**3. Results and Discussion**

### *3.1. Chemical and Physical-Chemical Parameters*

Table 1 shows the results from the three-way ANOVA performed on the data from the chemical and physical-chemical analyses carried out on the Verdial de Badajoz virgin olive oils. The effect of the organic production was moderate (three out of seven parameters were affected), the harvesting method greatly influenced the parameters (all of them were affected), and the effect of the harvesting time was weak (only one out of seven parameters were affected) (Table 1).

**Table 1.** *p*-Values from a three-way ANOVA (type of production, harvesting method and harvesting time) performed on the chemical and physical-chemical data of Verdial de Badajoz olive oil.


3.1.1. Effect of the Organic Production (Organic vs. Conventional)

In the case of the type of production system (Organic vs. Conventional), the effect was significant on the peroxide value (PV), the total phenols and the oxidative stability of oils (Table 1). The values averaged over the six week period for the Organic and Conventional oils are shown in Figure 1. Compared with the Conventional group, the Organic one had lower values for PV (9.63 ± 1.93 vs. 11.26 <sup>±</sup> 2.12 mEq O2 kg<sup>−</sup>1), and higher for the total phenols (166.7 <sup>±</sup> 15.0 vs. 149.0 <sup>±</sup> 12.2 mg kg<sup>−</sup>1) and the oxidative stability (26.3 ± 2.1 vs. 22.9 ± 2.0 h). However, most quality parameters (free acidity and the extinction coefficients) were not affected (*p* > 0.730 for all of them) (Table 1).

**Figure 1.** Mean values and standard deviation for the chemical and physical-chemical parameters over the six week period for the Organic ( ), Conventional ( ) and Ground ( ) oils. Different letters indicate statistical differences (Tukey test's *p* < 0.05)

The lack of a marked effect on most of the quality parameters might be expected as the agronomic practices were very similar (no irrigation in both cases, and little vs. no use of phytosanitary chemicals) except for fertilisation. Currently, there is no general agreement on what the effect of the organic practices on the virgin olive oil quality is, apparently because of the difficulty of dealing with a relatively weak effect without excluding other environmental sources of variation. In this sense, a three-year study on the Leccino and Frantoio olive cultivars showed non-consistent differences in the oil quality parameters and phenol content due to the organic practices, suggesting that genotype and year-to-year variations in climate have a stronger effect [9]. However, both a decrease and an increase in oil quality have been reported in other studies. On the one hand, a decrease in oil quality (free acidity and *K*270) was reported and attributed to infestation and fungal infection in the organic fruits as a consequence of the absence of pesticides [21]. Likewise, lower phenol contents have been reported in organic Picual and Hojiblanca oils than in the conventional ones [13]. On the other hand, an increase in oil quality or/and in phenol content was also reported as a result of the organic practices. A study on Picual oil showed lower values for PV, greater stability and higher phenol content in the organic oil than in the conventional one [22], which is in line with our results for Verdial de Badajoz oil. More recent research on Leccio and Frantoio olive oils found no effect on the quality parameters but showed an increase in the phenol content, which was attributed to the decreased availability of soil nitrogen when using organic practices, as phenol content increases with decreased soil nitrogen availability [23], suggesting that, under controlled environmental conditions, the effect of the agronomic practices on plant metabolism is clear [8]. Similarly, organic practices on Kolovi olive orchards resulted in an increase in phenols, including luteolin, which was proposed as a marker for organic production [24]. Our results seem to confirm those results and show that the increase in phenols also happens in Verdial de Badajoz oil from unirrigated orchards. The increase in the total phenols (which have antioxidant activity) could explain the increase in oil stability and the decrease in PV. In any case, the moderate effect of the organic production on these parameters might indicate that no large differences might be expected in the volatile compounds.

#### 3.1.2. Effect of the Harvesting Method

The harvesting method affected all the parameters included in Table 1. Not only did the Ground oils (from ground-picked fruits) reach the highest and therefore worst values in all the quality parameters, but also the lowest in the total phenol content and oxidative stability (Figure 1). All the Ground oils, taken over the six-week period, exceeded considerably the 0.8% limit for free acidity for the extra virgin olive oil stated in European Commission Regulation (61/2011) [25], values being in the 2.4–5.3 range, and moreover all except the oil produced in week 2 exceeded the maximum allowance at least in one more quality parameter. Conversely, the two types of oils from tree-collected fruits (Conventional and Organic oils) were within the allowance limits for all the quality parameters. These results match those reported in Picual samples [26], with all the oils from ground-picked fruits exceeding the limit for free acidity and all the ones from tree-picked olives being below it.

With respect to the total phenol content, ground-picked olives, which are more exposed to microbiological infection than tree-picked olives, might have yielded lower phenol concentrations due to the phenol decline that the microbiological activity causes, in particular in oleuropein derivatives [27]. Furthermore, since phenols protect oil from oxidation [28], a decrease in them would facilitate a decline in the oxidative stability of oil. These results show a considerable repercussion of the harvesting method on the Verdial de Badajoz oil quality over, regardless of the harvesting time, and confirm the fact that fruits collected from the ground result in poor quality olive oils [10,26]. Thus, clear differences in the volatile compound profile between the Ground oils and the others might be expected.

#### 3.1.3. Effect of the Harvesting Time

With respect to the harvesting time, the ANOVA revealed a slight effect, PV being the only parameter significant affected (Table 1). PV showed significant fluctuations throughout the six-week period instead of a steady trend (data not shown). Previous studies have reported that increased ripeness causes an undesirable increase in the values of the quality parameters, although with significant fluctuations, and a decrease in the oxidative stability and phenol content [12,29]. However, in our study harvesting was adjusted to real circumstances, where orchards management is set to harvest first the orchards which maturate first, as it is usually done in the commercial mills to achieve the best overall results, which could explain the lack of a clear trend. Our results show that in real conditions the harvesting time itself is not a critical quality factor for the Verdial de Badajoz oil as long as harvesting management is adequately set. The slight effect of harvesting time may anticipate slight changes in the volatile compound profile.

#### *3.2. Volatile Compounds*

A total of 145 volatile compounds were identified or tentatively identified in the headspace of the Verdial de Badajoz olive oils: 26 aldehydes, 13 ketones, 30 alcohols, 12 acids, 24 esters, 19 acyclic hydrocarbons, 13 cyclic hydrocarbons, four ethers and four other compounds (Table 2). The results from the three-way ANOVA show that few compounds were significantly affected by the organic production (34 out of 145), most of them (105 out of 145) were affected by the harvesting method and over half of them (83 out of 145) by the harvesting time (Table 2). Differences appeared not only in the lipoxygenase (LOX)-derived compounds (Table 3) but also in the fermentation (Table 4) and oxidation (Table 5) ones, as well as in all the chemical families of compounds (Table 2).


**Table 2.** Significance levels from a three-way ANOVA performed on the volatile compounds data \* from the oil extracted from Verdial de Badajoz fruits farmed organically or conventionally (Prod.) and collected from the threes or from the ground (Meth.) over a six-week period (Week).


**Table 2.** *Cont.*

\* The compound was identified by comparing it with: <sup>a</sup> the MS and LRI of the reference compound; <sup>b</sup> MS and LRI from literature; <sup>c</sup> the MS and retention time of the reference compound. Bold: compounds included on Tables 3–5.


**Table 3.** Results (means and significance \*) for the most representative C5 and C6 LOX volatile compounds of the Verdial de Badajoz oil headspace significantly affected by the organic production, harvesting method and/or harvesting time according to Table 2.

\* Different letters in the same row (A–C) or the same column (a–c) indicate statistical differences (Tukey test's *p* < 0.05). Results are expressed as AU × 10<sup>−</sup>6.

**Table 4.** Results (means and significance \*) for the most representative fermentation compounds of the Verdial de Badajoz oil headspace significantly affected by the organic production, harvesting method and/or harvesting time according to Table 2.


\* Different letters in the same row (A–C) or the same column (a–c) indicate statistical differences (Tukey test's

*p* < 0.05). Results are expressed as AU × 10<sup>−</sup>6.


**Table 5.** Results (means and significance \*) for the most representative oxidation compounds of the Verdial de Badajoz oil headspace significantly affected by the organic production, harvesting method and/or harvesting time according to Table 2.

\* Different letters in the same row (A–C) or the same column (a–c) indicate statistical differences (Tukey test's *p* < 0.05). Results are expressed as AU × 10<sup>−</sup>6.

Regarding the type of production (Organic vs. Conventional), the modest effect found (Table 2) might be expected since the oils were just moderately different in the chemical and physical-chemical parameters (Table 1). Previous work on the effect of organic practices on the volatile compounds of olive oil has reported no consistent differences in a three-year study [9], but also a general rise [13,21], which could indicate that there might be further agronomic factors influencing the results.

Conversely, in the case of the harvesting method, the noticeable effect on the volatile compounds might be expected since the oils were markedly different in the chemical and physical-chemical parameters (Table 1). For most compounds, the largest abundances appeared in the Ground oils (Tables 3–5), which could be due to the mechanical damage caused by the drop of the fruits. The damage accelerates the decay process and makes easier the access of microorganisms to the fruits, whose infection results in changes in the volatile compound profile [27].

With regard to the harvesting time, the effect (Table 2) was stronger than expected taking into account the weak influence on the chemical and physical-chemical parameters (Table 1). In any case, a weak effect of the harvesting time was reported for Picual and Hojiblanca oils from irrigated orchards [13]. Our results for oil from unirrigated orchards suggest that the volatile compounds might be more affected by the harvesting time than what the chemical analyses may reveal.
