A method for rapid detection of aflatoxin in peanuts and products thereof

Abstract

This invention relates to a rapid detection method for aflatoxins in peanuts and their products, belonging to the field of food inspection and testing technology. The rapid detection method of this invention mainly utilizes thin-layer chromatography to detect aflatoxins B1, B2, and G1 in peanuts or their products. Using the method described in this invention, rapid detection of these three aflatoxins in peanuts or their products can be achieved with high accuracy and low cost.

Description

Technical Field

[0001] This invention belongs to the field of food inspection and testing technology, and relates to a rapid method for detecting aflatoxin in peanuts and their products. Background Technology

[0002] Peanuts hold a vital position in the world's grain and oilseed food industry, serving as a major oilseed and cash crop with a wide cultivation range. In recent years, with the increasing pursuit of nutritional health, the health benefits of peanuts have been continuously discovered and appreciated. Studies have shown that their rich unsaturated fatty acids have cholesterol-lowering effects, and their abundant resveratrol has anti-tumor, anti-aging, and cardiovascular disease prevention effects. The red skin of peanuts has blood-tonifying and hemostatic functions. Furthermore, peanuts also possess nutritional value such as lung-nourishing, stomach-strengthening, and memory-enhancing properties, making them a truly natural health food. As a nutrient-rich crop, peanuts are rich in protein, unsaturated fatty acids, trace elements, and substances with anti-aging effects, making them very popular in my country. Consumption is particularly high during the peanut harvest season and holidays such as New Year's Day and the Spring Festival, making peanuts a staple snack in many Chinese households. However, as a crop, peanuts are highly susceptible to contamination by fungal toxins. Among known fungal toxins, aflatoxin exhibits the most significant toxicity to peanuts and their processed products.

[0003] Aflatoxin is produced by the putrefactive fungus Aspergillus flavus and is easily contaminated in grains, oils, processed foods, and even animal feed. It is highly toxic and carcinogenic, and its effects are difficult to detect, posing a significant safety hazard. Aflatoxins are a group of complex structures with difuran rings and oxanaphenones, and 17 polymorphic compounds, including B1, B2, G1, G2, M1, and P1, have been identified.

[0004] Current technologies for aflatoxin detection mainly include high-performance liquid chromatography (HPLC) and immunoassay. However, these methods require cumbersome pretreatment processes such as extraction and immunoaffinity column purification, and the instruments used often cost hundreds of thousands or even millions of dollars, making them unsuitable for rapid detection. Immunoassay is suitable for in-house testing by companies or for initial screening of large batches of samples by regulatory authorities. However, its biggest drawback is the susceptibility to false positives due to complex sample matrices. Once a positive result is detected, instrumental confirmation is still required. Summary of the Invention

[0005] The main objective of this invention is to provide a rapid detection method for aflatoxin in peanuts and their products. This method is simple to operate, low in cost, and highly accurate, making it suitable for rapid screening.

[0006] The present invention employs the following technical solutions to achieve the above objectives: A rapid method for detecting aflatoxins in peanuts and their products mainly involves thin-layer chromatography (TLC) to detect aflatoxins B1, B2, and G1. The TLC method primarily includes the following steps: Peanut or its product test solution and aflatoxin reference solution were spotted separately on the same silica gel G thin-layer plate. Methanol-dimethylformamide was used as the developing solvent, and the plate was developed upwards. The plate was then removed and dried. Methanol-ethyl acetate-chloroform was used as the developing solvent, and the plate was developed in the reverse direction to 2 / 3 of the plate. The plate was then removed, dried, and heated with 10% sulfuric acid ethanol solution as the colorimetric reagent until the spots were clear. The plates were then examined under visible light.

[0007] Specifically, the thin-layer chromatography operation method is as follows: Peanut or its product test solution and aflatoxin reference solution were spotted separately onto the same silica gel G thin-layer plate. Methanol-dimethylformamide (9:1, v / v) was used as the developing solvent, and the plate was developed upwards to 1.0 cm from the top. The plate was then removed and air-dried. Then, methanol-ethyl acetate-chloroform (0.5:4:5.5, v / v / v) was used as the developing solvent, and the plate was developed in reverse to 2 / 3 of the distance from the other end. The plate was removed, air-dried, and sprayed with 10% sulfuric acid ethanol solution. The plate was heated at 105°C until the spots were clear and examined under visible light.

[0008] The preparation method of the peanut or its product test solution described above includes the following steps: Step 1, Extraction: Take peanuts or their products, add methanol-water mixed solution for ultrasonic extraction, then add petroleum ether-n-hexane mixed solution for continued ultrasonic treatment, filter, let the extract stand to separate into layers, and take the lower layer solution I; Step 2, purification: Add chloroform-dimethylformamide mixed solution to the lower layer solution I, shake and let stand to separate the layers, take the lower layer solution II, remove the organic solvent, evaporate to dryness, and redissolve in methanol to obtain the peanut butter test solution.

[0009] Specifically, In step 1, the volume ratio of methanol to water is 80:20; the volume ratio of petroleum ether to n-hexane is 70:30.

[0010] In step 2, the volume ratio of chloroform to dimethylformamide is 70:10.

[0011] The aflatoxin reference solution mentioned above is a mixed reference solution of aflatoxin B1, B2, and G1; The mixed reference solution is prepared by dissolving aflatoxin B1, B2, and G1 standards in methanol to prepare a 10 ng / ml mixed reference solution.

[0012] The present invention has the following beneficial effects: Peanuts and their products are rich in oils and proteins, resulting in a complex detection matrix. Furthermore, aflatoxin is a fat-soluble component, and the oils in peanuts can interfere with its extraction and purification, leading to unstable recovery rates and poor reproducibility. Additionally, other fat-soluble components in peanuts can be co-extracted with aflatoxin, further interfering with detection. This invention optimizes the extraction and detection conditions for aflatoxin in peanuts and their products. In sample processing, an optimized extraction solvent and method are used to effectively remove interfering components, eliminate matrix effects, and improve recovery rates. In thin-layer chromatography, an optimized developing solvent and method are employed, enabling thorough separation of the four aflatoxins in peanuts and their products with good separation and no mutual interference.

[0013] The method for detecting aflatoxin in peanuts or their products provided by this invention can meet the needs of rapid on-site screening, is simple to operate, has low cost, and can also ensure the accuracy of detection. Attached Figure Description

[0014] Figure 1 Thin-layer chromatography results 1, from left to right: peanut butter test sample of Comparative Example 1, mixed reference standard, peanut butter test sample of Example 3, peanut butter test sample of Example 1, and peanut kernel test sample of Example 2; Figure 2 Thin-layer chromatography result 2, from left to right: peanut butter test sample and mixed reference sample 2 (comparative example 2). Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0016] Example 1 Step 1, Extraction: Weigh 20g of a commercially available brand of peanut butter and place it in a 250ml stoppered conical flask. Add 100ml of methanol-water (80:20, v / v) mixed solution and sonicate for 10-15 minutes. Then add 100ml of petroleum ether-n-hexane (70:30, v / v) mixed solution and continue sonicating for 10-15 minutes. Filter the solution and transfer it to a separatory funnel. Allow it to stand and separate into layers. Take the lower layer solution I. Step 2, purification: Add chloroform-dimethylformamide (70:10, v / v) mixed solution to the lower layer solution I, shake and let stand to separate the layers, take the lower layer solution II, remove the organic solvent under reduced pressure, evaporate to dryness, and redissolve in methanol to obtain the peanut butter test solution; Step 3, Preparation of reference solution: Take aflatoxin B1, B2 and G1 standards, dissolve them in methanol, and prepare a mixed reference solution of 10 ng / ml; Step 4, Thin-layer chromatography detection: Spot the peanut butter test solution and the mixed reference solution separately onto the same silica gel G thin-layer plate. Use methanol-dimethylformamide (9:1, v / v) as the developing solvent and develop upward to 1.0 cm from the top. Then remove and air dry. Then use methanol-ethyl acetate-chloroform (0.5:4:5.5, v / v / v) as the developing solvent and develop the thin-layer plate in reverse to 2 / 3 of the distance from the other end. Remove, air dry, spray 10% sulfuric acid ethanol solution onto the thin-layer plate, heat at 105℃ until the spots are clear, and examine under visible light.

[0017] Example 2 Step 1, Extraction: Take commercially available peanut kernels of a certain brand, crush them, weigh 20g of peanut powder, place it in a 250ml stoppered conical flask, add 100ml of methanol-water (80:20, v / v) mixed solution, sonicate for 10~15min, then add 100ml of petroleum ether-n-hexane (70:30, v / v) mixed solution, continue sonicating for 10~15min, filter, transfer the extract to a separatory funnel, let it stand to separate into layers, and take the lower layer solution I; Step 2, purification: Add chloroform-dimethylformamide (70:10, v / v) mixed solution to the lower layer solution I, shake and let stand to separate the layers, take the lower layer solution II, remove the organic solvent under reduced pressure, evaporate to dryness, and redissolve in methanol to obtain the peanut kernel test solution; Step 3, Preparation of reference solution: Take aflatoxin B1, B2 and G1 standards, dissolve them in methanol, and prepare a mixed reference solution of 10 ng / ml; Step 4, Thin-layer chromatography detection: Spot the peanut kernel test solution and the mixed reference solution separately onto the same silica gel G thin-layer plate. Use methanol-dimethylformamide (9:1, v / v) as the developing solvent and develop upward to 1.0 cm from the top. Then remove and air dry. Then use methanol-ethyl acetate-chloroform (0.5:4:5.5, v / v / v) as the developing solvent and develop the thin-layer plate in reverse to 2 / 3 of the distance from the other end. Remove, air dry, spray 10% sulfuric acid ethanol solution onto the thin-layer plate, heat at 105℃ until the spots are clear, and examine under visible light.

[0018] Example 3 Step 1, Extraction: Weigh 20g of peanut butter, add 1ml of the reference solution prepared in Step 3, mix well, and let stand for 24h; then place in a 250ml stoppered conical flask, add 100ml of methanol-water (80:20, v / v) mixed solution, sonicate for 10-15min, then add 100ml of petroleum ether-n-hexane (70:30, v / v) mixed solution, continue sonicating for 10-15min, filter, transfer the extract to a separatory funnel, let stand to separate layers, and take the lower layer solution I; Step 2, purification: Add chloroform-dimethylformamide (70:10, v / v) mixed solution to the lower layer solution I, shake and let stand to separate the layers, take the lower layer solution II, remove the organic solvent under reduced pressure, evaporate to dryness, and redissolve in methanol to obtain the peanut butter test solution; Step 3, Preparation of reference solution: Take aflatoxin B1, B2 and G1 standards, dissolve them in methanol, and prepare a mixed reference solution of 10 ng / ml; Step 4, Thin-layer chromatography detection: Spot the peanut butter test solution and the mixed reference solution separately onto the same silica gel G thin-layer plate. Use methanol-dimethylformamide (9:1, v / v) as the developing solvent and develop upward to 1.0 cm from the top. Then remove and air dry. Then use methanol-ethyl acetate-chloroform (0.5:4:5.5, v / v / v) as the developing solvent and develop the thin-layer plate in reverse to 2 / 3 of the distance from the other end. Remove, air dry, spray 10% sulfuric acid ethanol solution onto the thin-layer plate, heat at 105℃ until the spots are clear, and examine under visible light.

[0019] Comparative Example 1 Step 1, Preparation of test solution: Weigh 20g of peanut butter and place it in a 250ml stoppered conical flask. Add 100ml of methanol-dimethylformamide-water (80:5:15, v / v / v) mixed solution and sonicate for 10-15min. Then add 100ml of petroleum ether-n-hexane (70:30, v / v) mixed solution and continue sonicating for 10-15min. Filter and transfer the extract to a separatory funnel. Let it stand to separate the layers. Take the lower layer solution I, remove the organic solvent under reduced pressure, evaporate to dryness, and redissolve in methanol to obtain the peanut butter test solution. Step 2, Preparation of reference solution: Take aflatoxin B1, B2 and G1 standards, dissolve them in methanol, and prepare a mixed reference solution of 10 ng / ml; Step 3, Thin-layer chromatography detection: The peanut butter test solution, aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 reference solutions were spotted separately onto the same silica gel G thin-layer plate. Methanol-dimethylformamide (9:1, v / v) was used as the developing solvent, and the plate was developed upwards to 1.0 cm from the top. The plate was then removed and dried. Then, methanol-ethyl acetate-chloroform (0.5:4:5.5, v / v / v) was used as the developing solvent, and the plate was developed in reverse to 2 / 3 of the way from the other end. The plate was removed, dried, and sprayed with 10% sulfuric acid ethanol solution. The plate was heated at 105°C until the spots were clear and examined under visible light.

[0020] Comparative Example 2 Step 1, Extraction: Weigh 20g of peanut butter, add 1ml of the reference solution prepared in Step 3, mix well, and let stand for 24h; then place in a 250ml stoppered conical flask, add 100ml of methanol-water (80:20, v / v) mixed solution, sonicate for 10-15min, then add 100ml of petroleum ether-n-hexane (70:30, v / v) mixed solution, continue sonicating for 10-15min, filter, transfer the extract to a separatory funnel, let stand to separate layers, and take the lower layer solution I; Step 2, purification: Add chloroform-dimethylformamide (70:10, v / v) mixed solution to the lower layer solution I, shake and let stand to separate the layers, take the lower layer solution II, remove the organic solvent under reduced pressure, evaporate to dryness, and redissolve in methanol to obtain the peanut butter test solution; Step 3, Preparation of reference solution: Take aflatoxin B1, B2 and G1 standards, dissolve them in methanol, and prepare a mixed reference solution of 10 ng / ml; Step 4, Thin-layer chromatography detection: Spot the peanut butter test solution and the mixed reference solution separately onto the same silica gel G thin-layer plate. Use chloroform-acetone (10:2, v / v) as the developing solvent, and develop upward to 1.0 cm from the top. Remove and air dry. Then use ether as the developing solvent and develop in the reverse direction to 0.5 cm from the other end. Remove and air dry. Spray 10% sulfuric acid ethanol solution onto the thin-layer plate and heat at 105℃ until the spots are clear. Examine under visible light.

[0021] Results and Analysis Aflatoxin levels in commercially available peanut products were detected according to the methods described in Examples 1-3 and Comparative Examples 1-2, respectively. The results are shown in the table below. Figure 1-2 As shown.

[0022] since Figure 1 As can be seen from the examples, no aflatoxin was detected in Examples 1-2, and no spots were found at the corresponding positions of the reference standard, indicating that there were no interfering components. The test solution in Comparative Example 1 was different from that of the present invention, and no aflatoxin mixed reference solution was added, but spots appeared at the corresponding positions of the reference standard, indicating that its test solution contained interfering components, which would cause inaccurate detection.

[0023] since Figure 2 As can be seen from the above, the developing agent in Comparative Example 2 is the aflatoxin reverse development method disclosed in the prior art, which is significantly different from the developing agent used in the reverse development of this invention. It cannot detect multiple aflatoxins, has poor separation, and the spots overlap, making it impossible to accurately detect aflatoxins.

Claims

1. A method for rapid detection of aflatoxin in peanuts and their products, characterized in that, The method is to detect aflatoxin using thin-layer chromatography, which includes the following steps: Peanut or its product test solution and aflatoxin reference solution were spotted separately on the same silica gel G thin-layer plate. Methanol-dimethylformamide was used as the developing solvent, and the plate was developed upwards. The plate was then removed and dried. Methanol-ethyl acetate-chloroform was used as the developing solvent, and the plate was developed in the reverse direction to 2 / 3 of the plate. The plate was then removed, dried, and heated with 10% sulfuric acid ethanol solution as the colorimetric reagent until the spots were clear. The plates were then examined under visible light.

2. The method as described in claim 1, characterized in that, The volume ratio of methanol to dimethylformamide in the thin-layer chromatography method is 9:

1.

3. The method as described in claim 1, characterized in that, In the thin-layer chromatography method, the volume ratio of methanol-ethyl acetate-chloroform is 0.5:4:5.

5.

4. The method according to any one of claims 1-3, characterized in that, The preparation method of the peanut and its product test solution includes the following steps: Step 1, Extraction: Take peanuts or their products, add methanol-water mixed solution for ultrasonic extraction, then add petroleum ether-n-hexane mixed solution for continued ultrasonic treatment, filter, let the extract stand to separate into layers, and take the lower layer solution I; Step 2, purification: Add chloroform-dimethylformamide mixed solution to the lower layer solution I, shake and let stand to separate the layers, take the lower layer solution II, remove the organic solvent, evaporate to dryness, and redissolve in methanol to obtain the peanut butter test solution.

5. The method as described in claim 4, characterized in that, In step 1, the volume ratio of methanol to water is 80:

20.

6. The method as described in claim 4, characterized in that, In step 1, the volume ratio of petroleum ether to n-hexane is 70:

30.

7. The method according to any one of claims 1-3, characterized in that, The aflatoxin reference solution is a mixed reference solution containing aflatoxin B1, B2, and G1.

8. The method as described in claim 7, characterized in that, The mixed reference solution is prepared by dissolving aflatoxin B1, B2, and G1 standards in methanol to prepare a 10 ng / ml mixed reference solution.

Images