*2.5. Zymogram Screening for Yeast Enzymatic Activities*

72 yeast isolates, belonging to the yeast species most frequently found in oil samples from decanter, were screened for enzymatic activities of potential interest in terms of olive oil quality as reported by Romo-Sánchez et al. [16]. The enzymatic activities screened were cellulase, polygalacturonase, ß-glucosidase, peroxidase, and lipase. The substrates used were, respectively, carboxymethylcellulose (CMC), polygalacturonic acid, cellobiose, H2O2, and CaCl2/Tween 80 (all purchased to Sigma Aldrich). Each isolate was grown in YPD (yeast extract 10 g/L; peptone 20g/L, D-glucose 20 g/L) broth at 30 ◦C for 24 h. To check for lipase activity, cultures were inoculated into 0.1% olive oil integrated with 0.01% Tween 80 broth. Cultures for checking cellulase and ß-glucosidase activity were then grown in a yeast nitrogen base (YNB) broth at 30 ◦C for 6 h under shaking conditions (100 rpm) for consumption of residual carbon source. Aliquots of 5 µL at 10<sup>6</sup> CFU/mL were spotted on agar plates containing YP (yeast extract 10 g/L; peptone 20 g/L, agar 15 g/L) and 1% of each specific substrate as single carbon source. All plates were incubated at 28 ◦C for 3 days, except for lipase activity for 7 days. The activity was detected for clear halo for polygalactunorase, according to Fernández González et al., [33], appearance of white precipitation areas for lipase, [34] or growth for cellulase and ß-glucosidase [35]. Peroxidase activity was assessed by oxygen bubble production from H2O2.

## *2.6. Yeast Inoculation into Commercial EVOO*

Some isolates belonging to the dominant yeast species (*C. adriatica*, *N. wickeramii*, *N. molendini*-*olei*, *Y. terventina*) and possessing some enzymatic activities were inoculated into a commercial filtered extra-virgin olive oil (EVOO). Each pure isolate was grown in YPD medium until the early stationary phase and then yeast cells were inoculated in order to have a final concentration of 10<sup>6</sup> cell/mL. The inoculated oil and samples without inoculum as control were placed in sterile glass tubes and bottles, in the dark at a temperature of 15 ◦C for 180 days until microbial and chemical analysis.
