Study On Extraction Condition And Assay Method Of Natamycin

Natamycin(Pimaricin)is a kind of macrolide antifungal antibiotic. Because it is one kind highly effective anti-fungus antibiotic which can special suppresses the yeast and the mildew, and it is widely used in food industries and medical treatment.In this study, the work focused on that made some measures to increase output of natamycin. The major research contents and results were as follows:A RP-HPLC method and an ultraviolet spectrophotometric method for the analysis of Natamycin in fermentation broth were developed. The sample was separated by methyl alcohol, and natamycin was quantified by the absorbance at 303 nm in the ultraviolet spectrophotometric method. The sample was separated at 25℃on a Dikma Technologies C18(5μm, 250×4.6 mm)eluted at a flow rate of 1 mL/min with a mobile Phase of methanol-water(60/40, V/V). Natamycin was detected and quantified by the absorbance at 303 nm in the RP-HPLC. The first one was simple and rapid, but was disturbed by somethings at a time. The second one was accurate but waste of time.The optimization of the flask fermentation condition was studied. pH, inoculum concentration and the optimal medium for dissolve oxygen on the yield of natamycin fermentation were studied. The result was that a 300 mL flask containing 45 mL medium was cultivated at 28℃and the inoculum concentration was 6%, the agitation speed was 200 r/min, pH 7.5. Natamycin was extracted with methanol and response surface methodology was employed to optimized the process variables of the major factors (pH, rotational speed,liquid-to-solid ratio). The results showed that all the parameters had significant effects on natamycin yield(P<0.05), the interaction of pH and liquid-to-solid ratio was very significant(P=0.0103), but the interaction of pH and rotational speed, rotational speed and liquid-to-solid ratio were not significant. The optimum process parameters were obtained: pH10, rotational speed 420 r/min, liquid-to-solid ratio 5:1. Under these conditions, the natamycin yield was 81.02%. The final natamycin production reached 3.34 g/L, with the former's production 2.54 g/L compared with 31.5% increase. After crystallize natamycin average yield was 77.0%, purity was 88.3%, titer≥8.52×103 IU/mg.The batch fermentation in a 10 L bioreactor were studied. Initial glucose concentration: 40 g/L, magnesium phosphate 0.2%, after fermentation time 48 h added sodium propionate 0.2%, liquid volume: 6 L/10 L, inoculum size: 10% of inoculum was inoculated in 10 L cans within, temperature: 28℃, defoamer: bubble enemy, ventilation: 2 L/min, speed: 200 r/min, fermentation period: 96 h. Natamycin yield of the batch fermentation was 3.78 g/L. The natamycin yield of batch fermentation was 13.2% higher than that of the shake flask fermentation.