Pediocin 5 production and plasmid stability during continuous free and immobilized cell cultures of Pediococcus acidilactici UL5

Continuous production of pediocin 5 from Pediococcus acidilactici UL5 was investigated in MRS medium at different pH and dilution rates during continuous free cell (FC) and immobilized cell (IC) fermentations. Pediocin 5 activity from FC operated at a dilution rate of 0.31 h(-1) largely increased from 128 to 2048 AU ml(-1) as pH decreased from 7.0 to 5.0. Pediocin 5 activity in IC at a dilution rate of 0.93 h(-1) was much less affected by pH, varying from 256 AU ml(-1) at pH 7.0 to 512 AU ml(-1) at pH 5.0. At the optimum pH 5.0, the dilution rate greatly influenced pediocin 5 activity both in FC and IC. Pediocin 5 production during continuous FC culture decreased with time for all dilution rates tested except 0.31 h(-1) and average activity over 144 h cultures reached a maximal value of 4915 AU ml(-1) at a dilution rate of 0.26 h(-1). For IC, pediocin 5 production was stable with time and increased with the dilution rate from 256 to 1024 AU ml(-1) in the range of 0.47-2.28 h(-1). Three Listeria strains were tested for their ability to screen low bacteriocin-producing variants (Bac(v)(+)) of Bac(+) cells in FC and IC cultures by using a modified deferred antagonism method. Ten to 28% of Bac(v)(+) cells appeared after 144 h of FC cultures at dilution rates in the range 0.09-0.42 h(-1) and pH control set points of 5.0-7.0 while almost no Bac(v)(+) cell was detected during 192 h IC culture in the same pH range and for dilution rates varying from 0.47 to 2.28 h(-1). The Bac(v)(+) cells isolated produced eight- to 64-fold less pediocin 5 than the Bac(+) cells. Although electrophoresis analysis showed no apparent difference in the plasmid profiles of Bac(v)(+) and Bac(+) cells, the Bac(-) mutant obtained by acriflavine treatment had lost the pMJ5 plasmid encoding for bacteriocin production. The decreased quantity of plasmid DNA in Bac(v)(+) cells suggests that the decreased pediocin 5 activity of Bac(v)(+) cells resulted from a decrease in plasmid copy number.