2.5. Fatty Acid Composition
Fatty acid composition is considered an essential characteristic for olive oil: firstly, for the detection of adulteration and fraud; secondly, it has previously been used as a criterion for classifying olive oils [
37,
38].
Table 4 shows the methyl ester fatty acid composition in the two studied olive oil samples.
Qualitatively, the analyzed olive oils present a similar fatty acids profile; in contrast, quantitatively, differences were recorded. Overall, 11 fatty acids have been detected in the EVOOs in this study. Oleic acid (C18:1) is the most abundant, followed by linoleic acid (C18:2), palmitic acid (C16:0), stearic acid (C18:0), and linolenic acid (C18:3). This finding agrees with results reported by [
23], who showed that oleic acid (C18:1), linoleic acid (C18:2), and palmitic acid (C16:0) are the major fatty acids in olive oil and vary significantly depending on the cultivar genotype. These fatty acids are very important criteria for determining the quality and authenticity of olive oil. The results recorded in
Table 4 revealed that in the two analyzed EVOOs, the amount of oleic acid represents more than 55%, the palmitic acid quantity was less than 20%, and the linoleic acid level did not exceed 21%. Oleic acid is a principal monounsaturated fatty acid; the highest value was observed in EVOO from the Gousalani (68.73 ± 0.651%), and the lowest percentage was recorded in EVOO from Nourgou (65.33 ± 0.588%). On the other hand, the latter is characterized by the highest content of the major saturated fatty acid (C16:0) (17.48 ± 0.50%), and the lowest percentage is recorded in olive oil produced from the Gousalani cultivar (13.32 ± 0.29%). Concerning the stearic acid, another main saturated acid, the amounts obtained are comparable for the two analyzed EVOOs. Linoleic acid (C18:2) is an important fatty acid for human nutrition. It is the first polyunsaturated fatty acid in olive oil correlated negatively to oxidative stability. Other fatty acids are considered minor. The determination of these minor fatty acids is very necessary for the characterization of olive varieties [
39] and for the precise knowledge of lipid profiles during the authentication of the varietal origin of olive oils [
40]. According to the results from ANOVA, the three fatty acids C14:0, C17:0, and C17:1 did not show any significant variation among the EVOO samples analyzed. In contrast, a significant variation was observed in the level of C18:0 (
p < 0.05). The concentration of C18:1 and C18:3 revealed a very significant difference (
p < 0.01), and highly significant variation (
p < 0.001) was recorded in the other detected fatty acids (C16:0, C16:1, C18:2, C20:0, C20:1). This difference is mainly explained by the genetic factor.
2.7. Phenolic Composition
The EVOO samples analyzed present a high amount of phenolic substances, as shown in
Table 5, where the cumulative concentration of individual phenolic content is evaluated as a function of genotype cultivar. The results from ANOVA showed that the effect of genotype factor on phenolic composition was statistically highly significant (
p < 0.001). The olive oil extracted from the Gousalani cultivar showed a higher value in total phenols (516.16 mg/kg) than the Nourgou oil (348.03 mg/kg).
Our data agree with those mentioned in previous studies, which showed that olive oil produced from different Tunisian varieties could be considered a potential source of phenolic compounds [
8,
49]. According to several authors, the content of phenolic compounds is highly dependent from the cultivar under consideration [
6,
50]. Ref. [
51] reported that genetic factors have an impact on olive oil quality, mainly its phenolic profile.
Table 6 shows that 19 compounds were recorded in the two olive oils analyzed. A remarkable difference was recorded for the different phenolic substances depending on olive oil genotype. In this regard, ref. [
52] reported that there is a difference in both qualitative and quantitative profiles of phenolic constituents from the analyzed olive fruit samples collected from various areas.
EVOO samples were characterized by appreciable concentrations of phenolic alcohols, phenolic acids, secoiridoids, verbascoside, flavonoids, and phenolic aldehydes. The common simple phenols detected in the olive oils analyzed were secoiridoids (i.e., oleuropein aglycone, oleuropein, and ligstroside aglycone). These findings were in accordance with the results of [
53], who revealed that secoiridoids, oleuropein derivatives, are the most predominant among phenolic antioxidants of extra virgin olive oils.
EVOO extracted from the Gousalani cultivar was characterized by higher values than the Nourgou cultivar. In fact, oleuropein aglycone (164.99 mg/kg) dominated the three others secoiridoids. Several authors have reported that secoiridoids were the most abundant phenolic fraction in olive oils produced from some Tunisian varieties: Sehli, Baldi, Chétoui, Chemchali, Besbessi, Touffehi, Zalmati, Jemri, Neb Jmel, Tounsi, and Fakhari [
32,
54,
55]. The second major phenolic group detected was phenolic alcohols, mainly hydroxytyrosol and tyrosol. According to [
30,
56], they were derived from hydrolysis of oleuropein aglycone and ligstroside aglycone. The value of these two phenolic compounds is very important, as these antioxidant substances contribute to the prevention of blood lipids from oxidative stress [
44].
The content of hydroxytyrosol of analyzed EVOO varied from 2.56 to 6.19 mg/kg for the Nourgou and Gousalani genotypes, respectively. On the other hand, a high value of tyrosol is recorded in the Nourgou oil (7.14 mg/kg), and a low value is observed in the Gousalani oil (4.77 mg/kg). The phenolic alcohols present in the studied EVOO were high compared to the corresponding values for olive oil samples analyzed by [
23].
In addition to these phenolic components, phenolic acids (e.g., caffeic, vanillic, p-coumaric, ferulic, and o-coumaric) were also detected in the analyzed oils. Gousalani olive oil was characterized by a high content of o- coumaric acid (12.60 mg/kg), and the olive oil produced from the Nourgou cultivar presents high amounts of vanillic acid (1.46 mg/kg) and p-coumaric acid (2.53 mg/kg).
The other identified phenolic acids were observed in low quantities. These findings were also confirmed by [
57], who admitted that phenolic acids were recorded as minor metabolites in olive oil. Considering the phenolic profile, flavonoids were the most frequent class of phenols, with seven compounds detected and quantified: catechin, luteolin-7-
O-glucoside, rutin, luteolin-4-
O-glucoside, luteolin, apigenin, and diosmetin.
The observed major constituent of the flavonoids group was luteolin-4-O-glucoside. Its content ranged from 4.53 mg/kg in Nourgou oil to 26.83 mg/kg in Gousalani oil. Regarding the other detected phenolic compounds, they were characterized by variable concentrations, likely depending on genetic factors. In addition to these phenolic compounds, other phenols were detected in EVOO samples analyzed in this study, such as vanillin, which was present in very low quantities in both oils under investigation (varied from 0.47 to 0.83 mg/kg). Verbascoside was also detected in the olive oils studied. It was present in considerable amount; the highest content was found in Nourgou oil (7.00 mg/kg), and the lowest value was noted in Gousalani oil (4.63 mg/kg).
Overall, statistical analysis revealed a significant variation of the majority of individual phenolic compounds, which depended mainly on the genotype (
p < 0.001), except for catechin and diosmetin (
Table 6).
A multivariate statistical approach was used to better discriminate the two EVOO samples. The results of both unsupervised and supervised investigations are reported as
Figure 1A (heat map from unsupervised hierarchical clustering analysis) and
Figure 1B (score plot from OPLS-DA prediction modelling). Overall,
Figure 1 clearly demonstrates a huge diversity in terms of the phytochemical fingerprint when comparing the two genotypes; in particular, the majority of phytochemicals were better represented in the Gousalani oil sample (
Figure 1A). Interestingly, the OPLS-DA prediction model showed more than acceptable model parameters related to both goodness of fitting and prediction (both higher than 90%), thus confirming the ability of the annotated phytochemicals to discriminate the two EVOO samples.
Finally, the best phytochemicals in terms of cultivar discrimination were extrapolated through a VIP approach, considering as a minimum cut-off a VIP score > 1. The results are reported in
Table 7. Interestingly, we found 10 features mostly associated with cultivar discrimination, with o-coumaric acid, verbascoside, and hydroxytyrosol possessing the highest prediction abilities (
Table 7), exclusively related with their between-group separation ability. On the other hand, three additional phenolics were particularly able to also explain a within-group separation, namely oleuropein aglycone, vanillic acid, and vanillin (
Table 7).
Previous works [
58] have affirmed that the development of metabolomic profiling approaches for phenolic fingerprints could enhance the understanding of the biological and nutritional properties of olive oil, as well as promote their use for traceability and authenticity purposes. Furthermore, according to [
59], in addition to their impacts on food quality, the application of untargeted metabolomics profiling of sterols and polyphenols coupled to multivariate chemometrics appears to be a very promising tool to discriminate different EVOO samples of different geographical origins (Tunisian vs. Italian). The amount of phenolic compounds in olive leaves, fruits, and oil is not stable and varies widely [
60]. The content of polyphenols and oil in olive drupes varied significantly according to many factors such as cultivar or variety genotype [
61], plant individuals and population [
60], the region of its production, environmental and pedoclimatic conditions, agricultural practices, ripening stage of olives, and the fruit processing method [
62,
63], together with extraction methods, extraction solvents [
64], and the systems used to separate oil from olive pastes. The conditions of storage are also important.
2.8. In Vitro Antioxidant Activity Characterization
The antioxidant characteristic should not be evaluated in vitro by means of a single antioxidant assay model, due mostly to the huge chemical variability of antioxidant metabolites together with the complexity of the food matrix [
65]. In our study, the antioxidant potential was assessed as both reducing power and radical scavenging by using FRAP and ORAC methods, respectively.
An examination of
Table 8 shows that the results from ANOVA revealed that the effect of genotype factor on antioxidant activity was very significant (
p < 0.01) with the FRAP assay and highly significant (
p < 0.001) with the ORAC method.
The antioxidant capacity evaluated by FRAP test ranged from 15.66 to 50.00 mg gallic acid equivalent/100 g for the Nourgou and Gousalani olive oils, respectively. Regarding the ORAC radical scavenging capacity, different results were observed between the two EVOO samples analyzed. The highest antioxidant potential was recorded in the Gousalani oil extract (8796 mM TEAC), whereas the lowest activity was shown in the Nourgou oil extract (2024.5 mM TEAC).
Based on the classification proposed by [
66], which suggested four categories of ORAC quality, these two olive oils analyzed in this study can be characterized by a top quality of ORAC activity. The ranges were the following: 1–4: low-quality EVOO; 4–8: intermediate-quality EVOO; 8–12: high-quality EVOO; >12: top-quality EVOO.
FRAP and ORAC tests revealed that the two EVOOs are characterized by powerful antiradical activity and good reducing power capacity. Our results agreed with several studies that have proven that olive oil is characterized by a significant antioxidant activity. This is comparable to reference antioxidants such as BHT, as measured by various methods like the DPPH test, FRAP test, ABTS test, and β-carotene bleaching test [
67,
68]. According to [
69], leaves, pulp, and stone extracts from Tunisian olive cultivars ‘Zarrazi’ and ‘Chemlali’, cultivated in situ, and two oleaster trees from the natural ecosystem in southern Tunisia showed high antioxidant capacity according to DPPH and ABTS assays. Equally, ref. [
70] reported that the Tunisian Chetoui variety had an important antioxidant property (which varied between extracts from different organs), as shown by various methods such as DPPH, FRAP, ORAC, and β-carotene-linoleic acid bleaching assays. Ref. [
71] reported that a Tunisian commercial olive oil shows very high antioxidant inhibition activity (84.96%) in comparison with olive oils produced from various Algerian regions. Virgin olive oil is a promising source of bioactive and natural antioxidants that can proceed through different mechanisms to confer an effective defense system against free radical attack [
72]. According to [
73], olive oil contains bioactive compounds (with strong antioxidant potential). It was clear that EVOOs are antioxidant in nature and can be used as a healthy substitute in place of other cooking oils.
2.9. Correlation Analyses
Olive oil is one of the edible oils with a high price in the fats and oils industry. Several antioxidant assays, in vitro and in vivo, revealed that olive oils are good sources of antioxidants. A correlation analyses among the various parameters was investigated especially to analyze how the differences revealed by antioxidant capacity values estimated by the two methods can be explained on the basis of the concentrations of the phytochemical compounds, or in particular which group of metabolites were responsible for the antioxidant property variability. The results of the correlation are of great importance to determine if the antioxidant activity was controlled by several direct and indirect metabolites.
The results obtained showed a considerable relationship between several molecules detected and the antioxidant capacity evaluated. Estimates of the correlation are shown in
Figure 2.
The correlation coefficients (
Figure 2) of each compound revealed the presence of significant and positive correlation within the two methods, FRAP and ORAC. A positive and significant correlation was observed between the chlorophyll pigments; the total phenols; the total sterols; the fatty acids (mainly (C16:0), (C16:1), (C17:0), (C18:0), and (C18:3)); the phenolic alcohols (hydroxytyrosol), the secoiridoids (oleuropein aglycone, oleuropein, ligstroside aglycone), the phenolic acids (caffeic, ferulic,
o-coumaric), the flavonoids (rutin, luteolin-4-
O-glucoside, luteolin), and the antioxidant activity with the two methods. High correlation coefficients were found, with R values higher than 0.6, ranging from 0.81222 to 0.963 for the FRAP assay and from 0.89548 to 0.99833 for the ORAC test.
These results agree with those reported by [
74], which stated that the antioxidant activities are mainly correlated to the health-promoting substances of olive oil, such as phenolic components, pigments, squalene, tocopherols, and sterols. These bioactive metabolites make olive oil one of the most important healthy edible oils worldwide. In the same context, ref. [
75] revealed a positive correlation between the total phenol and flavonoids content and DPPH radical-scavenging activity; they reported that this finding could be ascribed to the highest contents of the total phenols and flavonoids, particularly ortho-hydroxylated phenolics, such as hydroxytyrosol and oleuropein aglycone. According to [
76], a linear positive relationship between the total phenols content and antioxidant activity was observed, and the higher contents of total phenols in Tunisian oil were positively correlated with antioxidant activities in comparison with the other olive oils analyzed. The correlation analysis carried out on the different phenolic classes revealed that the total phenolic, flavonoid, and ortho-di-phenolic content were correlated with radical scavenging activity [
77]. It has been demonstrated that bioactive phenolic compounds of olive oil are mainly responsible for its antioxidant or free-radical-scavenging activity [
78].