A method for the production of carbon-13 labeled vomitoxin and a toxin-producing culture medium employed

Abstract

The application belongs to the technical field of toxin preparation, and discloses a preparation method of carbon 13 labeled vomitoxin and a toxin production culture medium used in the method. The preparation method of the carbon 13 labeled vomitoxin comprises the following steps: S1, spore production culture: inoculating activated fusarium in a spore production culture medium, culturing at 20-30 DEG C for 5-7 days, and then collecting spores; S2, toxin production culture: transferring the collected spores into a toxin production culture medium for culture, culturing for 7-14 days under shock conditions, and obtaining a fermentation liquor containing carbon 13 labeled vomitoxin; and S3, vomitoxin extraction: adding sodium chloride to a saturated state, then adding ethyl acetate, shock extraction, centrifugal collection of the upper ethyl acetate phase, and combination of the ethyl acetate phases to obtain a crude extract containing carbon 13 labeled vomitoxin. The method has simple production process steps, high preparation efficiency, low production cost, high labeling rate, and can also be used as a raw material to prepare various carbon 13 labeled vomitoxin derivatives.

Description

Technical Field

[0001] This invention relates to the field of toxin preparation technology, and in particular to a method for preparing carbon-13 labeled vomitoxin and the toxin-producing culture medium used. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin, belongs to the trichothecene family of compounds. Vomitoxin is primarily produced by *Fusarium graminearum* and *Fusarium culmorum*. These fungi mostly grow slowly in grain crops during the cold, humid season and harvest time. Vomitoxin often coexists with other mycotoxins, such as 3-ACDON, 15-ACDON, ZON, NIV, and FX. When humans and animals ingest grains and their products containing this toxin, it can easily lead to liver damage and harm their health. Therefore, screening and hazard assessment of vomitoxin in grains and their products are essential.

[0003] Currently, methods for detecting vomitoxin include thin-layer chromatography, gas chromatography, liquid chromatography, and liquid chromatography-mass spectrometry. These methods require a large amount of standard substances for accurate determination of toxin concentration in samples. However, isotope internal standards combined with mass spectrometry can eliminate matrix interference through precise determination of isotope abundance and accurate weighing of the added internal standard. Furthermore, isotope internal standards can eliminate differences in sample pretreatment, enabling quantitative determination of the analyte.

[0004] Therefore, the existing technology needs further improvement. Summary of the Invention

[0005] To address the current lack of mature methods for producing isotope-labeled vomitoxin standards in China, this invention provides a low-cost, simple, and efficient method for producing carbon-13 labeled vomitoxin, along with a toxin-producing culture medium. This method is suitable for the industrial production of vomitoxin standards.

[0006] To address the above problems, this application provides the following technical solution:

[0007] In a first aspect, this application provides a method for preparing carbon-13 labeled vomiting toxin, which includes the following steps:

[0008] S1. Sporulation culture: Activated Fusarium is inoculated into a sporulation culture medium and cultured at 20-30℃ for 5-7 days, after which spores are collected.

[0009] S2. Toxin production culture: The collected spores are transferred to a toxin production culture medium and cultured at 20-30℃ and 120-200r / min for 7-14 days to obtain a fermentation broth containing carbon-13 labeled vomitoxin.

[0010] S3. Extraction of vomitoxin: Add sodium chloride to the fermentation broth until saturated, then add ethyl acetate, shake and extract for 0.5-1.5 h, centrifuge and collect the upper ethyl acetate phase, repeat the extraction at least twice, combine the ethyl acetate phases to obtain a crude extract containing carbon-13 labeled vomitoxin.

[0011] Preferably, the conditions for sporulation culture are: culture at 25℃ for 6 days; and the conditions for toxin production culture are: culture at 25℃ with shaking at 160 r / min for 10 days.

[0012] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the spore concentration inoculated during the toxin-producing culture is 10. 3~ 10 4 The volume ratio of added ethyl acetate to the original mixture was 1:1.

[0013] Preferably, in step S2, the final spore concentration in the culture reaches 1×10⁻⁶ at the time of inoculation. 4 per mL.

[0014] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the sporulation culture medium is prepared from the following components: sodium carboxymethyl cellulose (CMC) 10 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and peptone 1 g / L.

[0015] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the toxin-producing culture medium is prepared from the following components:

[0016] The mixture consisted of 10–20 g / L of C-13-labeled carbon source, 0.5–2 g / L of nitrogen source, 0.1–1 g / L of magnesium sulfate heptahydrate, 0.5–2 g / L of potassium dihydrogen phosphate, 0.1–1 g / L of potassium chloride, 0.01–0.05 g / L of ferrous sulfate heptahydrate, 0.01–0.04 g / L of manganese chloride, 0.05–0.15 g / L of ε-PL, and 20–500 μL of trace element solution, with the remainder being water as solvent.

[0017] Optionally, in the method for preparing the carbon-13 labeled vomitoxin, the toxin-producing culture medium is prepared from the following components: 10 g / L of C-13 labeled carbon source, 1 g / L of ammonium chloride, 0.5 g / L of magnesium sulfate heptahydrate, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of potassium chloride, 0.02 g / L of ferrous sulfate heptahydrate, 0.1 g / L of ε-PL, 0.04 g / L of manganese chloride, and 100 μL of trace element mixture, with the remainder being water as solvent.

[0018] Optionally, the carbon source is one or more of 13C-glucose, 13C-D-mannose, 13C-sodium acetate, and 13C-sucrose-13C-glycerol. The nitrogen source is ammonium chloride or ammonium sulfate.

[0019] More preferably, the nitrogen source is ammonium chloride, and the carbon source is 13C-glucose.

[0020] Optionally, in the method for preparing the carbon-13 labeled vomitoxin, the trace element mixture contains the following components at the following concentrations: zinc sulfate heptahydrate 0.5 g / L, copper sulfate heptahydrate 0.025 g / L, manganese sulfate monohydrate 0.052 g / L, boric acid 5 mg / L, and sodium molybdate dihydrate 5 mg / L.

[0021] Preferably, the trace element mixture contains the following components at the following concentrations: zinc sulfate heptahydrate 0.5 g / L, copper sulfate heptahydrate 0.025 g / L, manganese sulfate monohydrate 0.052 g / L, boric acid 5 mg / L, and sodium molybdate dihydrate 5 mg / L.

[0022] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the following step is further included after step S3:

[0023] S4. Crude purified vomiting toxin: Purified by high-performance liquid chromatography using an ODS-BP column. The mobile phase was 0.1 vol% formic acid-water-methanol, with mobile phase A being 0.1 vol% formic acid-water and mobile phase B being methanol.

[0024] The elution procedure was as follows: elution of phase B at 5% isocratic concentration for 5 min, elution of phase B at 5%-100% gradient for 40 min, and elution of phase B at 100% isocratic concentration for 10 min. The eluent with a retention time of approximately 31.7 min was collected, which was the crude pure solution of carbon-13 labeled vomitoxin.

[0025] Optionally, the crude vomitoxin solution can be transferred to a rotary evaporator, concentrated under reduced pressure at 35°C, freeze-dried, and then reconstituted with 50% acetonitrile water to obtain the crude extract for later use.

[0026] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the following step is further included after step S4:

[0027] S5, Purification of Vomiting Toxins:

[0028] The crude pure solution of vomiting toxin obtained in the previous step was subjected to rotary evaporation to obtain freeze-dried product and then reconstituted. The reconstituted crude toxin extract was then purified again by high pressure preparative liquid chromatography using an ODS-BP column. The mobile phase A was 0.1 vol% formic acid water and the mobile phase B was methanol.

[0029] The elution program was as follows: 5% isocratic elution of phase B for 5 min, 5%-53% gradient elution for 10 min, 53% isocratic elution for 12 min, 53%-100% gradient elution for 0 min, 100% isocratic elution for 5 min, and the eluent with a retention time of about 23.6 min was collected and contained carbon-13 labeled vomitoxin.

[0030] Secondly, this application also provides a toxin-producing culture medium used in the preparation of the aforementioned carbon-13 labeled vomitoxin, the toxin-producing culture medium comprising the following components: 10-20 g / L of a C-13 labeled carbon source, 0.5-2 g / L of a nitrogen source, 0.1-1 g / L of magnesium sulfate heptahydrate, 0.5-2 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of potassium chloride, 0.01-0.05 g / L of ferrous sulfate heptahydrate, 0.01-0.04 g / L of manganese chloride, 0.05-0.15 g / L of ε-PL, and 20 μL-500 μL of a trace element mixture, the remainder being water as a solvent.

[0031] Preferably, the toxin-producing culture medium is prepared from the following components: 10 g / L of C-13 labeled carbon source, 1 g / L of ammonium chloride, 0.5 g / L of magnesium sulfate heptahydrate, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of potassium chloride, 0.02 g / L of ferrous sulfate heptahydrate, 0.1 g / L of ε-PL, 0.04 g / L of manganese chloride, and 100 μL of trace element mixture, with the remainder being water as solvent.

[0032] Thirdly, this application also provides a carbon-13 labeled vomitoxin, which is prepared using the aforementioned preparation method.

[0033] Fourthly, this application also provides the application of the carbon-13 labeled vomitoxin prepared by the above method in the preparation of 13C fully labeled vomitoxin derivatives.

[0034] The 13C-standard vomitoxin derivatives include: ketogenic derivatives, acetylated derivatives, glucose derivatives, and sulfate derivatives.

[0035] Various derivatives of 13C fully labeled vomitoxin can be prepared by synthetic processes using the high-labeling-rate C13-labeled vomitoxin obtained by the above method.

[0036] Fifthly, this application also provides a method for preparing a ketated derivative of 13C-standardized vomitoxin, the method comprising the following steps:

[0037] Oxaloyl chloride was added to freshly distilled dichloromethane, and the dichloromethane solution was slowly added under the protection of nitrogen at low temperature, followed by the slow addition of DMSO.

[0038] Subsequently, the aforementioned 13C fully standardized vomitoxin (molecular formula U-[13C15]-DON), dichloromethane, and DMSO solution were added within 1–10 minutes, and the solution was kept at -60°C. o C~80 o Stir for 10-20 minutes under temperature C, then add triethylamine;

[0039] After 1-10 minutes, once the mixture has gradually returned to room temperature, water is added. The mixture is then extracted with dichloromethane, dried with anhydrous sodium sulfate, concentrated, and the crude extract is purified by silica gel column chromatography to obtain a ketogenic derivative of 13C-standard vomitoxin.

[0040] The ketogenic derivatives of the 13C-standard vomitoxin include: 13C-standard 3-keto-vomitoxin (U-[13C15]-3keto-DON), 13C-standard 15-keto-vomitoxin (U-[13C15]-15keto-DON), and 13C-standard 3,15-diketo-vomitoxin (U-[13C15]-3,15diketo-DON).

[0041] Sixthly, this application also provides a method for preparing an acetylated derivative of 13C-standard vomitoxin, the method comprising the following steps:

[0042] The 13C-labeled vomitoxin (chemical formula U-[13C15]-DON) prepared by the aforementioned method was dissolved in dichloromethane, pyridine and DMAP were added, followed by the addition of acetic anhydride, and the reaction mixture was stirred overnight. The pH of the reaction was adjusted to 4-5, and then extracted with dichloromethane. The extract was dried with Na2SO4, filtered, and the solvent was evaporated. The crude extract was purified by column chromatography to obtain the acetylated derivative of vomitoxin.

[0043] The acetylated derivatives of the above-mentioned 13C-labeled vomitoxin include: 13C-labeled 3-acetyl-vomitoxin (U-[13C15]-3Ac-DON), 13C-labeled 15-acetyl-vomitoxin (U-[13C15]-15Ac-DON), and 13C-labeled 3,15-diacetyl-vomitoxin (U-[13C15]-3,15Ac-DON).

[0044] Seventhly, this application also provides a method for preparing 13C-standard 3-glucosyl-vomiting toxin, the method comprising the following steps:

[0045] Step 1: Dissolve the 13C-labeled vomitoxin prepared by the aforementioned method in dichloromethane, add 13C-labeled acetylglucosamine (2,3,4,6-tetraacetyl-1-bromo-α-D-[13C6]-glucopyranoside) and silver carbonate to the solution, stir at room temperature in the dark for 20-30 h, and separate the reaction solution by silica gel column chromatography to obtain 13C-labeled 3-acetylglucosyl-vomitoxin (U-[13C21]-DON-3G-Ac).

[0046] Step 2: Dissolve 13C-labeled 3-acetylglucosyl-vomiting toxin (U-[13C21]-DON-3G-Ac) in 0.5 mL of tetrahydrofuran aqueous solution. After cooling to 0°C, add potassium hydroxide aqueous solution (KOHaq). Stir the resulting suspension at room temperature for 3-5 h, add acid to adjust the pH to 4-5, and finally extract with ethyl acetate. Combine the organic layers, dry and concentrate with Na2SO4, and purify the crude extract by silica gel column chromatography to obtain 13C-labeled 3-glucosyl-vomiting toxin (U-[13C21]-DON-3G-Ac).

[0047] In the aqueous solution of tetrahydrofuran, the ratio of tetrahydrofuran to water is (3~5):1.

[0048] Eighthly, this application also provides a method for preparing 13C fully labeled 3-sulfate ester-vomiting toxin, the method comprising the following steps:

[0049] Step 1: Dissolve the 13C-labeled vomitoxin prepared by the aforementioned method and 1,2-dimethylimidazole in anhydrous dichloromethane to obtain a mixture; add sulfonylimidazolium trifluoromethanesulfonate (molecular formula: C8H) to the above mixture. 10 Cl3F3N2O6S2 (CAS: 1185733-70-3). After stirring at room temperature for 10-15 hours, the reaction mixture was diluted with dichloromethane, washed with water, dried with Na2SO4, and concentrated under reduced pressure. The 13C-3-sulfate-based vomitoxin intermediate (U-[13C21]-DON-3S-INT-01) was purified by silica gel column chromatography.

[0050] Step 2: Dissolve the 13C-3-sulfate-vomiting toxin intermediate obtained in the previous step in anhydrous acetonitrile solution, and add ammonium formate (HCOONH4) and zinc powder. Stir the resulting suspension at room temperature for 40-50 hours, filter with diatomaceous earth and evaporate, and finally purify by reversed-phase chromatography to obtain 13C full-standard 3-sulfate-vomiting toxin (U-[13C21]-DON-3S).

[0051] The aforementioned ketogenic derivatives, acetylated derivatives, glucose derivatives, and sulfate derivatives of vomitoxin are all cryptic fungal toxins. In order to prevent and control the contamination of such cryptic fungal toxins, while doing a good job in the detection of routine fungal toxins, it is also necessary to increase the detection of various cryptic toxins of vomitoxin. However, at present, there is a lack of standard samples of the aforementioned cryptic toxins of vomitoxin on the market. Therefore, it is very important to synthesize the aforementioned ketogenic derivatives, acetylated derivatives, glucose derivatives, and sulfate derivatives of vomitoxin with full 13C standard.

[0052] The present invention has the following beneficial effects:

[0053] 1. This invention provides a low-cost, simple, and efficient method for producing carbon-13 labeled vomitoxin. The method has simple production steps, high preparation efficiency, low production cost, and high labeling rate. The carbon-13 labeled vomitoxin obtained has strong practicality and is suitable for the industrial production of isotope-labeled vomitoxin standards, and has good market application prospects. Detailed Implementation

[0054] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0055] Example 1: A method for preparing carbon-13 labeled vomitoxin

[0056] 1. Experimental materials

[0057] Toxin-producing strains: Fusarium strains capable of producing deoxynivalenol were used. Options included: Fusarium strains purchased from the China Agricultural Microbial Culture Collection Center (ACCC38871 and ACCC39294), Fusarium strains purchased from the China Industrial Microbial Culture Collection Center (CICC2697), and Fusarium strains purchased from the China General Microbial Culture Collection Center (CGMCC3.4598 and CGMCC3.3489). In this embodiment, Fusarium strain CGMCC3.4598 was used as an example for subsequent experiments.

[0058] 2. Culture medium

[0059] (1) Culture medium for activating microorganisms: Potato glucose agar medium (i.e. PDA medium) was used: potato 200.0 g / L, glucose 20.0 g / L, agar 20.0 g / L, pH natural, sterilized at 121℃ for 20 min.

[0060] (2) Sporulation medium:

[0061] The formula is as follows: sodium carboxymethyl cellulose (CMC) 10g / L, sodium nitrate 1g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.5g / L, peptone 1g / L, pH at rest, sterilized at 121℃ for 20min.

[0062] (3) Toxin-producing culture medium: 10 g / L of C-13 labeled glucose, 1 g / L of ammonium chloride, 0.5 g / L of magnesium sulfate heptahydrate, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of potassium chloride, 0.02 g / L of ferrous sulfate heptahydrate, 0.1 g / L of ε-PL, 0.04 g / L of manganese chloride, and 100 μL of trace element mixture, pH 6.5; the remainder is water.

[0063] The formula for the trace element mixture is as follows: 5g zinc sulfate heptahydrate, 0.25g copper sulfate heptahydrate, 0.52g manganese sulfate monohydrate, 50mg boric acid, 50mg sodium molybdate dihydrate, and 100mL ultrapure water.

[0064] Various culture media were prepared according to the above formula for subsequent experiments.

[0065] 3. Preparation method of carbon-13 labeled vomitoxin

[0066] The preparation method includes the following steps:

[0067] (1) Activation of strain: Take a loop of Fusarium graminearum strain from the slant culture stored at 4℃ and transfer it to a PDA plate. Place the plate in a 25℃ incubator and incubate in the dark for 5 days.

[0068] (2) Sporulation culture: The activated Fusarium hyphae were inoculated into the sporulation culture medium and cultured at 25°C for 6 days at 160 r / min. The spores were then collected.

[0069] (3) Toxin production culture: The collected spores are transferred to a toxin production medium for culture until the final spore concentration reaches 1×10⁻⁶. 4 The samples were cultured at 25°C with shaking at 160 rpm for 10 days to obtain carbon-13 labeled vomitoxin ferments.

[0070] (4) Toxin extraction: Sodium chloride was added to the culture medium after toxin production culture until saturation, and then ethyl acetate was added in an equal volume of 1:1. The mixture was shaken and extracted for 1 hour. The upper ethyl acetate phase was collected by centrifugation. This extraction was repeated 3 times. The ethyl acetate phases were combined to obtain the crude extract of vomitoxin fermentation product containing carbon 13 labeling. The crude extract was then redissolved in 50% acetonitrile water.

[0071] (5) Purification of toxins:

[0072] ① Crude purity of toxins:

[0073] Purification was performed using high-performance liquid chromatography (HPLC) with an ODS-BP column. The mobile phase A was 0.1 vol% formic acid in water, and the mobile phase B was methanol.

[0074] Elution procedure: 5% isocratic elution of phase B for 5 min, 5%-100% gradient elution for 40 min, and 100% isocratic elution for 10 min. Collect the eluent and detect DON labeled with carbon-13 at a retention time of approximately 31.7 min. Combine the product phases to obtain a crude pure solution.

[0075] The crude pure liquid was transferred to a rotary evaporator, concentrated under reduced pressure at 35°C, freeze-dried, and then reconstituted with 50% acetonitrile water for later use.

[0076] ② The purity of the toxin:

[0077] The reconstituted sample was purified by high-performance liquid chromatography (HPLC) using an ODS-BP column. The mobile phase A was 0.1 vol% formic acid in water, and the mobile phase B was methanol.

[0078] Elution program: Phase B content 5% isocratic elution for 5 min, 5%-53% gradient elution for 10 min, 53% isocratic elution for 12 min, 53%-100% gradient elution for 10 min, 100% isocratic elution for 5 min. Collect the eluent and analyze the residue with a retention time of approximately 23.6 min to determine if it contains carbon-13 labeled DON.

[0079] The final results showed that the toxin recovery rate was 67% and the purity was 95%.

[0080] 4. Experimental Results and Analysis

[0081] The preparation method of this embodiment yielded 67% of the 13-labeled vomiting toxin, with a labeling rate of 92% and a purity of 95%.

[0082] Example 2: Optimization of Toxin-Producing Culture Medium

[0083] 1. Optimization of different 13C-carbon sources

[0084] 1.1 Experimental Design

[0085] (1) Set up different experimental groups. The toxin-producing medium of each experimental group uses different carbon sources labeled with C-13. The contents of other components (ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, ferrous sulfate heptahydrate) are shown in Table 1 below. Add 500mL of ultrapure water, and make up to 1L with purified water. Adjust the pH to 6.5 with 1M sodium hydroxide. Set up 3 parallel experiments for each group. Then sterilize at 121℃ for 15min to obtain 13C-liquid toxin-producing medium.

[0086] Culture temperature: 20-30℃, shake culture at 120-200r / min for 7-14 days, and detect the concentration and labeling rate of carbon-13 labeled vomitoxin fermentation product.

[0087] The DON concentration was determined by HPLC at a wavelength of 218 nm. Chromatographic conditions included a C18 column (150 mm × 4.6 mm, 5 μm), a column temperature of 35 °C, an injection volume of 10 μL, and a mobile phase of acetonitrile + water (10 + 90). DON exhibits a specific absorption peak at 218 nm. The DON content in the sample was calculated based on the peak area, compared to the retention time of standard DON.

[0088] The method for determining the C-13 labeling rate in DON is as follows: HPLC-MS is used to scan the C-13 labeled DON sample in negative ion scanning mode using ESI. Qualitative analysis is performed based on the characteristic mass-to-charge ratio m / z, daughter ion fragments, and retention times of different DON molecules under mass spectrometry conditions. Then, the total labeling amount of DON is obtained by summing the peak areas, and the C-13 labeling rate of the product with full carbon labeling is calculated.

[0089] Carbon-13 labeled vomitoxin was prepared using the toxin-producing culture medium described above, following the method in Example 1.

[0090] (2) Concentration and labeling rate of carbon-13 labeled vomitoxin fermentation products.

[0091] Table 1. Group settings

[0092]

[0093] 1.2 Experimental Results:

[0094] Table 2. Toxin yield and labeling rate for each group

[0095]

[0096] The data in the table show that the toxin concentration and C-13 labeling rate of experimental group 4 are higher than those of other experimental groups. This result indicates that using the toxin-producing culture medium of Example 4 to culture Fusarium graminearum can improve the yield and labeling rate of vomitoxin fermentation products.

[0097] 2. Optimization of 13C-glucose concentration

[0098] 2.1 Experimental Design:

[0099] (1) Take C-13 labeled glucose, ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, ferrous sulfate heptahydrate, and trace element mixture, add 500 mL of ultrapure water, and make up to 1 L with purified water. Adjust the pH to 6.5 with 1 M sodium hydroxide. Set up 3 parallel experiments for each group. Then sterilize at 121 °C for 15 min to obtain 13C-liquid toxin-producing culture medium.

[0100] Carbon-13 labeled vomitoxin was prepared using the toxin-producing culture medium described above, following the method in Example 1.

[0101] The DON concentration was determined by HPLC at a wavelength of 218 nm. Chromatographic conditions included a C18 column (150 mm × 4.6 mm, 5 μm), a column temperature of 35 °C, an injection volume of 10 μL, and a mobile phase of acetonitrile + water (10 + 90). DON exhibits a specific absorption peak at 218 nm. The DON content in the sample was calculated based on the peak area, compared to the retention time of standard DON.

[0102] The method for determining the C-13 labeling rate in DON is as follows: HPLC-MS is used to scan the C-13 labeled DON sample in negative ion scanning mode using ESI. Qualitative analysis is performed based on the characteristic mass-to-charge ratio m / z, daughter ion fragments, and retention times of different DON molecules under mass spectrometry conditions. Then, the total labeling amount of DON is obtained by summing the peak areas, and the C-13 labeling rate of the product with full carbon labeling is calculated.

[0103] (2) Concentration and labeling rate of carbon-13 labeled vomitoxin fermentation products.

[0104] Table 3. Formulation of toxin-producing culture medium for each experimental group

[0105]

[0106] 2.2 Experimental Results:

[0107] Table 4 Results of each experimental group for 13C-glucose concentration optimization

[0108]

[0109] As can be seen from the data in Table 4, the toxin concentration and C-13 labeling rate of experimental group 2 are higher than those of experimental groups 1 and 3. This indicates that using the toxin-producing medium of experimental group 2 to culture Fusarium graminearum can improve the yield and labeling rate of vomitoxin fermentation products. Therefore, using 13C-glucose as the carbon source is more conducive to improving the yield and labeling rate of vomitoxin fermentation products.

[0110] 3. Optimization of different nitrogen sources in toxin-producing culture media

[0111] 3.1 Experimental Design:

[0112] Take C-13-labeled glucose, different nitrogen sources (ammonium chloride, ammonium sulfate, sodium nitrate, potassium nitrate), magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, and ferrous sulfate heptahydrate, add them to 500 mL of ultrapure water, and bring the volume to 1 L with purified water. Adjust the pH to 6.5 using 1M sodium hydroxide. Set up three replicates for each experiment (see Table 5 below for the specific formulations of each experimental group). Then, sterilize at 121℃ for 15 min to obtain 13C-liquid toxin-producing medium. Incubate at 20-30℃ with shaking at 120-200 r / min for 7-14 days.

[0113] Carbon-13 labeled vomitoxin was prepared using the toxin-producing culture medium described above, following the method in Example 1.

[0114] The concentration and labeling rate of carbon-13 labeled vomitoxin ferment were determined, and the detection method is described in the aforementioned experiment.

[0115] Table 5. Group settings

[0116]

[0117] 3.2 Experimental Results:

[0118] Table 6

[0119]

[0120] As can be seen from Table 6, the toxin concentration and C-13 labeling rate of experimental group 1 (Example 7) were higher than those of experimental groups 2, 3 and 4. This indicates that using ammonium chloride as a nitrogen source can improve the yield and labeling rate of vomitoxin fermentation products.

[0121] 4. Optimization of ε-PL (ε-polylysine) concentration in the toxin-producing culture medium

[0122] 4.1 Experimental Methods

[0123] Take C-13 labeled glucose, ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, and ferrous sulfate heptahydrate (see Example 1 for component content), add 500 mL of ultrapure water, and bring the volume to 1 L with purified water. Add the following concentrations of ε-PL, with no ε-PL added as a control. Adjust the pH to 6.5 using 1M sodium hydroxide. Set up three replicates for each experiment. Then, sterilize at 121℃ for 15 min to obtain 13C-liquid toxin-producing medium. Incubate at 20-30℃ with shaking at 120-200 rpm for 7-14 days.

[0124] Carbon-13 labeled vomitoxin was prepared using the toxin-producing culture medium described above, following the method in Example 1.

[0125] The concentration and labeling rate of carbon-13 labeled vomitoxin ferment broth were determined.

[0126] Table 7

[0127]

[0128] 4.2 Experimental Results and Analysis

[0129] As can be seen from Table 7, the concentration and labeling rate of vomitoxin ferment broth were highest when the ε-PL concentration was 0.1 g / L.

[0130] 5. Effects of different manganese ions on the concentration of vomitoxin fermentation products

[0131] 5.1 Experimental Methods

[0132] (1) Take C-13 labeled glucose, ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, and ferrous sulfate heptahydrate (see Example 1 for the content of each component), add 500 mL of ultrapure water, and bring the volume to 1 L with purified water. Add 0.01 g of the following different metal ions respectively. Adjust the pH to 6.5 with 1 M sodium hydroxide. Set up 3 replicates for each group of experiments. Then sterilize at 121℃ for 15 min to obtain 13C-liquid toxin-producing medium. Culture temperature: 20-30℃, shake culture at 120-200 r / min for 7-14 days.

[0133] Carbon-13 labeled vomitoxin was prepared using the toxin-producing culture medium described above, following the method in Example 1.

[0134] The concentration and labeling rate of carbon-13 labeled vomitoxin ferment broth were determined.

[0135] (2) Multiple test groups were set up according to the method in (1). The only difference between the test groups was the addition of different concentrations of manganese chloride (see Table 9 for details). The carbon-13 labeled vomitoxin was prepared using the above-mentioned toxin-producing culture medium according to the method in Example 1. The concentration and labeling rate of the carbon-13 labeled vomitoxin ferment were detected.

[0136] 5.2 Experimental Results and Analysis

[0137] Table 8. Effects of different manganese ions on toxin yield and labeling rate.

[0138]

[0139] (1) As can be seen from the data in Table 8, the addition of manganese chloride resulted in the highest concentration and labeling rate of vomitoxin fermentation product. Further investigation is needed to determine the optimal concentration of manganese chloride.

[0140] Table 9. Effects of different manganese chloride concentrations on toxin yield and labeling rate.

[0141]

[0142] (2) As can be seen from the data in Table 9, the concentration and labeling rate of vomitoxin fermentation product were highest when the concentration of added manganese chloride was 0.04 g / L.

[0143] Example 3: Optimization of the inoculation method in the preparation of carbon-13 labeled vomitoxin

[0144] 3.1 Optimization of vaccination methods

[0145] (1) Test methods

[0146] In this embodiment, experimental group 1 and experimental group 2 were set up. Experimental group 1 was operated according to the PDA mycelial inoculation method, and experimental group 2 was operated according to the CMC spore inoculation method. Other methods and steps are the same as in Example 1. The experimental results are shown in Table 10 below.

[0147] The PDA mycelial inoculation method is as follows: use an inoculation loop to pick up mycelia that have been cultured on a PDA plate for 6 days and transfer them to a toxin-producing medium.

[0148] CMC spore inoculation is as follows: Pass the CMC culture medium through two layers of sterile gauze, take 5 mL of the bacterial suspension, centrifuge at 6000 rpm for 5 min, take 4 mL of the supernatant and discard it, add 3 mL of sterile water, repeat the above centrifugation operation twice, and dilute the remaining 1 mL of bacterial suspension with sterile water as appropriate, then transfer it to the toxin-producing culture medium to achieve a final spore concentration of 1×10⁻⁶. 4 per mL.

[0149] (2) Experimental results and analysis

[0150] Table 10

[0151]

[0152] As can be seen from the experimental results in Table 10, the concentration and labeling rate of deoxynivalenol (i.e., vomitoxin) were the highest when CMC spores were used for inoculation, and CMC spore inoculation is the preferred method for inoculating the strain.

[0153] 3.2 Optimal spore concentration for inoculation

[0154] The following three experimental groups were set up, with different spore concentrations for each group (see table below for details). Other methods and operations are detailed in Example 1. Finally, the concentration and labeling rate of carbon-13 labeled vomitoxin in the fermented species were determined.

[0155] Table 11

[0156]

[0157] The results in Table 11 show that the spore inoculation concentration was 1×10⁻⁶. 4 The highest labeling rate of the vomitoxin marker was achieved at a concentration of 1 / mL.

[0158] Example 4 Synthesis of carbon-13 labeled vomitoxin derivatives

[0159] In this embodiment, the carbon-13 labeled DON prepared and purified in the previous embodiment was used as a raw material to synthesize other carbon-13 labeled related compounds according to the following method.

[0160] 4.1 Ketinated derivatives of 13C-standardized vomitoxin

[0161] This embodiment provides a method for synthesizing ketogenic derivatives of 13C-standardized vomitoxin, which include: 13C-standardized 3-keto-vomitoxin (U-[13C15]-3keto-DON), 13C-standardized 15-keto-vomitoxin (U-[13C15]-15keto-DON), and 13C-standardized 3,15-diketo-vomitoxin (U-[13C15]-3,15diketo-DON).

[0162] (1) Synthesis method

[0163] Oxaloyl chloride (4.47 mg, 35.2 μmol) was added to freshly distilled dichloromethane at -78°C. oUnder CN2 protection, a solution of dichloromethane (0.5 mL) in DMSO (6 μL) was slowly added. Then, over 5 minutes, U-[13C15]-DON (10 mg, 32 μmol), dichloromethane (0.5 mL), and DMSO (10 μL) solution were added. The reaction mixture was incubated at -78°C. o Stir for 15 minutes, then add triethylamine (20 μL). After 5 minutes, gradually return the reaction to room temperature, add water, extract the mixture with dichloromethane, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography to obtain 13C-standard 3-keto-vomiting toxin (U-[13C15]-3keto-DON), 13C-standard 15-keto-vomiting toxin (U-[13C15]-15keto-DON), and 13C-standard 3,15-diketo-vomiting toxin (U-[13C15]-3,15diketo-DON).

[0164] The synthesis steps are as follows:

[0165]

[0166] (2) Experimental results and analysis

[0167] The final yield was 2.98 mg of U-[13C15]-3keto-DON, with a yield of 30% and a purity of 96.7%; the final yield was 5.16 mg of U-[13C15]-15keto-DON, with a yield of 52% and a purity of 99.2%; the final yield was 1.59 mg of U-[13C15]-3,15diketo-DON, with a yield of 16% and a purity of 97.5%.

[0168] 4.213C Full-Standard Preparation of Acetylated Derivatives of Vomitoxin

[0169] This embodiment provides a method for synthesizing acetylated derivatives of 13C-standard vomitoxin, which include: 13C-standard 3-acetyl-vomitoxin (U-[13C15]-3Ac-DON), 13C-standard 15-acetyl-vomitoxin (U-[13C15]-15Ac-DON), and 13C-standard 3,15-diacetyl-vomitoxin (U-[13C15]-3,15Ac-DON).

[0170] (1) Synthesis method

[0171] 13C-labeled vomitoxin (U-[13C15]-DON) (10 mg, 32 μmol) was dissolved in dichloromethane (0.5 mL), and pyridine (1 mL) and DMAP (1 mg) were added, followed by the dropwise addition of acetic anhydride (16 mg, 160 μmol). The reaction mixture was stirred overnight, and the pH was adjusted to 4-5 with 1M HCl, followed by extraction with dichloromethane. After drying with Na2SO4, filtration, and solvent evaporation, the remaining residue was purified by column chromatography to obtain acetylated derivatives of vomitoxin: U-[13C15]-3Ac-DON, U-[13C15]-15Ac-DON, and U-[13C15]-3,15Ac-DON.

[0172]

[0173] (2) Experimental results and analysis

[0174] The final yield was 3.88 mg of U-[13C15]-3Ac-DON, with a yield of 34% and a purity of 95.2%; the final yield was 5.82 mg of U-[13C15]-15Ac-DON, with a yield of 51% and a purity of 96.3%; the final yield was 1.92 mg of U-[13C15]-3,15diAc-DON, with a yield of 15% and a purity of 98.6%.

[0175] 4.3 Synthetic steps of 13C-labeled 3-glucosyl-vomiting toxin

[0176] (1) Synthesis method

[0177] Step 1: 13C-labeled vomitoxin (10 mg, 32 μmol) was dissolved in 0.5 mL of dichloromethane. Acetyl glucoside (2,3,4,6-tetraacetyl-1-bromo-α-D-[13C6]-glucopyranoside) (0.26 g, 0.64 mmol) and silver carbonate (0.18 g, 0.64 mmol) were added to the solution, and the mixture was stirred in the dark at room temperature for 24 h. The reaction solution was separated by silica gel column chromatography to obtain 13C-labeled 3-acetylglucosyl-vomitoxin (U-[13C21]-DON-3G-Ac).

[0178]

[0179] Step 2: 13C-labeled 3-acetylglucosyl-vomiting toxin (U-[13C15]-DON-3G-Ac) was dissolved in 0.5 mL of tetrahydrofuran / water (4:1), cooled to 0 °C, and KOHaq (1 M) was added. The resulting suspension was stirred at room temperature for 4 hours. The reaction mixture was acidified to pH 4-5 with 1 M hydrochloric acid and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated. 13C-labeled 3-glucosyl-vomiting toxin (U-[13C21]-DON-3G) was purified by silica gel column chromatography.

[0180]

[0181] (2) Experimental results and analysis

[0182] The final yield was 7.39 mg of 13C-labeled 3-glucosyl-vomiting toxin, with a yield of 48% and a purity of 96.7%.

[0183] 4.4 Synthetic steps of 13C-3-sulfate ester-vomiting toxin

[0184]

[0185] (1) Synthesis method

[0186] Step 1: Dissolve 13C-labeled vomitoxin (10 mg, 32 μmol) and 1,2-dimethylimidazole (4.6 mg, 48 μmol) in anhydrous dichloromethane (0.5 mL), then add sulfonylimidazole trifluoromethanesulfonate (molecular formula: C8H). 10 Cl3F3N2O6S2 (CAS: 1185733-70-3) (18.2 mg, 40 μmol). After stirring at room temperature for 12 hours, the reaction mixture was diluted with dichloromethane, washed with water, dried over Na2SO4, and concentrated under reduced pressure. The 13C-3-sulfate-based vomiting toxin intermediate (U-[13C21]-DON-3S-INT-01) was purified by silica gel column chromatography.

[0187]

[0188] Step 2: Add HCOONH4 and zinc powder to an anhydrous acetonitrile (0.5 mL) solution of the 13C-3-sulfate-vomiting toxin intermediate. Stir the resulting suspension at room temperature for 48 hours, filter with diatomaceous earth, evaporate, and purify by reversed-phase chromatography to obtain 13C-3-sulfate-vomiting toxin (U-[13C21]-DON-3S).

[0189] (2) Experimental results and analysis

[0190] The final yield of 13C-3-sulfate-based vomiting toxin was 7.21 mg, with a yield of 55% and a purity of 98.9%.

[0191] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing carbon-13 labeled vomitoxin, characterized in that, Includes the following steps: S1. Sporulation culture: Activated Fusarium graminearum is inoculated into a sporulation culture medium and cultured at 20-30℃ for 5-7 days, after which spores are collected. S2. Toxin production culture: The collected spores are transferred to a toxin production culture medium and cultured at 20-30℃ and 120-200r / min for 7-14 days to obtain a fermentation broth containing carbon-13 labeled vomitoxin. In the toxin-producing culture, the inoculated spore concentration was 10. 3~ 10 4 cells / mL; S3. Extraction of vomitoxin: Add sodium chloride to the fermentation broth until saturated, then add ethyl acetate. The volume ratio of the added ethyl acetate to the original mixture is 1:

1. Shake and extract for 0.5-1.5 hours. Centrifuge and collect the upper ethyl acetate phase. Repeat this extraction at least twice. Combine the ethyl acetate phases to obtain a crude extract containing carbon-13 labeled vomitoxin. The toxin-producing culture medium is prepared from the following components: 10 g / L C-13 labeled glucose, 1 g / L ammonium chloride, 0.1-1 g / L magnesium sulfate heptahydrate, 0.5-2 g / L potassium dihydrogen phosphate, 0.1-1 g / L potassium chloride, 0.01-0.05 g / L ferrous sulfate heptahydrate, 0.04 g / L manganese chloride, 0.1 g / L ε-PL, and 20 μL-500 μL of trace element mixture, with the remainder being water as the solvent.

2. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that, The sporulation medium is prepared from the following components: sodium carboxymethyl cellulose (CMC) 10 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and peptone 1 g / L.

3. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that, The toxin-producing culture medium is prepared from the following components: 10 g / L C-13 labeled glucose, 1 g / L ammonium chloride, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L potassium chloride, 0.02 g / L ferrous sulfate heptahydrate, 0.1 g / L ε-PL, 0.04 g / L manganese chloride, and 100 μL of trace element mixture, with the remainder being water as the solvent.

4. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that, The trace element mixture contains the following components at the following concentrations: zinc sulfate heptahydrate 0.1~1 g / L, copper sulfate heptahydrate 0.01~0.05 g / L, manganese sulfate monohydrate 0.01~0.1 g / L, boric acid 1~10 mg / L, and sodium molybdate dihydrate 1~10 mg / L.

5. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that, The following steps are included after step S3: S4. Crude purified vomitoxin: Purified by high-performance liquid chromatography using an ODS-BP column. The mobile phase A was 0.1 vol% formic acid in water, and the mobile phase B was methanol. The elution procedure was as follows: elution of phase B at 5% isocratic concentration for 5 min, elution of phase B at 5%-100% gradient for 40 min, and elution of phase B at 100% isocratic concentration for 10 min. The eluent with a retention time of approximately 31.7 min was collected, which was the crude pure solution of carbon-13 labeled vomitoxin.

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