A pretreatment method for detecting aflatoxin in rice

Abstract

The present invention discloses a pretreatment method for detecting aflatoxin in rice, comprising the following steps: (1) preparing DES; (2) extracting aflatoxin; (3) preparing ionic liquid @β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material; (4) self-assembling an SPE column of IL@β-CD / rGO / MWCNTs material, and activating and pre-balancing; (5) extracting the extract A using an SPE column, and performing high performance liquid chromatography analysis on the eluent obtained by extraction to obtain a sample spectrum. The present invention has the advantages of simple extraction procedure, high efficiency, low price, good biocompatibility, non-volatile, and no by-products generated during the extraction process; secondly, the present invention has the advantages of short extraction time, high extraction efficiency, safety and environmental protection, small amount of extractant and sample, etc., which greatly reduces the cost of aflatoxin detection in rice, and has the advantages of simple operation and small amount, so it has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a pretreatment method for detecting aflatoxin in rice. Background Art

[0002] Aflatoxin (AF) is a highly toxic and carcinogenic derivative compound produced by Aspergillus flavus and Aspergillus parasiticus. There are many types of aflatoxins, and more than 20 types have been isolated and identified. According to the structure, chromatography and fluorescence characteristics, they are mainly divided into aflatoxin B group (AFB1, AFB2), G group (AFG1, AFG2) and their derivatives (AFM1, AFM2). The most common are the two major types of aflatoxins, group B and group G. Among them, AFB1 is the most harmful and carcinogenic, which is listed as a Class I carcinogen by FAO and WHO and is one of the main factors inducing malignant tumors such as primary hepatocellular carcinoma. For these reasons, China and other countries such as the European Union, Mexico and Brazil have set strict restrictions on AFs in grains and foods, with special attention to AFB1. For example, the EU stipulates that the total amount of AFs in imported rice and products must not exceed 4.0µg / kg, and the aflatoxin AFB1 in edible peanuts must not exceed 2.0µg / kg.

[0003] Rice is the main food crop for human consumption, and more than half of the world's population relies on rice as their staple food. As one of the three major staple foods, rice occupies an important position in the grain industry, so it is crucial to ensure the safety of rice. Studies have shown that rice is very susceptible to contamination by mold and the AFs it produces due to poor storage environment, simple grain storage equipment, and lack of scientific management methods. Aflatoxin infection of rice causes a large amount of AFs to be deposited in the rice, which not only reduces the seed germination rate, but also causes irreversible damage to people's bodies once these rice enter the market and are eaten by people. At the same time, because a lot of moldy rice is directly discarded, it also causes a lot of waste, causing huge economic losses to the grain industry. Therefore, the detection of aflatoxin in rice is crucial.

[0004] In the process of aflatoxin detection, the pretreatment process is a key step. Rice and other food crops are rich in sugars, amino acids, small molecules and other substances, which can easily produce matrix effects during the detection process, reducing the sensitivity of the detection and even causing false positives. Therefore, pretreatment of the detection object can fully eliminate the interference of the analyte or achieve the purpose of pre-concentrating the target, thereby improving the sensitivity, precision and accuracy of the detection. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a pretreatment method for detecting aflatoxin in rice. The present invention first uses a deep eutectic solvent (DES) to extract aflatoxin from rice, then uses an ionic liquid-modified β-cyclodextrin@graphene oxide-multi-walled carbon nanotube composite material to purify the aflatoxin extract, and finally combines it with HPLC technology to achieve ultra-sensitive detection of aflatoxin in rice. Compared with other detection methods, the amount of organic solvent used in the present invention is greatly reduced, and the detection is more sensitive.

[0006] The present invention is specifically implemented by the following technical solutions:

[0007] The present invention provides a pretreatment method for detecting aflatoxin in rice, which comprises the following steps:

[0008] (1) Preparation of DES: Mix hydrogen bond acceptor tetrabutylammonium bromide and hydrogen bond donor levulinic acid evenly to obtain a pure and uniform liquid;

[0009] (2) Aflatoxin extraction: the solution obtained in step (1) is mixed with water to obtain an extraction solvent, and the rice powder and the extraction solvent are mixed evenly at a solid-liquid ratio of 1:10-40 g / mL to obtain an aflatoxin extract A;

[0010] (3) Preparation of ionic liquid@β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite materials:

[0011] A. mixing graphene oxide, ionic liquid, β-cyclodextrin, water and multi-walled carbon nanotubes and performing a prepolymerization reaction to obtain a prepolymer;

[0012] B. mixing the prepolymer, the crosslinking agent and the reducing agent and performing a polymerization reaction, filtering, washing and freeze-drying the obtained product to obtain an ionic liquid @β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material, i.e., IL@β-CD / rGO / MWCNTs material;

[0013] (4) Self-assemble the SPE column of IL@β-CD / rGO / MWCNTs material using 1 mL SPE empty column tube and polypropylene gasket, and activate and pre-balance the SPE column;

[0014] (5) The extract A obtained in step (2) is extracted using the SPE column obtained in step (4), and the eluate obtained by the extraction is analyzed by high performance liquid chromatography to obtain a sample spectrum.

[0015] Furthermore, in step (1), the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 1:3.

[0016] Furthermore, in step (1), the hydrogen bond acceptor and the hydrogen bond donor are subjected to a co-melting treatment at 80 to 90° C. for 3 to 4 hours.

[0017] Furthermore, during the eutectic treatment in step (1), the rotation speed of the shaking table is 200 rpm.

[0018] Furthermore, in step (2), the solution and water are configured to form an extraction solvent with a volume ratio of 10 to 90%, preferably 40 to 70%.

[0019] Furthermore, in step (2), the rice powder is mixed with the extraction solvent and shaken in a shaker at 30 to 45° C. and 150 to 230 rpm for 1 to 3 hours.

[0020] Furthermore, the ionic liquid in A of step (3) is 1-butyl-3-methyl-imidazole acetate.

[0021] Furthermore, in step (3), the amount ratio of graphene oxide, ionic liquid, β-cyclodextrin, water and multi-walled carbon nanotubes in A is (30-50) mL: (5-15) g: (5-10) g: (80-120) mL: (0.10-0.20) g, and the concentration of graphene oxide is 2 mg / mL.

[0022] Furthermore, after the graphene oxide, ionic liquid, β-cyclodextrin, water and multi-walled carbon nanotubes are mixed in A of step (3), magnetic stirring is performed for 30 to 60 minutes, and then ultrasonic dissolution is performed for 30 to 60 minutes to perform a prepolymerization reaction.

[0023] Furthermore, in step (3) B, the cross-linking agent is one of glutaraldehyde, epichlorohydrin or citric acid; and the reducing agent is L-ascorbic acid.

[0024] Furthermore, the amount ratio of the prepolymer, the crosslinking agent and the reducing agent in B of the step (3) is (120-160) g: (4-6) mL: (2.5-3.5) g.

[0025] Furthermore, the polymerization reaction temperature in step (3) B is 80-90° C. and the reaction time is 12-24 hours.

[0026] Furthermore, the product in step (3) B is filtered, washed with ultrapure water for 3-5 times, and freeze-dried for 24 hours. The freeze-drying conditions are -40 to -50°C and 8 to 15 MPa.

[0027] Furthermore, in step (4), the amount of IL@β-CD / rGO / MWCNTs material used is 2-20 mg, preferably 12 mg.

[0028] Furthermore, in step (5), the solid-liquid ratio of IL@β-CD / rGO / MWCNTs material to extract A is 4-10:1 mg / ml, the extraction temperature is 20-45°C, the extraction time is 20-40 min, and the pH is 4-9.

[0029] Another aspect of the present invention provides an ionic liquid@β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material, which is obtained by the following steps:

[0030] A. mixing graphene oxide, ionic liquid, β-cyclodextrin, water and multi-walled carbon nanotubes and performing a prepolymerization reaction to obtain a prepolymer;

[0031] B. The prepolymer, the cross-linking agent and the reducing agent are mixed and then subjected to polymerization reaction. The obtained product is filtered, washed and freeze-dried to obtain an ionic liquid @β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material, namely, IL@β-CD / rGO / MWCNTs material.

[0032] Another aspect of the present invention provides the use of the above-mentioned ionic liquid@β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material in purifying flavonoids extract.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention firstly uses a new green extraction solvent to extract trace aflatoxin in rice. The extraction procedure is simple and efficient. Compared with traditional organic solvents, DES has the advantages of low raw material price, good biocompatibility, non-volatile, simple process operation, and no by-products produced during the extraction process, which fully reflects the characteristics of green chemistry. Secondly, the present invention prepares a new porous material to replace the expensive immunoaffinity column as an extractant. The IL@β-CD / rGO / MWCNTs new extraction material has a pore surface area and strong specific adsorption capacity. It can be reused for more than 10 times, integrating concentration and extraction, and has the advantages of simple and convenient operation, short extraction time, high extraction efficiency, safety and environmental protection, and small amount of extractant and sample. It greatly reduces the cost of aflatoxin detection in rice, and has the advantages of simple operation and small amount, so it has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Liquid phase diagram of aflatoxin standard;

[0036] Figure 2 Liquid phase diagram of aflatoxin detection after pre-treatment of rice samples using the method of the present invention;

[0037] Figure 3Effects of deep eutectic solvent (DES) water content (a), liquid-to-solid ratio (b), extraction temperature (c) and extraction time (d) on the extraction rate of aflatoxin in rice.

[0038] Figure 4 The influence of different conditions on the extraction effect; a is the amount of extractant, b is different temperatures, c is the extraction time and d is the extraction pH;

[0039] Figure 5 The influence of repeated use of IL@β-CD / rGO / MWCNTs on the extraction effect. DETAILED DESCRIPTION

[0040] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0041] Example 1: Preparation of ionic liquid @β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite (IL@β-CD / rGO / MWCNTs)

[0042] A: 80 mg of graphene oxide (concentration of graphene oxide is 2 mg / mL), 10 g of ionic liquid (1-butyl-3-methyl-imidazole acetate), 8 g of β-cyclodextrin, 100 mL of water and 0.10 g of multi-walled carbon nanotubes were mixed, magnetically stirred for 30 min, and then ultrasonically dissolved for 30 min to carry out a prepolymerization reaction to obtain a prepolymer.

[0043] B: 150 g of the prepolymer, 5 ml of the cross-linking agent (glutaraldehyde) and 3 g of the reducing agent (L-ascorbic acid) were mixed, magnetically stirred for 15 min, and after uniform dissolution, polymerization reaction was carried out at 90° C. for 12 h. After the reaction, the obtained product was filtered and washed with ultrapure water 5 times, and freeze-dried (freeze-drying conditions were -40 to -50° C., 8 to 15 MPa) for 24 h to obtain an ionic liquid @β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material.

[0044] Example 2: A pretreatment method for detecting aflatoxin in rice

[0045] (1) Preparation of DES: Mix the hydrogen bond acceptor tetrabutylammonium bromide and the hydrogen bond donor levulinic acid in a molar ratio of 1:2, put them into a sealed glass bottle, shake them at 200 rpm in a shaker at 90°C for 3 to 4 hours to obtain a pure and uniform liquid, and then let it stand at 30°C overnight.

[0046] (2) Aflatoxin extraction: The liquid obtained in (1) was mixed with water to prepare a 40% (volume ratio) extraction solvent. The rice powder and the extraction solvent were evenly mixed at a solid-liquid ratio of 1:20 g / mL. The mixture was shaken in a shaker at 40°C and 200 rpm for 3 h to obtain aflatoxin extract A.

[0047] (3) Use 1 mL SPE empty column tube and polypropylene gasket to self-assemble the SPE column of IL@β-CD / rGO / MWCNTs material obtained in Example 1. First, put a polypropylene gasket into the empty column tube, then load 12 mg of IL@β-CD / rGO / MWCNTs material, flick the tube wall by hand to make the material evenly distributed in the column tube, and finally place another polypropylene gasket on top of the material to compact the IL@β-CD / rGO / MWCNTs material so that the filling material remains at the bottom of the SPE tube.

[0048] (4) Rinse the SPE column prepared in step (3) with 3 mL of methanol and 3 mL of ultrapure water, respectively, to activate and pre-equilibrate it.

[0049] (5) Add 1 mL of the extract A obtained in step (2) to the SPE column activated in step (4), and perform extraction after the loading is completed, wherein the solid-liquid ratio of IL@β-CD / rGO / MWCNTs material to the extract A is 4-10:1 mg / ml, the extraction temperature is 20-45°C, the extraction time is 20-40 min, and the pH is 4-9. Subsequently, rinse the column with 4 mL of ultrapure water, then desorb with 3 mL of methanol, and then blow dry the desorbed liquid with nitrogen at 40°C. Finally, re-dissolve with 1 mL of methanol solution, sonicate for 5 min, filter the re-solution with a 0.22µm microporous filter membrane and store it in a sample bottle for high performance liquid chromatography analysis to obtain a sample spectrum. The liquid phase diagram of aflatoxin standard is shown in the figure below. Figure 1 As shown, the liquid phase diagram of aflatoxin detection after pre-treating the rice sample using the method of the present invention is as follows Figure 2 shown.

[0050] Example 3: Optimization and verification of pretreatment conditions for detecting aflatoxin in rice

[0051] Detection instrument: Waters liquid chromatograph is used, including e2695 separation chromatography system host, column oven and 2475 fluorescence detection, and 254 nm photochemical derivatizer.

[0052] Sample processing: The rice was dried at 50°C until constant weight, then ground with a grinder, passed through an 80-mesh sieve, and placed in a dryer for later use.

[0053] Extraction process: Accurately weigh 1g of rice, add a certain volume (5, 10, 20, 30, 40mL) of DES (water content of 20, 30, 40, 50, 60, 70, 80, 90%), and shake in a shaker at 150-230rpm at different temperatures (25, 30, 35, 40, 45℃) (time is 5, 10, 30, 60, 120min) to obtain aflatoxin extract A.

[0054] Extraction process: First, the SPE column filled with 12 mg IL@β-CD / rGO / MWCNTs was activated. After activation in the centrifuge tube, 1.0 mL of water sample was added and allowed to stand at 25°C for 30 min. Subsequently, the column was rinsed with 3-6 mL of ultrapure water, and then desorbed with 2-6 mL of methanol. The desorbed solution was then dried with nitrogen at 40°C. Finally, 1 mL of methanol solution was used for re-dissolution. After ultrasonication for 5 min, the re-solution was filtered with a 0.22 µm microporous membrane and stored in a sample bottle for HPLC analysis.

[0055] HPLC-FLD method determination: The peak areas of the four aflatoxins were determined using the following chromatographic conditions, and the contents of AFB1, AFB2, AFG1 and AFG2 were obtained by substituting the standard working curve equation (Table 1). Chromatographic conditions: chromatographic column: Waters XBrigdeTMC18 column (5μm, 250 × 4.6 mm); isocratic elution with acetonitrile: methanol: water (10:25:65); excitation wavelength of 360nm, emission wavelength of 450nm; column temperature of 35℃, injection volume of 20μL.

[0056] Table 1 Linear equation and range of aflatoxin

[0057]

[0058] Results and Analysis

[0059] Optimization of extraction conditions

[0060] Optimization of DES water content: According to the above extraction conditions, other experiments were the same, and the effects of DES water content of 20, 30, 40, 50, 60, 70, 80, and 90% on the extraction efficiency of the target compound were investigated. Figure 3 As shown in (a), when the moisture content is 60%, the extraction efficiency reaches the maximum value. If the moisture content continues to increase, the extraction efficiency will decrease.

[0061] Optimization of liquid-to-solid ratio: According to the above extraction conditions, other experiments were the same, and the effects of different liquid-to-solid ratios of 5, 10, 20, 30, and 40 mL / g on the extraction efficiency of the target compound were investigated. Figure 3As shown in (b), when the liquid-to-solid ratio reaches 10 mL / g, the extraction efficiency reaches its maximum value. If the liquid-to-solid ratio continues to increase, the extraction efficiency will decrease. This may be due to the results and physical properties of DES itself.

[0062] Optimization of extraction temperature: According to the above extraction conditions, other experiments were the same, and the effects of different extraction temperatures (20℃, 25℃, 35℃, 40℃, and 45℃) on the extraction efficiency of the target compound were investigated. Figure 3 As shown in (c), when the extraction temperature increases from 20°C to 30°C, the extraction efficiency of aflatoxin gradually increases, while when the temperature is further increased, the extraction rate of aflatoxin does not change much. From the perspective of energy saving, 30°C is selected.

[0063] Optimization of extraction time: According to the above extraction conditions, other experiments were the same, and the effects of different extraction times of 5, 10, 30, 60, and 120 min on the extraction efficiency of the target compound were investigated. Figure 3 As shown in (d), when the extraction time is 60 min, the extraction efficiency of the target compound is the highest. After further extending the extraction time, the extraction efficiency of the target compound does not change significantly, indicating that the extraction process has reached equilibrium.

[0064] Optimization of extraction conditions: In order to obtain higher extraction efficiency, IL@β-CD / rGO / MWCNTs solid phase extraction was used to extract extractants containing aflatoxin, including AFB1, AFB2, AFG1 and AFG2, and the effects of different extraction parameters on the recovery of aflatoxin in extract A were studied. Therefore, in order to establish a solid-liquid microextraction method for herbicide solid phase extraction using IL@β-CD / rGO / MWCNTs, the parameters controlling the performance, such as the optimization of solid-liquid ratio, temperature, time and pH, were investigated.

[0065] Investigation of extraction solid-liquid ratio: The selection of extractant is crucial in the process of liquid phase microextraction. A good extractant can often effectively extract the analyte from a large amount of matrix with a small amount, achieving the purpose of separation and enrichment at the same time. This study conducted experiments with solid-liquid ratios of 2:1, 4:1, 6:1, 8:1, 10:1 and 12:1 (mg / mL). Figure 4 aIt can be seen that when the solid-liquid ratio is 8:1, the extraction recovery rate reaches the maximum, so it is selected for the next experiment.

[0066] Extraction temperature investigation: Temperature has a certain influence on the extraction efficiency. This study conducted experiments on the extraction efficiency in the temperature range of 20-45℃. Figure 4 b It can be seen that when the extraction temperature is 30℃, the extraction recovery rate reaches the maximum, so the solid-liquid ratio of 8:1 and the extraction temperature of 30℃ are selected for the next experiment.

[0067] Extraction time investigation: In solid-liquid microextraction research, extraction time is also a key factor affecting extraction efficiency. This study studied the extraction efficiency of 10-120 min. Figure 4 c It can be seen that the extraction recovery rate basically reaches 100% at 30 minutes, that is, aflatoxin in extract A is completely extracted, so the extraction time is selected as 30 minutes.

[0068] Extraction pH investigation: pH is an important parameter that affects the ionic form of herbicides. Figure 4 d It can be seen that the extraction recovery rate is the highest at pH 6, so the extraction pH is selected as 6.

[0069] Material reuse research: Material reuse is an important indicator for evaluating the quality of materials. Figure 5 It can be seen that after IL@β-CD / rGO / MWCNTs was reused for 6 times, the recovery rate of four aflatoxin can still reach more than 80%.

Claims

1. A pretreatment method for detecting aflatoxin in rice, characterized in that The following steps are involved: (1) Preparation of DES: Mix hydrogen bond acceptor tetrabutylammonium bromide and hydrogen bond donor levulinic acid evenly to obtain a pure and uniform liquid; (2) Aflatoxin extraction: the uniform liquid obtained in step (1) is mixed with water to obtain an extraction solvent, and the rice powder and the extraction solvent are uniformly mixed at a solid-liquid ratio of 1:10-40 g / mL to obtain an aflatoxin extract A; (3) Preparation of ionic liquid@β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composites: A. mixing graphene oxide, ionic liquid, β-cyclodextrin, water and multi-walled carbon nanotubes and performing a prepolymerization reaction to obtain a prepolymer, wherein the ionic liquid is 1-butyl-3-methyl-imidazole acetate; B. mixing the prepolymer, the crosslinking agent and the reducing agent and performing a polymerization reaction, filtering, washing and freeze-drying the obtained product to obtain an ionic liquid @β-cyclodextrin-reduced graphene oxide-multi-walled carbon nanotube composite material, i.e., IL@β-CD / rGO / MWCNTs material; (4) Self-assemble the SPE column of IL@β-CD / rGO / MWCNTs material using 1 mL SPE empty column tube and polypropylene gasket, and activate and pre-balance the SPE column; (5) The extract A obtained in step (2) is extracted using the SPE column obtained in step (4). The column is first rinsed with 3-6 mL of ultrapure water and then desorbed with 2-6 mL of methanol. The eluate obtained by extraction is analyzed by high performance liquid chromatography to obtain a sample spectrum.

2. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (1), the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 1:

3.

3. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (1), the hydrogen bond acceptor and the hydrogen bond donor are eutectic treated at 80-90° C. for 3-4 hours.

4. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (2), the uniform liquid and water are mixed into an extraction solvent with a volume ratio of 10 to 90%. The rice powder and the extraction solvent are mixed and shaken in a shaker at 30 to 45° C. and a rotation speed of 150 to 230 rpm for 1 to 3 hours.

5. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (3), the usage ratio of graphene oxide, ionic liquid, β-cyclodextrin, water and multi-walled carbon nanotubes is (30-50) mL: (5-15) g: (5-10) g: (80-120) mL: (0.10-0.20) g, and the concentration of graphene oxide is 2 mg / mL.

6. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (3), the cross-linking agent is one of glutaraldehyde, epichlorohydrin or citric acid; the reducing agent is L-ascorbic acid; and the amount ratio of the prepolymer, the cross-linking agent and the reducing agent is (120-160) g: (4-6) mL: (2.5-3.5) g.

7. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (3), the polymerization reaction temperature is 80-90° C. and the reaction time is 12-24 hours.

8. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (4), the amount of IL@β-CD / rGO / MWCNTs material used is 2-20 mg.

9. A pretreatment method for detecting aflatoxin in rice as claimed in claim 1, characterized in that In step (5), the solid-liquid ratio of IL@β-CD / rGO / MWCNTs material to extract A is 4-10:1 mg / mL, the extraction temperature is 20-45°C, the extraction time is 20-40 min, and the pH is 4-9.

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