**1. Introduction**

Extra virgin olive oil (EVOO) is not just a product obtained from the fruit of the olive tree by mechanical extraction, but rather the result of complex changes in fruit components. Because of these changes, chemical compounds affecting the qualitative characteristics for sensory acceptability of extra virgin olive oil [1] may be produced. Pleasant sensory notes, characterizing extra virgin olive oil, are mainly originated from aldehydes, esters, alcohols, and ketones, which are responsible for oil sensory attributes such as "green" and "fruity" [2–6]. Nevertheless, several phenomena can alter the initial pleasant flavor, giving rise to unpleasant sensory notes, classified, according to the current olive oil regulations (EU Reg. 1348/2013), into four groups: "fusty", "musty", "winey–vinegary", and "rancid". Microorganisms associated with the olives may affect oil quality according to their metabolic activities. Indeed, as reported by Vichi et al. [7–9], oils from microbiologically contaminated olives exhibited a lower quality level and influences of olive microbiota on oil characteristics were greater than the effects exerted by malaxation time and temperature. Guerrini et al. [10] showed that sensory defects and specific volatile compounds (i.e., 2-butanone, butyric acid, 2-heptanol, octanoic acid, 1-octen-3-ol) were correlated to both yeast and mould concentrations detected in extracted and filtered oils. Yeasts

and moulds are present in extracted oil because, during olive crushing, microorganisms of olives pass on into oil through both solid particles of olive fruit and micro-drops of vegetation water [11–13]. Some yeast species occurring in newly unfiltered oil can remain viable and metabolically active during the conservation period and, according to their metabolic capabilities, can either improve or worsen the oil quality [9]. Enzymatic activities of yeasts isolated from either olives or olive oil have been reported to include β-glucosidase, β-glucanase, polyphenoloxidases, peroxidase, lipase and cellulase activities [11,14–17]. Enzymes such as ß-glucosidase are known to improve oil quality by increasing phenolic compound extractability, while others such as lipase, polyphenoloxidases, and peroxidase are known to cause detrimental effects [5,18–20]. Recent studies [17,21] demonstrated that the presence of some yeast species might be responsible for olive oil sensory decay during storage. In particular, laboratory experiments showed the presence of defects in olive oil treated with specific yeast strains of *Candida adriatica*, *Nakazawaea wickerhamii,* and *Candida diddensiae*, while other olive oil samples treated with other *Candida diddensiae* strains were defect-free after four months of storage [22,23]. By the way, yeasts belonging to various genera were isolated in commercial extra virgin olive oil (*Candida*, *Nakazawaea*, *Williopsis*, *Ogataea*, *Yamadazyma* and *Saccharomyces*) [11–14,24–27].

Despite these evidence regarding the presence of viable yeasts in oil and their potential impact on olive oil quality, only few studies have investigated the yeast species occurring in the different phases of the olive oil extraction process and their effects on the oil quality [16,23,28]. In particular, a recent study of Mari et al. [28]. Mari et al. showed that the yeast populations occurring in olive oil extraction processes are numerically significant and originate principally from the yeasts colonizing the oil extractive plants. In fact, this study showed that only three of the eleven dominant yeast species detected on the washed olives were also found in extracted oil at significant isolation frequencies (*Candida adriatica*, *Nakazawaea molendini-olei*, and *Nakazawaea wickerhamii*). On the contrary, some yeast species showed significant isolation frequencies only in extracted oil (*Yamadazyma terventina*), or in kneaded pastes and pomaces (*Zygotorulaspora mrakii*). The occurrence of different yeast species according to the source of isolation (pastes, extracted oil or pomaces) suggests a contamination of the plant during oil extraction that select specific yeast species [28]. Ciafardini et al. [29] found a lower species diversity based on the origin of isolation. These Authors found six different yeast species (*Kluyveromyces marxianus*, *Candida oleophila*, *Candida diddensiae*, *Candida norvegica*, *Wickerhamomyces anomalus* and *Debaryomyces hansenii*). Except from *K. marxianus* that was found only in the wash water and *W. anomalus* that was found only in the six-month stored olive oil, all the other species occurred in the wash water and in the kneaded paste as well as in the newly produced olive oil. Anyway, a selected microbiota, when numerically significant, could affect olive oil quality in different ways, based on the specific metabolic capabilities of each yeast species or even strain. Therefore, the aim of this study was to assess whether the yeast microbiota occurring in olive oil extraction process affects the quality of extra virgin olive oil and, in particular, the volatile compounds content

For this purpose, at first, yeast species present during different extractive processes carried out in the same crop season as well as the chemical and sensory characteristics of the resulting olive oils were investigated. Then, some isolates belonging to the yeast species present at higher frequency in the process and possessing some enzymatic activities were inoculated into a commercial olive oil in order to assess their effective effects on extra-virgin olive oil quality.

#### **2. Materials and Methods**

## *2.1. Sampling throughout Olive Oil Extraction Processes*

During the same crop season, 14 batches of approx. 200 kg olives, form Frantoio, Moraiolo, and mixed cultivars, were processed in a Tuscany oil mill (Azienda Agricola Buonamici s.r.l., Fiesole, Florence, Italy). Olives were collected and processed, within 4 h of harvesting, in three different harvest time at ten-day intervals: 6 at the beginning (HD1a, HD1b, HD1c, HD1d, HD1e and HD1f), 5 in the middle (HD2a, HD2b, HD2c, HD2d and HD2e) and 3 at the end (HD3a, HD3b and HD3c). Plant for oil

extraction (TEM, Florence, Italy) consisted of a cleaning and water washing system, an olive grinding cutter crusher (mod. FR350), a controlled-temperature vertical axis malaxation equipment (500 kg capacity) (mod. V500), a "decanter" (two-step mod. D1500) with 1500 kg/h maximum capacity and a cardboard filter press (15 µm cut-off). Plastic residue or "alperujo" from decanter was subjected to separation by centrifugation of stone fragments to obtain destoned pomace. Olives were crushed at 2500 rpm (crusher holes 6.5 mm in diameter); malaxation was carried out at half capacity under vacuum (residual pressure of 20 kPa) at 22 ± 1 ◦C for a mean time of 15 min. Decanter worked with a screw conveyor rotating at a slower speed than that of the bowl. Samples were collected in double in several steps of extraction processes (washed olives, crushed and kneaded pastes, oils from decanter, pomaces), for microbial, chemical and sensory analyses (filtered oils).
