Chemical characterization and oxidative stability of olive oils extracted from olive trees of Southern Brazil

The objective of this work was to characterize the chemical composition of olive (Olea europaea) oils produced in Southern of Brazil and correlate it with oxidative stability. Olive oils from the Arbequina, Coratina, Frantoio and Koroneiki cultivars were evaluated. A completely randomized experimental design was used, in a uniform arrangement, with three replicates. Acidity value, peroxide index, specific absorption, tocopherol content, phenolic compound content, carotenoid content, chlorophyll content, fatty acid profile, and oxidative stability were determined. The oils from the Coratina and Frantoio cultivars were classified as extra virgin-oils. The olive oil from the Coratina cultivar showed the highest levels of pigments, followed by the oil from Koroneiki. The oil from the Coratina cultivar also presents higher contents of phenolic compounds (1,725.5 mg kg-1) and tocopherols (437.8 mg kg-1). The major fatty acid in all samples is oleic acid.


Introduction
The consumption of extra-virgin olive oil has increased significantly due to its unique sensory characteristics and health benefits. Typical aroma, flavor, color, and functional properties allow distinguishing olive oil from other edible vegetable oils (Fadda et al., 2012).
Olive oil, as well as other oils and fats, besides contributing to the quality of certain foodstuffs, provides nutritional value as a good source of metabolic energy, as well as essential linoleic and α-linolenic fatty acids. Its composition is characterized by a high content of a monounsaturated fatty acid, the oleic acid (Krichene et al., 2010), associated with high levels of bioactive compounds such as tocopherols, phenolic compounds, phytosterol (mainly β-sitosterol) and pigments (chlorophylls and carotenoids), which contribute to its sensory characteristics, as well as the Pesq. agropec. bras., Brasília, v.52, n.12, p.1231-1240, dez. 2017 DOI: 10.1590/S0100-204X2017001200012 oxidative stability (Allalout et al., 2009;Krichene et al., 2010;Fregapane & Salvador, 2013). Important functions are also assigned to bioactive compounds, such as the participation in the prevention of cardiovascular diseases, cancers, degenerative diseases, and obesity (Fadda et al., 2012;Cicerale et al., 2013). However, olive oil is quite susceptible to lipid oxidation, a spontaneous and unavoidable phenomenon, with direct implication on the commercial value of the oils, either in pure oil or in products containing oil in their formulation, as food, cosmetics, and medicines (Silva et al., 1999).
Scientific interest in olive cultivation in the state of Rio Grande do Sul, Brazil, is due to suitable characteristics for olive oil production, especially from the Coratina, Frantoio, and Koroneiki cultivars, which have shown high and constant fruit productivity (Coutinho et al., 2009;Servili, 2015). Arbequina, one of the most suitable cultivars for olive production, presents good adaptation to Brazilian soil and exhibits vegetative vigor, precocity, and high yielding capacity (Coutinho et al., 2009). Therefore, quality, chemical composition, and stability of oils produced in Southern Brazil should be investigated.
The objective of this work was to characterize the chemical composition of olive oils produced in Southern of Brazil and correlate it with oxidative stability.

Materials and Methods
Experiments were conducted in the chromatography laboratory at Universidade Federal de Pelotas, located in the municipality of Capão do Leão, state of Rio Grande do Sul, Brazil. Olive oil samples were obtained from the Arbequina, Coratina, Frantoio, and Koroneiki cultivars during the 2013/2014 harvest, from adult plants of Olea europaea L., from the experimental unit of Embrapa Clima Temperado, located in the municipality of Pelotas, Rio Grande do Sul, Brazil (52º21'W, 31º52'S, at an altitude of 224 m). The soil of the region is classified as Luvissolo Hipocrômico órtico típico (Alfisols), a shallow clayey soil (Santos et al., 2006). The climate is characterized as humid with hot summers, according to the Köppen & Geiger classification system (1928) with an annual average temperature of 18.4°C and rainfall of 1,582 mm.
Oil extraction was performed by fruit rupture and centrifugation using the Abencor system, consisting of a MM-100 mill, a TB 100 thermobeater, and a CF-100 centrifuge (MC2, Ingenieria y Systemas, Sevilla, Spain) with subsequent decantation and filtration. Samples were packed in amber glasses and kept frozen at -80°C in an ultra freezer (Ilshin Cab, Co., Ltd., Tongjin-eup, South Korea) until the analysis was initiated. The olive fruits used in the oil extraction were obtained from a completely randomized experimental design, in a factorial arrangement, with three replicates.
The acidity and peroxide value of the samples were determined respectively, according to the America Oil Chemists' Society (AOCS) methods Ca 5a -40 (Firestone, 1998) and Cd 8-53 (Firestone, 1998). Specific absorption was measured at 232 and 270 nm (K 232 and K 270 ) according to the method recommended by International Olive Council (IOOC) (2010), and tocopherol content was determined according to the method described by Pestana et al. (2008), with minor modifications. Approximately 0.250 mg olive oil was diluted in isopropanol (high-performance liquid chromatography grade) up to 5 mL and the mixture was centrifuged for 6 min at 9,000 g in a microcentrifuge (NT800 Nova Técnica, Piracicaba, SP, Brazil). The organic phase was transferred to a 1.5-mL vial and samples between 10 to 20 μL were analysed using the high-performance liquid chromatography system (Shimadzu, Kyoto, Japan), consisting of the LC-10ATVP solvent mixing module, FCV-10ALVP degasser, DGU-14A reodine pump, SCL-10AVP system control, CTO-10ASVP column oven, and SIL-10AF automatic sampler. Tocopherol separation was performed using the Shimadzu reverse phase analytical column, Shim-Pack CLC-ODS (3.9 cm x 150 mm x 4 μm), having octadecyl groups as stationary phase, and a fluorescence detector with 290 and 330 nm of excitation and emission wavelength, respectively. Data were acquired and processed using the Class-VP software (Shimadzu, Kyoto, Japan). A mixture of acetonitrile: methanol: isopropanol in the ratio of 50:40:10 (v/v/v) was used as the starting mobile phase for 10 min, changing linearly at 1 mL min -1 constant flow to acetonitrile: methanol: isopropanol 30: 65: 5, (v/v/v) up to 12 min, and returning linearly to the initial mobile phase up to 15 min of analysis.
The identification and quantification of tocopherols were performed using external calibration curves of (β + γ)-and δ-tocopherol, and the results were expressed as milligrams of tocopherol per 100 g of sample.
The extration of phenolic compounds followed the method described by Montedoro et al. (1992), with modifications. An oil sample of 2.5 g was mixed with 2.0 mL methanol: water (70:30) mixture and 2 mL hexane, subjected to vigorous stirring for 1 min, followed by continuous agitation for 20 min, and centrifugation at 7,000 g at 4°C for 10 min in the 5430R microcentrifuge (Eppendorf Hamburg, Germany). The hydroalcoholic phase was collected and centrifuged again at 7,000 g at 4°C for 4 min, transferred to a 2-mL volumetric flask, and the volume completed with a methanol: water (70:30) mixture. Total phenolic compounds were determined, as described by Gambacorta et al. (2010), by mixing 100 μL of the hydroalcoholic extract with 100 μL of the Folin-Ciocalteu reagent at 2 mol L -1 in a falcon tube and, after 4 min, adding 800 μL 5% sodium carbonate. The mixture was kept in a water bath for 20 min at 40°C. Samples were subjected to absorbance measurements at 750 nm in 6705 UV/VIS spectrophotometer (Jenway, Staffordshire, UK). For total phenolic compound quantification, a gallic acid standard curve was constructed, with readings at 750 nm. The results were expressed as mg kg -1 of gallic acid.
The carotenoid content was determined as described by Rodrigues-Amaya (2001), with readings at 450 nm. The results were expressed as mg kg -1 β-carotene.
Total chlorophyll was determined at 630, 670, and 710 nm using the Cc 13d-55 methodology of AOCS (Firestone, 1998). For both analyses, a blend of isooctane: ethanol (3:1) was used as a reference, and the results were expressed as mg kg -1 . The both readings were performed on 6705 UV/VIS spectrophotometer (Jenway, Staffordshire, UK).
The olive oil samples were derivatized according Hartmann & Lago (1973). Fatty acids were quantified in a gas chromatograph (Perkin Elmer Clarus 500, Shelton, USA), fitted with a FID detector, using a polyethylene glycol ID Carbowax 20M column (0.25 μm, 30 m x 0.25 mm). The temperature gradient was maintained at 90°C for 1 min, increased at a 12°C min -1 rate up to 160°C for 3.5 min, then increased at a rate of 1.2°C min -1 up to 190ºC, with a linear increase of 15ºC min -1 up to 230°C for 15 min. The injector and detector temperatures were maintained at 250°C. Nitrogen at a rate of 1.5 mL min -1 was used as carrier gas (Zambiazi, 1997). Fatty acid methyl esters were identified by comparison with retention times of reference standards, and results were expressed as relative fatty acid percentage.
Oxidative stability was determined in the oil samples in the 743 Rancimat (Metrohm, Herisau, Switzerland) at 110°C, at an inflation rate of 10 L h -1 , according to EN ISO14112 (CEN, 2003).
Data were analyzed for normality by the Shapiro-Wilk test, homoscedasticity by the Hartley test, and residue independence by the graphic analysis. Subsequently, data were subjected to the analysis of variance through the F-test, at 5% probability. The effects of cultivars were compared by Tukey's test, at 5% probability, and correlations between dependent variables were analyzed using Pearson's correlation coefficient (SAS Institute Inc., Cary, NC).

Results and Discussion
Differences were observed in acidity values, except for the oils from the Coratina and Koroneiki cultivars (Table 1). In addition, only the oil from the Arbequina cultivar was not in line with the established legislation standards for extra-virgin olive oil (0.8%), and it was classified as lampante virgin olive oil because it features acidity higher than 2% (Brasil, 2012).
Several factors can lead to high acidity content, including harvesting process, fruit maturation index, Pesq. agropec. bras., Brasília, v.52, n.12, p.1231-1240, dez. 2017 DOI: 10.1590/S0100-204X2017001200012 olive quality, storage, and oil extraction. According to Cardoso et al. (2010), storage and oil extraction should not exceed 4 hours. The authors found high acidity content at the end of harvest, with values of 2.2 and 3.6%, respectively, in the Ascolano and Negroa cultivars. The high acidity may have been due to the injury in fruits and time of exposure of the olives at room temperature, favoring enzyme activity, and increasing acidity values.
Peroxide values of olive oils from the Coratina and Frantoio cultivars were within the limits allowed for the classification of extra-virgin olive oil, according to Brasil (2012) andIOOC (2012). The other samples presented values above the limits established for extra-virgin and virgin olive oils. These results were probably due to the longer exposure time of the fruits before oil extraction or the greater injury between harvest and processing. Cardoso et al. (2010) also found high peroxide values in the oil from the Negroa olive cultivar (25.5%), similar to the one found in oil from the Koroneiki cultivar.
Oils from the Coratina and Frantoio cultivars presented specific absorption coefficients (K 270 ) in accordance with the extra-virgin oil classification (Table 1). For the other samples, the specific absorption coefficients K 232 were within the limit for classification of extra-virgin olive oil. As the process of obtaining the oil was the same for all cultivars, it is assumed that differences in this parameter may be related to the characteristics of each cultivar. Dabbou et al. (2010) reported similar results to those obtained in this study for the oils from the Arbequina and Koroneiki cultivars.
Oil from the Coratina cultivar stood out due to the highest tocopherol content, with α-tocopherol as the major compound in all oils ( Table 2). Separation of γ and β-tocopherol was not possible by the reverse phase column (Figure 1), so that results were expressed as the sum of both compounds.
Oils of the Arbequina and Frantoio cultivars presented lower tocopherol content, which varies according to the cultivar (Table 2). It should be noted that this compound plays an important role in the olive oil stability (Allouche et al., 2007). This variation is confirmed in the literature, with reports of α-tocopherol content in virgin olive oil between 100 and 460 mg kg -1 , representing 96% of total tocopherols 3.0 5.6 2.5 6.7 (1) Means followed by equal letters, in the column, do not differ by Tukey's test, at 5% probability. K 232 and K 270 , specific absortion measured at 232 and 270 nm, respectively. n=3.  (Allouche et al., 2007;Gambacorta et al., 2010;Mansouri et al., 2014). Regarding phenolic compound content, olive oil from the Coratina cultivar presented higher values than those of the other samples. Possibly the cultivar, fruit maturation index, and storage at ambient or refrigerated temperatures were responsible for the different levels observed in this study. Phenolic compounds also contribute to the peculiar aroma and flavor of olive oil, which are believed to significantly enhance oxidative stability due to their antioxidant properties (Allouche et al., 2007).
The oil from the Coratina cultivar stood out from the others due to this higher pigment content. Confirming this result, Aparicio-Ruíz et al. (2009) reported the β-carotene levels for the oils from the Coratina (4.04 and 5.41 mg kg -1 ), Frantoio (4.03 and 4.80 mg kg -1 ), and Koroneiki (3.89 and 6.26 mg kg -1 ) cultivars close to those found in the olive cultivars from Southern Brazil.
Chlorophylls act as pro-oxidants in the presence of light; thus, variations in their oil contents, together with inadequate storage conditions, may affect the shelf life of the product (Bengana et al., 2013). According to the literature, the centrifugation process, which was used to obtain the Brazilian olive oils, produces high losses of pigments in relation to the pressing process used in other countries (Torres & Maestri, 2006). This may explain in part the lower content of pigments found in Brazilian olive oils.
As expected, it was verified that the major fatty acids were oleic, palmitic, and linoleic ( Table 3). The oleic fatty acid was the most representative in all samples, with higher content in olive oil from the Koroneiki cultivar (76.5 %) and lower content in olive oil from Figure 1. Typical chromatogram of δ-, β+γ-, and α-tocopherols separation of olive (Olea europaea) oil from the Arbequina cultivar by high-performance liquid chromatography using reverse phase RP-18CLS-ODS column and fluorescence detector, excitation at 290 nm, and emission at 330 nm. The peaks correspond to: 1, δ-tocopherol; 2, β + γ-tocopherol, and 3, α-tocopherol.
Differences in oxidative stability were verified between samples, and greater stability was observed for the olive oil from the Coratina cultivar (Table 3). Lower peroxide, acidity, and polyunsaturated fatty acid ratio, as well as higher contents of bioactive compounds, may have positively affected the oxidative stability of this olive oil.
These results corroborate those presented by Ramalho & Jorge (2006), who evaluated the oxidative stability of soy oil by Rancimat at 110°C. By adding the antioxidants α-tocopherol and rosemary extract to the oils, the authors demonstrated their protective effect on oxidation. Oils with higher oxidative stability also showed higher α-tocopherol contents.
A high positive correlation coefficient (r = 0.99, p<0.0001) was observed for carotenoids and (γ + β)tocopherol contents, confirming that an increase in carotenoids can lead to an increase in (γ + β)tocopherol (Table 4). Positive correlations were also  (1) Means followed by equal letters, in the column, do not differ by Tukey's test, at 5% probability. CV, coefficient of variation mean of three replicates.
Arbequina (59.2%). These results are in alignment with the values recommended by the Brazilian legislation (Brasil, 2012;IOOC, 2012). Dabbou et al. (2010) reported, respectively, 61.4 and 76.0% for oleic acid in olive oil from the Arbequina and Koroneiki cultivars grown in Southern Tunisia. Daskalaki et al. (2009) found values from 75.1 to 78.5% for olive oil from the Koroneiki cultivar produced in Greece. Therefore, depending on the cultivar and the origin region, the relative percentage of the fatty acids varies. Although the Arbequina cultivar is easily adaptable to the Brazilian soil and is well indicated for olive oil production, it is disadvantageous because it provides a oil with lower levels of oleic acid, which partially contributes to its lower stability (Mello & Pinheiro, 2012). In the present study, the oil of this cultivar showed the highest content of saturated fatty acid; the palmitic acid content was the highest (18.5%), close to the maximum value of 20% established by Brasil (2012) and IOOC (2012) (Figure 2).
The production of olive oil in Brazil is still small, but of great importance for a boost in the country's olive crop. Thus, this study provides data on oils obtained from the most expressive cultivars in the southern region of Rio Grande do Sul, Brazil. In this scenario, many possibilities open up, and there is great motivation for further investigative research.

Conclusions
1. The olive (Olea europaea) oils from the Coratina and Frantoio cultivars produced in Southern Brazil are classified as extra-virgin, in alignment with the quality parameters established for the product.