A method for determining the total amount of aflatoxin

Abstract

The present invention discloses a method for determining the total amount of aflatoxin, and belongs to the technical field of aflatoxin detection. The present invention establishes a simple method for determining the aflatoxin content in an environment based on a two-step color development reaction, so that colorless AFs react to generate a green product, and the method can simultaneously meet the requirements of naked eye analysis and ultraviolet visible spectrophotometer detection of the AFs content. A new strategy is provided for realizing the rapid detection of the total content of aflatoxin in an environment. The detection method of the present invention realizes the rapid qualitative detection of aflatoxin, the color change is sensitive, the method is simple, and the quantitative detection of the total amount of aflatoxin in the sample can be realized at the same time, which greatly improves the detection efficiency of aflatoxin and has a wide range of market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of aflatoxin detection, and in particular relates to a method for determining the total amount of aflatoxin. Background Art

[0002] Aflatoxin is a type of fungal toxin naturally present in food. It is a toxic secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. The toxicity of aflatoxin is 68 times that of arsenic and 10 times that of potassium cyanide. 1 mg of aflatoxin can induce cancer, and 20 mg can kill a person. It is rated as a Class I carcinogen by the International Agency for Research on Cancer. At present, more than 20 types of aflatoxin and its derivatives have been isolated, among which aflatoxin B1, B2, G1, and G2 are the most common and the most harmful. It is often found in daily foods such as peanuts, dried fruits, rice, grains and oils, posing a great threat to human dietary health. Considering the harm of aflatoxin to human health, countries around the world have increasingly stringent restrictions on the content of aflatoxin in food and feed, which also requires the continuous updating of the detection technology for the total content of aflatoxin B1, B2, G1, and G2, and the development in the direction of faster, more accurate, and more convenient.

[0003] The most common technologies for detecting aflatoxin content are chromatography and immunoassay. Chromatography, such as high-performance liquid chromatography-fluorescence detector or mass spectrometer, is considered the "gold standard" for detecting aflatoxin because of its good accuracy, but it requires expensive detection instruments, time-consuming sample preparation, a long detection process, requires professional operators, and has high detection costs. It is not suitable for screening a large number of samples in remote areas; immunoassay can achieve rapid screening of a large number of samples, and can even achieve on-site detection. Thanks to the specific recognition of antibodies, the accuracy of immunoassay is very high, and it can achieve sensitive detection of a single variety of toxins, but it cannot detect the total amount of toxins, and it is difficult to obtain high-quality antibodies, and the cost is expensive. Therefore, it is particularly important to develop a new method for detecting aflatoxin that is highly sensitive, fast, simple, and economical.

[0004] In recent years, colorimetric sensing analysis has emerged as a new force due to its unique color reaction that can realize visual detection and analysis. It can realize qualitative and quantitative analysis through color changes, and use ultraviolet spectrophotometer to measure the absorbance of samples to accurately analyze and determine the toxin content of samples. It has attracted much attention due to its simple operation, cheap reagents, and rapid reaction. Summary of the invention

[0005] The present invention establishes a simple method for determining the aflatoxin content in the environment based on a two-step color reaction, so that the colorless AFs react to generate a green product. The method can simultaneously meet the requirements of naked eye analysis and ultraviolet-visible spectrophotometer detection of the AFs content, providing a new strategy for realizing rapid detection of the total content of aflatoxin in the environment.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] A method for determining the total amount of aflatoxin comprises the following preparation steps:

[0008] (1) Sample pretreatment: After the sample is crushed, a solvent is added for constant temperature extraction, and the sample solution is obtained after filtration;

[0009] (2) Detection method: Take 30 μL of the sample solution and place it in a 2 mL test tube, add 170 μL of 0.01 M NaOH solution, shake well, and let it stand for 10 min, add 100 μL of buffer solution to the test tube and shake well, then add 50 μL of 4-aminoantipyrine solution and 150 μL of oxidant solution in sequence, let it stand for 1-2 min to fully react, and obtain a reaction solution;

[0010] (3) Add 350 μL of chloroform to the reaction solution and shake vigorously to allow the extraction to reach equilibrium. Use a UV-visible spectrophotometer to measure the absorbance of the extract and calculate the total amount of aflatoxin based on the standard curve.

[0011] Furthermore, the solvent in step (1) is a methanol solution with a mass concentration of 70%. The solid-liquid ratio of the sample to methanol is 1 g: 5-15 ml.

[0012] Furthermore, the constant temperature extraction method of step (1) is: constant temperature extraction in a 30°C water bath for 20-60 minutes.

[0013] Furthermore, in step (2), the buffer solution is a borax-sodium hydroxide solution with a concentration of 0.05 M and a pH of 9.34.

[0014] Furthermore, in step (2), the concentration of 4-aminoantipyrine in the final system extract is 2.5 mg / mL, and the mass concentration of the 4-aminoantipyrine solution used is 2%.

[0015] Furthermore, the oxidant in step (2) is potassium ferrocyanide, the concentration of potassium ferrocyanide in the final system extract is 0.235 mg / mL, and the mass concentration of the oxidant solution used is 8%.

[0016] The present invention establishes a two-step color development reaction based on 4-aminoantipyrine to detect aflatoxin. Since the main structure of aflatoxin B1, B2, G1, and G2 is a coumarin ring, the ring is rapidly opened under alkaline conditions to generate a phenolic hydroxyl group, and there is no substituent at the para position of the phenolic hydroxyl group, i.e., position 6. According to the Emerson reaction mechanism, 4-aminoantipyrine can replace the active hydrogen at the para position of the phenolic hydroxyl group in the presence of an oxidant to generate a doxyphenol condensate that changes color, thereby realizing rapid detection of aflatoxin. The reaction mechanism is as follows: Figure 1 As shown. AFB1, AFB2, AFG1, and AFG2 hydrolyze the ester bond under alkaline conditions to form a phenolic hydroxyl group on the benzene ring. When there is no substituent at the para position of the phenolic hydroxyl group, the active hydrogen at the para position can react with 4-aminoantipyrine to condense and form a double bond. The reaction product of phenol and 4-aminoantipyrine is red. Experiments have shown that the color development conditions of phenol are not suitable for aflatoxin. Therefore, a large number of experiments have been conducted to obtain color development conditions suitable for aflatoxin. In the Na3BO3-NaOH environment, the final product of this reaction is green. At the same time, the blank sample is yellow, and the color change can be used to directly determine whether there is aflatoxin in the sample without distinguishing which type it is. This achieves qualitative detection of aflatoxin.

[0017] Beneficial Effects

[0018] This detection method can realize rapid qualitative detection of aflatoxin, with sensitive color change and simple method. It can also realize quantitative detection of the total amount of aflatoxin in the sample, greatly improving the detection efficiency of aflatoxin and having broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the reaction mechanism diagram of the color development method of the present invention;

[0020] Figure 2 This is the effect of NaOH concentration and hydrolysis time on the first step reaction;

[0021] Figure 3 This is a graph showing the effect of buffer solution pH and reaction time on the second step reaction;

[0022] Figure 4 This is a graph showing the effect of 4-aminoantipyrine concentration on the absorbance of the reaction;

[0023] Figure 5 This is the graph showing the effect of the concentration of the oxidant on the reaction;

[0024] Figure 6 The UV-visible spectra of the doxyphenol products generated by the reaction of AFs with different concentrations. The upper left corner shows the color change of the reactants. The color gradually deepens from light yellow to green from left to right.

[0025] Figure 7 This is the linear relationship between the absorbance at 595 nm and the AFs concentration. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0027] Example 1

[0028] A method for determining the total amount of aflatoxin comprises the following preparation steps:

[0029] (1) Sample pretreatment: After the sample is crushed, a solvent is added for constant temperature extraction, and the sample solution is obtained after filtration;

[0030] (2) Detection method: Take 30 μL of the sample solution and place it in a 2 mL test tube. Add 170 μL of 0.01 M NaOH solution and shake well. Then let it stand for 10 min. Add 100 μL of buffer solution to the test tube and shake well. Then add 50 μL of 4-aminoantipyrine solution and 150 μL of oxidant solution in sequence. Let it stand for 1 min to allow for sufficient reaction.

[0031] (3) Add 350 μL of chloroform to the reaction solution and shake vigorously to allow the extraction to reach equilibrium. Use a UV-visible spectrophotometer to measure the absorbance of the extract and calculate the total amount of aflatoxin based on the standard curve.

[0032] The solvent in step (1) is a methanol solution with a mass concentration of 70%. The solid-liquid ratio of the sample to methanol is 1 g:5 ml.

[0033] The constant temperature extraction method of step (1) is: constant temperature extraction in a 30° C. water bath for 20 min.

[0034] The buffer solution in step (2) is a borax-sodium hydroxide solution with a concentration of 0.05 M and a pH of 9.34.

[0035] In step (2), the concentration of 4-aminoantipyrine in the final system extract is 2.5 mg / mL, and the mass concentration of the 4-aminoantipyrine solution used is 2%.

[0036] The oxidant in step (2) is potassium ferrocyanide, the concentration of potassium ferrocyanide in the final system extract is 0.235 mg / mL, and the mass concentration of the goose oxidant solution used is 8%.

[0037] Example 2

[0038] A method for determining the total amount of aflatoxin comprises the following preparation steps:

[0039] (1) Sample pretreatment: After the sample is crushed, a solvent is added for constant temperature extraction, and the sample solution is obtained after filtration;

[0040] (2) Detection method: Take 30 μL of the sample solution and place it in a 2 mL test tube. Add 170 μL of 0.01 M NaOH solution and shake well. Then let it stand for 10 min. Add 100 μL of buffer solution to the test tube and shake well. Then add 50 μL of 4-aminoantipyrine solution and 150 μL of oxidant solution in sequence. Let it stand for 2 min to allow for sufficient reaction.

[0041] (3) Add 350 μL of chloroform to the reaction solution and shake vigorously to allow the extraction to reach equilibrium. Use a UV-visible spectrophotometer to measure the absorbance of the extract and calculate the total amount of aflatoxin based on the standard curve.

[0042] The solvent in step (1) is a methanol solution with a mass concentration of 70%. The solid-liquid ratio of the sample to methanol is 1 g:15 ml.

[0043] The constant temperature extraction method of step (1) is: constant temperature extraction in a 30° C. water bath for 60 min.

[0044] The buffer solution in step (2) is a borax-sodium hydroxide solution with a concentration of 0.05 M and a pH of 9.34.

[0045] In step (2), the concentration of 4-aminoantipyrine in the final system extract is 2.5 mg / mL, and the mass concentration of the 4-aminoantipyrine solution used is 2%.

[0046] The oxidant in step (2) is potassium ferrocyanide, the concentration of potassium ferrocyanide in the final system extract is 0.235 mg / mL, and the mass concentration of the oxidant solution used is 8%.

[0047] Example 3

[0048] In the first and most critical step of the reaction, aflatoxin is decomposed under alkaline conditions. The factors that affect its decomposition efficiency are the concentration of NaOH and the decomposition time. Therefore, these two reaction conditions are optimized. Figure 2 As shown in the figure, the longer the hydrolysis time, the higher the absorbance of the final product at 595nm, indicating that the longer the time, the more complete the hydrolysis. At 3min, 5min, and 10min, although the absorbance increases, the increase is small, so 10 minutes has met the experimental requirements. However, the hydrolysis efficiency is low when the concentration of NaOH is low, and the pH of the final solution is high when the concentration is high, which has a greater impact on the reaction environment, so 0.01M is the optimal concentration of NaOH.

[0049] Example 4

[0050] Since pH has a decisive influence on this experiment, we adjusted it by adding 0.05M Na3BO3-NaOH solution, such as Figure 3As shown, when Na3BO3-NaOH is added to pH = 9.34, the product has a maximum absorbance at 595 nm. Figure 3 It can also be seen that after adding the oxidant, the absorbance decreases over time, which indicates that the indoxyl products produced by the reaction are unstable, and precipitation occurs after shaking, causing the absorbance to further decrease. Violent shaking should be avoided during the reaction, and the time point for adding chloroform should be within 2-5 minutes after adding the oxidant.

[0051] Example 5

[0052] As the core colorimetric reagent of this experiment, the concentration of 4-aminoantipyrine has a critical effect on the experiment. Figure 4 As shown, the absorbance of the product tends to be stable after the concentration of 4-aminoantipyrine is 2.5 mg / mL, and the absorbance of the blank sample increases slightly. From the perspective of saving reagents, 2.5 mg / mL is selected as the optimal concentration of 4-aminoantipyrine.

[0053] Example 6

[0054] Optimize the concentration of oxidant, such as Figure 5 As shown, with the increase of the amount of oxidant, the color change and absorbance of the product first increase and then decrease. The main reason is that potassium ferrocyanide solution is acidic. Too high a concentration will cause the pH in the system to decrease, thus affecting the reaction. Therefore, 0.235 mg / mL is selected as the optimal concentration of the oxidant.

[0055] Detection method sensitivity and total amount detection

[0056] A series of AFs standard solutions of different concentrations were added to the test tubes for color development reaction, e.g. Figure 6 As shown in the figure, as the concentration of AFs increases from 0 to 3 μg / mL, the color of the reaction solution changes from light yellow to green, and the absorption band of the extract at 595 nm increases systematically. The absorbance at 595 nm increases regularly with the concentration of AFs, as shown in the figure. Figure 7 As shown, a linear correlation of R2 = 0.9991 was obtained. The detection limit of the standard solution of AFs was calculated to be 0.09 μg / mL (3σ). The above results obtained from the standard solution show that the colorimetric method can detect the total amount of AFs in actual samples.

[0057] Spike recovery test

[0058] Five blank samples were taken and AFs standard solution was added at concentrations of 0.25, 0.5, 0.75, 1.5, and 3 μg / mL, respectively. The aflatoxin content was determined according to the method of the present invention. The results are shown in Table 1:

[0059] Table 1 Results of spike recovery determination

[0060]

[0061] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A method for determining the total amount of aflatoxin, characterized in that: The method comprises the following preparation steps: (1) Sample pretreatment: After the sample is crushed, a solvent is added for constant temperature extraction, and the sample solution is obtained after filtration; (2) Detection method: Take 30 μL of the sample solution and place it in a 2 mL test tube, add 170 μL of 0.01 M NaOH solution, shake well and let stand for 10 min, add 100 μL of buffer solution to the test tube and shake well, then add 50 μL of 4-aminoantipyrine solution and 150 μL of oxidant solution in sequence, let stand for 1-2 min to fully react and obtain a reaction solution; (3) Add 350 μL of chloroform to the reaction solution and shake vigorously to allow the extraction to reach equilibrium. Measure the absorbance of the extract using a UV-visible spectrophotometer and calculate the total amount of aflatoxin based on the standard curve. The buffer solution in step (2) is a borax-sodium hydroxide solution with a concentration of 0.05 M and a pH of 9.34; In step (2), the concentration of 4-aminoantipyrine in the final system extract is 2.5 mg / mL, and the mass concentration of the 4-aminoantipyrine solution used is 2%; The oxidant in step (2) is potassium ferrocyanide, the concentration of potassium ferrocyanide in the final system extract is 0.235 mg / mL, and the mass concentration of the oxidant solution used is 8%.

2. The method for determining the total amount of aflatoxin according to claim 1, characterized in that: The solvent in step (1) is a methanol solution with a mass concentration of 70%.

3. The method for determining the total amount of aflatoxin according to claim 1, characterized in that: The constant temperature extraction method in step (1) is: constant temperature extraction in a 30°C water bath for 20-60 minutes.

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