A rapid detection method of aflatoxin in edible oil

Abstract

The application relates to a rapid detection method of aflatoxin in edible oil, and belongs to the technical field of aflatoxin detection. The detection method comprises the steps of sample pretreatment and thin layer chromatography detection. The method can realize rapid detection of four kinds of aflatoxin in edible oil, is simple to operate, and is low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of aflatoxin detection and relates to a rapid detection method for aflatoxin in edible oil. Background Art

[0002] Grain and oil safety has always been a key concern in the video industry, with mycotoxins being a common source of contamination. Over 400 types of mycotoxins have been discovered, with aflatoxins garnering significant attention due to their high carcinogenicity. Oilseed crops are threatened by mycotoxin contamination at every stage of their production, from planting to edible oil processing. Peanuts and corn are particularly susceptible to aflatoxin contamination. my country's national food safety standard, "Limits of Mycotoxins in Food" (GB2761-2017), stipulates that the aflatoxin content in peanut oil and corn oil must not exceed 0.02 mg / kg.

[0003] Aflatoxins are metabolites of Aspergillus flavus and Aspergillus parasiticus. They are a class of chemically similar compounds. Several types have been isolated and identified, including B1, B2, G1, G2, M1, M2, P1, Q, H1, GM, and toxic alcohol. B1 is the most toxic and carcinogenic. Clinical trials have shown that aflatoxins can induce experimental liver cancer in experimental animals such as poultry, primates, and fish. The main manifestations of the disease include necrosis, cirrhosis, and hepatic hemorrhage. The harm caused by aflatoxins to organisms is directly related to their direct interaction with protein synthesis. The furan ring structure in aflatoxin is the most significant structure causing harm to humans.

[0004] At present, the detection methods of aflatoxin mainly include enzyme-linked immunosorbent assay, liquid chromatography, fluorescence spectrophotometry, etc. Summary of the Invention

[0005] The main purpose of the present invention is to provide a rapid detection method for aflatoxin, which is simple to operate and low in cost, can quickly detect aflatoxin in edible oil, and ensure the food safety of edible oil.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:

[0007] The invention discloses a rapid detection method for aflatoxin in edible oil, specifically comprising the step of determining aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 in the edible oil by thin layer chromatography.

[0008] Furthermore, the thin layer chromatography detection step is:

[0009] The edible oil test solution, aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 reference substances were spotted on the same silica gel G thin layer plate, and a methanol-ethyl acetate-dimethylformamide mixed solution was used as the developing agent. When the plate was developed upward to 1 / 3, the plate was taken out and air-dried. Then, a chloroform-acetone-methanol-acetic acid mixed solution was used as the developing agent, and the plate was developed upward. After the development was completed, the plate was taken out and air-dried, and 10% sulfuric acid ethanol solution was sprayed on the plate. The plate was heated at 105°C until the spots were clear and examined under visible light.

[0010] Furthermore, the volume ratio of methanol, ethyl acetate and dimethylformamide in the methanol-ethyl acetate-dimethylformamide mixed solution in the thin layer chromatography method is 3:8:0.5.

[0011] Furthermore, the volume ratio of chloroform, acetone, methanol and acetic acid in the chloroform-acetone-methanol-acetic acid mixed solution in the thin layer chromatography method is 6:2:1:0.1.

[0012] Furthermore, the preparation method of the edible oil test solution comprises the following steps:

[0013] Step 1, sample pretreatment: Take the edible oil to be tested, add a petroleum ether-n-hexane mixed solution, transfer to a separatory funnel, add a methanol-dimethylformamide-water mixed solution, shake, and then stand to separate the layers; transfer the upper layer solution to a second separatory funnel, add the methanol-dimethylformamide-water mixed solution again, shake, and then stand to separate the layers; repeat 2-3 times, combine the lower layer solutions after each separation, and concentrate under reduced pressure and evaporate to dryness to obtain the sample to be tested;

[0014] Step 2, preparation of the test solution: re-dissolve the sample obtained in step 1 in methanol to obtain the test solution.

[0015] Furthermore, the preparation method of the reference solution is:

[0016] Take aflatoxin B1, B2, G1, and G2 standards, dissolve them in methanol respectively, and prepare 10 ng / ml reference solution.

[0017] Furthermore, the volume ratio of petroleum ether to n-hexane in the petroleum ether-n-hexane mixed solution in step 1 is 8:2.

[0018] Furthermore, in the methanol-dimethylformamide-water mixed solution in step 1, the volume ratio of methanol, dimethylformamide and water is 6:0.1:4.

[0019] The present invention has the following beneficial effects:

[0020] The present invention uses thin-layer chromatography to simultaneously detect aflatoxins B1, B2, G1, and G2 in edible oils, effectively avoiding food safety issues caused by aflatoxins in edible oils. The present invention treats edible oil samples to effectively remove interfering components. In thin-layer chromatography, the detection method is optimized, and a fractional development method is used to develop the four aflatoxins, effectively separating them. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : The chromatogram in performance test 1, from left to right, is Example 1 test solution, Example 2 test solution, Example 3 test solution, mixed reference solution, mixed test solution 1, mixed test solution 2, and mixed test solution 3;

[0022] Figure 2 : The chromatogram in performance test 2, from left to right, is the test solution of comparative example 1, the mixed reference solution, and the mixed test solution;

[0023] Figure 3 : The chromatogram in performance test three, from left to right are the test solution of Comparative Example 2, the mixed reference solution, and the mixed test solution. DETAILED DESCRIPTION

[0024] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope of protection of the claims of this application.

[0025] Example 1 Detection of aflatoxin in corn oil

[0026] Step 1, corn oil sample pretreatment: take 50 g of edible corn oil to be tested, add 50 mL of petroleum ether-n-hexane (8:2, v / v) mixed solution, mix well, transfer to a separatory funnel, add 100 mL of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution, shake and let stand for stratification; transfer the upper layer solution to a second separatory funnel, add an equal volume of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution again, shake and let stand for stratification; repeat 2-3 times, combine the lower layer solutions after each stratification, concentrate under reduced pressure and evaporate to dryness to obtain the sample to be tested;

[0027] Step 2, preparation of the test solution: re-dissolve the sample obtained in step 1 in methanol to obtain the test solution.

[0028] Step 3, preparation of reference solution: take aflatoxin B1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B1 reference solution; take aflatoxin B2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B2 reference solution; take aflatoxin G1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G1 reference solution; take aflatoxin G2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G2 reference solution;

[0029] Step 4, thin layer chromatography detection: The prepared corn oil test solution and each reference solution were spotted on the same silica gel G thin layer plate, and a methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution was used as the developing solvent. When the plate was developed to 1 / 3, the plate was removed and air-dried. Then, a chloroform-acetone-methanol-acetic acid mixed solution (6:2:1:0.1, v / v / v) was used as the developing solvent, and the plate was developed upward. After development was completed, the plate was removed and air-dried. 10% sulfuric acid ethanol solution was sprayed on the plate, and the plate was heated at 105°C until the spots were clear. The plate was inspected under visible light.

[0030] Example 2 Detection of aflatoxin in peanut oil

[0031] Step 1, peanut oil sample pretreatment: take 50 g of edible peanut oil to be tested, add 50 mL of petroleum ether-n-hexane (8:2, v / v) mixed solution, mix well, transfer to a separatory funnel, add 100 mL of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution, shake and let stand for stratification; transfer the upper layer solution to a second separatory funnel, add an equal volume of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution again, shake and let stand for stratification; repeat 2-3 times, combine the lower layer solutions after each stratification, concentrate under reduced pressure and evaporate to dryness to obtain the sample to be tested;

[0032] Step 2, preparation of the test solution: re-dissolve the sample obtained in step 1 in methanol to obtain the test solution.

[0033] Step 3, preparation of reference solution: take aflatoxin B1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B1 reference solution; take aflatoxin B2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B2 reference solution; take aflatoxin G1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G1 reference solution; take aflatoxin G2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G2 reference solution;

[0034] Step 4, thin layer chromatography detection: The prepared peanut oil test solution and each reference solution were spotted on the same silica gel G thin layer plate, and a methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution was used as the developing solvent. When the plate was developed upward to 1 / 3, the plate was removed and air-dried. Then, a chloroform-acetone-methanol-acetic acid mixed solution (6:2:1:0.1, v / v / v) was used as the developing solvent, and the plate was developed upward. After the development was completed, the plate was removed and air-dried. 10% sulfuric acid ethanol solution was sprayed on the plate, and the plate was heated at 105°C until the spots were clear. The plate was inspected under visible light.

[0035] Example 3 Detection of aflatoxin in soybean oil

[0036] Step 1, soybean oil sample pretreatment: take 50 g of edible soybean oil to be tested, add 50 mL of petroleum ether-n-hexane (8:2, v / v) mixed solution, mix well, transfer to a separatory funnel, add 100 mL of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution, shake and let stand for stratification; transfer the upper layer solution to a second separatory funnel, add an equal volume of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution again, shake and let stand for stratification; repeat 2-3 times, combine the lower layer solutions after each stratification, concentrate under reduced pressure and evaporate to dryness to obtain the sample to be tested;

[0037] Step 2, preparation of the test solution: re-dissolve the sample obtained in step 1 in methanol to obtain the test solution.

[0038] Step 3, preparation of reference solution: take aflatoxin B1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B1 reference solution; take aflatoxin B2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B2 reference solution; take aflatoxin G1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G1 reference solution; take aflatoxin G2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G2 reference solution;

[0039] Step 4, thin layer chromatography detection: The prepared soybean oil test solution and each reference solution were spotted on the same silica gel G thin layer plate, and a methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution was used as the developing solvent. When the plate was developed to 1 / 3, the plate was removed and air-dried. Then, a chloroform-acetone-methanol-acetic acid mixed solution (6:2:1:0.1, v / v / v) was used as the developing solvent, and the plate was developed upward. After development was completed, the plate was removed and air-dried. 10% sulfuric acid ethanol solution was sprayed on the plate, and the plate was heated at 105°C until the spots were clear. The plate was inspected under visible light.

[0040] Comparative Example 1

[0041] Step 1, corn oil sample pretreatment: take 50 g of edible corn oil to be tested, add 50 mL of petroleum ether-n-hexane (8:2, v / v) mixed solution, mix well, transfer to a separatory funnel, add 100 mL of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution, shake and let stand for stratification; transfer the upper layer solution to a second separatory funnel, add an equal volume of methanol-dimethylformamide-water (6:0.1:4, v / v / v) mixed solution again, shake and let stand for stratification; repeat 2-3 times, combine the lower layer solutions after each stratification, concentrate under reduced pressure and evaporate to dryness to obtain the sample to be tested;

[0042] Step 2, preparation of the test solution: re-dissolve the sample obtained in step 1 in methanol to obtain the test solution.

[0043] Step 3, preparation of reference solution: take aflatoxin B1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B1 reference solution; take aflatoxin B2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B2 reference solution; take aflatoxin G1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G1 reference solution; take aflatoxin G2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G2 reference solution;

[0044] Step 4, thin layer chromatography: Spot the prepared corn oil test solution and each reference solution on the same silica gel G thin layer plate, use chloroform-ethyl acetate-dimethylformamide (5:5:0.5, v / v / v) mixed solution as the developing solvent. After development, take out and dry, spray 10% sulfuric acid ethanol solution on the thin layer plate, heat at 105°C until the spots are clear, and inspect under visible light.

[0045] Comparative Example 2

[0046] Step 1, corn oil sample pretreatment: take 50 g of corn oil to be tested, add 20 mL of petroleum ether, mix well, add 20 mL of methanol-water (7:3, v / v) mixed solution, ultrasonicate for 5 min, let stand, after separation, aspirate the lower layer solution, which is the test solution;

[0047] Step 2, preparation of reference solution: take aflatoxin B1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B1 reference solution; take aflatoxin B2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin B2 reference solution; take aflatoxin G1 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G1 reference solution; take aflatoxin G2 standard, dissolve it in methanol, and prepare a 100 ng / ml aflatoxin G2 reference solution;

[0048] Step 3, thin layer chromatography detection: The prepared corn oil test solution and each reference solution were spotted on the same silica gel G thin layer plate, and a methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution was used as the developing solvent. When the plate was developed upward to 1 / 3, the plate was removed and air-dried. Then, a chloroform-acetone-methanol-acetic acid mixed solution (6:2:1:0.1, v / v / v) was used as the developing solvent, and the plate was developed upward. After development was completed, the plate was removed and air-dried. 10% sulfuric acid ethanol solution was sprayed on the plate, and the plate was heated at 105°C until the spots were clear. The plate was inspected under visible light.

[0049] Performance Test 1

[0050] First, 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution prepared in each of the above embodiments was placed in a 10 mL volumetric flask, and methanol was added to the volume to obtain a mixed reference solution.

[0051] Secondly, 1 mL of the test solution of Example 1 and 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution were placed in a 10 mL volumetric flask, and methanol was added to the volume to obtain a mixed test solution 1; 1 mL of the test solution of Example 2 and 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution were placed in a 10 mL volumetric flask, and methanol was added to the volume to obtain a mixed test solution 2; 1 mL of the test solution of Example 2 and 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution were placed in a 10 mL volumetric flask, and methanol was added to the volume to obtain a mixed test solution 2;

[0052] For detection, the test solution, each mixed test solution, and mixed reference solution obtained in Examples 1 to 3 were spotted on the same silica gel G thin layer plate and detected by thin layer chromatography in the following manner:

[0053] Use methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution as developing agent, develop upward to 1 / 3, take out the thin layer plate and dry it; then use chloroform-acetone-methanol-acetic acid (6:2:1:0.1, v / v / v) mixed solution as developing agent, develop upward, after development is complete, take out and dry it, spray 10% sulfuric acid ethanol solution on the thin layer plate, heat at 105℃ until the spots are clear, and examine under visible light. Figure 1 .

[0054] Results and analysis:

[0055] since Figure 1 It can be seen that aflatoxin was not detected in the test solutions in Examples 1 to 3, while the mixed test solutions and mixed reference solutions to which aflatoxin was added had better aflatoxin separation and could show clear spots.

[0056] Performance Test 2

[0057] First, 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution prepared in each of the above embodiments was placed in a 10 mL volumetric flask, and methanol was added to the volume to obtain a mixed reference solution.

[0058] Next, take 1 mL of the test solution of Comparative Example 1 and 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution and place them in a 10 mL volumetric flask. Add methanol to the volume to obtain a mixed test solution.

[0059] Detection: The test solution, mixed test solution, and mixed reference solution obtained in Comparative Example 1 were spotted on the same silica gel G thin layer plate and thin layer chromatography was performed in the same manner as described in Comparative Example 1:

[0060] Use methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution as developing agent, develop upward to 1 / 3, take out the thin layer plate and dry it; then use chloroform-acetone-methanol-acetic acid (6:2:1:0.1, v / v / v) mixed solution as developing agent, develop upward, after development is complete, take out and dry it, spray 10% sulfuric acid ethanol solution on the thin layer plate, heat at 105℃ until the spots are clear, and examine under visible light. Figure 2 .

[0061] Results and analysis:

[0062] since Figure 2 It can be seen that according to the thin layer detection method described in Comparative Example 1, aflatoxin cannot be effectively separated, the separation degree is poor, and certain components in the test solution will interfere with the detection results.

[0063] Performance Test 3

[0064] First, 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution prepared in each of the above embodiments was placed in a 10 mL volumetric flask, and methanol was added to the volume to obtain a mixed reference solution.

[0065] Next, take 1 mL of the test solution of Comparative Example 2 and 1 mL of the aflatoxin B1, B2, G1, and G2 reference solution and place them in a 10 mL volumetric flask. Add methanol to the volume to obtain a mixed test solution.

[0066] Detection: The test solution, mixed test solution, and mixed reference solution obtained in Comparative Example 2 were spotted on the same silica gel G thin layer plate and thin layer chromatography was performed in the same manner as described in Comparative Example 2:

[0067] Use methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) mixed solution as developing agent, develop upward to 1 / 3, take out the thin layer plate and dry it; then use chloroform-acetone-methanol-acetic acid (6:2:1:0.1, v / v / v) mixed solution as developing agent, develop upward, after development is complete, take out and dry it, spray 10% sulfuric acid ethanol solution on the thin layer plate, heat at 105℃ until the spots are clear, and examine under visible light. Figure 3 .

[0068] Results and analysis:

[0069] since Figure 3 It can be seen that aflatoxin should not be detected in the sample in Comparative Example 2. Certain components in the test solution will interfere with the detection of aflatoxin. There are chromatographic peaks of other components at the aflatoxin peak position, which interferes with the test results.

Claims

1. A rapid detection method for aflatoxin in edible oil, characterized in that: The rapid detection method is to use thin layer chromatography to determine aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 in edible oil; The thin layer chromatography detection steps are: The edible oil test solution, aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 reference solution were spotted separately on the same silica gel G thin-layer plate. A mixed solution of methanol-ethyl acetate-dimethylformamide (volume ratio) of 3:8:0.5 was used as a developing solvent. When the plate was developed to 1 / 3 of the plate, the plate was removed and air-dried. The plate was then developed upward using a mixed solution of chloroform-acetone-methanol-acetic acid (volume ratio) of 6:2:1:0.

1. After development was complete, the plate was removed and air-dried. 10% sulfuric acid ethanol solution was sprayed on the plate, and the plate was heated at 105°C until the spots were clear. The plate was then examined under visible light. The preparation method of the edible oil test solution comprises the following steps: Step 1, sample pretreatment: Take the edible oil to be tested, add a mixed solution of petroleum ether and n-hexane in a volume ratio of 8:2, transfer to a separatory funnel, add a mixed solution of methanol, dimethylformamide, and water in a volume ratio of 6:0.1:4, shake, and allow to stand for separation. Store the lower layer in a separate container and keep the upper layer for later use; Step 2: Repeat step 1 for 2-3 times with the upper layer solution, combine the lower layer solutions after each layer separation, concentrate under reduced pressure and evaporate to dryness to obtain the sample to be tested; Step 3, preparation of test solution: re-dissolve the sample obtained in step 2 in methanol to obtain the test solution.

2. The rapid detection method according to claim 1, wherein The preparation method of the reference solution is: Take aflatoxin B1, B2, G1, and G2 standards, dissolve them in methanol respectively, and prepare 10 ng / ml reference solution.

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