**4. Discussion**

The olive oil accumulation on fruits during ripening follows the triglyceride-forming biosynthesis pathway up to the achievement of full drupe maturation. The olive oil yield in fruits is influenced by the choice of the right harvesting time for each variety and by several growing conditions, such as water availability [31]. In our study, an evident effect of varietal characteristics was observed, and the two Ottobratica clones differed for mean oil production at both harvesting times. Free acidity is generally the first parameter discussed to evaluate the quality of olive oil production; it is well-known that oil-free acidity can be affected by many factors, including fruit handling harvesting mean, storage, and processing, but also by the harvesting time. All of the oils produced in October were inside the limits for the extra virgin olive category [21], and, in particular, those of Ciciarello possessed the lowest mean acidity among the other minor accessions (*p* < 0.05). It is interesting to note that good oils from Ottobratica clones could be obtained at both harvesting times, without significant variations among each other (*p* > 0.05) and, different from Ottobratica, their reference variety. The oils of this last production in November denoted the previously discussed variability during the years and, on average, poor quality (free acidity of 1.18 ± 1.08%). Peroxides and extinction coefficients complied with the regulation limits for the extra virgin olive oil [21]. Fatty acid composition of samples was inside the limits imposed by European regulation for the extra virgin category [32]. The fatty acid composition did not largely vary between harvesting times in oils from each minor accession; only the oils of Tonda di Filogaso were different for stearic acid (means of 1.72% in October and 3.44% in November). Oleic acid was particularly abundant in oils from Ciciarello and Ottobratica Cannavà; for this chemical parameter, oils becoming to this clone were of higher quality with respect to those from Ottobratica cv.

The molecules responsible for olive oil color are pigments belonging to chlorophyll and carotenoid compounds. Their quantification is important to determine not only the sensorial characters and consumer acceptability of olive oils, but also their antioxidant potentiality; olive oil chlorophylls react as radical scavengers in dark storage and as prooxidant (sensitizer pigments) in light. Carotenes instead protect cells against the light, with oxygen and sensitizer pigment effects having the ability to quench singlet oxygen and excited sensitizer molecules. Moreover, they can also react as antioxidants under conditions

other than photosensitization [33]. The total pigment content varies among varieties, drupe ripening, or olive oil stocking before the extraction [34,35].

In particular, the total content can range from 2 to 40 mg/kg for chlorophylls and from zero to a few mg/kg for carotenoids [36,37]. Olive oil produced at the second harvesting time showed reduced amounts of both pigments in our study, according to the literature [38]. Among the oil samples from the different accessions, only those of Cannavà did not vary for total pigments. Total tocopherols were detected in oils from minor varieties at a higher content than those observed in oils obtained from other cultivars in Calabria [15]. The quantified total phenols were in the range of 460–778 mg/kg, according to Fabiani [39], manifesting a strong antioxidant potentiality. A positive correlation between the total phenol content and ABTS assay was indeed evidenced by a high Pearson coefficient (r = 0.7–0.9) in all of the oils, in particular those extracted in November, according to Sicari [40]. It confirms their usefulness in providing the minimum intake of 5 mg of hydroxytyrosol per serving of olive oil (total phenol content >250 mg/kg) that is required to manifest the antioxidant effect in a balanced diet.

Finally, a multivariate data analysis was performed to evidence the influence of varietal characteristics or drupe harvesting times to the olive oil quality (Table 4). Results showed an evident effect due to the olive origin, especially for the prevalent fatty acids (*p* < 0.00) in the olive oils. From this data elaboration that considered overall data for all of the three years of experimentation, it was noted that, among the other qualitative parameters, in particular the total carotene content was affected exclusively by the drupe ripening, as the literature [35] confirms.


**Table 4.** Multivariate analyses of qualitative characteristics of olive oils extracted from drupes produced from the four minor accessions and at two harvesting times.

\*\* Significance at *p* < 0.01; \* significance at *p* < 0.05; n.s., not significant.

#### **5. Conclusions**

This study allowed the characterization of the oil productions from four minor olive accessions grown in the same area of Calabria, with the aim to compare the qualitative differences measured during three years of observations, excluding climatic variables due to different environmental conditions. For some of these (oils from Tonda di Filogaso and Ciciarello cv), harvesting times significantly affected the results for free acidity and total pigments. All of the oil productions obtained in October possessed the chemical parameters to be classified as extra virgin olive oils. Comparing all of the studied accessions, olive oils from Ciciarello cv and Calipa and Cannavà clones also showed good quality when extracted in November. This is interesting for growing practices in the same studied area and for new knowledge about the potentiality of the two new clones obtained from the Ottobratica cultivar. In particular, the oils of the Ottobratica Cannavà clone showed better quality for free fatty acidity at both harvesting times, with oleic acid content and total antioxidants (polyphenols and tocopherols) with respect to the cultivar of reference, Ottobratica, largely diffused in the considered territory.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2304-8158/ 10/2/305/s1, Table S1: Ripening index of the olives at harvest times, Table S2: Qualitative characterization of the Tonda di Filogaso oils, Table S3: Qualitative characterization of the Ciciarello oils, Table S4: Qualitative characterization of the Ottobratica Calipa oils, Table S5: Qualitative characterization of the Ottobratica Cannavà oils, Table S6: Qualitative characterization of the Ottobratica oils.

**Author Contributions:** Conceptualization, R.M. and M.P.; Data curation, A.P., M.P., and R.M.; Formal analysis, A.D.B., R.R., and S.S.; Funding acquisition, M.P.; Methodology, A.P., A.D.B., and R.R.; Resources, M.P.; Supervision, A.P. and M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by AGER 2 PROJECT, grant number 2016-0105.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets presented in this study are available as Supplementary materials.

**Acknowledgments:** The authors thank the Olearia San Giorgio firm for sample supplying and Antonino Tramontana for assistance in conducting olive oil extraction.

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

#### **References**

