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Chapter III Material and methods
time 0 h and after 24 h of incubation at 37°C to enumerate surviving bacteria as described by
Xanthopoulos et al. (2000).
3.7.3 Determination of antimicrobial potential of probiotic strains
3.7.3.1 Production of H2O2
Overnight grown cultures (10 µl) were spotted onto ABTS-agar plates (2, 2'-azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid)) and incubated anaerobically at 37°C for 72 h.
After anaerobic incubation, plates were exposed to the atmosphere. Oxidative coloration of
ABTS by H2O2 was visible as a violet halo surrounding the colony of H2O2 producer
Lactobacillus strains, indicating H2O2 production (Marshall, 1979).
3.7.3.2 Screening for antagonistic activity
The agar spot test as described by Schillinger and Lucke (1987) was used for
screening the antagonistic activity of the selected Lactobacillus strains. 10 µl of overnight
Lactobacilli culture were spotted onto modified MRS agar (2 g/l glucose and 13 g/l agar) and
incubated at 37°C for 24 h. These plates were over-layered with MRS soft agar (7.5 g/l agar)
inoculated with ca. 1 x 10 8
CFU/ml of indicator strains. The agar spot test method of Uhlman
(1992) was used to test whether the inhibition zones observed in the screening for
antagonistic activity were due to the bacteriocin production or as a result of acid inhibition.
Briefly, cell-free neutralized supernatants were obtained from overnight producer cultures
grown in MRS broth at 37°C. After centrifuging the culture at 7,200 x g for 10 min, the
supernatants were neutralized with sterile 5 M NaOH and then boiled for 5 min to inactivate
residual viable cells. The supernatants were tested against the same indicator strains as
mentioned above.