Monitoring of the Fermentations

Physicochemical control of the fermentation brines was achieved through periodical determination of pH, NaCl concentration (%, wt/vol), titratable (expressed as lactic acid, g/100 mL) and combined acidities (expressed as undissociated organic salts, Eq/L), according to the methods described by Garrido Fernández et al. (1997).

The microbial populations adhered to fruits were studied by washing olives twice in phosphate buffer solution (PBS), to remove non-adhered cells, pitting and weighting the olives and immediately transferring (~10 g) into a stomacher bag containing 75 mL of a sterile saline solution (0.9%, NaCl). Then, the fruits were homogenized for 1 min at 300 rpm in a stomacher model Seward 400 (Seward Medical, Ltd., West Sussex, England). Suspensions of the samples or appropriated dilutions were then plated onto solid culture media using a Spiral System (model dwScientific, Don Whitley Scientific Limited, England). Enterobacteriaceae were counted on Crystal Violet Neutral-Red Bile Glucose (VRBD) agar (Merck, Darmstadt, Germany), LAB were spread onto de Man Rogosa and Sharpe (MRS) agar (Oxoid, Basingstoke, Hampshire, England) supplemented with 0.02% (wt/vol) sodium azide (Sigma, St. Luis, MI, USA), and yeasts were grown on yeast-malt-peptone-glucose medium (YM) agar (Difco, Becton and Dickinson Company, Sparks, MD, USA) supplemented with oxytetracycline and gentamicin sulfate (0.005%, wt/vol) as selective agents. The plates were incubated at 30°C for 48–72 h, counted using a Flash & Go (IUL, Barcelona, Spain) image analysis system and expressed as log₁₀ CFU/g.

Samples obtained from fermentation brines were diluted, if necessary, in sterile saline solution (0.9% NaCl) and plated on the culture media described above. After incubation as described for fruit samples, counts in brines were expressed as log₁₀ CFU/mL.