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Industrial preparation method for producing ginkgolide B through fermentation of ginkgo endophytic fungi

A technology of ginkgolide and endophytic fungi, which is applied in the field of biological metabolism synthesis and purification, can solve the problems of no industrial application prospect and low yield of ginkgolide B compound, and achieve large production capacity, low product cost and energy saving consumption effect

Pending Publication Date: 2021-11-30
陈开云 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ginkgolide B is fermented by endophytic fungi of Ginkgo plants, the reaction conditions are mild, there is no ginkgolic acid residue, less pollution, and the operation is relatively simple, but the yield of ginkgolide B compounds is low, and it does not have industrial application prospects

Method used

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Examples

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Effect test

preparation example Construction

[0064] According to one embodiment of the present application, there is provided an industrial preparation method for producing ginkgolide B by fermentation of ginkgo endophytic fungi, comprising the following steps:

[0065] S1: Using the conventional tissue separation method, the roots, stems, leaves, bark and fruits of Ginkgo biloba were taken respectively, soaked in ethanol for 1 min, rinsed with sterile water for 3 to 4 times, and then soaked in 2.5% sodium hypochlorite for 3 min; the tissues were cut into A small piece of 0.5cm×0.5cm is planted on the contact medium of the cut site, and the tissue imprinting method is used as a control to test whether the surface is thoroughly disinfected;

[0066] S2: Culture the medium in a constant temperature incubator at 28°C, observe regularly, and transfer the newly grown hyphae to the new solid medium with an inoculation needle, and transfer each strain after purification 2 to 3 times Preserve two of each on the inclined surface ...

Embodiment 1

[0161] The steps of the isolation, mutagenesis and preservation method of the original aspergillus fumigatus strain are as follows:

[0162] a. Using the conventional tissue separation method, take the root, stem, leaf, bark and fruit of Ginkgo biloba, soak them in 75% ethanol for 1 min, rinse them with sterile water for 3 to 4 times, and then soak them in 2.5% sodium hypochlorite for 3 min; Cut into small pieces of about 0.5cm×0.5cm, plant the contact culture medium on the cutting part, and use the tissue imprinting method as a control to check whether the surface disinfection is thorough.

[0163] b. Take a few slants of dominant strains cultured for a week, add the normal saline containing isoniatin respectively, then use the inoculation loop to scrape off the bacteria on the slant, shake it evenly, and pour the mixture into the sterile triangle Place in the bottle on a constant temperature shaker to resist shaking, and after shaking, filter through a funnel layered with st...

Embodiment 2

[0200] Strain preparation and industrial fermentation method:

[0201] i. Take the test tubes of the same size obtained in step e of Example 1, fill them with quantitative PDA medium respectively, and prepare basically the same slant for future use. Pick the activated strain and inoculate it on the medium slant. These slopes were placed in a 28°C incubator for 9 days;

[0202] ii. The culture medium formula of the liquid shake flask is PDA, pH 7. The initial pH value is 5.0. A total of 250ml was prepared, divided into 250mL Erlenmeyer flasks, 30ml per bottle, sterilized at 121°C for 30 minutes, then cooled to room temperature for use;

[0203] iii. Pick and transfer the cultured test tube slant strains into a 250ml Erlenmeyer flask equipped with shaker culture medium, and place it on a shaker for 24 hours; the shaker culture temperature is 28°C, and the rotation speed is 120 rpm;

[0204] iv. Prepare the seed culture medium according to the formula standard, pump the seed ...

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Abstract

The invention discloses an industrial preparation method for producing ginkgolide B by fermentation of ginkgo endophytic fungi, which comprises the following steps: separating an aspergillus fumigatus protovariant strain from ginkgo tree roots, branches, leaves or barks; using the aspergillus fumigatus protovariant strain as the start strain, combining the technologies of mutagenesis, test tube slant culture, seed culture, fermentation culture, membrane filtration, membrane concentration, extraction separation, macroporous resin purification, recrystallization and the like with a supercritical CO2 fluid drying technology and a full-automatic control technology, and producing the ginkgolide B compound through fermentation of the ginkgo endophytic fungi under the synergistic effect of the technologies of mutagenesis, test tube slant culture, seed culture, fermentation culture, membrane filtration, membrane concentration, extraction separation, macroporous resin purification, recrystallization and the like. The biosynthesis method of non-chemical synthesis and non-ginkgo leaf extraction of the bilobalide B compound is realized; the bilobalide B compound produced by using the technology disclosed by the invention is mild in reaction condition, relatively simple to operate, free of ginkgolic acid residue, low in product cost, high in yield, high in purity, low in residual solvent, low in energy consumption, large in productivity, small in environmental pollution and suitable for industrial large-scale production, and has good economic benefits and application prospects.

Description

technical field [0001] The invention belongs to the technical field of biological metabolism synthesis and purification, and in particular relates to an industrial preparation method for ginkgolide B fermented by ginkgo endophytic fungi. Background technique [0002] Ginkgo biloba is the most peculiar plant that has been discovered in the world and has made the greatest contribution to mankind. Ginkgo biloba has five major components and five major functions. Ginkgo biloba will be the most promising Chinese medicinal material in the world and can treat many problems in the world. Miscellaneous diseases: [0003] Ginkgolides A, B, and C: they are platelet activating factor (PAF) antagonists and the most important active ingredients in Ginkgo biloba. Ginkgolide B has the strongest physiological activity among ginkgolides, which is the strongest found so far. The platelet activating factor antagonist is clinically used for the treatment of thrombosis, acute pancreatitis and ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C12N13/00C12N15/01C12N1/14C07D493/22C12R1/68
CPCC12P17/181C12N13/00C12N15/01C12N1/14C07D493/22
Inventor 陈开云柏凤静
Owner 陈开云
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