Study On The Extraction, Isolation, Purification And Biological Formulation Of The Antibiotic From Streptomyces Strain Men-myco-93-63

Streptomyces Strain Men-myco-93-63 is a biological agent isolated from potato scab disease declined soil, which has evident antagonistic aginst many plant pathogens. The objectives of this study are to isolate and purify the antibiotic produced by the biological agent, and use the crude antibiotic to make a biological formulation.At first a primary antibiotic screening model using Verticillium dahliae, and Streptomyces scabies as indications was established. The fermentation liquid was pretreated with inorganic flocculants, then extracted with macoreticular absorbent HZ-803 and further extracted by ethyl acetate to, finally prepare the crude antibiotic. The crude antibiotic was analyzed by reverse-phase high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: column : WATERS Nova-pak C18 (7.8 mm X 300 mm 6 um), detect wave length: 240 nm, flow rate: 2.0 mL/min, mobile Phase: 30%-80%-100% methanol. The result of analysis HPLC indicated that biological agent produce 13 components against Streptomyces scabies, and 7 components against Verticillium dahliae. The crude antibiotic was further purified by preparative HPLC. The Pre-chromatographic conditions were as follows: column : WATERS Nova-pak C18 (19 mm X 300 mm 6 urn) , detect wave length : 240 nm, flow rate: 15 mL/min, mobile Phase: 70%-70%-100% methanol. The 7 peaks were appeared on the pre-HPLC, collecting peak VI. By the analysis of UV spectrum data between peak VI and component 13, the former is identical with the latter. At last, based on the physical and chemical properties, MS, IR and UV spectra data, the moeular-type of the bioactive component was identified as C20H38O4.In addition, an antibiotic isolation and extraction process for pilotscale experiment was developed. The whole process was divided into three steps: 1)Pretreatment and Solid-Liquid separation: Firstly the fermentation was pretreated with 0.08% PAM, then cenlrifeged at 4000 rpm for 20 min. After it was filtrated, the filtrate was regulated at adding acid to pH 4.0. 2) Concentration : The fermentation liquid was concentrated at 80℃ and precipitated with methanol to dissolve the extraction. 3)Gain crude antibiotic: the methanol solution was precipitated with 10% ethanol ,and the filtrate was concentrated at 40℃ to obtain the crude antibiotic was obtained. Inthe meantime two biological formulations were made -Emulsifying concentrate (EC) and Granule (G). The EC agent is not only effective aginst Verticillium dahliae and Streptomyces scabies, but also suppressive to vegetable root nematode, which can kill 30.3% of the vegetable root nematode in 24 h after application. The G agent can kill 41.9% of nematode in 24h after application.