Method for evaluating the bioavailability of pesticide residues in aquatic products

Abstract

The application provides an evaluation method of pesticide residue bioavailability of aquatic products, which comprises the following steps: simulating oral mastication, simulating gastric digestion and simulating intestinal digestion of aquatic product samples to obtain digested chyme; detecting pesticide residues in the digested chyme and the aquatic product samples by gas chromatography respectively, and calculating the bioavailability of pesticides in the aquatic products after gastrointestinal digestion; and further evaluating the bioavailability of pesticides across the cell membrane in the aquatic products by performing cell absorption and transport experiments. The method is simple, convenient and operable, and can be used to evaluate the bioavailability of pesticide residues in aquatic products in human body, and provides a reference for further evaluating the bioavailability of pesticide residues in aquatic products.

Description

Technical Field

[0001] This invention relates to the field of food safety technology, and in particular to a method for evaluating the bioavailability of pesticide residues in aquatic products. Background Technology

[0002] In the process of aquaculture, various organic pesticides, such as pyrethroids, organochlorines, organophosphates, and carbamates, are often sprayed to prevent parasites and other pests. Due to bioaccumulation, organic pesticides accumulate in organisms along the food chain and eventually enter the human body through food intake. However, the amount of harmful substances contained in food is not equal to the amount of harmful substances that the human body may absorb during digestion.

[0003] Previous studies primarily assessed the metabolism of harmful substances in humans through animal experiments. However, animal models are time-consuming, expensive, and raise ethical concerns. In recent years, in vitro models have emerged and, due to their simplicity, convenience, and ease of operation, have been applied in the food and pharmaceutical industries. In vitro digestion models mainly include static, semi-dynamic, and dynamic models. Semi-dynamic and dynamic models require specialized equipment, hindering widespread adoption among laboratories. Static models are widely used due to their ease of operation and lack of specialized equipment. However, many static models exist, differing from one another, and lack a unified standard. Therefore, establishing a method to directly evaluate the harmful components of a pesticide in aquatic products is crucial. Bioavailability, an indicator of maximum oral bioavailability, refers to the amount of absorbable substances released from the food matrix in the digestive tract. By measuring the amount of harmful substances contained in the food itself and the amount of harmful substances in the digestive juices after digestion, we can more intuitively reflect the absorption of harmful substances in the body and assess the health risks caused by the intake of a certain harmful substance through food. Summary of the Invention

[0004] This invention provides a method for evaluating the bioavailability of pesticide residues in aquatic products. This method is simple, convenient, and highly operable, and can be used to evaluate the bioavailability of pesticide residues in aquatic products to the human body, providing a reference for further assessment of the bioavailability of pesticide residues in aquatic products.

[0005] This invention provides a method for evaluating the bioavailability of pesticide residues in aquatic products, comprising: simulating oral chewing, gastric digestion, and intestinal digestion of aquatic product samples to obtain digested chyme; detecting pesticide residues in the digested chyme and aquatic product samples respectively using gas chromatography, and calculating the bioavailability of pesticides in aquatic products after gastrointestinal digestion; further comprising conducting cell absorption and transport experiments to further evaluate the bioavailability of pesticides in aquatic products after crossing cell membranes.

[0006] In this invention, by employing the above-mentioned in vitro simulated digestion method, the bioavailability of pesticides in aquatic products can be assessed more conveniently. In particular, the bioavailability of pesticides can be further assessed through cell absorption experiments.

[0007] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, the simulated oral chewing uses simulated saliva with a pH of 7.0±0.2 and a digestion time of 0.5 to 3 min; the simulated gastric digestion uses hydrochloric acid gradually added to simulated gastric juice and a digestion time of 1.5 to 3.5 h; and the simulated intestinal digestion uses simulated intestinal juice with a pH of 7.0±0.2 and a digestion time of 1.5 to 3 h.

[0008] In this invention, by employing the above-mentioned oral-gastrointestinal simulated digestion method, food can be fully digested. In order to better simulate the physiological environment of food during in vivo gastric digestion, the gastric pH is continuously adjusted during the in vitro simulated digestion process to make it more closely resemble the real digestion process.

[0009] The method for evaluating the bioavailability of pesticide residues in aquatic products according to the present invention includes:

[0010] 1) Sample pretreatment: The aquatic products are pretreated to obtain aquatic product samples;

[0011] 2) Preparation of electrolyte solution: The electrolyte solution is selected from one or more of KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6 and (NH4)2CO3, and the electrolyte solution is divided into oral electrolyte solution, gastric electrolyte solution and intestinal electrolyte solution;

[0012] 3) Simulated oral chewing: The aquatic product sample is minced into a paste to obtain meat paste; the meat paste is mixed with oral phase simulated digestive fluid and digested at 36-38℃ for 1-4 minutes to obtain oral phase digestion products; the oral phase simulated digestive fluid includes oral electrolyte solution and salivary amylase. If the food does not contain starch or has a very low starch content, amylase may not be added.

[0013] In this invention, it is preferable to mix 5g of minced meat with 1mL of 5× oral simulated digestion solution, add 0.05mL of 0.15M CaCl2(H2O)2 solution, and add water to a 1× concentration oral simulated digestion solution, mix evenly, and digest at 36-38℃ for 1-4 minutes. By adopting the above-mentioned preferred ratio of food to oral simulated digestion solution, this invention can better simulate oral chewing of the above-mentioned minced meat, thus more closely conforming to the human oral chewing process.

[0014] 4) Simulated gastric digestion: The oral phase digestion products are mixed with the gastric phase electrolyte solution, a portion of hydrochloric acid solution is added, followed by CaCl2(H2O)2 solution, water, pepsin solution, and gastric lipase solution. The remaining hydrochloric acid solution is then added in steps. After the acid addition is complete, digestion continues for 15–40 minutes to obtain gastric digested chyme.

[0015] In this invention, preferably, 10 mL of oral digestion product is taken, 2 mL of oral electrolyte solution is added, followed by 20% of the estimated total hydrochloric acid (concentration 1M) hydrochloric acid solution, 0.01 mL of 0.15M CaCl2(H2O)2 solution, and the water that is estimated to need to be added during the preliminary experiment is added; then, 1 mL of porcine pepsin solution is added to make the porcine pepsin activity reach 2000 U / mL; 1 mL of gastric lipase solution is added to make the gastric lipase activity reach 60 U / mL; timing begins, and immediately 10% of the estimated total hydrochloric acid hydrochloric acid hydrochloric acid solution (concentration 1M) is added, followed by 10% of the estimated total hydrochloric acid hydrochloric acid hydrochloric acid solution (concentration 1M) at regular intervals, the intervals being calculated based on the estimated gastric digestion time; after the acid addition is completed, digestion lasts for 15-40 minutes, and gastric digestion ends; by adopting the above-mentioned preferred oral simulated digestive solution and digestion pH setting, this invention can better match the physiological environment in the human stomach, thereby better simulating gastric digestion.

[0016] 5) Simulated intestinal digestion: The gastric digested chyme is mixed sequentially with NaOH solution and the intestinal electrolyte solution, a mixed solution of porcine pancreatic enzyme and bile salts is added, CaCl2(H2O)2 solution is added, and NaOH solution is added dropwise to adjust the pH to 7.0±0.2 for intestinal digestion to obtain digested chyme. In this invention, preferably 20 mL of gastric digested chyme is taken, and about 2 mL of 1M NaOH solution is added first, followed by 4 mL of 5× eSIF solution, then the above-mentioned mixed solution of porcine pancreatic enzyme and bile salts is added, followed by 0.01 mL of CaCl2(H2O)2 solution, and 1M NaOH solution is added dropwise to adjust the pH to 7.0±0.2; the intestinal digestion time is 2 hours. By using the above-mentioned preferred digestive enzymes and bile salts, this invention can better match the physiological environment of the human intestinal tract, thereby better simulating intestinal digestion.

[0017] 6) Detection of pesticide residues in undigested aquatic products; Take the aquatic product samples from step 1), mince and pre-treat them, and detect pesticide residues by chromatographic methods;

[0018] 7) Detection of pesticide residues in digested aquatic products; The digested food from step 6) is subjected to pesticide residue detection according to the chromatographic method described above;

[0019] 8) Calculate the bioavailability B after gastrointestinal digestion: Calculate the bioavailability B based on the test results obtained in steps 6)-7), and evaluate the bioavailability B.

[0020] 9) Cell uptake and transport experiment: NCM460 cells were used to perform cell viability experiments to analyze the effect of pesticides on cell viability; then, the transmembrane transport of pesticides was performed using Transwell, and the pesticide concentrations in the lower and upper chambers of the Transwell were calculated after the transport experiment to obtain the transmembrane transport efficiency C of pesticides.

[0021] 10) Calculate the bioavailability A of pesticide residues in aquatic products after gastrointestinal-cellular digestion and absorption, as follows: A = B × C, where B is the bioavailability of pesticide residues after gastrointestinal digestion of aquatic products, and C is the transmembrane transport efficiency of pesticide residues in intestinal digestate.

[0022] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, in step 1), the pretreatment includes washing and shelling; preferably, it also includes cooking the pretreated aquatic product samples. In the present invention, the aquatic products are washed, and then processed according to consumption habits, such as removing scales from fish and removing sand and shells from shellfish; before simulated digestion, the aquatic products are cooked according to dietary cooking habits; preferably, the processing methods for different aquatic products should be based on the characteristics of different varieties, and the original samples are retained and refrigerated at 2-5°C to facilitate the subsequent detection of pesticide residues in the food.

[0023] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, in step 2), the oral electrolyte solution includes 14-16 mM KCl, 3-4 mM KH2PO4, 13-15 mM NaHCO3, 0.4-0.6 mM MgCl2(H2O)6, and 0.04-0.08 mM (NH4)2CO3; and / or

[0024] The gastric phase electrolyte solution includes 6–8 mM KCl, 0.7–1.0 mM KH₂PO₄, 23–26 mM NaHCO₃, 45–48 mM NaCl, 0.10–0.14 mM MgCl₂(H₂O)₆, and 0.4–0.6 mM (NH₄)₂CO₃; and / or

[0025] The intestinal electrolyte solution comprises 6–8 mM KCl, 0.7–0.9 mM KH₂PO₄, 80–90 mM NaHCO₃, 36–40 mM NaCl, and 0.3–0.4 mM MgCl₂(H₂O)₆. Preferably, the electrolyte solution is preheated at 37°C before use.

[0026] In this invention, by using the above-mentioned electrolyte solution, especially at a preferred concentration, it is possible to better match the physiological environment of the human body, thereby better simulating digestion.

[0027] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, during simulated digestion, the pepsin activity is 1800–2200 U / mL; the gastric lipase activity is 50–70 U / mL; and / or, the pancreatic enzyme activity is 90–110 U / mL; and the bile salt concentration is 8–12 mM. In this invention, by employing the above-mentioned preferred enzyme activities and bile salt concentrations, digestion can be made more complete, thereby better simulating the human digestive process. In this invention, CaCl2(H2O)2 must be prepared and added before digestion, and the slight volume change caused by the addition of CaCl2(H2O)2 can be ignored.

[0028] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, in step 3), when the sample being tested contains starch, salivary amylase is added during the simulated oral digestion process.

[0029] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, in step 4), the total amount of hydrochloric acid added is the amount of hydrochloric acid required to achieve a pH of 1.5 to 2.5; and / or, in step 5), intestinal digestion is terminated by heating in a water bath at 95°C for 5 minutes; and / or, in steps 3) to 5), the digestion process is preferably carried out in a constant temperature environment of 37°C.

[0030] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, gastric digestion is terminated by adding 1M NaOH solution to the digestate and adjusting the pH to 8.0 (±0.2).

[0031] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, in step 9), a pesticide of a set concentration is added to the upper chamber of the Transwell; and / or, in step 7), the chromatographic method includes one of gas chromatography, liquid chromatography, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

[0032] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, gas chromatography is used to detect pyrethroid pesticides. More preferably, the gas chromatography detection conditions are: HP-5 column, 30m × 320μm × 0.25μm, injection port temperature of 250℃, electron capture detector (ECD), detector temperature of 300℃, programmed temperature ramp to 100℃ and hold for 2 min, then ramped at 6℃ / min to 270℃ and held for 10 min. In this invention, by employing the above-mentioned preferred detection method and conditions, higher sensitivity is achieved.

[0033] According to the method for evaluating the bioavailability of pesticide residues in aquatic products provided by the present invention, the ratio of pesticide residue content in the digested chyme obtained in step 7) to the pesticide residue content obtained in step 6) is calculated to obtain the bioavailability B of pesticides in aquatic products after gastrointestinal digestion; the product of the bioavailability B and the transmembrane transport efficiency C obtained in step 9) is calculated to obtain the bioavailability A of pesticide residues in aquatic products after gastrointestinal digestion and permeation through monolayer cells.

[0034] The beneficial effects of this invention are at least as follows: This invention provides a method for evaluating the bioavailability of pesticide residues in aquatic products. By calculating the bioavailability of pesticide residues in aquatic products before and after simulated digestion, it indirectly reflects the degree of digestion, absorption, and utilization of pesticide residues in aquatic products by the human body. Furthermore, this method is highly feasible and applicable to the vast majority of aquatic products. This method can further provide a basis for the bioavailability of harmful substances in humans or animals. It also has the following advantages: This method can be used to simultaneously detect the bioavailability of multiple pesticide residues in aquatic products; it is simple to operate and highly efficient; during the gastric digestion stage, hydrochloric acid is gradually added to adjust the pH, which more closely resembles the actual physiological environment of gastric digestion and is more operable; based on simulated gastrointestinal digestion, cell experiments are added to further evaluate the bioavailability of pesticides in aquatic products; the oral cavity stage is added during the simulated digestion process, and gastric lipase is added to the gastric phase, making it closer to the digestive process experienced by humans. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a flowchart illustrating the method for evaluating the bioavailability of pesticide residues in aquatic products according to the present invention.

[0037] Figure 2This is a chromatogram of deltamethrin pesticide residue in raw oysters before and after digestion in Example 1 of the present invention (A is raw oyster meat; B is raw oyster meat after intestinal digestion).

[0038] Figure 3 This is a chromatogram of deltamethrin pesticide residue in steamed oysters before and after digestion in Example 2 of the present invention (A is steamed oyster meat; B is steamed oyster meat after digestion).

[0039] Figure 4 This is a chromatogram of deltamethrin pesticide residue in roasted oysters before and after digestion in Example 3 of the present invention (A is roasted oyster meat; B is roasted oyster meat after digestion). Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product manual. Instruments and other equipment whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the methods described are conventional methods, and the raw materials described are all obtainable from publicly available commercial sources. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product manual.

[0042] In the following embodiments of the present invention,

[0043] Bioavailability B (after gastrointestinal digestion) (%) = Pesticide residue content in digested food ÷ Pesticide residue content in undigested food;

[0044] Transmembrane transport efficiency C (%) = pesticide concentration in the lower chamber of the Transwell ÷ pesticide concentration in the upper chamber;

[0045] Bioavailability A (after passing through the cell) (%) = Bioavailability B × Transmembrane transport efficiency C.

[0046] Example 1

[0047] This embodiment provides a method for evaluating the bioavailability of deltamethrin in raw oysters, specifically including the following steps:

[0048] Step 1: Sample pretreatment. Wash the oysters with their shells on using tap water, then remove the shells, extract the oyster meat, and mince it for later use.

[0049] Step 2: Prepare the electrolyte concentrate. As shown in Table 1, the electrolyte concentrate contains KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, and (NH4)2CO3. The final concentrations of each electrolyte solution in the oral cavity were KCl (15.1 mM), KH2PO4 (3.7 mM), NaHCO3 (13.6 mM), MgCl2(H2O)6 (0.5 mM), and (NH4)2CO3 (0.06 mM); the final concentrations of each electrolyte solution in the stomach were KCl (6.9 mM), KH2PO4 (0.9 mM), NaHCO3 (25 mM), NaCl (47.2 mM), MgCl2(H2O)6 (0.12 mM), and (NH4)2CO3 (0.5 mM); and the final concentrations of each electrolyte solution in the intestine were KCl (6.8 mM), KH2PO4 (0.8 mM), NaHCO3 (85 mM), NaCl (38.4 mM), and MgCl2(H2O)6 (0.33 mM).

[0050] Table 1 Electrolyte Concentrate Preparation Table

[0051]

[0052]

[0053] Step 3: Simulate oral chewing. Use an automatic meat grinder to simulate oral chewing, grinding the processed oysters into a paste. Weigh 5g of the meat paste and add 1mL of 5× oral electrolyte solution, 0.05mL of CaCl2(H2O)2 solution, and add water to a 1× concentration of simulated oral digestive solution. Mix well and digest at 37℃ for 2 minutes.

[0054] Step 4: Simulated gastric digestion. Take 10 mL of the oral phase digestion product, add 2 mL of electrolyte solution, and then add hydrochloric acid. The amount of hydrochloric acid added is based on the total amount of hydrochloric acid determined in the preliminary experiment. First, add 20% of the estimated total amount of hydrochloric acid solution (concentration of 1M), then add 0.01 mL of CaCl2(H2O)2 solution to bring the final concentration of the simulated gastric digestion solution to 0.15 mM. Add the amount of water that was estimated to need to be added during the preliminary experiment. Record stable pH data. Next, add 1 mL of porcine pepsin solution to achieve an activity of 2000 U / mL. Add 1 mL of gastric lipase solution to achieve an activity of 60 U / mL. Start timing and immediately add 10% of the estimated total amount of hydrochloric acid solution (concentration of 1M). Then, add 10% of the estimated total amount of hydrochloric acid solution (concentration of 1M) every 15 minutes. After the acid addition is complete, digestion lasts for 30 minutes, at which point gastric digestion is finished. The gastric digestion time is 2.5 hours.

[0055] Step 5: Simulated intestinal digestion. Take 20 mL of gastric digested chyme, first add approximately 2 mL of 1M NaOH solution, then add 4 mL of 5× intestinal electrolyte solution, followed by the above-mentioned mixed solution of porcine pancreatic enzymes and bile salts, and finally add 0.01 mL of CaCl2(H2O)2 solution. Adjust the pH to 7.0 (±0.2) by adding 1M NaOH solution dropwise. The intestinal digestion time is 2 hours.

[0056] Table 2. Composition of Simulated Digestive Fluid

[0057]

[0058]

[0059] Step Six: Detection of pesticide residues in undigested Pacific oysters. The Pacific oyster samples obtained in Step One were minced using a meat grinder. The samples were then processed using the QuEChERS rapid sample pretreatment technique, followed by extraction, purification, and concentration. Finally, the deltamethrin content in the Pacific oysters was detected by gas chromatography.

[0060] QuEChERS rapid sample pretreatment technique for extracting deltamethrin from oyster meat: Take 10g of oyster meat, add 5mL of water and mix well, add 15mL of 1% acetonitrile acetate for extraction and purification, then take 3mL and blow it dry with nitrogen at 45℃, and add 2mL of n-hexane to redissolve.

[0061] The gas chromatography detection conditions were as follows: HP-5 column (30m×320μm×0.25μm), injection port temperature 250℃, electron capture detector (ECD), detector temperature 300℃, programmed temperature increase to 100℃ and hold for 2 min, then increased to 270℃ at 6℃ / min and held for 10 min.

[0062] Step 7: Detection of pesticide residues in digested food.

[0063] QuEChERS rapid sample pretreatment technique for extracting deltamethrin from oyster meat: Take 15 mL of intestinal digestion fluid, add 15 mL of 1% acetonitrile acetate for extraction, purify, then take 4 mL and blow dry under nitrogen at 45℃, add 1.0 mL of n-hexane to redissolve, and concentrate 4 times.

[0064] The detection conditions are the same as those in step six, which are the same as those for gas chromatography.

[0065] Step 8: Calculate bioavailability after gastrointestinal digestion B: Calculate and evaluate bioavailability based on the results obtained in Steps 6 and 7.

[0066] Step Nine: Cell Absorption and Transport Assay. NCM460 cells were selected. First, cell viability was assessed by treating cells with 5, 10, 17.5, and 30 ng / mL deltamethrin for 24 hours, and the effect on cell viability was analyzed. Then, three treatment groups (2, 10, and 30 ng / mL) were used to conduct transmembrane transport experiments of deltamethrin using Transwell assays to obtain the transmembrane transport efficiency C of the pesticide.

[0067] Step 10: Calculate bioavailability. Calculate the product of the bioavailability B and the transmembrane transport efficiency C described in Step 7 to obtain the bioavailability A of pesticide residues in oysters after gastrointestinal digestion and permeation through monolayer cells.

[0068] Bioavailability A (%) of deltamethrin in Pacific oyster = deltamethrin content after permeation through cell membrane ÷ deltamethrin content in undigested Pacific oyster.

[0069] The bioavailability B of deltamethrin in oysters after gastrointestinal digestion, multiplied by the transmembrane transport efficiency C of deltamethrin, is the bioavailability A of deltamethrin residue in Pacific oysters after gastrointestinal digestion and cellular processes.

[0070] The bioavailability of deltamethrin in steamed oysters was calculated and is shown in Table 3.

[0071] Table 3. Bioavailability of deltamethrin in Pacific oyster.

[0072]

[0073] Example 2

[0074] This embodiment provides a method for evaluating the bioavailability of deltamethrin in steamed oysters, specifically including the following steps:

[0075] Step 1: Sample pretreatment. Wash the oysters with their shells on using tap water. According to dietary cooking habits, steam the oysters until cooked. In this experiment, steam for 5 minutes after the water boils, and then let them sit for 2 minutes.

[0076] Step 2: Prepare the electrolyte concentrate. As shown in Table 1 above, the electrolyte concentrate contains KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, and (NH4)2CO3. The final concentrations of each electrolyte solution in the oral cavity were KCl (15.1 mM), KH2PO4 (3.7 mM), NaHCO3 (13.6 mM), MgCl2(H2O)6 (0.5 mM), and (NH4)2CO3 (0.06 mM); the final concentrations of each electrolyte solution in the stomach were KCl (6.9 mM), KH2PO4 (0.9 mM), NaHCO3 (25 mM), NaCl (47.2 mM), MgCl2(H2O)6 (0.12 mM), and (NH4)2CO3 (0.5 mM); and the final concentrations of each electrolyte solution in the intestine were KCl (6.8 mM), KH2PO4 (0.8 mM), NaHCO3 (85 mM), NaCl (38.4 mM), and MgCl2(H2O)6 (0.33 mM).

[0077] Step 3: Simulate oral chewing. Use an automatic meat grinder to simulate oral chewing, grinding the processed oysters into a paste. Weigh 5g of the meat paste and add 1mL of 5× oral electrolyte solution, 0.05mL of CaCl2(H2O)2 solution, and add water to a 1× concentration of simulated oral digestive solution. Mix well and digest at 37℃ for 2 minutes.

[0078] Step 4: Simulated gastric digestion. Take 10 mL of the oral phase digestion product, add 2 mL of gastric electrolyte solution, and then add hydrochloric acid. The amount of hydrochloric acid added is based on the total amount of hydrochloric acid determined in the preliminary experiment. First, add 20% of the estimated total amount of hydrochloric acid solution (concentration of 1M), then add 0.01 mL of CaCl2(H2O)2 solution to bring the final concentration of the simulated gastric digestion solution to 0.15 mM. Add the amount of water that was estimated to need to be added during the preliminary experiment. Record stable pH data. Next, add 1 mL of porcine pepsin solution to achieve an activity of 2000 U / mL. Add 1 mL of gastric lipase solution to achieve an activity of 60 U / mL. Start timing and immediately add 10% of the estimated total amount of hydrochloric acid solution (concentration of 1M). Then, add 10% of the estimated total amount of hydrochloric acid solution (concentration of 1M) every 15 minutes. After the acid addition is complete, digestion lasts for 30 minutes, at which point gastric digestion is finished. The gastric digestion time is 2.5 hours.

[0079] Step 5: Simulated intestinal digestion. Take 20 mL of gastric digested chyme, first add approximately 2 mL of 1M NaOH solution, then add 4 mL of 5× intestinal electrolyte solution, followed by the above-mentioned mixed solution of porcine pancreatic enzymes and bile salts, and finally add 0.01 mL of CaCl2(H2O)2 solution. Adjust the pH to 7.0 (±0.2) by adding 1M NaOH solution dropwise. The intestinal digestion time is 2 hours.

[0080] Note: The composition of the simulated digestive fluid is shown in Table 2 above.

[0081] Step Six: Detection of pesticide residues in undigested Pacific oysters. The Pacific oyster samples obtained in Step One were minced using a meat grinder. The samples were then processed using the QuEChERS rapid sample pretreatment technique, followed by extraction, purification, and concentration. Finally, the deltamethrin content in the Pacific oysters was detected by gas chromatography.

[0082] QuEChERS rapid sample pretreatment technique for extracting deltamethrin from oyster meat: Take 10g of oyster meat, add 5mL of water and mix well, add 15mL of 1% acetonitrile acetate for extraction and purification, then take 3mL and blow it dry with nitrogen at 45℃, and add 2mL of n-hexane to redissolve.

[0083] The gas chromatography detection conditions were as follows: HP-5 column (30m×320μm×0.25μm), injection port temperature 250℃, electron capture detector (ECD), detector temperature 300℃, programmed temperature increase to 100℃ and hold for 2 min, then increased to 270℃ at 6℃ / min and held for 10 min.

[0084] Step 7: Detection of pesticide residues in digested food.

[0085] QuEChERS rapid sample pretreatment technique for extracting deltamethrin from steamed oyster intestinal digest: Take 15 mL of intestinal digest, add 15 mL of 1% acetonitrile acetate for extraction and purification, then take 4 mL and blow dry under nitrogen at 45 °C, add 1.0 mL of n-hexane to redissolve, and concentrate 4 times.

[0086] The detection conditions are the same as those in step six, which are the same as those for gas chromatography.

[0087] Step 8: Calculate bioavailability after gastrointestinal digestion B: Calculate and evaluate bioavailability based on the results obtained in Steps 6 and 7.

[0088] Step Nine: Cell Absorption and Transport Assay. NCM460 cells were selected. First, cell viability was assessed by treating cells with 5, 10, 17.5, and 30 ng / mL deltamethrin for 24 hours, and the effect on cell viability was analyzed. Then, three treatment groups (2, 10, and 30 ng / mL) were used to conduct transmembrane transport experiments of deltamethrin using Transwell assays to obtain the transmembrane transport efficiency C of the pesticide.

[0089] Step 10: Calculate bioavailability. Calculate the product of the bioavailability B and the transmembrane transport efficiency C described in Step 7 to obtain the bioavailability A of pesticide residues in oysters after gastrointestinal digestion and permeation through monolayer cells.

[0090] Bioavailability A (%) of deltamethrin in Pacific oyster = deltamethrin content after permeation through cell membrane ÷ deltamethrin content in undigested Pacific oyster.

[0091] The bioavailability B of deltamethrin in oysters after gastrointestinal digestion, multiplied by the transmembrane transport efficiency C of deltamethrin, is the bioavailability A of deltamethrin residue in Pacific oysters after gastrointestinal digestion and cellular processes.

[0092] The bioavailability of deltamethrin in steamed oysters was calculated and is shown in Table 4.

[0093] Table 4. Bioavailability of deltamethrin in steamed oysters

[0094]

[0095] Example 3

[0096] This embodiment provides a method for evaluating the bioavailability of deltamethrin in roasted oysters, specifically including the following steps:

[0097] Step 1: Sample pretreatment. Wash the oysters with tap water in their shells. Use an oven to roast the oysters at 200℃ for 20 minutes. After roasting, remove the oysters, remove the shells, and mince the oyster meat into a paste for later use.

[0098] Step 2: Prepare the electrolyte concentrate. As shown in Table 1 above, the electrolyte concentrate contains KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, and (NH4)2CO3. The final concentrations of each electrolyte solution in the oral cavity were KCl (15.1 mM), KH2PO4 (3.7 mM), NaHCO3 (13.6 mM), MgCl2(H2O)6 (0.5 mM), and (NH4)2CO3 (0.06 mM); the final concentrations of each electrolyte solution in the stomach were KCl (6.9 mM), KH2PO4 (0.9 mM), NaHCO3 (25 mM), NaCl (47.2 mM), MgCl2(H2O)6 (0.12 mM), and (NH4)2CO3 (0.5 mM); and the final concentrations of each electrolyte solution in the intestine were KCl (6.8 mM), KH2PO4 (0.8 mM), NaHCO3 (85 mM), NaCl (38.4 mM), and MgCl2(H2O)6 (0.33 mM).

[0099] Step 3: Simulate oral chewing. Use an automatic meat grinder to simulate oral chewing, grinding the processed oysters into a paste. Weigh 5g of the meat paste and add 1mL of 5× oral electrolyte solution, 0.05mL of CaCl2(H2O)2 solution, and add water to a 1× concentration of simulated oral digestive solution. Mix well and digest at 37℃ for 2 minutes.

[0100] Step 4: Simulated gastric digestion. Take 10 mL of the oral phase digestion product, add 2 mL of gastric electrolyte solution, and then add hydrochloric acid. The amount of hydrochloric acid added is based on the total amount of hydrochloric acid determined in the preliminary experiment. First, add 20% of the estimated total amount of hydrochloric acid solution (concentration of 1M), then add 0.01 mL of CaCl2(H2O)2 solution to bring the final concentration of the simulated gastric digestion solution to 0.15 mM. Add the amount of water that was estimated to need to be added during the preliminary experiment. Record stable pH data. Next, add 1 mL of porcine pepsin solution to achieve an activity of 2000 U / mL. Add 1 mL of gastric lipase solution to achieve an activity of 60 U / mL. Start timing and immediately add 10% of the estimated total amount of hydrochloric acid solution (concentration of 1M). Then, add 10% of the estimated total amount of hydrochloric acid solution (concentration of 1M) every 15 minutes. After the acid addition is complete, digestion lasts for 30 minutes, at which point gastric digestion is finished. The gastric digestion time is 2.5 hours.

[0101] Step 5: Simulated Intestinal Digestion. Take 20 mL of gastric digested chyme, first add approximately 2 mL of 1M NaOH solution, then add 4 mL of 5× intestinal electrolyte solution, followed by the above-mentioned mixed solution of porcine pancreatic enzymes and bile salts, and finally add 0.01 mL of CaCl2(H2O)2 solution. Adjust the pH to 7.0 (±0.2) by adding 1M NaOH solution dropwise. The intestinal digestion time is 2 hours. Note: The composition of the simulated digestive fluid is shown in Table 2 above.

[0102] Step Six: Detection of pesticide residues in undigested Pacific oysters. The Pacific oyster samples obtained in Step One were minced using a meat grinder. The samples were then processed using the QuEChERS rapid sample pretreatment technique, followed by extraction, purification, and concentration. Finally, the deltamethrin content in the Pacific oysters was detected by gas chromatography.

[0103] QuEChERS rapid sample pretreatment technique for extracting deltamethrin from oyster meat: Take 10g of oyster meat, add 5mL of water and mix well, add 15mL of 1% acetonitrile acetate for extraction and purification, then take 3mL and blow it dry with nitrogen at 45℃, and add 2mL of n-hexane to redissolve.

[0104] The gas chromatography detection conditions were as follows: HP-5 column (30m×320μm×0.25μm), injection port temperature 250℃, electron capture detector (ECD), detector temperature 300℃, programmed temperature increase to 100℃ and hold for 2 min, then increased to 270℃ at 6℃ / min and held for 10 min.

[0105] Step 7: Detection of pesticide residues in digested food.

[0106] QuEChERS rapid sample pretreatment technique for extracting deltamethrin from the digested intestinal chyme of roasted oysters: Take 15 mL of intestinal digestion fluid, add 15 mL of 1% acetonitrile acetate for extraction and purification, then take 4 mL and blow it dry with nitrogen at 45 °C, add 1.0 mL of n-hexane to redissolve, and concentrate 4 times.

[0107] The detection conditions are the same as those in step six, which are the same as those for gas chromatography.

[0108] Step 8: Calculate bioavailability after gastrointestinal digestion B: Calculate and evaluate bioavailability based on the results obtained in Steps 6 and 7.

[0109] Step Nine: Cell Absorption and Transport Assay. NCM460 cells were selected. First, cell viability was assessed by treating cells with 5, 10, 17.5, and 30 ng / mL deltamethrin for 24 hours, and the effect on cell viability was analyzed. Then, three treatment groups (2, 10, and 30 ng / mL) were used to conduct transmembrane transport experiments of deltamethrin using Transwell assays to obtain the transmembrane transport efficiency C of the pesticide.

[0110] Step 10: Calculate bioavailability. Calculate the product of the bioavailability B and the transmembrane transport efficiency C described in Step 7 to obtain the bioavailability A of pesticide residues in oysters after gastrointestinal digestion and permeation through monolayer cells.

[0111] Bioavailability A (%) of deltamethrin in Pacific oyster = deltamethrin content after permeation through cell membrane ÷ deltamethrin content in undigested Pacific oyster.

[0112] The bioavailability B of deltamethrin in oysters after gastrointestinal digestion, multiplied by the transmembrane transport efficiency C of deltamethrin, is the bioavailability A of deltamethrin residue in Pacific oysters after gastrointestinal digestion and cellular processes.

[0113] The bioavailability of deltamethrin in roasted oysters was calculated and is shown in Table 5.

[0114] Table 5. Bioavailability of deltamethrin in roasted oysters

[0115]

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the biological availability of a pesticide residue in an aquatic product, characterized by, It comprises: 1) sample pretreatment: pretreating aquatic products to obtain aquatic product samples; 2) preparation of electrolyte solution: the electrolyte solution is selected from one or more of KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6 and (NH4)2CO3, and the electrolyte solution is divided into oral electrolyte solution, gastric phase electrolyte solution and intestinal phase electrolyte solution; 3) oral cavity chewing simulation: the aquatic product sample is ground into a paste to obtain meat paste; the meat paste is mixed with oral cavity phase simulated digestive juice, and is digested at 36-38℃ for 1-4 min to obtain oral cavity phase digestion product; The oral cavity simulation digestive juice comprises oral cavity electrolyte solution and salivary amylase; 4) gastric digestion simulation: the oral cavity phase digestion product is mixed with the gastric phase electrolyte solution, part of hydrochloric acid solution is added, CaCl2(H2O)2 solution, water, pepsin solution and gastric lipase solution are added, and then the remaining hydrochloric acid solution is added step by step, after the addition of acid is completed, the digestion is continued for 15-40 min to obtain gastric digestion meat paste; 5) intestinal digestion simulation: the gastric digestion meat paste is sequentially mixed with NaOH solution and the intestinal phase electrolyte solution, a mixed solution of porcine pancreatic enzyme and bile salt is added, CaCl2(H2O)2 solution is added, and NaOH solution is added dropwise to adjust the pH to 7.0±0.2, intestinal digestion is carried out, and digestion meat paste is obtained; 6) detection of pesticide residues in undigested aquatic products: the aquatic product sample in step 1) is ground and pretreated, and the pesticide residues are detected by chromatography; 7) detection of pesticide residues in digestion meat paste of digested aquatic products: the digestion meat paste in step 5) is subjected to pesticide residue detection according to the chromatography method; 8) calculation of bioavailability B after gastrointestinal digestion: the detection results obtained in steps 6)-7) are used for calculation to obtain the bioavailability B; 9) cell absorption and transport experiment: NCM460 cells are used for cell viability experiment to analyze the influence of pesticides on cell viability; then the transmembrane transport experiment of pesticides is carried out by Transwell, the ratio of the pesticide concentration in the lower chamber of NCM460 cell Transwell to the pesticide concentration in the upper chamber is calculated, and the transmembrane transport efficiency C of the pesticide is obtained; 10) calculation of bioavailability A of pesticide residues in aquatic products after gastrointestinal-cell digestion and absorption: A=B×C, wherein B is the bioavailability of pesticide residues after gastrointestinal digestion of aquatic products, and C is the transmembrane transport efficiency of pesticide residues in intestinal digestion meat paste.

2. The method for evaluating the pesticide residue bioaccessibility of aquatic products according to claim 1, characterized in that, The pH value of the simulated saliva used in the oral cavity chewing simulation is 7.0±0.2, and the digestion time is 0.5-3 min; the simulated gastric digestion uses stepwise addition of hydrochloric acid to the simulated gastric juice, and the digestion time is 1.5-3.5 h; the pH value of the simulated intestinal fluid used in the intestinal digestion simulation is 7.0±0.2, and the digestion time is 1.5-3 h.

3. The method for evaluating the pesticide residue bioaccessibility of aquatic products according to claim 1, characterized in that, In step 1), the pretreatment comprises cleaning and shelling.

4. The method for evaluating the pesticide residue bioaccessibility of aquatic products according to claim 3, characterized in that, In step 1), the pretreated aquatic product sample is also subjected to cooking.

5. The method for evaluating the bioavailability of pesticide residues in aquatic products according to claim 1, wherein The oral electrolyte solution comprises 14-16 mM KCl, 3-4 mM KH2PO4, 13-15 mM NaHCO3, 0.4-0.6 mM MgCl2(H2O)6, 0.04-0.08 mM (NH4)2CO3; and / or The gastric electrolyte solution comprises 6-8 mM KCl, 0.7-1.0 mM KH2PO4, 23-26 mM NaHCO3, 45-48 mM NaCl, 0.10-0.14 mM MgCl2(H2O)6, 0.4-0.6 mM (NH4)2CO3; and / or The intestinal electrolyte solution comprises 6-8 mM KCl, 0.7-0.9 mM KH2PO4, 80-90 mM NaHCO3, 36-40 mM NaCl, 0.3-0.4 mM MgCl2(H2O)6.

6. The method for evaluating the pesticide residue bioaccessibility of aquatic products according to claim 1, characterized in that, In the simulated digestion, the pepsin activity is 1800-2200 U / mL; the gastric lipase activity is 50-70 U / mL; and / or, the pancreatic enzyme activity is 90-110 U / mL; and the bile salt concentration is 8-12 mM.

7. The method for evaluating the pesticide residue bioaccessibility of aquatic products according to claim 1, characterized in that, In step 3), when the detected sample contains starch, salivary amylase is further added in the simulated oral digestion process.

8. The method for evaluating the pesticide residue bioaccessibility of aquatic products according to claim 1, characterized in that, In step 4), the total amount of hydrochloric acid added is the amount of hydrochloric acid required to reach pH 1.5-2.5; and / or, in step 5), the intestinal digestion is terminated by heating in a water bath at 95℃ for 5 min; and / or, in steps 3) to 5), the digestion process is carried out in a constant temperature environment at 37℃.

9. The method for evaluating the biological availability of a pesticide residue in an aquatic product according to any one of claims 1 to 8, characterized by, In step 9), a pesticide of a set concentration is added to the upper chamber of the Transwell; and / or, in step 7), the chromatography method comprises one of gas chromatography, liquid chromatography, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

10. The method for evaluating the pesticide residue bioaccessibility of an aquatic product according to claim 9, characterized by, The pyrethroid pesticide is detected by gas chromatography, and the detection conditions of the gas chromatography are as follows: a chromatographic column HP-5, 30 m x 320 μm x 0.25 μm, an injection port temperature of 250℃, an electron capture detector ECD, a detector temperature of 300℃, a programmed temperature of 100℃ for 2 min, then 6℃ / min to 270℃ for 10 min.

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