Antioxidant Activity and Anthocyanin Contents in Olives (cv Cellina di Nardo) during Ripening and After Fermentation

Abstract

The olive tree “Cellina di Nardò” (CdN) is one of the most widespread cultivars in Southern Italy, mainly grown in the Provinces of Lecce, Taranto, and Brindisi over a total of about 60,000 hectares.  Although this cultivar is mainly used for oil production, the drupes are also suitable and potentially marketable as table olives.  When used for this purpose, olives are harvested after complete maturation, which gives to them a natural black color due to anthocyanin accumulation. This survey reports for the first time on the total phenolic content (TPC), anthocyanin characterization, and antioxidant activity of CdN olive fruits during ripening and after fermentation. The antioxidant activity (AA) was determined using three different methods. Data showed that TPC increased during maturation, reaching values two times higher in completely ripened olives.  Anthocyanins were found only in mature olives and the concentrations reached up to 5.3 g/kg dry weight. AA was determined for the four ripening stages, and was particularly high in the totally black olive fruit, in accordance with TPC and anthocyanin amounts.  Moreover, the CdN olives showed a higher TPC and a greater AA compared to other black table olives produced by cultivars commonly grown for this purpose. These data demonstrate the great potential of black table CdN olives, a product that combines exceptional organoleptic properties with remarkable antioxidant capacity.

Introduction

The olive cultivar “Cellina di Nardò” (CdN), also known as “Leccese”, “Saracena”, “Visciola”, “Asciulo”, or “Muredda”, is an Italian variety that is widespread in Salento (Apulia, South Italy), especially in the Province of Lecce, although it is also grown in the territories of Taranto and Brindisi, covering a total of about 60,000 hectares. However, since 2013, Salento has been suffering from a devastating olive disease (the olive quick decline syndrome) caused by the bacteria Xylella fastidiosa [1,2] that has progressively destroyed hundreds of thousands of olive trees. Unfortunately, the CdN cultivar is sensitive to Xylella fastidiosa, and in the coming years is likely to disappear completely from the Salento area [3,4]. For this reason, a complete characterization of such traditional cultivars is required, as many plants are still cultivated in South Italy.

The CdN tree is vigorous; the branches at the top are erect, while the lateral ones almost pendulous, and the tree can reach a height of 20 m. The leaves have an elliptical elongated shape; the upper side of the leaf is dark green while the lower one is silvery gray. The blooming stage is quite early, and the inflorescence results in 15–20 flowers. The fruit is an elliptical drupe, slightly asymmetrical, with a color ranging from green to black; when ripening is completed, it has a reduced size, with a weight ranging from 1.5 to 2.0 g, and a low oil yield (15–17%).

The drupes have a high resistance to detachment and are not suitable for mechanical harvesting. On the contrary, this cultivar is valued by local farmers due to the slow vegetative growth, good fructification in adverse conditions, and good tolerance to cold and various pests. Despite the low oil yield and difficulties in harvesting, the CdN is cultivated mainly for oil production, which is characterized by an intense fruity flavor. The oil obtained from CdN and Ogliarola olives (another traditional cultivar widespread in Salento) is guaranteed by the brand “Terra d’Otranto” as a ‘‘Protected Designation of Origin”, and presents some specific characteristics as a consequence of the geographical influence, pedoclimatic conditions, agronomic techniques, and oil processing.

The olive fruit consists of water (about 50%) and fats (20%), and the remaining part is made up of nitrogenous compounds, cellulose, sugars, and secondary metabolites [5]. The secondary metabolites of fresh fruit and fermented olives can vary greatly. In fact, the different processes of extraction and purification can modify the chemical structure of the molecules due to exposure to oxygen or solvents or even pH changes, situations that can commonly occur in phenolic metabolism [6]. The proportion of phenolic compounds within the edible part of the olive is considerable and can reach concentrations ranging from 1% to 3% of the fresh weight of the pulp [7]. There is a complex mixture of phenolic compounds in olives, some of which are present at very low concentrations and as a consequence are difficult to identify [8]. Moreover, phenolic and secondary metabolites are not uniformly present in diverse parts of the fruit: most of them are present in the pulp (about 85–90%), followed by the peel (about 8–12%), and then by the seed (1–2%).

Among phenolic compounds, oleuropein is generally the most represented among the various olive cultivars, reaching concentrations up to 140 mg/g fresh weight (FW) [9]. Oleuropein belongs to the secoiridoid family, as well as other compounds usually found in olives like dimethyl oleuropein, verbascoside, ligstroside, and nüzhenide [10]. In particular, oleuropein, dimethyl oleuropein, and verbascoside have been found in the all parts of the olive fruit (pulp, skin, and seed); conversely, the presence of nüzhenide was reported only in the seed [11]. In the olive fruit there are also flavones such as luteolin-7-glucoside, flavonols such as quercetin 3-rutinoside [12], anthocyanins like cyanidin-3-rutinoside and cyanidin-3-glucoside, and phenolic acids such as hydroxybenzoic, gallic, ferulic, caffeic, vanillic, and syringic acid [13].

All these secondary metabolites are of great interest for human health because of their antioxidant activity and properties with respect to cancer prevention, inflammatory disorders, and cardiovascular diseases [14,15].

The health properties of CdN when used as table olive have not yet been reported, and the Xylella fastidiosa threat suggests their urgent investigation due to the extinction risk caused by the pathogen. Therefore, in this paper a characterization of the secondary metabolites during ripening stages was carried out. Moreover, six different table olive cultivars were compared to CdN cultivars to establish the best food in terms of antioxidant effects and phenolic compounds.

Results

The effects of water regimes on the plant water status, photosynthetic performance, metabolites fluctuations and fruit quality parameters were evaluated. All DIS treatments enhanced leaf tissue density, RDI and SDI generally did not affect leaf water status and maintained photosynthetic machinery working properly, while SDIAF treatment impaired olive tree physiological indicators. DIS treatments maintained the levels of primary metabolites in leaves, but SDIAF plants showed signs of oxidative stress.

Moreover, DIS treatments led to changes in the secondary metabolism, both in leaves and in fruits, with increased total phenolic compounds, ortho-diphenols, and flavonoids concentrations, and higher total antioxidant capacity, as well higher oil content.

Phenolic profiles showed the relevance of an early harvest in order to obtain higher oleuropein levels with associated higher health benefits.

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.

Figure 2

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

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.

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).

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).

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.

Open in a separate windowFigure 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.

Table 1

List of chemicalss and anthocyanins putatively identified by High-Performance Liquid Chromatography coupled to Electrospray Ionization Time-of-Flight Mass Spectrometry (HPLC ESI/MS-TOF) following extraction from CdN olive pulp at different stages of maturation.

N.	Compound	RT a (min)	(M−H)−	m/z Exp b	m/z Clc c	Diff. (ppm) d	Score e	Ref.f
1	* Quinic acid	2.82	C7H11O6	191.0510	191.0561	−5.89	90.44	[23,24,25]
2	Hydroxytyrosol glucoside	4.63	C14H19O8	315.1095	315.1085	−1.26	96.62	[22,23,25]
3	Secologanoside is. 1	4.85	C16H21O11	389.1095	389.1089	−1.11	88.91	[22,23,25]
4	Secologanoside is. 2	4.94	C16H21O11	389.1101	389.1089	−2.62	96.13	[24,26]
5	* Rutin	5.83	C27H29O16	609.1474	609.1461	−2.15	90.20	[24,26]
6	* Verbascoside	6.02	C29H35O15	623.2013	623.1618	−0.05	93.73	[24,25]
7	Elenoic acid glucoside	6.31	C17H23O11	403.1262	403.1246	−3.68	80.90	[24,26]
8	Oleuropein aglycon	7.02	C16H25O10	377.1459	377.1453	−1.23	92.94	[24]
9	* Quercitrin	8.85	C21H19O11	447.0960	447.0933	−6.05	89.44	[27]
10	Hydroxyoleuropein	9.82	C25H31O14	555.1773	556.1803	−2.04	97.55	[24,27]
11	* Luteolin 7 glucoside is. 1	10.03	C21H19O11	447.0952	447.0933	−3.93	77.64	[24,25]
12	* Luteolin rutinoside	10.95	C27H29O15	593.1517	593.1512	−0.87	97.79	[24]
13	* Luteolin 7 glucoside is. 2	11.87	C21H19O11	447.0948	447.0933	−3.03	96.13	[24,25,26]
14	* Oleuropein	12.21	C15H9O13	539.1772	539.1770	0.03	97.14	[23,24,25,27]
15	* Luteolin	12.53	C15H9O6	285.0419	285.0405	−4.87	97.08	[23,24,25,27]
16	* Quercetin	13.07	C15H9O7	301.0351	301.0354	1.10	96.04	[24,25]
17	Ligstroside	13.88	C25H31O12	523.1823	523.1821	−0.03	97.55	[26]
18	* Apigenin 7 glucoside	14.31	C15H9O5	269.0461	269.0455	−1.77	98.70	[23]
19	Diosmetin	14.72	C16H11O6	299.0566	299.0561	−1.43	98.50	[23]
20	** Cyanidin 3 glucoside	15.03	C21H21O11	449.1081	449.1078	0.66	92.21	[28]
21	** Cyanidin 3 rutinoside	15.82	C27H31O15	595.1658	595.1657	0.16	95.23	[28]

^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)⁺.

The metabolic profile during olive maturation (Figure 4) highlighted a great 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.

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).

Olive Extract	Cyanidin-3-Rutinoside (g/kg DW)
Stage 0	ND
Stage 2	Traces
Stage 4	3.22 b ± 0.22
Stage 7	4.62 a ± 0.06
Table olive (fermented)	1.16 c ± 0.16

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.

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).

Olive Extract	ORAC Test μmol TE/100 g FW	DPPH μmol TE/100 g FW	Superoxide Anion Test IC50 (µg FW)
Stage 0	11,412 b ± 1722	2888 d ± 234	3.15 a ± 0.13
Stage 2	13,565 b ± 2173	4212 c ± 351	2.15 b ± 0.35
Stage 4	15,990 a,b ± 486	6285 b ± 312	1.45 b,c± 0.49
Stage 7	18,788 a ± 3298	9062 a ± 302	1.05 c ± 0.07

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).

Black Table Olive Extract	ORAC Test μmol TE/100 g FW	DPPH μmol TE/100 g FW	Superoxide Anion Test (IC50 µg FW)
Cellina di Nardò	7415 a ± 353	2920 a ± 51	4.25 c ± 0.21
Kalamata	4717 b ± 96	2533 a ± 135	6.10 c ± 0.42
Leccino	3964 c ± 213	2612 a ± 686	9.55 c ± 1.34
Empeltre	2355 d ± 224	1209 b ± 247	22.00 b ± 2.83
Ogliarola	1676 e ± 87	1186 b ± 398	22.95 b ± 2.62
Blanqueta	1537 e ± 164	1299 b ± 416	42.21 a ± 11.60
Hojiblanca	747 f ± 10	1356 b ± 246	47.55 a ± 2.05

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.

Conclusion

Different treatments (or curing methods) that are necessary to remove the bitterness of the raw olive and to stabilize them to obtain edible table olives, causing a loss in phenolic substances which also results in a loss of anthocyanins and antioxidant activity. However, CdN black table olives were the richest in polyphenols, consequently possessing the best antioxidant activity among the analyzed black table olives and among other black table olives reported in literature.  Moreover, it is plausible that regular consumption of CdN table olives can give real returns in terms of prevention of oxidative stress.

Authors:  Alessio Aprile,^,  Carmine Negro,  Erika Sabella,  Andrea Luvisi,  Francesca Nicolì,  Eliana Nutricati,  Marzia Vergine,  Antonio Miceli,  Federica Blando,  and Luigi De Bellis

• Department of Biological and Environmental Sciences and Technologies (DiSTeBA), Salento University, Via Prov. le Lecce-Monteroni, 73100 Lecce, Italy;
• Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Research Unit of Lecce, Via Prov. le Lecce-Monteroni, 73100 Lecce,

Read the full study at Antioxidants MDPI