A kind of mycotoxin extractant and preparation method thereof

Abstract

The present application provides a mycotoxin extractant and a preparation method thereof, and also provides an application method thereof, which is particularly used for pre-treatment of samples, such as for the pre-treatment steps in mycotoxin detection of various samples such as grains, feed, Chinese medicinal materials or decoction pieces, and can be used simultaneously for the detection of aflatoxins, zearalenone, vomitoxin, ochratoxin A, fumonisins and T2 toxin, which can improve the efficiency and accuracy of detection.

Description

Technical Field

[0001] The present application relates to a mycotoxin extractant and a preparation method thereof, and belongs to the field of biochemical analysis and detection. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by fungi. One fungus may produce multiple toxins, and multiple fungi may produce one toxin. More than 1,000 mycotoxins have been discovered worldwide, and more than 20 have been isolated and identified. Among them, the most harmful ones are aflatoxin AFLA, vomitoxin (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisins (FUM), T-2 toxin, fumonisins and ergot toxins. Mycotoxins are widely present in grain, oil, food and feed, as well as in Chinese medicinal materials or decoction pieces. If Chinese medicinal materials are not dried in time after collection, stored improperly or handled poorly during preparation and processing, they may be contaminated by various fungi and produce mycotoxins.

[0003] Mycotoxins will eventually enter our food chain through contaminated food, feed, and Chinese medicine, thereby showing carcinogenicity, genotoxicity, and teratogenicity to humans and animals. Due to the widespread contamination of mycotoxins and the severity of their harm, my country has stipulated limits on mycotoxins in food, feed, Chinese medicinal materials, or decoction pieces, and there is an urgent need for efficient and accurate methods to detect mycotoxins in various samples.

[0004] However, the complexity of the samples poses challenges to testing. For example, in addition to the wide variety of agricultural products, the matrix background of traditional Chinese medicine is also complex. Pigments, mucus, starch, protein and different active ingredients in traditional Chinese medicine (such as flavonoids, anthraquinones, phenanthroquinone compounds, etc.) may affect the test results. Therefore, different extraction steps are needed to eliminate the influence of the matrix.

[0005] For example, feed includes both feed raw materials and various compound feeds. Different feeds have different raw material compositions, such as starch feed, oil feed, sugar feed and protein feed, etc. Compound feed has complex ingredients and usually contains feed additives. Therefore, different samples usually require different extractants to obtain more accurate results.

[0006] Therefore, people are pursuing a universal method that can detect mycotoxins in various samples efficiently and accurately at the same time. Existing universal extraction methods mainly use organic solvents such as methanol, ethanol, acetonitrile, etc. as extractants, but such extraction methods are not only difficult to remove the influence of the matrix, but also easy to cause environmental pollution. In order to solve the above problems, it is very necessary to develop a safe, environmentally friendly, highly accurate mycotoxin extractant that is universally applicable to various samples. Summary of the invention

[0007] The present application firstly provides a mycotoxin extractant, comprising yeast extract, polyethylene glycol, polydimethylsiloxane and buffer salt.

[0008] In some embodiments, the yeast extract is yeast extract powder and yeast extract.

[0009] In some embodiments, the buffer salt comprises a phosphate, a carbonate, a borate, or an organic acid salt.

[0010] In some embodiments, the average molecular weight of polyethylene glycol is 2000-4000 Daltons, preferably polyethylene glycol 2000 or polyethylene glycol 4000.

[0011] In some embodiments, the extractant is prepared by: completely dispersing polydimethylsiloxane in polyethylene glycol, and then mixing with yeast extract and buffer salt; dissolving with deionized water to obtain the extractant when used;

[0012] In some embodiments, the contents of yeast extract powder, polyethylene glycol, polydimethylsiloxane and buffer salt in the extractant are 4% to 6% (mass fraction), 2% to 4% (mass fraction), 0.03% to 0.07% (mass fraction) and 0.015 to 0.025 mol / L, respectively; and the pH of the extractant is 6.8 to 7.6.

[0013] In some embodiments, the contents of yeast extract powder, polyethylene glycol, polydimethylsiloxane and buffer salt in the extractant are 4.8% to 5.2% (mass fraction), 2.8% to 3.2% (mass fraction), 0.04% to 0.06% (mass fraction) and 0.018 to 0.022 mol / L, respectively; and the pH of the extractant is 7.0 to 7.4.

[0014] In order to mix quickly and thoroughly, polydimethylsiloxane must first be dispersed in polyethylene glycol and then mixed with other reagents, otherwise it will be difficult to mix evenly.

[0015] The present application also provides use of the above-mentioned extractant in extracting fungal toxins from samples.

[0016] In some embodiments, the sample includes agricultural products (such as grains), feed, and traditional Chinese medicine (such as Chinese medicinal materials or decoction pieces).

[0017] In some embodiments, the cereals include wheat, barley, oats, rye, buckwheat, paddy, rice, millet, soybean, broad bean, pea, mung bean, red bean, kidney bean, sorghum, corn, millet, and millet products thereof.

[0018] In some embodiments, the feed includes soybean meal, DDGS (Distillers Dried Grains with Solubles), rice husks, cottonseed meal, and the like.

[0019] In some embodiments, the Chinese medicinal materials or decoction pieces include Polygala tenuifolia, Coix seeds, Platycladus chinensis seeds, American ginseng, etc.

[0020] In some embodiments, the mycotoxins include: aflatoxins, zearalenone, deoxynivalenol, ochratoxin A, fumonisins, T2 toxin, fumonisins, and / or ergot toxins.

[0021] The present application also provides a method for detecting mycotoxins in a sample, comprising the steps of extracting the mycotoxins in the sample using the above-mentioned extractant, and then performing a detection step.

[0022] In some embodiments, the sample includes agricultural products (such as grains), feed, and traditional Chinese medicine (such as Chinese medicinal materials or decoction pieces).

[0023] In some embodiments, the cereals include wheat, barley, oats, rye, buckwheat, paddy, rice, millet, soybean, broad bean, pea, mung bean, red bean, kidney bean, sorghum, corn, millet, and millet products thereof.

[0024] In some embodiments, the feed includes soybean meal, DDGS (Distillers Dried Grains with Solubles), rice husks, cottonseed meal, and the like.

[0025] In some embodiments, the Chinese medicinal materials or decoction pieces include Polygala tenuifolia, Coix seeds, Platycladus chinensis seeds, American ginseng, etc.

[0026] In some embodiments, the mycotoxins include: aflatoxins, zearalenone, deoxynivalenol, ochratoxin A, fumonisins, T2 toxin, fumonisins, and / or ergot toxins.

[0027] In some embodiments, the method comprises adding an extractant to the sample, shaking, standing and then centrifuging; diluting before detection, and then using a colloidal gold detection strip for detection.

[0028] In some embodiments, the steps of extracting mycotoxins from a sample using the above extractant are as follows: weigh 5 g of the powder sample into a clean extraction container, add 15 ml of the extractant, and shake vigorously for at least 1.5-2 minutes. If particles are present, let stand for at least 1 minute to obtain a sample extract. Pipette 1.0-1.5 mL of the extract into a centrifuge tube and centrifuge for at least 10 seconds.

[0029] Based on the above situation, the inventors have developed a mycotoxin extractant. Its advantages are that first, it does not contain organic solvents and is environmentally friendly; second, the extractant is in powder form, which is easy to transport and store; third, the sample liquid after extraction can be used for aflatoxin, zearalenone, vomitoxin, ochratoxin A, fumonisin and T2 toxin detection strips at the same time, saving operation time and steps; fourth, yeast extract is a by-product of yeast and is commonly used in the field of food and biological fermentation. Yeast extract is made by leaching soluble substances in yeast cells through processes such as autolysis, enzymolysis, and concentration. It is rich in vitamins, enzymes, peptides, nucleic acids and other nutrients, and thus helps to extract intercellular or intracellular mycotoxins at the same time. DETAILED DESCRIPTION

[0030] In order to make the present invention described in this application more fully understood, the following examples are described. It should be appreciated that these examples are only for illustrative purposes and should not be construed as limiting the present invention in any way.

[0031] The present application provides a mycotoxin extractant, characterized in that it includes yeast extract, polyethylene glycol, polydimethylsiloxane and buffer salt. In some embodiments, the contents of yeast extract powder, polyethylene glycol, polydimethylsiloxane and buffer salt in the extractant are 4% to 6% (mass fraction), 2% to 4% (mass fraction), 0.03% to 0.07% (mass fraction) and 0.015 to 0.025 mol / L, respectively, and the pH of the extractant is 6.8-7.6. More preferably, the contents of yeast extract, polyethylene glycol, polydimethylsiloxane and buffer salt are 4.8% to 5.2%, 2.8% to 3.2%, 0.04% to 0.06% and 0.018 to 0.022 mol / L, respectively. In some embodiments, the pH of the extractant is 7.0 to 7.4. In some embodiments, the yeast extract is preferably yeast extract powder.

[0032] In some embodiments, the extractant is prepared as follows: first, polydimethylsiloxane is completely dispersed in polyethylene glycol, and then mixed with yeast extract and buffer salt; when used, it is dissolved in deionized water to obtain the extractant.

[0033] The present application also provides the use of the above extractant in detecting mycotoxins in a sample. In some embodiments, the sample may include agricultural products, feed, or traditional Chinese medicine.

[0034] The present application also provides a method for detecting mycotoxins in a sample, comprising the steps of extracting the mycotoxins in the sample using the above-mentioned extractant, and then performing a detection step. Specifically, the method comprises: adding the above-mentioned extractant to the sample, shaking, standing, and then centrifuging; diluting before detection, and then using a colloidal gold detection strip for detection.

[0035] Example 1 Experimental process

[0036] 1. Preparation method of mycotoxin extractant:

[0037] Weigh 30.0g of polyethylene glycol 4000 and 0.5g of polydimethylsiloxane, and disperse the polydimethylsiloxane completely in the polyethylene glycol 4000. Then add 50.0g of yeast extract powder and 0.02 mol of phosphate, mix well, and dissolve in 1L of deionized water before use.

[0038] 2. Mycotoxin extraction method:

[0039] Weigh 5g of powder sample into a clean extraction container, add 15ml of extraction reagent to 5g of powder sample, and shake vigorously for 1.5 minutes. If particles are present, let stand for 1 minute to obtain sample extract. Transfer 1.0mL of extract to a centrifuge tube and centrifuge for 10 seconds to form a test solution.

[0040] 3. Mycotoxin detection method:

[0041] The test solution was mixed with a dilution buffer (0.1 M PBS buffer) to obtain a diluted test solution. The fungal toxins in the sample were detected using a colloidal gold test strip. The test solution was added to the test strip and kept warm at 45°C. After completion, the quantitative results were read using a detector.

[0042] Example 2 Effect of oscillation time on test results in the extraction method

[0043] Wheat samples with negative mycotoxin residues were selected, and a total of 6 mycotoxins, including aflatoxin, zearalenone, vomitoxin, ochratoxin A, fumonisin, and T2 toxin, were spiked at a concentration of 100 μg / kg. The spiked samples of each drug were divided into 5 parts, and extracted and tested according to the method of Example 1 of the present application, wherein the oscillation time was 0.5 minutes, 1 minute, 1.5 minutes, 2 minutes, and 2.5 minutes, respectively, and repeated three times. The test results were averaged, and the oscillation time and test results were shown in Table 1, where accuracy = (average value of the test results / spiked concentration) * 100%.

[0044] Table 1 Effect of oscillation time on the results in the extraction method

[0045]

[0046]

[0047] As can be seen from Table 1, as the oscillation time increases, the accuracy of the test results increases first and then decreases. When the oscillation time is 1.5-2 minutes, the accuracy is the highest, which is 97.7-100.0%. Therefore, 1.5-2 minutes of oscillation can be selected as the optimal oscillation time. An oscillation time that is too short (such as less than 1.5 minutes) or too long (such as more than 2.5 minutes) will affect the accuracy of the test results.

[0048] Example 3 Effects of the Mycotoxin Extractant of the Present Application and Traditional Extractants on the Detection Results

[0049] Mycotoxin-negative samples of wheat, corn, rice, and soybean were selected for mycotoxin spikes. The types of mycotoxins and the corresponding spike concentrations are shown in Tables 2-4. The mycotoxin extractant of Example 1 of the present application and 70% methanol and 50% ethanol were used to extract the samples according to the method of Example 1, and then the colloidal gold detection strips and detectors were used for detection. Three samples were tested for each concentration of each spiked toxin, and the results were averaged. The spike concentrations and test results are shown in Tables 2-4.

[0050] Table 2 Test results of the extraction reagents in this application

[0051]

[0052]

[0053]

[0054] Table 3 Detection results of 70% methanol

[0055]

[0056]

[0057] Table 4 50% ethanol test results

[0058]

[0059]

[0060]

[0061] As shown in Tables 2-4, the accuracy of the sample extraction using the mycotoxin extraction reagent described in the present application and the detection using the colloidal gold detection strip and the detector is 98.0-100.0%; the accuracy of the sample extraction using 70% methanol is 93.0-96.0%; the accuracy of the sample extraction using 50% ethanol is 80.0-85.0%. Therefore, the extraction reagent described in the present application has the highest accuracy.

[0062] Example 4 Extraction effect of the mycotoxin extractant and BSA extractant of the present application

[0063] Positive samples with known mycotoxin content (see Table 5 for specific sample information) were selected, and the mycotoxin extractant and BSA extractant of Example 1 of the present application (the difference between the two is that 5% BSA is used to replace the yeast extract powder in the present application) were used to extract the samples according to the method of Example 1, and then the colloidal gold test strip and the detector were used for detection. Each sample was tested in 3 parallels, and the results were averaged. The spiked concentration and the test results are shown in Table 5.

[0064] Table 5 Extraction effect of the mycotoxin extractant and BSA extractant of the present application

[0065]

[0066]

[0067] As shown in Table 5, the accuracy of the sample extraction using the mycotoxin extraction reagent described in the present application and the detection using the colloidal gold detection strip and the detector is 97.9-99.8%, and the accuracy of the sample extraction using the BSA extraction agent is 73.7-92.1%. Therefore, the extraction effect of the extraction reagent described in the present application is better.

[0068] Example 5 The extraction effect of the extraction reagent of the present application on various toxins in the grain matrix

[0069] Wheat, corn, rice, soybean and sorghum samples with negative mycotoxin residues were selected and spiked with aflatoxin, zearalenone, vomitoxin, ochratoxin A, fumonisin and T2 toxin (see Table 6 for the spike concentration corresponding to each toxin). Each sample was divided into 3 parts and extracted using the extraction reagent and extraction method of Example 1 of the present application. Then, the samples were tested using colloidal gold test strips and a detector. The test results were averaged, and the spike concentrations and test results are shown in Table 6.

[0070] Table 6 Extraction effect of mycotoxins in different sample types

[0071]

[0072]

[0073] As can be seen from Table 6, the extraction rates of different toxins in wheat, corn, rice, soybean and sorghum by the fungal toxin extraction reagent described in the present application are 97.0-99.6%, 97.0-100.1%, 98.0-100.0%, 97.0-99.7% and 97.0-100.0%, respectively. Therefore, the extraction reagent has a good extraction effect on different toxins in different grain samples.

[0074] Example 6 Extraction effect of the extraction reagent of the present application on various toxins in feed matrix

[0075] Soybean meal, cottonseed meal, DDGS, rice husk and piglet feed samples with negative mycotoxin residues were selected and spiked with aflatoxin, zearalenone, vomitoxin, ochratoxin A, fumonisin and T2 toxin (see Table 7 for the spike concentration corresponding to each toxin). Each sample was divided into 3 parts and extracted using the extraction reagent and extraction method of Example 1 of the present application. Then, the colloidal gold test strips and the detector were used for detection. The test results were averaged, and the spike concentrations and test results are shown in Table 7.

[0076] Table 7 Extraction effect of mycotoxins in different feed matrices

[0077]

[0078]

[0079] As can be seen from Table 7, the extraction rates of different toxins in soybean meal, cottonseed meal, DDGS, rice husk and piglet feed by the fungal toxin extraction reagent described in the present application are 98.7-99.7%, 98.3-99.7%, 98.3-99.3%, 98.3-99.3% and 98.0-99.5%, respectively. Therefore, the extraction reagent has a good extraction effect on different toxins in different feed samples.

[0080] Example 7 The extraction effect of the extraction reagent of the present application on various toxins in different traditional Chinese medicine matrices

[0081] Polygala tenuifolia, coix seed, cypress seed, American ginseng and tangerine peel samples with negative fungal toxin residues were selected, and spiked with aflatoxin, zearalenone, vomitoxin, ochratoxin A, fumonisin and T2 toxin (see Table 8 for the spiked concentration corresponding to each toxin). Each sample was divided into 3 parts, extracted using the extraction reagent and extraction method of Example 1 of the present application, and then tested using colloidal gold test strips and a detector. The test results were averaged, and the spiked concentrations and test results are shown in Table 8.

[0082] Table 8 Extraction effect of mycotoxins in different sample types

[0083]

[0084]

[0085] As can be seen from Table 8, the extraction rates of different toxins in Polygala tenuifolia, Coix seeds, Platycladus orientalis seeds, American ginseng and dried orange peel by the fungal toxin extraction reagent described in the present application are 98.3-100.0%, 97.0-99.4%, 97.0-99.4%, 97.0-99.5% and 98.0-100.6%, respectively. Therefore, the extraction reagent has a good extraction effect on different toxins in different Chinese medicine samples.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application is described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should all be included in the scope of the claims and specification of the present application. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall within the scope of the claims.

Claims

1. A mycotoxin extractant, characterized in that: Includes yeast extract, polyethylene glycol, polydimethylsiloxane and buffer salts.

2. The extractant according to claim 1, characterized in that The contents of the yeast extract, polyethylene glycol, polydimethylsiloxane and buffer salt are 4% to 6%, 2% to 4%, 0.03% to 0.07% and 0.015 to 0.025 mol / L respectively; and the pH of the extractant is 6.8 to 7.

6.

3. The extractant according to claim 1, characterized in that The preparation method of the extractant is as follows: firstly, polydimethylsiloxane is completely dispersed in polyethylene glycol, and then mixed with yeast extract and buffer salt; when used, it is dissolved in deionized water to obtain the extractant.

4. Use of the extractant according to any one of claims 1 to 3 in extracting mycotoxins from a sample.

5. The use according to claim 4, characterized in that The samples include agricultural products, feed, or traditional Chinese medicine; wherein the agricultural products include grains; the grains include wheat, barley, oats, rye, buckwheat, paddy, millet, soybeans, broad beans, peas, mung beans, red beans, kidney beans, sorghum, corn, millet, and their products; the traditional Chinese medicine includes Chinese medicinal materials or decoction pieces.

6. The use according to claim 4, characterized in that The mycotoxins include: aflatoxins, zearalenone, deoxynivalenol, ochratoxin A, fumonisins, T2 toxin, fumonisins and / or ergot toxins.

7. A method for detecting mycotoxins in a sample, characterized in that: The method comprises the steps of extracting the fungal toxins in the sample using the extractant according to any one of claims 1 to 3, and then performing a detection step.

8. The method according to claim 7, characterized in that The samples include agricultural products, feed, or traditional Chinese medicine; wherein the agricultural products include grains; the grains include wheat, barley, oats, rye, buckwheat, paddy, millet, soybeans, broad beans, peas, mung beans, red beans, kidney beans, sorghum, corn, millet, and their products; the traditional Chinese medicine includes Chinese medicinal materials or decoction pieces.

9. The method according to claim 7, characterized in that The mycotoxins include: aflatoxins, zearalenone, deoxynivalenol, ochratoxin A, fumonisins, T2 toxin, fumonisins and / or ergot toxins.

10. The method according to claim 7, characterized in that The method comprises adding the extractant according to any one of claims 1 to 3 to the sample, shaking, standing and then centrifuging; diluting before detection, and then using a colloidal gold detection strip for detection.

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