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Papaya treatment with putrescine maintained the overall quality and promoted the antioxidative enzyme activities of the stored fruit
Scientia Horticulturae 268 (2020) 109367
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Papaya treatment with putrescine maintained the overall quality and promoted the antioxidative enzyme activities of the stored fruit
T
Aliya Hanifa, Saeed Ahmada,*, M. Jafar Jaskania, Rashid Ahmadb a b
Institute of Horticultural Sciences, University of Agriculture Faisalabad Pakistan, Pakistan Department of Agronomy, University of Agriculture Faisalabad Pakistan, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: Carica papaya Catalase Peroxidase Superoxide dismutase Total antioxidants Total phenolic contents Shelf life
Papaya is an emerging, profit generating fruit of Pakistan having high nutritional value. It has very limited shelf life which limits its long distance transport and resulted in high postharvest losses. Putrescine has great potential to maintain the firmness, quality of fruit and reduce the losses. Therefore, the role of putrescine for balancing fruit firmness, enzyme activities and variations in biochemical properties of Red lady papaya fruit were evaluated during storage. Mature unripe papaya fruit were subjected to different concentrations of PUT (0 mM, 1 mM, 2 Mm, 3 mM) and then stored at 12 °C temperature and 90–95 % RH for 28 days. Fruit firmness, weight loss, antioxidant enzyme activities (CAT, SOD and POD), total phenolic, total antioxidants and other biochemical attributes were studied on weekly basis. Fruit firmness was substantially higher in putrescine treated fruits along with less weight loss % during storage. TSS and ripening index were higher in control fruit, while they were lower with PUT treatment. 2 mM PUT suppressed the decay incidence during whole storage period which was almost 2.9 times less than control fruit, similarly the activity of CAT enzyme was maximum (6.94 U/mg protein), POD (1.07-fold higher than control) and SOD was also higher in the same treatment. Total antioxidants and total phenolic contents were at upper limits in fruit treated with 2 mM PUT during storage. It can be concluded that 2 mM PUT is helpful for extending the shelf life of papaya fruit by suppressing fruit softening, fruit decaying and by enhancing the enzyme activities and maintaining good keeping quality during storage.
1. Introduction Papaya, Carica papaya L., is important fruit plant which grows rapidly and produces fruit throughout the year (Paul and Duarte, 2011). Papaya is called powerhouse of essential nutrients. It is an excellent source of ascorbic acid, carbohydrates, fibers and vitamins. It contains high amount of papain which aids in digestion (Oloyede, 2005). It contains and is rich in phytochemicals, total phenolics and antoixidative enzymes. However, postharvest losses in papaya are very high, because it is highly perishable and being climacteric in nature gets deteriorated easily due to rapid respiratory activities even after harvest and creates hurdle in its long distance transport. Postharvest losses of fruit and vegetables occur at various stages during transport, in storage houses or at market level (wholesale, retail). Fruit softening and low temperature injury are the major constraints in long distance transport of Red lady papaya (Nunes et al., 2006). However, microbial decay, physical injury and loss of firmness are the major postharvest defects of papaya which limits its shelf life and shipment to distant places (Hernandez et al., 2006).
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Externally applied polyamines have great potential for lengthening the postharvest life and preserving the texture of many fruit e.g. strawberry, plum, apricot and mango (Martinez-Romero et al., 2002; Malik and Singh, 2005 and Khosroshahi et al., 2007). Polyamines are positively charged organic molecules, light in weight and are existed in nearly all respiring organisms for regulating their various biological processes like growth and development (Barman et al., 2011). Putrescine (PUT), spermine (SPM), cadaverine (CAD) and spermidine (SPD) are biologically active forms of polyamines that can regulate various physical, physiological and biochemical processes of fruit (Malik and Singh, 2005; Khan et al., 2007). It was elucidated by Valero et al. (2002) that polyamines can retain the quality of fruit by suppressing ethylene production, lowering respiration rate, delaying color change, maintaining firmness and inducing resistance against mechanical damage. Postharvest treatment of climacteric and non-climacteric fruit with polyamines increases their shelf life with good quality maintained (Wang et al., 1993). Storage life of fruit is primarily relied on its ripening because it regulates the ultrastructure of cell wall (Singh et al., 2013).
Corresponding author. E-mail address: [email protected] (S. Ahmad).
https://doi.org/10.1016/j.scienta.2020.109367 Received 31 October 2019; Received in revised form 11 March 2020; Accepted 12 March 2020 Available online 19 March 2020 0304-4238/ © 2020 Elsevier B.V. All rights reserved.
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2.2.4. Respiration For measuring the rate of respiration one fruit from each replicate was kept in the air tight glass jar for 1 h. The accumulated CO2 was measured by the CO2 analyzer as described by Razzaq et al., 2013a, and expressed as mmol CO2 kg−1 h−1.
Physiological changes during ripening causes oxidative stress leading to reduce the ability of antoixidative enzymes for eliminating free radicles (Jimenez et al., 2002). However oxidative stress can be prevented by antoixidative enzymes such as catalase, superoxide dismutase and peroxidases (Hodges et al., 2004). Polyamines have great potential for regulating the activities of antoixidative enzyme system for protecting the covering sheaths of cell from damage, due to oxidative stress and consequently, the fruit ripening is delayed (Razzaq et al., 2014). Polyamines are good anti senescent compounds which can retard fruit softening and maintain fruit quality during storage (Khan et al., 2007). In spite of the fact that papaya is profoundly nutritious and therapeutically significant, is neglected by the potential growers due to huge postharvest losses particularly the “Red lady papaya” (mostly grown in Pakistan). As for as we know, there is no information available regarding the role of putrescine for maintaining the quality of papaya fruit in terms of fruit firmness, decay, weight loss, vitamin C, acidity, antoixidative enzymes (CAT, SOD and POD), total antioxidants and phenolic contents during storage which provides a strong ground for the proposed investigations. This study can provide a great help to the progressive growers to overcome the problems of long distance transport.
2.3. Biochemical attributes for fruit quality 2.3.1. TSS, TA, ripening index Total soluble solids were measured from juice sample of papaya fruit using a digital refrectrometer. Titratable acidity was measured by adopting the procedure of Hortwitz (1960). TA was detected by titrating 10 ml juice and 10 ml distill water against 1/10 N NaOH using 2.3 drops of phenolphthalein (indicator) and expressed in %.
TA(%) =
Ripening index was measured by simply taking ratio of TSS and TA (TSS/TA). 2.3.2. Ascorbic acid Ascorbic acid content was measured by titrating 5 ml aliquot which was prepared by dissolving 90 ml of 0.4 % oxalic acid with 10 ml papaya juice against 2, 6-dichlophenolindophenol dye. Titration method was stopped when sample color was changed into light pink color and disappeared after 15 s. (AOAC, 2000).
2. Material and methods 2.1. Experimental procedure Mature unripe “Red Lady” papaya fruit with 25 % yellow color were harvested from a local orchard located at Garh Fateh Shah, Tehsil Sammundri, Faisalabad, Punjab, Pakistan. Uniform sized, disease free, and healthy looking papaya fruit were selected and transported to the lab of Postharvest Research Centre, Ayyub Agriculture Research Institute, Faisalabad. Fruit was initially washed with running tap water then with chlorine containing water and allowed to dry at room temperature. This whole research was programmed according to completely randomized design containing four treatments each of which was replicated four times (15 fruit in each replicate). Fruit were dipped in 10 l solution of 0 mM, 1 mM, 2 mM and 3 mM PUT separately for 10 min, air dried, and placed in storage chambers at 12 °C temperature and 90 ± 5 % relative humidity for 28 days. During the entire storage, fruit weight loss, decay, firmness, TSS, acidity, sugars, total antioxidants, total phenolic contents, ripening index and antoixidative enzyme activities were studied on weekly basis.
Ascorbic acid(A.A) =
1 × C1×A × 100 C2×B×C
Where C1 = dye used against papaya aliquot A = quantity of aliquot after adding oxalic acid C2 = dye used for the titration of standard ascorbic acid C = papaya juice taken for titration B = quantity of aliquot utilized for the estimation of AA 2.3.3. Sugars Sugars in papaya fruit were measured by adopting the titration procedures of Hortwitz (1960). Total sugars (%) = 25 × (A/B) Reducing sugars % = 6.25 × (A/B) Non- reducing sugars = (total sugars – reducing sugars) × 0.95
2.2. Estimation of quality attributes 2.2.1. Fruit weight loss % For checking percentage of weight reduction, fruit weight of all treatments were taken using digital weighing machine on weekly basis and then % weight loss was checked by taking the variation in weight at 0 day and final day weight and conveyed as weight loss percentage compared to primary weight at 0 day.
2.3.4. Total antioxidants and total phenolic contents The total phenolic content was measured following the procedure of Razzaq et al., 2013b taking Folin- Ciocalteu reagent. The prepared sample and reagent were vortexed, incubated, and centrifuged 13000 × g to get the supernatant which was used to check the absorbance at 765 nm and 517 nm by spectrophotometry. The final readings were shown as mg GAE/ 100 g. The antioxidant activity was executed by using the method of Brand-Williams et al. (1995). 50 μl of the supernant was mixed with DPPH solution (0.004 % methanol) in the microtubes and incubated under darkness. Absorbance was noted at 517 nm and the antioxidants were documented in terms of DPPH scavenging activity. % DPPH Inhibition was calculated as under:
2.2.2. Fruit decay % In order to check fruit decaying, number of decayed fruit (fruit showing 2, 3 spots of fungal attack) were calculated on visual basis during every visit on weekly basis and expressed as
Decay% =
0.1 NaOH used × 0.067×100 mL of juice used
decayed fruit ×100 Total fruit
I (%) = (Ab-As)/Ab × 100 Where Ab = blank mixture As = sample mixture
2.2.3. Firmness N Firmness of fruit was measured with a Penetrometer (Model DFM50, Ametek Inc., USA) using 8 mm spherical tip. Fruit skin was peeled from a single point and tip of penetrometer was inserted in fruit on both sides of the fruit. Firmness was noted by taking the mean of two readings and expressed as Newton (N).
2.4. Activities of antioxidative enzymes Enzyme activities were measured by following the method of Liu 2
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et al. (2009). For this purpose 1 g of papaya pulp was mixed with phosphate buffer (pH 7.2) and homogenized using pestle and mortar. This mixture was poured in eppendorfs, centrifuged for 10 min and the supernant was collected for additional estimation of other enzymes. The activity of CAT enzyme was assessed by standard method of Liu et al. (2009). 100 μl of the enzyme extract was mixed 50 mM phosphate buffer (pH 5), 5.9 mM H2O2 and the reaction was started. The change in the reaction mixture was noted after every 20 s at a wavelength of 240 nm and expressed as U mg−1 protein where one Unit is equal to change in absorbance of 0.01Unit per min. The POD activity was followed by the method previously described, but the reaction mixture for POD activity contained phosphate buffer (50 mM), H2O2 (40 mM), guaiacol (20 mM) and 0.1 ml of the enzyme extract. Absorbance was noted at 470 nm and expressed as U mg−1 protein. The activity of SOD enzyme was measured by the standard procedure of Liu et al. (2009). For this purpose distilled water (800 μL), phosphate buffer with pH 5 [500 μl (50 mM)], 100 μl NBT (20 mM), 200 μl methionine (22 mM), 200 μL Triton-X (0.1 mM), 100 μl riboflavin (0.6 mM) as an enzyme-substrate were mixed with the enzyme extract (100 μL) in test tubes. Absorbance was noted at 560 nm wavelength and expressed as U mg−1 protein. 2.5. Protein content The content of protein was assessed by the method of Bradford (1976) in which bovine serum albumin was used as standard. The protein content was used for the estimation of the antoixidative enzyme activities. 2.6. Statistical analysis The experiment was programmed under Completely Randomized design (CRD) with 2 factor factorial arrangement. The acquired observations were analyzed by using computer based statistical software and further compared by using Least significance Difference test (Steel et al., 1997).
Fig. 1. Effect of the postharvest application of putrescine during papaya storage, on weight loss % (A), Decay % (B), and fruit firmness (C). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for Fruit weight loss%: Treatment = 0.31**, storage period = 0.35**, Interaction = 0.71**; Decay %: Treatment = 1.23**, Storage period = 1.381**, Interaction = 2.76**; Fruit firmness: Treatment = 0.33**, storage period = 0.36**, interaction = 0.739**.
3. Results 3.1. Quality attributes 3.1.1. Weight loss % Generally the weight loss was increased with storage time (Fig. 1A). Unexpectedly, the weight reduction was more in fruit subjected to the higher dose of 3 mM PUT (10.64 %) in the course of the whole storage. As for the untreated fruit is concerned, slow rate of weight reduction was observed in the first 7 days, then it increased until the 28th day. Results for weight reduction were statistically significant (P ≤ 0.005) among treatment as well as their interrelation with storage time. The lowest weight loss was in fruit from the 2 mM PUT (4.62 %), followed by the 1 mM PUT (8.20 %), control (9.11 %) and 3 mM PUT (10.64 %). After completion of storage under controlled conditions, average reduction in weight loss was almost 2 times less in 2 mM PUT treated fruit in comparison to untreated fruit.
which was almost 2.9 times less then control fruit. 3.1.3. Fruit firmness N Fruit firmness was progressively declined with storage duration (Fig. 1C). However, PUT treated fruit were firmer than control fruit even after the completion of 28-day storage. 2 mM PUT was more effective in maintaining the fruit firmness during the four-week storage. The highest percentage of fruit softening (11.95 N) was associated with the highest treatment concentration, followed by the control (11.39 N), 1 mM PUT (12.36 N) and 2 mM PUT (14.95 N). At the end of storage mean firmness were 1.31 folds more in 2 mM treated fruit in comparison to the control treatment.
3.1.2. Fruit decay % Fruit decay increased with progression in storage regardless of the applied treatments. However, it was less noticeable in the PUT treated fruit (Fig. 1B). During the first week of storage decay percentage was less in an all treatments, then linearly increased up to 4 weeks. At the end of storage, the highest decay was in the control fruit (17.22 %) followed by the 3 mM PUT (10.12 %), 1 mM PUT (10.18 %) and the lowest in those treated at 2 mM PUT (6.45 %), at this concentration disease was almost negligible. Interaction of the PUT treatments and storage time revealed significant results which indicated that 2 mM PUT suppressed the decay incidence during the whole storage period
3.1.4. Respiration The rate of respiration was increased during the entire period of storage with respect to the all treatments applied. However, putrescine treated fruit showed less respiratory activity as compared to the control fruit. Among all the treatments, 2 mM putrescine treatment was most effective in suppressing the respiration rate (Fig. 6). At the end of storage, lowest respiration rate (7.07 mmol/ kg/ h) was exhibited in control fruit, followed by 1 mM PUT (7.42 mmol/ kg/ h), 3 mM PUT (7.46 mmol/ kg/ h), and the highest (8.1 mmol/ kg/ h) was recorded in control fruit. 3
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Fig. 2. Effect of the postharvest application of putrescine during papaya storage on TSS (A), TA (B) and ripening index (c). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD value for TSS: Treatment = 0.114** storage period = 0.127**, Interaction = 0.255**, TA: Treatment = 3.2**Storage period = 2.82**, Interaction = NS; Ripening index: Treatment = 7.15**, storage period = 7.99**, interaction = 15.98*.
Fig. 3. Effect of the postharvest application of putrescine during papaya storage on ascorbic acid (A), total antioxidant (B) and total phenolic contents (c). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for ascorbic acid: Treatment = 2.05**, storage period = 2.29**, Interaction = NS, Total antioxidant: Treatment = 1.097**, Storage period = 1.22**, Interaction = 2.45**; total phenolic: Treatment = 1.15**, storage period = 1.293**, interaction = 2.587*.
3.1.5. TSS In the present study, maximum accumulation of TSS (8.62°Brix) was recorded in those fruit that were not subjected to any application of PUT in the course of entire storage. However, TSS accumulation was minimum with higher concentration of PUT after completion of storage. Significant interaction (P ≤ 0.05) was recorded among PUT treatments and the time of storage considering TSS accumulation. The fruit of control treatment gave the highest percentage of TSS (8.62°Brix) followed by 1 mM (7.425°Brix), 2 mM (7.075°Brix) and the lowest accumulation was recorded in case of 3 mM PUT (6.9°Brix) application after termination of the storage period. Overall results revealed the ability of PUT for suppressing the accumulation of TSS in stored papaya fruit as compared to the control fruit.
3.1.7. Ripening index Ripening index of papaya fruit were increased over the time of storage. In case of the untreated papaya fruit it remained at the upper limit throughout the storage compared to the remaining treatments, while treated one showed lower ripening index. All concentrations of putrescine showed slow increase in ripening index in 0–7 day of storage while control treatment resulted in rapid increase of ripening index. Once the storage finished, highest index was found in untreated fruit, followed by 1, 2 and 3 mM PUT treatments. Interaction among SA and storage days depicted significance (P ≤ 0.05) of PUT treatment for controlling ripening index of papaya fruit (Fig. 2C). 3.1.8. Ascorbic acid (mg/100 g) With increase in storage duration, amount of ascorbic acid were reduced in all treatments. However, this decline was less prominent in the fruit subjected to the PUT applications. Statistical analysis of the entire data showed important variations among treatment, time of storage and their interrelation (Fig. 3A). The highest value of ascorbic acid was noticed in fruit of 2 mM PUT treatments (13.54 mg/100 g) while the lowest values were recorded in case of control treatment in the whole storage period. Positive interaction (P ≤ 0.05) was found among PUT treatments and the storage period, which shows that ascorbic acid contents declined in all 4 treatments with increased interval of storage.
3.1.6. Titratable acidity The titratable acidity expressed a behavior entirely opposite to the TSS accumulation (Fig. 2B). Statistical analysis of the data proved that significant variations (P ≤ 0.05) exist among PUT treatment, time of storage and their relation with each other. The highest TA percentage was obtained in fruit dipped in 3 mM PUT (0.056 %), followed by 2 mM (0.054 %), 1 mM (0.048 %) and the untreated ones (0.041 %). After the end of the storage period, the TA value corresponding to the highest concentration applied was of 0.056 % which was significantly higher than untreated fruit. 4
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3.1.9. Total antioxidants (%DPPH inhibition) The total antioxidants increased during the first 3 weeks of storage, then decreased during the last week irrespective of the all treatments. However, fruit treated with 2 mM PUT showed the highest total antioxidant compared to the remaining treatments during 28 days of storage. The 2 and 3 mM PUT concentrations were similar with no significant differences among each other but, there were significant differences (P ≤ 0.05) with the control fruit. The highest % DPPH inhibition (62.25) was recorded in 2 mM PUT treatment while the lowest (53.5) was in untreated fruit at the end of storage period (Fig. 3B). 3.1.10. Total phenolic contents (mg GAE 100 g−1) The total phenolic contents depicted the same trend as total antioxidants (Fig. 3C). These were maximum in 2 mM PUT treatments while minimum in control fruit. The total phenolic contents were increased up to 21st day then decreased up to 28 day of storage. At the end of storage period mean total phenolic contents were maximum (54.5 mg GAE 100 g−1) in 2 mM PUT and minimum (47.5 mg GAE 100 g−1) in control fruit. Interaction effect of PUT treatments and the time of storage also depicted positive results regarding the TPC. Overall result suggested that 2 mM PUT treatment performed best for increasing the total phenolic contents as compared to the control treatment after 28 days of storage. 3.1.11. Total sugars (%) The sugar contents of the stored papaya fruit showed linear increase in case of the fruit treated with 2 mM PUT while all other treatments showed different pattern. There was non-significant (P ≥ 0.05) difference among the fruit of control and 1 mM PUT treatments regarding the sugar percentage. At the end of the experiment, total sugars were minimum (10.46 %) in 2 mM PUT treatment while maximum (15.63 %) in control treatment (Fig. 4A). Interaction data regarding total sugars also indicated that 2 mM PUT application performed substantially better than others in controlling the accumulation of total sugars with continuation of the storage. Generally, the accumulation of total sugars was slow during 3rd week of storage compared to other days regarding all treatments.
Fig. 4. Effect of the postharvest application of putrescine during papaya storage on Total sugars (A), Reducing sugars (B), and non reducing sugars (C). Vertical bars indicate the standard error of means. LSD Values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for Total sugars: Treatment = 0.295**, storage period = 0.331**, Interaction = 0.06**; Reducing sugars: Treatment = 0.591*,Storage period = 0.66**, Interaction = NS; Non-reducing sugars: Treatment = 0.516**, storage period = 0.577**, interaction = 1.15 *.
3.1.12. Reducing sugars (%) The reducing sugars of all treatments were increased from 0 to 7 days, then reach to the almost same level on the 14th day. The fruit of 1 mM PUT and control treatment showed almost alike response towards the reducing sugars throughout the storage with negligible differences (Fig. 4B). Once the storage finished, reducing sugars were highest in case of 1 mM PUT and lowest in 2 mM PUT treated papaya. Interaction of the PUT treatments and the time of storage resulted in positive relation regarding reducing sugars. Maximal amount of reducing sugars (10.32 Brix) was recorded in lower dose of PUT while minimal amount (8.0435 Brix) was revealed by fruit subjected to 2 mM PUT during last week of cold storage.
compared to the control fruit during the entire storage (Fig. 5A). The SOD activity in the control fruit was increased up to 21 days then suddenly decline during the last 7 days of storage. The highest SOD activity (1.19 U/mg protein) in control fruit was achieved at day 21 which was then decreased to 1.15 U/mg protein at 28th day. However, the fruit treated with 2 mM PUT showed higher SOD activity during the entire 4 weeks as compared to the all other treatments. The maximum SOD activity (1.317 U/mg protein) was recorded in 2 mM PUT treated fruit which was 1.14-folds higher than the untreated fruit at the end of storage.
3.1.13. Non-reducing sugars (%) Unlike the total and reducing sugars, non-reducing sugars were decreased during the first week then almost stabled up to the completion of the storage period in the fruit exposed to 2 mM PUT (Fig. 4C). At the end of storage, average non-reducing sugars of the untreated papaya fruit were almost 1.6 times more than treated fruit with 2 mM PUT. Maximum increase in non-reducing sugars of both untreated and 1 mM treated papaya fruit were observed during the last week of storage however, the 2 and 3 mM PUT treatment showed stability regarding non-reducing sugars.
3.1.14.2. Peroxidase activity. With the continuation of storage, peroxidase activity was also increased regardless of the treatments. The increasing trend of the POD activity was distinguished (P ≤ 0.05) in PUT treated fruit while, less distinguishable in the untreated fruit. Averaged over PUT treatment, the mean POD activity was significantly higher (1.07-fold) in 2 mM PUT treated fruit when compared with untreated fruit during the whole storage (Fig. 5B). Interaction of treatment into storage period depicted a clear difference in the mean POD activity during the storage period.
3.1.14. Antoixidative enzyme activities 3.1.14.1. Superoxide dismutase activity. Postharvest putrescine application significantly (P ≤ 0.05) increased the superoxide dismutase (SOD) activity in the pulp of stored papaya fruit as
3.1.14.3. Catalase activity. Exogenously applied putrescine also increased the catalase activity in papaya fruit as compared to the untreated fruit (Fig. 5C). The fruit dipped in 2 mM PUT solution and 5
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control fruit after 28 days of storage. 4. Discussion Weight loss of papaya fruit was continuously decreased throughout the storage. The reasons behind weight loss could be the rapid respiratory activities, loss of water through the skin and the consumption of stored metabolites during metabolic activities. Less weight loss was recorded in the putrescine treated fruit as compared to the control fruit, which might be associated with ability of PUT to stabilize the cell membrane (Mirdehghan et al., 2007) and also the suppression of respiration (Valero et al., 1998b). The suppression in the rate of respiration in putrescine treated mangoes has been reported previously by Razzaq et al. (2014). Similar results were reported in mango by Jawandha et al. (2012) and in pomegranate (Barman et al., 2011). Our results are also in accordance with Shiri et al. (2012) who reported reduced weight loss in PUT treated table grapes during long term storage. Fruit decay was significantly less in PUT treated fruit which may be due to the anti-pathogenic properties of polyamines. Walters (2003) has reviewed the anti-pathogenic abilities of polyamines and reported that polyamines have ability to make strong bonding with phenols and HCAA (hydroxycinnamic acid amide) both of which induce resistance against pathogen ultimately reducing decay incidence. Another factor for reducing the decay of PUT treated papaya fruit may also be associated with the strong defense mechanism against fungal attack. In this study firmness was declined with the passage of time during storage however, less reduction was noticed in PUT treated fruit. Putrescine a positively charged compound has tendency to make bonding with carboxyl group of cell wall resulting in strengthening of cell wall. This PUT-Carboxyl group bonding becomes hindrance in the way of cell wall degrading enzyme which ultimately results in retarded fruit softening (Valero et al., 2002). Similarly, reduced fruit softening by PUT treatment was reported in plum (Khan et al., 2007) mango (Razzaq et al., 2014) and grapes (Barman et al., 2011). Our results are also in line with Khosroshahi et al. (2007) who reported that retention of fruit firmness during storage of various fruit and vegetable is one of the key roles being played by putrescine. In the present study total soluble solids, ripening index and sugars were increased in papaya fruit during storage while reverse situation was observed in case of acidity and ascorbic acid. However both the increasing and decreasing trends were retarded by PUT application. The lower TSS, sugars and ripening index in PUT treated fruit as compared to control may be due to suppression of ethylene biosynthesis which directly affects metabolism of sugar in fruit. These results are supported by Bal (2012) who documented that soluble solid contents were higher in PUT treated fruit when stored at low temperature. The breakdown of starch and accumulation of total sugars are the major changes that happen during the process of ripening and storage. Our results indicated that total sugars were increased with expanded storage period however, more prominent increase was noticed in control fruit. Less accumulation of total sugars in PUT treated papaya fruit is due to delayed senescence and ripening, because of suppression of activities of amylase and phosphorylase enzymes which are stimulated in ripening (Hubbard et al., 1990). Externally applied PUT increase the endogenous level of polyamines that might reduce the accumulation of total sugars, reduced respiration as suggested by Valero et al. (2002). The acidity of fruit is another important parameter which determines the quality of fruit for consumer’s point of view. The titratable acidity of papaya fruit was declined in the course of entire storage duration however; this decline was less in PUT treated papaya fruit in comparison to control fruit. Reduction in acidity along with storage might be due to utilization of acids during the process of respiration (Zokaee et al., 2007 and Ishaq et al., 2009). Less decline in acidity of PUT treated fruit was also reported by Ghasemnezhad et al. (2010). Ascorbic acid contents of papaya fruit during storage were declined
Fig. 5. Effect of the postharvest application of putrescine during papaya storage on superoxide dismutase (A), peroxidase (B), and catalase (C). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for SOD: Treatment = 0.01**, storage period = 0.127**, Interaction = 0.025**; POD: Treatment = 0.08 **, Storage period = 0.089**, Interaction = 0.178**; CAT: Treatment = 0.141**, storage period = 0.157**, interaction = 0.314*.
Fig. 6. Effect of the postharvest application of putrescine, on respiration rate during papaya storage. Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = highly significant (P < 0.01). LSD value for respiration: Treatment = 0.11** storage period = 0.121** and Interaction = 0.035**.
untreated expressed lower catalase activity during first 7 days while, higher for the rest of storage. The activity of catalase enzyme in the pulp of all fruit was increased from day 7 till 28th day however this increase was more pronounced in the PUT treated fruit. The Maximum catalase activity (6.94 U/mg protein) was found in fruit that were dipped in 3 mM PUT which was significantly (P ≤ 0.05) higher than the 6
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Acknowledgements
regardless of the applied treatments, however, less decline was noticed in PUT treated fruit as compared to the control fruit. Reduction of ascorbic acid contents in control fruit may be due to respiration and ripening related processes; while lower rate in PUT treated fruit could be due to suppression of ascorbate oxidase activity by PUT (Malik et al., 2006). Reduction in ascorbic acid contents were also reported by Yahia et al. (2001) in tomato and bell pepper. The total antioxidants are a good way of assessing nutritional value of fruit after storage. Results of the present study revealed that total antioxidants and total phenolic contents were increased initially then decreased just like ascorbic acid contents. Thus it can be concluded that antioxidants and phenolic contents are correlated. PUT treated fruit showed higher level of antioxidants and total phenolic which may be due to its anti-senescence properties because the activity of total antioxidants generally increased with senescence (Ghasemnezhad et al., 2010). Such correlation between TPC and total antioxidants was reported by (Palafox-Carlos et al., 2012) in mango. The catalase activity prevent the cells against oxidative damage caused by stress by breakdown of H2O2 into O2 and water (Ghasemnezhad et al., 2010). Increase in catalase activity is important for preventing cell damage while lower CAT activity shows weakened system of cells to remove H2O2 (Ng et al., 2005). Catalase activity was significantly higher in PUT treated fruit as compared to control in present study. Similar results were reported in apricots by Saba et al. (2012). The superoxide dismutase activity was higher in the PUT treated fruit as compared to the control fruit during entire storage period. The increase in SOD activity catalyzes the breakdown of hydrogen peroxide and provides protection to the cell against oxidative stress (Verma and Mishra, 2005). Higher SOD activity in PUT treated fruit may be due to bonding between PUT and antoixidative enzymes as reported in mango (Razzaq et al., 2014). Peroxidase enzyme activity limits the entry of pathogen to the cell tissue by creating a barrier and making cross linkages in the cell wall (Almardo et al., 2008). In this experiment higher POD activity was recorded in PUT treated papaya fruit during cold storage. Similar findings were also reported by Saba et al. (2012) in PUT treated apricots.
We are grateful to Mr. Rashid (owner of papaya orchard) for providing us good quality papaya fruit. We also appreciate Post-Harvest Research Centre, Ayyub Agriculture Research Institute, Faisalabad, Pakistan for providing us technical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2020.109367. References Almardo, L., Gomez Ros, L.V., Navaro, S.B., Bru, R., Barcelo, A.R., Pedreno, M.A., 2008. Class 3 peroxidases in plant defence system. J. Exp. Bot. 60, 377–390. AOAC, 2000. Vitamins and Other Nutrients, Official Methods of Analysis, 17th ed. AOAC, Washington, D.C, pp. 16–20 Chapter 45. Bal, E., 2012. Effect of postharvest putrescine and salicylic acid on cold storage duration and quality of sweet cherries. J. Faculty Agric. 7 23–21. Barman, k., Ram, A., Pal, R.K., 2011. Putrescine and carnauba wax pretreatments alleviate chilling injury, enhance shelf life and preserve pomegranate fruit quality during cold storage. Sci. Hortic. 130, 795–800. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248–254. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25–30. Ghasemnezhad, M., Shiri, M.A., Sanavi, M., 2010. Effect of chitosan coatings on some quality indices of apricot (Prunus armeniaca L.) during cold storage. Casp. J. Environ. Sci. 8, 25–33. Hernandez, Y., Lobo, M.G., Gonzalez, M., 2006. Determination of vitamin C in tropical fruits: a comparative evaluation of methods. Food Chem. 96, 654–664. Hodges, D.M., Lester, G.E., Munro, K.D., Toivonen, P.M.A., 2004. Oxidative stress: importance for postharvest quality. HortScience 39, 924–929. Hortwitz, W., 1960. Official and Tentative Methods of Analysis. Association of Official Agricultural Chemists, Washington, DC, pp. 314–320. Hubbard, N.L., Pharr, D.M., Huber, S.C., 1990. Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric. Plant Physiol. 94, 201–208. Ishaq, S., Rathore, H.A., Majeed, S., Awan, S., Zulfiqar, S., 2009. The studies on the physic chemical and organoleptic characteristics of apricot (Prunus arminiaca L.) produced in Rawlakot, Azad Jammu Kashmir during storage. Pak. J. Nutr. 8, 856–860. Jawandha, S.K., Gill, M.S., Singh, N.P., Gill, P.P.S., Singh, N., 2012. Effect of postharvest treatment of putrescine on storage of mango cv. Langra. Afr. J. Agric. Res. 7, 6432–6436. Jimenez, A., Cressen, G., Kular, B., Firmin, J., Robinson, S., Verhoeyen, M., 2002. Changes in oxidative process and components of the antioxidant system during tomato fruit ripening. Planta 214, 751–758. Khan, A.S., Singh, Z., Abbasi, N.A., 2007. Pre-storage putrescine application suppresses ethylene biosynthesis and retards fruit softening during low tem-perature storage in ‘Angelino’ plum. Postharvest Biol. Technol. 46, 36–46. Khosroshahi, M.R.Z., Esna-Ashari, M., Ershadi, A., 2007. Effect of exogenous putrescine on post-harvest life of strawberry (Fragaria ananassa Duch.) fruit, cultivar Selva. Sci. Hortic. 114, 27–32. Liu, D., Zou, J., Meng, Q., Zou, J., Jiang, W., 2009. Uptake, accumulation and oxida-tive stress in garlic (Allium sativum L.) under lead phytotoxicity. Ecotoxicology 18, 134–143. Malik, A.U., Singh, Z., 2005. Pre-storage application of polyamines improves shelf-life and fruit quality in mango. J. Hort. Sci. Biotechnol. 80, 363–369. Malik, A.U., Singh, Z., Tan, S.C., 2006. Exogenous application of polyamines improves shelf life and fruit quality of mango. Acta Hortic. 699, 291–296. Martinez-Romero, D., Serrano, M., Carbonell, A., Brugos, L., Riquelme, F., Valero, D., 2002. Effects of postharvest putrescine treatment on extending shelf life and reducing mechanical damage in apricot. J. Food Sci. 67, 1706–1712. Mirdehghan, S.H., Rahemi, M., Castillo, S., Martínez-Romero, D., Serrano, M., Valero, D., 2007. Pre-storage application of polyamines by pressure or immersion improves shelf-life of pomegranate stored at chilling temperature by increasing endogenous polyamine levels. Postharvest Biol. Technol. 44, 26–33. Ng, T.B., Gao, W., Li, L., Niu, S.M., Zhao, L., Liu, J., Liu, F., 2005. Rose (Rosa rugosa)flower extract increases the activities of antioxidant enzymes and their gene expression and reduces lipid peroxidation. Biochem. Cell Biol. 83, 78–85. Nunes, M.C.N., Emond, J.P., Brecht, J.K., 2006. Brief deviations from set point temperatures during normal airport handling operations negatively affect the quality of papaya (Carica papaya) fruit. Postharvest Biol. Technol. 41, 328–340. Oloyede, O., 2005. Chemical profile of unripe pulp of Carica papaya. Pak. J. Nutr. 4, 379–381. Palafox-Carlos, H., Yahia, E., Islas-Osuna, M.A., Gutierrez-Martinez, P., Robles-Sánchez, M., González-Aguilar, G.A., 2012. Effect of ripeness stage of mango fruit (Mangifera indica L., cv. Ataulfo) on physiological parameters and antioxidant activity. Sci. Hortic. 135, 7–13.
5. Conclusion In conclusion, postharvest application of putrescine maintained the better quality of papaya fruit during the 4 weeks of storage. However, fruit subjected to 2 mM PUT showed higher firmness, less decay, less weight loss, suppressed respiration, higher level of total phenolic contents, total antioxidants and antoixidative enzyme activities of “Red lady” papaya fruit during storage. In general, this study suggested that the prestorage dipping of papaya fruit in PUT @ 2 mM performed best to maintain its quality and can be used for its prolonged storage effectively. Author contributions Aliya Hanif: PhD scholar and she conducted all research projects, analyzed the data and wrote the manuscript. Saeed Ahmad: PhD supervisor and guided in all executing the experiment, data analysis and written the manuscript. Muhammad Jafar Jaskani: Committee member help in experiment. Rashid Ahmad: Committee member and help to data analysis and writing. Declaration of Competing Interest None. 7
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Biological Approach, 3rd edition. McGraw Hill Book Co. Inc., New York, USA. Valero, D., Martinez-Romero, D., Serrano, M., Riquelme, F., 1998b. Influence of postharvest treatment with putrescine and calcium on endogenous polyamines, firmness and abscisic acid in Lemon (Citrus lemon L. Burm cv. Verna). J. Agric. Food Chem. 46, 2102–2109. Valero, D., Martinez-Romero, D., Serrano, M., 2002. The role of polyamines in the improvement of the shelf life of fruit. Trends Food Sci. Technol. 13, 228–234. Verma, S., Mishra, N.S., 2005. Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defense system. J. Plant Physiol. 162, 669–677. Walters, D.R., 2003. Polyamines and plant disease: review. Phytochemistry 64, 97–107. Wang, C.Y., Conway, W.S., Abbott, J.A., Kramer, G.F., 1993. Postharvest infiltration of polyamines and calcium influences ethylene production and texture changes in Golden Delicious apples. J. Am. Soc. Hortic. Sci. 118, 801–806. Yahia, E.M., Contreras-Padilla, M., Gonazalez-Aguilar, G., 2001. Ascorbic acid content in relation to ascorbic acid oxidase activity and polyamine content in tomato and bell pepper fruits during development, maturation and senescence. LWT-Food Sci. Technol. 34, 452–457. Zokaee, K.M.R., Ashari, M.E., Ershadi, A., 2007. Effect of exogeneous putrescine on post harvest life of strawberry (Fragaria ananasa Duch) fruit cultivar Selva. Hortic. Sci. 114, 27–32.
Paull, R.E., Duarte, O., 2011. Tropical Fruits, vol. I CAB International, Wallingford, England. Razzaq, K., Khan, A.S., Malik, A.U., Shahid, M., 2013a. Ripening period influences fruit softening and antioxidative system of ‘Samar Bahisht Chaunsa’ mango. Sci. Hortic. 160, 108–114. Razzaq, K., Khan, A.S., Malik, A.U., Shahid, M., Ullah, S., 2013b. Foliar application of zinc influences the leaf mineral status, vegetative and reproductive growth, yield and fruit quality of ‘Kinnow’ mandarin (Citrus reticulata Blanco.). J. PlantNutr. 36, 1–17. Razzaq, K., Khan, A.S., Malik, A.U., Shahid, M., Ullah, S., 2014. Role of putrescine in regulating fruit softening and antoixidative enzyme systems in ‘Samar Bahisht Chaunsa’ mango. Post Harvest Biol. Technol. 96, 23–32. Saba, K.M., Arzani, K., Barzegar, M., 2012. Postharvest polyamine application alleviates chilling injury and affects apricot storage ability. J. Agric. Food Chem. 60, 8947–8953. Shiri, M., Ghasemnezhad, A., Bakhshi Davood, M., Sarikhani, H., 2012. Effect of postharvest putrescine application and chitosan coating on maintaining quality of table grape cv. Shahroudi during long term storage. J. Food Process. Preserv. 1–9. Singh, Z., Singh, R.K., Sane, V.A., Nath, P., 2013. Mango – postharvest biology and biotechnology. Crit. Rev. Plant Sci. 32, 217–236. Steel, R.G.D., Torrie, J.H., Dicky, D.A., 1997. Principles and Procedures of Statistics: A
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Papaya treatment with putrescine maintained the overall quality and promoted the antioxidative enzyme activities of the stored fruit
T
Aliya Hanifa, Saeed Ahmada,*, M. Jafar Jaskania, Rashid Ahmadb a b
Institute of Horticultural Sciences, University of Agriculture Faisalabad Pakistan, Pakistan Department of Agronomy, University of Agriculture Faisalabad Pakistan, Pakistan
A R T I C LE I N FO
A B S T R A C T
Keywords: Carica papaya Catalase Peroxidase Superoxide dismutase Total antioxidants Total phenolic contents Shelf life
Papaya is an emerging, profit generating fruit of Pakistan having high nutritional value. It has very limited shelf life which limits its long distance transport and resulted in high postharvest losses. Putrescine has great potential to maintain the firmness, quality of fruit and reduce the losses. Therefore, the role of putrescine for balancing fruit firmness, enzyme activities and variations in biochemical properties of Red lady papaya fruit were evaluated during storage. Mature unripe papaya fruit were subjected to different concentrations of PUT (0 mM, 1 mM, 2 Mm, 3 mM) and then stored at 12 °C temperature and 90–95 % RH for 28 days. Fruit firmness, weight loss, antioxidant enzyme activities (CAT, SOD and POD), total phenolic, total antioxidants and other biochemical attributes were studied on weekly basis. Fruit firmness was substantially higher in putrescine treated fruits along with less weight loss % during storage. TSS and ripening index were higher in control fruit, while they were lower with PUT treatment. 2 mM PUT suppressed the decay incidence during whole storage period which was almost 2.9 times less than control fruit, similarly the activity of CAT enzyme was maximum (6.94 U/mg protein), POD (1.07-fold higher than control) and SOD was also higher in the same treatment. Total antioxidants and total phenolic contents were at upper limits in fruit treated with 2 mM PUT during storage. It can be concluded that 2 mM PUT is helpful for extending the shelf life of papaya fruit by suppressing fruit softening, fruit decaying and by enhancing the enzyme activities and maintaining good keeping quality during storage.
1. Introduction Papaya, Carica papaya L., is important fruit plant which grows rapidly and produces fruit throughout the year (Paul and Duarte, 2011). Papaya is called powerhouse of essential nutrients. It is an excellent source of ascorbic acid, carbohydrates, fibers and vitamins. It contains high amount of papain which aids in digestion (Oloyede, 2005). It contains and is rich in phytochemicals, total phenolics and antoixidative enzymes. However, postharvest losses in papaya are very high, because it is highly perishable and being climacteric in nature gets deteriorated easily due to rapid respiratory activities even after harvest and creates hurdle in its long distance transport. Postharvest losses of fruit and vegetables occur at various stages during transport, in storage houses or at market level (wholesale, retail). Fruit softening and low temperature injury are the major constraints in long distance transport of Red lady papaya (Nunes et al., 2006). However, microbial decay, physical injury and loss of firmness are the major postharvest defects of papaya which limits its shelf life and shipment to distant places (Hernandez et al., 2006).
⁎
Externally applied polyamines have great potential for lengthening the postharvest life and preserving the texture of many fruit e.g. strawberry, plum, apricot and mango (Martinez-Romero et al., 2002; Malik and Singh, 2005 and Khosroshahi et al., 2007). Polyamines are positively charged organic molecules, light in weight and are existed in nearly all respiring organisms for regulating their various biological processes like growth and development (Barman et al., 2011). Putrescine (PUT), spermine (SPM), cadaverine (CAD) and spermidine (SPD) are biologically active forms of polyamines that can regulate various physical, physiological and biochemical processes of fruit (Malik and Singh, 2005; Khan et al., 2007). It was elucidated by Valero et al. (2002) that polyamines can retain the quality of fruit by suppressing ethylene production, lowering respiration rate, delaying color change, maintaining firmness and inducing resistance against mechanical damage. Postharvest treatment of climacteric and non-climacteric fruit with polyamines increases their shelf life with good quality maintained (Wang et al., 1993). Storage life of fruit is primarily relied on its ripening because it regulates the ultrastructure of cell wall (Singh et al., 2013).
Corresponding author. E-mail address: [email protected] (S. Ahmad).
https://doi.org/10.1016/j.scienta.2020.109367 Received 31 October 2019; Received in revised form 11 March 2020; Accepted 12 March 2020 Available online 19 March 2020 0304-4238/ © 2020 Elsevier B.V. All rights reserved.
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2.2.4. Respiration For measuring the rate of respiration one fruit from each replicate was kept in the air tight glass jar for 1 h. The accumulated CO2 was measured by the CO2 analyzer as described by Razzaq et al., 2013a, and expressed as mmol CO2 kg−1 h−1.
Physiological changes during ripening causes oxidative stress leading to reduce the ability of antoixidative enzymes for eliminating free radicles (Jimenez et al., 2002). However oxidative stress can be prevented by antoixidative enzymes such as catalase, superoxide dismutase and peroxidases (Hodges et al., 2004). Polyamines have great potential for regulating the activities of antoixidative enzyme system for protecting the covering sheaths of cell from damage, due to oxidative stress and consequently, the fruit ripening is delayed (Razzaq et al., 2014). Polyamines are good anti senescent compounds which can retard fruit softening and maintain fruit quality during storage (Khan et al., 2007). In spite of the fact that papaya is profoundly nutritious and therapeutically significant, is neglected by the potential growers due to huge postharvest losses particularly the “Red lady papaya” (mostly grown in Pakistan). As for as we know, there is no information available regarding the role of putrescine for maintaining the quality of papaya fruit in terms of fruit firmness, decay, weight loss, vitamin C, acidity, antoixidative enzymes (CAT, SOD and POD), total antioxidants and phenolic contents during storage which provides a strong ground for the proposed investigations. This study can provide a great help to the progressive growers to overcome the problems of long distance transport.
2.3. Biochemical attributes for fruit quality 2.3.1. TSS, TA, ripening index Total soluble solids were measured from juice sample of papaya fruit using a digital refrectrometer. Titratable acidity was measured by adopting the procedure of Hortwitz (1960). TA was detected by titrating 10 ml juice and 10 ml distill water against 1/10 N NaOH using 2.3 drops of phenolphthalein (indicator) and expressed in %.
TA(%) =
Ripening index was measured by simply taking ratio of TSS and TA (TSS/TA). 2.3.2. Ascorbic acid Ascorbic acid content was measured by titrating 5 ml aliquot which was prepared by dissolving 90 ml of 0.4 % oxalic acid with 10 ml papaya juice against 2, 6-dichlophenolindophenol dye. Titration method was stopped when sample color was changed into light pink color and disappeared after 15 s. (AOAC, 2000).
2. Material and methods 2.1. Experimental procedure Mature unripe “Red Lady” papaya fruit with 25 % yellow color were harvested from a local orchard located at Garh Fateh Shah, Tehsil Sammundri, Faisalabad, Punjab, Pakistan. Uniform sized, disease free, and healthy looking papaya fruit were selected and transported to the lab of Postharvest Research Centre, Ayyub Agriculture Research Institute, Faisalabad. Fruit was initially washed with running tap water then with chlorine containing water and allowed to dry at room temperature. This whole research was programmed according to completely randomized design containing four treatments each of which was replicated four times (15 fruit in each replicate). Fruit were dipped in 10 l solution of 0 mM, 1 mM, 2 mM and 3 mM PUT separately for 10 min, air dried, and placed in storage chambers at 12 °C temperature and 90 ± 5 % relative humidity for 28 days. During the entire storage, fruit weight loss, decay, firmness, TSS, acidity, sugars, total antioxidants, total phenolic contents, ripening index and antoixidative enzyme activities were studied on weekly basis.
Ascorbic acid(A.A) =
1 × C1×A × 100 C2×B×C
Where C1 = dye used against papaya aliquot A = quantity of aliquot after adding oxalic acid C2 = dye used for the titration of standard ascorbic acid C = papaya juice taken for titration B = quantity of aliquot utilized for the estimation of AA 2.3.3. Sugars Sugars in papaya fruit were measured by adopting the titration procedures of Hortwitz (1960). Total sugars (%) = 25 × (A/B) Reducing sugars % = 6.25 × (A/B) Non- reducing sugars = (total sugars – reducing sugars) × 0.95
2.2. Estimation of quality attributes 2.2.1. Fruit weight loss % For checking percentage of weight reduction, fruit weight of all treatments were taken using digital weighing machine on weekly basis and then % weight loss was checked by taking the variation in weight at 0 day and final day weight and conveyed as weight loss percentage compared to primary weight at 0 day.
2.3.4. Total antioxidants and total phenolic contents The total phenolic content was measured following the procedure of Razzaq et al., 2013b taking Folin- Ciocalteu reagent. The prepared sample and reagent were vortexed, incubated, and centrifuged 13000 × g to get the supernatant which was used to check the absorbance at 765 nm and 517 nm by spectrophotometry. The final readings were shown as mg GAE/ 100 g. The antioxidant activity was executed by using the method of Brand-Williams et al. (1995). 50 μl of the supernant was mixed with DPPH solution (0.004 % methanol) in the microtubes and incubated under darkness. Absorbance was noted at 517 nm and the antioxidants were documented in terms of DPPH scavenging activity. % DPPH Inhibition was calculated as under:
2.2.2. Fruit decay % In order to check fruit decaying, number of decayed fruit (fruit showing 2, 3 spots of fungal attack) were calculated on visual basis during every visit on weekly basis and expressed as
Decay% =
0.1 NaOH used × 0.067×100 mL of juice used
decayed fruit ×100 Total fruit
I (%) = (Ab-As)/Ab × 100 Where Ab = blank mixture As = sample mixture
2.2.3. Firmness N Firmness of fruit was measured with a Penetrometer (Model DFM50, Ametek Inc., USA) using 8 mm spherical tip. Fruit skin was peeled from a single point and tip of penetrometer was inserted in fruit on both sides of the fruit. Firmness was noted by taking the mean of two readings and expressed as Newton (N).
2.4. Activities of antioxidative enzymes Enzyme activities were measured by following the method of Liu 2
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et al. (2009). For this purpose 1 g of papaya pulp was mixed with phosphate buffer (pH 7.2) and homogenized using pestle and mortar. This mixture was poured in eppendorfs, centrifuged for 10 min and the supernant was collected for additional estimation of other enzymes. The activity of CAT enzyme was assessed by standard method of Liu et al. (2009). 100 μl of the enzyme extract was mixed 50 mM phosphate buffer (pH 5), 5.9 mM H2O2 and the reaction was started. The change in the reaction mixture was noted after every 20 s at a wavelength of 240 nm and expressed as U mg−1 protein where one Unit is equal to change in absorbance of 0.01Unit per min. The POD activity was followed by the method previously described, but the reaction mixture for POD activity contained phosphate buffer (50 mM), H2O2 (40 mM), guaiacol (20 mM) and 0.1 ml of the enzyme extract. Absorbance was noted at 470 nm and expressed as U mg−1 protein. The activity of SOD enzyme was measured by the standard procedure of Liu et al. (2009). For this purpose distilled water (800 μL), phosphate buffer with pH 5 [500 μl (50 mM)], 100 μl NBT (20 mM), 200 μl methionine (22 mM), 200 μL Triton-X (0.1 mM), 100 μl riboflavin (0.6 mM) as an enzyme-substrate were mixed with the enzyme extract (100 μL) in test tubes. Absorbance was noted at 560 nm wavelength and expressed as U mg−1 protein. 2.5. Protein content The content of protein was assessed by the method of Bradford (1976) in which bovine serum albumin was used as standard. The protein content was used for the estimation of the antoixidative enzyme activities. 2.6. Statistical analysis The experiment was programmed under Completely Randomized design (CRD) with 2 factor factorial arrangement. The acquired observations were analyzed by using computer based statistical software and further compared by using Least significance Difference test (Steel et al., 1997).
Fig. 1. Effect of the postharvest application of putrescine during papaya storage, on weight loss % (A), Decay % (B), and fruit firmness (C). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for Fruit weight loss%: Treatment = 0.31**, storage period = 0.35**, Interaction = 0.71**; Decay %: Treatment = 1.23**, Storage period = 1.381**, Interaction = 2.76**; Fruit firmness: Treatment = 0.33**, storage period = 0.36**, interaction = 0.739**.
3. Results 3.1. Quality attributes 3.1.1. Weight loss % Generally the weight loss was increased with storage time (Fig. 1A). Unexpectedly, the weight reduction was more in fruit subjected to the higher dose of 3 mM PUT (10.64 %) in the course of the whole storage. As for the untreated fruit is concerned, slow rate of weight reduction was observed in the first 7 days, then it increased until the 28th day. Results for weight reduction were statistically significant (P ≤ 0.005) among treatment as well as their interrelation with storage time. The lowest weight loss was in fruit from the 2 mM PUT (4.62 %), followed by the 1 mM PUT (8.20 %), control (9.11 %) and 3 mM PUT (10.64 %). After completion of storage under controlled conditions, average reduction in weight loss was almost 2 times less in 2 mM PUT treated fruit in comparison to untreated fruit.
which was almost 2.9 times less then control fruit. 3.1.3. Fruit firmness N Fruit firmness was progressively declined with storage duration (Fig. 1C). However, PUT treated fruit were firmer than control fruit even after the completion of 28-day storage. 2 mM PUT was more effective in maintaining the fruit firmness during the four-week storage. The highest percentage of fruit softening (11.95 N) was associated with the highest treatment concentration, followed by the control (11.39 N), 1 mM PUT (12.36 N) and 2 mM PUT (14.95 N). At the end of storage mean firmness were 1.31 folds more in 2 mM treated fruit in comparison to the control treatment.
3.1.2. Fruit decay % Fruit decay increased with progression in storage regardless of the applied treatments. However, it was less noticeable in the PUT treated fruit (Fig. 1B). During the first week of storage decay percentage was less in an all treatments, then linearly increased up to 4 weeks. At the end of storage, the highest decay was in the control fruit (17.22 %) followed by the 3 mM PUT (10.12 %), 1 mM PUT (10.18 %) and the lowest in those treated at 2 mM PUT (6.45 %), at this concentration disease was almost negligible. Interaction of the PUT treatments and storage time revealed significant results which indicated that 2 mM PUT suppressed the decay incidence during the whole storage period
3.1.4. Respiration The rate of respiration was increased during the entire period of storage with respect to the all treatments applied. However, putrescine treated fruit showed less respiratory activity as compared to the control fruit. Among all the treatments, 2 mM putrescine treatment was most effective in suppressing the respiration rate (Fig. 6). At the end of storage, lowest respiration rate (7.07 mmol/ kg/ h) was exhibited in control fruit, followed by 1 mM PUT (7.42 mmol/ kg/ h), 3 mM PUT (7.46 mmol/ kg/ h), and the highest (8.1 mmol/ kg/ h) was recorded in control fruit. 3
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Fig. 2. Effect of the postharvest application of putrescine during papaya storage on TSS (A), TA (B) and ripening index (c). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD value for TSS: Treatment = 0.114** storage period = 0.127**, Interaction = 0.255**, TA: Treatment = 3.2**Storage period = 2.82**, Interaction = NS; Ripening index: Treatment = 7.15**, storage period = 7.99**, interaction = 15.98*.
Fig. 3. Effect of the postharvest application of putrescine during papaya storage on ascorbic acid (A), total antioxidant (B) and total phenolic contents (c). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for ascorbic acid: Treatment = 2.05**, storage period = 2.29**, Interaction = NS, Total antioxidant: Treatment = 1.097**, Storage period = 1.22**, Interaction = 2.45**; total phenolic: Treatment = 1.15**, storage period = 1.293**, interaction = 2.587*.
3.1.5. TSS In the present study, maximum accumulation of TSS (8.62°Brix) was recorded in those fruit that were not subjected to any application of PUT in the course of entire storage. However, TSS accumulation was minimum with higher concentration of PUT after completion of storage. Significant interaction (P ≤ 0.05) was recorded among PUT treatments and the time of storage considering TSS accumulation. The fruit of control treatment gave the highest percentage of TSS (8.62°Brix) followed by 1 mM (7.425°Brix), 2 mM (7.075°Brix) and the lowest accumulation was recorded in case of 3 mM PUT (6.9°Brix) application after termination of the storage period. Overall results revealed the ability of PUT for suppressing the accumulation of TSS in stored papaya fruit as compared to the control fruit.
3.1.7. Ripening index Ripening index of papaya fruit were increased over the time of storage. In case of the untreated papaya fruit it remained at the upper limit throughout the storage compared to the remaining treatments, while treated one showed lower ripening index. All concentrations of putrescine showed slow increase in ripening index in 0–7 day of storage while control treatment resulted in rapid increase of ripening index. Once the storage finished, highest index was found in untreated fruit, followed by 1, 2 and 3 mM PUT treatments. Interaction among SA and storage days depicted significance (P ≤ 0.05) of PUT treatment for controlling ripening index of papaya fruit (Fig. 2C). 3.1.8. Ascorbic acid (mg/100 g) With increase in storage duration, amount of ascorbic acid were reduced in all treatments. However, this decline was less prominent in the fruit subjected to the PUT applications. Statistical analysis of the entire data showed important variations among treatment, time of storage and their interrelation (Fig. 3A). The highest value of ascorbic acid was noticed in fruit of 2 mM PUT treatments (13.54 mg/100 g) while the lowest values were recorded in case of control treatment in the whole storage period. Positive interaction (P ≤ 0.05) was found among PUT treatments and the storage period, which shows that ascorbic acid contents declined in all 4 treatments with increased interval of storage.
3.1.6. Titratable acidity The titratable acidity expressed a behavior entirely opposite to the TSS accumulation (Fig. 2B). Statistical analysis of the data proved that significant variations (P ≤ 0.05) exist among PUT treatment, time of storage and their relation with each other. The highest TA percentage was obtained in fruit dipped in 3 mM PUT (0.056 %), followed by 2 mM (0.054 %), 1 mM (0.048 %) and the untreated ones (0.041 %). After the end of the storage period, the TA value corresponding to the highest concentration applied was of 0.056 % which was significantly higher than untreated fruit. 4
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3.1.9. Total antioxidants (%DPPH inhibition) The total antioxidants increased during the first 3 weeks of storage, then decreased during the last week irrespective of the all treatments. However, fruit treated with 2 mM PUT showed the highest total antioxidant compared to the remaining treatments during 28 days of storage. The 2 and 3 mM PUT concentrations were similar with no significant differences among each other but, there were significant differences (P ≤ 0.05) with the control fruit. The highest % DPPH inhibition (62.25) was recorded in 2 mM PUT treatment while the lowest (53.5) was in untreated fruit at the end of storage period (Fig. 3B). 3.1.10. Total phenolic contents (mg GAE 100 g−1) The total phenolic contents depicted the same trend as total antioxidants (Fig. 3C). These were maximum in 2 mM PUT treatments while minimum in control fruit. The total phenolic contents were increased up to 21st day then decreased up to 28 day of storage. At the end of storage period mean total phenolic contents were maximum (54.5 mg GAE 100 g−1) in 2 mM PUT and minimum (47.5 mg GAE 100 g−1) in control fruit. Interaction effect of PUT treatments and the time of storage also depicted positive results regarding the TPC. Overall result suggested that 2 mM PUT treatment performed best for increasing the total phenolic contents as compared to the control treatment after 28 days of storage. 3.1.11. Total sugars (%) The sugar contents of the stored papaya fruit showed linear increase in case of the fruit treated with 2 mM PUT while all other treatments showed different pattern. There was non-significant (P ≥ 0.05) difference among the fruit of control and 1 mM PUT treatments regarding the sugar percentage. At the end of the experiment, total sugars were minimum (10.46 %) in 2 mM PUT treatment while maximum (15.63 %) in control treatment (Fig. 4A). Interaction data regarding total sugars also indicated that 2 mM PUT application performed substantially better than others in controlling the accumulation of total sugars with continuation of the storage. Generally, the accumulation of total sugars was slow during 3rd week of storage compared to other days regarding all treatments.
Fig. 4. Effect of the postharvest application of putrescine during papaya storage on Total sugars (A), Reducing sugars (B), and non reducing sugars (C). Vertical bars indicate the standard error of means. LSD Values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for Total sugars: Treatment = 0.295**, storage period = 0.331**, Interaction = 0.06**; Reducing sugars: Treatment = 0.591*,Storage period = 0.66**, Interaction = NS; Non-reducing sugars: Treatment = 0.516**, storage period = 0.577**, interaction = 1.15 *.
3.1.12. Reducing sugars (%) The reducing sugars of all treatments were increased from 0 to 7 days, then reach to the almost same level on the 14th day. The fruit of 1 mM PUT and control treatment showed almost alike response towards the reducing sugars throughout the storage with negligible differences (Fig. 4B). Once the storage finished, reducing sugars were highest in case of 1 mM PUT and lowest in 2 mM PUT treated papaya. Interaction of the PUT treatments and the time of storage resulted in positive relation regarding reducing sugars. Maximal amount of reducing sugars (10.32 Brix) was recorded in lower dose of PUT while minimal amount (8.0435 Brix) was revealed by fruit subjected to 2 mM PUT during last week of cold storage.
compared to the control fruit during the entire storage (Fig. 5A). The SOD activity in the control fruit was increased up to 21 days then suddenly decline during the last 7 days of storage. The highest SOD activity (1.19 U/mg protein) in control fruit was achieved at day 21 which was then decreased to 1.15 U/mg protein at 28th day. However, the fruit treated with 2 mM PUT showed higher SOD activity during the entire 4 weeks as compared to the all other treatments. The maximum SOD activity (1.317 U/mg protein) was recorded in 2 mM PUT treated fruit which was 1.14-folds higher than the untreated fruit at the end of storage.
3.1.13. Non-reducing sugars (%) Unlike the total and reducing sugars, non-reducing sugars were decreased during the first week then almost stabled up to the completion of the storage period in the fruit exposed to 2 mM PUT (Fig. 4C). At the end of storage, average non-reducing sugars of the untreated papaya fruit were almost 1.6 times more than treated fruit with 2 mM PUT. Maximum increase in non-reducing sugars of both untreated and 1 mM treated papaya fruit were observed during the last week of storage however, the 2 and 3 mM PUT treatment showed stability regarding non-reducing sugars.
3.1.14.2. Peroxidase activity. With the continuation of storage, peroxidase activity was also increased regardless of the treatments. The increasing trend of the POD activity was distinguished (P ≤ 0.05) in PUT treated fruit while, less distinguishable in the untreated fruit. Averaged over PUT treatment, the mean POD activity was significantly higher (1.07-fold) in 2 mM PUT treated fruit when compared with untreated fruit during the whole storage (Fig. 5B). Interaction of treatment into storage period depicted a clear difference in the mean POD activity during the storage period.
3.1.14. Antoixidative enzyme activities 3.1.14.1. Superoxide dismutase activity. Postharvest putrescine application significantly (P ≤ 0.05) increased the superoxide dismutase (SOD) activity in the pulp of stored papaya fruit as
3.1.14.3. Catalase activity. Exogenously applied putrescine also increased the catalase activity in papaya fruit as compared to the untreated fruit (Fig. 5C). The fruit dipped in 2 mM PUT solution and 5
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control fruit after 28 days of storage. 4. Discussion Weight loss of papaya fruit was continuously decreased throughout the storage. The reasons behind weight loss could be the rapid respiratory activities, loss of water through the skin and the consumption of stored metabolites during metabolic activities. Less weight loss was recorded in the putrescine treated fruit as compared to the control fruit, which might be associated with ability of PUT to stabilize the cell membrane (Mirdehghan et al., 2007) and also the suppression of respiration (Valero et al., 1998b). The suppression in the rate of respiration in putrescine treated mangoes has been reported previously by Razzaq et al. (2014). Similar results were reported in mango by Jawandha et al. (2012) and in pomegranate (Barman et al., 2011). Our results are also in accordance with Shiri et al. (2012) who reported reduced weight loss in PUT treated table grapes during long term storage. Fruit decay was significantly less in PUT treated fruit which may be due to the anti-pathogenic properties of polyamines. Walters (2003) has reviewed the anti-pathogenic abilities of polyamines and reported that polyamines have ability to make strong bonding with phenols and HCAA (hydroxycinnamic acid amide) both of which induce resistance against pathogen ultimately reducing decay incidence. Another factor for reducing the decay of PUT treated papaya fruit may also be associated with the strong defense mechanism against fungal attack. In this study firmness was declined with the passage of time during storage however, less reduction was noticed in PUT treated fruit. Putrescine a positively charged compound has tendency to make bonding with carboxyl group of cell wall resulting in strengthening of cell wall. This PUT-Carboxyl group bonding becomes hindrance in the way of cell wall degrading enzyme which ultimately results in retarded fruit softening (Valero et al., 2002). Similarly, reduced fruit softening by PUT treatment was reported in plum (Khan et al., 2007) mango (Razzaq et al., 2014) and grapes (Barman et al., 2011). Our results are also in line with Khosroshahi et al. (2007) who reported that retention of fruit firmness during storage of various fruit and vegetable is one of the key roles being played by putrescine. In the present study total soluble solids, ripening index and sugars were increased in papaya fruit during storage while reverse situation was observed in case of acidity and ascorbic acid. However both the increasing and decreasing trends were retarded by PUT application. The lower TSS, sugars and ripening index in PUT treated fruit as compared to control may be due to suppression of ethylene biosynthesis which directly affects metabolism of sugar in fruit. These results are supported by Bal (2012) who documented that soluble solid contents were higher in PUT treated fruit when stored at low temperature. The breakdown of starch and accumulation of total sugars are the major changes that happen during the process of ripening and storage. Our results indicated that total sugars were increased with expanded storage period however, more prominent increase was noticed in control fruit. Less accumulation of total sugars in PUT treated papaya fruit is due to delayed senescence and ripening, because of suppression of activities of amylase and phosphorylase enzymes which are stimulated in ripening (Hubbard et al., 1990). Externally applied PUT increase the endogenous level of polyamines that might reduce the accumulation of total sugars, reduced respiration as suggested by Valero et al. (2002). The acidity of fruit is another important parameter which determines the quality of fruit for consumer’s point of view. The titratable acidity of papaya fruit was declined in the course of entire storage duration however; this decline was less in PUT treated papaya fruit in comparison to control fruit. Reduction in acidity along with storage might be due to utilization of acids during the process of respiration (Zokaee et al., 2007 and Ishaq et al., 2009). Less decline in acidity of PUT treated fruit was also reported by Ghasemnezhad et al. (2010). Ascorbic acid contents of papaya fruit during storage were declined
Fig. 5. Effect of the postharvest application of putrescine during papaya storage on superoxide dismutase (A), peroxidase (B), and catalase (C). Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = Highly significant (P < 0.01). LSD for SOD: Treatment = 0.01**, storage period = 0.127**, Interaction = 0.025**; POD: Treatment = 0.08 **, Storage period = 0.089**, Interaction = 0.178**; CAT: Treatment = 0.141**, storage period = 0.157**, interaction = 0.314*.
Fig. 6. Effect of the postharvest application of putrescine, on respiration rate during papaya storage. Vertical bars indicate the standard error of means. LSD values NS = Non-significant (P > 0.05); * = Significant (P < 0.05); ** = highly significant (P < 0.01). LSD value for respiration: Treatment = 0.11** storage period = 0.121** and Interaction = 0.035**.
untreated expressed lower catalase activity during first 7 days while, higher for the rest of storage. The activity of catalase enzyme in the pulp of all fruit was increased from day 7 till 28th day however this increase was more pronounced in the PUT treated fruit. The Maximum catalase activity (6.94 U/mg protein) was found in fruit that were dipped in 3 mM PUT which was significantly (P ≤ 0.05) higher than the 6
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Acknowledgements
regardless of the applied treatments, however, less decline was noticed in PUT treated fruit as compared to the control fruit. Reduction of ascorbic acid contents in control fruit may be due to respiration and ripening related processes; while lower rate in PUT treated fruit could be due to suppression of ascorbate oxidase activity by PUT (Malik et al., 2006). Reduction in ascorbic acid contents were also reported by Yahia et al. (2001) in tomato and bell pepper. The total antioxidants are a good way of assessing nutritional value of fruit after storage. Results of the present study revealed that total antioxidants and total phenolic contents were increased initially then decreased just like ascorbic acid contents. Thus it can be concluded that antioxidants and phenolic contents are correlated. PUT treated fruit showed higher level of antioxidants and total phenolic which may be due to its anti-senescence properties because the activity of total antioxidants generally increased with senescence (Ghasemnezhad et al., 2010). Such correlation between TPC and total antioxidants was reported by (Palafox-Carlos et al., 2012) in mango. The catalase activity prevent the cells against oxidative damage caused by stress by breakdown of H2O2 into O2 and water (Ghasemnezhad et al., 2010). Increase in catalase activity is important for preventing cell damage while lower CAT activity shows weakened system of cells to remove H2O2 (Ng et al., 2005). Catalase activity was significantly higher in PUT treated fruit as compared to control in present study. Similar results were reported in apricots by Saba et al. (2012). The superoxide dismutase activity was higher in the PUT treated fruit as compared to the control fruit during entire storage period. The increase in SOD activity catalyzes the breakdown of hydrogen peroxide and provides protection to the cell against oxidative stress (Verma and Mishra, 2005). Higher SOD activity in PUT treated fruit may be due to bonding between PUT and antoixidative enzymes as reported in mango (Razzaq et al., 2014). Peroxidase enzyme activity limits the entry of pathogen to the cell tissue by creating a barrier and making cross linkages in the cell wall (Almardo et al., 2008). In this experiment higher POD activity was recorded in PUT treated papaya fruit during cold storage. Similar findings were also reported by Saba et al. (2012) in PUT treated apricots.
We are grateful to Mr. Rashid (owner of papaya orchard) for providing us good quality papaya fruit. We also appreciate Post-Harvest Research Centre, Ayyub Agriculture Research Institute, Faisalabad, Pakistan for providing us technical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2020.109367. References Almardo, L., Gomez Ros, L.V., Navaro, S.B., Bru, R., Barcelo, A.R., Pedreno, M.A., 2008. Class 3 peroxidases in plant defence system. J. Exp. Bot. 60, 377–390. AOAC, 2000. Vitamins and Other Nutrients, Official Methods of Analysis, 17th ed. AOAC, Washington, D.C, pp. 16–20 Chapter 45. Bal, E., 2012. Effect of postharvest putrescine and salicylic acid on cold storage duration and quality of sweet cherries. J. Faculty Agric. 7 23–21. Barman, k., Ram, A., Pal, R.K., 2011. 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5. Conclusion In conclusion, postharvest application of putrescine maintained the better quality of papaya fruit during the 4 weeks of storage. However, fruit subjected to 2 mM PUT showed higher firmness, less decay, less weight loss, suppressed respiration, higher level of total phenolic contents, total antioxidants and antoixidative enzyme activities of “Red lady” papaya fruit during storage. In general, this study suggested that the prestorage dipping of papaya fruit in PUT @ 2 mM performed best to maintain its quality and can be used for its prolonged storage effectively. Author contributions Aliya Hanif: PhD scholar and she conducted all research projects, analyzed the data and wrote the manuscript. Saeed Ahmad: PhD supervisor and guided in all executing the experiment, data analysis and written the manuscript. Muhammad Jafar Jaskani: Committee member help in experiment. Rashid Ahmad: Committee member and help to data analysis and writing. Declaration of Competing Interest None. 7
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