Rapid detection method for aflatoxin in edible oil

Abstract

The invention relates to a method for rapidly detecting aflatoxin in edible oil, and belongs to the technical field of aflatoxin detection. The detection method comprises the steps of sample pretreatment, thin-layer chromatography detection and the like. The method provided by the invention can realize rapid detection of four aflatoxins in edible oil, and has the advantages of simple operation and low cost.

Description

Technical Field

[0001] The present 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 an important issue focused on in the food field. Among them, mycotoxins are relatively common factors for grain and oil pollution. More than 400 kinds of mycotoxins have been discovered so far. Among them, aflatoxin has become the focus of people's attention due to its high carcinogenicity. All links from the planting of oil crops to the processing of edible oil are threatened by mycotoxin pollution. Among them, the proportion of peanut and corn contaminated by aflatoxin is particularly high. The national food safety standard of China, "Limits of Mycotoxins in Foods" (GB2761-2017), clearly stipulates that the content of aflatoxin in peanut oil and corn oil shall not exceed 0.02 mg / kg.

[0003] Aflatoxin is a metabolite of Aspergillus flavus and Aspergillus parasiticus. It is a class of compounds with similar chemical structures. More than a dozen varieties such as B1, B2, G1, G2, M1, M2, P1, Q, H1, GM, and toxicol have been isolated and identified. Among them, B1 is the most toxic and carcinogenic substance. In clinical trials, it has been found that aflatoxin can cause experimental animals such as poultry, primates, and fish to develop experimental liver cancer, and the main disease manifestations are necrosis, liver cirrhosis, liver hemorrhage and other lesions. The harm caused by aflatoxin to organisms is directly related to the direct organization of protein synthesis by aflatoxin. The furan ring structure in aflatoxin is the most important structure that causes harm to the human body.

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

[0005] The main purpose of the present invention is to provide a rapid detection method for aflatoxin. This method is simple to operate, 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 purpose:

[0007] A rapid detection method for aflatoxin in edible oil, specifically using thin-layer chromatography to determine aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 in edible oil.

[0008] Further, the detection steps of the thin-layer chromatography are as follows:

[0009] Spot the test sample solution of edible oil, and reference substances of aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 on the same silica gel G thin-layer plate respectively. Use a mixed solution of methanol - ethyl acetate - dimethylformamide as the developing agent, and develop upward. When it reaches 1 / 3, take out the thin-layer plate and let it dry. Then use a mixed solution of chloroform - acetone - methanol - acetic acid as the developing agent and develop upward again. After the development is completed, take it out and let it dry. Spray a 10% sulfuric acid - ethanol solution on the thin-layer plate, heat it at 105°C until the spots are clear, and examine it under visible light.

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

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

[0012] Further, the preparation method of the test sample solution of edible oil includes the following steps:

[0013] Step 1, sample pretreatment: Take the edible oil to be tested, add a mixed solution of petroleum ether - n-hexane, transfer it to a separatory funnel, add a mixed solution of methanol - dimethylformamide - water, shake it, and let it stand for stratification; transfer the upper layer solution to a second separatory funnel, add the mixed solution of methanol - dimethylformamide - water again, shake it, and let it stand for stratification; repeat 2 - 3 times, combine the lower layer solutions after each stratification, concentrate it under reduced pressure until it is evaporated to dryness to obtain the test sample;

[0014] Step 2, preparation of the test sample solution: Redissolve the test sample obtained in Step 1 with methanol to obtain the test sample solution.

[0015] Further, the preparation method of the reference substance solution is as follows:

[0016] Take the standard substances of aflatoxin B1, B2, G1, and G2, dissolve them with methanol respectively, and prepare a reference substance solution with a concentration of 10 ng / ml.

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

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

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

[0020] In the present invention, thin-layer chromatography is used to simultaneously detect aflatoxins B1, B2, G1, and G2 in edible oil. Four aflatoxins can be detected simultaneously, effectively avoiding the food safety problems caused by aflatoxins in edible oil. In the present invention, the edible oil sample is processed to effectively remove interfering components. In thin-layer detection, the detection method is optimized. The four aflatoxins are developed by the method of developing in batches, and effective separation of the four aflatoxins can be achieved. Description of the Drawings

[0021] Figure 1 : Chromatogram in Performance Test 1. From left to right, they are the test solution of Example 1, the test solution of Example 2, the test solution of Example 3, the mixed reference solution, the first mixed test solution, the second mixed test solution, and the third mixed test solution;

[0022] Figure 2 : Chromatogram in Performance Test 2. From left to right, they are the test solution of Comparative Example 1, the mixed reference solution, and the mixed test solution;

[0023] Figure 3 : Chromatogram in Performance Test 3. From left to right, they are the test solution of Comparative Example 2, the mixed reference solution, and the mixed test solution. Detailed Embodiments

[0024] The present invention will be further clarified below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope protected by the claims of this application.

[0025] Detection of Aflatoxins in Corn Oil in Example 1

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

[0027] Step 2, preparation of the test solution: The sample to be tested obtained in Step 1 is redissolved with methanol to obtain the test solution.

[0028] Step 3, Preparation of reference substance solutions: Take aflatoxin B1 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin B1 reference substance solution; take aflatoxin B2 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin B2 reference substance solution; take aflatoxin G1 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin G1 reference substance solution; take aflatoxin G2 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin G2 reference substance solution;

[0029] Step 4, Thin-layer chromatography detection: Spot the prepared corn oil test sample solution and each reference substance solution on the same silica gel G thin-layer plate respectively. Use a mixed solution of methanol - ethyl acetate - dimethylformamide (3:8:0.5, v / v / v) as the developing agent, and develop upward. When it reaches 1 / 3, take out the thin-layer plate and dry it in the air; then use a mixed solution of chloroform - acetone - methanol - acetic acid (6:2:1:0.1, v / v / v) as the developing agent, develop upward. After the development is completed, take out and dry it. Spray 10% sulfuric acid ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine it under visible light.

[0030] Example 2 Detection of Aflatoxin in Peanut Oil

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

[0032] Step 2, Preparation of test sample solution: Redissolve the test sample obtained in Step 1 with methanol to obtain the test sample solution.

[0033] Step 3, Preparation of reference substance solutions: Take aflatoxin B1 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin B1 reference substance solution; take aflatoxin B2 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin B2 reference substance solution; take aflatoxin G1 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin G1 reference substance solution; take aflatoxin G2 reference substance, dissolve it in methanol respectively to prepare a 100 ng / ml aflatoxin G2 reference substance solution;

[0034] Step 4, Thin-layer chromatography detection: Spot the prepared peanut oil test solution and each reference solution on the same silica gel G thin-layer plate respectively. Use the mixed solution of methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) as the developing agent. When the development reaches 1 / 3, take out the thin-layer plate and dry it. Then use the mixed solution of chloroform-acetone-methanol-acetic acid (6:2:1:0.1, v / v / v) as the developing agent and develop upward. After the development is completed, take out and dry it. Spray 10% sulfuric acid ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine under visible light.

[0035] Example 3 Detection of Aflatoxin in Soybean Oil

[0036] Step 1, Pretreatment of soybean oil sample: Take 50 g of the to-be-detected edible soybean oil, add 50 mL of the mixed solution of petroleum ether-n-hexane (8:2, v / v), mix well and transfer it to a separatory funnel. Add 100 mL of the mixed solution of methanol-dimethylformamide-water (6:0.1:4, v / v / v), shake and let it stand for stratification. Transfer the upper layer solution to the second separatory funnel, add the same volume of the mixed solution of methanol-dimethylformamide-water (6:0.1:4, v / v / v) again, shake and let it 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 to-be-detected sample.

[0037] Step 2, Preparation of test solution: Redissolve the to-be-detected sample obtained in Step 1 with methanol to obtain the test solution.

[0038] Step 3, Preparation of reference solutions: Take aflatoxin B1 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin B1 reference solution; take aflatoxin B2 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin B2 reference solution; take aflatoxin G1 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin G1 reference solution; take aflatoxin G2 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin G2 reference solution.

[0039] Step 4, Thin-layer chromatography detection: Spot the prepared soybean oil test solution and each reference solution on the same silica gel G thin-layer plate respectively. Use the mixed solution of methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) as the developing agent. When the development reaches 1 / 3, take out the thin-layer plate and dry it. Then use the mixed solution of chloroform-acetone-methanol-acetic acid (6:2:1:0.1, v / v / v) as the developing agent and develop upward. After the development is completed, take out and dry it. Spray 10% sulfuric acid ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine under visible light.

[0040] Comparative Example 1

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

[0042] Step 2, preparation of test solution: Re - dissolve the sample to be tested obtained in Step 1 with methanol to obtain the test solution.

[0043] Step 3, preparation of reference solution: Take aflatoxin B1 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin B1 reference solution; Take aflatoxin B2 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin B2 reference solution; Take aflatoxin G1 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin G1 reference solution; Take aflatoxin G2 reference standard, dissolve it with methanol respectively to prepare a 100 ng / ml aflatoxin G2 reference solution;

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

[0045] Comparative Example 2

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

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

[0048] Step 3, Thin-layer chromatography detection: Spot the prepared corn oil test solution and each reference solution on the same silica gel G thin-layer plate respectively. Use a mixed solution of methanol-ethyl acetate-dimethylformamide (3:8:0.5, v / v / v) as the developing agent, and develop upward. When it reaches 1 / 3, take out the thin-layer plate and dry it; then use a mixed solution of chloroform-acetone-methanol-acetic acid (6:2:1:0.1, v / v / v) as the developing agent, develop upward. After the development is completed, take out and dry it. Spray 10% sulfuric acid ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine it under visible light.

[0049] Performance test 1

[0050] First, take 1 mL of each of the aflatoxin B1, B2, G1, and G2 reference solutions prepared in the above embodiments and place them in a 10 mL volumetric flask, and make up the volume with methanol to obtain a mixed reference solution;

[0051] Second, take 1 mL of the test solution of Example 1, 1 mL of aflatoxin B1, B2, G1, and G2 reference solutions and place them in a 10 mL volumetric flask, and make up the volume with methanol to obtain a mixed test solution 1; take 1 mL of the test solution of Example 2, 1 mL of aflatoxin B1, B2, G1, and G2 reference solutions and place them in a 10 mL volumetric flask, and make up the volume with methanol to obtain a mixed test solution 2; take 1 mL of the test solution of Example 2, 1 mL of aflatoxin B1, B2, G1, and G2 reference solutions and place them in a 10 mL volumetric flask, and make up the volume with methanol to obtain a mixed test solution 2;

[0052] Detection: Spot the test solutions, each mixed test solution, and the mixed reference solution obtained in Examples 1-3 on the same silica gel G thin-layer plate, and perform thin-layer chromatography detection in the following manner:

[0053] Using a mixed solution of methanol - ethyl acetate - dimethylformamide (3:8:0.5, v / v / v) as the developing agent, perform ascending development. When it reaches 1 / 3, take out the thin-layer plate and let it dry; then use a mixed solution of chloroform - acetone - methanol - acetic acid (6:2:1:0.1, v / v / v) as the developing agent for ascending development. After the development is completed, take out and let it dry. Spray a 10% sulfuric acid - ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine it under visible light. The results are shown in Figure 1 。

[0054] Results and analysis:

[0055] From Figure 1 it can be seen that in Examples 1 - 3, aflatoxins were not detected in the test solution. For each mixed test solution and the mixed reference solution added with aflatoxins, the separation degree of aflatoxins was good, and clear spots could be shown.

[0056] Performance Test Two

[0057] First, take 1 mL of the reference solutions of aflatoxin B1, B2, G1, and G2 prepared in each of the above embodiments and place them in a 10 mL volumetric flask, and make up the volume with methanol to obtain a mixed reference solution;

[0058] Secondly, take 1 mL of the test solution of Comparative Example 1 and 1 mL of the reference solutions of aflatoxin B1, B2, G1, and G2 and place them in a 10 mL volumetric flask, and make up the volume with methanol to obtain a mixed test solution;

[0059] Detection: Spot the test solution, the mixed test solution, and the mixed reference solution obtained in Comparative Example 1 on the same silica gel G thin-layer plate, and perform thin-layer chromatography detection in the manner described in Comparative Example 1:

[0060] Using a mixed solution of methanol - ethyl acetate - dimethylformamide (3:8:0.5, v / v / v) as the developing agent, perform ascending development. When it reaches 1 / 3, take out the thin-layer plate and let it dry; then use a mixed solution of chloroform - acetone - methanol - acetic acid (6:2:1:0.1, v / v / v) as the developing agent for ascending development. After the development is completed, take out and let it dry. Spray a 10% sulfuric acid - ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine it under visible light. The results are shown in Figure 2 。

[0061] Results and analysis:

[0062] From Figure 2 it can be known that according to the thin-layer detection method described in Comparative Example 1, aflatoxins cannot be effectively separated, the separation degree is poor, and some components in the test solution will interfere with the detection results.

[0063] Performance Test Three

[0064] First, take 1 mL of the aflatoxin B1, B2, G1, and G2 reference substance solutions prepared in each of the above embodiments respectively and place them in a 10 mL volumetric flask. Add methanol to make up the volume to obtain a mixed reference substance solution.

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

[0066] Detection: Spot the test substance solution, the mixed solution to be tested, and the mixed reference substance solution obtained in Comparative Example 2 on the same silica gel G thin layer plate, and perform thin layer chromatography detection in the manner described in Comparative Example 2:

[0067] Use a mixed solution of methanol - ethyl acetate - dimethylformamide (3:8:0.5, v / v / v) as the developing agent. When the development reaches 1 / 3, take out the thin layer plate and dry it. Then use a mixed solution of chloroform - acetone - methanol - acetic acid (6:2:1:0.1, v / v / v) as the developing agent and perform upward development. After the development is completed, take out and dry it. Spray 10% sulfuric acid ethanol solution on the thin layer plate, heat it at 105 °C until the spots are clear, and examine under visible light. The results are shown in Figure 3 .

[0068] Results and analysis:

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

Claims

1. A rapid detection method for aflatoxin in edible oil, characterized in that, The rapid detection method is to determine aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 in edible oil by thin-layer chromatography.

2. The rapid detection method according to claim 1, characterized in that, The detection steps of the thin-layer chromatography method are as follows: Spot the test solution of the edible oil sample, and the reference substances of aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 on the same silica gel G thin-layer plate respectively. Use a mixed solution of methanol-ethyl acetate-dimethylformamide as the developing agent, and develop upward. When it reaches 1 / 3, take out the thin-layer plate and dry it in the air; then use a mixed solution of chloroform-acetone-methanol-acetic acid as the developing agent and develop upward. After the development is completed, take it out and dry it. Spray 10% sulfuric acid ethanol solution on the thin-layer plate, heat it at 105 °C until the spots are clear, and examine it under visible light.

3. The rapid detection method according to claim 2, wherein In the methanol-ethyl acetate-dimethylformamide mixed solution in the thin-layer chromatography method, the volume ratio of methanol, ethyl acetate, and dimethylformamide is 3:8:0.

5.

4. The rapid detection method according to claim 2, wherein, In the chloroform-acetone-methanol-acetic acid mixed solution in the thin-layer chromatography method, the volume ratio of chloroform, acetone, methanol, and acetic acid is 6:2:1:0.

1.

5. The rapid detection method according to claim 2, wherein, The preparation method of the test solution of the edible oil sample includes the following steps: Step 1, sample pretreatment: Take the edible oil to be tested, add a mixed solution of petroleum ether-n-hexane, transfer it to a separating funnel, add a mixed solution of methanol-dimethylformamide-water, shake it, and let it stand for stratification; transfer the upper layer solution to a second separating funnel, add a mixed solution of methanol-dimethylformamide-water again, shake it, and let it stand for stratification; repeat 2-3 times, combine the lower layer solutions after each stratification, concentrate it under reduced pressure and evaporate to dryness to obtain the sample to be tested; Step 2, preparation of the test solution: Re-dissolve the sample to be tested obtained in Step 1 with methanol to obtain the test solution.

6. The rapid detection method according to claim 5, characterized in that, In the petroleum ether-n-hexane mixed solution in Step 1, the volume ratio of petroleum ether to n-hexane is 8:

2.

7. The rapid detection method according to claim 5, characterized in that, In the methanol-dimethylformamide-water mixed solution in Step 1, the volume ratio of methanol, dimethylformamide, and water is 6:0.1:

4.

8. The rapid detection method according to claim 2, characterized in that, The preparation method of the reference solution is as follows: Take the standard substances of aflatoxin B1, B2, G1, and G2, dissolve them with methanol respectively, and prepare a reference solution with a concentration of 10 ng / ml.

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