*3.3. VOOs Characteristics*

Different features influence the definition of VOO quality. From a consumer point of view, the hedonistic aspects are relevant although also the nutritional ones may be of concern. The main impact on the nutritional score of VOO is ascribable to the FA (fatty acid) composition and particularly to the amount of oleic acid [42]. The phenolic compounds, together with other bioactive ones, also play a key role, as proven by the specific health claim adopted by the European Union [43]. On the other hand, while phenolic compounds are responsible for the organoleptic features of VOOs [44], it is not true for oleic acid. Finally, from a producer and seller point of view, compliance with the mandatory quality parameters defined by the EU law is of primary concern [31]. Starting from these considerations, and in the view of the valorization of the oils from Oliva Rossa, the VOOs were characterized for their fatty acid composition as well as the basic quality parameters and the minor compounds. Given the sampling issues related to the crop years 2016/2017 and 2018/2019—which may bias the comparison—only the results of the years 2017/2018 and 2019/2020 are reported and discussed. Table 1 reports the FA composition of the Oliva Rossa VOOs.

**Table 1.** Fatty acid composition of the Oliva Rossa virgin olive oils (VOOs) extracted during fruit ripening in two different harvest seasons (mean ± SD, *n* = 3).


\* Pi (mean, *n* = 3) of the drupes at the moment of oil extraction; SFA, total saturated fatty acids; MUFA, total monounsaturated fatty acids; PUFA, total polyunsaturated fatty acids; O/L, oleic acid over linoleic acid ratio. Different letters for each parameter indicate significant differences according to two-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S2–S4: three subsequent samplings.

> The palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acids were the most abundant, as is typical in olive oils. Oleic acid, the main monounsaturated fatty acid, ranged from about 71% to just over 76%. The global mean value was roughly 73%, which is similar to the data reported elsewhere [24,25]. The crop year, the sampling time and the

interaction among those factors showed an effect on the C18:1 content. The VOOs extracted during 2017/2018 had significantly higher values of C18:1 than those of 2019/2020. Further, while in 2017/2018 an increase in C18:1 was observed during ripening, the opposite was found in 2019/2020. The second most abundant fatty acid, linoleic acid, ranged between around 8% to 13.51%. A mean value of about 8% was reported in the Italian monovarietal oils databank [24].

Linoleic acid content was generally higher in 2019/2020 with respect to 2017/2018; also, a significant effect of the interaction between harvest year and sampling was observed. In particular, while in 2017/2018 a decrease in linoleic acid was reported during ripening, in 2019/2020 it was the opposite. The palmitic acid content showed a significant difference among the samples with an opposite trend in the studied harvest seasons. Indeed, the highest content was found at S4 in 2017/2018 while in S2 in 2019/2020. The stearic acid content was on average around 2.60% and it showed a regular decrease in content during 2017/2018, while again an opposite trend was observed in 2019/2020. Finally, other significant differences were observed for all the minor fatty acids. The content of some minor fatty acids (namely, miristic, arachic and gadoleic) in 2019/2020 oils deserves special attention, as they were close to the maximum value allowed by official regulations [31]. Altogether, it comes out that Oliva Rossa VOOs were richer in MUFA (total monounsaturated fatty acids) in 2017/2018 whilst richer in PUFA (total polyunsaturated fatty acids) in 2019/2020. Giving a clear explanation of the observed differences could be a difficult task considering the complex effect of single factors (genetic, environmental and agronomic), their interactions or independence, as reviewed by Inglese and others [39]. However, differences in the oleic, palmitic and linoleic acid content were mostly related to the course of temperatures during the year. In particular, lower temperatures could be correlated with a higher content of oleic acid and, on the opposite, with a lower content of palmitic and/or linoleic acids [39]. In the 2017/2018 crop season, the mean temperatures registered during fruits ripening were actually lower than those registered in the 2019/2020 season (Figure S1) and could justify the differences observed in terms of fatty acids composition.

From a technological point of view, it is a fact that the different composition in fatty acids could in turn influence the oxidative stability of the product. In particular, values of O/L equals or higher than 7 have been suggested as an indication of good stability [45]. From Table 1, it could be observed that the ratio was different between the years and, in 2017/2018, it was always higher than 7.

The results of the basic chemical analyses of the VOOs are reported in Table 2.


**Table 2.** Basic quality parameters of the Oliva Rossa VOOs extracted during fruit ripening in two different harvest seasons (mean ± SD, *n* = 3).

\* Pi (mean, *n* = 3) of the drupes at the moment of oil extraction; FFA, free fatty acids; PV, peroxide value; K232 and K270, specific absorption at 232 and 270 nm, respectively. Different letters for each parameter indicate significant differences according to two-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S2–S4: three subsequent samplings.

> FFA is commonly the benchmark parameter for the vertical differentiation of olive oils into a specific product class (extra, virgin and lampante) [9]. The mean oil acidity ranged from 0.61% to 0.34%, being on average higher in the harvest season 2017/2018. The observed values could be considered quite high for freshly extracted oils. During maturation, a significant decrease in FFA was observed in 2019/2020. This decreasing

trend was also reported by other authors [16]. Moving to the oxidation assessment, PV and K232 are generally considered markers of primary oxidation, while K270 is a marker of secondary oxidation products. Differences were highlighted considering both the harvest season and the sampling time. A specular trend was observed between the years. The oils extracted in 2019/2020 were affected by a higher extent of primary oxidation (PV and K232) but, on the average, by a less pronounced secondary oxidation (K270). This might be linked to the significantly higher amount of PUFA in oils from 2019/2020 and to the significantly lower value of O/L (Table 1), indices of a higher susceptibility to oxidation.

On the whole, from Table 2, it could be stated that the Oliva Rossa VOOs still were classified as extra virgin olive oil (based on the reported parameters) till S3, which, in turn, corresponded to the maximum oil content (Figure 2). Obviously, these results cannot be generalized for all the VOOs obtained from this cultivar because the quality parameters are mostly influenced by the technological aspects of olive processing. Hence, it should be clear that such conclusions are related to the experimental conditions of the present study.

The profile of the minor compounds of the oils is reported in Table 3. Minor compounds are fundamental molecules of VOO, which, in turn, influence its stability to oxidation, its nutritional and healthy aspects and its organoleptic features [44,46]. The oils had a high-medium content of TPC, ranging from about 800 to 350 mg kg<sup>−</sup>1. These results agree with the few available data in the literature [24,25]. The phenolic content decreased significantly during ripening, as is well known, in both the harvest seasons [37,47,48]. Moreover, the effect of the harvest season on the TPC was evident, with the 2017/2018 season having a general higher content with respect to the 2019/2020 season. The tocopherols content was also remarkable. The content of α-tocopherol decreased during ripening, without significant differences in 2017/2018. In 2019/2020, an outstanding content of α-tocopherol was reported at S2. The total amount of tocopherols followed the general trend reported for α-tocopherol considering that the latter represents roughly 90% of the total tocopherols in olive oils. The sum of the β- and γ-tocopherols ranged from about 6 to about 3 mg kg−<sup>1</sup> with the highest significant value reached at S2 in 2019/2020.


**Table 3.** Minor compounds (mg kg−1) of the Oliva Rossa VOOs extracted during fruit ripening in two different harvest seasons (mean ± SD, *n* = 3).

\* Pi (mean, *n* = 3) of the drupes at the moment of oil extraction; TPC, total phenolic compounds. Different letters for each parameter indicate significant differences according to two-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S2–S4: three subsequent samplings.

> Pigments are important compounds of olive oils, which affect the product stability and also could give useful information about fruit ripening and authenticity [46,49]. During ripening, the pigment content decreased although we observed a parabolic trend, especially considering the harvest season 2017/2018. Ripening is generally reported as the main source of variability in pigment content despite other factors [48,50], even if technology could also have an impact [51]. The ratio between chlorophylls and carotenoids (data not shown) was always very close to unity, as reported in other studies and suggested for authenticity purposes [49].

> The profile of the phenolic compounds by HPLC for the harvest season 2017/2018 is reported in Table 4. In Figure S2, the relative chromatograms can be observed.


**Table 4.** Identified phenolic compounds (mg GAE kg<sup>−</sup>1) of the Oliva Rossa VOOs extracted during fruit ripening in the 2017/2018 harvest season (mean ± SD, *n* = 3).

GAE, gallic acid equivalents. 3,4-DHPEA, hydroxytyrosol; *p*-HPEA, tyrosol; 3,4-DHPEA-EDA, decarboxymethyl oleuropein-aglycone di-aldehyde; 3,4-DHPEA-EDA-CARB, carboxymethyl oleuropein-aglycone di-aldehyde; *p*-HPEA-EDA, decarboxymethyl ligstroside-aglycone di-aldehyde; *p*-HPEA-EA, ligstroside-aglycon. Different letters for each parameter indicate significant differences according to one-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S2–S4: three subsequent samplings.

The identified compounds were those commonly found in VOOs, belonging to the classes of phenolic alcohols (3,4-DHPEA, *p*-HPEA), phenolic acids (vanillic and syringic acids), flavonoids (luteolin and apigenin), lignans (pinoresinol) and, most importantly, secoiridoid derivatives (3,4-DHPEA-EDA, *p*-HPEA-EDA and *p*-HPEA-EA). Phenolic acids were found in very little amounts, according to the literature data [44].

Similarly, a minor contribution of phenolic alcohols was observed. It is known that phenolic alcohols originate from the more complex secoiridoid moieties mostly during VOO storage [44]. The di-aldehydic forms of 3,4-DHPEA and *p*-HPEA were the most abundant phenols in all the maturity stages, followed by a remarkable amount of pinoresinol. At the first sampling (S2), the oils showed the highest significant content of almost all the identified phenolic compounds. During ripening, the profile changed with a sharp reduction in 3,4-DHPEA-EDA and a less pronounced decrease in *p*-HPEA-EDA. At S3, the oils were characterized on one hand by the significantly higher amount of luteolin and 3,4-DHPEA-EDA-CARB while on the other by the significant lower content of *p*-HPEA-EDA and pinoresinol. At S4, the lowest significant amount of phenolic compounds was observed. Some phenols showed a parabolic trend during ripening, having a maximum or minimum content in correspondence to the technological optimum (S3); it was also the case for 3,4-DHPEA-EDA-CARB, *p*-HPEA-EDA, pinoresinol and luteolin. Such a trend could be linked to the complex pattern of biochemical and chemical phenomena, which could affect the relative amount of phenols in the VOOs and, to a lesser extent, to the natural variability of the sampled material.

Although no organoleptic assessment of the oils was carried out in this study, the relationship between phenolic compounds and the sensory features of the product is well known and confirmed by numerous works [5,44,52]. *p*-HPEA-EDA, also known as oleocanthal, have been proved to be the major compound responsible for pungent notes while other secoiridoid derivatives are more strongly linked to bitterness [44]. Considering the TPC (Table 3), the phenolic profile (Table 4) and the similarities with the data reported for the Coratina cultivar olive oils [11,28,53], which are well known to be strongly bitter and pungent, it might be supposed that the VOOs from the Oliva Rossa landrace have noteworthy bitter and pungent notes, although such a hypothesis should be verified by an in-depth study. To date, the few results available in the literature seem to confirm such a hypothesis [24].

The headspace volatile profile of the oils extracted in 2017/2018 is reported in Table 5.


**Table 5.** Identified volatile compounds (mg OE kg<sup>−</sup>1) of the Oliva Rossa VOOs extracted during fruit ripening in the 2017/2018 harvest season (mean ± SD, *n* = 3).

OE, 1-octanol equivalents. Different letters for each parameter indicate significant differences according to one-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S2–S4: three subsequent samplings.

The volatile compounds found in Oliva Rossa oils were those usually found in goodquality VOOs [4]. The most abundant ones were C5 and C6 aldehydes, alcohols and esters deriving from the well-known lipoxygenase pathway (LOX) [4], together with a remarkable amount of 3-ethyl-1,5-octadiene.

Regardless of the ripening stage of the drupes, (*E*)-2-hexenal was the most abundant compound. (*E*)-2-hexenal was reported to be strongly correlated with the bitter, almond, green, green apple-like, fatty, almond-like and cut-grass notes of the VOOs [4]. In decreasing order, 3-ethyl-1,5-octadiene was the following one. It was already detected in VOOs, although generally in quite lower concentrations [35]. The remarkable amount found in Oliva Rossa oils might be a typical trait of this olive landrace. Indeed, 3-ethyl-1,5-octadiene was already identified as one of the markers for varietal discrimination in Turkish olive oils [54]. It is worthy to note that the results reported here refer to only one crop season and thus further studies are needed to corroborate such evidence.

During ripening, significant differences in the volatile profile were observed [4,5]. Less ripe drupes (S2) gave oils with less total volatile compounds, whose profile was dominated by (*E*)-2-hexenal, 3-ethyl-1,5-octadiene, C5 compounds (1-penten-3-one, (*Z*)-2-penten-1-ol, 1-penten-3-ol) and (*Z*)-3-hexen-1-ol. In particular, (*Z*)-3-hexen-1-ol, which was significantly higher at S2 with respect to the other stages, was already reported as one of the indicators of the early stage of ripeness [4].

At the technological optimum (i.e., S3), the highest amount of total volatile compounds was observed. In detail, the oils were characterized by the significantly higher content of pentanal, 3-ethyl-1,5-octadiene, 1-penten-3-one, (*E*)-2-hexenal and (*Z*)-2-penten-1-ol. It is reported that the maximum content of (*E*)-2-hexenal is reached when the drupes' pigmentation changes from green to purple and then decrease with ripening [4,5]. However, this trend could be even cultivar dependent.

At S3, a noticeable content of acetic acid was found, too. Acetic acid could originate from the fermentation process and might be linked to some organoleptic defects such as wine-vinegary [55]. However, the detection of such defects involves many other volatile compounds [55]. At S4, a decrease in the total volatile compounds was observed with respect to S3. The oils were characterized by an increase in the acetate esters, which was associated with a decrease in the acetic acid content. A significantly higher content of 2-methyl butanal, 3-methyl butanal and 1-penten-3-ol was reported, too. The correlation among the volatile compounds and sensory features of the VOOs is well known [4,5,56]. Based on this, and considering the data reported in Table 5, it could be supposed that Oliva Rossa oils have notes of fruity, green, cut-leaves and almond, which is in accordance with the few data available in the literature [24].

#### **4. Conclusions**

Oliva Rossa is an old olive landrace from the Apulia region mostly unknown and not yet valorized. Aiming at its valorization, some useful results have been reported. First, depending on the crop year, the evolution of the drupes' pigmentation may vary significantly and the half-varaison of the skin color could not match the technological optimum (i.e., the maximum oil content). At its optimum, the drupes had a remarkable amount of phenolic compounds. A medium to high content of oleic acid was observed although significant differences along the years were highlighted, likely due to the different climatic conditions. When a high content of PUFA was reported, there also was significant primary oxidation observed. Thus, with respect to the European limits concerning the primary oxidation markers, this aspect deserves attention, even at the technological step, which has not been considered in this study. The oils had a remarkable amount of minor compounds, which were affected by ripening. Depending on the crop year, the noteworthy levels of MUFA and TPC could lead to oils with a good stability. The phenolic profile in the considered crop year was dominated by secoiridoids derivatives, which might indicate a product with remarkable pungent and bitter notes. Similarly, based on the available data, the volatile profile was dominated by C6 and C5 compounds arising from the LOX pathway. A distinct trait seemed to be the high content of 3-ethyl-1,5-octadiene. Further investigations are needed to confirm the results obtained and to estimate the environmental effect on the oil composition of Oliva Rossa.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2304-8 158/10/2/369/s1, Figure S1: Monthly average temperature (◦C) and rainfall (mm) recorded at the nearest weather station of Turi (Bari, Italy) for four years (2016–2019). The field including Oliva Rossa trees is about at 8 km from the weather station. Figure S2: UHPLC-DAD phenolic profile of Oliva Rossa VOOs extracted at different sampling times in 2017/2018 harvest season. IS, internal standard; (1) 3,4-DHPEA; (2) *p*-HPEA; (3) Vanillic acid; (4) Syringic acid; (5) 3,4-DHPEA-EDA; (6) 3,4-DHPEA-EDA-CARB; (7) *p*-HPEA-EDA; (8) Pinoresinol; (9) Luteolin; (10) *p*-HPEA-EA; (11) Apigenin.

**Author Contributions:** Conceptualization, G.S., C.S. and F.C.; methodology, G.S., R.S. and G.M.; formal analysis, G.S.; investigation, G.S. and R.S.; data curation, G.S. and F.C.; writing—original draft preparation, G.S., G.M. and F.C.; writing—review and editing, G.S., R.S., G.M., C.S. and F.C.; funding acquisition, F.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the AGER 2 Project, grant no. 2016-0105.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

