C. difficile and C. perfringens colonies were suspended in phosphate
buffered saline (PBS, 0.01 M phosphate buffer and 0.154 M
NaCl) to obtain an OD600 of 0.220, and 200 ml of the suspension
were added to 5 ml BHI and 1 ml of probiotic supernatant harvested
after 48 h of incubation. MRS broth at pH 6.8 and pH 3.9was used as
control for the supernatant with and without pH neutralization,
respectively. Growth was measured spectrophotometrically at 0 h,
24 h and 48 h after anaerobic incubation at 37 C. The difference in
OD600 measurements in the control samples between 0 h and 48 h
of incubation was defined as 100% growth. Differences in OD600
measurements at 0 h, 24 h and 48 h of incubation of the test
samples were calculated as percentages of the value obtained from the control. Inhibition was defined as a reduction of growth of at
least 70% compared to the respective control samples. Each testwas
performed in duplicate.