Preparation method for mycotoxin in feed

Abstract

The invention belongs to the technical field of feed safety detection, and discloses a feed mycotoxin preparation method, which comprises the following specific steps: 1, raw material treatment; step 2, ultrasonic extraction; step 3, primary concentration; step 4, molecularly imprinted solid-phase extraction; step 5, SFC purification; and step 6, post-processing and detecting. According to the method, the ultrasonic-assisted extraction and molecular imprinting solid-phase extraction technologies are integrated, so that the target mycotoxin can be efficiently extracted in a complex matrix, the influence of other interfering substances is remarkably reduced, and high-selectivity adsorption and separation of the mycotoxin are realized by adopting the magnetic nanoparticle-loaded MIP technology; compared with a traditional preparation method, the high-purity standard substance extracted by the method provides an accurate and repeatable calibration basis for subsequent feed detection and quantitative analysis, and the accuracy and reliability of a detection result are ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed safety detection, and specifically relates to a preparation method for feed mycotoxins. Background Art

[0002] With the rapid development of animal husbandry and aquaculture, feed safety issues have attracted increasing attention. As one of the common contaminants in feed, the toxicity and carcinogenicity of mycotoxins have been widely confirmed. It not only poses a threat to animal health but also may pose potential risks to human health through the food chain. Therefore, accurately detecting the residual levels of mycotoxins in feed is of great significance for ensuring the safety of animal products and public health.

[0003] Currently, the detection methods for mycotoxins in feed mainly rely on modern analytical techniques such as high-performance liquid chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). However, the accuracy and reliability of these detection methods largely depend on the quality of reference substances. Therefore, a preparation method for feed mycotoxins is needed to obtain high-purity mycotoxin reference standards, enabling them to provide accurate calibration bases for detection methods, thereby achieving quantitative analysis and risk assessment of mycotoxins in feed. Currently, when preparing mycotoxins, due to the presence of various impurities and other interfering substances in the raw materials, the purity of the prepared mycotoxins is low, and the safety risks of preparing mycotoxins are high. Therefore, it is necessary to improve and optimize them. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method for feed mycotoxins to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A preparation method for feed mycotoxins, and the specific steps of this preparation method are as follows:

[0006] Step 1, raw material treatment: Select verified contaminated feed and microbial strains that produce mycotoxins to ensure that the raw materials contain the target mycotoxins and perform pretreatment;

[0007] Step 2, ultrasonic extraction: Utilize ultrasonic-assisted technology to mix the raw materials with green organic solvents to further break the cell structure and promote the release of mycotoxins from the matrix;

[0008] Step 3, preliminary concentration: Concentrate the extract through a rotary evaporator under reduced pressure to remove most of the solvents and obtain a crude extract rich in the target mycotoxins;

[0009] Step 4, Molecularly imprinted solid-phase extraction: For the target mycotoxin, design a template molecule based on its structural characteristics, synthesize MIP particles with specific binding sites, and prepare the MIP particles on the surface of magnetic nanoparticles to form magnetic MIP;

[0010] Step 5, SFC purification: Through supercritical fluid chromatography technology, and using supercritical CO2 as an environmentally friendly solvent, supplemented with a small amount of modifier to optimize the separation;

[0011] Step 6, Post-treatment and detection: Use high-performance liquid chromatography-mass spectrometry to quantify and confirm the structure of the purified mycotoxin to ensure that the purity meets the standard requirements.

[0012] Preferably, the contaminated feed samples should be collected from feed batches known to have a risk of mycotoxin contamination, and at least 5 representative samples should be collected and mixed evenly to reduce individual differences;

[0013] The microbial strains should be selected as known high-yield mycotoxin strains identified by molecular biology, and cultured using a standard medium under the conditions of a temperature of 28-30 °C and a relative humidity of more than 80%.

[0014] Preferably, the specific steps of the raw material treatment are as follows:

[0015] A1, Pre-freeze the collected feed samples to enhance the brittleness of the samples;

[0016] A2, Grind and mill at low temperature to ensure that the final powder particle size is uniform, with the target of an average particle size not exceeding 0.5 mm;

[0017] A3, Use an ultrasonic crusher to treat the homogenized powder, set the ultrasonic power at about 200-300 W, control the time within 5-10 minutes, and the interval between each treatment is 30 seconds;

[0018] A4, Mix the crushed sample with n-hexane in a ratio of 1:10, stir for 30 minutes to fully dissolve the oil;

[0019] A5, Centrifuge to separate the solid and liquid, and discard the upper layer of solvent;

[0020] A6, Repeat the degreasing operation 2-3 times to confirm that the oil content is less than 1%.

[0021] Preferably, the specific steps of the ultrasonic extraction stage are as follows:

[0022] B1, First prepare a 70% ethanol aqueous solution as a green organic solvent, and then mix the pretreated sample with the 70% ethanol solution in a ratio of 1:10;

[0023] B2, placing the solution mixture in the container of the ultrasonic extractor and setting the parameters;

[0024] B3, set the ultrasonic frequency to 40kHz and the ultrasonic power to 200-300W;

[0025] B4, use a water bath to maintain a constant temperature and extract at 25-35°C for 30 minutes;

[0026] B5, after the extraction is completed, turn off the equipment, filter out impurities by centrifugal filtration, and finally collect the supernatant.

[0027] Preferably, the specific steps of the molecular imprinting solid phase extraction are:

[0028] C1, after the prepared magnetic MIP particles are uniformly dispersed, they are loaded into a special solid phase extraction column to form a magnetic MIP extraction material;

[0029] C2, reconstitute the crude extract extracted and concentrated by ultrasonication in a buffer, adjust the pH to 4.5-5.0, and allow the buffer to slowly pass through the magnetic MIP column;

[0030] C3, using low concentration ethanol aqueous solution to wash away nonspecifically bound impurities, and after elution, using an external magnetic field to quickly adsorb and separate the magnetic MIP particles from the solution;

[0031] C4, collect the final eluate and test the concentration of mycotoxins.

[0032] Preferably, in the post-processing and detection stage, the purified mycotoxins are quantified and their structures are confirmed by high performance liquid chromatography to ensure that the purity meets the standard requirements and are stored in a low temperature and inert gas environment.

[0033] Preferably, the method is carried out in a closed system during the preparation of mycotoxins, from extraction, concentration, magnetic MIP separation to SFC purification, and key parameters are monitored online in real time.

[0034] Preferably, the MIP particles use the target mycotoxin as a template molecule, and are free radical polymerized in a solvent with functional monomers and cross-linking agents to form a polymer network. The magnetic nanoparticles are surface silanized to provide rich functional groups, and MIP is synthesized in situ on their surface to obtain magnetic MIP particles.

[0035] The beneficial effects of the present invention are as follows:

[0036] 1. By integrating ultrasonic-assisted extraction and molecularly imprinted solid-phase extraction technologies, the method of the present invention can efficiently extract target mycotoxins from complex matrices, significantly reducing the influence of other interfering substances. By adopting the MIP technology loaded with magnetic nanoparticles, highly selective adsorption and separation of mycotoxins are achieved, thereby obtaining high-purity standards. Compared with traditional preparation methods, the high-purity standards extracted by this method provide an accurate and reproducible calibration basis for subsequent feed detection and quantitative analysis, ensuring the accuracy and reliability of detection results.

[0037] 2. The present invention performs ultrasonic-assisted extraction with green organic solvents and operates under low-temperature conditions, effectively avoiding the degradation of mycotoxins caused by high temperatures and significantly reducing the environmental pollution risk of organic solvents. At the same time, during the preparation operation, the method is fully operated within a closed automated system, which not only improves the operation efficiency but also reduces the direct contact risk between experimental personnel and highly toxic substances, ensuring experimental safety and environmental friendliness.

[0038] 3. Through an online automated monitoring system, the present invention monitors the temperature, pressure, pH, and flow rate in key steps such as extraction, enrichment, and purification in detail, shortening the entire preparation time and reducing manual operation errors. Moreover, compared with traditional preparation methods, the magnetic MIP solid-phase extraction used in this method reduces the use of a large amount of solvents and sample treatment steps, improving the repeatability and stability of the overall process. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the preparation flow chart of the mycotoxin of the present invention;

[0040] Figure 2 is the flow chart of the molecularly imprinted solid-phase extraction step of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] As Figures 1 to 2 shown, the embodiments of the present invention provide a preparation method for feed mycotoxins. The specific steps of this preparation method are as follows:

[0043] Step 1, raw material treatment: Select verified contaminated feed and microbial strains that produce mycotoxins to ensure that the raw materials contain target mycotoxins (such as aflatoxin, fumonisin, zearalenone, etc.), and perform pretreatment;

[0044] Step 2, ultrasonic extraction: Using ultrasonic-assisted technology, the raw materials are mixed with a green organic solvent (such as ethanol or its mixed solvent system) to further break the cell structure and promote the release of mycotoxins from the matrix.

[0045] Step 3, preliminary concentration: The extract is concentrated by a rotary evaporator under reduced pressure to remove most of the solvent, obtaining a crude extract rich in the target mycotoxin.

[0046] Step 4, molecularly imprinted solid-phase extraction: For the target mycotoxin, a template molecule is designed based on its structural characteristics, MIP particles with specific binding sites are synthesized, and the MIP particles are prepared on the surface of magnetic nanoparticles to form magnetic MIP.

[0047] Step 5, SFC purification: Through supercritical fluid chromatography (SFC) technology, and using supercritical CO2 as an environmentally friendly solvent, supplemented with a small amount of modifier (such as methanol or acetonitrile) to optimize the separation.

[0048] By integrating ultrasonic-assisted extraction and molecularly imprinted solid-phase extraction technologies, the method can efficiently extract the target mycotoxin from complex matrices, significantly reducing the influence of other interfering substances. By using the MIP technology loaded on magnetic nanoparticles, high-selectivity adsorption and separation of mycotoxins are achieved, thus obtaining high-purity standards. Compared with traditional preparation methods, the high-purity standards extracted by this method provide an accurate and reproducible calibration basis for subsequent feed detection and quantitative analysis, ensuring the accuracy and reliability of the detection results.

[0049] Step 6, post-treatment and detection: High-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used to quantify and confirm the structure of the purified mycotoxin to ensure that the purity meets the standard requirements.

[0050] Among them, the contaminated feed samples should be collected from feed batches known to have a risk of mycotoxin contamination, and at least 5 representative samples should be collected and mixed evenly to reduce individual differences.

[0051] After the sample collection and mixing, the initially collected samples need to be quickly detected to ensure that the target toxins (such as aflatoxin, fumonisin, zearalenone, etc.) are present in the samples and the concentration meets the requirements of subsequent process extraction.

[0052] The microbial strains should be selected as known high-yield mycotoxin strains identified by molecular biology and cultured using a standard medium under the conditions of a temperature of 28 - 30 °C and a relative humidity of more than 80%.

[0053] Control the culture period within 7 to 10 days so as to collect the culture when the mold is in the high mycotoxin production period. The collected culture should be pretreated as soon as possible to prevent degradation.

[0054] Among them, the specific steps of the raw material treatment are as follows:

[0055] A1. Pre-freeze the collected feed sample (it can be frozen in a liquid nitrogen environment for 3 to 5 minutes) to enhance the brittleness of the sample.

[0056] A2. Conduct crushing and grinding under low-temperature conditions to ensure that the final powder particle size is uniform, with the target of an average particle size not exceeding 0.5 mm.

[0057] A3. Use an ultrasonic crusher to treat the homogenized powder, set the ultrasonic power at about 200–300 W, control the time within 5 to 10 minutes, and have a 30-second interval for each treatment.

[0058] A4. Mix the crushed sample with n-hexane at a ratio of 1:10 (w / v), and stir for 30 minutes to fully dissolve the oil.

[0059] A5. Separate the solid and liquid by centrifugation (for example, 4000 rpm, 10 minutes), and discard the upper-layer solvent.

[0060] A6. Repeat the degreasing operation 2 - 3 times to confirm that the oil content is less than 1%.

[0061] First, homogenize the sample, then break the cell wall by crushing to release the mycotoxins bound inside the cells, and finally reduce the contents of oil and macromolecular interfering substances in the feed sample through degreasing operations to improve the selectivity of subsequent solvent extraction.

[0062] Among them, the specific steps of the ultrasonic extraction stage are as follows:

[0063] B1. First, prepare a 70% ethanol aqueous solution as a green organic solvent, and then mix the pretreated sample with the 70% ethanol solution at a ratio of 1:10 (w / v).

[0064] B2. Place the solution mixture in the container of the ultrasonic extractor and set the parameters.

[0065] B3. Set the ultrasonic frequency to 40 kHz and the ultrasonic power to 200 - 300 W.

[0066] B4. Use a water bath to maintain a constant temperature and extract for 30 minutes within the range of 25 - 35 °C.

[0067] B5. After the extraction is completed, turn off the equipment, filter out the impurities by centrifugation, and finally collect the supernatant (this extract contains a high concentration of mycotoxins).

[0068] 70% ethanol can not only dissolve mycotoxins well, but also moderately dissolve other components inside cells, which is conducive to the release of toxins after cell structure disruption. At the same time, it has low toxicity and is more environmentally friendly.

[0069] Ultrasonic-assisted extraction is carried out with green organic solvents and operated under low-temperature conditions, effectively avoiding the problem of mycotoxin degradation caused by high temperature. At the same time, the risk of environmental pollution by organic solvents is greatly reduced. At the same time, during the preparation operation, the whole process is operated in a closed automated system, which not only improves the operation efficiency but also reduces the direct contact risk of experimental personnel with highly toxic substances, ensuring experimental safety and environmental friendliness.

[0070] Among them, the specific steps of the molecularly imprinted solid-phase extraction are as follows:

[0071] C1, After the prepared magnetic MIP particles are evenly dispersed, they are filled into a special solid-phase extraction column to form a magnetic MIP extraction material;

[0072] C2, The crude extract obtained by ultrasonic extraction and concentration in the early stage is reconstituted in a buffer solution, and the pH is adjusted to 4.5 - 5.0, and then the buffer solution is slowly passed through the magnetic MIP column;

[0073] C3, Use a low-concentration ethanol aqueous solution to wash away non-specifically bound impurities, and after elution, quickly adsorb and separate the magnetic MIP particles from the solution by using an external magnetic field;

[0074] In the elution stage, a weakly polar eluent can be used first to wash away non-specifically bound impurities, and in the way of gradually increasing the solvent polarity in a gradient manner, gradually switch to a solution with a higher organic solvent ratio to desorb the firmly bound target toxin, ensuring that the target toxin is effectively dissociated under mild conditions without damaging its structure.

[0075] C4, Collect the final eluate and test the concentration of the mycotoxin.

[0076] Among them, in the post-treatment and detection stage, the purified mycotoxin is quantified and its structure is confirmed by high-performance liquid chromatography (LC-MS / MS) to ensure that the purity meets the standard requirements, and it is stored under low temperature and inert gas environment.

[0077] Among them, in the preparation process of the mycotoxin, from extraction, concentration, magnetic MIP separation to SFC purification, all are completed in a closed system, and key parameters (such as temperature, pressure, pH and flow rate) are monitored in real time online.

[0078] Through an online automated monitoring system, the temperature, pressure, pH, and flow rate in key steps such as extraction, enrichment, and purification are monitored in detail, shortening the overall preparation time and reducing manual operation errors. Moreover, compared with traditional preparation methods, the magnetic MIP solid-phase extraction used in this method reduces the use of a large amount of solvents and sample treatment steps, improving the repeatability and stability of the overall process.

[0079] Among them, the MIP particles use the target mycotoxin as a template molecule, cooperate with a functional monomer and a crosslinking agent to carry out free radical polymerization in a solvent to form a polymer network. The magnetic nanoparticles are treated by surface silanization to provide abundant functional groups and in-situ synthesize MIP on their surface to obtain magnetic MIP particles.

[0080] The magnetic nanoparticles are prepared by the co-precipitation method to prepare Fe3O4 nanoparticles, and the target particle size is controlled within the range of 50-200 nm to ensure a large specific surface area and good magnetic response. Then, the Fe3O4 particles are dispersed in a solution containing TEOS and reacted under alkaline conditions to form a uniform SiO2 coating layer to improve surface activity and provide binding sites for subsequent MIP polymerization; the MIP particles use the target mycotoxin as a template molecule, and according to the structural characteristics of the template molecule, methacrylic acid or other suitable functional monomers are added in a molar ratio, and then a crosslinking agent (such as divinylbenzene) is added to ensure the formation of a firm and highly selective polymerization network; finally, the previously modified magnetic Fe3O4 nanoparticles are dispersed in the MIP precursor solution and placed in a constant temperature water bath at 60-70 °C under nitrogen protection for a polymerization reaction for 4-6 hours to uniformly form the MIP layer on the surface of the magnetic particles.

[0081] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0082] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing feed mycotoxins, characterized in that: The specific steps of the preparation method are: Step 1, raw material processing: select verified contaminated feed and culture microbial strains that produce mycotoxins, ensure that the raw materials contain target mycotoxins, and perform pretreatment; Step 2, ultrasonic extraction: using ultrasound-assisted technology, the raw materials are mixed with green organic solvents to further break up the cell structure and promote the release of mycotoxins from the matrix; Step 3, preliminary concentration: concentrating the extract by a reduced pressure rotary evaporator to remove most of the solvent to obtain a crude extract rich in the target mycotoxin; Step 4, molecular imprinting solid phase extraction: for the target mycotoxin, the template molecule is designed based on its structural characteristics, MIP particles with specific binding sites are synthesized, and the MIP particles are prepared on the surface of magnetic nanoparticles to form magnetic MIP; Step 5, SFC purification: through supercritical fluid chromatography technology, using supercritical CO2 as an environmentally friendly solvent, supplemented by a small amount of modifier to optimize separation; Step 6. Post-processing and testing: Use high performance liquid chromatography-mass spectrometry to quantify and confirm the structure of the purified mycotoxins to ensure that the purity meets the standard requirements.

2. The method for preparing feed mycotoxins according to claim 1, characterized in that: The contaminated feed samples should be collected from feed batches known to have mycotoxin contamination risks, and no less than 5 representative samples should be collected and mixed evenly to reduce individual differences; The microbial strains shall be selected from strains known to produce high mycotoxins identified by molecular biology, and shall be cultured using standard culture medium at a temperature of 28 to 30° C. and a relative humidity of more than 80%.

3. The method for preparing feed mycotoxins according to claim 1, characterized in that: The specific steps of the raw material processing are: A1, pre-freeze the collected feed samples to enhance the brittleness of the samples; A2, grinding and crushing at low temperature to ensure uniform particle size of the final powder, with the goal of an average particle size not exceeding 0.5 mm; A3, use ultrasonic crusher to treat the homogenized powder, set the ultrasonic power to about 200-300W, control the time to 5-10 minutes, and each treatment interval is 30 seconds; A4, mix the crushed sample with n-hexane in a ratio of 1:10 and stir for 30 minutes to fully dissolve the oil; A5, separate the solid and liquid by centrifugation and discard the upper solvent; A6, repeat the degreasing operation 2-3 times to confirm that the oil content is less than 1%.

4. The method for preparing feed mycotoxins according to claim 1, characterized in that: The specific steps of the ultrasonic extraction stage are: B1, first prepare 70% ethanol aqueous solution as green organic solvent, then take the pretreated sample and mix it with 70% ethanol solution in a ratio of 1:10; B2, placing the solution mixture in the container of the ultrasonic extractor and setting the parameters; B3, set the ultrasonic frequency to 40kHz and the ultrasonic power to 200-300W; B4, use a water bath to maintain a constant temperature and extract at 25-35°C for 30 minutes; B5, after the extraction is completed, turn off the equipment, filter out impurities by centrifugal filtration, and finally collect the supernatant.

5. The method for preparing feed mycotoxins according to claim 1, characterized in that: The specific steps of the molecular imprinting solid phase extraction are: C1, after the prepared magnetic MIP particles are uniformly dispersed, they are loaded into a special solid phase extraction column to form a magnetic MIP extraction material; C2, reconstitute the crude extract extracted and concentrated by ultrasonication in a buffer, adjust the pH to 4.5-5.0, and allow the buffer to slowly pass through the magnetic MIP column; C3, using low concentration ethanol aqueous solution to wash away nonspecifically bound impurities, and after elution, using an external magnetic field to quickly adsorb and separate the magnetic MIP particles from the solution; C4, collect the final eluate and test the concentration of mycotoxins.

6. The method for preparing feed mycotoxins according to claim 1, characterized in that: In the post-processing and detection stage, the purified mycotoxins are quantified and their structures are confirmed by high performance liquid chromatography to ensure that the purity meets the standard requirements and are stored in a low temperature and inert gas environment.

7. The method for preparing feed mycotoxins according to claim 1, characterized in that: In this method, the preparation process of mycotoxins, from extraction, concentration, magnetic MIP separation to SFC purification, is completed in a closed system, and key parameters are monitored online in real time.

8. The method for preparing feed mycotoxins according to claim 1, characterized in that: The MIP particles use the target mycotoxin as a template molecule, and are combined with functional monomers and cross-linking agents to perform free radical polymerization in a solvent to form a polymer network. The magnetic nanoparticles are treated with surface silanization to provide rich functional groups, and MIP is synthesized in situ on their surface to obtain magnetic MIP particles.

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