Postharvest Fruit Detachment in Oil Palm Bunches with Ethephon and Ethylene Gas Application

The processing of oil palm fresh fruit bunches (FFB), together with loose fruits, in the current mill operation contributes to oil loss and high free fatty acids (FFA), affecting crude palm oil quality. Fruit detachment induced by ethephon and ethylene may mitigate the current processing issues. This study shows that a 0.50% (v/v) ethephon application by the evaporation method induced the highest fruit detachment of 30.8 ± 1.1% after 24 h at room temperature, with the FFA content in the extracted crude palm oil at 0.34 ± 0.09%. Ethephon application was effective on bunches between 14 and 28 kg, and fruit detachment was higher in ripe and underripe bunches at 24.1 ± 0.9% and 23.2 ± 0.1%, respectively. A significant fruit detachment of 47.2 ± 2.4% was achieved when the bunches were also stripped mechanically, but the FFA content increased almost 4-fold, from 1.0 ± 0.2% to 3.8 ± 1.2%. The application of ethylene gas at 750 ppm yielded 29.4 ± 1.9% fruit detachment. The findings present the possibility of using ethylene as an indirect method for minimizing oil loss without increasing the FFA content in future crude palm oil production systems.


Introduction
Conventionally, oil palm (Elaeis guineensis) milling involves the processing of fresh fruit bunches (FFB) that arrive from the estates and are transported to the mill. FFB and loose fruits (LF) on the ground are collected and transported to mills for crude palm oil (CPO) production. The harvesting, transporting, and dumping of FFB from ramp to mill conveyors often damages the fruits and triggers the release of endogenous lipases, the enzymes responsible for the hydrolysis of triglycerides to free fatty acid (FFA) [1,2]. The bruises caused by the bunch-to-bunch impact and the bunch-to-conveyor friction further elevate the formation of FFA, depending on the conveyor length and mill design. In practice, achieving a balance between maximizing the oil yield and maintaining oil quality within the commercial limits of <5% FFA [3] are critical to plantation and mill operations.
FFB mixed with LF ends up in the sterilizer cage, where sterilization deactivates the lipolytic enzymes and restricts the FFA content from increasing further in the fruits. However, sterilizing FFB and LF together in batches under the same conditions is unfavorable, as it leads to the overcooking of the LF. Oil seepage is higher in overcooked fruits, and the oil is either absorbed by the empty fruit bunches (EFB), or lost in the sterilizer condensate, resulting in a lower oil extraction rate (OER) [4]. The OER is one of the key factors in determining the performance of a mill. Therefore, efforts to reduce oil loss due to empty fruit bunch (EFB) absorption, while maintaining good CPO quality during the sterilization step, are needed. Furthermore, the CPO extracted from the LF collected from the field have significantly higher FFA due to the delays in LF collection and processing.
In the future, for oil palm mill design, the most ideal process would be to sterilize the fruits, which are detached from the FFB without EFB [5]. On top of that, separate LF collected from the estates, and overripe FFB, should also be processed separately to avoid spoilage of the good CPO quality, since the LF and overripe bunches are commonly the largest contributor to high FFA content and low CPO quality. Currently, there are no suitable mechanical methods for removing oil palm fruits from the bunches before sterilization, as it tends to be too harsh and can cause significant damage to the resulting detached fruits. Alternatively, fruit detachment can be induced by treating the bunches with ethylene, a natural plant hormone that promotes ripening in climacteric fruits [6][7][8][9][10] and initiates fruits abscission [11,12]. It was demonstrated that the postharvest treatment of oil palm FFB with exogenous ethylene was able to enhance fruit detachment and increase oil yields with lower FFA content [13].
Commercially, ethylene is available either as pure gas or from a synthetic releasing compound, such as ethephon (2-chloroethanephosphonic acid) [14]. The hydrolysis of ethephon at greater than pH 5 yields ethylene, chloride, and phosphate [15]. The use of ethephon for inducing fruit abscission has been demonstrated in peaches [15], mangoes [16], and grapes [17]. Although some studies have shown that ethephon was able to loosen oil palm fruits, and significantly reduce the force needed for fruit detachment, the details of the underlying mechanisms were not known [18,19].
In this study, we further investigate the factors affecting ethephon efficacy on postharvest oil palm FFB processing, and the optimum application dosage on the detachment rate. A study was also conducted to compare the effectiveness of using ethephon and ethylene gas for oil palm fruit detachment.

Fruit Materials
'Tenera' variety oil palm fresh fruit bunches were harvested from commercial fields in Banting and Carey Island, Malaysia. Only bunches that had undergone color change (from deep violet to yellowish orange), and weighing between 14 and 28 kg, were selected for this study. Bunch ripeness was determined according to the number of empty sockets resulting from the freshly detached fruits. The bunches were transported to the research facility within two hours of harvesting.

Ethephon Treatment
The first phase of the study was designed to determine the optimal conditions for ethephon treatment, including the application method, dosage, and incubation period. Ethephon treatment was performed on individual ripe bunches, and each treatment consisted of five replicates (n = 5). The bunches were individually placed inside a covered incubation box (V = 150 L), slightly elevated from the bottom using a metal rack.
Ethephon was prepared as an aqueous solution (200 mL, pH 9) and the initial dosage was fixed at 0.50% (v/v). Two application methods were tested, namely, spraying and evaporation. In spraying, ethephon was applied directly onto the bunches using a hand sprayer, while for evaporation, ethephon was poured into the box and allowed to evaporate. No ethephon was applied to the control bunches (untreated). The bunches were incubated at room temperature for 24 h. Once a suitable application method was established, the ethephon dosage was varied at 0.25% (v/v), 0.50% (v/v), and 1.00% (v/v). The decomposition of ethephon into ethylene was measured using a portable gas detector (PG610, Henan Inte Electrical Equipment Co., Ltd., Zhengzhou, China). The dosage with the highest fruit detachment was used for the remainder of the study. Subsequent treatments were carried out with 6 h, 12 h, and 24 h incubation periods.

Bunch Ripeness and Bunch Size
The second phase of the study was conducted to evaluate the effects of bunch ripeness and bunch size on ethephon-induced fruit detachment. An internal ripeness standard Agriculture 2021, 11, 1030 3 of 10 was used that consisted of four categories, namely, unripe: 0 empty sockets, under-ripe: 1-9 empty sockets, ripe: 10-49 empty sockets, and overripe: >50 empty sockets. Ethephon treatment was carried out under optimal conditions (as determined in 2.2), and each ripeness category was comprised of five bunches with three replicates (n = 15). In the following treatment, the size of ripe bunches was determined according to the bunch weight as either: small: 14-18 kg; medium: 19-23 kg; or large: 24-28 kg. Ethephon treatment was performed on individual bunches under optimal conditions and each size category consisted of five replicates (n = 5).

Fruit Detachment (%) and FFA Content (%)
After the incubation period, the detached fruits were stripped manually from bunches by hand and weighed. Fruit detachment (%) was determined for each treatment according to the formula (total weight of detached fruits/total weight of bunches × 100%). Approximately 1 kg of detached fruits were collected and sterilized at 120 • C (HVE-50, Hirayama, Kasukabe, Japan) for 90 min. Crude palm oil was then pressed from the mesocarp separated from the nut, centrifuged, and the acidity value was determined using a rapid FFA analyzer (PalmOil Tester, CDR Florence, Italy).

Mechanical Bunch Stripping
In the third phase of the study, oil palm bunches treated with ethephon, and control bunches under the optimal conditions from 2.2, were subjected to mechanical bunch stripping using a rotating thresher (L 183 cm × W 112 cm × H 172 cm) with a speed of 1000 rpm. Fruit detachment (%) and the FFA content without threshing (manual), and with threshing (mechanical), were determined, as described earlier.

Ethylene Gas Treatment
The fourth phase of the study was designed to explore the feasibility and reproducibility of ethylene as an ethephon alternative for fruit detachment. The same setup used for ethephon treatment was replicated by using ethylene gas, incubated for 24 h. Each treatment consisted of five replicates (n = 5). Ethylene gas (Gaslink, Puchong Selangor, Malaysia) was transferred into the box by a fixed pressure at a 0.3 bar and a flow of 0.1 L/min. Ethylene concentration was varied at 500 ppm, 750 ppm, and 1250 ppm, which was calculated based on the ethylene concentration detected during the previous ethephon treatment at 0.25% (v/v), 0.5% (v/v), and 1.00% (v/v).

Statistical Analysis
All results were presented as means of the replicates and data accuracy was determined using standard deviation. The statistical analysis of a one-way analysis of variance (ANOVA) with a post-hoc Tukey test (p = 0.05) between the control and treatment groups were determined using Minitab software version 20.4.0.0.

Effect of Ethephon Treatment on Fresh Fruit Bunches
Ethephon was used in the first phase of the study to evaluate the application method, dosage, and incubation period. As shown in Figure 1A (column), fruit detachment was significantly higher (p < 0.001) for the ethephon-treated bunches, for both spraying (28.5 ± 2.1%) and evaporation (28.5 ± 0.4%), in comparison to the control (9.7 ± 3.6%). Visibly, most of the detached fruits came from the outer layer, while the loosened fruits in the middle and inner layers were trapped between the spikelets on the bunch. The total fruits from a fresh fruit bunch typically consist of 50% from the total bunch weight. The middle and inner layers will be made up of 22-25% of the total fruits (internal data). The results in Figure 1B show that the FFA content was lower (p = 0.001) in the detached fruits from the ethephon-treated bunches, whether by spraying (0.6 ± 0.1%), or the evaporation method (0.7 ± 0.3%), as opposed to the control (1.9 ± 0.8%). A higher proportion of fruits with minimal bruising were observed in both treatment groups, which most likely contributed to the low FFA content in the extracted crude palm oil. However, ethephon did not have any effect on the FFA content if the fruits were bruised prior to treatment (unpublished data). Similar results were reported previously [19], where the spraying of a bunch with ethephon had loosened fruits mainly from the outer layer while the FFA content was not influenced. However, a separate study found that treated fruits from bunches fumigated with gaseous ethylene had a lower FFA content than control fruits [13]. Ethylene induced ripening, and abscission activated the senescence pathway and cell-wall deterioration by multiple enzymes, such as expansin, polygalacturonase mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase [20,21]. The senescence process in oil palm fruits will start 160 days after anthesis and once abscission occurs [22]. This alludes that ethephon-treated bunches will not likely have an increase in the FFA content in the fruits once abscised, provided no damages occur that will release lipases and hydrolyze the triglycerides.  Although both application methods produced comparable fruit detachment (p > 0.999), with relatively low FFA content (p = 0.770), the evaporation method was preferred as it avoids direct contact of the ethephon solution with the oil palm bunches. This eliminates the risk of having any residual ethephon, chloride, and phosphate in the extracted crude palm oil.
Significantly higher (p < 0.001) fruit detachment was achieved for all three ethephon dosages in relation to the control, as shown in Figure 1C. The highest fruit detachment was achieved for 0.50% (v/v) and 1.00% (v/v) ethephon, with 30.8 ± 1.1% and 32.0 ± 3.4% detachment, respectively. The loosened fruits trapped in the middle and inner layers between the spikelets in the bunch could not be removed during manual bunch stripping. Thus, increasing the ethephon dosage from 0.50% (v/v) to 1.00% (v/v) did not significantly improve (p = 0.860) fruit detachment. Therefore, 0.50% (v/v) ethephon was established as the optimum dosage to be used in subsequent studies.
Ethephon decomposed into ethylene at pH 6-9, but at a faster rate at pH 9 [23]. Hence, pH 9 was maintained throughout this study. The decomposition of ethephon into ethylene was evident based on the initial accumulation of ethylene inside the incubation box, as shown in Figure 1D. The ethylene levels increased steadily with time, and maximized at 900 ppm, 1500 ppm, and 2330 ppm for 0.25% (v/v), 0.50% (v/v), and 1.00% (v/v) dosages. From the data, it was observed that with ethylene concentration at the 1500 mL/L for 0.5% (v/v), the detachment of fruits was optimum at 30.8 ± 1.1%. The subsequent decline in the ethylene levels could be an indicator of ethephon exhaustion while exogenous ethylene was being absorbed by the bunches to bind to the ethylene receptors to induce abscission. Up to 70 mL/L of ethylene was detected for the control after 24 h, most likely attributed to the endogenous ethylene released during postharvest ripening. This explains the fruit detachment of 5.2 ± 2.5% observed in the untreated bunches after 24 h ( Figure 1E). Similarly, the release of endogenous ethylene after postharvest fruit abscission has been reported in bananas, [24], apples [25], and peaches [26], mainly during the ripening process. However, it was unlikely that the small amount of endogenous ethylene had any significant impact in this study, as enhanced fruit detachment was only achieved in the ethephontreated bunches.
The effects of different incubation periods on fruit detachment and FFA content are shown in Figure 1E,F. The results reveal that the minimum incubation period needed to achieve the minimum fruit detachment at 27.6 ± 4.4%, was 12 h with 0.5% (v/v) ethephon. Although fruit detachment increased slightly to 30.8 ± 1.1% when the incubation period was prolonged to 24 h, the difference was not significant (p = 0.233). There was no presentable FFA data at 6 h, as the number of detached fruits collected was insufficient for oil extraction. The FFA content difference in the ethephon-treated bunches (0.23 ± 0.12%) was insignificant (p = 0.758) to the control (0.30 ± 0.08%) after 12 h. When the incubation period was extended to 24 h, the FFA level increased slightly (p = 0.420) to 0.34 ± 0.09% for the ethephon-treated bunches, while the control increased significantly (p < 0.001) to 0.71 ± 0.05%. On the basis of these results, the incubation periods of 12 h and 24 h were proven to be equally effective in terms of fruit detachment and FFA content. However, when considering convenience and commercial feasibility, 24 h would be ideal, as the bunches can be treated with ethephon immediately after harvesting and the incubation can be carried out overnight. The detached fruits will then be ready for processing the following morning, within the usual mill operating hours.

Effects of Bunch Ripeness and Bunch Size on Ethephon-Induced Fruit Detachment
A further study was performed to investigate if oil palm bunch ripeness and size would affect ethephon-induced fruit detachment. As shown in Figure 2A, fruit detachment was significantly higher (p < 0.001) in the ethephon-treated bunches compared to the control for every ripeness category. Unripe oil palm bunches were responsive to ethylene, similar to other climacteric fruits, such as tomatoes, bananas, and pears, which are usually harvested at the mature green stage and subjected to postharvest ripening [27,28]. Between the treatments, the highest fruit detachment was observed in underripe and ripe bunches, at 24.1 ± 0.9% and 23.2 ± 0.1%, respectively. Fruit detachment was lower in overripe bunches, as most of the outer layer fruits had detached prior to ethephon treatment. The control untreated bunches showed a high percentage of detachment due to the natural senescence activity that causes abscission [29]. The results suggest that ethephon-induced fruit detachment can be advantageous in commercial application, as oil palm bunches with mixed ripeness (predominantly ripe) cannot be avoided during harvesting.
extraction. The FFA content difference in the ethephon-treated bunches (0.23 ± 0.12%) was insignificant (p = 0.758) to the control (0.30 ± 0.08%) after 12 h. When the incubation period was extended to 24 h, the FFA level increased slightly (p = 0.420) to 0.34 ± 0.09% for the ethephon-treated bunches, while the control increased significantly (p < 0.001) to 0.71 ± 0.05%. On the basis of these results, the incubation periods of 12 h and 24 h were proven to be equally effective in terms of fruit detachment and FFA content. However, when considering convenience and commercial feasibility, 24 h would be ideal, as the bunches can be treated with ethephon immediately after harvesting and the incubation can be carried out overnight. The detached fruits will then be ready for processing the following morning, within the usual mill operating hours.

Effects of Bunch Ripeness and Bunch Size on Ethephon-Induced Fruit Detachment
A further study was performed to investigate if oil palm bunch ripeness and size would affect ethephon-induced fruit detachment. As shown in Figure 2A, fruit detachment was significantly higher (p < 0.001) in the ethephon-treated bunches compared to the control for every ripeness category. Unripe oil palm bunches were responsive to ethylene, similar to other climacteric fruits, such as tomatoes, bananas, and pears, which are usually harvested at the mature green stage and subjected to postharvest ripening [27,28]. Between the treatments, the highest fruit detachment was observed in underripe and ripe bunches, at 24.1 ± 0.9% and 23.2 ± 0.1%, respectively. Fruit detachment was lower in overripe bunches, as most of the outer layer fruits had detached prior to ethephon treatment. The control untreated bunches showed a high percentage of detachment due to the natural senescence activity that causes abscission [29]. The results suggest that ethephon-induced fruit detachment can be advantageous in commercial application, as oil palm bunches with mixed ripeness (predominantly ripe) cannot be avoided during harvesting. The results in Figure 2B show that fruit detachment decreased slightly with increasing bunch size, from small (30.4 ± 6.3%), and medium (27.6 ± 4.3%), to large (26.8 ± 4.2%). However, the difference was not significant (p = 0.496). The decreasing ratio of ethylene to bunch (ethylene/kg) with increasing bunch size could explain the small differences in the fruit detachment observed. Since the bunches selected for this study were limited to a range between 14 and 28 kg, the impact of bunch size may be more apparent outside this range. Another reason could be due to the lower ratio of outer fruits to inner fruits, where The results in Figure 2B show that fruit detachment decreased slightly with increasing bunch size, from small (30.4 ± 6.3%), and medium (27.6 ± 4.3%), to large (26.8 ± 4.2%). However, the difference was not significant (p = 0.496). The decreasing ratio of ethylene to bunch (ethylene/kg) with increasing bunch size could explain the small differences in the fruit detachment observed. Since the bunches selected for this study were limited to a range between 14 and 28 kg, the impact of bunch size may be more apparent outside this range. Another reason could be due to the lower ratio of outer fruits to inner fruits, where larger oil palm bunches often have multiple layers of fruits attached to the spikelets forming a compact structure [30], causing the penetration and effectiveness of ethylene to be reduced.

Mechanical Bunch Stripping on Ethephon-Treated Bunches
Manual stripping by human labor is considered ineffective commercially because of the large scale of oil palm bunches involved. A mechanical thresher was used to release the detached fruits from the spikelets at a fast-rotating speed. Figure 3A shows that fruit detachment in the ethephon-treated bunches increased significantly (p < 0.001), from 28.5 ± 0.4% before threshing (manual) to 47.2 ± 2.4% after threshing (mechanical). The comparatively higher force used during threshing resulted in additional fruits being Agriculture 2021, 11, 1030 7 of 10 detached from the outer layer, while some portions of the fruits trapped in the middle and inner layers were also dislodged. Although the FFA content increased (p = 0.026) almost 4-fold, from 1.0 ± 0.2% to 3.8 ± 1.2% ( Figure 3B), due to the increased bruising in the detached fruits, this was still within the commercial oil quality limit, which is commonly set at a 5% maximum [3]. These results prove that the ethephon-treated bunches can be mechanically stripped to further increase fruit detachment without compromising overall oil quality. It must be emphasized, however, that the entirety of this study was carried out in a controlled environment and the bunches were not subjected to the harsh handling otherwise encountered in actual plantation and mill conditions. Achieving similar fruit detachment and FFA content in a commercial setting will only be feasible if optimum facilities and machineries are put in place to overcome such handling issues.

Mechanical Bunch Stripping on Ethephon-Treated Bunches
Manual stripping by human labor is considered ineffective commercially because of the large scale of oil palm bunches involved. A mechanical thresher was used to release the detached fruits from the spikelets at a fast-rotating speed. Figure 3A shows that fruit detachment in the ethephon-treated bunches increased significantly (p < 0.001), from 28.5 ± 0.4% before threshing (manual) to 47.2 ± 2.4% after threshing (mechanical). The comparatively higher force used during threshing resulted in additional fruits being detached from the outer layer, while some portions of the fruits trapped in the middle and inner layers were also dislodged. Although the FFA content increased (p = 0.026) almost 4-fold, from 1.0 ± 0.2% to 3.8 ± 1.2% ( Figure 3B), due to the increased bruising in the detached fruits, this was still within the commercial oil quality limit, which is commonly set at a 5% maximum [3]. These results prove that the ethephon-treated bunches can be mechanically stripped to further increase fruit detachment without compromising overall oil quality. It must be emphasized, however, that the entirety of this study was carried out in a controlled environment and the bunches were not subjected to the harsh handling otherwise encountered in actual plantation and mill conditions. Achieving similar fruit detachment and FFA content in a commercial setting will only be feasible if optimum facilities and machineries are put in place to overcome such handling issues.

Comparison between Ethephon and Ethylene Treatment
Ethephon can be costly and acidic-buffered conditions are recommended for stability. The usage of ethephon will generate waste and needs to be disposed of. Hence, the usage of ethylene gas was explored. Table 1 depicted the concentration of ethylene in ppm after conversion from the average ethylene amount released from three different ethephon concentrations. Figure 4 illustrates that both ethephon and ethylene application produced comparable results (p > 0.05), where 750 ppm and 1250 ppm ethylene yielded 29.4 ± 1.9% and 30.1 ± 2.2% fruit detachment, respectively. In addition, increasing the ethylene from 750 ppm to 1250 ppm did not significantly improve fruit detachment (p = 1.00). Ethylene concentration at 750 ppm should be considered in the future, as approximately 66% of the chemical costs could be saved. If ethephon is substituted with ethylene gas, approximately

Comparison between Ethephon and Ethylene Treatment
Ethephon can be costly and acidic-buffered conditions are recommended for stability. The usage of ethephon will generate waste and needs to be disposed of. Hence, the usage of ethylene gas was explored. Table 1 depicted the concentration of ethylene in ppm after conversion from the average ethylene amount released from three different ethephon concentrations. Figure 4 illustrates that both ethephon and ethylene application produced comparable results (p > 0.05), where 750 ppm and 1250 ppm ethylene yielded 29.4 ± 1.9% and 30.1 ± 2.2% fruit detachment, respectively. In addition, increasing the ethylene from 750 ppm to 1250 ppm did not significantly improve fruit detachment (p = 1.00). Ethylene concentration at 750 ppm should be considered in the future, as approximately 66% of the chemical costs could be saved. If ethephon is substituted with ethylene gas, approximately 266% of the total chemical costs could be further reduced (internal study). Further potential cost savings could be achieved, as there will be no wastewater generated by using ethylene gas in the process. All these factors imply that ethylene has the potential to be used as an alternative fruit detachment agent in future up-scaling studies. ethylene gas in the process. All these factors imply that ethylene has the potential to be used as an alternative fruit detachment agent in future up-scaling studies.

Conclusions
The postharvest treatment of oil palm fresh fruit bunches with ethephon produced high-quality detached fruits that reduced the free fatty acid content in the extracted crude palm oil. Application of ethephon by the evaporation method effectively induced fruit detachment in underripe, ripe, and overripe bunches, irrespective of the bunch size. Fruit detachment was enhanced further when the ethephon-treated bunches were stripped mechanically. Ethylene gas, as an ethephon alternative, also effectively induced fruit detachment by providing a competitive advantage over ethephon in terms of cost reduction and wastewater elimination. These findings can potentially pave the way for new milling technologies to improve the efficiency of the separate processing of high-quality loose fruits. A combination of mechanization stripping coupled with a sterilization process must be integrated in automating the separation of detached fruits from the empty fruit bunch in order to maintain the low level of free fatty acids in the extracted crude palm oil.

Conclusions
The postharvest treatment of oil palm fresh fruit bunches with ethephon produced high-quality detached fruits that reduced the free fatty acid content in the extracted crude palm oil. Application of ethephon by the evaporation method effectively induced fruit detachment in underripe, ripe, and overripe bunches, irrespective of the bunch size. Fruit detachment was enhanced further when the ethephon-treated bunches were stripped mechanically. Ethylene gas, as an ethephon alternative, also effectively induced fruit detachment by providing a competitive advantage over ethephon in terms of cost reduction and wastewater elimination. These findings can potentially pave the way for new milling technologies to improve the efficiency of the separate processing of high-quality loose fruits. A combination of mechanization stripping coupled with a sterilization process must be integrated in automating the separation of detached fruits from the empty fruit bunch in order to maintain the low level of free fatty acids in the extracted crude palm oil.  Data Availability Statement: The data within this paper are available from the corresponding author upon reasonable request.