*2.2. Olives' Fermentation*

Non-edible olives, 24 kg, were cleaned with cold water and inserted into a plastic vessel with a non-pre-acidified brine and 5% *m*/*m* NaCl, in a final working volume of 75%. No sterilization procedures were adopted on the non-edible olives before starting the natural fermentation, and no starter strains of yeast or bacteria were inoculated. The olive-brine mixture was aerated manually every 3 days to facilitate the growth of aerobic microorganisms. The fermentation was carried out for 60 days and the pH was monitored during all the process. Neither sodium chloride nor acids or bases were added to the brine after fermentation starting [18–21].

Brine samples were withdrawn for analysis at regular time intervals. The samples (1 mL) were aseptically transferred to 9 mL sterile 1/4 Ringer's solution. Decimal dilutions in the same Ringer's solution were prepared and used for the enumeration of lactic acid bacteria, yeasts and molds, and for enterobacteria.

The lactic acid bacteria amount was estimated on de Man-Rogosa-Sharpe agar (MRS Agar, Oxoid, Basingstoke, UK) added with 50 mg/L of Nystatin (Sigma, Saint-Quentin Fallavier, France) at 25 ◦C for 72 h.

The population of yeasts and molds was estimated on Yeast Glucose Chloramphenicol Agar (YGC, bioMérieux, Lyon, France) at 25 ◦C for 48 h.

Violet Red Bile Glucose agar (VRBGA; Oxoid, Basingstoke, UK) was used for enterobacteria counts, incubated at 37 ◦C for 24 h.

The analyses were carried out in triplicate and the plates were subjected to microbiological numbering by CFU counting.
