2.8. Preparation of cell free supernatant and well diffusion method
In order to obtain cell free supernatants (CFS), each LAB strain
was cultivated in MRS broth at 37 C for 24 h with 5% CO2. CFS were
obtained by centrifuging the cultures at 8078 g in the cold (4 C)
for 20 min followed by exposure to chloroform for 20 min. In order
to eliminate the inhibitory effects attributed to the organic acids,
these fractions with biological activity were neutralized by the
addition of 1 mol l1 sodium hydroxide. All strains that showed
antilisterial activity were tested by the well diffusion assay. TSAwas
first shown with the indicator organisms and then wells of 8mm of
diameter were made. Then, 100 ml of CFS and neutralized CFS from
each LAB were poured into different wells. The diameters of inhibition
zones around each well were measured after incubation at
37 C for 24 h with 5% CO2. All the experiments were carried out in
duplicate