Effect of Spanish style processing on the phenolic compounds and antioxidant activity of Algerian green table olives

S. Mettouchia, R. Sacchib, Z.E.D. Ould Moussaa, A. Paduanob, M. Savaresec and A. Tamendjaria,*

aLaboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000, Algérie

bLaboratory of MolecularGastronomy, Faculty di Agraria, UniversitaDegliStudi Di Napoli Federico II. Naples, Italy

cCRIOL, Centro Ricerche per l’Industria Olearia, c/o Industria Olearia Biagio Mataluni; via Badia, zona industriale, 82016 Montesarchio (BN), Italy

*Corresponding author: abderezakt@yahoo.fr

 

SUMMARY

The study was carried out on seven Algerian olive cultivars to report the effect of Spanish style processing on individual and total phenolic compounds and the changes that occur in antioxidant capacity. The results indicate that the treatment leads to losses in phenolic contents which are cultivar dependent. Sigoise is the least affected variety (12.25%) and Azzeradj from Seddouk the most affected one (94.80%). The phenolic profile shows drastic changes after processing. Hydroxytyrosol is dominant in processed olives (14.42–545.42 mg·100 g−1) while oleuropein is the major phenolic compound in fresh olives (994.27 mg·100 g−1). As a consequence to the loss in phenolic content, substantial reductions in the antioxidant activities of the extracts are noted. They are estimated to be 13.12–92.75% in scavenging activity against the DPPH radical, 37.78–93.98% in reducing capacity, 59.45–97.94% in the hydrogen peroxide radical and 7.26–51.66% in the inhibition bleaching of β-carotene. Among the processed varieties, only Sigoise presented a positive value of RACI (relative antioxidant capacity index).

 

RESUMEN

Efecto del procesamiento sobre el contenido fenólico y la actividad antioxidante de aceitunas verdes de mesa argelinas. El estudio se llevó a cabo con siete variedades de aceitunas argelinas y se investigó el efecto de la elaboración al estilo español sobre los compuestos fenólicos individuales y totales; así como los cambios que se producen en la capacidad antioxidante. Los resultados obtenidos muestran que el procesamiento conduce a pérdidas en el contenido fenólico, lo cual es dependiente de la variedad. Sigoise es la variedad menos afectada (12.25%) y Azzeradj de Seddouk la más (94.80%). El perfil fenólico muestra cambios drásticos después de su procesamiento. El hidroxitirosol es el polifenol predominante en aceitunas procesadas (14.42–545.42 mg·100 g−1), mientras que la oleuropeína es el compuesto fenólico mayoritario en las aceitunas frescas (994.27 mg·100 g−1). En consonancia con la pérdida en estos polifenoles, se detectaron reducciones sustanciales de la actividad antioxidante de los extractos. Se estima en 13.12 a 92.75% la actividad de eliminación de radicales DPPH 37.78–93.98% en la reducción de la capacidad, 59.45 a 97.941% en el radical peróxido de hidrógeno y 7.26–51.66% en la inhibición de blanqueo del β-caroteno. Entre las variedades procesadas, sólo Sigoise presentó un valor positivo de RACI (Indice Relativo de Capacidad Antioxidante).

 

Submitted: 29 March 2015; Accepted: 06 July 2015

Citation/Cómo citar este artículo: Mettouchi S, Sacchi R, Ould Moussa ZED, Paduano A, Savarese M, Tamendjari A. 2016. Effect of Spanish style processing on phenolic compounds and antioxidant activity of Algerian green table olives. Grasas Aceites 67 (1): e114. doi: http://dx.doi.org/10.3989/gya.0378151.

KEYWORDS: Antioxidant activity; Phenolic compounds; RACI; Spanish style processing; Table olives

PALABRAS CLAVE: Aceitunas de mesa; Actividad antioxidante; Compuestos fenólicos; Estilo español de procesamiento; RACI

Copyright: © 2016 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence.


 

CONTENT

1. INTRODUCTIONTOP

Table olives (Olea europaea) and olive oil are the main constituents of the Mediterranean diet. According to the statistical data (IOC, 2013), the worldwide production of table olives is estimated at more than 2.3 million tons (2013–2014 season). The Algerian production is on the rise, from 67.600 tonnes (average of 2001–2006) to 139.700 tonnes (2007–2013). With this production, Algeria contributes with 3.8 and 6%, respectively, to world production. Table olives possess important biological proprieties, linked to their predominance in monounsaturated fat contents, and to antioxidant compounds like phenolics and tocopherols which have health benefits (Bianchi, 2003). Phenolic compounds have been shown to beneficially alter lipid composition, platelet and cellular function, as well as reduce oxidative damage and inflammation (Cicercale et al., 2010). Soni et al. (2006) reported the action of olive pulp extract on gastrointestinal disorders due to their antibacterial activity.

Several factors are known to affect the qualitative and quantitative phenolic profiles of table olives. These bioactive compounds are closely affected by cultivar (Vinha et al., 2005), degree of maturation (Malheiro et al., 2011), growing conditions (Marsilio et al., 2006), fruit size (Amiot et al., 1990) and processing methods (Sahan et al., 2013).

Olives cannot be consumed directly after harvest due to their extreme bitterness and they must undergo various processes. Three kinds of table olives are of economic importance in the international market: Spanish style green olives in brine, Greek style naturally black olives in brine, and California black ripe olives. Spanish style green olives or “alkali-treated green olives in brine” are the most widely distributed (El Khaloui and Nouri, 2007).

Many studies have been carried out regarding the influence of different processing methods of table olives on the levels of total and single phenolics (Romero et al., 2004; Pereira et al., 2006), but very few of them (Ben Othman et al., 2009) have been aimed at assessing the impact of this processing on the antioxidant capacity of the fruits. However, to our knowledge, no studies have been carried out about changes induced by Spanish style processing on Algerian green table olives. Therefore, this study is undertaken to investigate the evolution of phenolic compounds after Spanish style processing and to evaluate the result that those changes have on the antioxidant capacity of the finished product in order to determine the effect of this kind of processing on Algerian olive cultivars.

2. MATERIAL AND METHODSTOP

2.1. Plant materialTOP

Olive fruits of seven Algerian cultivars: Azzeradj from Seddouk, Gordal, Sevilla, Sigoise, Taffahi, Bouchouk and Azzeradj from Tazmalt were harvested in October, 2012, at the green maturation stage. Four trees were selected and tagged and the olive fruits were hand-picked from different parts of the olive tree. After sorting and sizing, three kilograms of olives per variety were used.

2.2. ProcessingTOP

Olives were debittered in an alkali solution (15 g·L−1) during 8 to12 h until the lye had penetrated two thirds of the pulp. Three washing waters are more than sufficient (twice for 4 h and once for 12 h) to eliminate excess alkali. Then, the olives were fermented in brine (8% NaCl) where a lactic fermentation reduces the pH to 4.5.

Samples of fresh and processed olives were freeze-dried at −58 °C (Christ, Alpha 1–4 LD plus, Osterode am Harz, Germany), ground in electric blender (IKA model A 11 B, Staufen, Germany) and stored at −18 °C until analysis.

2.3. Analysis of phenolic compoundsTOP

2.3.1. ExtractionTOP

Phenolic compounds were extracted according to Mc Donald et al. (2001). Freeze dried olive pulps (5 g) were homogenized in 25 mL of methanol/water (80:20, v/v). The residue was extracted twice; extracts were combined, and washed with hexane. The extracts were filtered, and then kept cold until analysis.

2.3.2. Total phenolic compoundsTOP

The total phenolic content of the extracts was determined with Folin Ciocalteu reagent according to Borzello et al. (2000). Total phenol values were expressed as mg gallic acid equivalents per 100 g Dry Weight (mg GAEq·100 g−1 DW).

2.3.3. HPLC separation and identification of phenolic compoundsTOP

The presence and amount of phenolic compounds in the olive extracts were studied by reversed phase HPLC analysis using a binary gradient elution. The analysis was performed by reversed phase HPLC on a LC-10ADVP Shimadzu (Milan, Italy) liquid chromatography equipped with an SPD M10AVP diode array detector (Shimadzu). The chromatographic separation was achieved on a Spherisorb S5 ODS-3 (250 mm×4.6 mm i.d.) reversed-phase column (Phenomenex, Macclesfield, UK). The solvent system used was a gradient of solvent A (water: trifluoroacetic acid, 97:3, v/v), and solvent B (acetonitrile: methanol, 80:20, v/v). A step gradient from 5% to 98% B (45 min) was applied at a flow rate of 1 mL·min−1. Peak quantification was carried out at 279 nm. The main phenolic compounds were identified by comparison with relative retention times of pure compounds, when available, or by comparing the relative elution order and UV spectra with those reported in the literature (Brenes et al., 2000; Rovellini and Cortesi, 2002). The identity of each peak was confirmed by LC-MS, performed on an LC-10AD VP Shimadzu (Milan, Italy) liquid chromatograph on-line with an LCMS-2010EV Shimadzu (Milan, Italy) mass spectrometer, equipped with an electrospray ionization (ESI) interface. A Discovery HS C18 column (5 μm, 150 mm×2.1 mm i.d., Supelco, St. Louis, MO, USA), at a flow rate of 0.35 mL·min−1, was used. The solvent system used was a gradient of solvent A (water+formic acid 0.25%), and solvent B (methanol+formic acid 0.25%), with a step gradient from 5% to 55% B (45 min). The ESI mass spectra (m/z 60–900) in the negative ion mode were obtained under the following conditions: interface voltage 4 kV; nebulizer gas flow 1.5 L·min−1; block heater temperature 250 °C; curved desolvation line temperature and voltage of 300 °C and −5 V, respectively; Q-Array voltage 0 V DC and 150 V RF; detector voltage 1.5 kV. Some operating parameters (interface voltage and Q-Array voltage) were then modified in order to obtain a moderate fragmentation of the de-protonated molecular ions: an interface voltage of 5 kV and a Q-Array voltage of −50 V DC and 150 V RF were used (Savarese et al., 2007).

2.4. Antioxidant activityTOP

2.4.1. Reducing powerTOP

The Ferric Reducing Power of the extracts was measured as reported by Zhan et al. (2006) using ferric chloride. The absorbance was then measured at 700 nm and the reducing power was expressed as mg quercetin, butylated hydroxyanisole and butylated hydroxytoluene equivalents per 100 g Dry Weight (mg QE, BHAEq and BHTEq·100 g−1 DW).

2.4.2. DPPH free radical scavenging activityTOP

The procedure reported by Boskou et al., (2006) was adapted. An aliquot of the appropriate dilution of the extract (0.5 mL) was added to a 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) solution (2 mL) and kept in the dark for 30 min. The absorbance was measured at 515 nm and the antiradical activity was expressed as mg quercetin and trolox equivalents per 100 g Dry Weight (mgQEq andTEq·100 g−1 DW).

2.4.3. Hydrogen peroxide radical scavenging assayTOP

The hydrogen peroxide radical scavenging activity was determined according to Hemalatha et al. (2013), the absorbance was measured at 230 nm after 10 min of incubation at 37 °C. The percentage of hydrogen peroxide scavenging is calculated as follows:

% scavenged H2O2 = (AC-AT)/AC*100

Where: Ac is the absorbance of the control and AT is the absorbance of the test.

2.4.4. β-Carotene bleaching assayTOP

The effect of extracts on the β-carotene- linoleic acid emulsion was determined by applying the method reported by Nsimba et al. (2008). The absorbance of the tested samples was repeatedly measured every15 min at 470 nm. The total antioxidant activity was calculated based on the following equation:

Where: AA% is antioxidant activity, A0 and At; emulsion absorbance at t=0 and after incubation time t (t=105 min), A00 and A0t: absorbance values for negative control at t=0 and after the same incubation time.

2.5. Statistical analysisTOP

All experiments were conducted in triplicate and a statistical analysis was done using Statistica 5.5 with the analysis of variance (ANOVA/MANOVA) to determine the significant differences at a level of confidence of (P<0.05). Correlation coefficients were calculated using the Pearson coefficient.

3. RESULTS AND DISCUSSIONTOP

3.1. Phenolic compoundsTOP

3.1.1. Total phenolic contentTOP

Phenolic contents (Table 1) differ significantly (p<0.05) among the investigated olive cultivars, and between fresh and processed olives for the same cultivar. Their content are higher in fresh olives (346 mg·100 g−1 in Bouchouk to 2406 mg·100 g−1 in Azzeradj from Seddouk), than in processed ones (124 mg·100 g−1 in Azzeradj from Seddouk to 1688 mg·100 g−1 in Sigoise).

Table 1. Total and simple phenolic contents of fresh and processed table olives
Azz Sed Gordal Sevilla Sigoise Teffahi Bouchouk Azz Taz
Total phenolic content (mg GAEq/100 g DW) F: 2406.87±15.1m 2141.3±8.97k 1950.50±12.8j 1923.85±1.02l 1651.62±9.18h 346.50±4.86c 1220.25±14.20g
P: 124.93±3.92a 545.06±4.23e 727.15±7.84f 1688±2.55i 475.85±11.65d 283.43±1.41b 359.05±1.18c
Simple phenolic content (mg/100 g DW)
Oleuropein F: 994.26±26.64g 836.42±40.56f 563.66±15.43e 433.03±0.00d 270.16±81.29c 0.00±0.00a 427.03±24.71d
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 184.29±49.27d 0.00±0.00a 0.00±0.00a 0.00±0.00a
Oleuropein-der F: 375.63±0.23f 164.62±5.53d 164.51±11.34d 167.79±0.00d 105.45±21.04b,c 78.75±10.93b,c 170.43±9.72d
P: 0.00±0.00a 233.23±13.68e 129.15±54.85cd 124.43±24.02c,d 79.84±11.19b,c 56.65±6.21b 0.00±0.00a
Hydroxytyrosol F: 255±72.15g 159.39±16.05c 168.98±5.01e 445.5±0.00f 29.79±0.00a 23.21±0.68a 135.342±11.37b
P: 105.97±12.2bc 45.68±1.49a 545.42±13.24f 98.80±19.8b 14.49±1.49a 100.60±1.65b 38.22±0.00a
Hydroxytyrosol-Glu F: 141.64d±7.06 257.97±31.75e 172.93±16.54de 99.00±0.00c 38.51±0.00a,b,c 61.35±1.77b,c 174.81±23.86
P: 0.00a±0.00 80.23±5.18c 81.03±7.90c 71.10±9.58bc 14.66±2.85ab 16.72±1.56a,b 64.52±0.00b,c
Verbascoside F: 391.08±5.50d 200.84±12.32c 393.30±6.63d 407.54±0.00d 138.57±0.00b 0.00±0.00a 207.03±20.06c
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 131.58±18.34b 0.00±0.00b 0.00±0.00a 0.00±0.00a
Verbascosid-der F: 215.57±42.27b,c 151.44±14.17b,c 270.12±3.35c 306.47±0.00d 106.54±14.57a,b 0.00±0.00a 104.40±6.37a,b
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 100.57±25.48a,b 0.00±0.00a 0.00±0.00a 0.00±0.00a
Quercetine 3-Galactosid F: 205.29±0.61c 356.65±26.98e 188.22±19.59c 256.77±0.00d 109.47±16.68b 0.00±0.00a 187.73±12.76c
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 120.92±32.78c 0.00±0.00a 0.00±0.00a 0.00±0.00a
Tyrosol F: 53.23±0.55e 84.08±2.10g 80.13±1.94fg 76.60±0.00f 25.44±0.00b 22.26±0.16b 48.72±0.47e
P: 37.27±0.73c 43.65±1.09d 106.49±0.26h 35.84±8.76c 24.38±0.00b 27.60±4.23b 24.35±0.00b
Ligstroside F: 334.89±86.71e 164.39±3.95d 140.06±3.90cd 70.31±0.00ab 86.02±3.91b,c 65.30±8.64a,b 114.52±3.79d
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 52.68±16.76a,b 51.10±0.00a,b 0.00±0.00a 0.00±0.00a
Acid benzoic/ac cinnamic-der F: 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a
P: 0.00±0.00a 195.22±14.81f 154.37±4.11e 0.00±0.00a 37.16±2.55b 51.26±2.35c 133.94±0.00e
Benzoic acid-der F: 0.00±0.00a 476.08±7.01f 195.75±37.6c 471.13±0.00f 0.00±0.00a 97.98±7.16b 0.00±0.00a
P: 312.23±26.32e 0.00±0.00a 295.45±34.64de 258.66±51.56d 101.69±20.31b 195.80±3.32c 0.00±0.00a
Hydroxytyrosol-elenolic acid F: 295.68±17.74f 98.90±2.35c 159.82±5.79e 121.96±0.00d 0.00±0.00a 0.00±0.00a 110.84±7.71c,d
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 74.102±21.88b 0.00±0.00a 0.00±0.00a 0.00±0.00a
Acid caffeic-der F: 133.57±10.95b,c 221.95±33.78e 212.91±16.13de 146.25±0.00b,c 142.95±35.82b,c 107.26±1.80b 136.23±27.20b,c
P: 35.37±3.6a 197.86±22.65c,d,e 141.38±3.27bc 156.4±59.13b,c,d 0.00±0.00a 56.42±3.08a 0.00±0.00a
Flavonoids F: 231.51±34.72g 215.93±8.10fg 103.54±16.1cd 149.81±41.63d,e 137.74±33.06d,e 0.00±0.00a 185.90±4.38e,f
P: 0.00±0.00a 0.00±0.00a 0.00±0.00a 142.72±0.00d,e 47.09±10.49a,b 0.00±0.00a 76.93±0.00b,c
F: fresh olives; Azz Sed: Azzeradj from Seddouk; Der: derivative; P: Processed olives; Azz Taz: Azzeradj from Tazmalt. Results represent averages of three replicates. Different letters in the same line, and between fresh and processed olives for the same cultivar indicate significant difference (p<0.05).

The results obtained for fresh olives are similar to those obtained for Portuguese cultivars (Pereira et al. 2006), Tunisian cultivars (Ben Othman et al. 2009), and Italian cultivars Piscopo et al. (2014). However, processed olives contained higher amounts of phenolic compounds than those studied by Blekas et al. (2002) and similar contents to those reported by Ben Othman et al. (2009).

A drastic decrease in phenolic content was noted after processing. Losses in those compounds differ largely among the cultivars, from 12.25% (Sigoise) to 94.80% (Azzeradj from Seddouk). This trend confirms that processing influence phenolic behavior differently between cultivars according to their phenolic profile. In fact, the diffusion of phenols from olive pulp to brine depends on cultivar characteristics, fruit skin, permeability, type of phenols present in the olives and their ability to diffuse outside the fruit (Kiai and Hafidi, 2014).

3.1.2. HPLC identification of phenolic compoundsTOP

The HPLC analysis of phenolic compounds (Table 1) showed that olive flesh revealed different phenolic compound compositions for the seven analyzed cultivars with a drastic difference between fresh and processed olives.

The main phenolic compound detected in fresh olives was oleuropein; its proportion ranged from 25.43% (Sevilla) to 39.10% (Gordal). The oleuropein level was variety dependant and Azzeradj from Seddouk presented the highest amount (994.27 mg·100 g−1), while in Bouchouk this compound was not detected. These significant differences could be explained by the cultivated variety, cultivars with large fruit size (data not shown) have higher amounts of oleuropein (Azzeradj from Seddouk and Gordal) than the small size ones (Sigoise and Bouchouk) as reported previously by Amiot et al. (1990). The analyzed cultivars showed higher amounts of oleuropein compared to Tunisian cultivars (Ben Othman et al., 2009) and Italian cultivars (Piscopo et al., 2014). The results confirmed the effect of cultivar and geographical origin on the phenolic content of olives.

Verbascoside was the second most abundant phenolic compound; its values ranged from 391.08 to 407.548 mg·100 g−1 for Azzeradj from Seddouk and Sigoise, respectively. The amounts of ligstroside vary between 65.30 mg·100 g−1 (Bouchouk) and 334.89 mg·100 g−1 (Azzeradj from Seddouk).

Similarly to the evolution of oleuropeine, verbascoside and ligstroside contents showed a decline in processed olives; their contents were under quantification limits in processed olives for the majority of cultivars, excepted for Sigoise. The results obtained confirm previous data (Boskou et al., 2006; Kiai and Hafidi, 2014) which reported a decrease in oleuropein and verbascoside contents, similarly to an increase in hydroxytyrosol. Processing according to Spanish style causes hydrolysis of phenol complexes due to NaOH treatment, leading to the liberation of simple phenols. Tyrosol showed a slight decrease as well after processing. This observation was not in agreement with the findings of Sahan et al. (2013) who observed an increase in the tyrosol content arising from ligstroside hydrolysis. In addition, fresh olives are rich in quercetin -3 galactoside (109.47 mg·100 g−1 in Teffahi to 356.65 mg·100 g−1 in Gordal). These values are much higher than the amount reported by Piscopo et al. (2014) in Italian cultivar; with the exception of Bouchouk (0.00 mg·100 g−1).

In processed olives, the first major phenolic compound was hydroxytyrosol. The highest content was recorded for Sevilla (545.42 mg·100 g−1 DW equivalent to 109 mg·100 g−1 FW), this result is higher than that of Sahan et al., (2013) (26.45 mg·100 g−1 FW). The lowest level was observed in Teffahi (14.49 mg·100 g−1). It is known that hydroxytyrosol derives from the hydrolysis of oleuropein and verbascoside. An increase in the content of this compound was observed only in Sevilla and Bouchouk varieties. In contrast, for other varieties, a decrease was noted. This might be explained by the diffusion of this polar compound to the brine, or its oxidation during de-bittering (Pasqualone et al., 2014).

Caffeic acid, which is derived from the hydrolysis of verbascoside, was not detected in processed olives. Rodriguez et al. (2008) demonstrated that phenolic acids as caffeic, p-coumaric and ferulic acids are metabolized by L-plantarum, which contain a phenolic acid decarboxylase, to their corresponding vinyl derivatives. Romero et al. (2004) showed that simple phenolic compounds are converted by lactic acid bacteria and de-polymerized.

3.4. Antioxidant activityTOP

3.4.1. Reducing powerTOP

The ferric reducing power of the phenolic extracts of fresh and processed olives are represented in Table 2 (The three standards used recorded the same level of significance).The fresh olives of Azzeradj from Seddouk exhibited the strongest reducing capacity (4531.90 mg QEq/100 g; 7784.4 mg BHAEq·100 g−1; 9180.82 mg BHTEq·100 g−1) while Bouchouk recorded the lowest one (522.45 mg QEq·100 g−1; 887.40 mg BHAEq·100 g−1; 1058.66 mg BHTEq·100 g−1).

Table 2. Antioxidant activities of methanolic extracts of fresh and processed olives
  SA DPPH (mg Eq/100g DW) Reducing Power (mg Eq/ 100g DW) SA H2O2 (%) IBBC (%)
Cultivar Quercetin Eq Trolox Eq α-tocopherol Eq Quercetin Eq BHA Eq BHT Eq
Azz Sed F 1972.62±6.71kl 5458.53±18.57kl 14365.51±48.86kl 4531.90±255.67l 7784.40±439.15l 9180.82±517.93l 85.02±0.69g 72.17±3.39ef
Azz Sed P 143.00±0.21a 395.71±0.58a 1041.40±1.54a 283.36±0.66a 486.73±1.13a 574.85±1.39a 1.75±0.39a 34.88±2.03a
Gordal F 1853.34±27.34l 5128.46±75.64l 13496.85±199.07l 3748.66±104.43k 6439.02±179.39k 7594.11±211.57k 88.89±2.22h 71.69±0.84ef
Gordal P 503.64±2.47d 1393.65±6.84d 3667.74±18.01d 928.44±23.08c 1594.77±39.65c 1868.54±47.16c 2.49±0.39a 75.14±0.93ef
Sevilla F 1453.74±32.30i 4022.72±89.39i 10586.83±235.26i 3063.5±294.56i 5262.15±505.96i 6206.11±596.72i 88.67±9.67i 73.95±1.27ef
Sevilla P 783.90±1.12f 2169.18±3.09f 5708.74±8.14f 1556.35±0.00e 2673.32±2.625e 3154.86±0.99e 24.21±3.51d 45.48±4.15b
Sigoise F 1660.05±44.18k 4593.61±122.26k 12089.25±321.77k 3723.16±100.42j 6395.23±172.49j 7542.47±203.43j 68.09±0.65f 81.29±1.86f
Sigoise P 1693.05±24.60j 4684.93±68.08j 12329.58±179.16j 2316.28±12.27g 3978.64±21.07g 4687.18±24.99g 21.24±0.93d 75.25±3.47ef
Teffahi F 1384.64±9.17h 3831.49±25.37h 10083.55±66.78h 3104.11±1789.52h 5331.90±81.01h 6288.38±95.54h 13.90±3.77c 78.36±2.88ef
Teffahi P 646.79±14.10e 1789.77±39.03e 4710.23±102.72e 997.018485±8.12d 1712.57±13.94d 2026.75±16.80d 12.89±0.26bc 62.65±2.43d
Bouchouk F 180.24±2.75a 498.76±7.60a 1312.62±20.01a 522.45±11.34b 897.40±19.48b 1058.39±22.98b 10.68±1.30b 61.81±0.25d
Bouchouk P 271.54±3.21b 751.39±8.87b 1977.47±23.36b 576.74±23.8b 990.66±40.88b 1155.19±48.66b 4.33±1.69a 53.75±11.10c
Azz Taz F 881.94±16.96g 2440.45±46.93g 6422.68±123.51g 1946.18±45.91f 3342.93±78.86f 3942.62±93.01f 19.79±2.30d 75.08±1.52ef
Azz Taz P 453.27±1.99c 1254.26±5.50c 3300.92±14.48c 863.89±10.54c 1483.90±18.10c 1747.40±21.40c 30.45±7.24e 67.90±3.47de
F: fresh olives; Azz Sed: Azzeradj from Seddouk; IBBC: inibition of bleaching of beta-carotene.
P: Processed olives; Azzj Taz: Azzeradj from Tazmalt.
Results represent average of three replications.
Different letters in the same line, and between fresh and processed olives for the same cultivar indicate significant difference (p<0,05).

Processing caused a decrease in the reducing capacity of extracts. The values ranged from 283.36 QEq·100 g−1, 486.72 BHAEq·100 g−1, 574.85 mg BHTEq·100 g−1 (Azzeradj from Seddouk) to2316.28 mg QEq·100 g−1, 3978.64 mg BHAEq·100 g−1, 4687.47 mg BHTEq·100 g−1 (Sigoise). So that, decreases in reducing power after processing were estimated to 37.78% (Sigoise), 52.42% (Sevilla), 55.25% (Azzeradj from Tazmalt), 68.39% (Teffahi), 76.09% (Gordal) and 93.98% (Azzeradj from Seddouk).

The results showed that a reduction in reducing power is related to a loss in phenolic content. A significante correlation (r=0.98) (Table 3) was obtained between the two parameters. Azzeradj from Seddouk recorded the highest phenol content loss (9.80%) and showed the highest reducing capacity loss (93.98%). In contrast, Sigoise, in which the phenolic content was more preserved, showed the lowest reducing power reduction (37%). No change is noted in reducing power for Bouchouk, which is probably due to its preservation of phenolic compounds.

Table 3. Correlation coefficients for the relationship between antioxidant activities and phenolic content of the extracts
Var TP Oleuro Hydroxy DPPHQ DPPH-T DPPH-αT RP-Q RP-BHA RP-BHT Querc Ligst vebasco Tyrosol hydroxy-
glu
Ac-
hydroxy
B-carot H2O2
Var 1.000
TP −0.429 1.000
Oleuro −0.488 0.900* 1.000
Hydroxy −0.335 0.336 0.261 1.000
DPPHQ −0.533* 0.955* 0.858* 0.414 1.000
DPPH-T −0.490 0.967* 0.830* 0.357 0.939* 1.000
DPPH-αT −0.490 0.967* 0.830* 0.357 0.939* 1.000* 1.000
RP-Q −0.444 0.980* 0.905* 0.408 0.979* 0.947* 0.947* 1.000
RP-BHA −0.444 0.980* 0.905* 0.408 0.979* 0.947* 0.947* 1.000* 1.000
RP-BHT −0.444 0.980* 0.905* 0.408 0.979* 0.947* 0.947* 1.000* 1.000* 1.000
Querc −0.328 0.866* 0.830* 0.113 0.733* 0.866* 0.866* 0.787* 0.787* 0.787* 1.000
Ligst −0.427 0.786* 0.911* 0.099 0.750* 0.694* 0.694* 0.786* 0.786* 0.786* 0.689* 1.000
Verbasco −0.333 0.850* 0.754* 0.118 0.714* 0.821* 0.821* 0.752* 0.752* 0.752* 0.828* 0.749* 1.000
Tyrosol −0.473 0.451 0.371 0.595* 0.392 0.486 0.486 0.390 0.390 0.390 0.522 0.229 0.474 1.000
Hydroxy-Glu −0.265 0.704* 0.779* 0.211 0.546* 0.613* 0.613* 0.638* 0.638* 0.638* 0.819* 0.644* 0.671* 0.631* 1.000
Ac-Hydroxy 0.051 −0.422 −0.470 0.085 −0.358 −0.434 −0.434 −0.378 −0.378 −0.378 −0.508 −0.459 −0.494 0.104 −0.153 1.000
B-Carot 0.096 0.678* 0.510 −0.105 0.574* 0.587* 0.587* 0.637* 0.637 0.636* 0.560* 0.434 0.563* −0.14 0.519 −0.103 1.000
H2O2 −0.459 0.824* 0.875* 0.413 0.829* 0.774* 0.774* 0.838* 0.838 0.838* 0.703* 0.722* 0.658* 0.460 0.732* −0.335 0.456 1.000
*Significant linear correlation at p<0.05.
TP: Total phenolic.
DPPH-Q (Q: quercetin eq), DPPH-T (T:trolox eq), DPPH-αT (α-tocopherol eq).
RP=reducing power, RP-Q (quercetin eq), RP-BHA (BHA eq); PR-BHT (BHT eq).

The potential anti-oxidant index (PAOXI) values of extracts are given in Table 4. Lower PAOXI indicates a lower phenolic content and a better efficiency (Sun and Tanumihardjo, 2007). Our results indicated that all the samples (fresh and processed olives) are efficient because of their PAOXI values of <1. However, the differences noted among PAOXI values might be related to the differences in the phenolic profile of each cultivar. The five varieties processed according to the Spanish style exhibited a better efficiency than their corresponding fresh olives. They are classified as: Azzeradj from Tazmalt=Azzeradj from Seddouk>Sevilla>Teffahi>Bouchouk. This indicates that the processing had a positive effect on phenolic profiles (in some cultivars), which permits the transformation of glycoside to aglycone forms, which is more effective.

Table 4. PAOXI values of extracts corresponding to reducing power, scavenging activity and inhibition bleaching of β-carotene
RP SAH2O2 IBBC
Azzsed F: 0.514d F: 0.286b F: 0.336g
P: 0.429a P: 0.626c P: 0.035a
Gordal F: 0.560e F: 0.237a,b F: 0.299f
P: 0.597f P: 2.209f P: 0.072b
Sevilla F: 0.594f F: 0.208a F: 0.263e
P: 0.468b P: 0.317b P: 0.161c
Sigoise F: 0.584e,f F: 0.319b F: 0.267e
P: 0.805h P: 0.858d P: 0.248e
Teffahi F: 0.527d F: 1.053e F: 0.210d
P: 0.480b,c P: 0.369b P:0.076b
Bouchouk F: 0.648g F: 0.323b F: 0.055a,b
P: 0.505c,d P: 0.575c P: 0.054a,b
Azz Taz F: 0.632g F: 0.578c F: 0.161c
P: 0.419a P: 0.106a P: 0.054b
F: fresh olives P: Processed olives.
RP: Reducing Power SAH2O2 : Scavenging activity against H2O2 IBBC: Inhibition Bleaching of β-carotene.
Different letters in the same column indicate significant difference (p<0.05).

3.4.2. DPPH radical scavenging activityTOP

The scavenging capacity of the extracts (Table 2) showed no statistically significant difference between Gordal, Azzeradj from Seddouk and Sigoise for fresh olives. These cultivars exhibited the highest scavenging capacities. Differences noted among the other varieties should certainly be related to the differences in the phenolic profiles of cultivars.

The effect of processing on scavenging capacity differs among the cultivars; as a consequence of the decline in phenolic content, a reduction was noted for six cultivars with remarkable differences in loss percentages: 92.75% (Azzeradj from Seddouk), 72.82% (Gordal), 53.28% (Teffahi), 48.60% (Azzeradj from Tazmalt), 46.1% (Sevilla) and only13.12% for Sigoise, which was the best efficient extract among processed olives. This in agreement with the results of Sahan et al. (2013) for the Gemlik cultivar. On the contrary, Bouchouk showed an increase (33.57%) in antioxidant activity after processing, which can be attributed to the increase in cinnamic acid derivatives (100%), hydroxytyrosol (76.93%), benzoic acid derivatives (49.96%) and tyrosol (19.35%), confirming that the antioxidant effect does not depend only on the phenolic content but also on the phenolic profile. On the other hand, Brenes and de Castro (1998) claimed that the antioxidant activity of hydroxytyrosol is higher than that of oleuropein. Velkov et al. (2007) ranked the phenols of green olives according to their scavenging activity as: dihydrocaffeic acid>hydroxytyrosol>caffeic acid>oleuropein.

According to the Folin Ciocalteu assay, Gordal showed a higher phenolic content than Teffahi, while it presented the lowest antioxidant capacity; these contradictory results reflect the influence of the nature of phenolic compound on antioxidant activity.

A graphic representation% DPPH inhibition=f (C (mg·mL−1)) revealed a perfect linearity for all samples (0.993≤r≤0.997) (data not shown) which indicates that the extract scavenging effect on DPPH radical increases with increasing concentrations. The scavenging capacity of fresh olives according to the effective concentrations (EC50) (Table 2) followed the order: Azzeradj from Seddouk>Sevilla>Sigoise>Gordal>Teffahi>Azzeradj from Tazmalt>Bouchouk. For the processed olives the order was: Sigoise>Sevilla>Gordal>Teffahi>Azzeradj from Tazmalt>Bouchouk>Azzeradj from Seddouk. The results obtained for fresh olives (except for Bouchouk and Azzeradj from Tazmalt) are in good agreement with those of Arslan and Ozcan (2011) and Malheiro et al. (2011). Otherwise, Ilias et al. (2011) revealed an effective concentration two times lower for Sigoise from Tlemcen; this can be related to differences in composition linked to the geographical origin (Vinha et al., 2005).

The effective concentration (EC50) of extracts exhibited an inverse relationship with phenol contents, showing a significant negative correlation (r=−0.772). Phenolic compounds of olive extracts are good hydrogen donors.

Sigoise processed olives were more effective in scavenging DPPH radical, showing smaller EC50 values (0.38 mg/mL) than those found by Sousa et al. (2008) for the Portuguese Alcaparra variety. These results confirm the superiority of Sigoise processed cultivar in antioxidant activity, which can be related to its higher quercetine-3 galactosid and caffeic acid contents than other processed olives. The high EC50values obtained for Bouchouk and Azzeradj from Seddouk were justified by their polyphenol contents.

3.4.3. Hydrogen peroxide radical scavenging assay (SAH2O2)TOP

Inhibition percentages of hydrogen peroxide in fresh and processed green olives (Table 2) at a concentration of 3.571 mg·mL−1 showed significant differences (p<0.05). Fresh olives of the Gordal and Sevilla varieties exhibited the strongest scavenging activities (88.89 and 88.67%) similar to that of the Chemlal cultivar analyzed by Nadour et al. (2012) but at a concentration of 0.25 mg·mL−1. Bouchouk exerts the lowest one (10.68%).

Losses in scavenging activities after processing were estimated to be 97.94, 97.19, 72.69, 68.8 and 59.45%, respectively, for Azzeradj from Seddouk, Gordal, Sevilla, Sigoise and Bouchouk. After processing, Teffahi recorded statistically the same activity and Azzeradj from Tazmalt showed an increase of 35.0%. This may be related to the formation of more effective phenols after processing, and mainly to the generation of phenolic acids (133.94 mg·100 g−1) (hydroxycinnamic and hydroxybenzoic acid derivatives).

The antioxidant activity of flavonoids against H2O2 was not significant (r=0.404) (Table 3). However, if we consider separately fresh samples and processed samples, this coefficient was 0.814 and 0.361, respectively. Thus, the loss in this coefficient estimated at 2.25 explains the negative effect of processing by Spanish style on the flavonoid contents of green olives.

Potential antioxidant index (PAOXI) values of the extracts (Table 4) confirmed losses in scavenging activities by the increase of PAOXI values for the cultivars Sevilla, Bouchouk, Azzeradj from seddouk, and Sigoise. Although Gordal showed a very low efficiency of its phenolic compounds, requiring the implication of more than two phenolic fractions to enrich by one scavenging activity unity. The PAOXI value obtained for Azzeradj from Tazmalt is in agreement with the increase in scavenging activity, recording the lowest PAOXI value among the studied cultivars.

3.4.4. β- carotene bleaching assayTOP

The percentages of inhibition bleaching of β-carotene (IBBC) for fresh and processed olive extracts at the concentration of 7.14 mg·mL−1 are given in Table 2. No significant differences (p≤0.05) were noted between fresh and processed olives except for Azzeradj from Seddouk and Sevilla. This indicates that the phenolic compounds of green olives (fresh and processed) react strongly in an organic medium, probably due to their partition coefficient.

Losses in antioxidant activity after processing varied according to the cultivar: Azzeradj from Seddouk (51.66%), Sevilla (38.5%), Teffahi (20.04%), Bouchouk (13.01%), Azzerad from Tazmalt (9.56%), Sigoise (7.26%). However, an increase was recorded for Gordal which is probably due to the increase in phenolic acids, such as in cinnamic acid derivatives. Sroka and Cisowski (2003) postulated that phenolic acids are the weakest inhibitors in lipid peroxidation. Otherwise, contrary to the findings of Han et al. (2012), who reported a synergism action between flavonoids and β-carotene accordingly at the water/lipid interfaces, our results show a decline in flavonoid content and similar antioxidant activity after processing. A moderate correlation was established between β-carotene bleaching assay and total phenolics (r=0.67) (Table 3), this means that lipid peroxidation inhibitory activity could be partially correlated to the phenolic content.

The PAOXI values obtained (Table 4) clearly showed that the majority of extracts had better activities in an emulsion medium than in an aqueous one. This may be explained by the “Polar paradox” phenomenon characterized by the accumulation of a polar antioxidant in the oil-water interface, thus protecting the lipids from oxidation (Hayes et al., 2011).

3.4.5. Evaluation of table olive total antioxidant capacityTOP

To get a complete picture of the ranking of the antioxidant capacities of table olives, a relative antioxidant capacity index (RACI) was calculated by integrating the antioxidant capacity values generated from the different tests. RACI is the mean value of standard scores transformed from the initial data generated with different methods (Sun and Tanumihardjo, 2007). Results of the classification of samples (Figure 1) revealed the superiority of the fresh olives of Azzeradj from Seddouk in the totality of tests, giving a RACI value of +1.53. The processed olives of the same variety present the lowest RACI value (−1.41). The results showed that among the processed olives, only Sigoise denoted a positive value of RACI.

Figure 1. Relative antioxidant capacity index (RACI) of extracts of fresh and processed olives.

 

4. CONCLUSIONTOP

In conclusion, the results revealed a significant influence of Spanish style processing on the phenolic compounds and antioxidant capacity of green table olives extracts of seven Algerian cultivars. This effect differs greatly among the cultivars; Sigoise is the most conservative variety and Azzeradj from Seddouk is the most dissipating one.

The results obtained for the antioxidant activity assessed by the four assays showed that among the studied cultivars, the fresh olives of the Azzeradj variety from Seddouk exhibited the highest phenolic content and exerted the strongest antioxidant activities, but they were also the most affected by the processing treatment (loss of 94.80% in phenolic content), which caused a loss of 93.74, 92.75, 97.94, 51.66% in reducing capacity, scavenging capacity against DPPH radical, hydrogen peroxide radical and in the bleaching test, respectively. Instead, Sigoise, the most representative cultivar of the Algerian Market, was the least affected by the processing. The antioxidant activity of processed olives recorded significant losses for the aqueous medium in contrast to the organic medium. Fresh olives of Azzeradj from Seddouk exhibited the highest RACI value, confirming their superiority in antioxidant capacity.

The data from this study show that olive extracts and mainly extracts of fresh olives of Azzeradj from seddouk may constitute a good source of healthy compounds. It would be interesting to use other methods of preparation that could preserve them.

As far as we know, this is the first report considering the antioxidant potential of Algerian green olive cultivars. Further studies are needed to focus on phenolic loss reduction as a result of Spanish style processing of green table olives.

ACKNOWLEDGEMENTSTOP

The authors are grateful to the staff of ITAFV (Institut d’Arboriculture Fruitière et de la Vigne) Takerietz (Bejaia, Algeria) and the company of KHODJA & CO Seddouk (Bejaia, Algeria) for providing the samples.

 

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