Statistical methodologies, including Plackett-Burman design and Box-Behnken design, were employed to optimize the fermentation conditions for the production of active substances against aquatic pathogen Vibrio harveyi by marine-derived Aspergillus flavipes strain HN4-13. The optimal crucial fermentation values for maximum production of active substances against V. harveyi were obtained as follows: X1 (peptone) = 0.3%, X2 (KCl) = 0.25%, and X3 (inoculum size) = 4.5%. The predicted diameter of inhibitory zone against V. harveyi was 23.39 mm, and the practical value reached 23.71 ± 0.98 mm with a 62.3% increase. Bioassay-guided fractionation resulted in the acquisition of two compounds whose structures were identified as questin (1) and emodin (2). Questin exhibited the same antibacterial activity against V. harveyi as streptomycin (MIC 31.25 µg/mL). This is the first time to report questin as a potential antibacterial agent against aquatic pathogen V. harveyi.
Keywords: Aspergillus flavipes; Bioassay-guided fractionation; Fermentation optimization; Questin; Response surface methodology; Vibrio harveyi.