The most active fraction was dissolved in Milli Q water,
filtered through 0.22 mm filters, and then separated by Reversephase
high-pressure liquid chromatography (RP-HPLC) on a
Waters C18 column (4.6 mm  250 mm) (XBrideg, Ireland).
Sample was injected at a volume of 60 ml (50 mg/ml). Peptides
were eluted with eluent A (water containing 0.1% trifluoroacetic
acid (TFA)) for 5 min, then with a linear gradient of acetonitrile
(40e100% for 60 min) containing 0.1% TFA at a flow rate of 1 ml/
min. On-line UV absorbance scans were performed between 200
and 300 nm at a rate of one spectrum per second with a resolution
of 1.2 nm. Chromatographic analyses were completed
with Millennium software. The antibacterial activities of the
eluted peaks were determined. The liquid chromatographic system
consisted of a Waters 600E automated gradient controller
pump module, a Waters Wisp 717 automatic sampling device
and a Waters 996 photodiode array detector. Spectral and chromatographic
data were stored on a NEC Image 466 computer.
Millennium software was used to plot, acquire and analyse
chromatographic data.