**3. Results**

#### *3.1. Total Phenolic Content during Maturation and after Fermentation Processes*

To evaluate metabolic profiles and antioxidant activities, olives belonging to homogeneous classes of maturity were sampled according to Guzman et al. [16]. Their fresh and dry weight has been determined and the pulp percentage was calculated (data not shown). The TPC during maturation was quantified by the Folin–Ciocolteau method and the results are reported in Figure 2.

As reported in Figure 2, there is a progressive increase in the amount of total phenolic contents during maturation. Green and immature olives, corresponding to Stage 0, have the lowest number of polyphenols, equal to 14.0 mg of gallic acid equivalent (GAE)/g dry weight pulp. In Stage 2, the TPC value increased, reaching 26.18 mg (GAE)/g DW, whereas in Stage 4 TPC was 29.68 mg (GAE)/g DW. When the olives reached full maturity (stage 7), a further increase in total phenolic substances was recorded: 31.80 mg GAE/g DW.

**Figure 2.** Total phenolic contents (mg GAE/g DW) in Cellina di Nardò olives at four di fferent stages of the maturation process. Results are expressed as mg of GAE/g dried olive pulp. Same letters mean no statistical di fferences between averages (Duncan test, *n* = 3, *p* = 0.05). GAE: gallic acid equivalent; DW: dry weight.

Following fermentation/curing, the CdN olives were compared with other six commercial black table olives for the presence of phenolic compounds (Figure 3). It was found that among the seven black table olives, the CdN table olives were the richest in phenolic compounds, with a TPC equal to 13.08 mg/g DW. Since these table olives showed a TPC of 31.80 mg GAE/g DW at full maturity—Stage 7 (Figure 2)—the fermentation process drastically reduced the TPC. Kalamata olives also showed a high content of phenols (10.84 mg GAE/g DW). The lowest level of polyphenols was observed in Hojablanca cultivar (1.19 mg GAE/g DW) (Figure 3).

To identify the principal phenolic compounds in CdN olive extracts, a reverse-phase HPLC/MS-TOF was used. The identification was carried out by comparing the retention times, UV absorbance, and molecular masses with literature data and analytical standard when available.

Representative chromatograms of olive extracts during maturation are reported in Figure 4 (A–D) and the list of identified compounds are reported in Table 1.

**Figure 3.** Total phenolic contents (mg GAE/g DW) in seven commercial black table olives. Results are expressed as mg of gallic acid equivalent (GAE)/g dried olive pulp. Same letters indicate no statistical differences between averages (Duncan test, *n* = 3, *p* = 0.05).



**a** RT, Retention time; **b** *m*/*z* Exp, mass to charge experimental; **c** *m*/*z* Clc, mass to charge calculated; **d** Diff., difference between the observed mass and the theoretical mass of the compound (ppm); **e** Isotopic abundance distribution match: a measure of the probability that the distribution of isotope abundance ratios calculated for the formula matches the measured data; **f** Ref., References. \* Confirmed by authentic chemical standard. \*\* These peaks were identified in positive ion mode (M−H)<sup>+</sup>.

The metabolic profile during olive maturation (Figure 4) highlighted a grea<sup>t</sup> variability among the four analyzed stages. The green olives (Stage 0) had a predominance of substances represented by the peaks 2, 6, 14, 15, and 17, corresponding to hydroxytyrosol glucoside, verbascoside, oleuropein, luteolin, and ligstroside, respectively. During drupe maturation, starting from Stage 4, the quantity of these compounds decreases. Conversely, the concentrations of the anthocyanins cyanidin 3 glucoside and cyanidin 3 rutinoside increase after Stage 4.

**Figure 4.** Representative chromatograms of Cellina di Nardò olive extracts during the maturation process. Detection at 280 nm. For the identification of the peaks and relative compounds, see Table 1.

#### *3.2. Anthocyanin Quantification in CdN Olives*

Anthocyanins were identified by 520 nm UV absorbance and molecular weight, and confirmed by authentic chemical standard spectra; the quantification was performed using the calibration curve obtained using the standard cyanidin 3-rutinoside (Table 2).

**Table 2.** Anthocyanin contents in Cellina di Nardò olive extracts during maturation stages and after fermentation. Data are reported as g/kg DW of cyanidin 3-rutinoside. Same letters indicate no statistical differences between averages (Duncan test, *n* = 3, *p* = 0.05).


With the maturation progresses, it is evident that there is a variation of anthocyanin contents: in green fruits (corresponding to Stage 0), there are no anthocyanins; some traces begin to appear in olives belonging to Stage 2; in the olives of Stage 4, anthocyanins are detectable and 3.22 g/kg DW were reported, while fully ripe olives showed 4.62 g/kg DW. The quantity of anthocyanin in fermented CdN olives was lower: 1.16 g/kg DW.

#### *3.3. Antioxidant Activity of Olive Extracts*

To evaluate the antioxidant properties of the olive fruit extracts during ripening stages, three different antioxidant assays were carried out (ORAC, DPPH, and superoxide anion scavenging activity). Data are reported in Table 3.

**Table 3.** Antioxidant activity detected in extracts of Cellina di Nardò olives at four different maturation stages. Results are expressed as μmol Trolox Equivalents/100 g FW (ORAC and DPPH tests) and as Inibitory Concentration (IC50, μg of FW olive pulp). Same letters indicate no statistical differences between averages (Duncan test, *n* = 3, *p* = 0.05).


As reported in Table 3, the three antioxidant in vitro assays provided similar results among stages. The olive extracts with the highest antioxidant activities were the olives of the Stage 7 (complete maturation). Conversely, the antioxidant activity was lower at earlier maturation stages. However, the ratio and the increase in the antioxidant activity between the olives of Stage 0 and those of Stage 7 were different among the antioxidant assays. The ratio between CdN Stage 0 and CdN Stage 7 in ORAC assay was less than two-fold, whereas in DPPH and superoxide anion assays the ratio was about three-fold. The same analyses were conducted on commercial black table olives, and the results are reported in Table 4.


**Table 4.** Antioxidant activity detected in commercial black table olives of seven different cultivars. Results are expressed as μmol of Trolox Equivalents (TE)/100 g FW (ORAC and DPPH tests) and as IC50 per μg FW (superoxide anion assay) of olive pulp. Same letters indicate no statistical differences between averages (Duncan test, *n* = 3, *p* = 0.05).

Whereas the fully ripened CdN olive extracts showed an antioxidant activity of 18,788 ± 3298, 9062 ± 302, and 1.05 ± 0.07, respectively in ORAC, DPPH, and superoxide anion assay, the antioxidant activities of table CdN olives dropped dramatically (an about three-fold reduction) after the fermentation process. However, despite this drastic decrease, the antioxidant activity of CdN olives was the highest among the analyzed table olives. Kalamata and Leccino had similar antioxidant activities of CdN using the DPPH and superoxide anion assays, but the values reported by ORAC assay were statistically different among CdN, Kalamata, and Leccino. Blanqueta and Hojiblanca black table olives showed the lowest antioxidant activities.
