Optimization Of Fermentation Culture On Embellisia Oxytropis And Study On Primary Active Component

Endophyte which can produce various bioactive substances, including insecticidal, antimicrobial and antitumor compounds and so on, is a special and important group of microorganisms with biological diversity. It is also the important resources to seek new antibiosis and antitumor drugs.In recent years, the endophyte have be paied attention gradually, for it had a great variety and the few development characteristics. Moreover, the researcher discovered the active compound which endophyte produced not only had a great variety, but also has many new undeveloped substances. Therefore, the endophyte had received more attentions because of their enormous potential as to be new drugs. The research of anti-tumor endophyte had been just recently commenced, for the confinement of research method and device, we had not utilized the endophyte as drugs, so the research of endophyte had not only brought great social effects but also have actual significance.In this studay, the optimization of fermentation culture condition on Embellisia oxytropis, isolated from Oxytropis kansuensis(Leguminosae; Oxytropis DC.), was investigated. The active component from Embellisia oxytropis was isolated and analyzed. The results were as follow:Various media and conditions were used to study the effects on mycelia of Embellisia oxytropis by solid culture. The result was analyzed and showed that: the optimum culture conditions was pH6.5, 20℃, and the optimum medium was corn powder, yeast powder.Various media and conditions were used to study the effects on mycelia by shaking culture and 1g9(3)⁴ orthogonal experiment. The result was analyzed by SPSS method and direct-viewing analysis method. The magnitude of range indicated that the factors which effected the mycelia growth were carbon source, nitrogen source, KH₂PO₄ and MgSO₄ in sequence. The result showed that: corn powder was the optimum carbon source and potato is the optimum nitrogen source. The optimum medium was: corn powder 5%, potato 3%, KH₂PO₃ 0.1%, MgSO₄ 0.08%, V_B110μg/100ml, V_B215μg/100mL. The optimum fermented condition was: culture temperature 24℃, speed of revolution 100n/min, pH 6.5, the amount of inoculation 12%, and liquid volume 30%. Under this condition, dried mycelia weight was 0.573g per 100mL fermented liquid.The pre-disposal treatment had been carried on for the mycelia which were from the liquid in-depth fermentation. The mycelia were filtrated by Buchner funnel, flushed mycelia by distilled water, dried under 45℃and pulverized. Concentrate the extracting solution which extracted by Soxhlet's for 48h, by means of extraction between H₂O:CH₂CL₂=3:1 twice, rotary evaporating the upper abstraction, pH=7. The upper abstraction were isolated by 732 type cation exchang resin, eluted with NH₄OH 1mol/L(pH=7). The eluent was concentrated and freeze-dried. The alkaloid was detected by TLC.Culture solution was pretreated by removaling hybridprotein, pigment, polycose and metal ion. Disposaled culture solution was separated by D101 macroporous resin and 732 type cation exchang resin. It was eluted by absolute alcohol and 1mol/L NH₄OH respectively, purified by silica gel column chromatography. The result which detected by TLC showed that the extraction existed in portion of absolute alcohol and 1mol/L NH₄OH. The concentrated eluent which were freeze-dried was pallide-flavens cluster crystals existing.Comparing the detection of IR, MS and the reference, the extracted substance is a kind of alkaloid. It has a structure of Indolizidine alkaloids. The foundation which further studying on the chemical composition of Embellisia oxytropis has been settled.