Microbiological Analyses

Ten grams of sourdough was homogenized in 90 ml of sterile physiological solution (9 g/L NaCl) in a Stomacker 400 Lab Blender (PBI International, Milan, Italy) (1 min agitation, 1 min pause, and 1 min agitation). Total mesophilic bacteria were determined on Plate Count Agar (PCA) after incubation at 28°C for 48 h. Yeasts and molds were counted on YPD agar (20 g/L peptone, 20 g/L dextrose, 10 g/L yeast extract, and 20 g/L agar) after incubation at 30°C for 72 h. Enterococci were enumerated on Slanetz and Bartley medium (SB) after incubation at 37°C for 48 h. Fecal and total coliforms were counted on Violet Red Bile Lactose agar (VRBLA) after incubation for 48 h at 44 and 37°C, respectively. LAB were counted and isolated by using de Man, Rogosa, Sharpe (MRS) medium, maltose-added MRS (m-MRS), and sourdough bacteria (SDB) agar medium (Vera et al., 2009). Media for LAB were supplemented with 0.1 g/L cycloheximide (Sigma-Aldrich, Milan, Italy). Plates were incubated at 28°C for 72 h under anaerobic conditions using an anaerobic system (Anaerogen). All media and supplements, when not differently specified, were provided by Oxoid (Milan, Italy).