3.1. Fatty Acids
Optimal harvesting time is important due to the tendency for the production of high yield oil and high-quality extra virgin olive oil. But, these requirements are not easy to meet.
Presented results followed the previous research of the physico-chemical characteristics of oil extracted from the pulp of the olive Buza during ripening in the year 1998 in the same orchard [9
]. The climatic conditions during olive harvesting in the crop years 1998 and 1999 were different, and therefore also the changed acidity and peroxide value (see Table 1
). In the year 1999, a higher average temperature and significantly lower rainfall than in the year 1998 were noted, when a higher polyunsaturated fatty acids (PUFAs) in all olive picking dates were reported. However, the level of monounsaturated fatty acids (MUFAs) and oleic acid in olive oil did not change significantly during ripening (see Table 2
), which is in accordance with earlier results [9
We found that olive oil from Buza has a significantly higher level of oleic acid than Drobnica, which corresponds with results from the multi-year data ranging from 1992 to 2009 in Istria where the level of oleic acid in the Buza variety was 74% [20
] or near 74% [11
]. In Drobnica, cultivated in Dalmatia, oleic acid was found to be lower (near or under 70%) [7
Changes in the fatty acid composition of the extracted oil have been reported to be associated with olive fruit maturation [27
]. Stearic acid (C18:0) was minimally changed in Drobnica (from 2.39 ± 0.10 to 1.98 ± 0.26), while in Buza it was reduced (from 2.12 ± 0.16 to 1.67 ± 0.39) with maturation. The ripening process changed significantly the myristoleic, linoleic, linolenic, eicosenoic and lignoceric acids in oil from Buza, and oleic and linolenic acid in oil from Drobnica. More differences between Buza and Drobnica oils were found in November, as a result of its variability and also due to differences in the ripening process. Palmitic acid content decreased during ripening in Buza oils, from 13.44 ± 0.25 to 12.07 ± 1.58, while it increased in Drobnica oils: from 15.11 ± 1.17 to 18.48 ± 3.37. In addition, linoleic acid and oleic acid showed the opposite trend: almost unchanged linoleic acid in Drobnica oil and a significant increase of the same in Buza in November. The same trend was found in PUFAs.
The ratio of MUFAs/PUFAs was attributed the nutritional properties and oxidative stability of olive oils. This ratio was higher in Buza in September-October (9.26 ± 0.07 to 9.23 ± 1.20), however, in November it notably dropped down (to 6.85 ± 0.72) under the value of Drobnica (7.38 ± 0.58). An increase in PUFAs negatively affects the olive oil stability observed in November in Buza. On the other hand, this trend is not attributed to Drobnica.
Ipek et al. [29
] reported the association between fatty acid traits and simple sequence repeat (SSR) markers. Significant associations were found between five SSR markers and the stearic, oleic, linoleic, and linolenic acids of olive oil. Also, very high associations (P
< 0.001) were indicated between ssrOeUA-DCA14 and stearic acid; and between GAPU71B and oleic acid, so that these markers could be used for marker-assisted selection of olives.
3.2. Unsaponifiable Compounds
Phytosterols, alkanols, squalene, tocopherols are the main groups of components analysed in the unsaponifiable fraction. The sterols and alcohols profile is used for characterisation of virgin olive oils and detection of the adulteration of olive oil with similar vegetable oils or virgin olive oil with olive-pomace oil [30
The amount of unsaponifiable matter in olive oil varies from 1% to 5% in ripe olives [32
]. The higher content of unsaponifiables is probably associated with olive processing as well as the subsequent loss of their physiological activities and quality. As shown in Table 3
, the level of unsaponifiable matter in laboratory extracted oil in Drobnica ranged from 1.90 ± 0.09% to 2.58 ± 0.11%, and from 1.47 ± 0.08% to 2.37 ± 0.11% in Buza. Unsaponifiable components can provide information about the adulteration of vegetable oils as well as their variety and even the geographical origin, as has been analysed in some publications [33
Total sterols significantly changed during ripening in both cultivars studied. Their content decreased from September to November in Drobnica from 2388.36 ± 444.42 mg/kg to 968.68 ± 31.71 mg/kg, and in Buza from 1354.47 ± 90.58 mg/kg to 850.19 ± 94.23 mg/kg. Although the level of total sterols varied between cultivars, only β-sitosterol in Buza changed significantly during ripening. As a predominant phytosterol in both oils, β-sitosterol ranged from 95.24 ± 2.73% to 98.27 ± 1.18% in Drobnica and from 93.95 ± 0.30% to 98.09 ± 0.25% in Buza. Our results of the total sterols, β-sitosterol and squalene showed the opposite trend than that described by Fernández-Cuesta et al. [39
]. Squalene is the major olive oil hydrocarbon accounting for more than 90% of the hydrocarbon fraction [33
]. In the oils from Buza and Drobnica, notable differences were found in the squalene content as shown in Table 3
. Squalene was increased in Drobnica from September (6927.46 ± 1878.75 mg/kg) to October (9696.52 ± 299.22 mg/kg), after which it decreased in November (5078 ± 1598.01 mg/kg). Buza showed a rise in squalene content during all periods observed (5383.73 ± 576.25 mg/kg–7696.5 ± 503.15 mg/kg). Fernández-Cuesta et al. [39
] reported that the level of squalene increased during the period of maturation (September–November) in the cultivars Picual and Arbequina grown in Cordoba (Spain), which significantly increased from September (4102 mg/kg) to November (4673 mg/kg). However, they found no difference in the fruit flesh between November and December. Squalene also had no effect on the oil oxidative stability in the case of Drobnica and Lastovka [7
]. Beltrán et al. [40
] studied 28 olive cultivars from the World Olive Germplasm Collection of Instituto de Investigación y Formación Agraria, Pesquera (IFAPA) in Cordoba, where from 110 to 839 mg/100 g of squalene was found in virgin olive oils. The difference in squalene content was explained by the genetic variability. It is worth noticing that the sterol content of the oil varies even within the fruits or nuts collected from the same tree [41
Tocopherols and polar phenolic compounds are responsible for the oxidative stability of the olive oil. In general, tocopherols decreased during the ripening process [42
]. We found that the concentration of α-tocopherol was altered significantly only in Buza. A decrease of α-tocopherol was also observed in both cultivars during ripening (in Buza it ranged from 141.74 ± 7.70 to 64.97 ± 15.42 mg/kg, and in Drobnica from 154.64 ± 54.66 to 77.55 ± 5.60 mg/kg). The concentration of α-tocopherol in virgin olive oils depends on many factors (cultivars, geographic area, oil processing, irrigation, etc.) including genetic factors [43
]. The level of rainfall has an effect on α-tocopherol content, thus, the drier crop years indicated a greater tocopherol concentration. However, this effect was dependent upon the cultivar [43
Despite the lower contribution of α-tocopherol in comparison to phenolic compounds in maintaining oxidative stability, α-tocopherol is still important, especially as oils ageing progresses [44
n-alkanes from nC22 to nC33 is characteristic for olive oil which makes it different from other vegetable oils. The profiles of aliphatic alcohols in olive oil also depend on the origin and fruit variety, so it is linked to the authenticity of the extra virgin olive oil. The Greek extra virgin olive oils were typically characterised by high levels of nC23 and nC25. However, the carbon number profile of Italian and Spanish oils was not characterised by a single profile [34
The level of total alkanols was significantly different in Buza during ripening. As shown in Table 3
, in both cultivars significant differences in nC23, nC24, nC26 and nC28 were found in all examined ripening stages, while nC22 was changed significantly only in Buza. Hexacosanol and tetracosanol were predominant aliphatic alcohols in both cultivars: hexacosanol was the highest in September (67.97 ± 1.44% in Drobnica and 63.14 ± 5.77% in Buza) and October (48.49 ± 1.10% in Drobnica and 64.18 ± 2.25% in Buza) and tetracosanol was the highest in November (48.25 ± 4.34% in Drobnica and 41.42 ± 0.40% in Buza). It is observed that Drobnica has an adequate trend of declining hexacosanol with ripening different from Buza. In addition, Drobnica had a higher amount of total alkanols in relation to Buza.
Koprivnjak et al. [18
] have used fatty acids and hydrocarbons to compare the Croatian varieties Bianchera, Carbonazza and Busa with the Italian Leccino variety. They have concluded that low values of aliphatic hydrocarbons nC24 characterise autochthonous Croatian varieties of cultivar, while higher values of nC25 and nC35 hydrocarbons characterise the Italian Leccino. We found lower values of nC24 at early stages of olive ripening. Our results also suggested that changes in aliphatic alcohols are bigger in Buza than in the Drobnica cultivar during ripening.
Based on these results, it is also concluded that sterols and alcohols rather than fatty acids are both very important compounds for the chemical authentication of virgin olive oil varieties [45
In many cultivars, hexacosanol was found as the most predominant of all fatty alcohols. Giuffrè [46
] found hexacosanol to be the major alkanol in olive oil in three autochthonous cultivars (Cassanese, Ottobratica, Sinopolese) and seven allochthonous cultivars (Coratina, Itrana, Leccino, Nocellara Messinese, Nociara, Pendolino and Picholine) with regard to olive oils from Southwest Calabria. Next, hexacosanol has found to be the most common aliphatic alcohol in monovarietal virgin olive oil from Tunisian cultivars (Jdallou, Chemlali Sfax, Swabâa, El Hor, and Oueslati) [47
] in the olive oil of cultivars grown in Central Italy, including the Leccino cultivar [48
]; in Coratina from the Apulia Region in the Southeast of Italy; in Koroneki from Crete [49
]; in Arbequina, Picual, and Manzanilla [50
]; and in pomace olive oil [51
López-López et al. [32
] observed the significant effects of processing on unsaponifiable matter (β-sitosterol, Δ5-avenasterol, total sterols, docosanol, tetracosanol) in the Manzanilla and Hojiblanca cultivars. In addition, Ranalli et al. [52
] reported on the greater concentration of fatty alcohols in oil from cultivars grown in central Italy (500 mg/kg) as compared with those grown in southwest Calabria. On the other hand, Apparicio and Luna [49
] noted that in the olive oil of Coratina grown in Apulia, the level of total aliphatic alcohols, 63 mg/kg (in two-phase extraction) and 58 mg/kg (in three phase extraction), is less than those grown in southwest Calabria. Our results have shown that the highest amount of aliphatic alcohols in oil is obtained from Drobnica (236.06 mg/kg), whereas it was determined to be the lowest in October in Buza (42.81 mg/kg). Angerosa et al. [53
] studied the influence of rainfall on the synthesis of oil on the Italian cultivar Frantoio of varying geographic origin. By applying statistical procedures, the authors have found that the amounts of sterols, squalene, oleic acid and some triacylglycerols were explained by the autumn temperatures, the relative humidity of the summer months and the rainfall over the whole year.
3.3. Phenolic Compounds
Phenolic compounds, α-tocopherol and β-carotene are reported as the main groups of compounds with antioxidant properties that correlated to the oxidative stability of virgin olive oils [54
The results presented in Table 4
show a significant change in total phenols and their antioxidant activity during ripening in both cultivars. Drobnica had a higher content of total phenols during the examined ripening period (437.67 ± 10.50 mg/kg, 316.34 ± 4.70 mg/kg and 273.26 ± 7.14 mg/kg, respectively) as compared with Buza (374.98 ± 10.49 mg/kg, 289.96 ± 9.79 mg/kg and 250.09 ± 5.52 mg/kg, respectively). A high level of phenolics is also connected with the high antioxidant activity of Drobnica, which ranged from 71.18 ± 0.98% to 57.31 ± 0.96% and in Buza from 71.48 ± 1.16% to 61.14 ± 0.48%. The concentration of hydroxytyrosol (HYTY) and tyrosol (TY) was significantly changed in both cultivars during ripening, while the concentration of luteolin (Lut) and apigenin (Apig) was significantly altered only in oil from Buza. The higher level of HYTY was found in Buza in September, while in November it was determined in Drobnica. The level of oleuropein (OL) was higher in Drobnica in September and October, however, in November it was almost the same in both cultivars.
Bilušić et al. [7
] reported that monovarietal extra virgin olive oil (EVOO) from Drobnica contained the highest amount of total phenols and major secoiridoid derivatives (oleocanthal, oleacein, oleuropein aglycon, and ligstroside aglycon) compared to other studied monovarietal EVOOs. This also affected the highest antioxidant activity of Drobnica oil and it’s very long (23 h) oxidative stability as determined by the Rancimat method. Similarly, Dalmatian monovarietal olive oils were analysed in relation to the harvest period (Buhavica, Drobnica, Lastovka and Oblica) [8
]. It was observed that the late harvest contained an extremely high concentration of oleocanthal + oleacein (966 mg/kg) in EVOO from Drobnica, and that oils from Drobnica and Lastovka (Korčula Island, South Dalmatia, position 42°57′31.62′′ N) had the longest oxidative stability (20.95 and 18.65 h, respectively). This study showed that the level of phenolics depends on the cultivar, however, the authors did not determine a significant change of phenolic secoiridoids in oil from Drobnica during the harvest period.
Lukić et al. [56
] studied the concentrations of phenols and volatiles in virgin olive oil from a late-ripening olive cultivar Istarska bjelica from October to December in the crop season 2015 grown in an unirrigated olive orchard located at the position 45°13′32.99′′ N, 13°35′38′′ E (Poreč, Croatia). They found that TY, HYTY increased during ripening, while other important phenolic compounds were decreased such as a dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA or oleacein), a dialdehydic form of decarboxymethyl elenolic acid linked to tyrosol (p-HPEA-EDA or oleocanthal), and oleuropein and ligstroside aglycones. Total secoiridoids and total phenols also were decreased during ripening. In addition, the authors found significant changes in the interaction between malaxation temperature, malaxation time and ripening both in relation to simple and complex phenols. Similarly, these authors studied the Dalmatian cultivar Oblica from September to November 2015 grown in Kaštel Stari at the position 43°32′59.99′′ N, 16°20′59.99′′ E [14
]. The above-mentioned results have confirmed the existence of a geographic and genotype difference between these two Croatian cultivars. This was especially observed in the opposite trend in p-HPEA-EDA, which is increased during ripening in Oblica. Malaxation temperature, malaxation time and ripening also affected phenolics composition in the oils.
According to the results based on the ripening process from November to January, Bengana et al. [57
] suggested that a maturation index of 2.4, found in November, was the most appropriate for the harvesting of olives in order to obtain the high-quality EVOOs from the Chemlal cultivar grown in an orchard located in the Haizar area in north-central Algeria. The highest HYTY, TY, phenolic alcohols and secoiridoids were noted in November. However, significant variation in olive-oil yield, carotenoids, and tocopherol content was not observed.
The secoiridoid derivatives of HYTY and TY were the major phenolic compounds in oils from Ayvalık, Domat and Gemlik olive varieties collected at different ripening periods from August to December in Edremit (Balıkesir) in Turkey in 2006 [58
]. In all examined oils the greatest concentration of HYTY was found in October (0.80 mg/kg, 1.15 mg/kg, 0.63 mg/kg, respectively). The level of luteolin in oils increased with the ripening of Ayvalık, Domat and Gemlik olives, ranging between 0.27–2.28, 0.00–1.42, 0.28–1.74 mg/kg, respectively. On the other hand, the highest TY content was established in Gemlik oil in August. The level and profile of other determined phenolic compounds depended on varietal differences during ripening.
Gomez-Rico et al. [59
] studied the degree of ripening of the olive fruit on the biophenolic and volatile profiles of six different Spanish varieties (Arbequina, Cornicabra, Morisca, Picolimón, Picudo and Picual) and their corresponding virgin olive oils. They found that the ratio between biophenol content in the olive fruit and its resulting olive oil varied significantly for each of the cultivars studied, especially in Picudo and Picolimón (ranging from 2.3 to 28, respectively). Besides the statistical difference in oleuropein content in all varieties studied, demethyloleuropein was only found in the Arbequina variety during the ripening process. They also established a different concentration of HYTY and TY dependent upon variety: 2.9 and 2.1 mg/100 g HYTY in Arbequina, 2.8 and 2.1 mg/100 g in Cornicabra, 0.4 and 0.6 mg/100 g in Marisca, 0.8 and 0.6 mg/100 g in Picolimon, 1.8 and 2.2 mg/100 g in Picual olive varieties, respectively; and TY content 2.4 and 2.1 mg/100 g in Arbequina, 1.5 and 1.2 mg/100 g in Cornicabra, 5.5 and 6.4 mg/100 g in Morisca, 4.2 and 3.9 mg/100 g in Picolimon and 3.3 and 3.3 mg/100 g in unripe and ripe Picual olive varieties, respectively.
High content of lipophilic (>300 mg/kg) and hydrophilic phenols (>600 mg/kg) for Galega Vulgar and Cobrançosa olive oils corresponded with early ripening stages were found Peres et al. [60
]. Total phenols were decreased when the ripening index ranged from 2.5 to 3.5. The dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA or oleacen) was the major phenolic compound identified in both oils, and the concentration of hydroxytyrosol and tyrosol was very low due to the high levels of 3,4-DHPEA-EDA and p- HPEA-EDA as their esterified derivatives.
The mentioned results confirm that changes in fruit colour during development and ripening olive fruits are crucial to the antioxidant capacity of oils. During harvesting, the content of TY and HYTY undergoes change. In early harvest, they were higher, and in late harvesting, HYTY levels decreased by 50.40% [61
]. HYTY is one of the phenols with the highest antioxidant effect in olive oil [62
], which results in the higher oxidative stability of the oil. This is in accord with the results obtained by Martinez Nieto et al. [63
], who reported that tyrosol and hydroxytyrosol concentrations decreased with increasing olive ripeness in the Picual and Arbequina varieties.
A high correlation between the antioxidant capacity of the chloroplastic pigments and total phenolic compounds in the Arbequina variety reported by Fernandez-Orozco et al. [64
]. The antioxidant capacity was increased with a higher total chlorophyll and xanthophyll content, while low correlation was found with β-carotene content. That confirms the fact that the early ripening stage is rich in antioxidants.
All plants must obtain a number of macro and microelements from their environment to ensure successful growth and development of both vegetative and reproductive tissues. Nitrogen, sulphur, and phosphorus serve as constituents of proteins and nucleic acids, while magnesium and the micronutrients (except chlorine) may function as constituents of organic structures, predominantly of enzyme molecules, where they are involved in the catalytic functions of the enzymes [65
Our hypothesis on the different content of minerals in olive oils during ripening was based on (i) variable soil mineral content in orchards, and (ii) different physiological mineral requirements in fruit during the ripening process. We assumed that the mineral profile in the oils correlates with the mineral content in the olive fruit. However, in the literature we did not find any report about the variation of mineral content in oils obtained from olive fruit during ripening. To be able to compare the mineral levels in oils, it is important to emphasise that the olives were not treated with fertilisers or metal-containing pesticides, and wherein the method of oil preparation could not influence the increase of trace elements.
In some cases, traces of Fe and Cu in virgin olive oil may originate from the soil and fertilisers, agrochemicals or from contamination by processing equipment and storage containers.
presents the trace elements in the olive oil extracted from Drobnica and Busa olives during ripening. In both orchards, the soil is rich in Ca and Fe. The orchard where Drobnica grew was near the sea, which reflected in a greater Na concentration. On the other hand, the level of Ca was higher in Buza that was located six kilometres from the sea. During the ripening, significant differences in both varieties were found in the content of Na, Mg, Fe, Zn, Cu, Al and P, as well as K in Drobnica and Ca in Busa. In both cultivars were found the lowest values of Na, K, Mg, Cu, Al and P in October, after that, in November the level of Ca, Zn, Cu and Ni was increased in Drobnica and the level of Fe was decreased in Buza.
Maintenance of minerals by means of mineral nutrition is a prerequisite for providing co-factors for the many enzymes of the phenylpropanoid and flavonoid pathway. Mg and Mn ions ensure the functioning of phenylalanine ammonia lyase (PAL), of CoA-ligases, and of methyltransferases [66
]. This would mean that the Mg level can affect the level of phenolic compounds, especially in the early stage of ripening as presented in Table 4
and Table 5
. Three forms of the superoxide dismutase (SOD) exist in plants as enzyme antioxidants, classified by their active site of the metal ion as Cu/Zn, Mn, and Fe forms. Thus, the need for these metals exists during ripening. Besides its inclusion in the Mn-containing superoxide dismutase (SOD), Mn plays an important role in the water-splitting enzyme of photosynthesis associated with photosystem II [67
The water splitting site consists of a cluster of four Mn ions and a Ca ion surrounded by amino acid side chains, of which seven provide ligands to the metals [68
]. Photosynthesis is the most sensitive process for manganese deficiency in higher plants. In Mn deficient leaves not only the chlorophyll content is lower, but also the content of typical thylakolid membrane constituents (glycolipids and polyunsaturated fatty acids) can be depressed up to 50%, which can be attributed to the role of Mn in the biosynthesis of fatty acids and carotenoids and related compounds [65
Although we did not find data reports about the level of minerals during ripening in the olive fruit, there are such reports that are related to other fruits.
The effect of increasing avocado fruit maturity on mineral composition and phenolic content resulted in an influence on postharvest fruit quality and the ripening physiology of fruit. Reduced calcium and magnesium concentrations were found in fruits with increased maturity [69
]. An increase in the concentrations of calcium, magnesium and phosphorus with the ripening process of the asparagus was found. The green ripening state has a greater concentration of calcium, magnesium and phosphorus. The changes from the white asparagus into a green ripening state affect a decrease in the content of sodium, while no significant differences were established for potassium [70
Much evidence was also given as to the level of the trace elements in some commercial olive oils in relation to olive oil processing, geographic origin, harvest year and olive cultivars [71
]. In a total of 50 samples of monovarietal EVVO analysed from two Protected Designation of Origin (PDO) Spanish provinces, Granada and Jaén, there were found to be significant differences between Cu, Cr, Fe and Ni content according to the geographical origin of the oils but not for Mn content [72
]. The authors suggest that the trace element content of extra virgin olive oils based on their geographical origin can be used for their local characterisation.