Preparation method of actinomycin D and application thereof
A technology of actinomycin and actinomycetes, applied in the field of preparation of actinomycin D, to achieve the effect of broad-spectrum and high-efficiency antagonistic activity
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Embodiment 1
[0021] The source and isolation culture of embodiment one Streptomyces mutans TRM45540
[0022] From the 0-30cm soil samples in the ancient city of Loulan, Xinjiang (1400m above sea level, 89 ° 22' 22 " in east diameter, 40 ° 29' 55 " in north latitude), adopt the coating plate method, with 5%, 10%, 15%, 20 % and 25% sodium chloride in 8 kinds of media, 28 ℃ constant temperature culture, a total of 35 strains of actinomycetes of 7 genera were isolated, and finally separated and screened out The biocontrol bacteria TRM45540 with the best antibacterial effect.
Embodiment 2
[0023] The identification of embodiment two Streptomyces mutans TRM45540
[0024] In order to further confirm the taxonomic status of the biocontrol bacteria TRM45540, the present invention uses traditional classification methods and molecular classification methods to classify and identify the strain.
[0025] 1 Culture medium for morphology observation
[0026] Inorganic salt starch agar (ISP4) medium: starch 10g L -1 ,(NH 4 ) 2 SO 4 4g·L -1 ,MgSO 4 2g·L -1 ,K 2 HPO 4 2g·L -1 ,CaCO 3 1g·L -1 ,NaCl 40g·L -1 , agar 18g·L -1 , pH 7.2-7.4.
[0027] 2 primers
[0028] Synthesized by Aoke Biotechnology (Beijing) Co., Ltd. using actinomycetes universal primers. The sequences are: 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3')
[0029] 3 test method
[0030] 3.1 Morphological observation
[0031] Ordinary microscope observation: using the insert method, cultivate the biocontrol bacteria TRM45540 at 28°C. Use an optical microscope to observe...
Embodiment 3
[0052] Example 3 Utilize Streptomyces mutans TRM45540 to ferment and prepare Actinomycin D
[0053] Inoculate a small amount of mycelium in the ISP4 medium, cultivate at 28°C for 4 days, and obtain the seed liquid of Streptomyces vulbilis TRM45540, take 2mL (8% inoculum size) of the seed liquid and inoculate it in the corn flour fermentation medium for fermentation and cultivation. The fermentation conditions It is: rotation speed 180rpm, initial pH 7.2-7.4, liquid volume 250mL, fermentation temperature 28°C, fermentation 7d. Collect the fermentation broth, filter the fermentation broth with four layers of gauze, and load the sample to the macroporous resin chromatographic column by wet method (fill 2.0kg of D101 or AB-8 macroporous resin per 100L of fermentation filtrate); then use 2 times the column volume of water, Eluted with 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution, 95% ethanol solution; combined 70-95% ethanol eluate, concent...
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