The invention discloses a kind of preparation method of Isphamycin after streptomyces aureus fully ferments fermented liquid purified and add hydrochloric acid turn salt refining and form comprise the steps such as acidifying filtration precipitation coarse crystallization filtration extracting crystallization washing dry mixing; Compared with the prior art the present invention has optimized technical process reduced reactions steps shorten extracting cycle in acidifying and settling step adopted cryogenic freezing water for cooling controlled temperature of reaction between 0 ~ 5 ℃ reduced the difference of duomycin in leaching process to changing loss constant product quality chlorquatrimycin Ledermycin and the foreign matter content such as dehydration tetracycline low Isphamycin content reaches 91 ~ 92%.

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