Explore The Metabolites From A Marine Bacillus

In the present work, an antibiotic-producing marine bacterium was isolated from a seawater samplecollected from Yuhuan, Zhejiang, China, identified and named as Bacillus amyloliquefaciens ESB-2on thebasis of phenotypic characteristics and16SrRNA gene sequencing.Two compounds were got from the Bacillus amyloliquefaciens ESB-2. The structures of thesecompounds were identified as macrolactin B and macrolactin A by MS,1H NMR and13C NMR.Response surface methodology was applied to optimize the fermentation conditions for rapid andefficient accumulation of macrolactin A and macrolactin B. Eight fermentation conditions were examinedfor their significance on macrolactin A and macrolactin B production using Plackett–Burman factorialdesign. Further optimization was carried out using Box-Behnken design of experiments to study theinfluence of process variables. The optimized fermentation condition for maximum macrolactin Bproduction was Peptone5mg/ml, yeast extract1mg/ml, glucose24.0mg/L, FePO40.01mg/ml, temperature27.2℃, initial pH value6.0, medium volume57.6%, rotation speed150r/min, inoculation5%andfermented for4days. Under the optimized conditions, the concentration of macrolactin B was increasedeight-folds and reached13.6μg/ml. The optimized fermentation condition for maximum macrolactin Aproduction was peptone14.8mg/mL, yeast extract1mg/mL, FePO40.01mg/mL, temperature26.3℃,initial pH value6.0, medium volume72.4%, rotation speed150r/min, inoculation5%and fermented for2days. Under the optimized conditions, the concentration of macrolactin A reached21.63μg/ml, representinga2.4-fold increase compared to the original standard condition, which was also17%higher than previoushighest report of18.5μg/ml and three times higher in terms of daily productivity.Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to theisola-tion and purification of two macrolactin antibiotics from marine bacterium Bacillus amyloliquefaciensfor the first time using stepwise elution with a pair of two-phase solvent systems composed ofn-hexane–ethyl acetate–methanol–water at (1:4:1:4, v/v) and (3:4:3:4, v/v). The preparative HSCCCseparation was performed on150mg of crude sample yielding macrolactin B (10.6mg) and macrolactin A(19.4mg) in a one-step separation, with purities over95%as determined by HPLC. Our resultsdemonstrated that HSCCC was an efficient technique to separate marine antibiotics, which provide anapproach to solve the problem of their sample availability for drug development.