*3.3. Sensory Quality*

− í í After fruit storage, panelists observed a decrease in fruitiness and a major positive aroma sensory characteristic in the oil samples of both cultivars, (Figure 2). The highest decrease was determined in oils stored at −20 ◦C (approximately 3 intensity units compared to control oils). The taste characteristics of the oils, such as bitterness and pungency, were less altered after the prolonged fruit storage than the olfactory characteristics, although in most cases slightly lower intensities were determined compared to the control, except for IB+4 oil, which was similar to IB-control oil (Figure 2). García et al. [39] found that bitterness and sensory quality of Picual oils obtained from fruits stored at RT decreased rapidly and that the loss was slowed down during storage at 5 ◦C. Morelló et al. [40] found that Arbequina olive oil had a decreased intensity of bitterness and pungency when produced from fruits that have been frozen on the trees. Inarejos-García et al. [37] observed a larger reduction of bitterness, determined as K225, in Cornicabra olive oil produced from fruits stored for 5 days at 20 ◦C compared to that obtained from olives stored at 10 ◦C for a week. The same authors concluded that prolonged storage could be useful for modifying the taste of oils of phenol-rich cultivars, such as Spanish Cornicabra, characterized by intense bitter taste that could affect consumers' preferences. On the other hand, preserving the bitterness and pungency in IB and RO oils could be very important, since the mentioned sensorial characteristics were shown to be typical for these autochthonous monovarietal olive oils [18,19], especially because these cultivars are included in the production of Croatian oils under the protected denomination of origin (PDO) "Istra", which gives them an added value.

− **Figure 2.** Results of sensory analysis of (**a**) Istarska bjelica (IB) and (**b**) Rosinjola (RO) monovarietal virgin olive oils obtained from fresh fruits immediately after harvest (control) and oils obtained from fruits stored seven days at three different storage temperature (RT—room temperature, +4 ◦C and −20 ◦C) prior to production. Results are expressed as mean values of the medians of three technical repetitions.

− − − In the oil samples obtained from the fruits stored for seven days at RT and −20 ◦C negative sensory characteristics were determined (Figure 2). In RT oils of both cultivars a slight intensity (around 1) of the "viney/winegary" defect was noted, while the defect "frostbitten olives" was recognized as the main defect in the oils obtained from fruits stored at −20 ◦C, with the intensity of 2.3 in the case of IB and 2.9 in the case of RO cultivar oil respectively. IB and RO oils obtained from the fruits stored at RT and −20 ◦C could not be classified as extra virgin olive oils (the highest quality category), since, according to the intensity of the recognized defects, they belonged to the virgin olive oil category (EEC, 1991). Sensory defects were prevented by the storage at +4 ◦C (Figure 2), indicating that +4 ◦C was the most appropriate temperature for fruit storage in order to assure good sensorial quality of the obtained oils. "Viney/winegary" defect, and "fusty", "muddy sediment" and "musty" defects usually develop in oils because of the proliferation of particular microorganisms (lactic, acetic and enteric bacteria, fungi and *Pseudomonas*) on olive fruits during unsuitable storage conditions [3,4]. Kiritsakis et al. [41] reported that Koroneiki olives stored at 0 ◦C and 5 ◦C had no fungus development, while this was not the case at 7.5 ◦C, where the noticed increase in oil acidity was a result of fungal lipase activity [41], which can cause development of sensory defects. Garcia et al. [38] have found a different response of the sensory quality of different cultivars: Blanqueta olive oil developed defects more rapidly than Villalonga olive oil during 30 days of storage at ambient temperature and at 5 ◦C, and the development of off-flavors was more rapid at ambient than at low temperature.

− Freeze injuries are a consequence of olive fruit cell dehydration and destruction caused by ice crystals forming inside the parenchyma cells, which cause destruction of cell membranes, leading to cell death and high oxidation of cell contents [40]. This is the consequence of the contact between enzymes and their respective substrates, which may have an effect on the composition of the obtained olive oil [40]. Freeze injuries were not detected on olives of both cultivars stored at +4 ◦C, which is in agreement with the result for Koroneiki olives stored at 5 ◦C and 7 ◦C for 40 days [41]. On the other hand, freeze injuries in the form of fruit skin browning and shriveling were detected by visual inspection on olives stored at −20 ◦C, which finally resulted in olive oils with perceived "frostbitten olives" defect (Figure 2). Although some authors reported that severe freezing damage of olive fruits on trees during winter time could have negative influence on the sensory characteristics of olive

oil [42,43], there is little information on how controlled freezing temperatures during olive fruit storage influence the sensorial profile of obtained oils. Romero et al. [43] reported two different descriptions of the "frostbitten olives" defect, which depend on whether the temperature changes took place abruptly, with rapid freeze–thaw cycles, or gradually. They reported that oils were grouped based on the concentrations of volatile compounds into two clusters, characterized by different profiles. The first was characterized by descriptors such as "soapy" and "strawberry-like" and the characteristic presence of ethyl 2-methylbutanoate and ethyl propanoate, and the second by "wood" and "humidity" descriptors and high concentrations of pentanal and octanal. In this study, the sensory profile of the "frostbitten olives" defect perceived by the panelists was described using a descriptor "wet wood" (Figure 2), which was more similar to the second profile reported by Romero et al. [43], indicating that a gradual drop of temperature took place during the controlled freezing at −20 ◦C, with the formation of extracellular ice and evaporation of liquid water inside the cells. According to Romero et al. [43], as water is removed from the cells, ice continues to grow and damages the cells until they break down.

#### *3.4. FAEE and Waxes*

FAEEs are closely related to health conditions of the fruits and their concentration is higher in olives that underwent hydrolytic and fermentative processes that produce additional amounts of free fatty acids and alcohols [44]. Regarding the FAEE parameter, there were no significant differences among the treatments in RO oils. However, an increase in FAEE concentration was observed in IB-RT compared to the IB-control oil (Table 2), which was probably a result of the softening and damage of the fruit tissue during prolonged storage as a consequence of accelerated ripening of the fruits at higher storage temperature (Table 1). Jabeur et al. [44] have found an increase in FAEE concentration during Chemlali olive fruit storage at ambient temperature (12–18 ◦C) for 25 days in closed plastic bags and in open perforated plastic boxes, probably a consequence of microorganism fermentation activity. In the oil samples investigated in this study, total FAEE concentration ranged from 4 to 12 ppm and as such was below the maximum legal limit of ≤35 ppm set for EVOO [8]. Although the FAEE values did not surpass the maximum legal limit, they were in line with the results obtained by sensory analysis of the IB-RT oil, where a slight intensity of "viney/winegary" defect was determined (Figure 2). The correlation found between FAEE amounts and fermentative defects was probably due to their common origin [1,6]. On the other side, the intensities of non-fermentative defects, e.g., "frostbitten olives", determined in the oils obtained after frozen storage of the fruits of both cultivars (Figure 2), are not related to the concentrations of FAEE as reported by the literature [1].

The concentration of waxes (C246) in the investigated samples ranged from 15 to 50 ppm (Table 2). Although the obtained values did not surpass the maximum legal limit for EVOO of ≤150 ppm [8], the RT treatment showed a significant increase in the concentration of most waxes compared to the controls and the other two treatments in the oils from both cultivars. Storage at room temperatures may cause acceleration of fruit ripening [9], which is followed by fruit cuticle thinning and softening of fruit tissue [10]. As a consequence of those changes, waxes from the waxy surface layer of the cuticle of olive fruit could be more easily extracted into oil. The more mature, and possibly the more degraded olive fruits were (as in the case of IB fruits stored at RT, Table 1), the higher was the amount of waxes extracted, which supported the assertion that higher concentration of waxes could indicate lower quality of olive oil [6,44]. The storage of fruits at temperatures lower than RT resulted in lower concentration of waxes in the obtained oils (Table 2), probably due to the delay in fruit ripening.

*Foods***2020**, *9*, 1445

**Table 2.** Concentrations of ethyl esters and waxes (mg/kg) in Istarska bjelica (IB) and Rosinjola (RO) monovarietal virgin olive oils obtained from fresh fruits immediately after harvest (control) and oils obtained from fruits stored seven days at three different storage temperature (RT—room temperature, +4 ◦C and −20 ◦C) prior to production.


Results are expressed as mean values ± standard deviation of three technical repetitions. Mean values labeled with a different superscript letter, within the same column and same cultivar are statistically different (Tukey's test, *p* < 0.05). In case there were no statistically significant differences the letters were omitted. EE C16—ethyl palmitate, EE C18—ethyl stearate, FAEE—fatty acid ethyl esters. C246 =C42 +C44 +C46, C0246 =C40 +C42 +C44 +C46.
