Comparison of the effects of various attenuation methods on cell permeability and accessibility of intracellular enzymes in Lactococcus lactis strains

Three Lactococcus lactis cheese starter cultures were exposed to various cell attenuation methods including sonication, chemical treatments, heat-shock and freeze-shock. Treated cells were subsequently assessed for changes in cell permeability and accessibility to the intracellular peptidases Pep X and Pep N. Generally, for all three strains, as the duration of sonication increased, the levels of intracellular peptidases in the cell free extract increased. For the other treatments, cell pellets of strains were prepared and treated with isopropyl alcohol (IPA), sodium dodecyl sulphate (SDS), hexadecyltrimethylammonium bromide (CTAB), heated over the range 45-65 degrees C or freeze-shocked using liquid nitrogen. The effects of various treatments on cell permeability were assessed using flow cytometry after staining with the Live/Dead (R) BacLight (TM) reagent kit. Treatment of cell pellets with CrAB generated a unique and highly permeabilized sub-population having the highest levels of accessible intracellular enzyme activity. Treatment with SDS also resulted in highly permeabilized cells and enhanced accessibility to Pep X or Pep N. The findings in this study are significant for the production of permeabilized L lactis cell fractions having enhanced accessibility to intracellular enzymes. (C) 2008 Elsevier Ltd. All rights reserved.