Effect of storage temperature transfer on the internal browning and other fruit qualities of GP3 and MD2 pineapple clones after postharvest applications of ABA, chitosan and decrowning

Changes in pineapple storage temperature accelerate the increase in internal browning (IB) incidence. This study aims for postharvest applications with changes in storage temperature affecting IB incidence and severity. This research was conducted on ‘GP3’ and ‘MD2’ pineapple clones when they were ripe for export. The experimental were a completely randomized design of storage temperatures (T1: at 7°C for 30 days and T2: at 7°C for 28 days followed by 16°C for 2 days), clones (‘GP3’ and ‘MD2’), decrownings (crown and crownless), and coatings [chitosan 1%, ABA 50mg/L, ABA+chitosan mixture, and control (H2O)]. The results showed that T2 had a significant effect on the increase IB incidence in ‘MD2’ and IB severity in ‘GP3’ compared to T1. Ascorbate acid decreased in ‘MD2’ stored at T2, but had no effect on ‘GP3’. Soluble solid content (SSC) of ‘MD2’ was higher than ‘GP3’. Titratable acidity (TA), SSC/TA ratio, and mold severity were not significantly affected by treatments. The T2 had a significant effect on fruit temperature (FT), with an increase of 2.77-2.88ºC. An increase in FT was positively correlated with maturity, fruit weight loss, and skin dehydration. During the 28 to 30 days of storage, the T2 treatment increased in ripeness by 81.25%.


Introduction
Storage temperature affects the shelf life of pineapples.Storing pineapples at low temperatures can delay yellowing of the skin and freshness of the fruit, extending the shelf life of the fruit [1].'MD2', 'Josapine', and 'Morris' pineapple cultivars have a storage temperature of 10 ºC to prolong shelf life [2].According to [3], pineapples keep well at 6 ºC.The optimum storage temperature for pineapple is 7-12 ºC for the shelf life of fresh pineapple [4].
Prolonged storage at cold temperatures can induce internal browning (IB) before fruit rots.The incidence of IB is an important problem in pineapple postharvest.According to [15], damage due to IB in postharvest pineapples is of particular concern in Sri Lanka compared to damage to the core of the fruit which is a common problem due to high dosage of nitrogen application.After storage of the susceptible pineapple cultivar 'Trad-see-thong' at 10 ºC, the incidence of IB appeared on day 10 of 1230 (2023) 012065 IOP Publishing doi:10.1088/1755-1315/1230/1/012065 2 storage and accelerated on day 8 when the fruit was transferred at 25 ºC temperature for 1 day.In the resistant cultivar 'Pattavia', the incidence of IB appeared after 19 days of storage at 10 ℃, and the incidence rate accelerated until 15 days of storage during fruit transfer.At low temperatures, 'Pattavia' pineapple cultivar were less susceptible to IB than 'Trad-see-thong' cultivar [16].
Transfer of storage temperature in pineapple fruit can reduce the content of endogenous abscisic acid (ABA) and increase endogenous gibberellins (GA), the incidence of IB, and the activity of the enzyme polyphenol oxidase (PPO).According to [17], temperature transfer stored of 'Trad-see-thong' and 'Pattvia' pineapple cultivars for 1 day at 25 ºC from 21 days at 10 ºC had lower ABA and higher GA endogenous content, IB incidence, and PPO enzyme activity.
Ascorbic acid (AsA) content of pineapple affects resistance to IB.The higher the AsA content, the less physiological damage to IB.The AsA content of pineapple fruit can inhibit the physiological damage of IB.The AsA content of pineapple is negatively correlated with the physiological impairment of IB and the activity of the PPO enzyme [19][20][21][22].The inhibitory activity is related to the activity of AsA compounds as antioxidants (acidification and chelation of PPO enzyme) and ROS scavengers that protect cells from damage [22][23][24][25][26][27][28].
The resistance of pineapple to IB is also related to the GA content in the fruit; the higher the GA content, the higher the IB incidence.According to [21], crown pruning will increase the GA content in the fruit, and decrease the ABA and vitamin C content.This shows that the crown is the main source of ABA endogenous, and IB control depends on the continuous transfer of ABA from the crown.
The applications of ABA and chitosan are expected to be effective postharvest applications in extending the shelf life at cold temperatures and temperature transfer by controlling the incidence of IB in pineapples.The application of 50 mg/L ABA is expected to have the same IB suppression effectiveness as the postharvest application of 200 mg/L ABA [19] and 380 µM [20,21] at cold storage temperature.Chitosan coating is expected to increase the effectiveness of ABA in inhibiting IB in pruned pineapple clones 'GP3' and 'MD2' as well as fruits with intact crowns.

Method
The study was conducted on freshly harvested pineapples from the Great Giant Foods Co. Ltd. (GGF) at Terbanggi Besar, Central Lampung, Indonesia ('GP3' clone) and GGF at Labuhan Ratu, East Lampung, Indonesia ('MD2' clone) with an export standard ripeness level [0% yellow color on the skin of the fruit (SC-0)] with a weight of 825-1124 g/fruit.The experimental design used a 4 factorial completely randomized design, namely: storage temperatures (T1: constant storage temperature of 7 ºC for 30 days and T2: storage temperature at 7 ºC for 28 days followed by 16 ºC for 2 days), clones ('GP3' and 'MD2'), decrowning (crown and crownless), and coating [chitosan 1%, ABA (Phytotechlab, Lenexa, Kansas, USA) 50 mg/L, ABA+chitosan mix, and control (H2O)].Fruit coatings with the postharvest applications of ABA and ABA+chitosan blend had been sprayed all over the surface of the fruit till it receives wet (bathes) and the applications of H2O and chitosan had been dipped.In the ABA+chitosan treatment, the fruit changed into sprayed with ABA, and air-dried for half-hour, accompanied through spraying with chitosan.All pineapples that have been dealt with had been airdried for half-hour earlier than being packed in perforated cardboard packing containers with a ability of 10-11 pieces (GGF cardboard packaging), then the culmination had been saved in a storage room with a temperature of 7 ºC.

Incidence and severity of internal browning
Observations of internal browning (IB) incidence were based on the presence or absence of IB symptoms in cross-cut pineapple flesh and were calculated based on the incidence for whole fruit samples.Observation of IB severity based on IB symptom domains calculated from symptom scores.Category scores are based on changes from the American Standard [29] classification for pineapple cultivars as follows: score 0 (0%, no symptoms), 1 (1-5%; mild), 2 (6-10%; moderate), 3 (11-20%; moderately severe) and 4 (21-100%; severe).The percentage of severity of IB was calculated with the formula: IB severity (%) = ∑(ℎ .      ) .  ℎℎ  100% (1)

Fruit temperature
Fruit temperature (FT) measurement with a thermal imaging camera (FLIR E5xt with Wi-Fi).Fruit thermal image (FTI) measurements were carried out at room temperature (25 ºC).The measurements of FTI were presented in the form of images which were visualized in pseudo color.Fruit thermal images were analyzed using the matrix laboratory program.The average FT required a calibration equation obtained from the color-bar.The region of interest (ROI, 70 x 70 pixels) was defined as temperature representation of the fruit object.Fruit temperature was the average value of the pixels in the ROI.

Fruit weight loss
The percentage value for weight loss (FWL) was obtained from the percentage difference between the actual weight minus the initial weight to the initial weight.

Skin dehydration
In fruit skin dehydration (SD) observations, scores were determined by looking at the per-eye fold percentage of 30 fruit eye samples per fruit rotating upward in all directions.Values in the scoring categories were determined by calculating the percentage of symptoms.that is, 0 (0%; no symptoms), 1 (≤25%; mild), 2 (≤50%; moderate), and 3 (51-100%; severe).The percentage of SD was calculated using the formula:

The incidence of internal browning
Transfer of storage temperature of pineapple for 2 days from 7 ºC for 28 days followed by 16 ºC until 30 days of shelf life (T2) has a higher incidence of internal browning (IB) compared to a constant temperature of 7 ºC for 30 days (T1).There was an interaction between clone and decrowning stored at temperature T1 on the incidence of IB.Crown pruning of 'GP3' pineapple clone stored at T1 increased the incidence of IB, while pruning of 'MD2' clone stored at T1 decreased the incidence of IB, although not significantly (figure 1).
'GP3' pineapple clone whose crowns remained intact and stored at T1 had a lower incidence than pineapples whose crowns were pruned or stored at T2.In 'MD2' pineapple clone whose crowns were pruned and stored at temperature T1, the incidence of IB was lower than for fruit stored at temperature T2, but not significant for unpruned fruits stored at temperature T1.However, in terms of efficiency, pruning the 'MD2' fruit crowns stored in T1 can reduce packaging costs.The incidence of IB in pineapple fruit can be reduced to 0% at T1 storage temperature by treating pineapple clone 'GP3' with intact crown, 1% chitosan, and 50 mg/l of ABA in a mixture of ABA+chitosan and 50 mg/l of ABA.A pineapple clone 'MD2' with the crown cut off.Pineapple coating was unable to reduce the incidence of IB when stored in T2 (figure 1).

The severity of internal browning
Storage temperature affects the severity of internal browning (IB), typically within 30 days.Pineapples stored at a transfer temperature of 7 ºC for 28 days followed by 2 days at 16 ºC (T2) had a higher IB severity than pineapples stored at a constant temperature of 7 ºC (T1).The combination of storage temperature, clone and decrowning did not show significant differences in IB severity.Pruning of pineapple clone "MD2" stored on T2 resulted in a higher severity of IB than on T1.Pineapple storage at T2 temperature was not significant compared to T1 temperature for all combinations of clone and coating treatments.IB severity can be reduced to 0% only by T1 storage temperature treatment by postharvest treatment with 50 mg/L of ABA for pineapple clone 'GP3' with crown intact and 1% chitosan, 50 mg/L ABA, and ABA+chitosan mixture for pineapple clone 'MD2' with pruned crown (figure 2).

Ascorbic acid
The 'MD2' clone had a higher ascorbic acid (AsA) content than the 'GP3' clone.The storage transfer temperature of 'MD2' pineapple clone from 28 days at 7 ºC to 16 ºC for 2 (T2) had a decrease AsA content in comparison to pineapple stored at a consistent temperature of 7 ºC (T1), particularly 276.95 and 363.75 ppm, it was not on 'GP3' clone.Crown pruning of 'MD2' pineapple clone stored at T2 reduced AsA content, but could be maintained through coatings application.AsA content after H2O treatment (control) in 'GP3' clone pineapple stored at T2 temperature reduced 31.03ppm in comparison to fruit stored at T1 temperature, but could be maintained by coatings (figure 3).

Other fruit qualities
Storage of pineapples for 28 days at 7 ºC accompanied through 2 days at 16 ºC did not affect on the soluble solid content (SSC), titratable acidity (TA), sweetness (STR), and mold severity as compared to store at a constant temperature of 7 ºC for 30 days.'MD2' clone pineapple had higher SSC and sweetness as compared to 'GP3' clone pineapple.Storage of pineapples at temperature T2 increased the mold intensity of 'GP3' clone with pruned crowns.Mold severity on pruned 'GP3' pineapple stored at temperature T2 can be decreased through postharvest application of fruit coatings.There became no significant distinction in mold severity in coatings 'GP3' pineapple clone stored at temperature T2 with all combinations at temperature T1 (  Storage of pineapples at T2 affects fruit temperature (FT), shell color (SC), fruit weight loss (FWL) and skin dehydration (SD).Pineapple storage in T2, FT, SC, FWL and SD were higher than T1.Increases in fruit temperature were positively correlated with increases in SC, FWL and SD.Color index suppression of GP3 clones in pineapples stored at a temperature of T2 after postharvest application ABA was applied at a dose of 50 mg/L for fruits with pruned crown and ABA + chitosan mixture was applied for fruits with intact crowns (table 2).

Discussion
Pineapple stored for 28 days at 7 ºC followed by 2 days at 16 ºC (T2) had significantly higher the incidence of internal browning (IB) in 'MD2' pineapple clone (see figure 1) and the severity of IB in 'GP3' pineapple clone (see figure 2) compared to fruit stored at a constant temperature of 7 ºC for 30 days (T1).The results of this study are supported by several other studies which state that storage temperature shifts can affect IB.Additional storage time of pineapple for 1 day from constant temperature of 10 ºC to 25 ºC in 'Trad-see-thong' (IB susceptible) and 'Pattavia' (IB tolerant) pineapples exacerbated the incidence of IB [16,17].
According to [30], pineapple fruit stored at 13 ºC followed by storage at 25 ºC for 7 days on pineapple cultivars 'Smooth Cayenne' and PRI hybrid '73-50' exacerbated IB symptoms and polyphenol oxidase (PPO) enzyme activity compared to constant fruit stored at 25 ºC for 3 weeks.Based on the comparison of the results obtained with the results of other studies, the transfer of storage temperature from cold (7 or 10 ºC) to warmer temperatures (16 or 25 ºC) during transfer (2 or 1 day) on susceptible cultivar ('Tradsee-thong' type Queen), tolerant ('Pattavia' type Smooth Cayenne), and resistant ('MD2' type hybrid) can exacerbate the physiological damage of IB in pineapple.According to [31], increasing the storage temperature for 12 days of storage can double the IB after 14 days of storage in pineapple cultivars 'Morris' (Queen) and 'Josapine' ('Spanish' hybrid x 'Smooth Cayenne').According to [17], changes in pineapple storage temperature increased the incidence of IB in the pineapple cultivars of 'Trad-seethong' and 'Pattavia'.The incidence of IB accelerated from 2 to 8 days in the susceptible pineapple cultivar 'Trad-see-thong' and from 4 to 15 days after shifting the storage temperature from 10ºC to 25ºC for 1 day in the resistant cultivar 'Pattavia' [16].
The ascorbic acid (AsA) content of the 'MD2' pineapple clone was higher than that of the 'GP3' pineapple clone.'MD2' pineapple clone stored at T2 temperature reduced the content of AsA compared to fruit stored at T1 temperature, respectively 276.95 and 363.75 ppm.The decrease in AsA content of pineapple stored at T2 temperature could be suppressed by postharvest application coating to 'MD2' clone with crownless and 'GP3' clone with intact crown (see figure 3).This showed that there was a negative correlation between AsA levels and IB physiological damage in pineapple, the higher the AsA level, the lower the IB damage.AsA content in pineapple suppressed IB physiological damage, this was related to the activity of AsA compounds as antioxidants (acidification and PPO enzyme chelating).Transferring to storage temperature in pineapple can reduce the content of abscisic acid (ABA) endogenous and increase gibberellin (GA) endogenous, incidence of IB, and activity of polyphenol oxidase (PPO) enzymes [17].Pineapple fruit which is stored at 13 °C followed by storage for 7 days at 25 ºC, the fruit of the cultivar 'Smooth Cayenne' and PRI hybrid '73-50' show an increase in polyphenol oxidase (PPO) activity along with an increase in IB symptoms.
'MD2' clone pineapple had significantly higher AsA content than 'GP3' clone pineapple (see figure 3) with lower IB severity (see figure 2).AsA functions as an antioxidant and reactive oxygen species (ROS) scavenger, so it can protect cells from damage [23][24][25][26].AsA protects the fruit from softening and spoilage due to oxidative stress and biotic stress by increasing antioxidant levels [23].The AsA content of pineapple negatively correlates with the physiological damage to IB and the activity of the PPO enzyme [19][20][21][22]27].A delayed decrease in AsA concentration in plums can reduce the incidence of IB [31].An increase in AsA content can inhibit the incidence of IB in Trad-see-thong pineapple cultivars [28] and loquat fruits [33].
An interaction was found between cloning ('GP3' and 'MD2') and decrowning (crown and crownless) in IB incidence.Pruning of pineapple clone 'GP3' (type 'Smooth Cayenne') increased IB incidence and decreased IB incidence in clone 'MD2' (hybrid type), which was further exacerbated at T2 storage temperature.This is supported by data [21], where pruning of pineapple crowns can increase the incidence of IB in Queen type pineapple.The combination of cloning and coating did not show a significant difference in storage temperature.
This demonstrates that coating of pineapple clones did not affect the incidence and severity of IB and AsA at storage temperatures T1 and T2.Treatment of ABA 50 mg/L on 'GP3' clone pineapple whose crown remained intact and coating on 'MD2' clone pineapple pruned at T1 storage temperature reduced the incidence of up to 0% for 30 days of storage, although there was no significant difference in AsA content.This is supported by [34], there is no correlation between AsA and the incidence of IB.Other factors may affect the incidence of IB, apart from the AsA content in pineapple fruit, to the incidence of IB in the interaction of clone and coating treatment at a constant temperature of 7 ºC.
Storage of pineapples for 28 days at 7 ºC followed by 2 days at 16 ºC did not affect the soluble solid content (SSC), titratable acidity (TA), sweetness (STR), and mold intensity compared to storage at a constant temperature of 7 ºC for 30 days.'MD2' clone pineapple had higher SSC and sweetness compared to 'GP3' clone pineapple.Storage of pineapples at T2 temperature increased the strength of mold clone 'GP3' with a pruned crown.Mold severity in pruned 'GP3' clone stored in T2 can be reduced by post-harvest fruit coating.There was no significant difference in mold severity in coatings of 'GP3' pineapple clone stored in T2 for all combined treatments in T1 (see table 1).Storage of pineapples at T2 affects fruit temperature (FT), shell color (SC), fruit weight loss (FWL) and skin dehydration (SD).Pineapple storages in T2, FT, SC, FWL and SD were higher than T1.Increases in FT were positively correlated with increases in SC, FWL and SD.Color index inhibition of GP3 pineapple clone stored at T2 temperature after post-harvest application of 50 mg/L of ABA to fruits with pruned crown and ABA + chitosan mixture to fruits with intact crown (see table 2).

Conclusion
Temperature changes of pineapple stored from 7 ºC for 28 followed by 2 days at 16 ºC (T2) had a higher incidence in 'MD2' pineapple clone and the severity of internal browning in 'GP3' pineapple clone and lower AsA although not significant in 'GP3' pineapple clone compared to postharvest applications stored at a constant temperature of 7 ºC for 30 days (T1).The AsA content of 276.95 ppm affected the suppression of the IB severity in pineapple.The reduction of AsA in stored pineapples at T2 was inhibited by the application of postharvest coatings to the 'GP3' clone with crowns were intact and the 'MD2' clone with crownless.Crownless can only be carried out in storage of 'MD2' pineapple clone at temperature T1 to reduce the incidence and severity of IB to 0% for 30 days with a mixture of ABA 50 mg/L, chitosan 1%, and ABA+chitosan mix.In 'GP3' pineapple clone, the incidence and severity of IB were reduced to 0% by ABA 50 mg/L exogenous with intact crown stored at T1 for 30 days.Postharvest applicatios of clone and decrowning combinations did not affect T2 storage temperature for IB severity at 30 days.Storage of pineapple at T2 temperature increased fruit temperature compared to T1 temperature and was positively correlated with shell color, reduced fruit weight and skin dehydration, but had no effect on soluble solid content, titratable acidity, sweetness and mold.The 'MD2' pineapple clone had lower IB incidence and severity and higher AsA, SSC and sweetness compared to the 'GP3' pineapple clone.

Table 2 .
Pineapple qualities change of transfer temperature storage for 2 days on fruit weight loss (FWL), skin dehydration (SD), shell color (SC), and fruit temperature (FT)