Oil quality characterization of the Arauco variety from the main olive growing areas of Mendoza (Argentina)

2.1. Zones and plant material

Assays were carried out over two harvest years (2016 and 2017) on the ‘Arauco’ variety grown in orchards with similar management and ages, located in three important olive growing areas of the Mendoza province (see maps in Figure 1).

Zone 1 (Junín) is included in the Olive Germplasm Collection of Mendoza, which is situated in the experimental farm of INTA in Junín (33º06´S, 68º29´W, 653 m.a.s.l). The plantation was established in 1956. The region has a historical average temperature of 17.3 ºC, a rainfall of 275 mm (mainly in summer and far below olive crop water requirements) and a frost-free period from November to April. Trees from the olive collection are spaced 12 m x 12 m (traditional system) and fortnightly flood irrigated to replenish the soil water content over the growing seasons.

Zone 2 (Rivadavia) is located within a commercial olive orchard in Los Campamentos, Rivadavia (33º15´S, 68º26´W, 660 m.a.s.l). The plantation was established in 1980. The region has a historical average temperature of 18.5 ºC, a rainfall of 195 mm (mainly in summer) and a frost-free period from November to March. Trees are spaced 10 m x 10 m (traditional system) and drip irrigated to replenish the soil water content and thus avoid water deficit.

Zone 3 (Maipú) is situated within a commercial olive orchard in Russell, Maipú (33°0’S, 68°44’W, and 850 m.a.s.l). The plantation was established in 1986. The region has a historical average temperature of 19.5 ºC, a rainfall of 120.5 mm (mainly in summer) and a frost-free period from October to April. Trees are spaced 10 m x 10 m (traditional system) and flood irrigated to replenish the soil water content and avoid water deficit.

From each orchard, four trees were randomly selected before collecting olives with similar crown volume and high fruit load. Manual harvest was conducted during the first half of May, before frost events. A fruit sample of 20 kg per tree (replicate) was taken. One replicate was used for machine conditioning and the oil obtained was not considered for the study. From the other three replicates, a fruit subsample of 2 kg was randomly selected to perform fruit analysis (pulp oil concentration in fresh and dry basis, fruit weight, fruit humidity, pulp/pit ratio and maturity index). The rest of the fruits (18 kg) were milled to extract olive oil. Briefly, olive fruits were crushed with a hammer mill and the olive paste obtained was malaxated for 40 min until passing through the decanter (Spremolive New 20 kg·h^-1, Toscana Enologica Mori) without the addition of water. Afterwards, the olive oil obtained was filtered and stored in a cold and dark place until its analytical parameters were determined.

2.2. Morphological and chemical characteristics of the fruit

Fruit dry weight and water concentration were determined in a sample of 100 fruits which were fresh-weighed and oven-dried (60 °C) to a constant weight. Fruit dry weight was also measured. Next, water concentration was calculated from the difference between fresh and dry weight divided by fresh weight. Pulp/pit ratio was determined from a sample of 50 randomly selected and weighed fruits; then, their pits were manually separated and weighed. Another sample of 100 fruits was randomly selected to determine maturity index, according to Uceda and Frías (1975)Uceda M, Frias L. 1975. Épocas de recolección. Evolución del contenido graso del fruto y de la composición y calidad del aceite. II Semin. Oleíc. Int. Córdoba Esp. 25-46. . Industrial yield was calculated by dividing kilograms of olive oil obtained by kilograms of milled fruit and then multiplied by 100 to express it as percentage.

Pulp oil concentration was determined in triplicate according to Avidan et al. (1999)Avidan B, Ogrodovitch A, Lavee S. 1999. A reliable and rapid shaking extraction system for determination of the oil content in olive fruit. Acta Hortic. 474, 653-658. https://doi.org/10.17660/ActaHortic.1999.474.135 . In brief, 5 g of pulp were weighed and oven-dried (60 ºC) to constant weight. Dried-pulp was weighed and crushed in a mortar and transferred to a test tube containing 10 mL of petroleum ether. After 12 h of shaking, the samples were filtered, transferred to a new, previously-weighed test tube and air-evaporated. Later, samples were oven-dried (60 °C) to constant weight. Pulp oil concentration was calculated on fresh basis (POCfb) and dry basis (POCdb) from weight difference.

2.3. Analytical methods

Oil quality was determined through acidity (expressed as oleic acid) and extinction coefficients (K₂₃₂ and K₂₇₀) according to the International Olive Council (IOC, 2015aIOC, 2015a. IOC, International Olive Council. Determination of free fatty acids, cold method, in COI/T20/Doc No 34, Ed. , 2015bIOC, 2015b. Spectrphotometric investigation in the ultraviolet, in COI/T20/Doc No 19/Rev 3. ). In addition, oil oxidative stability was calculated by measuring oxidation induction time using Rancimat equipment (Metrohm Ltd., Herisau, Switzerland). In summary, a sample of oil (3 g) was force-oxidized by means of air flow (10 L.h^-1) and heat (110 ºC). As a result, the necessary time to reach the curve inflection point was obtained.

Total phenolic content was determined according to the International Olive Council method (IOC, 2017IOC, 2017. Determination of Biophenols in Olive Oils by HPLC, in COI/T20/Doc N°29/Rev1. ). Briefly, a portion of olive oil (2 g) was weighed and mixed together with the internal standard syringic acid. Next, it was extracted by an ultrasound bath at room temperature with a mix of methanol/water (80/20 v/v). Afterwards, it was centrifuged and the supernatant was filtered and injected into a liquid chromatograph Dionex Ultimate 3000 HPLC system (CA, USA) with a UV detector at 280 nm. A C₁₈ reverse-phase column, Roc^® Restek (250 x 4.6 mm; 5 μm) (Pennsylvania, USA) was used, employing a gradient of water 0.2% phosphoric acid ((v/v)/methanol/acetonitrile) as indicated by the official methodology.

Total flavonoids were determined by the modified method of Zhishen et al. (1999)Zhishen J. 1999. The determination of flavonoid contentes in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555-559. https://doi.org/10.1016/S0308-8146(98)00102-2 . A sample of olive oil (6 g) was added to 10 mL of methanol/water solution (4:1 v/v), the mixture was shaken for 10 min at 4000 rpm, and the supernatant was extracted and put in a flask. This process was repeated three times. In a 10 mL volumetric flask, 1 mL of sample was added to 4 mL of distilled H₂O. Next, 0.3 mL of sodium nitrite (5% p/v) were added. After 5 min, 0.3 mL of aluminum chloride (10% p/v) were added, and 6 min later, 2 mL of sodium hydroxide (1M) were added. The volumetric flask was filled to the mark with distilled water. Flavonoids were measured by spectrophotometry at 510 nm of absorbance against a blank.

Olive oil fatty acid composition was determined by the IOC method (2001)IOC, 2001. Preparation of the fatty acid methyl esteres from olive oil and olive-pomace oil, in COI/T20/Doc N° 24. . An oil sample was weighed and dissolved with heptane. Afterwards, it was derivatized with a methanolic potassium hydroxide solution. The solution was left to decant and the supernatant was analyzed with a Shimadzu GC 2010 Plus gas chromatography system (Shimadzu, Japan), using a hydrogen flame ionization detector and a ZB-FAME capillary column (60 m x 0.25 mm; 0.2 um) (Zebron, Phenomenex, USA). Detection and injection were set at 260 ºC and 240 ºC, respectively, using hydrogen as carrier gas. The amounts of fatty acids were expressed as relative area percentages and identified by comparing their retention times with those of standard solutions. The fatty acids detected were palmitic (C16:0), palmitoleic (C16:1), margaric (C17:0), margaroleic (C17:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), arachidic (C20:0), eicosenoic (C20:1) and behenic (C22:0).

2.4. Statistical analysis

Data of zones and harvest years were submitted for variance analysis and means separated using the Tukey-test (α=0.05) and InfoStat statistical software version 1.5 (Di Rienzo et al., 2002Di Rienzo JA, Guzman AW, Casanoves F. 2002. A multiple-comparisons method based on the distribution of the root node distance of a binary tree. J. Agric. Biol. Environ. Stat. 7, 129-142. https://doi.org/10.1198/10857110260141193 ). A regression analysis was applied to study the relationships among all the parameters analyzed in both fruits and oil.

3.1. Fruit characteristics

The fruit characteristics of the ‘Arauco’ variety from the three main olive growing zones of Mendoza for the two seasons are shown in Table 2.

Taking into account zones and crop seasons, fruits from the ‘Arauco’ variety showed fresh and dry weight of 5.1 g and 1.8 g, respectively, pulp/pit ratio of 7.8, water concentration of 64.4% and oil concentration of 15.8% and 50.1% on fresh and dry weight basis, respectively, and a maturity index of 1.5. ‘Arauco’ has a large size, pulp/pit ratio higher than 5, and slow change in skin color, characteristics which are highly appreciated for table olive elaboration (Bodoira et al., 2015Bodoira R, Torres M, Pierantozzi P, Taticchi A, Servili M, Maestri D. 2015. Oil biogenesis and antioxidant compounds from “Arauco” olive (Olea europaea L.) cultivar during fruit development and ripening: Oil biogenesis from “Arauco” olive cultivar. Eur. J. Lipid Sci. Technol. 117, 377-388. https://doi.org/10.1002/ejlt.201400234 ; Kailis and Harris, 2007Kailis S, Harris D. 2007. Producing table olives. Landlinks Press, Collingwood VIC 3066 Australia. https://doi.org/10.1071/9780643094383 ), which constitutes the main industrial use of this variety in Argentina.

Fruit characteristics were significantly different among zones with the exception of pulp oil concentration. Across seasons, the fruits collected from Zone 2 showed higher fresh weight (6.4 g) and dry weight (2.2 g), pulp/pit ratio (8.9) and were more mature (2.3) than the fruits collected from Zones 1 and 3 (average 4.4 g, 1.6 g, 7.3, 1.1, following the same sequence as Zone 2). The fruit water concentration from Zones 1 and 2 was on average 65.4% higher than Zone 3 (62.6%). During oil processing, the oil extraction yield showed a similar pattern, with the highest value in Zone 2, intermediate in Zone 1, and the lowest in Zone 3. The earlier maturation and higher fruit size and pulp/stone ratio observed in Zone 2 seem to be more associated with fruit load and orchard management than with environmental conditions.

In all environments, except Zone 2 (MI = 2.3), fruits were harvested with a maturity index lower than 1.5 prior to the occurrence of severe early frost, which served to ensure high-quality oil. Bodoira et al. (2015)Bodoira R, Torres M, Pierantozzi P, Taticchi A, Servili M, Maestri D. 2015. Oil biogenesis and antioxidant compounds from “Arauco” olive (Olea europaea L.) cultivar during fruit development and ripening: Oil biogenesis from “Arauco” olive cultivar. Eur. J. Lipid Sci. Technol. 117, 377-388. https://doi.org/10.1002/ejlt.201400234 evaluated the olive variety ‘Arauco’ in the San Juan province and determined maximum oil concentration at a low maturity index (below 1) and maturity index between 1 and 2 to achieve maximum oil quality. In a previous study carried out in Mendoza on the ‘Arauco’ variety, it was observed that harvest before mid-May led to obtaining maximum oil quality by reducing olive exposure to severe frost events (Trentacoste et al., 2020Trentacoste ER, Banco AP, Piccoli PN, Monasterio RP. 2020. Olive oil characterization of cv. ‘Arauco’ harvested at different times in areas with early frost in Mendoza, Argentina. J. Sci. Food Agric. 100, 953-960. https://doi.org/10.1002/jsfa.10029 ). Similarly, Morelló et al. (2003)Morelló J-R, Motilva M-J, Ramo T, Romero M-P. 2003. Effect of freeze injuries in olive fruit on virgin olive oil composition. Food Chem. 81, 547-553. https://doi.org/10.1016/S0308-8146(02)00488-0 evaluated the effects of frost on the Arbequina variety oil and determined a significant decrease in phenol content and pigments due to frost.

3.2. Oil characteristics

Basic oil quality parameters (phenolic content, oxidative stability, acidity, extinction coefficients) and total flavonoids are shown in Table 3. All measured samples were classified as extra virgin olive oil according to IOC, 2015aIOC, 2015a. IOC, International Olive Council. Determination of free fatty acids, cold method, in COI/T20/Doc No 34, Ed. ; IOC, 2015bIOC, 2015b. Spectrphotometric investigation in the ultraviolet, in COI/T20/Doc No 19/Rev 3. ; IOC, 2017IOC, 2017. Determination of Biophenols in Olive Oils by HPLC, in COI/T20/Doc N°29/Rev1. . On average, the ‘Arauco’ variety presented 0.18% acidity, 0.09 extinction coefficients K₂₇₀, 434 mg·kg^-1 phenolic content and 54 mg·kg^-1 total flavonoids with non-significant differences among zones, in contrast to oxidative stability (8.9 h) and extinction coefficient K₂₃₂ (1.04). The oil from Zone 3 showed the highest oxidative stability (10.4 h), and the oil from Zone 2 the highest extinction coefficient (K₂₃₂=1.2).

It is worth noting that the phenol content in ‘Arauco’ oil was above 400 mg·kg^-1, regardless of zone. According to Montedoro et al. (1992)Montedoro G, Servili M, Baldioli M, Miniati E. 1992. Simple and hydrolyzable phenolic compounds in virgin olive oil. 1. Their extraction, separation, and quantitative and semiquantitative evaluation by HPLC. J. Agric. Food Chem. 40, 1571-1576. https://doi.org/10.1021/jf00021a019 , ‘Arauco’ oil could be classified as a variety within the “medium” category (i.e. 200-500 mg·kg^-1) in relation to phenolic content. The phenolic content range observed here coincides with Monasterio et al. (2017)Monasterio RP, Olmo-García L, Bajoub A, Fernández-Gutiérrez A, Carrasco-Pancorbo A. 2017. Phenolic Compounds Profiling of Virgin Olive Oils from Different Varieties Cultivated in Mendoza, Argentina, by Using Liquid Chromatography-Mass Spectrometry. J. Agric. Food Chem. 65, 8184-8195. https://doi.org/10.1021/acs.jafc.7b02664 , who evaluated oil from eight olive varieties from Mendoza province, including ‘Arauco’. The authors determined the phenolic content in ‘Arauco’ oil to be within the range of 233 to 404 mg·kg^-1. In addition, the phenolic content in the oil from ‘Arauco’ olives grown in Mendoza was similar to that of ‘Arauco’ oil from San Juan (433 mg·kg^-1) (33ºS, Bodoira et al., 2015Bodoira R, Torres M, Pierantozzi P, Taticchi A, Servili M, Maestri D. 2015. Oil biogenesis and antioxidant compounds from “Arauco” olive (Olea europaea L.) cultivar during fruit development and ripening: Oil biogenesis from “Arauco” olive cultivar. Eur. J. Lipid Sci. Technol. 117, 377-388. https://doi.org/10.1002/ejlt.201400234 ) and higher than that from La Rioja province (166 mg·kg^-1) (28ºS, Ceci and Carelli, 2010Ceci LN, Carelli AA. 2010. Relation Between Oxidative Stability and Composition in Argentinian Olive Oils. J. Am. Oil Chem. Soc. 87, 1189-1197. https://doi.org/10.1007/s11746-010-1598-6 ). These results reveal a possible latitudinal gradient where phenols increase with increasing latitude in the Southern hemisphere in relation to a decrease in temperature (Mousa et al., 1996Mousa YM, Gerasopoulos D, Metzidakis I, Kiritsakis A. 1996. Effect of Altitude on Fruit and Oil Quality Characteristics of `Mastoides’ Olives. J. Sci. Food Agric. 71, 345-350. https://doi.org/10.1002/(SICI)1097-0010(199607)71:3<345::AID-JSFA590>3.0.CO;2-T ), which is greater than the thermal gradient observed among the zones studied.

The fatty acid composition of olive oils from the three main growing zones of Mendoza during the two seasons is shown in Table 3. All samples were within IOC legal limits. On average across zones and seasons, the ‘Arauco’ variety presented the following fatty acid profile: 16.29±2.25% palmitic acid, 1.59±0.54% palmitoleic acid, 0.04±0.01% margaric acid, 0.09±0.01% margaroleic acid, 2.42±0.22% stearic acid, 65.47±6.15% oleic acid, 12.13±3.74% linoleic acid, 0.49±0.11% arachidonic acid, 0.98±0.18% linolenic acid, 0.3±0.05% eicosenoic acid, and 0.18±0.02% behenic acid. The contents in palmitic, palmitoleic, margaric, stearic, oleic and linoleic acids were significantly different among zones. Conversely, the contents in margaroleic acid, arachidonic acid, linolenic acid, eicosenoic acid and behenic acid were not significantly different among zones.

The oil from Zone 2 presented the highest contents in palmitic, palmitoleic, margaric and linoleic acids, and the lowest oleic and stearic acids contents. (the latter not significantly different from Zone 1) with respect to oils from Zones 1 and 3. Maximum temperature during fruit development would partially explain the lower oleic acid content in Zone 2 and a reverse trend for palmitic and linoleic acids. García-Inza et al. (2014)García-Inza GP, Castro DN, Hall AJ, Rousseaux MC. 2014. Responses to temperature of fruit dry weight, oil concentration, and oil fatty acid composition in olive (Olea europaea L. var. ‘Arauco’). Eur. J. Agron. 54, 107-115. https://doi.org/10.1016/j.eja.2013.12.005 carried out a manipulation experiment over the fruit-growth period in which fruits were exposed to increments of 5 ºC and 10 ºC above ambient temperature. The authors observed that the contents in palmitic and linoleic acids in oil increased linearly with fruit temperature, while oleic acid content decreased. In addition, greater fruit maturity from Zone 2 could contribute to explaining the fatty acid pattern. Gómez del Campo and García (2012)Gómez-del-Campo M, García JM. 2012. Canopy Fruit Location Can Affect Olive Oil Quality in ‘Arbequina’ Hedgerow Orchards. J. Am. Oil Chem. Soc. 89, 123-133. https://doi.org/10.1007/s11746-011-1900-2 found that more mature fruits produced oil with lower oleic acid content; while the polyunsaturated fatty acid percentage was greater than in less mature fruits.

In the ‘Arauco’ oils studied, the oleic acid content was higher than 57%, above the IOC limit (55%). The oleic acid content in oil from ‘Arauco’ olives grown in Mendoza was similar to the oleic acid range observed in ‘Arauco’ oil from San Juan (33ºS, Bodoira et al., 2015Bodoira R, Torres M, Pierantozzi P, Taticchi A, Servili M, Maestri D. 2015. Oil biogenesis and antioxidant compounds from “Arauco” olive (Olea europaea L.) cultivar during fruit development and ripening: Oil biogenesis from “Arauco” olive cultivar. Eur. J. Lipid Sci. Technol. 117, 377-388. https://doi.org/10.1002/ejlt.201400234 ) and higher than the 53.7-54.3% obtained in La Rioja and Catamarca provinces (28ºS, Ceci and Carelli, 2007Ceci LN, Carelli AA. 2007. Characterization of Monovarietal Argentinian Olive Oils from New Productive Zones. J. Am. Oil Chem. Soc. 84, 1125-1136. https://doi.org/10.1007/s11746-007-1140-7 ; Rondanini et al., 2011Rondanini DP, Castro DN, Searles PS, Rousseaux MC. 2011. Fatty acid profiles of varietal virgin olive oils (Olea europaea L.) from mature orchards in warm arid valleys of Northwestern Argentina (La Rioja). Grasas Aceites 62, 399-409. https://doi.org/10.3989/gya.125110 ). For the same variety, pooled results reveal a possible latitudinal gradient where oleic acid content increases with increasing latitude in the Southern hemisphere in relation to a decrease in temperature, as previously described by Rondanini et al. (2011)Rondanini DP, Castro DN, Searles PS, Rousseaux MC. 2011. Fatty acid profiles of varietal virgin olive oils (Olea europaea L.) from mature orchards in warm arid valleys of Northwestern Argentina (La Rioja). Grasas Aceites 62, 399-409. https://doi.org/10.3989/gya.125110 .

The main monounsaturated fatty acid was oleic, which ranged from 59.1 to 71.7%; the main saturated fatty acid was palmitic, ranging from 14.6 to 18.1%; and the main polyunsaturated fatty acid was linoleic, which ranged from 8.0 to 16.2%. Together, they accounted for 93.4 to 94.3% of the fatty acid content. Monounsaturated fatty acid/polyunsaturated fatty acid (MUFA/PUFA), oleic/linoleic acid, and unsaturated/saturated fatty acid (UFA/SFA) ratios were calculated (Table 3). On average, the ‘Arauco’ variety showed the following values for the three zones and two seasons: MUFA/PUFA = 5.6, oleic/linoleic = 5.9, and UFA/SFA = 4.2. These values were lower than those around 4.4 which were observed for the same ratios estimated in previous studies on the Arbequina olive variety in Junín (Zone 1) Mendoza, (Lémole et al., 2018Lémole G, Weibel AM, Trentacoste ER. 2018. Effect of shading in different periods from flowering to maturity on the fatty acid and phenolic composition of olive oil (cv. Arbequina). Sci. Hortic. 240, 162-169. https://doi.org/10.1016/j.scienta.2018.06.005 ). In addition, Zone 3 presented the highest MUFA/PUFA and oleic/linoleic ratios, which were significantly different from the other zones; while the UFA/SFA ratio was similar in both Zones 1 and 3 and significantly higher than in Zone 2.

In addition, linear regressions were studied among all the oil traits analyzed. Some interesting relations were identified between oxidative stability (OS) and fatty acids, or their ratios. Thus, a positive and closer relationship was found between the OS and MUFA/PUFA ratio (R²=0.96), than between the OS and UFA/SFA ratio (R²=0.66). Bhatnagar et al. (2009)Bhatnagar AS, Prasanth Kumar PK, Hemavathy J, Gopala Krishna AG. 2009. Fatty Acid Composition, Oxidative Stability, and Radical Scavenging Activity of Vegetable Oil Blends with Coconut Oil. J. Am. Oil Chem. Soc. 86, 991-999. https://doi.org/10.1007/s11746-009-1435-y determined that OS is negatively related to PUFA. Our results showed that the induction time to oxidation in ‘Arauco’ oils was more related to monounsaturated fatty acids than to total phenolic and total flavonoid compounds. This could be explained by the fact that all the samples showed similarly high phenolic contents. Given that the highest content in monounsaturated and polyunsaturated acids in olive oils is due to oleic and linoleic acids, respectively, OS was positively related to the oleic/linoleic ratio (R²=0.96). Nevertheless, not every monounsaturated acid plays such an important role given that palmitoleic acid (C16:1) displayed negative relationships with OS (R²=0.79). Other negative relationships were found between OS and linoleic and palmitic acids (R²=0.90 and R²=0.72, respectively). Martínez et al. (2014)Martínez M, Fuentes M, Franco N, Sánchez J, de Miguel C. 2014. Fatty Acid Profiles of Virgin Olive Oils from the Five Olive-Growing Zones of Extremadura (Spain). J. Am. Oil Chem. Soc. 91, 1921-1929. https://doi.org/10.1007/s11746-014-2528-9 determined that linoleic acid is highly susceptible to oxidation due to its key role in the lipoxygenase pathway as the precursor of many volatile compounds. In addition, stearic acid (C18:0) showed a positive relationship with OS (R²=0.93). Bhatnagar et al. (2009)Bhatnagar AS, Prasanth Kumar PK, Hemavathy J, Gopala Krishna AG. 2009. Fatty Acid Composition, Oxidative Stability, and Radical Scavenging Activity of Vegetable Oil Blends with Coconut Oil. J. Am. Oil Chem. Soc. 86, 991-999. https://doi.org/10.1007/s11746-009-1435-y evaluated blends with coconut oil, and observed that an increase in saturated fatty acids improved their oxidative stability.