28 deefication of anfungal compounds 281 Purication The water/salt solubie extract from the fermented hydrolysate was first fractionated by ultra ditration (Ultrafree MCcentrifugal filter units, Millipore) ing membrane sizes of 50, 30, 10, and 3 kDa cut-off, following the manufacturer's instructions. Fractions were tested for the antifungal activity on P. roqueforti DPPMAFI as described in 26. An aliquot of the 3 kDa partially purified fraction (coresponding to 10 mg of peptides) was further automatically fractionated 32 fractions for cach run) by Reversed- Phase Fast Perfor- mance Liquid Chromatography (RP-FPLC), using a Resource RPC col- umn and an AKTA FPLC equipment, with the UV detector operating at 214 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Fractions were separated as deseribed in Rizzello et al. (2011). Solvents were removed from collected fractions by freeze drying Fractions were redissolved in 600 l sterile water and assayed for the antifungal ac- tivity and the peptide concentration. Proteins and peptides concentration in the extract and purified fractions was determined by the Bradfced (radford, 1976) and ophthaldialdehyde (OPA) methods (Church et al., 1983), respectively. ...
正在翻譯中..