The kinetics of the production of inhibitory substances were
investigated by inoculating 10 ml of an overnight culture of selected
Lactobacillus isolates into 100 ml of MRS broth followed by incubation
at 30 C. Samples were taken at appropriate time intervals
and measured for changes in pH, bacterial growth (600 nm; Hitachi
U 1100 spectrophotometer, Tokyo, Japan) and antibacterial activity
(CFS) against L. monocytogenes ATCC 19115. For this purpose the
agar well diffusion bioassaywas used by measuring the diameter of
the inhibition zone around the wells, and antimicrobial activitywas
expressed as arbitrary units (AU) per ml. One AU is defined as the
reciprocal value of the highest dilution showing a clear zone of
growth inhibition (Ghanbari, Rezaei, Soltani, et al., 2009).
In a separate experiment, the inhibitory effect of CFSs against
L. monocytogenes ATCC 19115 as indicator strainwas also examined
in a liquid medium. For this purpose, 20 ml of each filter-sterilized
cell-free supernatant were added to a 100 ml culture of the indicator
organism at early exponential phase (aged 4 h). These experiments
were also repeated with stationary phase cells. The
optical density at 600 nm and the viable cell count were monitored
hourly during an observation period of 20 h. Indicator cells incubated
without CFSs served as negative-control.