The fruit for the experiment was harvested on a farm in Bollullos Par del Condado in 2017 (Huelva, Andalusia, Spain). The olive grove covers 8 ha and includes 1200 trees, primarily of the cultivars ‘Arbequina’, ‘Picual’, and ‘Verdial’. The trees were planted between 2005 and 2007 at distances of 6 x 7 m and irrigated according to a deficient regimen.

The ‘Arbequina’ fruit was collected in the second week of October (mean color index (CI): 34.5); the ‘Picual’ fruit in the first week of November (CI: 28.5); and the ‘Verdial’ fruit in the last week of November (CI: 20.81). To simultaneously evaluate the effects of the collection and conservation methods on fruit quality, 40 kg of olives were harvested with each collection method, namely a Manual Inverted Umbrella (MIU), referred to as Recollection Method 1 (R1), and the traditional method with nets, Recollection Method 2 (R2). From this 40 kg set, half was randomly taken and placed in a refrigerated room at 5 ºC, referred to as Storage Method 1 (S1); while the other half was stored indoors, but outside the refrigerator at room temperature, Storage Method 2 (S2), as is usually done with olives before processing. The physiological characteristics of the olives were evaluated at times: 0, 4, 8, and 14 days of storage (ST).

The MIU was designed to catch and collect the olives into boxes, preventing them from touching the ground (Figure 1a). A detailed description of the device can be found in Plasquy et al. (2019). The boxes where the picked olives were collected have a capacity of 20 kg, placed under the umbrella, and removed when full using a system of pulls and ropes. The harvesting team consisted of two operators who detached the fruit with branch shakers and two men who handled the MIU and the boxes. The traditional way of harvesting (R2) to which the R1 was compared, used nets that were placed under the tree (Figure 1b). Two operators with branch shakers stepped on the nets and moved around the tree to detach the fruit. Once picked, the nets with the fallen fruit were dragged to the next tree and spread open by two other men after which the operators continued to detach the fruit with the branch shakers. This continued until the nets became too heavy to displace them to the next tree. At that moment the collected olives were transferred into the same type of boxes used in R1.

The cooling room was installed in an existing outhouse on-site at the farm (Figure 1c). The existing construction measured 4.3 x 4.4 m on the ground floor. The inclination of the roof served to install a ceiling at 2.5 m, after which two separate spaces were created. Behind the existing barn gate of 3.0 m width, a separation wall was mounted with 10-cm thick extruded polystyrene sandwich insulating panels and a cooling door of 1,2 m width. The inside walls and the ceiling were isolated with insulating panels of 10 cm. The floor was covered with the same panels and finished with wooden boards of 2.5 cm (OSB) and a rubber protector. On the upper level a cold group was placed, formed by a compressor (EMBRACO, UNJ 9232) and the evaporator (LU-VE, SHDN 25-80) was mounted on the ceiling of the cooling room. A thermostat was mounted on the outside to have visual control of the inside temperature. The capacity of the cooling room amounted to 6.000 kg. The system was able to keep the chamber at 5 ºC (± 1 °C). To maintain this temperature, the compressor started every 5 to 10 min and ran for approximately 4 min. The cost to install the cooling room amounted to 2.500 €, including the purchase and installation of the refrigerating group by a technician, the isolation panels, the iron structure, and the finishing of the floor. The energy consumption amounted to 16 kWh, taking into account a consumption of 2 kW for the different motors present, working one-third of the time.

For each cultivar, decay incidence, weight loss, firmness, respiration rate, and ethylene production were analyzed by one-way ANOVA to determine the effect of the four treatments at each storage time (ST). The same variables were analyzed by two-way ANOVA to determine the effect of the treatments and the ST, and by three-way ANOVA to detect the effect of the three independent factors (four ST, two R, and two S). When a significant (P < 0.05) effect due to treatment or ST was detected, the Tuckey test was applied to distinguish mean values (P < 0.05).

The parameters of skin color did not show normal distributions and were not studied using parametric tests. Each value was identified by the mean value of 100 replicates ± standard deviation (SD). The nonlinear curve fit of the histograms of the IC (OriginPro 9.1.0, OriginLab Corp. Northampton, USA) are presented for 0 and 14 storage days in the four treatments tested (two R and two S) in each cultivar.

In all the studied cultivars both the ST and the use of different treatments affected decay incidence in one way at least, which was very significant (P <0.01), considering all the ST and each sampling date independently for this period (Figure 1). During the progress of the ST, the values for this variable increased differently according to the treatment applied. The recollection method turned out to be decisive for the changes in decay incidence in the stored fruit. Thus, the treatments using the R1 showed significantly lower decay than the harvest using nets, regardless of the storage temperature or the cultivar tested. The cold storage also showed an effect on ‘Arbequina’ and ‘Picual’ cultivars for this parameter. ‘Verdial’ olives presented no significant differences due to the different storage temperatures. However, systematically, the treatments with cold storage obtained lower decay values than their counterparts. The interaction between the R and the S only caused an effect on ‘Arbequina’ olives for this variable. During the ST decay incidence significantly increased in all the treatments and cultivars. However, each variety showed different behavior in this period. Whereas in ‘Arbequina’ fruit the differences between the treatments R2S2 and the rest increased throughout this period. The differences among the rest of combined treatments decreased and were not significant from the third sampling date (day 8). Similarly, the differences in decay incidence in ‘Picual’ olives between R2S2 and the other treatments increased over the examined storage time. The three lasting treatments stayed akin with respect to the decay incidence until R1S1 exhibited the significantly lowest values for this parameter at day 14. In contrast, ‘Verdial’ fruit was affected by R throughout the entire storage period, except for the last sampling date (day 14), when, as for ‘Picual’ olives, the treatment R1S1 showed the significantly lowest values for this parameter.

The effect on this parameter was extremely significant for the three varieties (P < 0.001) according to the treatments applied, the progress of storage time, and the interactions of both factors (Table 1). As in the case of decay incidence, the values for this variable increased differently during storage according to the combination of treatments applied. However, for this parameter, the storage temperature was the only factor that exerted a significant effect on the weight loss of the fruit, regardless of the cultivar tested. Cold storage significantly delayed the increase in this parameter during storage and this effect was increased significantly as the storage time became greater.

According to chromatic values (L*, a* and b*) cold storage delayed fruit ripening. This effect was most evident in the olives, ‘Picual’ and ‘Verdial’, which exhibited similar variations in these chromatic parameters from the second sampling date after 4 storage days, and then the differences increased compared to the cold-stored ones, which only showed slight differences from the initial values (Table 2). The ‘Arbequina’ olives maintained similar color parameters over 8 days of storage, regardless of the harvest or storage methods. However, after 14 days of storage, the olives stored under ambient conditions presented obvious changes in these parameters. While L* and b* values decreased, a* increased. This means that the skin of this fruit was darker, red and blue and less shiny, green and yellow. Consequently, these olives exhibited a darker purple (red + blue) skin color than those stored at 5 °C.

The non-linear curve fits of the CI histogram values obtained from the different treatments in the three cultivars tested after 0 and 14 storage days showed that during the storage period the fruit was separated into three groups (Figures 2, 3, 4). This three-modal profile evidenced that these distributions cannot be statistically evaluated by parametrical tests to know whether the effect of the different factors is significant. However, these histograms showed clear evidence that after 14 days of storage, the treatments kept at ambient temperature, regardless of the method used for harvesting, exhibited a higher frequency in the group with the lowest values for this parameter; whereas the cold-stored ones maintained higher frequency values in the group with the highest values for this chromatic formula. As CI diminished according to the progression of fruit ripening, it seems clear that the use of cold storage delayed this process.

Similarly, as in the cases of weight loss and incidence of decay, the fruit of the ‘Picual’ and ‘Verdial’ cultivars tested showed similar behavior in changes in firmness, and were affected in an extremely significant way by the treatments applied, storage time, and interactions between these two factors. In the case of ‘Arbequina’ the effect of the treatment was only significant at day 14. (Table 3). All the fruit exhibited a decrease in the firmness parameter during storage time, but it was more evident in the fruit stored at ambient temperature, regardless of the method used for harvesting. The firmness of ‘Verdial’ fruit was not affected by the R. Both ‘Arbequina’ and ‘Picual’ fruit firmness harvested by R1 showed significantly higher mean values for this parameter than the R2 harvested fruit. Cold storage allowed for maintaining significantly higher mean values for firmness in the fruit of the three cultivars. As storage time progressed, the effect of factors R and S increased their effects on fruit firmness. The ‘Arbequina’ and ‘Verdial’ olives did not show a significant effect of R or storage time. However, both cultivars, as well as ‘Picual’ were significantly affected by the combined effect of the three factors (ST, R, and S).

The production of CO₂ followed the same profile as the other studied parameters, showing a highly significant effect due to storage time, the combination of treatments applied, and the interaction of both of them (Table 4). Storage method resulted in the most important cause of variability for this parameter for the three varieties considered. This effect increased with the progression of the storage time. The fruit stored at 5 ºC showed a significantly higher respiration rate than the one stored at ambient temperature when this parameter was tested at 20 ºC. This effect increased with the progression of storage time. Only ‘Arbequina’ olives showed a significant effect due to R, which did not significantly interact with the progression of storage time. From the beginning of the storage period, the fruit harvested with R2 exhibited a higher respiration rate than the others. ‘Picual’ olives turned out to be especially sensitive to the combinations of the factors tested, but was not affected by the individual action of the different R.

All the varieties were affected at least very significantly (P < 0.01) in the amount of synthesized ethylene due to storage time and the four different treatments applied. Except for ‘Arbequina’ olives, which did not show significant differences in the amounts of synthesized ethylene induced by the combination of the effect due to storage time and treatment, the rest of the varieties were also affected by the interaction of these two factors. The recollection was mainly responsible for these effects. For all the varieties tested, the fruit harvested with nets showed higher amounts of synthesized ethylene than when picked with MIU. The storage determined an extremely significant effect on the amount of synthesized ethylene (P < 0.001) in ‘Picual’ and ‘Verdial’ olives, while in ‘Arbequina’ olives this effect was very near to being significant (P < 0.063). ‘Picual’ and ‘Verdial’ fruit was also, at least, significantly (P < 0.05) affected by the interaction of ST and R, ST and S, R and S, and for the triple interaction of these factors. Both varieties showed a different profile in the changes of these parameters during storage time. From the initial storage time (day 0) all the varieties tested showed higher values for this parameter when they were harvested with nets (R2). However, from the next sampling date (day 4) they showed different profiles. Whereas in ‘Arbequina’ olives these differences decreased during the following two sampling dates and increased in the last one, in ‘Picual’ olives the differences had increased on the second and the third sampling dates and decreased on the last one. Finally, in ‘Verdial’ olives these differences had decreased by the second sampling date of storage, but, subsequently, only increased in the fruit stored at room temperature.

The fact that the effect of recollection was more evident than storage is not surprising, although the fruit harvested using both systems did not show differences in the incidence of physiological deterioration at the moment of harvesting (data not shown). Yousfi et al. (2012). compared the effect of ‘Arbequina’ olives picked using two different harvesting systems (hand and mechanical with an adapted wine grape harvester) during cold storage. Although they did not appreciate any differences in the appearance of the fruit immediately after harvesting, subsequently they found an exponential increase in decay incidence in mechanically-harvested fruit stored at 3 ºC. In contrast, hand-harvested fruit was not significantly decayed after two weeks of cold storage. In the present study, the differences between R1 and R2 can be explained because the use of R2 inevitably implies the involuntary treading on of the fallen fruit by the operators and the dragging over the ground of the fruit on the nets during its displacement to the next tree until reaching the filling of the boxes. This infers a serious risk of infection with soil pathogens, given the micro-wounds that this activity inevitably provokes. The effect of cold storage (5 ºC), maintaining initial physiological integrity has been observed in different works, using different olive cultivars (Castellano et al., 1993; García et al., 1996; Rinaldi et al., 2010). In this study, the effect of S1 proved to be more evident with the progression of the storage period, whereas the effect of R1 was already noticeable from the second sampling date (4 days). Cold storage requires the combined effect of a careful harvest to maintain a minimum amount of decay in the stored fruit.

As expected, the storage temperature determined the level of fruit transpiration and, consequently, the olives maintained under refrigeration showed lower weight loss than the ones kept in ambient conditions. In contrast, the harvesting method did not show any effect on this parameter. Yousfi et al. (2012) found the same effect of low temperature delaying the progress of weight loss during the storage of ‘Arbequina’ olives, and they also observed a significant effect due to the recollection method because the use of an adapted grape harvester turned out to be more damaging to fruit than when picked by hand. The results presented in this study should be placed between these two extremes. They are higher than the ones obtained by hand-harvest and lower than when compared with the adapted grape harvester.

As for weight loss, the observed shift in skin color was only produced by storage temperature. Room temperature accelerated changes in the skin color, while refrigeration delayed it. This fact was previously observed in ‘Picual’ olives by Castellano et al. (1993) and by Yousfi et al., (2012) using ‘Arbequina’ fruit. In this paper, the appearance of the three model profiles when stored at room temperature typified this gradual color shift in the three studied varieties.

The observed changes in firmness showed a similar process as discussed by Yousfi et al. (2012). As the storage time progressed, this parameter decreased in all the combinations of treatments, but fruit refrigeration delayed this softening. Furthermore, the damaging action of the harvest method used is related to this process. While in the study by Yousfi et al. (2012) the internal partial breaking of the cellulose wall of the olive mesocarp cells was a consequence of the mechanical harvesting method, in this study the responsible agent was related to the use of nets.

Low temperature produces a decrease in the respiration rate. Maintaining the fruit at 5 ºC produced a reduction in the metabolism of the fruit cells; while fruit kept at room temperature continued its normal activity, including respiration (Rinaldi et al., 2010). The concentration of substrates for respiration (sugar, fatty acid, etc.) is limited for detached fruit. The olives stored at room temperature consumed these soluble substrates continuously; while refrigerated fruit still disposed of a higher concentration of these metabolites when brought at 20 ºC for respiration measurements. These processes explain the higher CO₂ production levels in refrigerated fruit. Meanwhile, the higher respiration rate showed by ‘Arbequina’ olives harvested with nets could be attributed to its damaging action, which can accelerate this parameter. Segovia-Bravo et al. (2011) found a higher CO₂ production in naturally bruised Manzanilla olives than in the healthy ones. Similarly, Morales-Sillero et al. (2015) observed that the fruit of two varieties (Manzanilla de Sevilla and Manzanilla Cacereña), harvested with a grape straddle harvester, showed a higher respiration rate than the hand-picked ones. They related this fact to the damaging action of this harvest method. Jimenez et al. (2017) demonstrated that the internal damage at morphological and histological levels differed between these varieties. In this study, ‘Arbequina’ olives seemed to be more sensitive to the action of the nets during harvest than ‘Picual’ and ‘Verdial’ olives.

The significantly higher amounts of ethylene synthesized by R2 harvested fruit indicated that this method was more damaging to the olive physiology than R1. This result coincides with that obtained by Morales-Sillero et al. (2017), who studied the ethylene production of Manzanilla and Cacereña olives stored at 2 ºC, which were harvested by hand or mechanically, using a grape straddle harvester. They found a significantly higher ethylene biosynthesis in the mechanically harvested fruit and identified this with the fact that this harvesting method was more detrimental to fruit integrity. Other causes for the increase in ethylene biosynthesis could be the natural progression of fruit ripening (Ardilla et al., 2012; Hyodo, 2018). When the R2S1 ‘Arbequina’ olives were placed at 20 ºC to measure their ethylene production it could be considered normal that they experimented a higher synthesis than the R1 ones because they were more damaged, but they showed significantly lower values for this parameter than R2S2 fruit, which was also damaged but with a higher level of ripening because it was maintained at a higher temperature during storage. R1 ‘Arbequina’ olives showed the same behavior during the first three sampling dates because they were not damaged but the value increased because ripening had progressed. Finally, on the last sampling date, the olives stored at a higher temperature showed a significantly higher ethylene production, evidencing a higher level of ripening. The profile of ‘Picual’ olives only differed from the one shown by ‘Arbequina’ olives in the maxima values exhibited by R2 olives in the third sampling date, followed by a clear reduction in this parameter during the last moment of storage. This was probably due to a particular characteristic of this variety, in which ethylene biosynthesis would only coincide with ripening at the beginning of this process. R1S2 ‘Picual’ olives did not show increasing values for this parameter at the end of the storage period when the ripening level of these olives was probably more evident. In contrast, the progression of ripening was better controlled by the cold storage in R1S1 olives, which showed the lowest ethylene production without changes throughout the storage period. The low values for ethylene production shown by S1 ‘Verdial’ olives could be related to better control of the ripening process due to cold storage. In contrast, fruit maintained at ambient temperature from the second sampling date showed increasing values for this parameter, which could be related to an accelerated ripening. Each variety showed a particular profile of ethylene production, due to its characteristics, such as sensitivity to the harvest system, response to cold storage, or the progression of ripening.