3.2. Oil Quality Parameters
The effects of geographical origin, processing system and harvesting time on the quality indices of olive oil including free acidity, peroxide value, K232 and K270, were investigated. Results (
Table 2) showed a very highly significant effect of the interactions geographical origin * processing system * harvesting time, geographical origin * processing system and geographical origin * harvesting time on this set of variables
. Moreover, processing system * harvesting time revealed a significant effect on these quality indices
. As per each single factor, the geographical origin and the processing system showed a very highly significant effect
and the harvesting time had a significant effect on this set of variables
.
The
Table 2 shows that between interactions, the three-way interaction geographical origin * processing system*harvesting time and its associated error accounted for the highest partial
(24%). As per individual factors, highest partial
(30% of the between subject’s variance) was attributed to the geographical origin and its associated error. It is worth noting that the three studied factors and their interactions showed sufficient power to detect such effects (the power statistics > 0.80). Several previous studies showed significant effects of geographical origin, processing system and harvesting time on the olive oil quality parameters [
10,
36]. However, Di Giovacchino et al. [
37] showed no significant differences between processing systems in free acidity, peroxide value, K232, K270.
To go further in the analysis of the effect of the geographical origin, processing system and harvesting time on each of the quality parameters, the tests of Between-Subjects effects were used (
Table 3). Results showed that the observed statistical power is higher than 0.8 for all significant effects. Accordingly, the tests of Between-Subjects effects have sufficient power to detect such effects.
The interaction geographical origin * processing system was the main contributor to total variance of the free acidity accounting for 21.37%. Moreover, the interaction geographical origin * harvesting time to total variance of peroxide value (18.75%) and K232 (10.69%). In addition, the interaction geographical origin * processing system*harvesting time to total variance of K270 accounting for 22.28% (
Table 3).
As shown in
Table 3, the free acidity was significantly affected by the interaction geographical origin*processing system. This revealed that whatever is the geographical origin, the processing system has a significant effect on the free acidity. In addition, the peroxide value was significantly affected by the three-way interaction geographical origin * processing system * harvesting time. This means that the interaction among the two factors (geographical origin * processing system) is different across the levels of the third factor (harvesting time).
Similarly, K270 was significantly affected by the three-way interaction geographical origin * processing system * harvesting time. The interaction among the two factors (geographical origin * processing system) is different across the three harvesting times.
The effect of each factor on the quality parameters of the olive oil was assessed. The effect of the geographical origin was significant on free acidity, peroxide value and K270 (
Table 4). These results are in disagreement with those obtained by Lazzez et al. [
38] that stated that the fruit ripening is the main factor influencing the olive oil qualitative parameters; and, the geographical origin has only a minor effect on these parameters.
Mean comparisons showed that Hasbaya oil recorded the lowest free acidity, Zgharta-Koura oil the lowest peroxide value and Jezzine oil the lowest K232 and K270. However, the oil from Akkar and Jezzine registered the highest free acidity exceeding 0.8% limit established by the IOC regulation for extra virgin olive oil [
39], which represented an advanced level of degradation. According to Ben Temime et al. [
40], the significant differences in olive oil qualitative parameters between geographical origins are not due to the cultivation area in itself, but to other factors affecting olive fruits quality such as olive fly attacks, mechanical damage during olive harvesting and transport, long delay between harvesting and processing, among others.
When considering only the two phases system that is present only in Akkar and Jezzine, the multivariate analyses of data collected from this system showed a significant effect of the geographical origin on the olive oil quality parameters . Yet, the tests of Between-Subjects effects revealed a significant effect of the region only on the K270 parameter ; with higher values in Akkar region (0.18 vs. 0.12). However, if considering only the olive oil samples obtained through the press system, MANOVA shows a very high significant effect of the geographical origin on the olive oil quality . The Tests of Between-Subjects effects reveals a significant effect of geographical origin on free acidity and peroxide value with respectively and . The highest acidity was recorded in Jezzine (1.66%) significantly higher than Akkar, Zgharta-Koura and Hasbaya (1.09%, 0.81% and 0.54%, respectively), with Akkar showing significantly higher acidity than Hasbaya. Regarding peroxide value, Jezzine showed significantly higher peroxide value than Hasbaya and Zgharta-Koura (15.24, 11.58, 7.70 meq O2/kg of oil). Note that Akkar (14.04 meq O2/kg of oil) recorded significantly higher peroxide value than Zgharta-Koura (8.49 meq O2/kg of oil). Also, for the 3-phases processing system alone, MANOVA reveals a very high significant effect of the geographical origin on the olive oil quality . The Tests of Between-Subjects Effects reveals a significant effect of geographical origin on free acidity ; with acidity in Jezzine (0.91%) significantly higher than that in Zgharta-Koura, Akkar and Hasbaya (0.70%, 0.54% and 0.39%, respectively). Note that oils obtained from Zgharta-Koura showed significantly higher acidity than those from Hasbaya. The results of these three comparisons on the same processing systems in different geographical origins confirm that free acidity, peroxide value and K270 are highly dependent on the geographical origins.
As per the processing system, the effect was significant on free acidity and K270 (
Table 5). These results are in partial agreement with those obtained by Ben Hassine et al. [
36], who indicated that the free acidity, the peroxide value, the K232 and the K270 are significantly affected by the processing system. Conversely, Salvador et al. [
41] demonstrated that while oxidative stability and antioxidant content differed significantly between processing systems; free acidity, peroxide value, K232 and K270 didn’t show significant differences.
In the present study, Sinolea system recorded the lowest free acidity; and the 2-phases system, the highest one exceeding, together with the press system, the limit of 0.8% established by the IOC regulation for extra virgin olive oil [
39]. These results are in disagreement with many previous studies that showed a highest free acidity in the press system and a lowest one in the centrifugation systems [
36,
42,
43]. The high level of free acidity observed in the present study in the 2-phases system is probably related to strong infection with the olive fruit fly (
Bactrocera olea) in both regions where the 2-phases is present: Chadra in Akkar and Bisri in Jezzine. The two regions consist of valleys with very high relative humidity and annual high infection with olive fruit fly. Indeed, several previous studies specified that the attack of the olive fly affects negatively the olive oil quality, leading to an increase in free acidity [
44,
45]. Previous studies stated that, when poor quality olives are industrially processed with either press or 3-phases centrifugation systems the centrifugation system, the latter gave oils with lower free acidity [
46]. In the present study, the 2-phases system was unable to reduce sufficiently the free acidity maybe due to the very high infection with the olive fruit fly [
44].
Press system and 3-phases decanter recorded the lowest K270 that depends on the presence of secondary oxidation products (conjugated trienes). The higher values observed in 2-phases system also may be due to the high attack of olive fruit fly indicated above; and the higher values in sinolea may be due to the observed high temperature that was used in those mills during the oil processing. These results are in agreement with those described by Gómez-Caravaca et al. [
44] who reported an increase in oxidation products in olive infested by the olive fruit fly; and with those stated by Ranalli et al. [
47] who also reported an increase in these products with higher malaxation temperatures.
The results (
Table 6) showed that the effect of harvesting time was only significant on peroxide value. It was noticeable that the peroxide value increased significantly in the late harvesting time to 14.91 Meq O
2/kg as compared to the early and intermediate with values of 12.57 and 11.51 Meq O
2/kg, respectively. However, other studies reported an increase only in free acidity along ripening [
48,
49] due to the progressive activation of the lipolytic activity and to the fact that the olives are more sensitive to pathogenic infections and mechanical damage, which results in oils with higher acidity values [
50]. Conversely to the results obtained in the present study, a decrease in peroxide value, K232 and K270 was observed in ‘Sayali’ olive oils [
49] and in other monovarietal olive oils from Tunisia at late harvesting [
51]. Bengana et al. [
52] reported higher values of all quality indices at late harvest of olive oils from ‘Chemlal’ variety cultivated in Algeria.
3.3. Fatty Acid Composition
MANOVA results performed on the set of the five main fatty acids of the olive oil (C16:0, C18:0, C18:1, C18:2 and C18:3) revealed a significant effect of the three-way interaction geographical origin * processing system * harvesting time on the fatty acid composition of olive oil
. On the other hand, only the geographical origin and the harvesting time revealed a very highly significant effect on the set of the main fatty acids in olive oil
(
Table 2).
The harvesting time and its associated errors accounted for high percentages of the between subject’s variance expressed as partial
(37%). In a previous four years study to determine the optimal harvesting period for ‘Chemlali’ olives, Lazzez et al. [
38] also reported that the harvesting time is the factor showing the highest effect on the composition of olive oil in comparison with crop year and growing area.
However, the Tests of Between-Subjects effects revealed that the geographical origin was the main contributor to total variance of C16:0, C18:0 and C18:1. However, the interaction geographical origin * processing system * harvesting time was the main contributor to total variance of C18:3; and, the interaction geographical origin * processing system the main contributor to total variance of C18:2 (
Table 3). These results are in agreement with those obtained by Bajoub et al. [
53] on the ‘Picholine Maroccaine’ monovarietal olive oil in Morroco, who reported a significant effect of geographical origin on all fatty acids except on the minor fatty acids, heptadecenoic and myristic acids. Also, there are several studies on the use of fatty acid composition for geographical characterization of olive oils from northern countries of the Mediterranean basin [
41,
54,
55]. On the other hand, the interaction geographical origin * processing system affected significantly all the main fatty acids in the olive oil (
Table 3). This means that whatever is the geographical origin, the main fatty acids of the olive oil are affected by the processing system.
Moreover, the interaction processing system * harvesting time affected significantly the C16:0 (
Table 3). This reveals that independently of the processing system, C16:0 is affected by the harvesting time.
Regarding the interaction geographical origin * processing system * harvesting time, it was only significant for C18:3 (
Table 3). The mentioned three-way interaction shows that the interaction among the two factors (geographical origin*processing system) is different across the three harvesting times.
As for the effect of each single factor, the mean comparisons showed that C16:0 and C18:2 contents were significantly higher in North Lebanon (Akkar and Zgharta-Koura) than in South Lebanon (Hasbaya and Jezzine). However, C18:1 was significantly higher in South Lebanon (Hasbaya and Jezzine) than in North Lebanon (Akkar and Zgharta-Koura). C18:0 was significantly higher in Jezzine and Zgharta-Koura than in Akkar and Hasbaya. However, the content of C18:3 was not affected by the geographical origin (
Table 4). According to Beltrán et al. [
56] the air temperature during oil biosynthesis could affect the amount of polyunsaturated fatty acids (linoleic and linolenic fatty acids) by means of the regulation of desaturase enzymes activities. For instance, Issaoui et al. [
57] in Tunisia and Mailer et al. [
8] in Australia both showed a higher content of C18:1 in cooler regions (high altitudes) and higher contents of C16:0 and C18:2 in warmer regions (low altitudes). The results obtained in the present study agree with these observations as the high C18:1 content was observed in the olive samples proceeding from Jezzine and Hasbaya where the olive fruits were harvested at altitudes up to 1000 and 1050 m, respectively; and, the high content of C16:0 and C18:2 were observed in oils from Zgharta-Koura and Akkar as the fruits were harvested from lower altitudes, up to 350 and 700 m respectively. Although, Serhan et al. [
24] also previously reported strong negative correlation between altitude and C16:0, additional studies on several years and involving different regions in North and South Lebanon are essential to prove these hypotheses.
It is worth noting that, the effect of processing system on the fatty acid composition was not significant in the present study (
Table 5), in concordance with the results obtained by Gimeno et al. [
42] and by Serhan et al. [
24] while comparing traditional and centrifugation processing systems in north Lebanon; but, in partial agreement with those obtained by Salvador et al. [
41] who reported slight differences in fatty acid composition due to the processing system, although the differences were significant only in case of C16:0, C16:1 and C18:3.
However, regarding the harvesting time, the effect was only significant on C16:0 whose content decreased significantly after the intermediate harvesting time (
Table 6). These results are in agreement with those reported by Cimato [
5] where the delay in harvesting tended to increase the content of unsaturated fatty acids, especially linoleic, at the expense of palmitic acid. However, these results are partially in agreement with those obtained by Baccouri et al. [
51] on Tunisian monovarietal olive oil and by Fuentes de Mendoza et al. [
48] in a three successive years study on ‘Morisca’ and ‘Carrasqueña’ olive varieties, who reported a decrease in palmitic and linoleic acids along ripening.
3.4. Total Phenols
The effect of the studied factors and their interactions on total phenols content, determined by the Folin-Ciocalteu method, was assessed. Results showed that the interaction geographical origin * processing system * harvesting time showed a highly significant effect on total phenols (
) (
Table 3). This three-way interaction geographical origin * processing system * harvesting time means that whatever is the geographical origin, the processing system has a significant effect on the total phenols content for the three harvesting times. Indeed, the interaction among the two factors (geographical origin * processing system) is different across the early, intermediate and late harvest.
As per each factor alone, very highly significant effects of geographical origin and processing system were observed on the total phenols content
, with the later showing the highest contribution (15.92%) and the former the second one (13.19%) (
Table 3). Mean comparisons showed that total phenols in Hasbaya was significantly higher than in Akkar and Jezzine (235.50, 208.42 and 193.53 mg GAE/Kg of oil, respectively); while, the total phenols in Zgharta-Koura recorded an intermediate value (217.88 mg GAE/Kg of oil) (
Table 4). These results don’t match those shown by Baccouri et al. [
51] that reported no difference in phenolic compounds according to the geographical origin in monovarietal olive oils from Tunisia; but they match those shown by Ben Temime et al. [
40] and Youssef et al. [
58] that reported different phenolic composition in ‘Chétoui’ and ‘Oueslati’, respectively, due to different climate and soil characteristics. Moreover, regarding the processing system, the oil from 2-phases system recorded significantly higher total phenols than 3-phases and press systems (240.91, 207.02 and 195.86 mg GAE/Kg of oil, respectively); however, sinolea system recorded an intermediate value (225.89 mg GAE/Kg of oil) (
Table 5). Salvador et al. [
41] previously reported that among all quality and compositional parameters of olive oil, phenolic compounds and oxidative stability stand as the main parameters affected by the processing system. In fact, it was demonstrated that the 2-phases decanter preserves more of the phenolic compounds in comparison to the 3-phases decanter where the added water causes large amounts of phenols to be eliminated with the olive mill waste water [
42] (12, 16). Moreover, the high amount of O
2 dissolved in the pastes during the process of press and sinolea systems due to contact with the air result in a loss of phenolic compounds due to the activation of endogenous enzymes, polyphenoloxidase and peroxidase, that oxidize the phenolic compounds and consequently reduce their concentration in the produced oil [
14].
However, regarding the harvesting time, the total phenols content decreased along ripening although the difference was not significant. This decrease in total phenols with the progress of ripening was previously well reported [
35,
49].
3.5. Oil Oxidative Stability (OSI)
To understand the effect of the three studied factors on the OSI, a three-way ANOVA was run. Results showed a highly significant effect (
) of the three-way interaction geographical origin * processing system * harvesting time (
Table 3); which means that regardless of the geographical origin, the processing system has a significant effect on the OSI for the three-harvesting time. Indeed, the interaction among the two factors (geographical origin*processing system) is different across the early, intermediate and late harvest.
As per each factor alone, the OSI was extremely highly significantly affected by the geographic origin and by the processing system
; and, highly significantly affected by the harvesting time
. It is worth to note that Hasbaya oil showed significantly higher OSI (10.14 h) followed by Zgharta-Koura, Akkar and Jezzine 8.01, 7.97, and 6.42 h, respectively) (
Table 4). Interestingly, this was the same tendency observed in total phenols, in agreement with previous results showing a high positive correlation (r = 0.937) between total phenols in oils from different locations and OSI [
59].
However, the 3-phases and the sinolea systems registered significantly higher OSI (9.87 and 9.84 h, respectively) in comparison with 2-phases and press systems (7.07 and 6.76 h, respectively) (
Table 5). Although the 2-phases registered the highest phenolic content, the lower OSI recorded in this system could be mainly due to the higher free acidity registered in oils from this system. In previous studies, Rotondi et al. [
60] have found a high positive correlation between higher free acidity and shorter shelf life of olive oil.
As per the harvesting time, the OSI decreased along ripening in parallel to the decrease of total phenols. The difference was only significant between the first and the last harvesting time (
Table 6).