Method for detecting aflatoxin B1 in edible oil

Abstract

The invention relates to a method for detecting aflatoxin B1 in edible oil. The method comprises the following steps: 1) preparing PdO nano-enzyme particles by taking palladium nitrate as a raw material; 2) drawing an aflatoxin B1 standard curve and obtaining a linear equation; (3) extracting aflatoxin B1 in to-be-detected edible oil, and preparing to-be-detected liquid; 4) replacing the aflatoxin B1 standard solution with the to-be-measured solution, and measuring the absorbance value of the to-be-measured solution at 652 nm; and 5) substituting the absorbance value into a linear equation, and calculating the concentration of aflatoxin B1 brought into the edible oil to be detected. According to the detection method, the aflatoxin B1 in the edible oil can be rapidly and efficiently detected, and meanwhile, the preparation method of the palladium oxide nano-enzyme particles is simple, low in cost, high in stability, environmentally friendly and suitable for large-scale preparation. In the detection process, the dosage of the required nano enzyme is small, the detection sensitivity is high, the response time is short, and the detection time is greatly shortened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food analysis, and particularly relates to a detection method of aflatoxin B1 in edible oil. BACKGROUND

[0002] Aflatoxins (AF) are a group of secondary metabolites produced by Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Aspergillus tamarii, etc. AFB1 is one of the most toxic and carcinogenic toxins, and has the widest pollution range. AFB1 is commonly found in peanuts, corn, rice, sorghum, milk and vegetable oil. Humans are mainly exposed to AFB1 by ingesting contaminated food. Long-term exposure can cause aflatoxin poisoning, and the clinical manifestations include liver damage, jaundice, digestive system dysfunction, and even death in severe cases. Therefore, effective monitoring and control of AFB1 content in food is of great significance for reducing public health risks. At present, there are various analysis methods for AFB1 detection, including thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Although the above technologies have high sensitivity and good repeatability, they usually require complex sample pretreatment procedures, and the instrument cost is high and the operation time-consuming, which limits their application in large-scale screening. Therefore, developing a sensitive and accurate method for detecting AFB1 in food is still an important challenge.

[0003] In recent years, with the rapid development of nanotechnology, enzyme mimics based on nanomaterials (i.e., nanozymes) have attracted widespread attention due to their advantages such as low cost, ease of preparation, and tunable catalytic performance. Among the many nanozymes, materials with peroxidase-mimicking activity have shown significant potential in constructing dual-mode biosensors for the detection of aflatoxin B1. For example, Qian et al. (https: / / doi.org / 10.1016 / j.bios.2020.112443) developed a colorimetric / electrochemical dual-signal aptamer sensor based on gold nanoparticles (AuNPs). These nanoparticles possess both peroxidase-like activity and the ability to promote silver deposition, which can be used for the detection of aflatoxin B1. In addition, Lu et al. (https: / / doi.org / 10.1021 / acsami.1c04751) utilized the photothermal effect and peroxidase-like activity of Prussian blue nanoparticles (PBNPs) to design a multimodal detection method for in-situ generation of PBNPs, achieving a triple signal response of photothermal, colorimetric, and fluorescence. While the aforementioned methods demonstrate excellent sensitivity, primarily due to the application of detection techniques such as electrochemical, photothermal, or fluorescence, they typically rely on specialized equipment or require fluorescent labeling, limiting their practical applicability. Therefore, developing a dual-mode detection strategy for aflatoxin B1 that combines ease of operation with high sensitivity remains a significant challenge. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a rapid, efficient, low-pollution, and low-cost method for detecting aflatoxin B1 in edible oils, specifically achieved through the following technical solution: A method for detecting aflatoxin B1 in edible oils includes the following steps: 1) Palladium oxide nanozyme particles were prepared using palladium nitrate as a raw material; 2) Aflatoxin B1 standard solutions of different concentrations were mixed with acetate-sodium acetate buffer, H2O2, 3,3',5,5'-tetramethylbenzidine and PdO nanozyme suspension, and incubated. The absorbance at 652 nm was measured. A standard curve was plotted with aflatoxin B1 concentration on the x-axis and absorbance difference on the y-axis, and the linear equation was obtained. 3) Process the edible oil to be tested, extract aflatoxin B1 from the edible oil to be tested, and prepare the test solution; 4) Replace the aflatoxin B1 standard solution in step 2) with the test solution in step 3), repeat the operation in step 2), and measure the absorbance value of the test solution at 652 nm. 5) Substitute the absorbance value obtained in step 5) into the linear equation obtained in step 2) to calculate the concentration of aflatoxin B1 in the edible oil to be tested.

[0005] Furthermore, the palladium oxide nanozyme particles in step 1) are prepared using the following steps: 1-1) Prepare a precursor by dissolving palladium nitrate in a certain volume of pure water and stirring with magnetic force until a homogeneous solution is formed; 1-2) Slowly add NaOH to the homogeneous solution from step 1-1) to adjust the pH of the solution to 12, ensuring complete precipitation to obtain a suspension; 1-3) Stir the suspension produced in step 1-2) at 70°C until a dark brown solution is produced; 1-4) The dark brown solution generated in step 1-3) is transferred to an autoclave and kept at 160°C for 12 hours. After cooling, centrifugation, washing and drying, palladium oxide nanozyme particles are obtained.

[0006] Further, in step 2), the pH of the acetate-sodium acetate buffer solution is 3.0-5.0, and the concentration is 0.1-0.3 M; the final concentration of H2O2 is 0.05-0.2 mM; the final concentration of 3,3',5,5'-tetramethylbenzidine is 100-200 μM; and the final concentration of the PdO nanozyme suspension is 10-50 μg / mL.

[0007] Furthermore, in step 2), the incubation conditions are 25-37℃, and the incubation time is 2-10 minutes.

[0008] Further, the method for processing the edible oil to be tested in step 3) is as follows: take the edible oil to be tested, add methanol-water extract to extract AFB1, then add sodium chloride to promote phase separation, homogenize and centrifuge and filter, and concentrate or dilute the filtrate to ensure that the extracted aflatoxin B1 is within the detection linear range.

[0009] Furthermore, the volume ratio of methanol to water is 7-6:3-2.

[0010] This invention provides a rapid and efficient detection method for aflatoxin B1 in edible oils without requiring expensive and complex instruments such as chromatography-mass spectrometry. Furthermore, the palladium oxide nanozyme particles are simple to prepare, low in cost, highly stable, and environmentally friendly, making them suitable for large-scale production. The low amount of nanozyme required during detection significantly reduces costs. The method also boasts high sensitivity and a short response time, allowing for visible color changes within minutes, greatly shortening detection time. Its visualization capabilities enable on-site quantitative or qualitative detection, offering speed, convenience, and significant practical value and market potential. Attached Figure Description

[0011] Figure 1 This is a scanning electron microscope image of the PdO nanozyme prepared in Example 1 of the present invention; Figure 2This is a transmission electron microscope image of the PdO nanozyme prepared in Example 1 of the present invention; Figure 3 This is the aflatoxin B1 standard curve established in Example 2 of the present invention; Figure 4 The images show the 300-800nm ​​ultraviolet scans of samples with different concentrations in this detection system in Example 3 of the present invention. Detailed Implementation

[0012] The present invention will be further described below with reference to specific embodiments.

[0013] Example 1: Preparation of PdO nanozymes 1) Dissolve 1g of palladium nitrate in 80mL of purified water to prepare a precursor, and stir magnetically until a homogeneous solution is formed; 2) Slowly add NaOH to the homogeneous solution to adjust the pH of the solution to 12, ensuring complete precipitation; 3) Stir the suspension produced in step 2) at 70°C for 1 hour; 4) Transfer the dark brown solution generated in step 3) to a 100 mL Teflon-lined stainless steel autoclave and maintain it at 160 °C for 12 h; 5) After cooling, collect the PdO by centrifugation, wash it with pure water and ethanol at least three times in sequence, and finally dry it under vacuum at 60°C. Grind the collected precipitate to obtain PdO nanoparticles.

[0014] The PdO nanoparticles prepared above were analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), such as... Figure 1 and Figure 2 As shown, the PdO surface exhibits a rough granular texture. EDS analysis revealed that Pd and O are uniformly distributed in the material, indicating that PdO was successfully synthesized on the surface.

[0015] Example 2: Application of PdO nanozyme in the detection of aflatoxin B1 in walnut oil (1) Establish a standard curve for aflatoxin B1 1) Add different volumes of aflatoxin B1 standard solution to acetate-sodium acetate buffer (pH 3.5, 0.2 M); H2O2 (final concentration 0.1 mM); 3,3',5,5'-tetramethylbenzidine (final concentration 150 μM); and PdO (final concentration 20 μg / mL). 2) After incubating at 25°C for 5 minutes, terminate the reaction with an ice bath. 3) Take 200 μL of each mixture and measure its absorbance A at 652 nm using a full-wavelength scanning microplate reader. At the same time, record the absorbance A0 of the blank without the standard. Calculate the standard curve equation of the difference between aflatoxin B1 concentration and absorbance ΔA. The equation of the standard curve is ΔA = -0.672 C. AFB1 +0.386, as Figure 3 Where ΔA is the absorbance difference, and C AFB1 The concentration of aflatoxin B1 is expressed in μg / L; the correlation coefficient R is given. 2 =0.975, the detection linear range is 5-125 μg / L, and the detection limit is 1 μg / L.

[0016] (2) Walnut oil treatment Take 5 g of crude walnut oil and add 25 mL of extraction buffer (methanol-water, volume ratio 7:3) to extract AFT. Then add 1 g of sodium chloride to promote phase separation. Homogenize at high speed for 2 min, then centrifuge at 6000 r / min for 15 min and filter. Concentrate or dilute the filtrate to ensure that aflatoxin B1 is within the detection linear range.

[0017] (3) Determination of aflatoxin B1 in walnut oil to be tested Replace the aflatoxin B1 standard solution in step (1) with the walnut oil sample treated in step (2), repeat the operation in step (1), and measure its absorbance value at 652 nm. The absorbance value was 0.325. (4) Calculation of aflatoxin B1 concentration in walnut oil: Substitute the absorbance value measured in step (3) into the linear equation obtained in step (1) to calculate the concentration of aflatoxin B1 in the walnut oil to be tested as 0.091 μg / L. -1 .

[0018] Example 3: Application of PdO nanozyme in the detection of aflatoxin B1 in peanut oil 1) Take 5 g of peanut oil and add 25 mL of extraction solution (methanol-water, volume ratio 7:3) to extract AFT. Then add 1 g of sodium chloride to promote phase separation. Homogenize at high speed for 2 min, then centrifuge at 6000 r / min for 15 min and filter. Concentrate or dilute the filtrate to ensure that aflatoxin B1 is within the detection linear range.

[0019] 2) Mixed acetate-sodium acetate buffer (pH 3.5, 0.2 M), PdO nanozyme (final concentration 20 μg / mL), TMB (final concentration 150 μM), and H2O2 (final concentration 100 μM) were added to the peanut oil sample treated in step 1).

[0020] 3) Incubate at 25℃ for 5 minutes.

[0021] 4) Scanning with ultraviolet wavelengths (300-800nm) Figure 4 The absorption peak of aflatoxin B1 in samples is weakened at 652 nm, so the presence of aflatoxin B1 can be preliminarily identified by colorimetric reaction and ultraviolet wavelength scanning.

Claims

1. A method for detecting aflatoxin B1 in edible oil, characterized in that, Includes the following steps: 1) Palladium oxide nanozyme particles were prepared using palladium nitrate as a raw material; 2) Aflatoxin B1 standard solutions of different concentrations were mixed with acetate-sodium acetate buffer, H2O2, 3,3',5,5'-tetramethylbenzidine and PdO nanozyme suspension, and incubated. The absorbance at 652 nm was measured. A standard curve was plotted with aflatoxin B1 concentration on the x-axis and absorbance difference on the y-axis, and the linear equation was obtained. 3) Process the edible oil to be tested, extract aflatoxin B1 from the edible oil to be tested, and prepare the test solution; 4) Replace the aflatoxin B1 standard solution in step 2) with the test solution in step 3), repeat the operation in step 2), and measure the absorbance value of the test solution at 652 nm. 5) Substitute the absorbance value obtained in step 5) into the linear equation obtained in step 2) to calculate the concentration of aflatoxin B1 in the edible oil to be tested.

2. The method for detecting aflatoxin B1 in edible oil as described in claim 1, characterized in that, The palladium oxide nanozyme particles in step 1) were prepared using the following steps: 1-1) Prepare a precursor by dissolving palladium nitrate in a certain volume of pure water and stirring with magnetic force until a homogeneous solution is formed; 1-2) Slowly add NaOH to the homogeneous solution from step 1-1) to adjust the pH of the solution to 12, ensuring complete precipitation to obtain a suspension; 1-3) Stir the suspension produced in step 1-2) at 70°C until a dark brown solution is produced; 1-4) The dark brown solution generated in step 1-3) is transferred to an autoclave and kept at 160°C for 12 hours. After cooling, centrifugation, washing and drying, palladium oxide nanozyme particles are obtained.

3. The method for detecting aflatoxin B1 in edible oil as described in claim 1, characterized in that, In step 2), the pH of the acetate-sodium acetate buffer solution is 3.0-5.0, and the concentration is 0.1-0.3 M; the final concentration of H2O2 is 0.05-0.2 mM; the final concentration of 3,3',5,5'-tetramethylbenzidine is 100-200 μM; and the final concentration of the PdO nanozyme suspension is 10-50 μg / mL.

4. The method for detecting aflatoxin B1 in edible oil as described in claim 1, characterized in that, In step 2), the incubation conditions are 25-37℃ and the incubation time is 2-10 minutes.

5. The method for detecting aflatoxin B1 in edible oil as described in claim 1, characterized in that, The method for processing the edible oil to be tested in step 3) is as follows: Take the edible oil to be tested, add methanol-water extract to extract AFB1, then add sodium chloride to promote phase separation, homogenize and centrifuge and filter, concentrate or dilute the filtrate to ensure that the extracted aflatoxin B1 is within the detection linear range.

6. The method for detecting aflatoxin B1 in edible oil as described in claim 5, characterized in that, The volume ratio of methanol to water is 7-6:3-2.

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