Piptoporus sp. A-9 for producing triterpenoid and application thereof
A technology for triterpenoids and compounds, which is applied in the directions of microorganism-based methods, fungi, microorganisms, etc., can solve the problems of long culture period and the like, and achieve the effects of low production cost, high application value and simple production method.
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Embodiment 1
[0031] Example 1 Isolation and Purification of the Triterpenoid-producing Trichomona Strains
[0032] The strains were collected from the fruiting bodies of basidiomycetes in the wild. Cut the fresh fruiting body with a sterile scalpel, tear off a little bacterial flesh with ophthalmic tweezers, inoculate it on a PDA plate, and culture it at 30°C. When the diameter of the mycelium mat reaches 2-3 cm, pick a little mycelium from the edge of the mycelium and inoculate it on a new PDA plate, and transfer it repeatedly until the pure strain is obtained. The target strain was re-screened by shake flask fermentation and further isolated and screened to obtain a stable triterpenoid-producing strain, which was named Piptoporus sp. A-9.
[0033] The above-mentioned strain Trichomona A-9 has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee on May 25, 2017. The preservation address is: No. 3, Yard 1, Beichen West Road...
Embodiment 2
[0036]Example 2 Sequencing of 18S rDNA and ITS sequence of Piptoporus sp. A-9CGMCC No.13895 strain
[0037] The triterpenoid-producing strain obtained in Example 1, that is, the CGMCC No. 13895 strain, was entrusted to Beshang Biological Company to perform 18S rDNA sequencing.
[0038] The experimental method is: pick the slant culture, extract the genome as a template, use the universal primers NS1 and NS8 to amplify the 18srRNA gene, the fragment size is about 1.8kb, and use the universal primers ITS1 and ITS4 to amplify the ITS sequence, the fragment size is about 750bp .
[0039] Take 5 μl for agarose gel electrophoresis, use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No. DV805A) to cut the gel and recover the target fragment, and perform DNA sequencing on the recovered product.
[0040] Sequencing results: the 18S rDNA sequence length of CGMCC No.13895 strain is 1679bp, and its nucleotide sequence is shown in SEQ ID NO.1; the ITS sequence fragment size of CGMC...
Embodiment 3
[0043] Example 3 Application of Trichotila A-9CGMCC No.13895 in the Preparation of Triterpenoids
[0044] The sequence of steps involved in applying the method is as follows:
[0045] (1) Strain selection: select Piptoporus sp. A-9CGMCC N0.13895;
[0046] (2) slant culture activation: inoculate the strains on the slant medium, and culture it statically for 100 hours under the condition of 30° C., and set aside;
[0047] (3) Seed culture: the bacterial strain cultivated in step (2) is put into 90-100mL (500mL Erlenmeyer flask) liquid seed medium with an inoculation loop under aseptic conditions, and the rotation speed is 160 rpm. Minutes, on a shaker with a rotation radius of 40mm, culture at 30°C for 80 hours to obtain a seed solution;
[0048] (4) shake flask fermentation: with the inoculum amount of 8~10%, inoculate seed liquid in the shake flask that 90-100mL (500mL Erlenmeyer flask) fermentation medium is housed, 30 ℃, rotating speed is 160 rev / Minutes, shaking the fla...
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