Enzymatic activities

The o-nitrophenyl-ß-D-galactopyranoside (Sigma-Aldrich, Steinheim, Germany) substrate was used to determine the ß-galactosidase activity as described by Vinderola and Reinheimer [19]. The proteolytic activity in sterile skimmed milk was evaluated by the o-phthaldialdehyde (OPA) method [20]. Briefly, after incubation of the strains in sterile skimmed milk at 37°C for 48 h, proteins in 1 ml milk culture samples were precipitated. After centrifugation and filtration of the resulting supernatants, the o-phthaldialdehyde reagent (Sigma-Aldrich, Steinheim, Germany) was added and the proteolytic activity was measured by OD reading at 340 nm (Jasco, Great Dunmow, UK). The results, which were the average of triplicate samples, were expressed as μg/ml of α-amino groups released in the medium. Glycine (Fluka, Steinheim, Germany) was used as standard to build the calibration curve (concentration range 0–75 μg/ml). Aminopeptidase activity was evaluated as described by Gatti et al. [21]. Briefly, cells were resuspended at an OD₅₅₀ among 1.2–1.5 and incubated with 0.656 mM/l solutions of Phe-Pro-βNa, Arg-βNa, Lys-βNa, Leu-βNa and Pro-βNa substrates (Bachem Feinchemikalien AG, Bubendorf, Switzerland) for 30 min at 40°C. The activities were evaluated after OD₅₈₀ reading and results expressed as μM/l β-naphthylamine/ml culture/h. The determinations were performed in duplicate in independent experiments.