A method for detecting tert-butyl-4-hydroxyanisole and tannic acid

Abstract

The application discloses a method for detecting tert-butyl-4-hydroxyanisole and tannic acid, which uses Cu2O cubes as a template and cerium ions as a donor to prepare two-dimensional Cu-tetrakis(4-carboxyphenyl) porphyrin (TCPP) nanosheets (Cu2O-Ce-TCPP) under ultraviolet irradiation. The Cu2O-Ce-TCPP shows significantly enhanced laccase-like nanoscale enzyme activity under ultrasonic stimulation. Under the action of ultrasonic waves, the oxidation activity of Cu2O-Ce-TCPP to the substrate 2,4-dichlorophenol is increased by 4 times. Due to the strong laccase-like activity, the Cu2O-Ce-TCPP can oxidize to synthesize the phenolic antioxidant tert-butyl-4-hydroxyanisole (BHA), and the oxidation of BHA is selectively inhibited by tannic acid (TA), so that a rapid detection method for BHA and TA is established. The method is applied to the detection and analysis of BHA and TA in food, and the results are consistent with the results detected by GB5009.32-2016 National Food Safety Standard for Determination of 9 Antioxidants in Food and LY / T 1642-2005 Analysis Test Method of Tannic Acid. The method can only selectively and rapidly oxidize BHA, and TA selectively inhibits the system, and the method has the characteristics of high sensitivity, strong specificity, simple operation, rapidness and the like.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a method for detecting tert-butyl-4-hydroxyanisole and tannic acid. Background Technology

[0002] Butylated p-hydroxyanisole (BHA) is a commonly used synthetic phenolic antioxidant in my country, primarily used in oils, fried flour products, instant rice and noodle products, puffed foods, and biscuits. my country's GB 2760-2014 "National Food Safety Standard for the Use of Food Additives" stipulates that the maximum usage of BHA in oil-containing foods is 0.2 g / kg (based on its content in the oil). Studies have shown that excessive use of BHA can harm human health, and BHA has potential carcinogenicity. Currently, the effective national standard GB / T 5009.30-2003 specifies the detection method for BHA in pastries and vegetable oils. This method requires the preparation of a chromatography column for purification followed by solvent analysis, which is cumbersome, results in high reagent and material consumption, and has a low recovery rate.

[0003] Tannic acid, also known as tannin, is a class of bioactive high-molecular-weight polyphenolic compounds. Due to its unique chemical and physiological properties, tannic acid is widely used in food, medicine, cosmetics, leather tanning, metallurgy, and dyeing. Currently, methods for detecting tannic acid include spectrophotometry, titration, protein preprecipitation, electrochemical analysis, chemiluminescence, high-performance liquid chromatography, fluorescence analysis, and atomic absorption spectrophotometry.

[0004] Nanozymes are nanomaterials possessing the catalytic activity of natural enzymes. They are low-cost, easy to store, and their enzyme activity can be regulated and controlled. Laccase, a polyphenol oxidase with a copper ion catalytic center, can catalyze the oxidation of various phenolic and phenolic amine substrates through electron transfer, producing harmless water. Its unique environmentally friendly properties have made it a focus of nanozyme research. However, due to its low enzyme activity, existing reports show a limited range of phenolic substances it can oxidize, primarily chlorophenols and phenols, with a few reports on the oxidation of bisphenol A. Ultrasound (US)-driven studies of enzyme-mimicking activity are relatively rare, especially for "laccase-like" nanozymes. While ultrasound, as a non-invasive therapeutic method, is widely studied in sonodynamic therapy, particularly in cancer treatment, through cavitation or sonoluminescence mechanisms, its application in driving laccase activity is almost nonexistent. Summary of the Invention

[0005] This invention discloses a method for detecting tert-butyl-4-hydroxyanisole and resveratrol. The method uses Cu₂O cubes as templates and cerium ions as donors to prepare two-dimensional Cu-tetra(4-carboxyphenyl)porphyrin (TCPP) nanosheets (Cu₂O-Ce-TCPP) under ultraviolet light irradiation. Cu₂O-Ce-TCPP exhibits significantly enhanced laccase-like nanozyme activity under ultrasonic stimulation. Under ultrasonic irradiation, the oxidation activity of Cu₂O-Ce-TCPP against the substrate 2,4-dichlorophenol is increased by 4-fold. Due to its strong laccase-like properties, Cu2O-Ce-TCPP can oxidize and synthesize the phenolic antioxidant tert-butyl-4-hydroxyanisole (BHA). Tannic acid (TA) selectively inhibits the oxidation of BHA, thus establishing a rapid detection method for BHA and TA. This method was applied to the detection and analysis of BHA and TA in food, and the results were consistent with those of GB5009.32-2016 National Food Safety Standard for the Determination of Nine Antioxidants in Food and LY / T 1642-2005 Test Method for Tannic Acid Analysis. The method of this invention can only selectively and rapidly oxidize BHA, while TA selectively inhibits the system. The method has the characteristics of high sensitivity, strong specificity, simple operation, and rapid speed.

[0006] The method for detecting tert-butyl-4-hydroxyanisole and resveratrol according to the present invention includes the following steps:

[0007] (1) Cu2O-Ce-TCPP nanozyme and 4-aminoantipyrine (4-AP) were added to the standard solution of tert-butyl-4-hydroxyanisole (BHA) and diluted with pH 6.2 MES buffer solution to obtain BHA solution with concentration range of 1.80-180 μg / mL. The solution was sonicated for 10-20 min, centrifuged, and the supernatant was placed at a wavelength of 503 nm to measure the absorbance. The quantitative relationship between absorbance and BHA concentration was established, a standard curve was plotted, and the regression equation was obtained.

[0008] (2) Cu2O-Ce-TCPP nanozyme, 4-aminoantipyrine (4-AP), and tert-butyl-4-hydroxyanisole (BHA) were added to the tannic acid (TA) standard solution and diluted to volume with pH 6.2 MES buffer solution. The concentration range of the TA solution was 0.667-66.7 μg / mL. The solution was sonicated at 300 W for 15-20 min, centrifuged, and the supernatant was placed at a wavelength of 503 nm to measure the absorbance. The quantitative relationship between absorbance and TA concentration was established, a standard curve was plotted, and the regression equation was obtained.

[0009] (3) Extract and purify the BHA in the sample to obtain the sample assay solution. Add Cu2O / Ce-TCPP nanozyme and 4-AP to the sample assay solution and make up the volume with pH 6.2 MES buffer solution. Sonicate for 10-20 min, centrifuge, take the supernatant and place it at a wavelength of 503 nm to measure the absorbance. Substitute the absorbance into the regression equation in step (1) to obtain the BHA content in the sample.

[0010] (4) Extract and purify the TA in the sample to obtain the sample assay solution. Add Cu2O / Ce-TCPP nanozyme, 4-AP and BHA to the sample assay solution, and make up the volume with pH 6.2 MES buffer solution. Sonicate for 10-20 min, centrifuge, take the supernatant and place it at a wavelength of 503 nm to measure the absorbance. Substitute the absorbance into the regression equation in step (1) to obtain the TA content in the sample.

[0011] The Cu2O-Ce-TCPP nanozyme was prepared as follows:

[0012] a. Preparation of Cu2O nanoparticles: 10-15 mL NaOH (2 mol / L), 100-120 mL CuCl2 (0.01 mol / L) and 4-5 g polyvinylpyrrolidone (PVP) are stirred and mixed for 30-40 min. Then, 0.5-0.7 mol / L ascorbic acid (AA) is added, and the mixture is stirred in a 55℃ water bath for 5-6 h. After washing with deionized water, the nanoparticles are dried under vacuum.

[0013] b. Preparation of Cu2O-Ce nanoparticles: Take 30-40 mg of Cu2O prepared in step (1), add 10-15 mL of deionized water and 10-15 mL of ethanol mixture, then add 10-15 mL of Ce(NO3)3 (0.1 mol / L) solution, sonicate for 20-30 min, and then stir under ultraviolet light for 1-1.5 h to obtain the final product.

[0014] c. Preparation of Cu2O-Ce-TCPP nanozyme: Add 10-15 mL of tetrakis(4-carboxyphenyl)porphyrin (TCPP, 0.025 mmol / L DMF solution) to the Cu2O-Ce solution prepared in step (2), stir in a 60℃ water bath for 6-8 h, centrifuge, wash alternately with ethanol and deionized water, and vacuum dry to obtain the product.

[0015] The sample assay solution is prepared as follows:

[0016] Solid samples: Weigh 5.00g of uniformly pulverized sample into an Erlenmeyer flask, add 8-10mL of anhydrous ethanol, vortex mix for 1-2min, then sonicate for 15-20min, allow to stand and separate into layers, and transfer the supernatant to a 50mL centrifuge tube. Extract the residue twice with 8-10mL of anhydrous ethanol each time. Combine the supernatants in a centrifuge tube, add 0.8-1.2g of neutral alumina, vortex for 1-2min, centrifuge at 3000r / min for 5-10min, transfer the supernatant to a 25mL volumetric flask, dilute to volume with anhydrous ethanol, and shake well to obtain the BHA test sample solution.

[0017] Oils: Accurately weigh 2g of vegetable oil or oily food into a 25mL colorimetric tube, add 5-7mL of 95% ethanol solution, vortex to mix thoroughly, let stand for a moment, and heat in a water bath at 85-95℃ for 10-15s to promote separation; transfer the supernatant to a concentration bottle, and extract twice more with 5-7mL of 95% ethanol solution, combine the extracts and dilute to 25mL to obtain the BHA test sample solution;

[0018] Weigh 0.5-5g (accurate to 0.01g) of the pulverized sample into a stoppered Erlenmeyer flask, add 10-60mL of acetone solution, extract by sonication for 45min, filter with filter paper, wash the residue with a small amount of acetone solution, filter again, combine the filtrates, add deionized water to make up to 10-100mL, and obtain the TA test sample solution.

[0019] The dosage of 100µg / mL Cu2O / Ce-TCPP nanozyme was 50-100µL, the dosage of 1mg / mL 4-AP was 100-150µL, the dosage of 150µg / mL BHA solution was 50-100µL, the ultrasonic power was 300-350W, and the centrifugation conditions were 5-10 min centrifugation time and 1000-3000 r / min centrifugation rate.

[0020] The advantages of this invention are:

[0021] 1. This invention introduces tetratetra(4-carboxyphenyl)porphyrin (TCPP), which has acoustic sensitivity, into Cu2O-Ce nanocomposite. By enhancing the laccase-like activity of the synthesized nanozyme through ultrasound, it can selectively oxidize and synthesize the phenolic antioxidant tert-butyl-4-hydroxyanisole (BHA). Tannic acid (TA) selectively inhibits the oxidation of BHA, thereby establishing a new method for rapid detection of BHA and TA.

[0022] 2. Under ultrasound, the oxidation activity of Cu2O-Ce-TCPP on the substrate 2,4-dichlorophenol increased by 4 times. The Michaelis-Menten kinetic equation showed that the synthetic Cu2O-Ce-TCPP simulated laccase activity under ultrasound with the smallest Michaelis constant. Ultrasound greatly improved the affinity between the nanozyme and the substrate, even exceeding the affinity of natural laccase.

[0023] 3. The method established in this invention is used for the detection of BHA and TA in food. It is reliable, simple and rapid, and the detection results are consistent with those of relevant national standard methods. Attached Figure Description

[0024] Figure 1 The images show SEM images of Cu2O synthesized in Example 1 (left) and TEM images of Cu2O-Ce-TCPP (right).

[0025] Figure 2 shows the UV-Vis absorption spectra of 2,4-dichlorophenol oxidized by Cu2O-Ce-TCPP in Example 1 under ultrasonic and non-ultrasonic conditions.

[0026] Figure 3 The Michaelis dynamics curves are those of Example 1 without ultrasonic action.

[0027] Figure 4 shows the Michaelis dynamics curves of the ultrasonic effect in Example 1.

[0028] Figure 5 The linear UV-Vis absorption spectrum (left) and regression equation (right) of Cu2O-Ce-TCPP oxidation of BHA in Example 1 are shown.

[0029] Figure 6 The left figure shows the linear UV-Vis absorption spectrum of TA in Example 1, and the right figure shows the regression equation.

[0030] Figure 7 The results show the effects of coexisting substances on BHA.

[0031] Figure 8 The results show the effect of coexisting antioxidant TA. Detailed Implementation

[0032] The technical solution of the present invention will be described in further detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0033] Example 1: Determination of BHA in corn oil samples and TA in pig compound feed

[0034] 1. Preparation of Cu2O nanoparticles: 12 mL NaOH (2 mol / L), 100 mL CuCl2 (0.01 mol / L) and 4 g polyvinylpyrrolidone (PVP) were stirred and mixed for 35 min. Then 0.6 mol / L ascorbic acid (AA) was added, and the mixture was stirred in a water bath at 55℃ for 6 h. After washing with deionized water, the nanoparticles were dried under vacuum.

[0035] 2. Preparation of Cu2O-Ce nanoparticles: Take 35 mg of Cu2O prepared in step (1), add 15 mL of deionized water and 12 mL of ethanol mixture, then add 15 mL of Ce(NO3)3 (0.1 mol / L) solution, sonicate for 25 min, and then stir under ultraviolet light for 1.5 h to obtain the final product.

[0036] 3. Preparation of Cu2O-Ce-TCPP nanozyme: Add 15 mL of tetrakis(4-carboxyphenyl)porphyrin (TCPP, 0.025 mmol / L DMF solution) to the Cu2O-Ce solution prepared in step (2), stir in a 60℃ water bath for 6 h, centrifuge, wash alternately with ethanol and deionized water, and vacuum dry to obtain the product. Figure 1 and Figure 2 Electron micrographs of Cu2O and Cu2O-Ce-TCPP.

[0037] 4. Evaluation of Cu2O-Ce-TCPP nanozyme laccase activity: Using 2,4-dichlorophenol (2,4-DP) as a substrate and 4-aminoantipyrine (4-AP) as a colorimetric reagent, the mimic enzyme activity of laccase was determined. Figure 3 In the presence of 4-AP, 2,4-DP can be oxidized by Cu₂O-Ce-TCPP to a quinone analog red product, producing a distinct absorption peak at 503 nm. Furthermore, after ultrasonic treatment (135 W) for 10 min, the absorbance of Cu₂O-Ce-TCPP increased significantly by approximately four times. Michaelis-Menten catalytic kinetic parameters were also determined. Figure 4 The Michaelis constants for ultrasonic treatment and non-ultrasonic treatment were 0.02 mM, respectively. -1 and 7.5mM -1 The Michaelis constant of natural laccase is 0.79 mM. -1 This indicates that ultrasonic treatment greatly enhances the affinity between Cu2O-Ce-TCPP nanozymes and their substrates.

[0038] 5. Preparation of BHA working curve: Add 100µL of 0.1mg / mL Cu2O-Ce-TCPP nanozyme, 100µL of 1mg / mL 4-aminopyridine (4-AP), and BHA standard solution with concentrations ranging from 1.80 to 180μg / mL to a 5mL stoppered colorimetric tube. Add MES buffer (30mmol / L, pH=6.8) and dilute to the mark. Sonicate at 300W for 20min. Measure the absorbance A at 503nm. Plot a standard curve with BHA concentration on the x-axis and absorbance A on the y-axis. The regression equation is shown below. Figure 5 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0039] 6. Preparation of TA working curve: Add 100µL of 0.1mg / mL Cu2O-Ce-TCPP nanozyme, 100µL of 1mg / mL 4-aminopyridine (4-AP), 150μg / mL BHA, and TA standard solution with concentrations ranging from 0.667 to 66.7μg / mL to a 5 mL stoppered colorimetric tube. Add MES buffer (30mmol / L, pH=6.8) and dilute to the mark. Sonicate at 300W for 20 min. Measure the absorbance A at 505nm. Plot the standard curve with TA concentration on the x-axis and absorbance A on the y-axis. See [link to standard curve]. Figure 6 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0040] Table 1. Linear equation, correlation coefficient, relative standard deviation, and linear range

[0041]

[0042] 7. Method Specificity Study: BHA was mixed with other antioxidants to form a co-antioxidant mixture. The effect of coexisting antioxidants on BHA in the above detection system was examined. The concentration of BHA was 10 mg / kg, and the concentration of the interfering substances was 50 mg / kg. Figure 7The results show the effects of coexisting antioxidants (tert-butylhydroquinone TBHQ), 2,4,5-trihydroxyphenylbutanone (THBP), nordihydroguaiacol (NDGA), propyl gallate (PG), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-methylphenol (BHT), dodecyl gallate (DG), glucose, maltose, sucrose, starch, and ascorbic acid on BHA. The figures show that the Cu₂O-Ce-TCPP system exhibits good selectivity and specificity in oxidizing BHA, with only BHA showing a significant oxidation reaction and other substances showing almost none. The method for determining BHA demonstrates good selectivity and specificity. The selectivity for TA determination was also investigated by mixing TA with other potentially coexisting substances and examining their effects on the detection system. The TA concentration was 10 mg / g, and the concentrations of the interfering substances were 5 mg / g. Figure 8 The results show the effects of coexisting substances glucose (GA), ascorbic acid (AA), chlorogenic acid (CGA), resveratrol (Res), tea polyphenols (GTP), maltose, sucrose, starch, and rutin on TA.

[0043] 8. Determination of BHA in corn oil samples

[0044] (1) Sample preparation: Accurately weigh 2g of corn oil into a 25mL colorimetric tube, add 6mL of 95% ethanol solution, vortex for 1min to mix thoroughly, let stand for a while, and heat in a 90℃ water bath for 10s to promote separation; transfer the supernatant to a concentration bottle with a pipette, and extract twice more with 6mL of 95% ethanol solution, combine the extracts and make up to 25mL to obtain the sample solution to be tested;

[0045] (2) Sample determination: Add 100µL of 0.1mg / mL Cu2O-Ce-TCPP nanozyme, 100µL of 1mg / mL 4-aminopyridine (4-AP) and 1mL of the sample solution to be tested in step (3) to a 5 mL stoppered colorimetric tube, add MES buffer solution (30mmol / L, pH=6.8) to make up to the mark, sonicate at 300w for 20min, measure absorbance A at 503nm wavelength, substitute into the regression equation in step (1), and calculate the content of BHA in the sample as 6.05mg / kg;

[0046] (3) Recovery and precision experiments: Two different concentrations of BHA standard solution were added to the soybean blended oil sample; each concentration was measured in parallel 3 times, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2. The spiked recovery rate of BHA was found to be 96.1% to 103.9%, and the RSD was 2.5% to 3.0%. This method has good accuracy and precision.

[0047] Table 2. Spike recoveries and RSDs of samples (n = 3)

[0048]

[0049] 9. Determination of TA in pig compound feed

[0050] (1) Sample preparation: Weigh 5g of the crushed sample (accurate to 0.01g) into a stoppered Erlenmeyer flask, add 60mL of acetone solution, extract by sonication for 45min, filter with filter paper, wash the residue with a small amount of acetone solution, filter, combine the filtrates, add deionized water to make up to 100mL, and obtain the sample solution to be tested.

[0051] (2) Sample determination: Add 100µL of 0.1mg / mL Cu2O-Ce-TCPP nanozyme, 100µL of 1mg / mL 4-aminopyridine (4-AP), 150μg / mL BHA and 100µL of the sample solution to be tested in step (3) to a 5 mL stoppered colorimetric tube, add MES buffer solution (30mmol / L, pH=6.8) to make up to the mark, sonicate at 300w for 15-20min, measure absorbance A at 505nm wavelength, substitute into the regression equation in step (2), and calculate the content of TA in the sample as 1.43mg / g.

[0052] Example 2: Determination of BHA in walnut oil samples and resveratrol in Polygonum cuspidatum

[0053] 1. Preparation of Cu2O nanoparticles: Same as in Example 1;

[0054] 2. Preparation of Cu2O-Ce nanoparticles: Same as in Example 1;

[0055] 3. Preparation of Cu2O-Ce-TCPP nanozymes: Same as in Example 1;

[0056] 4. BHA working curve creation: Same as in Example 1;

[0057] 5. TA working curve creation: Same as Example 1;

[0058] 6. Determination of BHA in walnut oil samples

[0059] (1) Sample preparation: Accurately weigh 2g of walnut oil into a 25mL colorimetric tube, add 7mL of 95% ethanol solution, vortex for 1min to mix thoroughly, let stand for a while, and heat in an 85℃ water bath for 15s to promote separation; transfer the supernatant to a concentration bottle with a pipette, and extract twice more with 7mL of 95% ethanol solution, combine the extracts and make up to 25mL to obtain the sample solution to be tested;

[0060] (2) Sample determination: Same as in Example 1, the BHA content in the walnut oil sample was 21.81 mg / kg.

[0061] 5. Determination of TA in Smilax glabra

[0062] (1) Sample preparation: Weigh 0.5g (accurate to 0.01g) of the crushed sample into a stoppered Erlenmeyer flask, add 10mL of 85% ethanol solution, extract in an 80℃ water bath for 45min, shake occasionally, cool and filter with filter paper, wash the residue with a small amount of 85% ethanol solution, filter, combine the filtrates, add deionized water to make up to 10mL, and obtain the sample solution to be tested;

[0063] (2) Sample determination: The content of TA in Smilax glabra was 13.90 mg / g.

[0064] Example 3: Determination of BHA in walnut oil and TA in green walnut husk

[0065] 1. Preparation of Cu2O nanoparticles: Same as in Example 1;

[0066] 2. Preparation of Cu2O-Ce nanoparticles: Same as in Example 1;

[0067] 3. Preparation of Cu2O-Ce-TCPP nanozymes: Same as in Example 1;

[0068] 4. BHA working curve creation: Same as in Example 1;

[0069] 5. TA working curve creation: Same as Example 1;

[0070] 6. Determination of BHA in walnut oil samples

[0071] (1) Sample preparation: Same as in Example 1;

[0072] (2) Sample determination: The BHA content in the walnut oil sample was 15.75 mg / kg.

[0073] 7. Determination of TA in green walnut husk

[0074] (1) Sample preparation: Same as in Example 1;

[0075] (2) Sample determination: The TA content in the green walnut skin was 190.12 mg / g.

[0076] Example 4: Determination of BHA in instant noodles and TA in raisins

[0077] 1. Preparation of Cu2O nanoparticles: Same as in Example 1;

[0078] 2. Preparation of Cu2O-Ce nanoparticles: Same as in Example 1;

[0079] 3. Preparation of Cu2O-Ce-TCPP nanozymes: Same as in Example 1;

[0080] 4. BHA working curve creation: Same as in Example 1;

[0081] 5. TA working curve creation: Same as Example 1;

[0082] 6. Determination of BHA in instant noodles:

[0083] (1) Sample preparation: Weigh 5.00g of uniformly pulverized instant noodle sample into a 100mL conical flask, add 9mL of anhydrous ethanol, vortex mix for 1.5min, then sonicate for 20min, let stand to separate the layers, and transfer the supernatant into a 50mL centrifuge tube. Extract the residue twice with 9mL of anhydrous ethanol each time. Combine the supernatants into a 50mL centrifuge tube, add 1g of neutral alumina, vortex for 2min, centrifuge at 3000r / min for 10min, transfer the supernatant into a 25mL volumetric flask, dilute to volume with anhydrous ethanol, shake well, and obtain the sample solution to be tested.

[0084] (2) Sample determination: Same as in Example 1, the BHA content in instant noodles was 5.89 mg / kg.

[0085] 5. Determination of TA in raisins

[0086] (1) Sample preparation: Same as in Example 1;

[0087] (2) Sample determination: The TA content in raisins was 43.13 mg / kg.

[0088] Examples 1-4 were compared using the method of the present invention with the methods of the national standard GB5009.32-2016 Food Safety Standard - Determination of Nine Antioxidants in Food and LY / T 1642-2005 Tannic Acid Analysis Test Method. The results are shown in Table 3. It can be seen from the results that the results of the two methods are consistent.

[0089] Table 3. Comparison results of the above methods: BHA / TA values

[0090]

[0091] The BHA and TA determination method established in this invention has the advantages of fewer processing steps, speed and simplicity, no need for sample purification treatment according to GB5009.32-2016, short time, low processing cost, simple operation, and no need for large-scale instruments and equipment, which makes it highly advantageous in actual detection.

Claims

1. A method for detecting tert-butyl-4-hydroxyanisole and tannic acid, characterized in that, Includes the following steps: (1) Cu2O-Ce-TCPP nanozyme and 4-aminoantipyrine were added to the standard solution of tert-butyl-4-hydroxyanisole and diluted with pH 6.2 MES buffer solution to obtain a concentration range of 1.80-180 μg / mL. The solution was sonicated for 10-20 min, centrifuged, and the supernatant was placed at a wavelength of 503 nm to measure the absorbance. The quantitative relationship between absorbance and concentration of tert-butyl-4-hydroxyanisole was established, a standard curve was plotted, and the regression equation was obtained. (2) Cu2O-Ce-TCPP nanozyme, 4-aminoantipyrine, and tert-butyl-4-hydroxyanisole were added to the tannic acid standard solution and diluted to volume with pH 6.2 MES buffer solution. The concentration range of the tannic acid solution was 0.667-66.7 μg / mL. The solution was sonicated at 300 W for 15-20 min, centrifuged, and the supernatant was placed at a wavelength of 503 nm to measure the absorbance. The quantitative relationship between absorbance and tannic acid concentration was established, a standard curve was plotted, and the regression equation was obtained. (3) Extract and purify the tert-butyl-4-hydroxyanisole from the sample to obtain the sample assay solution. Add Cu2O / Ce-TCPP nanozyme and 4-aminoantipyrine to the sample assay solution, and make up the volume with pH 6.2 MES buffer solution. Sonicate for 10-20 min, centrifuge, take the supernatant and place it at a wavelength of 503 nm to measure the absorbance. Substitute the absorbance into the regression equation in step (1) to obtain the tert-butyl-4-hydroxyanisole content in the sample. (4) Extract and purify the tannic acid from the sample to obtain the sample assay solution. Add Cu2O / Ce-TCPP nanozyme, 4-aminoantipyrine, and tert-butyl-4-hydroxyanisole to the sample assay solution, and make up to volume with pH 6.2 MES buffer solution. Sonicate for 10-20 min, centrifuge, and take the supernatant. The absorbance was measured at a wavelength of 503 nm. The absorbance was substituted into the regression equation in step (1) to obtain the tannic acid content in the sample. The Cu2O-Ce-TCPP nanozyme was prepared as follows: a. Preparation of Cu2O nanoparticles: 10-15 mL of 2 mol / L NaOH, 100-120 mL of 0.01 mol / L CuCl2, and 4-5 g of polyvinylpyrrolidone were stirred and mixed for 30-40 min. Then, 0.5-0.7 mol / L ascorbic acid was added, and the mixture was stirred in a 55℃ water bath for 5-6 h. After washing with deionized water, the nanoparticles were vacuum dried to obtain the final product. b. Preparation of Cu2O-Ce nanoparticles: Take 30-40 mg of Cu2O prepared in step (1), add 10-15 mL of deionized water and 10-15 mL of ethanol mixture, then add 10-15 mL of 0.1 mol / L Ce(NO3)3 solution, sonicate for 20-30 min, and then stir under ultraviolet light for 1-1.5 h to obtain the product. c. Preparation of Cu2O-Ce-TCPP nanozyme: Add 10-15 mL of tetra(4-carboxyphenyl)porphyrin solution prepared with 0.025 mmol / L DMF to the Cu2O-Ce solution prepared in step (2), stir in a 60℃ water bath for 6-8 h, centrifuge, wash alternately with ethanol and deionized water, and vacuum dry to obtain the nanozyme.

2. The method for detecting tert-butyl-4-hydroxyanisole and tannic acid according to claim 1, characterized in that: The sample assay solution is prepared as follows: ① Solid samples: Weigh 5.00g of uniformly pulverized sample into an Erlenmeyer flask, add 8-10mL of anhydrous ethanol, vortex mix for 1-2min, then sonicate for 15-20min, allow to stand and separate into layers, and transfer the supernatant to a 50mL centrifuge tube. Extract the residue twice with 8-10mL of anhydrous ethanol each time. Combine the supernatants in a centrifuge tube, add 0.8-1.2g of neutral alumina, vortex for 1-2min, centrifuge at 3000r / min for 5-10min, transfer the supernatant to a 25mL volumetric flask, dilute to volume with anhydrous ethanol, and shake well to obtain the tert-butyl-4-hydroxyanisole test sample solution. ② Oils: Accurately weigh 2g of vegetable oil or oily food into a 25mL colorimetric tube, add 5-7mL of 95% ethanol solution, vortex to mix thoroughly, let stand for a moment, and heat in a water bath at 85-95℃ for 10-15s to promote separation; transfer the supernatant to a concentration bottle, and extract twice more with 5-7mL of 95% ethanol solution, combine the extracts and dilute to 25mL to obtain the tert-butyl-4-hydroxyanisole sample solution to be tested; ③ Weigh 0.5-5g of the pulverized sample, accurate to 0.01g, into a stoppered Erlenmeyer flask, add 10-60mL of acetone solution, extract by sonication for 45min, filter with filter paper, wash the residue with a small amount of acetone solution, filter again, combine the filtrates, add deionized water to make up to 10-100mL, and obtain the tannic acid test sample solution.

3. The method for detecting tert-butyl-4-hydroxyanisole and tannic acid according to claim 1, characterized in that: The dosage of 100 μg / mL Cu2O / Ce-TCPP nanozyme was 50–100 μL, the dosage of 1 mg / mL 4-aminoantipyrine was 100–150 μL, the dosage of 150 μg / mL tert-butyl-4-hydroxyanisole solution was 50–100 μL, the ultrasonic power was 300–350 W, and the centrifugation conditions were 5–10 min centrifugation time and 1000–3000 r / min centrifugation rate.

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