Fractionation of bifidobacterial cell wall proteins.

The bifidobacterial cell wall

proteins were extracted from 50 ml of a bacterial culture in stationary growth

phase as reported by Hardie and Williams (13). Bifidobacterial cells were collected

by centrifugation for 10 min at 5,000 rpm at 4°C and then washed in 50

mM Tris-HCl (pH 7.6). The bacteria were resuspended in 2 ml of protoplast

buffer (50 mM Tris-HCl [pH 7.6], 1 M sucrose, 1.4 mM phenylmethylsulfonyl

fluoride, 15 mg/ml lysozyme). The suspension obtained was incubated for 90 min

at 37°C. Subsequently, the cell suspension was centrifuged for 3 min at 4,000 rpm

at 4°C, and the supernatant, containing the cell wall proteins, was collected and

stored at 20°C.