Preparation of concentrated culture filtrate. L. coryniformis subsp. coryniformis
Si3 was inoculated to a concentration of 105 cells/ml of 800 ml of MRS broth
in 1,000-ml Erlenmeyer flasks, plugged with cotton to allow air access, and
incubated as a still culture at 30°C for 48 h. The culture was then centrifuged
(15,000 3 g, 10 min), followed by filter sterilization (0.45-mm pore size; Millipore).
The sterile cell-free supernatant was freeze-dried and resuspended (to a
15-fold concentration) in either 10 mM acetic acid (HAc) or 20 mM citratephosphate
buffer (pH 3.4). Culture filtrate from the type strain of L. coryniformis
subsp. coryniformis (ATCC 25602) was used as a control and prepared in the
same manner as that from strain Si3.