Five beads of the deep frozen cultures were placed in 500 mL of MRS broth (Oxoid Ltd., Basingstoke, UK) and incubated for 48 h at 37 C under anaerobic conditions in plastic jars containing AnaeroGen (Oxoid Ltd., Basingstoke, UK). The final broth was transferred under aseptic conditions into 50 mL sterile centrifuge tubes(Sarstedt Ltd., Leicester, UK) and centrifuged at 3000 g for 5 min.After centrifugation, the supernatant was discarded and the harvested
cells in the form of pellets were washed twice using phosphate buffer saline pH 7.0.