are used to simulate the liver to examine the bioavailability of soybean oil metabolized lipids on hepatocytes. Furthermore, HepG2 cells are designed to adhere completely to the cell wall and exhibit the appropriate cell morphology to accurately simulate the liver. The entire model is reasonable, accurate, and objectively scientific.

[0030] 3) This invention provides a method for evaluating the bioavailability of soybean oil. This method involves collecting HepG2 cells from an in vitro co-culture model, staining them with Oil Red O, and determining the triglyceride content of the cells to assess the bioavailability of soybean oil. This evaluation method is not only objective and scientific, but it can also evaluate the bioavailability of different soybean oils, providing a new approach for objectively and scientifically evaluating the bioavailability of soybean oil at different metabolic stages (enzyme digestion, small intestinal absorption, nutrient transport, etc.).

[0031] 4) The method of this invention can provide a basis for formulating scientific and healthy standards for the intake of edible oils, and has important practical value. Attached Figure Description

[0032] Figure 1 A schematic diagram of the process for evaluating the bioavailability of soybean oil based on an in vitro simulated digestion / cell co-culture model established in this invention;

[0033] Figure 2 Evaluation indicators for the cell co-culture model established in this invention;

[0034] Figure 3 Oil Red O staining images of HepG2 cells under the cell co-culture model of this invention (a. blank control group, b. experimental group);

[0035] Figure 4 This invention investigates the effect of soybean oil digestion products on intracellular TG content in HepG2 cells under a cell co-culture model. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0037] Example 1

[0038] This embodiment describes a method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model.

[0039] The bioavailability of soybean oil cannot be directly calculated, therefore a suitable method needs to be designed for evaluation. Studies have shown that dietary lipids are first hydrolyzed in the small intestine into glycerol, monoglycerides, and non-esterified fatty acids, which can be absorbed by the small intestine and enter the intestinal mucosal epithelial cells. Medium- and short-chain fatty acids do not require esterification and can directly enter the portal vein and then reach the liver. Long-chain fatty acids and monoglycerides are resynthesized into triglycerides, which combine with cholesterol and phospholipids to form chylomicrons. Small intestinal cells transport these chylomicrons extracellularly into the bloodstream. In the blood, chylomicrons are broken down into triglycerides and taken up by other cells in the body. Therefore, this invention constructs an in vitro digestive system based on the digestive characteristics of soybean oil to simulate the soybean oil digestion process. The bioavailability of soybean oil is reflected by the degree of Oil Red O staining and TG content in HepG2 cells of a cell co-culture model.

[0040] The soybean oil used in this embodiment was non-GMO soybean oil produced by COFCO Group Co., Ltd. An in vitro digestion system was established for the soybean oil, such as... Figure 1 As shown: The establishment of this system takes into account that soybean oil is generally not consumed directly, but is ingested by the human body through cooking. Studies have shown that salivary mucin exists in the oral cavity. Under the action of teeth, upper and lower jaws, and swallowing, dietary oils are emulsified. Dietary oils are digested in the gastrointestinal tract in the form of emulsion. Therefore, in order to make the experimental state of soybean oil close to that of human ingestion, this embodiment uses whey protein isolate (WPI) as an emulsifier to treat soybean oil to simulate the cooking and oral digestion of soybean oil.

[0041] In this embodiment, the specific operation of the soybean oil emulsification method is as follows: Weigh 10g of soybean oil and add it to 90g of 1% WPI solution. After thorough stirring, homogenize with a homogenizer until no oil droplets float on the surface of the liquid, thus preparing a 10% soybean oil emulsion stock solution. When using, prepare the soybean oil emulsion stock solution into a soybean oil emulsion and use it immediately. The WPI solution is prepared by dissolving 1g of WPI in 100mL of phosphate buffer (5mM), stirring with an electric mixer for 20 minutes, sealing and placing it in a 4℃ refrigerator overnight to allow it to fully hydrate. The next day, remove it and readjust the pH of the WPI aqueous solution to 7.0 to prepare a 1% WPI solution for use.

[0042] When the soybean oil enters the stomach for digestion, simulating gastric juice and preventing the salts in the simulated gastric juice from reacting with the soybean oil emulsion is crucial to the experimental results. In this embodiment, the simulated gastric juice, in addition to pepsin, potassium chloride, and concentrated hydrochloric acid, also contains potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, magnesium chloride hexahydrate, ammonium carbonate, and calcium chloride dihydrate. This ensures that the pH of the simulated gastric juice remains almost constant during simulated digestion and prevents the salts in the gastric juice from reacting with the soybean oil under acidic conditions. The preparation method of the simulated gastric juice in this embodiment is as follows: Dissolve 3.2g pepsin, 0.5g potassium chloride, 0.12g potassium dihydrogen phosphate, 2.1g sodium bicarbonate, 2.76g sodium chloride, 0.02g magnesium chloride hexahydrate, 0.05g ammonium carbonate, 1.3mL concentrated hydrochloric acid, and 0.02g calcium chloride dihydrate, then bring the volume to 1L and adjust the pH to 2.0. The digestion method of soybean oil emulsion in simulated gastric juice is as follows: simulated gastric juice is mixed with the prepared soybean oil emulsion stock solution, with a total volume of 20 mL and a final soybean oil concentration of 1%. The mixture is then shaken at 37°C for 2 hours in a constant temperature water bath to obtain the gastric digestion products.

[0043] Next is intestinal digestion. Similar to gastric digestion, the simulation and absorption of intestinal fluid are crucial. In this embodiment, the simulated intestinal fluid is prepared as follows: 6.79g dipotassium hydrogen phosphate, 8.76g sodium chloride, 5g taurine bile salts, and 1.6g pancreatic lipase are dissolved and brought to a final volume of 1L, and the pH is adjusted to 7.0. The digestion method in the simulated intestinal fluid is as follows: 10mL of simulated gastric digestion products are added to 10mL of simulated intestinal fluid. Before adding the simulated intestinal fluid, the mixture is incubated in a 37°C water bath for 10 minutes to allow the temperature to reach equilibrium. The mixture is then shaken at 37°C for 2 hours in a water bath to obtain the intestinal digestion products. Considering that fatty acids in soybean oil cannot be directly absorbed and utilized by cells, in order to make the simulated intestinal digestion products more similar to human digestion products and to achieve the absorption of fatty acids in soybean oil, this embodiment extracts fatty acids from the above-mentioned intestinal digestion products, dissolves them in dimethyl sulfoxide (DMSO), then dissolves them in fatty acid-free bovine serum albumin (BSA) solution and links them with fatty acid-free bovine serum albumin (BSA) to obtain soybean oil digestion products that can be absorbed by small intestinal epithelial cells. The specific method for extracting fatty acids from the intestinal digestion products in this embodiment is as follows: Take 3 mL of the above-mentioned intestinal digestion products, add 5 times the volume of extraction reagent (chloroform:methanol:deionized water = 2:2:1), then shake the mixture for 3 minutes and centrifuge to obtain the lower chloroform layer. In order to fully extract fatty acids from the intestinal digestion products, after the first extraction, the upper aqueous phase is transferred to another centrifuge tube, and 0.1 mol / L HCl solution is added to adjust the pH value to below 1.5. The above fatty acid extraction steps are repeated. Then, the chloroform layers from the first and second extractions are combined and dried with nitrogen gas. Finally, the product obtained by blowing it dry with nitrogen was dissolved in 100 μL of dimethyl sulfoxide, and 0.9 mL of BSA (10%) solution was added. The mixture was shaken and mixed for 3 minutes to obtain soybean oil digest products that can be absorbed by small intestinal epithelial cells.

[0044] To simulate the digestion and absorption of soybean oil digestion products in the intestine, this embodiment establishes a cell co-culture model. Caco-2 cells and HT29 cells are used to simulate the intestine, and HepG2 cells are used to simulate the liver. The specific establishment process is as follows: When Caco-2 cells and HT29 cells are cultured and grown to more than 80% confluence with the bottom of the culture dish, the two cell lines are digested and counted separately with 0.25% trypsin (containing EDTA) digestion solution. The cell suspension density of the two cell lines is adjusted, and they are seeded in Transwell polycarbonate membrane 24-well plates for continuous culture and incubation. On day 20 of incubation, HepG2 cells are digested and counted with 0.25% trypsin (containing EDTA) digestion solution. The cell suspension density is adjusted to 1.5 × 104 cells / mL, and they are seeded on the basal side of the Transwell plate for continued culture and incubation. During incubation, the transmembrane resistance of the simulated intestinal epithelial cell monolayer and the alkaline phosphatase (AP / BL) activity were measured periodically. The incubation period continued until the transmembrane resistance of the simulated intestinal epithelial cell monolayer formed by Caco-2 and HT29 cells exceeded 400 Ω / cm. 2 A positive alkaline phosphatase activity (AP / BL) ratio of greater than 2 indicates that HepG2 cells adhere completely to the culture vessel, thus confirming the successful establishment of the cell co-culture model. Figure 2 As shown, after incubation with HepG2 cells for 24 hours, the transmembrane resistance of the simulated intestinal epithelial cell monolayer formed by Caco-2 cells and HT29 cells reached 424.42 ± 3.88 Ω / cm. 2 The alkaline phosphatase activity (AP / BL) ratio was 2.34 ± 0.22, and HepG2 cells adhered completely to the culture vessel and exhibited the expected cell morphology, thus the cell co-culture model was successfully established. Experiments showed that the optimal model was achieved when the Caco-2 cell concentration was 7:3 compared to the HT29 cell concentration. At this ratio, Caco-2 cells could form complete small intestinal epithelial cell membranes and villous structures, while HT29 cells secreted mucus similar to that of human small intestinal mucosa during culture. This effectively simulated both the tight junctions and villous structures of the small intestinal epithelium and the mucus present on the surface of small intestinal epithelial cells. Conversely, an excessively high proportion of Caco-2 cells resulted in the absence of a mucus layer, while an excessively high proportion of HT29 cells prevented the formation of a complete intestinal epithelial cell membrane.

[0045] The cell co-culture model described in this embodiment was cultured as follows: Soybean oil digestion products, which can be absorbed by small intestinal epithelial cells, were added to Transwell plates inoculated with Caco-2 cells. Fresh cell culture medium was added to the basal side, and the plates were statically cultured at 37°C, containing 5% carbon dioxide and 90% relative humidity for cell incubation and transport. The cell culture medium was DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. After 72 hours of cell incubation and transport, HepG2 cells from the cell co-culture model were collected for Oil Red O staining and total triglyceride content determination. A co-culture model without soybean oil digestion products was used as a blank control. Wherein:

[0046] Oil Red O staining was performed as follows: The original culture medium in the cell co-culture model was aspirated, the cells were rinsed twice with PBS, and then 4% paraformaldehyde solution was added. The cells were fixed at room temperature for 30 minutes, the formaldehyde solution was aspirated, and the cells were rinsed three times with PBS. Oil Red O staining solution was then added, and the cells were stained at room temperature for 1 hour. After washing away any excess stain with PBS, the stain was observed under an inverted microscope. Results are as follows: Figure 3 As shown, lipid accumulation occurred in HepG2 cells treated with soybean oil digestion products, and obvious red lipid droplets were visible after Oil Red O staining.

[0047] The total triglyceride content was determined as follows: the original culture medium of the cell co-culture model was discarded, the cells were washed twice with PBS, and then digested with 0.25% trypsin digestion solution. After the cells were completely detached, the cell suspension was aspirated into an EP tube, centrifuged, and the supernatant was discarded. The cells were washed once with PBS and then resuspended in 100 μL of PBS and pipetted to form a homogeneous cell suspension. Under ice-water bath conditions, the cells were sonicated for 3-5 seconds every 30 seconds, and this was repeated 5 times to obtain a cell homogenate. The triglyceride content was then measured using a cell triglyceride assay kit.

[0048] TG content (mmol / gport) = [(sample OD value - blank OD value) / (calibration OD value - blank OD value)] × calibrator concentration ÷ protein concentration of the sample to be tested (gport / L), where the protein concentration was determined using a BCA protein concentration kit. In this example, the total triglyceride content determination experiment included three parallel experiments for both the blank control and treatment groups, and the results are shown in Table 1 and... Figure 4 As shown.

[0049] Table 1. Results of total triglyceride content determination in HepG2 cells, a cell co-culture model.

[0050]

[0051] The results showed that, compared with the control group, the intracellular TG content in the soybean oil digestion product group was significantly increased, reaching 8.8 ± 0.49 times that of the control group. This indicates that after fatty acids in soybean oil digestion products are transported through a simulated intestinal epithelial cell membrane, lipid accumulation occurs in HepG2 cells, leading to an increase in intracellular triglyceride content. Furthermore, the greater the lipid deposition, the higher the triglyceride content, and the higher the utilization rate of soybean oil digestion products. This can provide a basis for formulating scientific and healthy standards for edible oil intake.

[0052] In summary, this invention establishes for the first time an in vitro digestion simulation system for soybean oil, and creatively uses Caco-2 and HT29 cells to simulate small intestinal epithelial cells and HepG2 cells to simulate liver cells. It objectively and scientifically simulates different metabolic stages of soybean oil digestion, small intestinal absorption, and nutrient transport. The bioavailability of soybean oil is evaluated by measuring lipid accumulation within HepG2 cells. This successfully achieves an objective and scientific evaluation of soybean oil bioavailability through in vitro experiments, providing a basis for formulating scientific and healthy standards for edible oil intake. The method for evaluating soybean oil bioavailability in this invention involves collecting HepG2 cells from an in vitro co-culture model, performing Oil Red O staining, and measuring triglycerides in the cells to assess the bioavailability. This evaluation method is not only objective and scientific, but also takes into account the different components of different edible oils, which affect the amount of lipid deposition in cells. By measuring the amount of lipid deposition, it can also indirectly reflect the differences in bioavailability among different edible oils, demonstrating good versatility and significant practical value.

[0053] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for evaluating the bioavailability of soybean oil based on an in vitro digestion / cell co-culture model, characterized by: Comprising the following steps: S1: Soybean oil emulsification: Soybean oil is emulsified and homogenized using WPI emulsifier to obtain uniform soybean oil emulsion; S2: Gastric digestion: The emulsified soybean oil emulsion is digested in simulated gastric juice to obtain soybean oil gastric digestion product; S3: Intestinal digestion: The soybean oil gastric digestion product is digested in simulated intestinal juice to obtain soybean oil intestinal digestion product; S4: Preparation of intestinal absorbable product: The fatty acids in the soybean oil intestinal digestion product are extracted, dissolved in dimethyl sulfoxide and connected with fat-free bovine serum albumin to obtain the intestinal absorbable soybean oil in vitro digestion product; S5: Cell co-culture: The original culture solution of the cell co-culture model is aspirated, the obtained intestinal absorbable soybean oil in vitro digestion product is added to the chamber of the Transwell plate, fresh cell culture medium is added to the basal side, and cell incubation and transport are carried out; S6: Determine physiological activity: Collect HepG2 cells in the cell co-culture model, perform oil red O staining and total triglyceride content determination, obtain lipid deposition amount, and evaluate the bioavailability of soybean oil; Wherein: In step S1, the soybean oil emulsification is: WPI is dissolved in phosphate buffer solution to obtain WPI solution, which is placed in a 4℃ refrigerator overnight to allow it to be fully hydrated; the next day, the pH of the WPI solution is adjusted to 7.0, soybean oil is added, stirred and homogenized until no oil droplets float on the surface of the liquid, and a soybean oil stock emulsion is obtained; the mass concentration of the WPI solution is 1%; the mass fraction concentration of the soybean oil stock emulsion is 10%; In step S2, the gastric digestion is: the soybean oil stock emulsion is added to the simulated gastric juice for dilution, the concentration of the soybean oil emulsion is controlled at 1%, and the gastric digestion product is obtained by incubating in a constant temperature water bath shaker at 37℃ for 2 hours; the pH value of the simulated gastric juice is 2.0, and its components consist of the following ingredients by weight: pepsin 3.2 parts, potassium chloride 0.5 parts, potassium dihydrogen phosphate 0.12 parts, sodium bicarbonate 2.1 parts, sodium chloride 2.76 parts, magnesium chloride hexahydrate 0.02 parts, ammonium carbonate 0.05 parts, concentrated hydrochloric acid 1.3 parts, calcium chloride dihydrate 0.02 parts; In step S3, the intestinal digestion is: the gastric digestion product is incubated in a 37℃ constant temperature water bath for 10 minutes to allow the temperature to reach equilibrium, the simulated intestinal juice is added, and the intestinal digestion product is obtained by incubating in a constant temperature water bath shaker at 37℃ for 2 hours; the pH value of the simulated intestinal juice is 7.0, and its components consist of the following ingredients by weight: dipotassium hydrogen phosphate 6.79 parts, sodium chloride 8.76 parts, bovine bile salt 5 parts, pancreatic lipase 1.6 parts; In step S4, the extraction of fatty acids is: take the intestinal digestion product, add 5 times the volume of extraction reagent, shake and mix for 3 minutes, then centrifuge to take the lower chloroform layer, dry it with nitrogen, dissolve it with dimethyl sulfoxide, add BSA solution, shake and mix to obtain the soybean oil in vitro digestion product; the extraction reagent is chloroform-methanol-deionized water solution, and the volume ratio of each component is 2:2:1; In step S5, the cell co-culture is as follows: S5-1: Caco-2 cells and HT29 cells are cultured and grown to more than 80% coverage of the bottom of the culture dish; S5-2: The two types of cells in step S5-1 are digested with 0.25% trypsin digestion solution and counted, and the density of the cell suspension is adjusted to Caco-2 cell concentration: HT29 cell concentration = 7:3; S5-3: The cell suspension in step S5-2 is inoculated into a Transwell plate for culture, and cultured continuously for 20 days to form an intestinal epithelial cell membrane; S5-4: The HepG2 cells were digested with 0.25% trypsin solution and counted, the cell suspension density was adjusted, and then inoculated on the basal side of the Transwell plate in step S5-3, and cultured for 24 hours to obtain a cell co-culture model, the transmembrane electrical resistance value of the intestinal epithelial cell membrane reached 424.42±3.88 Ω / cm 2 , the ratio of alkaline phosphatase activity AP / BL was 2.34±0.22, and the HepG2 cells grew completely adherent The oil red O staining in step S6 is as follows: the original culture medium in the cell co-culture model is aspirated, washed twice with PBS, then 4% paraformaldehyde solution is added, the cells are fixed at room temperature for 30 minutes, then the formaldehyde solution is aspirated, washed three times with PBS solution, then oil red O staining solution is added, stained at room temperature for 1 hour, then the floating color is washed off with PBS, and then photographed and observed under an inverted microscope; The determination of total triglyceride content in step S6 is as follows: the original culture medium in the cell co-culture model is aspirated, washed twice with PBS, then digested with 0.25% trypsin digestion solution, after the cells are completely detached, the cell suspension is aspirated into an EP tube, centrifuged, and the supernatant is discarded; then washed once with PBS, resuspended with 100 μL PBS, and blown into a uniform cell suspension; under ice water bath conditions, every 30 seconds, break in an ultrasonic crusher for 3-5 seconds, repeat 5 times, prepare the cell homogenate, and then determine using a cell triglyceride determination kit.

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