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Hirsutella sinensis ZJB18002 and application thereof

A technology of ZJB18002 and Trichosporium chinensis, which is applied to Cordyceps sinensis Trichosporium chinensis ZJB18002 and its application field, can solve the problems of long growth cycle and low mannitol content in Trichospora mycelium, and achieve the effect of great application prospect.

Active Publication Date: 2018-12-25
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of the long growth cycle of wild Cordyceps sinensis and the low mannitol content of the mycelia of Mortierella chinensis cultured by artificial fermentation, and creatively apply the methods of morphology and molecular biology to isolate and identify a new strain of Mortierella chinensis. Strain L0106 (Hirsutella sinensis L0106), expanded by artificial fermentation, and using mutagenesis technology to increase the oxalic acid content of Trichospora sinensis

Method used

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  • Hirsutella sinensis ZJB18002 and application thereof
  • Hirsutella sinensis ZJB18002 and application thereof
  • Hirsutella sinensis ZJB18002 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Screening culture and identification of wild Cordyceps sinensis Trichosporum sinensis

[0026] 1. Screening and cultivation

[0027]Excavated 45 Cordyceps sinensis complete fungus complexes with thick and plump fruiting bodies and bulky insects from the hillside at an altitude of 4000-4300 meters in Yushu, Qinghai. The host insects are bat moth larvae. Put them in a 15°C incubator for maintenance. Use a cleaning brush to remove impurities on the surface of the fruit body, rinse with sterile purified water, and then disinfect with 0.1% mercuric chloride. In the ultra-clean bench, use a sterile scalpel to cut open the inner sclerotium of the worm body, and pick out the upper, middle, and lower parts of the fruit body. The tissues of the three parts were placed on the sterilized slant medium (50 μg / mL) containing gentamicin, cultured in a constant temperature incubator at 16°C, and observed and recorded every day. The isolated tissue began to germinate afte...

Embodiment 2

[0030] Embodiment 2, the morphological identification of Cordyceps sinensis isolate

[0031] The growth and single colony morphology of the wild-type bacterium L0106 in Example 1 was observed on the slant medium, and the microorganisms on the fixed slide were observed with a scanning electron microscope to understand the morphology of the microorganisms. After identification, the mycelium grows slowly, and the slope needs to be cultivated for about 40 days. The mycelium is dense, difficult to pick, and has a snow-white bulge. No longer round, like vermicompost, many folds, fluffy hyphae outward, such as figure 1 shown. Electron microscope observation of the mycelium morphology showed that the mycelium was well developed, slender, with a transverse septum, and a diameter of 1.5-2.0 μm. The sporophytes that formed spores could be observed, and the results were as follows: figure 2 shown. After morphological identification, it was preliminarily judged that the colony formed b...

Embodiment 3

[0032] Embodiment 3, the molecular biological identification of Cordyceps sinensis isolate

[0033] (1) 18S rDNA primer design

[0034] Primers:

[0035] p1:5'-TCCGTAGGTGAACCTGCCG-3' and

[0036] p2:5'–TCCTCCGCTTATTGATATGC-3'. Primers were synthesized by Shanghai Shenggong Company.

[0037] (2) Genome extraction

[0038] The genomic DNA of the wild-type cell L0106 in Example 1 was extracted by using a rapid nucleic acid extractor and a microbial genome extraction kit (MP Company, USA).

[0039] (3) 18S rDNA sequence amplification

[0040] Using genomic DNA as a template, PCR amplification was performed using universal primers (BioRad, USA, PTC200 amplification instrument). The reaction conditions were: pre-denaturation at 95°C for 5 minutes, cycle parameters were denaturation at 94°C for 45s, annealing at 55°C for 60s, and 72°C. Extended for 90s, repeated 35 cycles, continued to extend for 10min at 72°C, and finally identified the PCR product by 0.9% agarose gel electrop...

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Abstract

The invention discloses Hirsutella sinensis ZJB18002 and application thereof in production of cordycepic acid, and provides a novel slant medium for isolating Hirsutella sinensis, a biological identification method of Hirsutella sinensis, and a method to acquire a strain with 2-3 times higher yield of cordycepic acid through mutagenesis. An effective means to widen the biological application rangeof Hirsutella sinensis is provided, and the methods and the medium above have a major application prospect.

Description

(1) Technical field [0001] The invention relates to the screening and application of a high-yield strain of Mortierella sinensis oxalic acid from the "Bailing" production fungus Cordyceps sinensis anamorph. (2) Background technology [0002] Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is a complex of Cordyceps sinensis parasitizing on the larvae of Lepidoptera (Lepidoptera) bat moth (Hepialus armoricanus Oberthur) larvae and larval corpses (including subunits and worm bodies ). Cordyceps sinensis is a kind of precious traditional fungal medicinal resources, which has the characteristics of diverse metabolites and biological activities, and has shown great application and development prospects in the field of biomedicine. Cordyceps sinensis has attracted much attention because of its wide and obvious medicinal effects, and is highly respected all over the world. Chinese medicine believes that Cordyceps sinensis enters the two meridians of the lung and kidney, whic...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P7/18C12R1/645
CPCC12P7/18C12N1/145C12R2001/645
Inventor 柳志强郑裕国易明张博秦祥田许峰滕毅袁水金金美英
Owner ZHEJIANG UNIV OF TECH
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