Since intact endospores treated with the bacteriocin did not
produce viable counts, a treatment with trypsin (which inactivates
AS-48) was applied in order to determine whether the bacteriocin
adsorbed on the spores would be responsible for inactivation
during the germination and outgrowth phases, or if intact spores
could be inactivated directly by the bacteriocin before germination.
Intact endospores of G. stearothermophilus CECT 43 were inoculated
in TSB broth with or without enterocin AS-48 (0.75 mg/ml) and
incubated at 30 C. After 5 min incubation, the samples were supplemented with trypsin (Sigma, Madrid) (1 mg/ml in 0.1 M phosphate buffer) to a final concentration of 100 mg/ml, or with
phosphate buffer alone. After 20 min of further incubation at 30 C
(with or without trypsin), spore suspensions were serially diluted
in sterile saline solution and plated for viable counts.