2.2. Free Acidity
Oil acidity is a simple and effective parameter to evaluate and classify a commercial-grade olive oil [23
]. The extraction of olive oil from fresh undamaged fruits carried out following a correct crushing procedure gives oils with very low acidity [23
]. However, during extraction and storage the olive oil can be altered by the release of free fatty acids as a consequence of the hydrolysis of triglycerides, thus increasing the free acidity.
The acidity values of Neb Jmel and Oueslati olive oils, extracted without (control) and with the addition of 3% olive leaves, are reported in Table 1
. The Neb Jmel oils showed very low values (0.6%). The addition of leaves during oil extraction did not affect the free acidity in this variety. Regarding Oueslati, acidity of oils (1%) was higher in comparison with the Neb Jmel ones. However, the addition of leaves influenced Oueslati oil quality, decreasing free acidity to 0.6%. Our results agree with those reported by Ben Mansour et al. [24
] for the Neb Jmel variety, whereas the free acidity of Oueslati oils was higher in comparison with the findings of Ouni et al. [25
] on the same variety. This behaviour could be related to the use of olives at an advanced stage of maturation. Indeed, a late harvest of olives may alter oil acidity by increasing the lipolytic enzyme activities [26
]. However, in our case the addition of olive leaves was responsible for the decrease of free acidity likely due to the presence of antioxidant compounds. Consequently, fortified Oueslati oils could still be classified as extra virgin olive oils as the free acidity value was lower than 0.8%.
2.3. Peroxide Value
Peroxides are intermediate products of oil oxidation which originate a complex mixture of volatile compounds such as aldehydes, ketones, hydrocarbons, alcohols and esters. These compounds are responsible for the alteration of the organoleptic characteristics [27
], dramatically reducing oil shelf-life as well as consumer acceptance. Furthermore, also light and high temperatures are well-known factors generally promoting peroxide formation [28
shows that the peroxide value of Neb Jmel oils was well below the established limit (<20 milliequivalents (meq) O2
/kg) for all the categories of olive oil [22
]. In contrast, a high peroxide value was observed in the Oueslati control oil (34 meq O2
/kg), so that it could no longer be classified as an extra virgin olive oil. However, the addition of leaves during oil extraction and processing prevented the oxidation and the formation of peroxides, reducing the peroxide value to half. Contrary to our results, Malheiro et al. [17
] reported that leaf addition increased the peroxide value. Such discrepancy could be explained with differences in the relative presence of additional antioxidants and of leaf residues. These constituents, through gas exchanges occurring during the respiration process, may have increased the availability of oxygen, thus inducing peroxidation.
2.4. Chlorophylls and Carotenoids
Chlorophylls and carotenoids play important roles in olive oils. They interfere with the oxidative stability, acting as antioxidants in the dark or as prooxidants when exposed to light [29
]. Furthermore, these compounds are responsible for the yellow-green pigmentation of olive oils, increasing consumer acceptability. Chlorophyll and carotenoid concentrations of olive oils and leaves are reported in Table 1
and Table 2
The Neb Jmel oil showed a higher chlorophyll value (Table 1
) in comparison with Oueslati. However, Oueslati leaves (Table 2
) displayed the highest chlorophyll (829.29 μg/g) and carotenoid (44.3 μg/g) concentrations, highlighting the role that the addition of chlorophyll could have had in the oxidative stability of the olive oil. This can further explain the difference with the results reported by Malheiro et al. [17
] concerning the peroxide value, as in their study olive leaf addition did not affect the chlorophyll content.
In the present work, the addition of leaves (3%) to Neb Jmel and Oueslati oils enhanced chlorophyll and carotenoid concentrations. In fact, chlorophyll reached the same level in both fortified oils, even if Oueslati oil was the one that showed the highest carotenoid concentration. Therefore, significant effects of leaf addition on oil pigments were observed in both varieties (Table 1
and Table 2
). The addition of leaves also turned the olive oils greener, this visual observation being clear in both oils, and was very likely associated with the increased pigment concentration. The increase in chlorophyll concentrations makes the fortified oils interesting from a nutritional point of view due to the antioxidant activity of chlorophyll and its potential to exert chemopreventive actions against carcinogens [30
2.5. Total Phenolics
Phenolics are important components for olive oil quality and organoleptic characteristics. Moreover, they are very effective antioxidants playing an important role in human diet and health [3
]. Current evidences strongly support the contribution of phenols to the prevention of cancer, cardiovascular and neurodegenerative diseases. The shelf-life of an oil is also correlated with its natural antioxidant amount [16
]. Indeed, phenolics delay the oxidative degradation process, thus extending the shelf-life of the product [3
shows that total phenolic concentration of the Neb Jmel oil (736 mg GA eq/kg oil) was significantly higher than that of Oueslati (528 mg GA eq/kg oil). In the literature, it was reported that the total phenolic concentration of Neb Jmel olive oils varies from 562 to 1167 mg GA eq/kg oil [24
], whereas for Oueslati it changes from 100 to 859 mg GA eq/kg oil [25
]. The effect of the geographic location on phenols can be evidenced from the different behaviour showed by each variety [24
]. Indeed, Neb Jmel oil (from the north of Tunisia) showed a higher phenol concentration than Oueslati (from central Tunisia). The present data confirm previous findings on the effect of the variety on phenol concentration in oils [24
Oueslati leaves showed a higher phenolic concentration than Neb Jmel ones (Table 2
). It is worth noting that leaf addition, increasing total phenolic concentration by 44 and 10% in Oueslati and Neb Jmel oils, respectively, (Figure 1
), determined the same mean value in the fortified products. Similar findings were also confirmed by other studies [32
2.6. Total Flavonoids
Flavonoids are plant secondary metabolites with different phenolic structures. These compounds are used mostly to generate pigments which play an important role in the colours of plants. During the past decade, many studies have reported their beneficial effects on human health [34
]. Indeed, flavonoids display important anti-inflammatory, antiallergic and anticancer activities as well as antiviral properties [34
]. In this study, total flavonoid concentration was determined in both the oils and leaves of the two Tunisian olive cultivars Neb Jmel and Oueslati (Table 2
, Figure 1
). The results point out that Oueslati leaves showed a concentration of total flavonoids two-fold higher than Neb Jmel. However, in Oueslati oil the total flavonoid content was about half than that found in Neb Jmel one (Figure 1
). The effect of leaf addition during oil extraction on these compounds was remarkable. Our results showed an increase in total flavonoids by 22% in Neb Jmel oils and by 160% in Oueslati, thus determining a not significant difference between the two fortified oils. This was likely due to the highest level of total flavonoids of Oueslati leaves. According to the present research, the findings of Ebrahimi et al. [36
] reported values ranging from 156 to 361 mg rutin eq/kg for refined and crude olive oils, respectively.
2.8. Phenolic Compounds
The analysis of the phenolic profile of olive leaf extracts and oils from the two varieties is reported in Table 3
and Table 4
, respectively. Thirteen phenolic compounds, among which phenolic acids, phenolic alcohols and secoiridoids, were identified and quantified. As regards olive leaf extracts, oleuropein derivatives were the most represented, followed by phenolic acids and phenolic alcohols (Table 3
With minor changes compared to leaves, oleuropein derivatives were still the most abundant compounds in oils, followed by phenolic alcohols and phenolic acids (Table 4
). In particular, Oueslati leaves showed a three-fold higher content in oleuropein derivatives than Neb Jmel ones, whereas Neb Jmel oil exhibited the highest concentration of these compounds. With leaf addition, oleuropein derivative concentration increased by 9 and 48% in Neb Jmel and Oueslati oils, respectively, confirming that olive leaves are a source of oleuropein derivatives [39
]. Consistent with other reports, we found that oleuropein derivative amounts changed depending on the variety.
During the past few years, the biological activities of olive oil phenolics, namely oleuropein derivatives, have been thoroughly investigated. Several studies have attempted to elucidate the performance of oleuropein derivatives as antioxidant compounds. As the excessive presence of reactive oxygen species has been suggested to participate in the aetiology of several diseases [40
], the focus on powerful antioxidants able to counteract the free-radical attack has become increasingly important. The antioxidant actions of oleuropein have been mostly assigned to its free-radical scavenging activity. Considering all together, the addition of olive leaves during oil extraction process suggests that it could be a means for improving oil quality.
Although olive fruits are rich in secoiridoids, hydroxytyrosol and tyrosol represent the two most important phenolic alcohols of both olive leaves [41
] and oils [42
]. Leaf composition of the Oueslati variety was characterised by a four-fold higher amount of hydroxytyrosol and tyrosol compared to Neb Jmel (Table 3
). However, the oils, and likely the fruits, were mostly endowed with these phenolic alcohols in the Neb Jmel variety, showing concentrations of hydroxytyrosol and tyrosol of 3.57 and 17.97 mg/kg, respectively (Table 4
). Obviously, the difference in the amounts of phenolic alcohols (hydroxytyrosol and tyrosol) depends not only on the variety, but also on the organ—leaf or fruit—considered. Our findings are in agreement with previous studies on phenolics in olive oil [17
]. Oueslati oil, after 3% leaf addition, did not show any significant change in the hydroxytyrosol and tyrosol contents in comparison with the control (Table 4
). These results suggest that a very high content of phenolics and related compounds could have ended up in olive mill wastewater. Indeed, many investigations clearly showed the occurrence of a high content of phenolic compounds in olive mill wastewater [43
]. The hydrophilic character of polyphenols was likely responsible for the solubilisation of the most part of phenols into the water phase during oil extraction.
Phenolic acids found at considerable concentrations in Neb Jmel and Oueslati leaves (Table 3
) were represented by gallic, protocatechuic, p
-hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, vanillin, p
-coumaric and ferulic acids. In the oils of the two varieties, the identified phenolic acids did not overcome the value of 2 mg/kg (Table 4
), which agrees with the results reported by Kelebek et al. [47
]. In both olive varieties, no significant effect of leaf addition was registered.
Tocopherols occur in vegetable oils, playing an important role during oxidative processes. Two identified compounds (α- and γ-tocopherol) were quantified in this study. The quantitative profile of α- and γ-tocopherol is shown in Table 2
and Figure 2
Tocopherol concentration in olive leaves is reported in Table 2
. α-Tocopherol was present at higher amounts in Neb Jmel leaves (82.37 μg/g DW) in comparison with Oueslati, which showed an eight-fold lower amount (10.12 μg/g DW); γ-tocopherol was found in trace amounts in the leaves of both varieties.
In control oils (Figure 2
), the α-isomer reached values of 257.8 and 283.6 mg/kg in Oueslati and Neb Jmal, respectively, not showing any significant difference between the two cultivars. Following leaf addition, α-tocopherol concentration showed a slight increase in the Neb Jmel oil, where it reached the value of 328.08 mg/kg. This result could be related to the higher presence of this compound in the corresponding leaves (Table 2
). In contrast, the lower amount of α-tocopherol detected in Oueslati leaves (10.12 μg/g) did not determine any improvement in the oil following leaf addition (Figure 2
). According to Malheiro et al. [17
], the amount of α-tocopherol in oils was not significantly influenced when leaf addition was less than 5%.
Concerning γ-tocopherol (Figure 2
), the amount was very low compared to the α-isomer (1.17–28.73 mg/kg), being it more represented in the Neb Jmel oil. Leaf addition (3%) did not significantly affect γ-tocopherol concentrations due to the fact that in the leaves of both varieties this isomer was detected in trace amounts (Table 2
Consistent with previous reports [48
], we found that olive leaves can be used as an alternative source to improve the chemical composition of olive oils, mainly the α-tocopherol concentration. Likewise, the present results agree with previous studies on the influence of the cultivar on α-tocopherol concentration. Franco et al. [49
] reported very high levels of α-tocopherol in seven varieties of Spanish oils (217–345 mg/kg). In contrast, in the Portuguese olive oil studied by Cunha et al. [50
], values ranging from 93 to 260 mg/kg were found. Similar values to those reported in the present experiment were found in some studies performed on different Tunisian oils [51
Antioxidants, such as vitamin E (tocopherols), may prevent the detrimental effects of free radicals. In the Mediterranean diet, olive oil substantially contributes to the daily intake of these antioxidants. The health benefits of vitamin E are evidenced by the fact that the ingestion of fresh fruits and vegetables is inversely related to the extent of some cancers as well as to plasma lipid peroxidation [54
]. It should be highlighted that the two vitamin E isoforms have different health-related properties. In fact, γ-tocopherol is the less powerful antioxidant, although being capable of trapping peroxynitrites. For this reason, γ-tocopherol has been acknowledged as the “other” vitamin E important for human health [54