Preparation method of vomitoxin marked by carbon 13 and adopted toxin-producing culture medium

Abstract

The invention belongs to the technical field of toxin preparation, and discloses a preparation method of vomitoxin marked by carbon 13 and an adopted toxin-producing culture medium. The preparation method of the carbon 13 labeled vomitoxin comprises the following steps: S1, spore production culture: inoculating activated fusarium into a spore production culture medium, culturing for 5-7 days under the condition of 20-30 DEG C, and collecting spores; s2, toxin-producing culture: transferring the collected spores into a toxin-producing culture medium for culture, and culturing for 7-14 days under a shaking condition to obtain fermentation liquor containing the vomitoxin marked by carbon 13; and S3, extraction of vomitoxin: adding sodium chloride to a saturated state, then adding ethyl acetate, carrying out oscillation extraction, centrifugally collecting an upper ethyl acetate phase, and merging the ethyl acetate phase to obtain a crude extract containing the C13-labeled vomitoxin. The method has the advantages of simple production process steps, high preparation efficiency, low production cost and high labeling rate, and can be used as a raw material for preparing various carbon 13 labeled vomitoxin derivatives.

Description

Technical Field

[0001] The present invention relates to the technical field of toxin preparation, and in particular to a method for preparing carbon 13 labeled vomitoxin and a toxin-producing culture medium used therein. Background Art

[0002] Deoxynivalenol (DON), also known as vomintoxin, belongs to the trichothecenes. DON is mainly produced by Fusarium graminearum and Fusarium culmorum. Most of these fungi grow slowly in grain crops during cold, humid and harvest seasons. DON often coexists with other mycotoxins, such as 3-ACDON, 15-ACDON, ZON, NIV, FX, etc. When humans and animals ingest grains and their products containing this toxin, it is easy to cause liver damage and endanger health. Therefore, screening and hazard assessment of DON in grains and their products are imperative.

[0003] At present, the detection methods for vomitoxin include thin layer chromatography, gas chromatography, liquid chromatography, liquid chromatography-mass spectrometry, etc. These detection methods require a large amount of standard substances for the accurate determination of the toxin concentration in the sample. The isotope internal standard substance combined with mass spectrometry can eliminate matrix interference by accurately determining the isotope abundance and accurately weighing the added internal standard. Not only that, the isotope internal standard can also eliminate the differences in the sample pretreatment process and realize the quantitative determination of the analyte.

[0005] Therefore, the prior art needs to be further improved. Summary of the invention

[0006] In view of the problem that there is currently no mature method for producing isotope-labeled vomitoxin standards in China, the present invention provides a low-cost, simple and efficient method for producing carbon-13-labeled vomitoxin and a toxin-producing culture medium used therein, which is suitable for the industrial production of vomitoxin standards.

[0007] To solve the above problems, this application provides the following technical solutions: In a first aspect, the present application provides a method for preparing carbon-13 labeled vomitoxin, which comprises the following steps: S1. Spore culture: inoculate the activated Fusarium into the spore culture medium, culture at 20-30°C for 5-7 days, and collect the spores; S2. Toxigenic culture: The collected spores are transferred to toxigenic culture medium for culture at 20-30°C and 120-200 rpm for 7-14 days to obtain a fermentation liquid containing carbon-13 labeled vomitoxin; S3. Extraction of vomitoxin: Sodium chloride is added to the fermentation broth until saturated, and then ethyl acetate is added, and the mixture is shaken for extraction for 0.5-1.5 hours. The upper ethyl acetate phase is collected by centrifugation, and the extraction is repeated at least twice. The ethyl acetate phases are combined to obtain a crude extract containing carbon-13 labeled vomitoxin.

[0008] Preferably, the spore production culture conditions are: culturing at 25°C for 6 days; the toxin production culture conditions are: culturing at 25°C and 160r / min shaking for 10 days.

[0009] Optionally, in the method for preparing carbon 13 labeled vomitoxin, the concentration of spores inoculated in the toxin-producing culture is 10 3~ 10 4 / mL; the volume ratio of the added ethyl acetate to the original mixed solution was 1:1.

[0010] Preferably, in step S2, the final concentration of spores in the culture at the time of inoculation reaches 1×10 4 Pieces / mL.

[0011] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the spore-forming culture medium is composed of the following ingredients: 10 g / L sodium carboxymethyl cellulose (CMC), 1 g / L sodium nitrate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 1 g / L peptone.

[0012] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the toxin-producing culture medium is composed of the following components: 10~20g / L C-13 labeled carbon source, 0.5~2g / L nitrogen source, 0.1~1g / L magnesium sulfate heptahydrate, 0.5~2g / L potassium dihydrogen phosphate, 0.1~1 g / L potassium chloride, 0.01~0.05 g / L ferrous sulfate heptahydrate, 0.01~0.04g / L manganese chloride, 0.05~0.15g / L ε-PL, and 20μL-500μL of trace element mixture, and the rest is solvent water.

[0013] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the toxin-producing culture medium is composed of the following ingredients: 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 a trace element mixture, and the rest is solvent water.

[0014] Optionally, the carbon source is a combination of one or more of 13C-glucose, 13C-D-mannose, 13C-sodium acetate, 13C-sucrose-13C-glycerol. The nitrogen source is ammonium chloride or ammonium sulfate.

[0015] Further preferably, the nitrogen source is ammonium chloride, and the carbon source is 13C-glucose.

[0016] Optionally, in the preparation method of the carbon-13 labeled vomitoxin, the trace element mixture contains the following components in the following concentrations: 0.5 g / L zinc sulfate heptahydrate, 0.025 g / L copper sulfate heptahydrate, 0.052 g / L manganese sulfate monohydrate, 5 mg / L boric acid, and 5 mg / L sodium molybdate dihydrate.

[0017] Preferably, the trace element mixed solution contains the following components in the following concentrations: 0.5 g / L zinc sulfate heptahydrate, 0.025 g / L copper sulfate heptahydrate, 0.052 g / L manganese sulfate monohydrate, 5 mg / L boric acid, and 5 mg / L sodium molybdate dihydrate.

[0018] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the step S3 further includes the following steps: S4. Crude DON: Purify by high pressure preparative liquid chromatography using an ODS-BP preparative column, the mobile phase is 0.1 vol% formic acid water-methanol, the mobile phase A is 0.1 vol% formic acid water, and the mobile phase B is methanol; The elution procedure is: 5% isocratic elution of phase B for 5 minutes, 5%-100% gradient elution for 40 minutes, and 100% isocratic elution for 10 minutes. The eluate with a retention time of about 31.7 minutes is collected, which is the crude pure solution of carbon-13 labeled vomitoxin.

[0019] Optionally, the above-mentioned crude DON solution can be transferred to a rotary evaporator, concentrated under reduced pressure at 35° C. and freeze-dried, and then re-dissolved with 50% acetonitrile water to obtain a crude extract for later use.

[0020] Optionally, in the method for preparing carbon-13 labeled vomitoxin, the step S4 further includes the following steps: S5. Purification of DON: The crude pure solution of vomitoxin prepared in the previous step was subjected to rotary evaporation to obtain freeze-dried product and then reconstituted. The reconstituted crude toxin extract was purified again by high pressure preparative liquid chromatography using an ODS-BP preparative column, with mobile phase A being 0.1 vol% formic acid water and phase B being methanol; The elution procedure is: 5% isocratic elution of phase B for 5 minutes, 5%-53% gradient elution for 10 minutes, 53% isocratic elution for 12 minutes, 53%-100% gradient elution for 0 minutes, 100% isocratic elution for 5 minutes, and the eluate with a retention time of about 23.6 minutes is collected, which is carbon-13 labeled vomitoxin.

[0021] In a second aspect, the present application also provides a toxin-producing culture medium for use in the preparation of the above-mentioned carbon-13 labeled vomitoxin, the toxin-producing culture medium comprising the following components: 10~20g / L of C-13 labeled carbon source, 0.5~2g / L of nitrogen source, 0.1~1g / 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, 0.01~0.04g / L of manganese chloride, 0.05~0.15g / L of ε-PL, and 20μL-500μL of trace element mixture, and the rest is solvent water.

[0022] Preferably, the toxin-producing medium is composed of the following ingredients: 10 g / L C-13 labeled carbon source, 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, and the rest is solvent water.

[0023] In a third aspect, the present application also provides a carbon-13 labeled vomitoxin, which is prepared using the aforementioned preparation method.

[0024] In a fourth aspect, the present application also provides the use of carbon-13 labeled vomitoxin prepared by the above method in the preparation of 13C fully labeled vomitoxin derivatives.

[0025] The 13C fully labeled vomitoxin derivatives include: keto derivatives, acetylated derivatives, glucose derivatives and sulfate derivatives.

[0026] Various 13C-labeled vomitoxin derivatives can be prepared through a synthetic process using the high-labeling rate carbon 13-labeled vomitoxin prepared by the above method.

[0027] In a fifth aspect, the present application also provides a method for preparing a 13C-labeled ketotoxin derivative, the method comprising the following steps: Add oxalyl chloride to freshly distilled dichloromethane, slowly add dichloromethane solution under the protection of low-temperature nitrogen, and then slowly add DMSO dropwise; Then, the 13C-labeled vomitoxin (molecular formula: U-[13C15]-DON), dichloromethane and DMSO solution prepared above were added within 1 to 10 minutes at -60 o C~80 o C, and then add triethylamine; After 1 to 10 minutes, when the mixed solution gradually returns to room temperature, water is added, the mixture is extracted with dichloromethane, and then dried over anhydrous sodium sulfate. After concentration, the obtained crude extract is purified by silica gel column chromatography to obtain a keto derivative of vomitoxin with a 13C full label.

[0028] The 13C-labeled ketotoxin derivatives include: 13C-labeled 3-keto-DON (U-[13C15]-3keto-DON), 13C-labeled 15-keto-DON (U-[13C15]-15keto-DON) and 13C-labeled 3,15-diketo-DON (U-[13C15]-3,15diketo-DON).

[0029] In a sixth aspect, the present application also provides a method for preparing a 13C fully labeled acetylated derivative of vomitoxin, the method comprising the following steps: The 13C-labeled vomitoxin (chemical formula: U-[13C15]-DON) prepared by the aforementioned method is dissolved in dichloromethane, pyridine and DMAP are added, and then acetic anhydride is added dropwise. The reactants are stirred overnight, the reaction pH is adjusted to 4-5, and then extracted with dichloromethane; the extract is dried over Na2SO4, filtered, and the solvent is evaporated, and the obtained crude extract is purified by column chromatography to obtain an acetylated derivative of vomitoxin.

[0030] The above-mentioned 13C-labeled acetylated derivatives of 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).

[0031] In a seventh aspect, the present application also provides a method for preparing 13C fully labeled 3-glucosyl-deoxytoxin, the method comprising the following steps: Step 1: The 13C-labeled vomitoxin prepared by the above method is dissolved in dichloromethane, and 13C-labeled acetylglucose bromide (2,3,4,6-tetraacetyl-1-bromo-α-D-[13C6]-glucopyranoside) and silver carbonate are added to the solution, and stirred at room temperature in the dark for 20 to 30 hours. The reaction solution is separated by silica gel column chromatography to obtain 13C-labeled 3-acetylglucosyl-vomitoxin (U-[13C21]-DON-3G-Ac); Step 2: Dissolve 13C-labeled 3-acetylglucosyl-vomitoxin (U-[13C21]-DON-3G-Ac) in 0.5 mL of tetrahydrofuran aqueous solution, cool to 0°C, add potassium hydroxide aqueous solution (KOHaq), stir the resulting suspension at room temperature for 3-5 hours, add acid to pH 4-5, and finally extract with ethyl acetate. After combining the organic layers, dry them over Na2SO4 and concentrate them. Purify the resulting crude extract by silica gel column chromatography to obtain 13C-labeled 3-glucosyl-vomitoxin (U-[13C21]-DON-3G-Ac).

[0032] In the aqueous solution of tetrahydrofuran, the ratio of tetrahydrofuran to water is (3-5):1.

[0033] In an eighth aspect, the present application further provides a method for preparing 13C-labeled 3-sulfate-deoxytoxin, the method comprising the following steps: Step 1: Dissolve the 13C-labeled vomitoxin and 1,2-dimethylimidazole prepared by the above method in anhydrous dichloromethane to obtain a mixed solution; add sulfonylimidazolium trifluoromethanesulfonate (molecular formula: C8H 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 over Na2SO4, and concentrated under reduced pressure. Purification by silica gel column chromatography gave 13C-3-sulfate-DON intermediate (U-[13C21]-DON-3S-INT-01); Step 2: The 13C-3-sulfate-DON intermediate obtained in the previous step is dissolved in anhydrous acetonitrile solution, and ammonium formate (HCOONH4) and zinc powder are added. The resulting suspension is stirred at room temperature for 40-50 hours, filtered through diatomaceous earth and evaporated, and finally purified by reverse phase chromatography to obtain 13C-labeled 3-sulfate-DON (U-[13C21]-DON-3S).

[0034] The above-mentioned keto derivatives, acetylated derivatives, glucose derivatives and sulfate derivatives of vomitoxin are all hidden mycotoxins. In order to prevent and control the contamination of such hidden mycotoxins, while doing a good job in the detection of conventional mycotoxins, it is also necessary to increase the detection of various hidden toxins of vomitoxin. However, there is currently a lack of standard products for the above-mentioned hidden toxins of vomitoxin on the market. Therefore, it is very important to synthesize the above-mentioned keto derivatives, acetylated derivatives, glucose derivatives and sulfate derivatives of vomitoxin with a full 13C label.

[0035] The present invention has the following beneficial effects: 1. The present invention provides a method for producing carbon-13 labeled vomitoxin in a low-cost, simple and efficient manner. The method has simple production process steps, high preparation efficiency, low production cost and high labeling rate. The prepared carbon-13 labeled vomitoxin has strong practicality, is suitable for the industrial production of isotope-labeled vomitoxin standards, and has good market application prospects. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work 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. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0037] Example 1 A method for preparing carbon 13 labeled vomitoxin 1. Experimental Materials Toxin-producing strains: Fusarium that can produce deoxynivalenol is used, and the following can be selected: Fusarium purchased from the China Agricultural Microorganism Culture Collection Center with numbers ACCC38871 and ACCC39294, purchased from the China Industrial Microorganism Culture Collection Center with numbers CICC2697, purchased from the China General Microorganism Culture Collection Center with numbers CGMCC3.4598 and CGMCC3.3489. In this example, Fusarium with a collection number of CGMCC3.4598 is used as an example for subsequent experiments.

[0038] 2. Culture medium (1) Culture medium for bacterial activation: Potato glucose agar medium (PDA medium): 200.0 g / L potato, 20.0 g / L glucose, 20.0 g / L agar, natural pH, sterilized at 121°C for 20 min.

[0039] (2) Spore-forming culture medium: The formula is: sodium carboxymethyl cellulose (CMC) 10g / L, sodium nitrate 1g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.5g / L, peptone 1g / L, natural pH, sterilization at 121℃ for 20min.

[0040] (3) Toxigenic culture medium: 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, 100 μL of trace element mixture, pH 6.5; the rest is solvent water.

[0041] Among them, the formula of the trace element mixed solution is: 5g zinc sulfate heptahydrate, 0.25g copper sulfate heptahydrate, 0.52g manganese sulfate monohydrate, 50mg boric acid, 50mg sodium molybdate dihydrate, 100mL ultrapure water.

[0042] Prepare various culture media according to the above recipes for subsequent experiments.

[0043] 3. Preparation method of carbon 13 labeled vomitoxin The preparation method comprises the following steps: (1) Activation of strains: Take a ring of Fusarium graminearum strains from the slant stored at 4°C and spread it on a PDA plate. Place the plate in a 25°C incubator in the dark and incubate for 5 days.

[0044] (2) Spore culture: The activated mycelium of Fusarium is inoculated into the spore culture medium, cultured at 25°C and 160 r / min for 6 days, and the spores are collected.

[0045] (3) Toxicity culture: The collected spores were transferred into toxicity culture medium for culture until the final concentration of spores in the culture reached 1×10 4 / mL, and continue to culture at 25°C and 160r / min for 10 days to obtain carbon-13 labeled vomitoxin fermentation product.

[0046] (4) Toxin extraction: Sodium chloride was added to the culture medium after toxin production culture until saturated, and then ethyl acetate was added in an equal volume of 1:1. The mixture was extracted by shaking for 1 hour, and the upper ethyl acetate phase was collected by centrifugation. This extraction was repeated 3 times, and the ethyl acetate phases were combined to obtain the crude extract containing carbon 13 labeled vomitoxin fermentation products. The crude toxin extract was then re-dissolved in 50% acetonitrile water.

[0047] (5) Purification of toxins: ①Crude purity of toxins: The product was purified by high pressure preparative liquid chromatography using an ODS-BP preparative column, with mobile phase A consisting of 0.1 vol% formic acid in water and phase B consisting of methanol.

[0048] Elution procedure: 5% isocratic elution for 5 minutes at 5% B phase content, 5%-100% gradient elution for 40 minutes, and 100% isocratic elution for 10 minutes. Collect the eluate, and detect the carbon-13 labeled DON with a retention time of about 31.7 minutes. Combine the product phases to obtain a crude pure solution.

[0049] The crude pure liquid was transferred to a rotary evaporator, concentrated under reduced pressure at 35°C, and then freeze-dried. It was then reconstituted with 50% acetonitrile water for later use.

[0050] ②Purification of toxins: The reconstituted sample was purified by high pressure preparative liquid chromatography using an ODS-BP preparative column, with mobile phase A consisting of 0.1 vol% formic acid in water and phase B consisting of methanol.

[0051] Elution procedure: 5% isocratic elution for 5 minutes at 5% B phase content, 5%-53% gradient elution for 10 minutes, 53% isocratic elution for 12 minutes, 53%-100% gradient elution for 10 minutes, 100% isocratic elution for 5 minutes. Collect the eluate and detect that the carbon-13 labeled DON is detected at a retention time of about 23.6 minutes.

[0052] Finally, it was measured that the toxin recovery rate was 67% and the purity was 95%.

[0053] 4. Experimental results and analysis The yield of 13-labeled DON obtained by the preparation method of this example is 67%, the labeling rate is 92%, and the purity is 95%.

[0054] Example 2 Optimization of toxin-producing culture medium 1. Optimization of different 13C-carbon sources 1.1 Experimental design (1) Different experimental groups were set up. The toxin-producing culture medium of each experimental group used different carbon sources labeled with C-13. The content of other components (ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, ferrous sulfate heptahydrate) was different. The specific formula is shown in Table 1 below. 500 mL of ultrapure water was added and the volume was made up to 1 L with purified water. The pH was adjusted to 6.5 with 1 M sodium hydroxide. Three parallel experiments were set up for each group of experiments. Then, the culture medium was sterilized at 121 °C for 15 min to obtain 13C-liquid toxin-producing culture medium.

[0055] Culture temperature: 20-30°C, 120-200r / min shaking culture for 7-14 days, detect the concentration and labeling rate of carbon 13 labeled vomitoxin fermentation product.

[0056] The detection method of DON concentration is: HPLC detection, the detection wavelength is 218nm. Chromatographic conditions: chromatographic column is C18 (150mm×4.6mm, 5μm), column temperature is 35℃, injection volume is 10μL, mobile phase is acetonitrile + water (10+90). DON has a specific absorption peak at a wavelength of 218nm. Compared with the retention time of standard DON, the DON content in the sample is calculated based on the peak area. The method for determining the C-13 labeling rate in DON is as follows: using HPLC-MS, the C-13 labeled DON sample is scanned using ESI in negative ion scanning mode, and a qualitative analysis is performed based on the characteristic mass-to-charge ratio m / z, daughter ion fragments and retention time of different DON molecules under mass spectrometry conditions, and then the total labeling amount of DON is obtained by summing the peak areas, and then the full carbon labeled C-13 labeling rate of the product is calculated.

[0057] The toxigenic medium was used to prepare carbon 13 labeled vomitoxin according to the method of Example 1.

[0058] (2) Concentration and labeling rate of carbon-13 labeled vomitoxin fermentation products.

[0059] Table 1 Settings of each group

[0060] 1.2 Experimental results: Table 2 Toxin production and labeling rate of each group

[0061] It can be seen from the data in the table that the toxin concentration and C-13 labeling rate of experimental group 4 are higher than those of other experimental groups. This result shows that the use of the toxin-producing culture medium of Example 4 to cultivate Fusarium graminearum can increase the yield and labeling rate of vomitoxin fermentation products.

[0062] 2. Optimization of 13C-glucose concentration 2.1 Experimental design: (1) Take C-13 labeled glucose, ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, ferrous sulfate heptahydrate, and a mixed solution of trace elements, add 500 mL of ultrapure water, dilute to 1 L with purified water, adjust the pH to 6.5 with 1 M sodium hydroxide, set up 3 parallels for each experiment, and then sterilize at 121 °C for 15 min to obtain 13C-liquid toxin-producing culture medium.

[0063] The toxigenic medium was used to prepare carbon 13 labeled vomitoxin according to the method of Example 1.

[0064] The detection method of DON concentration is: HPLC detection, the detection wavelength is 218nm. Chromatographic conditions: chromatographic column is C18 (150mm×4.6mm, 5μm), column temperature is 35℃, injection volume is 10μL, mobile phase is acetonitrile + water (10+90). DON has a specific absorption peak at a wavelength of 218nm. Compared with the retention time of standard DON, the DON content in the sample is calculated based on the peak area. The method for determining the C-13 labeling rate in DON is as follows: using HPLC-MS, the C-13 labeled DON sample is scanned using ESI in negative ion scanning mode, and a qualitative analysis is performed based on the characteristic mass-to-charge ratio m / z, daughter ion fragments and retention time of different DON molecules under mass spectrometry conditions, and then the total labeling amount of DON is obtained by summing the peak areas, and then the full carbon labeled C-13 labeling rate of the product is calculated.

[0065] (2) Concentration and labeling rate of carbon-13 labeled vomitoxin fermentation products.

[0066] Table 3 Formula settings of toxin-producing culture medium for each experimental group

[0067] 2.2 Experimental results: Table 4 Results of each experimental group with optimized 13C-glucose concentration

[0068] It can be seen from the data in Table 4 that the toxin concentration and C-13 labeling rate of experimental group 2 are higher than those of experimental groups 1 and 3, indicating that the use of the toxin-producing culture medium of experimental group 2 to cultivate Fusarium graminearum can increase the yield and labeling rate of vomitoxin fermentation products. Therefore, the use of 13C-glucose as a carbon source is more conducive to increasing the yield and labeling rate of vomitoxin fermentation products.

[0069] 3. Optimization of different nitrogen sources for toxin-producing culture medium 3.1 Experimental design: Take C-13 labeled glucose, different nitrogen sources (ammonium chloride, ammonium sulfate, sodium nitrate, potassium nitrate), magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, ferrous sulfate heptahydrate, add 500mL ultrapure water, dilute to 1L with purified water, adjust the pH to 6.5 with 1M sodium hydroxide, and set 3 parallels for each group of experiments (see Table 5 below for the specific formula of each experimental group). Then sterilize at 121°C for 15min to obtain 13C-liquid toxin-producing medium. Culture temperature: 20-30°C, 120-200 r / min shaking culture for 7-14 days.

[0070] The toxigenic medium was used to prepare carbon 13 labeled vomitoxin according to the method of Example 1.

[0071] The concentration and labeling rate of the carbon-13 labeled vomitoxin fermentation product were detected. The detection method is similar to the above experiment.

[0072] Table 5 Settings for each group

[0073] 3.2 Experimental results: Table 6

[0074] As can be seen from Table 6, the toxin concentration and C-13 labeling rate of experimental group 1 (Example 7) are higher than those of experimental groups 2, 3, and 4, which indicates that the use of ammonium chloride as a nitrogen source can increase the yield and labeling rate of vomitoxin fermentation products.

[0075] 4. Optimization of ε-PL (ε-polylysine) concentration in toxin-producing culture medium 4.1 Experimental Methods Take C-13 labeled glucose, ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, ferrous sulfate heptahydrate (the content of each component is shown in Example 1), add 500 mL of ultrapure water, purify and dilute to 1 L, add the following concentrations of ε-PL, use no ε-PL as a control, adjust the pH to 6.5 with 1M sodium hydroxide, set 3 parallels for each group of experiments, and then sterilize at 121°C for 15 minutes to obtain 13C-liquid toxin-producing medium. Culture temperature: 20-30°C, 120-200r / min shaking culture for 7-14 days.

[0076] The toxigenic medium was used to prepare carbon 13 labeled vomitoxin according to the method of Example 1.

[0077] Detect the concentration and labeling rate of carbon 13 labeled vomitoxin fermentation products.

[0078] Table 7

[0079] 4.2 Experimental results and analysis It can be seen from Table 7 that when the ε-PL concentration is 0.1 g / L, the concentration and labeling rate of the DON fermentation product are the highest.

[0080] 5. Effects of different manganese ions on the concentration of vomitoxin fermentation products 5.1 Experimental Methods (1) Take C-13 labeled glucose, ammonium chloride, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium chloride, and ferrous sulfate heptahydrate (the content of each component is shown in Example 1), add 500 mL of ultrapure water, dilute 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 3 parallels for each group of experiments, and then sterilize at 121° C. for 15 min to obtain 13C-liquid toxin-producing medium. Culture temperature: 20-30° C., 120-200 r / min shaking culture for 7-14 days.

[0081] The toxigenic medium was used to prepare carbon 13 labeled vomitoxin according to the method of Example 1.

[0082] Detect the concentration and labeling rate of carbon 13 labeled vomitoxin fermentation products.

[0083] (2) Referring to the method of (1), multiple test groups were set up. The only difference among the test groups was the addition of different concentrations of manganese chloride (see Table 9 for details). The above-mentioned toxin-producing culture medium was used to prepare carbon-13 labeled vomitoxin according to the method of Example 1, and the concentration and labeling rate of the carbon-13 labeled vomitoxin fermentation product were detected.

[0084] 5.2 Experimental Results and Analysis Table 8 Effects of different manganese ions on toxin production and labeling rate

[0085] (1) From the data in Table 8, it can be seen that the concentration and labeling rate of vomitoxin fermentation products were the highest when manganese chloride was added. The optimal manganese chloride concentration was further explored.

[0086] Table 9 Effects of different manganese chloride concentrations on toxin production and labeling rate

[0087] (2) From the data in Table 9, it can be seen that when the added concentration of manganese chloride is 0.04 g / L, the concentration and labeling rate of vomitoxin fermentation product are the highest.

[0088] Example 3 Optimization of the inoculation method in the preparation method of carbon 13 labeled vomitoxin 3.1 Optimization of vaccination method (1) Test method In this embodiment, test group 1 and test group 2 are set up, test group 1 is operated according to the PDA mycelium inoculation method, and test group 2 is operated according to the CMC spore inoculation method. Other methods and steps refer to Example 1. The experimental results are specifically shown in Table 10 below.

[0089] The PDA mycelium inoculation method is: use an inoculation loop to pick up the mycelium cultured on the PDA plate for 6 days and transfer it to the toxin-producing culture medium.

[0090] CMC spore inoculation was as follows: CMC culture medium was passed through two layers of sterile gauze, 5 mL of bacterial suspension was absorbed, and centrifuged at 6000 r / min for 5 min. 4 mL of the upper clear layer was absorbed and discarded, and 3 mL of sterile water was added. The above centrifugation operation was repeated twice. The remaining 1 mL of bacterial suspension was appropriately diluted with sterile water and transferred to the toxin-producing culture medium so that the final spore concentration was 1×10 4 Pieces / mL.

[0091] (2) Test results and analysis Table 10

[0092] It can be seen from the experimental results in Table 10 that the concentration and labeling rate of deoxynivalenol (i.e., vomitoxin) are the highest when CMC spore inoculation is used, and it is preferred to use CMC spore inoculation to inoculate the strain.

[0093] 3.2 Optimal spore concentration for inoculation The following three test groups were set up, each of which was inoculated with a different spore concentration (see the table below for details). Other methods and operations were specifically described in Example 1. Finally, the concentration and labeling rate of carbon 13-labeled vomitoxin of the fermented species were determined.

[0094] Table 11

[0095] From the results in Table 11, it can be seen that the spore inoculation concentration is 1×10 4 When the concentration of vomitoxin marker was 0.1447 / mL, the labeling rate of vomitoxin marker was the highest.

[0096] Example 4 Synthesis of Carbon-13 Labeled Deoxynivalenol Derivatives In this example, the carbon-13 labeled DON prepared and purified in the previous example was used as a raw material, and other carbon-13 labeled related compounds were synthesized from it according to the following method.

[0097] 4.1 13C-labeled ketotoxin derivatives In this embodiment, a method for synthesizing 13C-labeled ketotoxin derivatives of vomitoxin is provided. The 13C-labeled ketotoxin derivatives of vomitoxin include: 13C-labeled 3-keto-vomitoxin (U-[13C15]-3keto-DON), 13C-labeled 15-keto-vomitoxin (U-[13C15]-15keto-DON) and 13C-labeled 3,15-diketo-vomitoxin (U-[13C15]-3,15diketo-DON).

[0098] (1) Synthesis method Oxalyl chloride (4.47 mg, 35.2 umol) was added to freshly distilled dichloromethane at -78 o Under the protection of C N2, a solution of DMSO (6 uL) in dichloromethane (0.5 mL) was slowly added. Then, a solution of U-[13C15]-DON (10 mg, 32 umol), dichloromethane (0.5 mL) and DMSO (10 uL) was added within 5 minutes. The reaction mixture was heated at -78 o C and stirred for 15 minutes, then triethylamine (20 uL) was added. After 5 minutes, the reaction was gradually returned to room temperature, water was added, the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain 13C-labeled 3-keto-vomitoxin (U-[13C15]-3keto-DON), 13C-labeled 15-keto-vomitoxin (U-[13C15]-15keto-DON), and 13C-labeled 3,15-diketo-vomitoxin (U-[13C15]-3,15diketo-DON).

[0099] The synthesis steps are as follows:

[0100] (2) Experimental results and analysis The final product was U-[13C15]-3keto-DON 2.98 mg, with a yield of 30% and a purity of 96.7%; the final product was U-[13C15]-15keto-DON 5.16 mg, with a yield of 52% and a purity of 99.2%; the final product was U-[13C15]-3,15diketo-DON 1.59 mg, with a yield of 16% and a purity of 97.5%.

[0101] 4.213C Preparation of acetylated derivatives of vomitoxin In this embodiment, a method for synthesizing 13C-labeled acetylated derivatives of vomitoxin is provided. The 13C-labeled acetylated derivatives of 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).

[0102] (1) Synthesis method 13C-labeled DON (U-[13C15]-DON) (10 mg, 32 umol) was dissolved in dichloromethane (0.5 mL), pyridine (1 mL) and DMAP (1 mg) were added, and then acetic anhydride (16 mg, 160 umol) was added dropwise. The reaction was stirred overnight, the reaction pH was adjusted to 4-5 with 1M HCl, and then extracted with dichloromethane. After drying over Na2SO4, filtering and evaporating the solvent, the remaining residue was purified by column chromatography to obtain acetylated derivatives of DON: U-[13C15]-3Ac-DON, U-[13C15]-15Ac-DON and U-[13C15]-3,15Ac-DON.

[0103]

[0104] (2) Experimental results and analysis The final product was U-[13C15]-3Ac-DON 3.88 mg, with a yield of 34% and a purity of 95.2%; the final product was U-[13C15]-15Ac-DON 5.82 mg, with a yield of 51% and a purity of 96.3%; the final product was U-[13C15]-3,15diAc-DON 1.92 mg, with a yield of 15% and a purity of 98.6%.

[0105] 4.3 Synthesis steps of 13C-labeled 3-glucosyl-DON (1) Synthesis method Step 1: 13C-labeled vomitoxin (10 mg, 32 umol) was dissolved in 0.5 mL of dichloromethane. 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 stirred at room temperature in the dark 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).

[0106]

[0107] Step 2: 13C-labeled 3-acetylglucosyl-DON (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 by adding 1 M hydrochloric acid and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated. 13C-labeled 3-glucosyl-DON (U-[13C21]-DON-3G) was obtained by purification by silica gel column chromatography.

[0108]

[0109] (2) Experimental results and analysis Finally, 7.39 mg of 13C-labeled 3-glucosyl-vomitoxin was obtained with a yield of 48% and a purity of 96.7%.

[0110] 4.4 Synthesis steps of 13C-3-sulfate-DON

[0111] (1) Synthesis method Step 1: Dissolve 13C-labeled vomitoxin (10 mg, 32 umol) and 1,2-dimethylimidazole (4.6 mg, 48 umol) in anhydrous dichloromethane (0.5 mL), and add sulfonylimidazolium trifluoromethanesulfonate (molecular formula: C8H 10 Cl3F3N2O6S2, CAS: 1185733-70-3) (18.2 mg, 40 umol). 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. Purification by silica gel column chromatography gave 13C-3-sulfate-DON intermediate (U-[13C21]-DON-3S-INT-01).

[0112]

[0113] Step 2: To a solution of the 13C-3-sulfate-DON intermediate in anhydrous acetonitrile (0.5 mL) was added HCOONH4 and zinc powder. The resulting suspension was stirred at room temperature for 48 hours, filtered through celite, evaporated, and purified by reverse phase chromatography to give 13C-3-sulfate-DON (U-[13C21]-DON-3S).

[0114] (2) Experimental results and analysis The final yield of 13C-3-sulfate-deoxytoxin was 7.21 mg, the yield was 55%, and the purity was 98.9%.

[0115] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for preparing carbon 13 labeled vomitoxin, characterized in that: The following steps are involved: S1. Spore culture: inoculate the activated Fusarium into the spore culture medium, culture at 20-30°C for 5-7 days, and collect the spores; S2. Toxigenic culture: The collected spores are transferred to toxigenic culture medium for culture at 20-30°C and 120-200 rpm for 7-14 days to obtain a fermentation liquid containing carbon-13 labeled vomitoxin; S3. Extraction of vomitoxin: Sodium chloride is added to the fermentation broth until saturated, and then ethyl acetate is added, and the mixture is shaken for extraction for 0.5-1.5 hours. The upper ethyl acetate phase is collected by centrifugation, and the extraction is repeated at least twice. The ethyl acetate phases are combined to obtain a crude extract containing carbon-13 labeled vomitoxin.

2. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that: In toxigenic cultures, the spore concentration inoculated was 10 3~ 10 4 / mL; the volume ratio of the added ethyl acetate to the original mixed solution was 1:

1.

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

4. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that: The toxin-producing medium is prepared by the following components: 10~20g / L C-13 labeled carbon source, 0.5~2g / L nitrogen source, 0.1~1g / 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, 0.01~0.04g / L manganese chloride, 0.05~0.15g / L ε-PL, and 20μL-500μL of trace element mixture, and the rest is solvent water.

5. The method for preparing carbon-13 labeled vomitoxin according to claim 4, characterized in that: The toxin-producing medium is prepared from the following ingredients: 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 a trace element mixture, and the rest is solvent water; Optionally, the carbon source is a combination of one or more of 13C-glucose, 13C-D-mannose, 13C-sodium acetate, and 13C-sucrose-13C-glycerol.

6. The method for preparing carbon-13 labeled vomitoxin according to claim 5, characterized in that: The nitrogen source is ammonium chloride, and the carbon source is 13C-glucose.

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

8. The method for preparing carbon-13 labeled vomitoxin according to claim 1, characterized in that: The S3 step further includes the following steps: S4. Crude DON: Purification was performed by high pressure preparative liquid chromatography using an ODS-BP preparative column, with mobile phase A being 0.1 vol% formic acid in water and phase B being methanol; The elution procedure is: 5% isocratic elution of phase B for 5 minutes, 5%-100% gradient elution for 40 minutes, and 100% isocratic elution for 10 minutes. The eluate with a retention time of about 31.7 minutes is collected, which is the crude pure solution of carbon-13 labeled vomitoxin.

9. A carbon-13 labeled vomitoxin, characterized in that: The preparation method according to claim 1 is used for preparation.

10. A toxin-producing culture medium for use in the preparation of carbon-13 labeled vomitoxin, characterized in that: The toxin-producing culture medium includes the following components: 10~20g / L of C-13 labeled carbon source, 0.5~2g / L of nitrogen source, 0.1~1g / 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, 0.01~0.04g / L of manganese chloride, 0.05~0.15g / L of ε-PL, and 20μL-500μL of trace element mixture, and the rest is solvent water.

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