1. Introduction
Over the past two decades, Portugal has experienced a significant surge in olive cultivation, establishing itself as one of the leading global producers, primarily focused on virgin olive oil (VOO) rather than table olives (TO), with an overall production increase of approximately 400% during this period (
Figure 1).
The increase in production has mostly been observed in the Alentejo region, marked by a steady expansion of the total olive cultivated area and sector modernization, mainly by orchard intensification. Portugal is currently leading a new era in olive oil production by employing large-scale farming with high-density hedgerow systems. This has enabled an increase in productivity and reduced production costs through improved mechanization of orchard management techniques. Nevertheless, small-scale producers still retain their place in the national scenario of olive oil production, in part due to the simple fact that these are the sole guardians of the valuable germplasm of the traditional olive cultivars that still characterize the sensorial profile of Portuguese VOO and TO. Traditional olive cultivars are usually associated with a specific region, where they have adapted and thrived throughout the centuries, so these are the cultivars with the most resilience and well-adapted features for a given environment.
The olive fruit is a valuable source of various nutraceuticals, including antioxidants, vitamins, and minerals, which strongly contribute to its health-promoting properties [
2]. Among these nutraceutical compounds, the phenolic compounds have been shown to play an important role in human health. Their value has been shown mainly through their modulation pathways associated with inflammatory reactions and oxidative stress regulation [
3,
4], with the olive fruit being well recognized as a significant source of phenolic compounds [
5]. There are numerous phenolic compounds present in the olive fruit, which can comprise up to 1−3% of the total fruit pulp fresh weight, ranging from simple monophenolics to more complex compounds with multiple aromatic rings [
6]. Among all, oleuropein (Ole), a secoiridoid glucoside, is the predominant phenolic compound in olive fruit [
7], which can reach up to 14% on dry weight (DW) basis in total composition [
8], whereas verbascoside (Verb) is the main hydroxycinnamic derivative present [
9]. Along with the fruit maturation process, Ole undergoes several hydrolysis processes, yielding different compounds, with hydroxytyrosol (H-tyr) being one of the most relevant [
10]. During this process, three phases may be distinguished: (1) a growth phase, where accumulation of Ole occurs; (2) a green maturation phase, where levels of Ole start to reduce; and (3) a black maturation phase, which is characterized by the appearance of anthocyanins, leading to a continuous falloff of Ole levels [
8]. It is in this last phase where H-tyr and tyrosol (Tyr) start to increase their concentration, mainly due to the hydrolysis of Ole and other secoiridoid derivatives. In olives, these compounds are of significant relevance, since they provide a primary source of protection to the plant against external agents, such as bacteria, fungi, and viruses [
11]. Furthermore, in human nutrition, these specific phenolic compounds play a major role due to their antioxidant and anti-inflammatory properties [
12].
Cultivar specificity is considered of great relevance since the specific genetic makeup of a cultivar determines its potential to synthesize and accumulate specific phenolic compounds [
13], with the concentration of phenolic composition being strongly related to this trait. Additionally, agricultural practices, such as irrigation and pruning, have been shown to significantly influence the concentration of bioactive compounds in both olive fruit [
5,
14] and VOO [
15,
16]. This has shown particular relevance when comparing organic and integrated practices [
17], as well as when considering the specific edaphoclimatic conditions of the olive orchard [
18]. Therefore, to affirm olive fruit as a functional food rich in bioactive compounds that promote health, these factors must be considered.
This study aims to investigate how cultivar specificity and fruit ripeness affect the phenolic profile of olive fruit, considering the importance of olives as a source of nutraceuticals in the Mediterranean diet. For this, seven distinct cultivars were considered, comprising five of the most relevant traditional Portuguese varieties, ‘Galega vulgar’ (Gv), ‘Redondil’ (Red), ‘Carrasquenha’ (Car), ‘Cobrançosa’ (Cob), and ‘Azeiteira’ (Az), alongside two Spanish varieties, ‘Picual’ (Pic) and ‘Arbequina’ (Arb), with seven sampling points throughout the fruit maturation process. Thus, we aim to acknowledge the effective concentration of phenolic compounds in various olive cultivars with high expression in Portugal throughout their maturation process. To eliminate other influencing factors in this study, all considered cultivars were sourced from the same orchard and subjected to similar agricultural practices. With this study, we aim to provide some preliminary new information regarding the potential of distinct cultivars as a source of important nutraceutical compounds, showing the olive fruit, among its distinct cultivars and ripening stages, as a functional food rich in bioactive phenolic compounds.
3. Results and Discussion
For this study, seven olive fruit cultivars were selected, five of them being among the most widespread Portuguese traditional cultivars, namely Gv, Cob, Car, Red, and Az, and the two most widespread cultivars worldwide, the Spanish Pic and Arb. The MI was measured, according to the IOC guidelines [
19] and following Equation 1, over a period of approximately 14 weeks (from 18 July to 30 October), with a total of seven sampling points distributed throughout this period (
Table 1).
Table 2 illustrates the MI evolution of each cultivar during ripening, highlighting the distinct maturation profiles for all cultivars, with Gv identified as the earliest maturing cultivar. As shown, Gv reached an MI of 4—fruits with fully black skin—at T7, followed by Az, Cob, and Pic, with MI between 2 and 3, also at T7. Car, Arb, and Red demonstrated a significantly delayed maturation process, with MI around 2—more than half of the fruit has a green color—at T7. As a more standardized and visual parameter, MI may provide us with useful information regarding both at a morphological and chemical level. MI may be used to predict the best harvesting period for specific cultivars, being able to relate not only information regarding fat accumulation content but also qualitative parameters, such as bioactive compounds [
20,
23]. Being MI, a value obtained by visual observation of the olive fruits, this parameter relates specifically to the natural maturation process of each cultivar. As shown in
Table 2, both T1 and T2 presented MI values of 0, with the first fruit coloration, changing from deep green to yellow-green skin, only starting to occur by the beginning of September. From T2 onward, it is clear that different cultivars follow a distinct maturation process. Gv is a well-known traditional Portuguese cultivar for its early ripening [
20,
24]; in contrast to others, it gains fully black skin coloration at a considerably early stage.
In addition to MI, other significant morphological changes occur during ripening, including fruit caliber, fruit pulp-to-stone ratio, and moisture content (
Table 3). These are important parameters to measure and consider when evaluating the cultivars and their potential in nutraceutical components, since they will influence the amount of these compounds per fruit unit. Measuring the amount of “beneficial compounds” by fruit unit can be useful when considering a functional food such as the olive fruit. From this, we may establish a daily intake of olive fruits to accomplish a specific set of nutraceuticals, taking into consideration the particular cultivar and its respective ripeness.
As shown (
Table 3), Red cultivar presented the highest fruit caliber on average mass at all sampling periods, with values as high as 4.72 ± 1.69 g at T6. Gv and Arb were the cultivars with the lowest fruit caliber during the ripening process. In agreement with this, Red also showed the highest fruit pulp-to-stone ratio, with values of 8.48 ± 4.91, respectively, at T6. These results clearly indicate that Red stands out as an exceptional cultivar for table olive production, owing to its high fruit caliber and pulp-to-stone ratio. Indeed, Red is a highly valued cultivar in traditional Portuguese table olives, along with the Car and Az cultivars, which are specifically associated with the PDO (Protected Designation of Origin) “Azeitonas de Conserva de Elvas e Campo Maior DOP” for table olives [
25]. In contrast, the Spanish cultivar Arb exhibited the lowest values for both fruit caliber and pulp-to-stone ratio; its sole purpose is to produce virgin olive oil in super-intensive orchards, as it yields low-caliber fruits with a high fat content [
26].
Simultaneously, fruit moisture was also measured (
Table 3), showing a notorious decreasing trend for all cultivars along with ripening, with the lowest moisture values observed at T5, the beginning of October. Despite the observed decreasing trend, fruit moisture may be influenced by several external factors, such as climate and agronomic practices. Therefore, for the quantification of phenolic compounds, all measurements were performed on a DW basis to avoid external interferences. First, the TPC were measured, and as shown in
Table 4, a general decreasing trend was observed during ripening for all cultivars. Arb showed the lowest concentration of TPC at all sampling points, while Pic, Car, and Cob consistently exhibited the highest TPC throughout all sampling periods. For all cultivars, the highest TPC was obtained at T1, showing significant differences from all other sampling points (
p-value < 0.05). At T1, all measure TCP presented no statistically significant differences (
p-value > 0.05) among all cultivars, except for Arb.
This data offers some preliminary understanding of how different cultivars affect the synthesis and accumulation of phenolic compounds during ripening. This information can be highly useful for studying the olive fruit as a functional food, particularly due to the incredibly high concentrations of TPC observed at early maturation stages (T1). Considering Cob as an example, at T1, the measured TPC were approximately 61.6 g GAE kg
−1, showing a significant decrease during ripening to about half this value at T7 (32.3 g GAE kg
−1). Additionally, taking into account the average fruit caliber, pulp-to-stone ratio, and moisture content (
Table 3), we can state that at T1, an average olive fruit from the Cob cultivar contains about 22.2 mg of TPC (GAE, on a DW basis) and about 33.7 mg at T7 (
Table 5), which represents approximately 3.2% of its total pulp mass. On the other hand, much lower changes were observed if we consider cultivars with reduced fruit caliber that are also less prone to the synthesis and bioaccumulation of phenolic compounds, such as Arb. In contrast to Cob, Arb showed the lowest TPC and fruit caliber, measuring about 6.9 mg of TPC (GAE, on a DW basis) per olive fruit at T1 and 7.9 mg at T7. Thus, to measure the TPC per olive fruit unit—our main goal in evaluating olives as a functional food source rich in important nutraceutical compounds—we considered the average weight of the pulp from 100 fruits per cultivar at each measuring point (T). As shown (
Table 5), a slight increasing trend was observed during ripening for most cultivars; however, this increase reached statistical significance (
p-value < 0.05) only for the Cob and Red cultivars. These cultivars showed increases of 52% and 33% from T1 to T7, respectively, which was mainly due to the increment in fruit caliber and pulp-to-stone ratio (
Table 3). For this reason, both Cob and Red cultivars proved to be the most suitable candidates for being considered a natural food source rich in bioactive compounds, with a higher TPC per fruit unit at T7.
Despite being a favorable overall indicator of the potential content in phenolic compounds, the TPC measurement by the Folin–Ciocalteu is a nonspecific method and thus can be affected by other non-phenolic reducing molecules. Furthermore, since it is a colorimetric method, it cannot be used as a measurement of the effective concentration of specific target phenolic compounds. To address this, a specific method using HPLC was used to measure the target phenolic compounds Tyr, H-tyr, Verb, and its precursor Ole, during fruit ripening. Ole, the primary phenolic compound found in olive fruit, serves as the precursor to numerous other bioactive compounds that are strongly linked to health-promoting properties, including H-tyr [
7]. Therefore, information about the content of Ole in the fruits and its biotransformation pathway during ripening is highly relevant [
27,
28]. Ole concentrations in olive fruit are known to vary greatly within cultivars [
29,
30]. Ranalli et al. [
31] reported the Ole concentrations in olive fruits from seven Italian cultivars during their growth and maturation, demonstrating that the cultivar factor can significantly influence Ole concentration, sometimes even more than the maturity index (MI). In our study, the levels of Ole, Verb, Tyr, and H-tyr were evaluated along with ripening for the seven cultivars under study (
Table 6). As we can observe from
Table 6, all cultivars presented quite distinct phenolic profiles. Alongside TPC, it is of major relevance to acknowledge this information so that we may be able to select the best harvest period for each cultivar to obtain the most proper and nutritious fruits according to the intended purpose. Among all the olive cultivars under study, we can observe a general decrease in Ole concentration during the fruit’s growth and maturation phase (
Table 6), with the cultivar being a major influencing factor for this trend. This decrease in Ole during ripening may be due to the rising activity of hydrolytic enzymes, like β-glucosidase and esterase, which first break down Ole into its simpler forms. Conversely to this, the biodegradation of Ole by its enzymatic hydrolysis will produce an increase in H-tyr, reaching its maximum value at fully ripe fruits (MI ≥ 4).
Analyzing
Table 6, which presents the HPLC analysis of specific phenolic compounds, we find that Gv, at T1, exhibits one of the highest concentrations of Ole at approximately 32 ± 2 g kg
−1, equaled only by Az and surpassed by Red, which showed concentrations of 33 ± 2 and 39 ± 1 g kg
−1, respectively. Nevertheless, at T7, Gv reached the lowest concentrations of Ole, measuring 2.6 ± 0.4 g kg
−1; this may be due to Gv’s earlier ripening process, as it is the only cultivar to achieve an MI of 4 at this stage. Thus, the present data reveals that Gv can exhibit comparatively high Ole concentrations at early ripening stages. However, due to its early maturation process, it reaches minimal levels much quicker than the other cultivars. Furthermore, it was from T1 to T2 that the most significant Ole concentration drop occurred for all cultivars, still at the growing fruit phase. Despite this general decreasing trend, some cultivars showed some positive peaks of Ole accumulation along with ripening. It is not uncommon to observe some Ole accumulation with increasing ripening. In fact, Fernández-Poyatos et al. [
32] reported a significant Ole increase for the ‘Royal’ cultivar, from 15 December (MI 2.5) to 15 January (MI 5.5), from about 0.3 to about 2.8 g kg
−1. But since this Ole increase was shown in the last sampling period, it was not possible to see the evolution trend from this point forward. Also, since sampling started already at an advanced ripening stage, it was not possible to assess the Ole concentration at an early ripening stage. Ferro et al. [
20] reported a similar behavior for the Cob cultivar, with its maximum Ole concentration being the last sampling point (harvest). For some cultivars, namely Gv, Red, Car, and Arb, an Ole increase at some of the early ripening stages was observed, mainly from T2 to T3, which may be attributed to the growth phase of the fruit that occurs with the start of fruit maturation [
33]. Nevertheless, only for the Car cultivar did the Ole increase show to be statistically significant (
p-value < 0.05). For all cultivars, both Ole and TPC showed a similar trend, highlighting the relevance that Ole presents in the phenolic fraction of the olive fruit, transversal to the cultivar variability. The decrease in TPC of olive fruits is a well-known process that occurs during ripening that has been documented by other authors on different cultivars [
33,
34,
35]. Despite this, several published reports demonstrate an increase in TPC on olive fruits as they ripen. Bouaziz et al. [
36] found that during the maturation of the Tunisian ‘Chemlali’ cultivar, TPC increased from 6 to about 16 g kg
−1 of equivalent pyrogallol from July to February. Similar results were observed by Gougoulias et al. [
37], who reported an increase from about 4 to about 12 g GAE kg
−1 along the maturation of the Greek ‘Amfissis’ cultivar. The diversity of results may help us understand the significant chemical variability and distinct behavior that each cultivar shows during ripening, which, together with all environmental factors, may influence the production and accumulation of phenolic compounds in the olive fruit. These sources of diversity highlight the necessity to deepen our understanding of different olive fruit cultivars, especially the traditional Portuguese ones examined in this study.
Considerable differences in the content of Tyr and H-tyr were found to occur in the fruits of different cultivars during ripening, revealing a general increasing trend but not shaped for all cultivars during the sampling period under study. H-tyr is considered an indicator for the maturation of olives, with an increase in their levels consistently correlating with the hydrolysis of the components with higher molecular weights, such as Ole [
7]. In our study, we found that the highest concentrations of H-tyr at T7 were in the Az, Cob, and Gv cultivars, measuring 1.03 ± 0.03, 0.72 ± 0.07, and 0.71 ± 0.06 g kg
−1, respectively. These cultivars also exhibited the highest MI at this stage, confirming the intrinsic relationship between H-tyr synthesis and fruit ripening, which may influence the production and accumulation of phenolic compounds in olive fruit.
Regarding Verb, of all cultivars, Cob shows by far the highest concentrations of this compound, reaching its maximum at T7, with values of 4.3 ± 0.6 g kg
−1, about four times more than Car, which showed the second-highest Verb concentrations. Gv showed the lowest concentrations for Verb, with 0.4 ± 0.1 g kg
−1. These divergencies serve as evidence of each cultivar’s uniqueness, differentiating the specific phenolic profiles shaped along ripeness. Despite the clear differences in phenolic profiles among olive cultivars presented in
Table 6, the nutraceutical relevance of these variations remains underexplored. Establishing direct connections between specific cultivars and their potential health benefits would enhance the practical implications and the nutritional relevance of specific olive fruit cultivars. For instance, the notably higher Verb content in the Cob cultivar suggests a greater potential for anti-inflammatory or antioxidant applications [
38], while the early but abundant Ole accumulation in Gv highlights its suitability for functional food formulations targeting cardiovascular health [
39,
40]. Furthermore, understanding the cultivar-dependent phenolic profiles could support more informed dietary recommendations, valorize traditional Portuguese cultivars, and contribute to the development of targeted functional products aligned with Mediterranean dietary patterns.
Figure 2 shows the chromatographic images of two different cultivars, Gv (
Figure 2a) and Cob (
Figure 2b), at distinct ripening stages (T1 and T7). With this we can highlight how these variables (cultivars and MI) may strongly affect the phenolic profile of the olive fruit. Cob has a noticeable increase in Verb and a slight decrease in Ole as it ripens, while Gv has a sharp drop in Ole but no significant changes in Verb (
Figure 2 and
Table 6).
As shown in
Table 6, of all the quantified phenolic compounds, Ole is by far the most abundant, with the most significant decrease occurring from T1 to T3, which corresponds to the start of fruit maturation. Considering Ole quantification per fruit unit, we can observe from
Figure 3 that along the entire ripening process, Red cultivar showed the highest Ole amounts per fruit unit, ranging from 19.7 mg to 22.7 mg per olive fruit, from T1 to T7, respectively. These results also reveal that although the concentration of Ole generally decreases during ripening (
Table 6), the total amount of specific nutraceutical compounds (such as Ole) can remain constant when measured per fruit unit (
Figure 3). From a nutritional standpoint, such data reinforces the relevance of choosing specific cultivars to ensure a consistent source of bioactive compounds, regardless of ripeness. Results observed from
Figure 3 also reinforce the factor cultivar as more relevant than the MI factor when considering the amount of Ole per fruit unit. Therefore, one may consider these insights to increment the use of olive fruit as a natural source of specific nutraceutical compounds.