Method for detecting butylated hydroxyanisole in edible oil and fat by combining laccase mimic enzyme with dispersive liquid-liquid microextraction

Abstract

The invention discloses a method for detecting butylated hydroxyanisole in edible oil and fat by combining laccase mimic enzyme with dispersive liquid-liquid microextraction, the method takes L-aspartic acid and copper chloride as precursors, copper-doped carbon dots (Cu / Asp-CDs) with laccase-like nano-enzyme activity are synthesized by a microwave method, the Cu / Asp-CDs have excellent laccase-like nano-enzyme activity, and the Cu / Asp-CDs can be used for detecting butylated hydroxyanisole in edible oil and fat. BHA (butylated hydroxyanisole) can be selectively oxidized into a quinone compound, then the quinone compound is complexed with 4-aminoantipyrine (4-AP) to form a red quinone imine dye, an ultraviolet absorption peak exists at 505 nm, and a practical analysis method is provided for monitoring food quality in industry by utilizing a colorimetric technology combining nano-enzyme with liquid-liquid microextraction. The synthetic antioxidant in the edible oil can be rapidly evaluated within 30 minutes.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a method for detecting butylated hydroxyanisole in edible oils using laccase-mimicking enzyme combined with dispersion microextraction. Background Technology

[0002] Butylated hydroxyanisole (BHA), also known as tert-butyl-4-hydroxyanisole, is a synthetic phenolic antioxidant (SPA). Due to its excellent efficacy, strong stability, wide availability, and cost-effectiveness, it is commonly used to preserve and extend the shelf life of food and is widely used in edible vegetable oils. However, numerous studies have shown that BHA is associated with potential toxicity, carcinogenicity, mutagenicity, and effects on the activity of human respiratory enzymes, raising public concern. Currently, my country's maximum legal limit for BHA in edible oils is 0.2 g / kg, while the EU and Japan prohibit its use in all food products. Therefore, it is necessary to strictly monitor the amount of BHA added to edible oils. The currently effective national standard GB / T 5009.30-2003 specifies the detection method for BHA in foods such as pastries and vegetable oils. This method requires the preparation of a chromatography column for purification, followed by solvent elution and gas chromatography determination. These steps are cumbersome, resulting in high reagent and material consumption and low recovery rates.

[0003] Carbon dot (CD) nanozymes have attracted much attention due to their unique small size, ease of synthesis, and excellent water dispersibility, and have been applied in various sensing and detection applications. Recent reports have explored the laccase-like activities of some copper-doped carbon dots; however, due to low enzyme activity, the phenolic substances that can be oxidized are limited, mainly chlorophenols, phenols, and aniline. Mimicking the active sites of natural laccases is an effective way to improve laccase-like activity. Addressing the current limitations in the catalytic capacity and substrate oxidation of laccase nanozymes, this invention synthesizes copper-dot laccase nanozymes from ultra-small green raw materials, significantly improving the activity and selectivity of laccase nanozymes for substrate oxidation. While colorimetric methods are simple and rapid, they also suffer from sample matrix interference; various pretreatment methods have been used to extract and purify SPAs from sample matrices. Dispersion-liquid microextraction (DLLME) technology based on green eutectic solvents (DESs) has been widely developed and researched due to its advantages such as flexible design, biodegradability, non-toxicity, simple preparation process, low volatility, and cost-effectiveness. Summary of the Invention

[0004] This invention discloses a method for detecting butylated hydroxyanisole (BHA) in edible oils using a laccase-mimicking enzyme combined with dispersion-liquid microextraction. The method uses L-aspartic acid and copper chloride as precursors, and synthesizes copper-doped carbon dots (Cu / Asp-CDs) with laccase-like nanozyme activity via microwave method. Cu / Asp-CDs exhibit excellent laccase-like nanozyme activity, selectively oxidizing BHA to quinone compounds, which then complex with 4-aminoantipyrine (4-AP) to form a red quinone imine dye with a UV absorption peak at 505 nm. Simultaneously, a eutectic solvent is prepared using choline chloride as a hydrogen bond acceptor and thymol as a hydrogen bond donor. A dispersion-liquid microextraction method is established for the separation and extraction of BHA from edible oil samples, thus establishing a rapid colorimetric detection method for BHA. When applied to the detection and analysis of BHA in edible oils, the recovery rate is 99.0%-105.1%, and the detection limit is 0.015 mg / kg. The results are consistent with GB5009.32-2016. The results of the determination of nine antioxidants in food according to national food safety standards are consistent; the colorimetric technique of nanoenzyme combined with liquid-liquid microextraction provides a practical analytical method for monitoring food quality in industry, and can rapidly evaluate synthetic antioxidants in edible oils within 30 minutes.

[0005] This invention relates to a method for detecting butylated hydroxyanisole (BHA) in edible oils using laccase-mimicking enzyme-linked dispersion microextraction, comprising the following steps: Includes the following steps: (1) Add 0.30-0.50 g L-aspartic acid, 0.30-0.50 g CuCl2·2H2O, 2.00-2.50 g citric acid, and 100-200 μL ethylenediamine to 30-50 mL of deionized water. Stir the mixture at room temperature for 30-40 min, react it in a microwave at 170-200℃ for 1-2 h, cool it naturally to room temperature, centrifuge, filter it through a 0.22 μm filter membrane, and vacuum dry it to obtain copper-doped carbon dots Cu / Asp-CDs; (2) After mixing Cu / Asp-CDs nanozyme solution, butylated hydroxyanisole solution of different concentrations, 4-aminoantipyrine solution and H2O2, PBS buffer solution of pH 7.0 was added and reacted for 5-10 min. The absorbance was measured at 505 nm wavelength to determine the linear relationship between butylated hydroxyanisole concentration and absorbance value and obtain the regression equation. (3) Add 8-12 parts by weight of deionized water to 1 part by weight of edible oil sample, add eutectic solvent, sonicate for 5-10 min, add Cu / Asp-CDs, 4-aminoantipyrine solution and H2O2, add pH 7.0 PBS buffer solution, vortex for 0.5-1 min, react for 5-10 min, centrifuge, form obvious layers, take out the upper layer, measure the absorbance of the eutectic solvent enriched phase of the lower layer at a wavelength of 505 nm, substitute the absorbance into the regression equation of step (1) to obtain the butylated hydroxyanisole content in the sample.

[0006] The preparation of the eutectic solvent involves mixing choline chloride and thymol in a molar ratio of 1:2-3 and stirring continuously at 70°C for 30-40 minutes.

[0007] The Cu / Asp-CDs nanozyme solution has a concentration of 5.0 μg / mL and an addition amount of 50-100 μL; the 4-aminoantipyrine solution has a concentration of 1 mmol / L and an addition amount of 50-100 μL; the H2O2 solution has a concentration of 50 mmol / L and an addition amount of 50-100 μL; and the pH 7.0 PBS buffer solution has a concentration of 0.1 mmol / L and an addition amount of 1-3 mL.

[0008] The centrifugation is performed at 8000-10000 r / min for 10-15 min. The advantages of this invention are: 1. This invention uses green amino acids and copper chloride as raw materials to prepare copper-doped carbon dots (Cu / Asp-CDs) with laccase-like activity via a one-step microwave method. The synthesized Cu / Asp-CDs are monodisperse ellipsoidal crystals with an average diameter of 2.8 nm and possess active copper centers similar to those of natural laccase. Enzyme kinetic experiments show that Cu / Asp-CDs achieve a maximum rate constant ( V max It is 30 times that of natural laccase, and its Michaelis constant is ( K m The activity of Cu / Asp-CDs is only about half that of natural laccase, indicating that it has a high affinity for laccase substrates and catalytic kinetics. Experimental results show that the catalytic mechanism of Cu / Asp-CDs is similar to that of natural laccase. Compared with natural laccase, Cu / Asp-CDs also exhibits better stability. Under a wide range of pH, temperature and storage conditions of more than 30 days, its catalytic activity can be maintained at more than 90%.

[0009] 2. The synthesized laccase-mimicking nanozyme can selectively oxidize butylated hydroxyanisole (BHA) into quinone compounds in the presence of H2O2, and then complex with 4-aminoantipyrine (4-AP) to form a red quinone imine dye with an ultraviolet absorption peak at 505 nm, thus establishing a rapid colorimetric detection method for BHA.

[0010] 3. The method established in this invention is used for the detection of BHA in edible oils. Combined with dispersive liquid-liquid microextraction, it not only eliminates sample matrix interference but also improves detection sensitivity, with a detection limit of 0.015 mg / kg. Compared with the GC method in the current national standard, it has higher detection sensitivity and has great advantages in terms of detection cost, operability, and time. The method also has good specificity. Attached Figure Description

[0011] Figure 1 TEM images (a, b) of Cu / Asp-CDs synthesized in Example 1 at different sizes; Figure 2 The image shows the XRD pattern of Cu / Asp-CDs synthesized in Example 1; Figure 3 This is a high-resolution XPS Cu 2p image of Cu / Asp-CDs synthesized in Example 1; Figure 4 The UV-Vis absorption spectrum of Cu / Asp-CDs oxidizing BHA+ H2O2+4-AP in Example 1; Figure 5 The fluorescence spectrum of the Cu / Asp-CDs + H2O2 system after the addition of PTA in Example 1; Figure 6 The Michaelis-Menten kinetics curves for the oxidation of 2,4-DP by (a) Cu / Asp-CDs and (b) natural laccase in Example 1 are shown. Figure 7 The Michaelis-Menten kinetics curves for the oxidation of 4-AP by (a) Cu / Asp-CDs and (b) natural laccase in Example 1 are shown. Figure 8 The catalytic stability of Cu / Asp-CDs and natural laccase in Example 1 under different (a) temperatures, (b) pH, and (c) storage times; Figure 9 The following are the (a) UV-Vis absorption spectra and (b) linear regression equations of BHA oxidized by Cu / Asp-CDs in Example 1. Figure 10 The results show the effects of coexisting ions, coexisting antioxidants, and unsaturated fatty acids on BHA detection. Figure 11 This is a diagram showing the effect of liquid-liquid microextraction. Detailed Implementation

[0012] 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.

[0013] Example 1: Determination of BHA in rapeseed oil samples 1. Preparation of Cu / Asp-CDs nanozymes: 0.30 g L-aspartic acid, 0.30 g CuCl2·2H2O, 2.00 g citric acid, and 100 μL ethylenediamine were added to 30 mL deionized water. The mixture was stirred at room temperature for 30 min, reacted at 200℃ for 1 h using a 1.2 kW microwave, and allowed to cool naturally to room temperature. The mixture was then centrifuged at 8000 rpm for 15 min, filtered through a 0.22 μm filter, and vacuum dried at 60℃ for 24 h to obtain copper-doped carbon dot Cu / Asp-CDs nanozymes. The prepared Cu / Asp-CDs nanozymes were analyzed by transmission electron microscopy (TEM), as shown below. Figure 1 As shown in (a,b), the prepared Cu / Asp-CDs are uniformly dispersed spherical nanoparticles with an average particle size of about 2.8 nm. The interplanar spacing of 0.23 nm observed in Cu / Asp-CDs matches the (100) crystal plane of graphite carbon. Figure 2 The X-ray diffraction (XRD) pattern of the prepared Cu / Asp-CDs nanozyme shows the (001) crystal plane of Cu / Asp-CDs at 2... θ A clear diffraction peak at 22° indicates the successful synthesis of highly crystalline Cu / Asp-CDs nanomaterials; X-ray photoelectron spectroscopy (XPS) analysis was used to determine the composition, valence state, and binding energy of the elements present in Cu / Asp-CDs, and the Cu 2p spectrum of Cu / Asp-CDs was obtained. Figure 3 The results showed that copper exists in the nanozyme structure in the form of Cu(I) and Cu(II). The peaks at binding energies of 931.1 eV and 950.9 eV belong to Cu(I), and the peaks at binding energies of 933.5 eV and 953.8 eV belong to Cu(II). The results indicate that the coexistence of Cu(II) and Cu(I) in Cu / Asp-CDs is beneficial to the electron transfer and cycling of Cu in the laccase activity of Cu / Asp-CDs.

[0014] 2. Evaluation of Cu / Asp-CDs nanozyme laccase activity: Using BHA as a substrate and 4-aminoantipyrine (4-AP) as a colorimetric reagent in the presence of H2O2, the mimic enzyme activity of laccase was determined. Figure 4In the presence of 4-AP, 2,4-DP can be oxidized by Cu / Asp-CDs to a quinone analog red product, producing a distinct absorption peak at 505 nm. The BHA+4-AP and BHA+4-AP+H2O2 systems showed no change. Since H2O2 significantly increased laccase-like activity, this experiment used terephthalic acid (PTA) as a probe to detect hydroxyl radicals (·OH). Figure 5 As shown, after co-incubation with H2O2 and Cu / Asp-CDs, the reaction with ·OH converts the non-fluorescent PTA into its fluorescent product, emitting light at around 440 nm. The addition of H2O2 promotes the generation of hydroxyl radicals, thereby enhancing the oxidation of BHA. Michaelis-Menten catalytic kinetics were determined using laccase-commonly used 2,4-chlorophenol (2,4-DP) and 4-AP as substrates. The results are shown in [Figure number missing]. Figure 6 , 7 As shown in Table 1, when 2,4-DP and 4-AP are used as substrates, the Cu / Asp-CDs nanozymes exhibit stronger substrate affinity and reaction rates than natural laccase. When 2,4-DP is used as a substrate, the catalytic rate of the Cu / Asp-CDs nanozymes is 30.2 times higher than that of natural laccase; when 4-AP is used as a substrate, the catalytic rate of the Cu / Asp-CDs nanozymes is 30.9 times higher than that of natural laccase. These results indicate that the Cu / Asp-CDs nanozymes synthesized in this invention possess excellent laccase-like activity.

[0015]

[0016] 3. Stability of Cu / Asp-CDs nanozymes: The stability of Cu / Asp-CDs under different pH, temperature, and storage time conditions was investigated. The results are as follows: Figure 8 As shown in Figure a, Cu / Asp-CDs retained 86% of their activity at pH 3.0 and 84% of their activity at pH 10.0, while laccase lost approximately 30% and 98% of its activity at pH 3.0 and pH 10.0, respectively. Therefore, compared to laccase, Cu / Asp-CDs are an effective catalyst over a wider pH range. Figure 8 As shown in b, at different temperatures (range 4~74℃), the laccase-like activity of Cu / Asp-CDs remained above 86%, and even at 74℃, it still retained 97% of its activity. In contrast, laccase activity decreased significantly after reaching 24℃, and was almost completely inactivated at 44℃ and above. Regarding the effect of storage time, as... Figure 8 As shown in c, Cu / Asp-CDs retained 84% of their activity after 33 days, while the activity of laccase decreased significantly after only 5 days, indicating that Cu / Asp-CDs have high stability.

[0017] 4. Preparation of BHA working curve: Add 100 µL of 5.0 μg / mL Cu / Asp-CDs nanozyme, 100 µL of 1 mmol / L 4-aminopyridine (4-AP), and 100 µL of 50 mmol / L H2O2 solution to a 5 mL stoppered colorimetric tube. Add BHA standard solution with a concentration ranging from 0.12 to 12.0 mg / L. Add 0.1 mmol / L pH 7.0 phosphate buffer to a final volume of 3 mL. React for 10 min, and measure the absorbance at 505 nm. The absorbance shows a linear relationship with the BHA concentration. See the UV-Vis absorption spectrum below. Figure 9 a. Plot a standard curve with BHA concentration on the x-axis and absorbance on the y-axis to obtain the regression equation, see [link to curve]. Figure 9 b; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2;

[0018] 5. Method Specificity Study: BHA was mixed with other antioxidants as a co-antioxidant. The effects of inorganic interfering substances (Na2CO3, MgCl2, K2SO4, Cu(NO3)2) and coexisting antioxidants, as well as unsaturated fatty acids (tert-butylhydroquinone (TBHQ), propyl gallate (PG), 2,6-di-tert-butyl-p-methylphenol (BHT), linseed oil, castor oil, oleic acid, and arachidic acid) on BHA were investigated. Figure 10 As can be seen, the Cu / Asp-CDs system exhibits good selectivity and specificity in the oxidation of BHA, and other substances do not significantly interfere with the BHA detection system. Therefore, the method for determining BHA demonstrates good selectivity and specificity.

[0019] 6. Determination of BHA in rapeseed oil samples (1) Preparation of eutectic solvent: 1 mol choline chloride and 2 mol thymol are mixed and stirred continuously at 70℃ for 30 min to obtain the solvent; (2) Sample determination: Accurately weigh 0.10 g of rapeseed oil, add 50% anhydrous ethanol to dilute to 1 mL, and simultaneously add 50 μL of the eutectic solvent prepared in step (1). Sonicate for 10 min, add 100 µL of 5.0 μg / mL Cu / Asp-CDs nanozyme, 100 µL of 1 mmol / L 4-AP, and 100 µL of 50 mmol / L H2O2 solution. Add 0.1 mmol / L pH 7.0 phosphate buffer solution to 3 mL, vortex for 0.5 min, react for 10 min, centrifuge at 8000 rpm for 10 min, and form obvious layers (e.g. Figure 11The upper layer was removed, and the absorbance of the eutectic solvent-enriched phase in the lower layer was measured at a wavelength of 505 nm. The absorbance was substituted into the regression equation in step (4) to obtain the BHA content in the sample as 8.10 mg / kg.

[0020] Example 2: Determination of BHA in corn oil samples 1. Preparation of Cu / Asp-CDs nanozymes: 0.50 g L-aspartic acid, 0.50 g CuCl2·2H2O, 2.50 g citric acid, and 200 μL ethylenediamine were added to 50 mL of deionized water. The mixture was stirred at room temperature for 40 min, reacted at 180℃ for 2 h under 1.2 kW microwave, and allowed to cool naturally to room temperature. The mixture was centrifuged at 10000 rpm for 10 min, filtered through a 0.22 μm filter membrane, and vacuum dried at 60℃ for 24 h to obtain copper-doped carbon dot Cu / Asp-CDs nanozymes.

[0021] 2. BHA working curve creation: Same as in Example 1; 3. Determination of BHA in corn oil samples: (1) Preparation of eutectic solvent: Same as in Example 1; (2) Sample determination: Same as in Example 1, the BHA content in the corn oil sample was 4.24 mg / kg.

[0022] Example 3: Determination of BHA in walnut oil 1. Preparation of Cu / Asp-CDs nanozymes: Same as in Example 1; 2. BHA working curve creation: Same as in Example 1; 3. Determination of BHA in walnut oil: (1) Preparation of eutectic solvent: 1 mol choline chloride and 2 mol thymol are mixed and stirred continuously at 70℃ for 30 min to obtain the solvent; (2) Sample determination: Same as in Example 1, the BHA content in walnut oil was 18.39 mg / kg.

[0023] (3) Recovery and precision experiments: Two different concentrations of BHA standard solution were added to the peanut oil sample; each concentration was measured in parallel three times, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 3. The spiked recovery rate of BHA was found to be 99.0% to 105.1%, and the RSD was 2.1% to 3.7%. This method has good accuracy and precision.

[0024]

[0025] The methods of the present invention used in Examples 1-3 were compared with those of the national standard GB5009.32-2016, "National Food Safety Standard: Determination of Nine Antioxidants in Food". The results are shown in Table 4. The results show that the two methods are consistent.

[0026] The BHA 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 butylated hydroxyanisole (BHA) in edible oils using laccase-mimicking enzyme combined with dispersion-liquid microextraction, characterized in that, Includes the following steps: (1) Add 0.30-0.50 g L-aspartic acid, 0.30-0.50 g CuCl2·2H2O, 2.00-2.50 g citric acid, and 100-200 μL ethylenediamine to 30-50 mL of deionized water. Stir the mixture at room temperature for 30-40 min, react it in a microwave at 170-200℃ for 1-2 h, cool it naturally to room temperature, centrifuge, filter it through a 0.22 μm filter membrane, and vacuum dry it to obtain copper-doped carbon dots Cu / Asp-CDs; (2) After mixing Cu / Asp-CDs nanozyme solution, butylated hydroxyanisole solution of different concentrations, 4-aminoantipyrine solution and H2O2, PBS buffer solution of pH 7.0 was added and reacted for 5-10 min. The absorbance was measured at 505 nm wavelength to determine the linear relationship between butylated hydroxyanisole concentration and absorbance value and obtain the regression equation. (3) Add 8-12 parts by weight of deionized water to 1 part by weight of edible oil sample, add eutectic solvent, sonicate for 5-10 min, add Cu / Asp-CDs, 4-aminoantipyrine solution and H2O2, add pH 7.0 PBS buffer solution, vortex for 0.5-1 min, react for 5-10 min, centrifuge, form obvious layers, take out the upper layer, measure the absorbance of the eutectic solvent enriched phase of the lower layer at a wavelength of 505 nm, substitute the absorbance into the regression equation of step (1) to obtain the butylated hydroxyanisole content in the sample.

2. The method according to claim 1, characterized in that: The preparation of the eutectic solvent involves mixing choline chloride and thymol in a molar ratio of 1:2-3 and stirring continuously at 70°C for 30-40 minutes.

3. The method according to claim 1, characterized in that: The concentration of Cu / Asp-CDs nanozyme solution was 5.0 μg / mL, and the addition volume was 50-100 μL; the concentration of 4-aminoantipyrine solution was 1 mmol / L, and the addition volume was 50-100 μL; the concentration of H2O2 solution was 50 mmol / L, and the addition volume was 50-100 μL; the concentration of pH 7.0 PBS buffer solution was 0.1 mmol / L, and the addition volume was 1-3 mL.

4. The method according to claim 1, characterized in that: Centrifugation is performed at 8000-10000 r / min for 10-15 min.

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