*3.1. Yeast Concentrations Occurring in Di*ff*erent Extractive Olive Oil Processes*

The yeast populations present in samples of olives as well as of pastes, oil from decanter and pomaces obtained from the extraction processes carried out in the same crop season at the beginning (HD1), in the middle (HD2) and the end (HD3) of harvesting, were quantified (Figure 1).

The yeast concentration in the olives was not statistically different in the three days of sampling showing an average value of (5.6 ± 1.9) × 10<sup>2</sup> UFC/g. On the contrary, the yeast concentrations in the kneaded pastes, oil from decanter and pomaces of the first harvesting day (HD1) were statistically lower than the values found in samples from the second and/or the third harvesting day (HD2 and HD3, respectively).

A multidimensional map of the yeast concentrations quantified in pastes, oil from decanter and pomaces of the various extraction processes was obtained by PCA. The sample loading and score plots are reported in Figure 2.

**Figure 1.** Yeast concentrations at different steps of the oil extraction processes carried out at the beginning (HD1), in the middle (HD2) and the end (HD3) of harvesting in the same crop season. Different letters indicate significant different concentrations within each step (ANOVA, *p* < 0.05).

**Figure 2.** Principal Component Analysis of the yeast concentrations in different samples (pastes, oil from decanter and pomaces) during olive oil extraction processes carried out at the beginning (HD1), in the middle (HD2) and the end (HD3) of harvesting in the same crop season. The scores (**A**) and variable loadings (**B**) for the two first principal components.

The model explained 93% of data variability along the first (PC1) and second (PC2) principal components. The extraction processes clustered together according to the same harvesting date. A comparison between the score plot and the loading plot pointed out that the extraction processes showing a higher contamination by yeasts (HD2 and HD3) were all positioned on the left side of the plot. The extraction processes of the third harvesting date (HD3) were located in the left upper quadrant, crushed pastes being characterized by a higher yeast contamination than in the other processes (HD1 and HD2).

## *3.2. Identification of the Yeast Species*

Overall, twelve yeast species belonging to seven genera, besides the yeast-like fungus *Aureobasidium pullulans*, were detected in the different samples collected during the three harvesting days (Table 1). The isolation frequencies of each species were calculated according to the type of sample (olives, pastes, oil, or pomaces) and the harvesting day in which the extraction processes were carried out (Table 1). The comparison among the isolation frequencies highlighted that during the olive oil extractive process some species were typically found in olive fruits whilst other species were associated to crushed and kneaded pastes or found only in oil and in pomaces.

*Foods***2019**, *8*, 457



Moreover, frequencies of yeast species in different samples varied with the harvesting day. Indeed, washed olives were characterized by a significant presence of three different species: *A. pullulans, Candida norvegica*, and *Rhodotorula glutinis* (Table 1) which were often below the detection threshold in other samples. In the crushed pastes *Candida norvegica* and *Rhodotorula mucilaginosa* attained higher percentages than the other species that occurred at frequencies below 1% in samples obtained the first (HD1) and the second (HD2) harvesting day, while in the samples processed on the third harvesting day *A. pullulans* was found at 44%. In the kneaded pastes, the predominant yeast species on the first harvesting day were *Candida kluyveri* and *Saccharomyces cerevisiae* whereas on the second and the third harvesting day *Zygotorulaspora mrakii* and *Nakazawaea wickerhamii*. In oil samples from decanter the yeast species showing significant isolation frequencies were: *Candida adriatica*, *Nakazawaea wickerhamii*, *Nakazawaea molendini-olei*, *Yamadazyma terventina,* and *Metschnikowia fructicola*, although the latter species was isolated only from oil samples of the first harvesting day and *Y. terventina* and *C. adriatica* only from oil samples of the second and third harvesting day. Finally, pomaces were characterized by the presence of the same yeast species isolated from oil samples with the exception of *Z. mrakii*, a species isolated mainly from kneaded pastes of the second and third harvesting day.
