Figure 3. Purification of the antimicrobial peptides secreted by Aeromonas veronii.

The eluted at 100% acetonitrile (ACN) upon solid phase extraction was loaded onto a C18 column (250 × 4 mm, Vydac). Elution was performed with a linear gradient of acetonitrile in acidified water (dotted line), and absorbance was monitored at 225 nm. Each individually collected fraction was tested for its antimicrobial activity against A. hydrophila (white bar) and D. nishinomyaensis (black bar). (a) Fractions containing antimicrobial active substances were further purified by two additional RP-HPLC (b,c). The fraction that is still active at the third step of chromatography was further analyzed by mass spectrometry (Fig. S2).