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A kind of biosynthesis method of t-2 toxin

A biosynthesis, T-2 technology, applied in the field of biosynthesis of T-2 toxin, can solve the problems of strong reagent toxicity, high cost, cumbersome and complicated treatment, etc., achieve simple sample processing method, reduce toxin cost, and simplify process steps Effect

Active Publication Date: 2016-06-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a biosynthetic synthesis method of T-2 toxin. The T-2 toxin produced by the method of the present invention can be used as a standard product, which effectively solves the problem of long synthetic process route and low yield. Low cost, high cost, cumbersome and complicated handling, strong reagent toxicity, serious environmental pollution, and difficult separation

Method used

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  • A kind of biosynthesis method of t-2 toxin
  • A kind of biosynthesis method of t-2 toxin
  • A kind of biosynthesis method of t-2 toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The recovery of embodiment 1 bacterial classification and the preparation of conidia liquid

[0025] Preparation of Potato Dextrose Agar Medium (also known as PDA Medium): Commercially available potatoes were washed with distilled water, peeled, and cut into 3 cm long and wide pieces with a knife. Take 200g of potato pieces, put them into a 1L beaker, add 400mL of distilled water, boil for 10min, filter with gauze, discard the potato pieces, keep the filtrate, add 20g of glucose; 20g of agar; after heating to dissolve, add distilled water to 1000mL. Sterilize under high-pressure steam at 121°C for 20 minutes for use, heat and dissolve in a microwave oven before use, pour the plate on an ultra-clean operating table, and seal it with a parafilm after cooling for use.

[0026] Preparation of conidia suspension medium (also known as CMC medium): Each 1 LCMC medium contains 7.5g sodium carboxymethylcellulose, 0.5g potassium dihydrogen phosphate, 0.5g yeast powder, 0.5g ammon...

Embodiment 2

[0030] Embodiment 2 toxin-producing cultivation and yield monitoring

[0031] Inoculation culture of corn toxin-producing medium: After 7 days of culture in a desktop refrigerated constant temperature shaker, take the CMC conidia culture medium cultured on a shaker, scrape off the conidia carefully with an inoculation loop in an ultra-clean bench, and mix to form a suspension , take the sterilized pipette tip with a pipette holster, carefully draw 2mL of conidia suspension in the aseptic area of ​​the alcohol lamp and add it to 90 sterilized conical flasks packed with corn, seal with a parafilm tight. After the inoculation is completed, place the Erlenmeyer flask in a sterilized mold incubator, away from light. The culture temperature was set at 15°C and the humidity was 50%. Shake the Erlenmeyer flask evenly every day to ensure sufficient oxygen and uniform distribution of toxins, and promote the production of toxins. On the 11th day, 16th day, 21st day and 28th day of cul...

Embodiment 3

[0037] The separation and purification of embodiment 3T-2 toxin

[0038] After 28 days of inoculation and cultivation of the corn toxin-producing medium, the medium was taken out from the mold incubator, and the corn was carefully dried overnight in a drying oven. The next day, each 100g of corn culture was stirred and soaked overnight with 400mL of 70% ethanol, extracted three times, and the 70% ethanol extract was recovered by suction filtration. After the filtrate is collected, adjust the water temperature of the rotary evaporator to 65 degrees Celsius, rotate each 1L of ethanol extract to about 100mL-300mL at 40-60℃ on the rotary evaporator, and collect the water phase. Shake the remaining turbid aqueous phase to mix the precipitate. Add it to a 2L separatory funnel, add dichloromethane according to the water phase: dichloromethane 2:1 (v / v), shake evenly, and let it stand until it slowly separates. The dichloromethane phase was collected, and the aqueous phase was repea...

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Abstract

The invention belongs to the technical field of veterinary pharmacology and relates to a biosynthesis method of a T-2 toxin. The biosynthesis method utilizes corn particles as raw materials and comprises the following steps of carrying out fusarium graminearum sherbakoff recovery, carrying out conidium production, inoculating the corn toxin-production medium with the conidia, carrying out culture by the corn toxin-production medium, and carrying out ethanol extraction, dichloromethane extraction, one-step column chromatography and crystallization on the medium in a toxin-production peak stage to obtain T-2 toxin crystals. The biosynthesis method has extraction efficiency of 89%. Nuclear magnetism and mass spectrum structure identification proves that the T-2 toxin is correct. The T-2 toxin has the content greater than or equal to 95.66% and has a stable period more than 4 months. Compared with the traditional extraction technology, the biosynthesis method has the advantages that the one-step column chromatography can separate and purify a large amount of the T-2 toxin; the processes are simple and fast; a yield is high; the T-2 toxin has stable properties and high purity; a T-2 toxin synthesis cost is greatly reduced; the T-2 toxin synthesis processes are simplified; and the T-2 toxin can be widely used in toxicology and metabolism test research fields related to various toxins.

Description

technical field [0001] The invention belongs to the fields of veterinary pharmacology and natural medicine chemistry, and specifically relates to a biosynthesis method of T-2 toxin. Background technique [0002] T-2 toxin (I), chemical name 4β-15-diacetyl-8α-(3-methylbutyryloxy)-3α-hydroxy-12,13-epoxytrichothecene-9-ene. Molecular formula C 24 h 34 o 9 , with a molecular weight of 466.22, is the representative toxin of trichothecene group A in fusarium toxins, and is also the most toxic toxin in fusarium toxins. T-2 toxin has many pathogenic effects such as skin allergy, hemorrhagic syndrome, sepsis (Wyatt, et al., 1972; Yagen, et al., 1976), hematopoietic inhibition, diarrhea, vomiting, immunosuppression and teratogenicity. Mutagenesis (Sydenhame et al., 1991; Desjardins et al., 1993). [0003] [0004] Due to its potential carcinogenicity and wide distribution around the world, scholars from various countries have long studied the toxicity and metabolism of T-2 tox...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/18C07D493/10C12R1/77
CPCY02P20/582
Inventor 袁宗辉汪潇黄玲利潘源虎王玉莲陈冬梅陶燕飞谢书宇戴梦红王旭彭大鹏郝海红程古月刘振利
Owner HUAZHONG AGRI UNIV
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