**4. Discussion**

Cellina di Nardò is an olive cultivar closely connected to the Salento region by a long cultural tradition. Many plants are over 100 years old, and some of them (the monumental ones) are more than 1000 years old. People of this region have developed an empathic sentiment towards these plants, especially since the *Xylella* epidemic in 2013 [1].

In addition to oil production, CdN is used, in different local food preparations and after appropriate tanning, to produce table olives with a unique and recognizable flavor. Moreover, this olive is harvested when it is completely black (fully ripened), whereas most of the commercial black table olives are darkened by oxidation. The intense black color of the CdN natural black table olive is due to the accumulation in the skin and pulp tissues of a considerable quantity of anthocyanins, pigments that belong to the large family of polyphenols. The anthocyanins, as with polyphenols in general, have considerable beneficial properties as a consequence of their ability to scavenge oxygen radicals. They have good antioxidant, anti-inflammatory, and preventive effects against various pathological states, such as cardiovascular diseases and tumors [29].

The results reported in this work strongly support the possibility to use this table olive as a functional food. We demonstrated that the fully maturation is the best harvest time to obtain a table olive with a high content in phenolic compounds, anthocyanins, and other health valuable compounds. In fact, to assess whether and how the phenolic components vary during maturation, the phenolic content was studied during different ripening stages. The results showed an increase in the total quantity of polyphenols in relation to the stage of maturation; from a value of total phenolic substances of 14.00 mg GAE/g DW (Stage 0) to a value of 31.80 mg of GAE/g DW (Stage 7). Therefore, a two-fold intensification of total phenolic compounds was observed. These data are in accordance with the values reported by other authors [5]. The same authors observed that phenolic fraction in fresh pulp is about the 2% of the total weight of the pulp. We detected a similar value in fully ripened CdN olives where the phenolic fraction represents the 1.6% of the pulp total weight.

The qualitative analysis of phenolic compounds during maturation (Figure 3 and Table 1) demonstrated a grea<sup>t</sup> variation of the phenolic composition during ripening. In particular, from Stage 4, olives start to accumulate anthocyanins (cyanidin-3-rutinoside and cyanidin-3-glucoside), so that at Stage 7, the anthocyanin level reached 4.62 g/kg DW. Amiot et al. [9] reported an increase in anthocyanins matching to the progress of fruit ripening. In fact, the anthocyanin biosynthesis starts when the oleuropein decreases as direct result of an increase of the enzymatic activity of phenylalanine-ammonium lyase during drupe maturation [9]. The anthocyanin content found in CdN olives is higher than the quantity of anthocyanins present in most of other commercial olive fruits [28], as reported by a study conducted on several Italian cultivars. The black olives of Frantoio, a cultivar widespread in Tuscany, has a quantity of anthocyanins equal to 1.25 g/kg of fresh pulp; Ciliegino olives have an even lower quantity of phenolic compounds: 0.50 g/kg of fresh pulp [30].

After fermentation process, the level of anthocyanins in CdN decreased to 1.16 g/kg DW, a reduction of about 75% from the freshly harvested olives.

The same trend was observed for the total phenolic content. In fact, after the fermentation process of CdN, we noticed a drop in phenolic content (about 60%) to 13.08 GAE mg/g DW as reported also by Zou and colleagues [31], who observed how fermentation processes can reduce the total phenolic content by 50%. In the Spanish method fermentation for example, as well as in the Greek method, the debittering phase with NaOH causes the higher loss in phenolic compounds, probably due to the polyphenol oxidation under alkaline conditions [32]. During this process the polyphenols undergo to chemical transformations (hydrolysis of oleuropein into hydroxytyrosol) and their concentrations usually decrease. The following processing steps such as lactic fermentation, on the contrary, do not seem to affect total phenolic compound reduction [32].

To produce table olives from the CdN cultivar, completely ripened drupes (Stage 7) are usually employed, determining a high phenolic content after fermentation which is certainly higher than that of the black table olives on the market (Figure 3). The value is even higher than others found in literature concerning black table olive varieties. For example, in the Chondrolia table olives a total amount of phenolic substances of about 5.4 mg GAE/g FW was reported, while in the Amfiss olives the value is even lower and is equal to 2.3 mg GAE/g of fresh pulp [33]. Ben Othman et al. [34] estimated that the total phenolic content in black table olives is about 4–8 mg GAE/g DW. These data sugges<sup>t</sup> the grea<sup>t</sup> potential of CdN cultivar to be used as a food reach in phenolic compounds.

The antioxidant capacities of fresh CdN olives were also investigated at four different maturation stages and after the fermentation process. CdN olives showed antioxidant properties (7415 μmol TE/100 g FW) higher than the average of other vegetables. About other fruits, an ORAC value of 4275 μmol TE/100 g fresh weight was reported for the Red Delicious apple [35], and a value of 1837 μmol TE/100 g fresh weight was reported for grapes [36], although the latter fruit is considered rich in antioxidant substances. Moreover, we observed an increase of the antioxidant activity positively correlated to the increase in phenolic substances (*R*<sup>2</sup> = 0.832, *R*<sup>2</sup> = 0.756 and *R*<sup>2</sup> = 0.957, respectively, for ORAC, DPPH, and the superoxide anion test) as already reported in Tunisian olives [37].
