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Aspergillus versicolor ZJUTE2 and application thereof in preparation of sterigmatocystin

A technology of ZJUTE2 and Aspergillus versicolor, applied in the field of microorganisms, can solve the problems of high cost, cumbersome process, unreported ST yield, etc., and achieve the effects of short production cycle, few process steps, and easy strains

Pending Publication Date: 2022-07-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the patent does not report the yield of ST, and its preparation process requires repeated use of silica gel column chromatography, gel column chromatography and preparation of liquid, etc. The process is relatively cumbersome and the cost is high

Method used

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  • Aspergillus versicolor ZJUTE2 and application thereof in preparation of sterigmatocystin
  • Aspergillus versicolor ZJUTE2 and application thereof in preparation of sterigmatocystin
  • Aspergillus versicolor ZJUTE2 and application thereof in preparation of sterigmatocystin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Separation of Cordyceps sinensis colonizing fungus--Aspergillus versicolor ZJUTE2

[0027] (1) Collection of Cordyceps sinensis

[0028] Cordyceps sinensis was collected from Nagqu, Tibet in May 2019, and was identified by Professor Yang Kai of Zhejiang University of Technology as a compound of Cordyceps sinensis Sacc. parasitic on the larvae of bat moths. The sample (numbered CS-ZJUT-201905) was stored in the Institute of Natural Medicine, Zhejiang University of Technology.

[0029] (2) Isolation of strains

[0030] Wash the surface of Cordyceps sinensis with sterile water, immerse the washed plant samples in a container with 75% ethanol, take it out after 2 minutes, rinse with sterile water for 5 times, and then immerse it in a container with 0.1% mercuric chloride aqueous solution After 1min, take it out, rinse with plenty of sterile water to remove the residual mercuric solution. Under sterile operating conditions, use sterile forceps and blades to cut...

Embodiment 2

[0033] Example 2. Molecular biological identification of strain ZJUTE2

[0034] (1) Extraction of genomic DNA

[0035] Use TSINGKE plant DNA extraction kit (universal, Beijing Qingke Biotechnology Co., Ltd.) to extract the genomic DNA of strain ZJUTE2. The specific steps are as follows:

[0036] 1) Place the Spin Column in the Collection Tube, add 250 μL of Buffer BL, and centrifuge at 12,000 rpm for 1 min to activate the silica membrane;

[0037] 2) Pick the strain ZJUTE2 monoclonal bacteria on the plate of Example 1 into a 1.5mL centrifuge tube, add 400μL BuffergP1, vortex for 1min, and take a 65°C water bath for 20min. During this period, you can take it out and mix it upside down to fully lyse;

[0038] 3) Add 150 μL Buffer gP2, vortex for 1 min, and ice bath for 5 min;

[0039] 4) Centrifuge at 12000rpm for 5min, transfer the supernatant to a new centrifuge tube;

[0040] 5) Add an equal volume of anhydrous ethanol in the supernatant, immediately and fully shake and mi...

Embodiment 3

[0058] Example 3. Aspergillus versicolor ZJUTE2 fermentation to prepare Aspergillus versicolor

[0059] 1. Strain activation

[0060] Take out the cryopreservation tube of Aspergillus versicolor ZJUTE2 strain stored in the refrigerator at -80°C, thaw it at 4°C, dip an appropriate amount of spore suspension from the cryopreservation tube with a sterile cotton swab in the ultra-clean bench, inoculate it into the PDA medium, and put it in the PDA medium. The cells were cultured for 7 days in a constant temperature incubator at 28°C for bacterial activation.

[0061] 2. Strain fermentation

[0062] After activation for 7 days, the bacterial strain spores were washed down with 10 mL of the sterile aqueous solution containing 2% Tween 80 by volume, and placed in 100 mL of sterile water containing 2% Tween 80 by volume to obtain a spore suspension (spore concentration was 1×10 6 Pipette 1mL of spore suspension respectively, inoculate into 50 1L conical flasks containing rice cultu...

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Abstract

The invention relates to aspergillus versicolor ZJUTE2 and application of the aspergillus versicolor ZJUTE2 in fermentation preparation of sterigmatocystin. The invention reports a method for preparing high-purity ST with high yield through fermentation culture of the strain for the first time, so that the yield of ST reaches 2.04 g / kg rice, and the purity reaches 99.41%; the preparation method has the advantages of simple fermentation conditions, easy culture of strains, few process steps, short production period and the like, and has the potential of industrial and large-scale production. The method has important significance for solving the industrial problem that the high-purity ST standard substance or reference substance depends on import for a long time.

Description

(1) Technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a colonizing fungus of Cordyceps sinensis and its application in the preparation of Aspergillus versicolor, in particular to an Aspergillus versicolor ZJUTE2 and its application in the preparation of Aspergillus versicolor by a microbial fermentation method. Help to solve the industry problem of high-purity Aspergillus versicolor standard material preparation. (2) Background technology [0002] Aspergillus versicolor (sterigmatocystin, ST) is produced by Aspergillus versicolor, Aspergillus flavus, Aspergillus nidulans, Aspergillus rubella, Aspergillus gibberellum, Aspergillus pyrogen, Aspergillus claus, Aspergillus tetragoni, Aspergillus mucor, Aspergillus parasiticus and other Aspergillus species. a toxic secondary metabolite, molecular formula C 18 H 12 O 6 , the relative molecular mass is 324, the melting point is 246 ° C, soluble in most non-polar so...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P17/18C12R1/66
CPCC12N1/14C12P17/181
Inventor 应优敏周莹冉坤单伟光
Owner ZHEJIANG UNIV OF TECH
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