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Effects of putrescine treatment on the quality attributes and antioxidant activities of ‘Nam Dok Mai No.4’ mango fruit during storage
Scientia Horticulturae 233 (2018) 22–28
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Effects of putrescine treatment on the quality attributes and antioxidant activities of ‘Nam Dok Mai No.4’ mango fruit during storage Bussarin Wannabussapawich, Kanogwan Seraypheap
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Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords: Ripening Postharvest Polyamine Reactive oxygen species
This research is aimed at investigating the benefits of the exogenous application of putrescine on the postharvest quality and antioxidant activities of ‘Nam Dok Mai No.4’ mango. Mangoes harvested at commercial maturity were dipped into 1, 2, and 4 mmol/L putrescine (PUT) for 20 min while distilled water was used as the control. Treated fruit were stored at 14 °C for 9 days and then transferred to storage at 25 °C for 9 days. The 2 mmol/L PUT proved to be the most effective in keeping mango fruit quality intact during fruit ripening. Fruit hardness and titratable acidity (TA) were observed to be higher in treated fruit. The PUT treatment also caused a reduction in weight loss and soluble solids content (SSC). Moreover, 2 mmol/L PUT treated fruit exhibited the maximum superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX) and glutathione reductase (GR) activities and total antioxidant contents of fruit during storage. These findings suggest that exogenous application of 2 mmol/L PUT could be an effective treatment for prolonging the storage life and enhancing antioxidant activities of ‘Nam Dok Mai No.4’ mango after harvest.
1. Introduction Mango (Mangifera indica L.) is one of the most popular fruits in both the domestic and international markets because of its attractive aroma and great taste. Furthermore, with regard to nutrition, mango is appreciated for its rich mineral and vitamin content, including vitamin C, vitamin A, carotenoid and polyphenolic compounds (Masibo and He, 2008). It is well known that mango is a climacteric fruit which ripens quickly and has a short postharvest life, a major hurdle in prolonging its supply period in the international market (Ding et al., 2007). Various methods have been used to extend shelf life of mango fruit such as modified atmosphere packaging (Kumpoun and Uthaibutra, 2010), cold-shock treatment (Zhao et al., 2006), hot water treatment (Yimyong et al., 2011) and application of polyamines (Razzaq et al., 2014, Jongsri et al., 2017). Interestingly, a safe and potentially effective method for maintaining the storage life and quality of mango is the application of polyamines. Polycationic nature of polyamines, small organic metabolites, causes them to interact with negatively charged molecules such as phospholipids, protein, and nucleic acid which leads to antioxidant properties and the ability to protect cell from abiotic stresses (Kusano et al., 2008). Polyamines are growth regulators associated with numerous metabolic processes in plants including fruit maturation, fruit ripening, fruit softening, and fruit senescence (Gill and Tuteja, 2010a).
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Exogenous putrescine (PUT) has been reported to inhibit ethylene production, delay fruit ripening, and maintain fruit hardness in Kiwi fruit (Petkou et al., 2003). Polyamine treatments have also been found to reduce weight loss and maintain hardness in apricot (Davarynejad et al., 2013). During fruit ripening, excessive production and accumulation of reactive oxygen species (ROSs) cause oxidative damage and consequently reduce antioxidant ability to eliminate ROSs such as superoxide, hydroxyl radical, and hydrogen peroxide. If not scavenged, ROSs rapidly react with various molecules including DNA and proteins, resulting in membrane lipid peroxidation and ultimately causing cell death (Blokhina et al., 2003). The formation of ROSs is scavenged by the stimulation of antioxidant defense enzymes, e.g., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), guaiacol peroxidase (GPOX), and glutathione reductase (GR) (Gill and Tuteja, 2010b). Polyamines also play a role in non-enzymatic and enzymatic antioxidant systems (Gupta et al., 2013). It has been found that PUT treatment increased DPPH scavenging capacity and phenolic compounds in ‘Lasgerdi’ and ‘Shahrodi’ apricots (Davarynejad et al., 2013). Moreover, SOD, CAT and POX exhibited higher activities in ‘Bagheri’ and ‘Asgarabadi’ apricots (Saba et al., 2012) and ‘Samar Bahisht Chaunsa’ mango (Razzaq et al., 2014). However, information on the association of PUT treatment, non-
Corresponding author. E-mail address: [email protected] (K. Seraypheap).
https://doi.org/10.1016/j.scienta.2018.01.050 Received 18 October 2017; Received in revised form 16 January 2018; Accepted 16 January 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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Table 1 Effect of putrescine treatments on weight loss, firmness, SSC and TA of mango after storage at 14 °C and shelf life at 25 °C. Storage and shelf life periods
Putrescine treatment
Weight loss (%)
Firmness (N)
0
Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L
0g 0g 0g 0g 4.65 4.52 3.73 4.36 5.99 5.81 4.84 5.65 7.78 7.46 6.10 7.32 9.93 9.61 8.49 9.57
8.77 8.75 8.84 8.78 8.72 8.73 8.73 8.70 8.72 8.81 8.11 7.27 6.49 6.78 7.00 6.30 5.69 5.60 6.21 5.77
9
9+3
9+6
9+9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.22 e 0.25 ef 0.20 f 0.17 ef 0.26 d 0.35 d 0.22 e 0.26 d 0.30 bc 0.39 c 0.41 d 0.31 c 0.30 a 0.45 a 0.27 b 0.37 a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.06 0.05 0.05 0.04 0.05 0.05 0.04 0.22 0.25 0.08 0.20 0.13 0.07 0.07 0.22 0.15 0.13 0.08 0.11
a a a a a a a a c bc b d fg ef de g f f g f
SSC (°Brix)
TA (%)
9.28 ± 0.23 h 9.38 ± 0.20 h 9.38 ± 0.21 h 9.16 ± 0.18 h 13.49 ± 0.34 g 13.57 ± 0.32 g 13.33 ± 0.47 g 13.33 ± 0.72 g 16.91 ± 0.09 abc 16.80 ± 0.31 abc 14.76 ± 0.58 ef 17.79 ± 0.22 a 17.75 ± 0.19 a 16.23 ± 0.23 cd 15.58 ± 0.19 de 17.23 ± 0.15 ab 17.75 ± 0.19 a 17.76 ± 0.31a 16.53 ± 0.36 bc 17.70 ± 0.21 a
2.39 2.28 2.45 2.38 1.40 1.45 1.48 1.35 1.12 1.24 1.46 1.07 0.18 0.26 0.35 0.16 0.12 0.12 0.17 0.11
± 0.06 a ± 0.10 a ± 0.02 a ± 0.08 a ± 0.04 b ± 0.06 b ± 0.05 b ± 0.03 bc ± 0.09 de ± 0.07 cd ± 0.02 b ± 0.06 e ± 0.02g ± 0.02 fg ± 0.01 f ± 0.02 g ± 0.01 h ± 0.01h ± 0.00 g ± 0.01h
a
Means in a column followed by a different letter for the same storage period were significantly different at p = 0.05 by Duncan’s Multiple Range Test while ns showed no significance. Data are accompanied by standard errors of the means (n = 4).
enzymatic and enzymatic antioxidant systems, and hydrogen peroxide content in mango was lacking. Therefore, the aim of this work was to investigate the postharvest roles of PUT on quality attributes and antioxidant enzyme activities including SOD, CAT, GPOX, APX, and GR as well as free radical scavenging capacity and hydrogen peroxide content of mango cultivar ‘Nam Dok Mai No.4’ after cold storage and further shelf life at 25 °C.
Weight loss (%) =
[(Initial weight − final weight)] × 100 Initial weight
2.2.2. Fruit hardness Fruit hardness was determined at 3 points on the mango (blossom end, middle and stem end of the fruit) by using a penetrometer (Hardness tester FHM-1, Takemura, Japan) with a 12 mm cylindrical probe at a test speed of 1 mm/s. Results were expressed as force in Newtons (N) (Chancharoenrit, 2002).
2. Materials and methods
2.2.3. Soluble solids content (SSC) The soluble solids content was measured with a hand-held refractometer (Atago N-1E, Atogo Co., Japan) and reported as °Brix.
2.1. Plant materials and fruit treatment Mango fruit (Mangifera indica L. cv. ‘Nam dokmai No.4’) were harvested 90–100 days after fruit set which is the physiologically mature stage (the weight range is 350–450 g and average soluble solids content is 9.3 ± 0.4°Brix) from Saichon Commercial Orchard in Nakhon Ratchasima province, Northeast, Thailand. Afterward, mangoes were selected based on uniformity of size, color, and disease-free. Then, fruit were immediately transported to the laboratory. Before the application of treatments (day 0), 32 mangoes were sampled to monitor fruit characteristics. After which, mangoes were randomly distributed into 4 replicate groups of 160 fruit each. Fruit were immersed for 20 min in 1, 2, and 4 mmol/L putrescine (PUT) while distilled water was used as the control. All solution contained tween-20 (0.2%) to improve the absorption of the polyamine, and mangoes were left to dry before storage. In order to simulate the storage and exporting procedure of the commercial production of mango fruit, all of the control and treated fruit were stored at 14 °C and 90 ± 5% relative humidity for 9 days, and after which, fruit were transferred to 25 ± 1 °C and 65 ± 5% relative humidity and randomly sampled at 3, 6, and 9 days of shelf life at 25 °C (room temperature). The mesocarp were collected, frozen in liquid nitrogen, and stored at −80 °C for analysis of enzyme activities and total protein.
2.2.4. Titratable acidity (TA) Titratable acidity (TA) method was applied from AOAC (1984). One hundred mL of distilled water was mixed with 10 g of sliced mango pulp. Afterward, the macerate was filtered and titrated with 0.1 mol/L NaOH using phenolphthalein as the indicator. The reading for TA was expressed as a percentage of citric acid and was calculated as follows:
%TA = NaOH (mL.) × 0.1 NaOH (mol/L) × 0.07 × 100 10g 2.2.5. Peel color Peel color of mango was measured by using colorimeter (Konica Minolta Sensing, Inc., Japan) as Lightness (L) and hue angle value. The measurement was made from three equatorial positions of the mango peel (blossom end, middle and stem end). 2.3. Enzyme extraction and antioxidant enzyme activity assay The activity of SOD, CAT, GPOX, APX, and GR was measured using 1 g of endogenous sample homogenized in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 4% (w/v) polyvinylpyrrolidone, 4 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl. The homogenate was centrifuged at 10000g for 15 min at 4 °C, and the supernatant was used to assay for antioxidant enzyme activities.
2.2. Fruit quality measurements 2.2.1. Weight loss In order to evaluate any weight loss during the storage period, the same samples were consistently measured. The weight loss was calculated by AOAC (1984) as the following equation:
2.3.1. SOD determination SOD activity was analyzed using the method of McCord and 23
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Fig. 1. Peel color change of mango (A), L value (B), Hue angle (C) in various concentration of putrescine during storage period at 14 °C for 9 days and shelf life at 25 ± 1 °C.
Fridovich (1969). The reaction mixture of 200 μL containing 50 mmol/L potassium phosphate buffer (pH 7.8), 0.2 mmol/L EDTA (pH 8.0), 0.02 mmol/L cytochrome C, 20 μL of the enzyme extract, and 0.1 mmol/L Xanthine was added to the final solution. One unit of SOD activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of reduction at 550 nm.
reaction mixture was recorded at 470 nm and expressed as units of enzyme per milligram of protein. 2.3.4. APX determination APX activity was analyzed using the method of Nakano and Asada (1981). The APX reaction solution of 200 μL contained 50 mmol/L potassium phosphate buffer (pH 7.0), 10 mmol/L hydrogen peroxide, 1 mmol/L EDTA (pH 8.0), 0.5 mmol/L ascorbic acid, and 20 μL of the enzyme extract. Changes in the absorbance of the reaction solution at 290 nm were determined. The APX activity was defined as units of enzyme per milligram of protein.
2.3.2. CAT determination CAT activity was analyzed according to the method of Beers and Sizer (1952). The reaction mixture of 200 μL contained 50 mmol/L potassium phosphate buffer (pH 7.0), 10 mmol/L hydrogen peroxide, and 20 μL of the enzyme extract. The enzyme activity was calculated from the absorbance at 240 nm and shown as units of enzyme per milligram of protein.
2.3.5. GR determination GR activity was determined by Smith et al. (1988). A total of 200 μL of the reaction mixture contained 100 mmol/L potassium phosphate buffer (pH 7.5), 0.5 mmol/L EDTA (pH 8.0), 0.75 mmol/L 5,5′ dithiobis (2-nitrobenzoic acid), 0.1 mmol/L NADPH, 0.5 mmol/L GSSG, and 20 μL of the enzyme extract. The increase in absorbance was monitored at 412 nm, and the activity of enzyme was expressed as units of enzyme per milligram of protein.
2.3.3. GPOX determination GPOX activity was determined according to MacAdam et al. (1992). The reaction mixture of 200 μL contained 50 mmol/L potassium phosphate buffer (pH 6.0), 0.2 mmol/L hydrogen peroxide, 2.5 mmol/L guaiacol, and 20 μL of the enzyme extract. The absorbance of the 24
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observed in 2 mmol/L PUT treated fruit compared to other treatments on days 9 + 3, 9 + 6 and 9 + 9. No significant differences in weight loss were observed between 1 mmol/L PUT, 4 mmol/L PUT, and control treatments. The treatment results revealed that 2 mmol/L PUT was able to slow down the rate of weight loss. The loss of weight is mainly due to evaporation of water from the peel. It could be suggested that PUT inhibited respiration rate and ethylene production, thus delaying postharvest weight loss (Valero et al., 1998). Our results are in agreement with the finding of Davarynejad et al. (2013) that postharvest application of PUT significantly reduced the weight loss in apricot fruit.
2.3.6. Protein contents The protein concentration in each sample was estimated from mesocarp tissue following the Bradford (1976) method and was expressed as mg/g FW while using bovine serum albumin (BSA) as the standard. The standard curve was linear between 0 μg/mL BSA and 100 μg/mL BSA (R2 = 0.995). 2.4. Hydrogen peroxide content The hydrogen peroxide content was assayed according to Jana and Choudhuri (1982). H2O2 content was extracted by homogenized mesocarp tissue 1.0 g with 100 mmol/L potassium phosphate buffer (pH 6.5) and 1 mmol/L hydroxylamine at 0 °C. After centrifugation at 4 °C and 10000g for 15 min, the supernatant was collected. The reaction mixture consisting of 600 μL supernatant and 90 μL of 0.3% (v/v) titanium sulphate in 20% H2SO4 (v/v) were freshly prepared before use and then centrifuged at 10000g for 10 min. The standard curves were prepared with H2O2 at a concentration ranging between 10 μmol and 100 μmol (R2 = 0.991). The supernatant was measured at 410 nm and the amount of H2O2 was calculated according to standard curve and express as μmol/g FW.
3.1.2. Hardness Fruit hardness, which rapidly decreased during storage time, is a major limiting factor for the marketing and storage life of mango fruit. Although during the first 9 days of low temperature storage, no significant differences were observed among the fruits in all treatments (Table 1). On day 9 + 3, 9 + 6, and 9 + 9, fruit hardness was found significantly higher in 2 mmol/L PUT treated fruits when compared to other treatments. The results from this experiment revealed that mango fruit treated with 2 mmol/L PUT significantly increased fruit hardness during storage time. It has been reported that the application of polyamines enhances fruit hardness in different fruits, including plums (Davarynejad et al., 2015), apricot (Martínez-Romero et al., 2002), pomegranates (Barman et al., 2011), and grape (Harindra Champa et al., 2015). The influence of polyamines on fruit hardness augmentation can be attributed to their capacity in cross-linking to the carboxyl group of pectin substances in the cell wall, resulting in rigidification (Abbott et al., 1989). This binding between polyamines and pectin also inhibited cell wall-degrading enzymes, such as pectin esterase, polygalacturonase (PG), and pectin methylesterase (PME), which beneficially minimize fruit hardness after harvest (Valero et al., 2002).
2.5. DPPH free radical scavenging assay The DPPH assay was determined according to the method of Choi et al. (2006) with some modifications. One gram of mango tissue was homogenized using 10 mL of 80% (v/v) ethanol. The homogenate was vortexed for 1 min and centrifuged at 10000g for 15 min at 4 °C. The supernatants were used for fruit extract analysis. 100 μL of fruit extract was mixed in 300 μL of 80% (v/v) ethanol and 400 μL of 0.2 mmol/L DPPH solution and then kept in the dark for 10 min. The absorbance was measured at 515 nm. An ethanol solution without DPPH radical served as the control. Results were expressed as percentage of inhibition of the DPPH radical based on the following formula:
%Inhibition =
1 − Absorbance (sample)) Absorbance (control)
3.1.3. Soluble solids content (SSC) and titratable acidity (TA) The SSC and TA contents are important attributes of the postharvest quality of climacteric fruit. Our experiment revealed that 2 mmol/L PUT treated fruit exhibits the lowest SSC and the highest TA content during storage period (Table 1). A variation of SSC contents was statistically significant (p < 0.05). It was found that 2 mmol/L PUT treated fruits had the lowest percentage of SSC on days 9 + 3, 9 + 6, and 9 + 9 compared with 1 mmol/L, 4 mmol/L, and control fruit. TA content decreased constantly throughout the storage period, but there were no significant differences on days 0–9. From day 9 + 3 to the end of storage time, 2 mmol/L PUT stimulated significantly higher TA than other treatments. On day 9 + 9, 2 mmol/L PUT treated fruits had the highest TA (0.17%) followed by 4 mmol/L PUT (0.11%), 1 mmol/L (0.12%), and control fruit (0.12%), respectively. The concentration of SSC and TA was significantly affected by the application of PUT. During the ripening process, the concentration of SSC continuously increased because of the hydrolysis of polysaccharides (Mahto and Dasb, 2013), and TA was associated with the concentration of organic acid. Respiration appeared to provoke the consumption of organic acid and reduced the TA in fruits (Khosroshahi et al., 2007). The particular effect of PUT on SSC and TA could be related to the reduction of respiration rate and a delayed in ripening process. Similar results have also been reported in plum (Davarynejad et al., 2015) and apricot (MartínezRomero et al., 2002).
× 100
2.6. Ferric reducing antioxidant power assay (FRAP assay) FRAP assay was measured according to the method by Benzie and Strain (1999) with modifications. The FRAP solution was prepared by mixing 25 mL of a 0.3 mol/L acetate buffer, pH 3.6, 2.5 mL of a 10 mmol/L 2,4,6-tris (1-pyridy)-5-triazine (TPTZ) solution in 40 mmol/ L HCl and 20 mmol/L FeCl3·6H2O solution. Fruit extracts of 150 μL was mixed with FRAP solution of 3000 μL for 30 min in a darkened condition, and to which, the absorbance was determined at 593 nm. The standard curve was linear between 200 μmol/L and 1000 μmol/L FeSO4. 7H2O (R2 = 0.996). Results were expressed as micromoles of ferrous equivalent Fe (II) per fresh weight. 2.7. Statistical analysis Experimental data were the mean ± standard errors. Statistical analysis was performed with the IBM SPSS Statistics 22 (IBM SPSS, USA), and the data were tested by one-way analysis of variance (ANOVA). The means were separated by Duncan’s Multiple Range Test, and differences at p ≤ 0.05 were considered to be significant.
3.1.4. Peel color During mango ripening, the peel color of mango fruit changes from green to yellow. The lightness (L value) and hue angle have been used as good indicators of peel color changes. During fruit ripening, the L value increased and hue angles decreased. Fruit treated with 2 mmol/L PUT had a notable delay in peel color change on days 9 + 3, 9 + 6, and 9 + 9 (Fig. 1A). The L value of 2 mmol/L PUT fruits was the lowest (Fig. 1B), and for the hue angles, 2 mmol/L PUT fruits showed the highest value when compared with other treatments (Fig. 1C). The
3. Results and discussion 3.1. Effects of exogenous putrescine treatment on fruit quality parameters 3.1.1. Weight loss Our study showed that the loss of weight increased progressively during storage period (Table 1). However, the lowest weight loss was 25
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Fig. 2. The activity of (A) SOD, (B) CAT, (C) GPOX, (D) APX, (E) GR and concentration of (F) H2O2 in mango fruit treated with exogenous PUT during storage at 14 °C for 9 days and shelf life at 25 ± 1 °C. All data presented as a mean of four biological replicates, and error bar represent ± standard error. According to the Duncan’s Multiple Range Test (p ≤ 0.05), statistically significant differences were indicated for each sampling time, shown in different letters.
polyamines might be involved with the antioxidant system in plants (Verma and Mishra, 2005). Our results found that SOD activity progressively decreased with the increased in days of storage for all the treatments. Two mmol/L PUT treated fruit showed the highest SOD activity from day 9 to day 9 + 9 (Fig. 2A). The superoxide radicals are converted into hydrogen peroxide as a result of SOD, which later is disintegrated into H2O and O2 by CAT and POX. CAT activity showed dramatic increases in all treatments following removal from low temperature storage up to day 9 + 6 of fruit ripening, but later on, their activities decreased at the end of storage (Fig. 2B). Mango fruit treated with 2 mmol/L PUT exhibited the highest CAT activity among treatments during shelf life at 25 °C. Similarly, GPOX activity in mesocarp tissue rose noticeably on day 9 + 3, reached its maximum on day 9 + 6, and gradually declined until the end of the storage period (Fig. 2C). The highest GPOX activity was found in 2 mmol/L PUT treated fruits followed by 1 mmol/L while control fruits had the lowest GPOX activity. APX activity exhibited a constant increase until day 9 + 6 and remained continual until a decline was observed on day 9 + 9 (Fig. 2D). The 2 mmol/L PUT treated fruit exhibited the highest APX activity over the period of day 9 + 6 to day 9 + 9 of the treatment. Similarly, GR activity showed a steady increase in all treatments upon removal from
2 mmol/L PUT treatment are capable of delaying mango peel color changes during 9 days of shelf life at 25 °C similar to observations make in grape (Harindra Champa et al., 2015) and apricot (Martínez-Romero et al., 2002). Hydrolytic activities that act on chloroplast thylakoid membranes have been observed to be reduced after polyamines application (Lester, 2000).
3.2. Effect of exogenous putrescine treatment on antioxidant enzyme activities During fruit ripening, production and accumulation of reactive oxygen species (ROSs), such as hydrogen peroxide and superoxide anion, continually increased. Thus, a continuous increase in oxidation is responsible for the stressful process of ripening (Jimenez et al., 2002). The generated ROSs causes oxidative stress to lipid membrane, nucleic acid, and proteins (Scandalios, 1993). Plants have defensive mechanisms for scavenging damage caused by ROSs. Such types of damage could be scavenged by plant enzymatic defenses including SOD, CAT, APX, POX, MDHAR, DHAR, GPOX, and GR and also by non-enzymatic antioxidants, for example ascorbic acid, beta-carotenoid and glutathione (Gill and Tuteja, 2010b). It has been suggested that 26
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Fig. 3. Content of (A) DPPH and (B) FRAP of mango fruit treated with exogenous PUT during storage at 14 °C for 9 days and shelf life at 25 ± 1 °C. All data presented as a mean of four biological replicates, and error bar represent ± standard error. According to the Duncan’s Multiple Range Test (p ≤ 0.05), statistically significant differences were indicated for each sampling time, shown in different letters.
the lowest among treatments from day 9 to day 9 + 9 (Fig. 2). It has been well established that an excess of hydrogen peroxide is considered an oxidative stress parameter. Mango ripening was found to be associated with enhanced hydrogen peroxide contents. In the present study, we found that the treatment with 2 mmol/L had the highest reduction in H2O2 content in mango fruit. A noticeably similar reaction was observed in zucchini fruit treated with 1 mmol/L PUT which resulted in having the highest influence in the reduction of H2O2 content (Palma et al., 2015). Japanese plum fruit also paralleled with our findings (Singh and Singh, 2012).
low temperature storage to day 9 + 6, but it began to decrease at the end of the storage period (Fig. 2E). Mango fruits treated with 2 mmol/L PUT had significant higher GR activity compared with other treatments. The results of our study revealed that the application of 2 mmol/L PUT effectively induced activities of antioxidant enzymes, including SOD, CAT, GPOX, APX and GR. Oxidative stress is associated with reduced antioxidant enzyme activities, leading to a short postharvest life of fruit. The accumulation of ROSs contributed to higher membrane deterioration and loss of tissue structure (Kumar et al., 2011). Polyamines may bind to the antioxidant enzymes or may directly scavenge ROSs (Duan et al., 2008; Groppa and Benavides, 2008). In agreement with our finding, previous studies have shown that application of exogenous polyamines (PUT and SPM) significantly increased the activities of CAT, SOD, and POX in two apricot cultivars (Saba et al., 2012). Similarly, higher activities of CAT, SOD, and POX during the storage period have been reported in PUT treated ‘Samar Bahisht Chaunsa’ mango fruit (Razzaq et al., 2014). In addition, zucchini treated with PUT resulted in higher APX and GR activities with respect to control (Palma et al., 2016).
3.4. Effect of exogenous putrescine treatment on total antioxidant capacity The data indicated that the antioxidant capacity analyzed by DPPH free radical scavenging assay slightly increased until day 9 + 6 and later decreased by the end of the storage time (Fig. 3A). There was a notable difference (p < 0.05) between PUT and control treatments. During shelf life at 25 °C, 2 mmol/L PUT treated fruit had the highest total antioxidant capacity, while control fruit had the lowest. The total antioxidant content using FRAP assay had a similar trend as DPPH radical assay (Fig. 3B). The total antioxidant increased gradually until day 9 + 6, but on day 9 + 9, it exhibited a slight reduction. It was noticed that the 2 mmol/L PUT treated fruit showed the highest total antioxidant content when compared to other treatments. The determination
3.3. Effect of exogenous putrescine treatment on hydrogen peroxide Levels of H2O2 content in all treatment tended to increase gradually during ripening. The H2O2 content in 2 mmol/L PUT treated fruit was 27
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of antioxidant capacity is one of the approaches that expresses the nonenzymatic antioxidant system and biological properties of fruit which plays a vital role in maintaining fruit quality during storage period. In this work, the application of 2 mmol/L PUT was effective in maintaining the total antioxidant contents during postharvest storage both assayed by the FRAP and DPPH methods. These antioxidants were well maintained by putrescine, which are basically anti-senescence agents (Seiler and Rual, 2005). In addition, 2 mmol/L PUT also maintained total antioxidant property by increasing the SOD, CAT, GPOX, APX, and GR as shown in our data. Previous works describe polyamines as being associated with maintaining the total antioxidant in several fruits such as pomegranate (Mirdehghan et al., 2007), plum (Davarynejad et al., 2015), kiwi (Jhalegar et al., 2012), and zucchini (Palma et al., 2015).
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Martínez-Romero, D., Serrano, M., Carbonell, A., Burgos, O.L., Riquelme, F., Valero, D., 2002. Effect of post –harvest putrescine treatment on extending shelf life and reducing mechanical damage in apricot. J. Food Sci. 67, 1706–1712. Masibo, M., He, Q., 2008. Major mango polyphenols and their potential significance to human health. Compr. Rev. Food Sci. Food Saf. 7, 309–319. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055. Mirdehghan, S.H., Rahemi, M., Serrano, M., Guillén, F., Martínez-Romero, D., Valero, D., 2007. The application of polyamines by pressure or immersion as a tool to maintain functional properties in stored pomegranate arils. J. Agric. Food Chem. 55, 755–760. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Palma, F., Carvajal, F., Ramos, M.J., Jamilena, M., Garrido, D., 2015. Effect of putrescine application on maintenance of zucchini fruit quality during cold storage: contribution of GABA shunt and other related nitrogen metabolites. Postharvest Biol. Technol. 99, 131–140. Palma, F., Carvajal, F., Jamilena, M., Garrido, D., 2016. Putrescine treatment increase the antioxidant response and carbohydrate content in zucchini fruit stored at low temperature. Postharvest Biol. Technol. 118, 68–70. Petkou, I., Pritsa, T., Sfakiotakis, E., 2003. Effect of dipping and pressure infiltration of putrescine on the propylene induced autocatalytic ethylene production and ripening of ‘Hayward’ Kiwi fruit. Acta Hortic. 610, 261–266. Razzaq, K., Khan, A.S., Malik, A.U., Shahid, M., 2014. Role of putrescine in regulating fruit softening and antioxidative enzyme systems in ‘Samar Bahisht Chaunsa’ mango. Postharvest 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. Scandalios, G.J., 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7–12. Seiler, N., Rual, F., 2005. Polyamine and apoptosis. J. Cell. Mol. Med. 9, 623–642. Singh, P.S., Singh, Z., 2012. Role of membrane lipid peroxidation, enzymatic and nonenzymatic antioxidative systems in the development of chilling injury in Japanese plums. J. Am. Soc. Hortic. Sci. 137, 473–481. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5’-dithiobis (2-nitrobenzoic acid). Anal. Biochem. 175, 408–413. Valero, D., Martínez-Romero, D., Serrano, M., Riquelme, F., 1998. Polyamine response to external mechanical bruising in two mandarin cultivars. HortSci 33, 1220–1223. Valero, D., Martínez-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. Yimyong, S., Datsenka, U.T., Handa, K.A., Seraypheap, K., 2011. Hot water treatment delays ripening-associated metabolic shift in ‘Okrong’ Mango Fruit during storage. J. Amer. Soc. Hort. Sci. 136, 441–451. Zhao, Z., Jiang, W., Cao, J., Zhao, Y., Gu, Y., 2006. Effect of cold-shock treatment on chilling injury in mango (Mangifera indica L. cv. ‘Wacheng’) fruit. J. Sci. Food Agric. 86, 2458–2462.
4. Conclusion Exogenous application of 2 mmol/L PUT was the most effective treatment to improve the postharvest quality and antioxidant activities of ‘Nam Dok Mai No.4′ mango during storage. Positive effects of PUT treatment were evidently confirmed from its capability to maintain high SOD, CAT, GPOX, APX, and GR enzyme activities and non-enzymatic antioxidant contents while reduce hydrogen peroxide content. These finding suggested that the delaying of mango fruit ripening after the PUT treatment is promising for manufacturers and can be economically beneficial as mango can potential reach a wider market. Future studies may focus on PUT induction of antioxidant activity and internal polyamines for reducing chilling injury during the low temperature storage of mango fruit. Acknowledgements Financial support for this research was provided by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and the Overseas Research Experience Scholarship (ORES) from the Graduate School and Faculty of Science. The author was a recipient of a Science Achievement Scholarship of Thailand. References AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed. AOAC, Washington, DC. Abbott, J.A., Conway, S.W., Sams, E.C., 1989. Postharvest calcium chloride infiltration affects textural attributes of apples. J. Am. Soc. Hortic. Sci. 114, 932–936. Barman, K., Asrey, R., 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. Beers, R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Benzie, I.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 91, 179–194. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248–254. Chancharoenrit, J., 2002. Effects of Hot Water Dips on Physiological Changes and Chilling Injury After Storage of ‘Hom Thong’ Variety of Banana. Master’s Thesis. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. Choi, Y., Lee, S.M., Chun, J., Lee, H.B., Lee, J., 2006. Influence of heat treatment on the antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom. Food Chem. 99, 381–387. Davarynejad, G., Zarei, M., Ardakani, E., Nasrabadi, M.E., 2013. Influence of putrescine application on storability: postharvest quality and antioxidant activity of two Iranian apricot (Prunus armeniaca L.) cultivars. Notulae Sci. Biol. 5, 212–219. Davarynejad, G.H., Zarei, M., Nasrabadi, M.E., Ardakani, E., 2015. Effects of salicylic acid and putrescine on storability: quality attributes and antioxidant activity of plum cv.‘Santa Rosa’. J. Food Sci. Technol. 52, 2053–2062. Ding, Z.S., Tian, S.P., Zheng, X.L., Zhou, Z.W., Xu, Y., 2007. Responses of reactive oxygen metabolism and quality in mango fruit to exogenous oxalic acid or salicylic acid under chilling temperature stress. Physiol. Plant. 130, 112–121. Duan, J., Li, J., Guo, S., Kang, Y., 2008. Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances short-term salinity
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Effects of putrescine treatment on the quality attributes and antioxidant activities of ‘Nam Dok Mai No.4’ mango fruit during storage Bussarin Wannabussapawich, Kanogwan Seraypheap
T
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Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords: Ripening Postharvest Polyamine Reactive oxygen species
This research is aimed at investigating the benefits of the exogenous application of putrescine on the postharvest quality and antioxidant activities of ‘Nam Dok Mai No.4’ mango. Mangoes harvested at commercial maturity were dipped into 1, 2, and 4 mmol/L putrescine (PUT) for 20 min while distilled water was used as the control. Treated fruit were stored at 14 °C for 9 days and then transferred to storage at 25 °C for 9 days. The 2 mmol/L PUT proved to be the most effective in keeping mango fruit quality intact during fruit ripening. Fruit hardness and titratable acidity (TA) were observed to be higher in treated fruit. The PUT treatment also caused a reduction in weight loss and soluble solids content (SSC). Moreover, 2 mmol/L PUT treated fruit exhibited the maximum superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX) and glutathione reductase (GR) activities and total antioxidant contents of fruit during storage. These findings suggest that exogenous application of 2 mmol/L PUT could be an effective treatment for prolonging the storage life and enhancing antioxidant activities of ‘Nam Dok Mai No.4’ mango after harvest.
1. Introduction Mango (Mangifera indica L.) is one of the most popular fruits in both the domestic and international markets because of its attractive aroma and great taste. Furthermore, with regard to nutrition, mango is appreciated for its rich mineral and vitamin content, including vitamin C, vitamin A, carotenoid and polyphenolic compounds (Masibo and He, 2008). It is well known that mango is a climacteric fruit which ripens quickly and has a short postharvest life, a major hurdle in prolonging its supply period in the international market (Ding et al., 2007). Various methods have been used to extend shelf life of mango fruit such as modified atmosphere packaging (Kumpoun and Uthaibutra, 2010), cold-shock treatment (Zhao et al., 2006), hot water treatment (Yimyong et al., 2011) and application of polyamines (Razzaq et al., 2014, Jongsri et al., 2017). Interestingly, a safe and potentially effective method for maintaining the storage life and quality of mango is the application of polyamines. Polycationic nature of polyamines, small organic metabolites, causes them to interact with negatively charged molecules such as phospholipids, protein, and nucleic acid which leads to antioxidant properties and the ability to protect cell from abiotic stresses (Kusano et al., 2008). Polyamines are growth regulators associated with numerous metabolic processes in plants including fruit maturation, fruit ripening, fruit softening, and fruit senescence (Gill and Tuteja, 2010a).
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Exogenous putrescine (PUT) has been reported to inhibit ethylene production, delay fruit ripening, and maintain fruit hardness in Kiwi fruit (Petkou et al., 2003). Polyamine treatments have also been found to reduce weight loss and maintain hardness in apricot (Davarynejad et al., 2013). During fruit ripening, excessive production and accumulation of reactive oxygen species (ROSs) cause oxidative damage and consequently reduce antioxidant ability to eliminate ROSs such as superoxide, hydroxyl radical, and hydrogen peroxide. If not scavenged, ROSs rapidly react with various molecules including DNA and proteins, resulting in membrane lipid peroxidation and ultimately causing cell death (Blokhina et al., 2003). The formation of ROSs is scavenged by the stimulation of antioxidant defense enzymes, e.g., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), guaiacol peroxidase (GPOX), and glutathione reductase (GR) (Gill and Tuteja, 2010b). Polyamines also play a role in non-enzymatic and enzymatic antioxidant systems (Gupta et al., 2013). It has been found that PUT treatment increased DPPH scavenging capacity and phenolic compounds in ‘Lasgerdi’ and ‘Shahrodi’ apricots (Davarynejad et al., 2013). Moreover, SOD, CAT and POX exhibited higher activities in ‘Bagheri’ and ‘Asgarabadi’ apricots (Saba et al., 2012) and ‘Samar Bahisht Chaunsa’ mango (Razzaq et al., 2014). However, information on the association of PUT treatment, non-
Corresponding author. E-mail address: [email protected] (K. Seraypheap).
https://doi.org/10.1016/j.scienta.2018.01.050 Received 18 October 2017; Received in revised form 16 January 2018; Accepted 16 January 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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Table 1 Effect of putrescine treatments on weight loss, firmness, SSC and TA of mango after storage at 14 °C and shelf life at 25 °C. Storage and shelf life periods
Putrescine treatment
Weight loss (%)
Firmness (N)
0
Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L Control 1 mmol/L 2 mmol/L 4 mmol/L
0g 0g 0g 0g 4.65 4.52 3.73 4.36 5.99 5.81 4.84 5.65 7.78 7.46 6.10 7.32 9.93 9.61 8.49 9.57
8.77 8.75 8.84 8.78 8.72 8.73 8.73 8.70 8.72 8.81 8.11 7.27 6.49 6.78 7.00 6.30 5.69 5.60 6.21 5.77
9
9+3
9+6
9+9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.22 e 0.25 ef 0.20 f 0.17 ef 0.26 d 0.35 d 0.22 e 0.26 d 0.30 bc 0.39 c 0.41 d 0.31 c 0.30 a 0.45 a 0.27 b 0.37 a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.06 0.05 0.05 0.04 0.05 0.05 0.04 0.22 0.25 0.08 0.20 0.13 0.07 0.07 0.22 0.15 0.13 0.08 0.11
a a a a a a a a c bc b d fg ef de g f f g f
SSC (°Brix)
TA (%)
9.28 ± 0.23 h 9.38 ± 0.20 h 9.38 ± 0.21 h 9.16 ± 0.18 h 13.49 ± 0.34 g 13.57 ± 0.32 g 13.33 ± 0.47 g 13.33 ± 0.72 g 16.91 ± 0.09 abc 16.80 ± 0.31 abc 14.76 ± 0.58 ef 17.79 ± 0.22 a 17.75 ± 0.19 a 16.23 ± 0.23 cd 15.58 ± 0.19 de 17.23 ± 0.15 ab 17.75 ± 0.19 a 17.76 ± 0.31a 16.53 ± 0.36 bc 17.70 ± 0.21 a
2.39 2.28 2.45 2.38 1.40 1.45 1.48 1.35 1.12 1.24 1.46 1.07 0.18 0.26 0.35 0.16 0.12 0.12 0.17 0.11
± 0.06 a ± 0.10 a ± 0.02 a ± 0.08 a ± 0.04 b ± 0.06 b ± 0.05 b ± 0.03 bc ± 0.09 de ± 0.07 cd ± 0.02 b ± 0.06 e ± 0.02g ± 0.02 fg ± 0.01 f ± 0.02 g ± 0.01 h ± 0.01h ± 0.00 g ± 0.01h
a
Means in a column followed by a different letter for the same storage period were significantly different at p = 0.05 by Duncan’s Multiple Range Test while ns showed no significance. Data are accompanied by standard errors of the means (n = 4).
enzymatic and enzymatic antioxidant systems, and hydrogen peroxide content in mango was lacking. Therefore, the aim of this work was to investigate the postharvest roles of PUT on quality attributes and antioxidant enzyme activities including SOD, CAT, GPOX, APX, and GR as well as free radical scavenging capacity and hydrogen peroxide content of mango cultivar ‘Nam Dok Mai No.4’ after cold storage and further shelf life at 25 °C.
Weight loss (%) =
[(Initial weight − final weight)] × 100 Initial weight
2.2.2. Fruit hardness Fruit hardness was determined at 3 points on the mango (blossom end, middle and stem end of the fruit) by using a penetrometer (Hardness tester FHM-1, Takemura, Japan) with a 12 mm cylindrical probe at a test speed of 1 mm/s. Results were expressed as force in Newtons (N) (Chancharoenrit, 2002).
2. Materials and methods
2.2.3. Soluble solids content (SSC) The soluble solids content was measured with a hand-held refractometer (Atago N-1E, Atogo Co., Japan) and reported as °Brix.
2.1. Plant materials and fruit treatment Mango fruit (Mangifera indica L. cv. ‘Nam dokmai No.4’) were harvested 90–100 days after fruit set which is the physiologically mature stage (the weight range is 350–450 g and average soluble solids content is 9.3 ± 0.4°Brix) from Saichon Commercial Orchard in Nakhon Ratchasima province, Northeast, Thailand. Afterward, mangoes were selected based on uniformity of size, color, and disease-free. Then, fruit were immediately transported to the laboratory. Before the application of treatments (day 0), 32 mangoes were sampled to monitor fruit characteristics. After which, mangoes were randomly distributed into 4 replicate groups of 160 fruit each. Fruit were immersed for 20 min in 1, 2, and 4 mmol/L putrescine (PUT) while distilled water was used as the control. All solution contained tween-20 (0.2%) to improve the absorption of the polyamine, and mangoes were left to dry before storage. In order to simulate the storage and exporting procedure of the commercial production of mango fruit, all of the control and treated fruit were stored at 14 °C and 90 ± 5% relative humidity for 9 days, and after which, fruit were transferred to 25 ± 1 °C and 65 ± 5% relative humidity and randomly sampled at 3, 6, and 9 days of shelf life at 25 °C (room temperature). The mesocarp were collected, frozen in liquid nitrogen, and stored at −80 °C for analysis of enzyme activities and total protein.
2.2.4. Titratable acidity (TA) Titratable acidity (TA) method was applied from AOAC (1984). One hundred mL of distilled water was mixed with 10 g of sliced mango pulp. Afterward, the macerate was filtered and titrated with 0.1 mol/L NaOH using phenolphthalein as the indicator. The reading for TA was expressed as a percentage of citric acid and was calculated as follows:
%TA = NaOH (mL.) × 0.1 NaOH (mol/L) × 0.07 × 100 10g 2.2.5. Peel color Peel color of mango was measured by using colorimeter (Konica Minolta Sensing, Inc., Japan) as Lightness (L) and hue angle value. The measurement was made from three equatorial positions of the mango peel (blossom end, middle and stem end). 2.3. Enzyme extraction and antioxidant enzyme activity assay The activity of SOD, CAT, GPOX, APX, and GR was measured using 1 g of endogenous sample homogenized in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 4% (w/v) polyvinylpyrrolidone, 4 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl. The homogenate was centrifuged at 10000g for 15 min at 4 °C, and the supernatant was used to assay for antioxidant enzyme activities.
2.2. Fruit quality measurements 2.2.1. Weight loss In order to evaluate any weight loss during the storage period, the same samples were consistently measured. The weight loss was calculated by AOAC (1984) as the following equation:
2.3.1. SOD determination SOD activity was analyzed using the method of McCord and 23
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Fig. 1. Peel color change of mango (A), L value (B), Hue angle (C) in various concentration of putrescine during storage period at 14 °C for 9 days and shelf life at 25 ± 1 °C.
Fridovich (1969). The reaction mixture of 200 μL containing 50 mmol/L potassium phosphate buffer (pH 7.8), 0.2 mmol/L EDTA (pH 8.0), 0.02 mmol/L cytochrome C, 20 μL of the enzyme extract, and 0.1 mmol/L Xanthine was added to the final solution. One unit of SOD activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of reduction at 550 nm.
reaction mixture was recorded at 470 nm and expressed as units of enzyme per milligram of protein. 2.3.4. APX determination APX activity was analyzed using the method of Nakano and Asada (1981). The APX reaction solution of 200 μL contained 50 mmol/L potassium phosphate buffer (pH 7.0), 10 mmol/L hydrogen peroxide, 1 mmol/L EDTA (pH 8.0), 0.5 mmol/L ascorbic acid, and 20 μL of the enzyme extract. Changes in the absorbance of the reaction solution at 290 nm were determined. The APX activity was defined as units of enzyme per milligram of protein.
2.3.2. CAT determination CAT activity was analyzed according to the method of Beers and Sizer (1952). The reaction mixture of 200 μL contained 50 mmol/L potassium phosphate buffer (pH 7.0), 10 mmol/L hydrogen peroxide, and 20 μL of the enzyme extract. The enzyme activity was calculated from the absorbance at 240 nm and shown as units of enzyme per milligram of protein.
2.3.5. GR determination GR activity was determined by Smith et al. (1988). A total of 200 μL of the reaction mixture contained 100 mmol/L potassium phosphate buffer (pH 7.5), 0.5 mmol/L EDTA (pH 8.0), 0.75 mmol/L 5,5′ dithiobis (2-nitrobenzoic acid), 0.1 mmol/L NADPH, 0.5 mmol/L GSSG, and 20 μL of the enzyme extract. The increase in absorbance was monitored at 412 nm, and the activity of enzyme was expressed as units of enzyme per milligram of protein.
2.3.3. GPOX determination GPOX activity was determined according to MacAdam et al. (1992). The reaction mixture of 200 μL contained 50 mmol/L potassium phosphate buffer (pH 6.0), 0.2 mmol/L hydrogen peroxide, 2.5 mmol/L guaiacol, and 20 μL of the enzyme extract. The absorbance of the 24
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observed in 2 mmol/L PUT treated fruit compared to other treatments on days 9 + 3, 9 + 6 and 9 + 9. No significant differences in weight loss were observed between 1 mmol/L PUT, 4 mmol/L PUT, and control treatments. The treatment results revealed that 2 mmol/L PUT was able to slow down the rate of weight loss. The loss of weight is mainly due to evaporation of water from the peel. It could be suggested that PUT inhibited respiration rate and ethylene production, thus delaying postharvest weight loss (Valero et al., 1998). Our results are in agreement with the finding of Davarynejad et al. (2013) that postharvest application of PUT significantly reduced the weight loss in apricot fruit.
2.3.6. Protein contents The protein concentration in each sample was estimated from mesocarp tissue following the Bradford (1976) method and was expressed as mg/g FW while using bovine serum albumin (BSA) as the standard. The standard curve was linear between 0 μg/mL BSA and 100 μg/mL BSA (R2 = 0.995). 2.4. Hydrogen peroxide content The hydrogen peroxide content was assayed according to Jana and Choudhuri (1982). H2O2 content was extracted by homogenized mesocarp tissue 1.0 g with 100 mmol/L potassium phosphate buffer (pH 6.5) and 1 mmol/L hydroxylamine at 0 °C. After centrifugation at 4 °C and 10000g for 15 min, the supernatant was collected. The reaction mixture consisting of 600 μL supernatant and 90 μL of 0.3% (v/v) titanium sulphate in 20% H2SO4 (v/v) were freshly prepared before use and then centrifuged at 10000g for 10 min. The standard curves were prepared with H2O2 at a concentration ranging between 10 μmol and 100 μmol (R2 = 0.991). The supernatant was measured at 410 nm and the amount of H2O2 was calculated according to standard curve and express as μmol/g FW.
3.1.2. Hardness Fruit hardness, which rapidly decreased during storage time, is a major limiting factor for the marketing and storage life of mango fruit. Although during the first 9 days of low temperature storage, no significant differences were observed among the fruits in all treatments (Table 1). On day 9 + 3, 9 + 6, and 9 + 9, fruit hardness was found significantly higher in 2 mmol/L PUT treated fruits when compared to other treatments. The results from this experiment revealed that mango fruit treated with 2 mmol/L PUT significantly increased fruit hardness during storage time. It has been reported that the application of polyamines enhances fruit hardness in different fruits, including plums (Davarynejad et al., 2015), apricot (Martínez-Romero et al., 2002), pomegranates (Barman et al., 2011), and grape (Harindra Champa et al., 2015). The influence of polyamines on fruit hardness augmentation can be attributed to their capacity in cross-linking to the carboxyl group of pectin substances in the cell wall, resulting in rigidification (Abbott et al., 1989). This binding between polyamines and pectin also inhibited cell wall-degrading enzymes, such as pectin esterase, polygalacturonase (PG), and pectin methylesterase (PME), which beneficially minimize fruit hardness after harvest (Valero et al., 2002).
2.5. DPPH free radical scavenging assay The DPPH assay was determined according to the method of Choi et al. (2006) with some modifications. One gram of mango tissue was homogenized using 10 mL of 80% (v/v) ethanol. The homogenate was vortexed for 1 min and centrifuged at 10000g for 15 min at 4 °C. The supernatants were used for fruit extract analysis. 100 μL of fruit extract was mixed in 300 μL of 80% (v/v) ethanol and 400 μL of 0.2 mmol/L DPPH solution and then kept in the dark for 10 min. The absorbance was measured at 515 nm. An ethanol solution without DPPH radical served as the control. Results were expressed as percentage of inhibition of the DPPH radical based on the following formula:
%Inhibition =
1 − Absorbance (sample)) Absorbance (control)
3.1.3. Soluble solids content (SSC) and titratable acidity (TA) The SSC and TA contents are important attributes of the postharvest quality of climacteric fruit. Our experiment revealed that 2 mmol/L PUT treated fruit exhibits the lowest SSC and the highest TA content during storage period (Table 1). A variation of SSC contents was statistically significant (p < 0.05). It was found that 2 mmol/L PUT treated fruits had the lowest percentage of SSC on days 9 + 3, 9 + 6, and 9 + 9 compared with 1 mmol/L, 4 mmol/L, and control fruit. TA content decreased constantly throughout the storage period, but there were no significant differences on days 0–9. From day 9 + 3 to the end of storage time, 2 mmol/L PUT stimulated significantly higher TA than other treatments. On day 9 + 9, 2 mmol/L PUT treated fruits had the highest TA (0.17%) followed by 4 mmol/L PUT (0.11%), 1 mmol/L (0.12%), and control fruit (0.12%), respectively. The concentration of SSC and TA was significantly affected by the application of PUT. During the ripening process, the concentration of SSC continuously increased because of the hydrolysis of polysaccharides (Mahto and Dasb, 2013), and TA was associated with the concentration of organic acid. Respiration appeared to provoke the consumption of organic acid and reduced the TA in fruits (Khosroshahi et al., 2007). The particular effect of PUT on SSC and TA could be related to the reduction of respiration rate and a delayed in ripening process. Similar results have also been reported in plum (Davarynejad et al., 2015) and apricot (MartínezRomero et al., 2002).
× 100
2.6. Ferric reducing antioxidant power assay (FRAP assay) FRAP assay was measured according to the method by Benzie and Strain (1999) with modifications. The FRAP solution was prepared by mixing 25 mL of a 0.3 mol/L acetate buffer, pH 3.6, 2.5 mL of a 10 mmol/L 2,4,6-tris (1-pyridy)-5-triazine (TPTZ) solution in 40 mmol/ L HCl and 20 mmol/L FeCl3·6H2O solution. Fruit extracts of 150 μL was mixed with FRAP solution of 3000 μL for 30 min in a darkened condition, and to which, the absorbance was determined at 593 nm. The standard curve was linear between 200 μmol/L and 1000 μmol/L FeSO4. 7H2O (R2 = 0.996). Results were expressed as micromoles of ferrous equivalent Fe (II) per fresh weight. 2.7. Statistical analysis Experimental data were the mean ± standard errors. Statistical analysis was performed with the IBM SPSS Statistics 22 (IBM SPSS, USA), and the data were tested by one-way analysis of variance (ANOVA). The means were separated by Duncan’s Multiple Range Test, and differences at p ≤ 0.05 were considered to be significant.
3.1.4. Peel color During mango ripening, the peel color of mango fruit changes from green to yellow. The lightness (L value) and hue angle have been used as good indicators of peel color changes. During fruit ripening, the L value increased and hue angles decreased. Fruit treated with 2 mmol/L PUT had a notable delay in peel color change on days 9 + 3, 9 + 6, and 9 + 9 (Fig. 1A). The L value of 2 mmol/L PUT fruits was the lowest (Fig. 1B), and for the hue angles, 2 mmol/L PUT fruits showed the highest value when compared with other treatments (Fig. 1C). The
3. Results and discussion 3.1. Effects of exogenous putrescine treatment on fruit quality parameters 3.1.1. Weight loss Our study showed that the loss of weight increased progressively during storage period (Table 1). However, the lowest weight loss was 25
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Fig. 2. The activity of (A) SOD, (B) CAT, (C) GPOX, (D) APX, (E) GR and concentration of (F) H2O2 in mango fruit treated with exogenous PUT during storage at 14 °C for 9 days and shelf life at 25 ± 1 °C. All data presented as a mean of four biological replicates, and error bar represent ± standard error. According to the Duncan’s Multiple Range Test (p ≤ 0.05), statistically significant differences were indicated for each sampling time, shown in different letters.
polyamines might be involved with the antioxidant system in plants (Verma and Mishra, 2005). Our results found that SOD activity progressively decreased with the increased in days of storage for all the treatments. Two mmol/L PUT treated fruit showed the highest SOD activity from day 9 to day 9 + 9 (Fig. 2A). The superoxide radicals are converted into hydrogen peroxide as a result of SOD, which later is disintegrated into H2O and O2 by CAT and POX. CAT activity showed dramatic increases in all treatments following removal from low temperature storage up to day 9 + 6 of fruit ripening, but later on, their activities decreased at the end of storage (Fig. 2B). Mango fruit treated with 2 mmol/L PUT exhibited the highest CAT activity among treatments during shelf life at 25 °C. Similarly, GPOX activity in mesocarp tissue rose noticeably on day 9 + 3, reached its maximum on day 9 + 6, and gradually declined until the end of the storage period (Fig. 2C). The highest GPOX activity was found in 2 mmol/L PUT treated fruits followed by 1 mmol/L while control fruits had the lowest GPOX activity. APX activity exhibited a constant increase until day 9 + 6 and remained continual until a decline was observed on day 9 + 9 (Fig. 2D). The 2 mmol/L PUT treated fruit exhibited the highest APX activity over the period of day 9 + 6 to day 9 + 9 of the treatment. Similarly, GR activity showed a steady increase in all treatments upon removal from
2 mmol/L PUT treatment are capable of delaying mango peel color changes during 9 days of shelf life at 25 °C similar to observations make in grape (Harindra Champa et al., 2015) and apricot (Martínez-Romero et al., 2002). Hydrolytic activities that act on chloroplast thylakoid membranes have been observed to be reduced after polyamines application (Lester, 2000).
3.2. Effect of exogenous putrescine treatment on antioxidant enzyme activities During fruit ripening, production and accumulation of reactive oxygen species (ROSs), such as hydrogen peroxide and superoxide anion, continually increased. Thus, a continuous increase in oxidation is responsible for the stressful process of ripening (Jimenez et al., 2002). The generated ROSs causes oxidative stress to lipid membrane, nucleic acid, and proteins (Scandalios, 1993). Plants have defensive mechanisms for scavenging damage caused by ROSs. Such types of damage could be scavenged by plant enzymatic defenses including SOD, CAT, APX, POX, MDHAR, DHAR, GPOX, and GR and also by non-enzymatic antioxidants, for example ascorbic acid, beta-carotenoid and glutathione (Gill and Tuteja, 2010b). It has been suggested that 26
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Fig. 3. Content of (A) DPPH and (B) FRAP of mango fruit treated with exogenous PUT during storage at 14 °C for 9 days and shelf life at 25 ± 1 °C. All data presented as a mean of four biological replicates, and error bar represent ± standard error. According to the Duncan’s Multiple Range Test (p ≤ 0.05), statistically significant differences were indicated for each sampling time, shown in different letters.
the lowest among treatments from day 9 to day 9 + 9 (Fig. 2). It has been well established that an excess of hydrogen peroxide is considered an oxidative stress parameter. Mango ripening was found to be associated with enhanced hydrogen peroxide contents. In the present study, we found that the treatment with 2 mmol/L had the highest reduction in H2O2 content in mango fruit. A noticeably similar reaction was observed in zucchini fruit treated with 1 mmol/L PUT which resulted in having the highest influence in the reduction of H2O2 content (Palma et al., 2015). Japanese plum fruit also paralleled with our findings (Singh and Singh, 2012).
low temperature storage to day 9 + 6, but it began to decrease at the end of the storage period (Fig. 2E). Mango fruits treated with 2 mmol/L PUT had significant higher GR activity compared with other treatments. The results of our study revealed that the application of 2 mmol/L PUT effectively induced activities of antioxidant enzymes, including SOD, CAT, GPOX, APX and GR. Oxidative stress is associated with reduced antioxidant enzyme activities, leading to a short postharvest life of fruit. The accumulation of ROSs contributed to higher membrane deterioration and loss of tissue structure (Kumar et al., 2011). Polyamines may bind to the antioxidant enzymes or may directly scavenge ROSs (Duan et al., 2008; Groppa and Benavides, 2008). In agreement with our finding, previous studies have shown that application of exogenous polyamines (PUT and SPM) significantly increased the activities of CAT, SOD, and POX in two apricot cultivars (Saba et al., 2012). Similarly, higher activities of CAT, SOD, and POX during the storage period have been reported in PUT treated ‘Samar Bahisht Chaunsa’ mango fruit (Razzaq et al., 2014). In addition, zucchini treated with PUT resulted in higher APX and GR activities with respect to control (Palma et al., 2016).
3.4. Effect of exogenous putrescine treatment on total antioxidant capacity The data indicated that the antioxidant capacity analyzed by DPPH free radical scavenging assay slightly increased until day 9 + 6 and later decreased by the end of the storage time (Fig. 3A). There was a notable difference (p < 0.05) between PUT and control treatments. During shelf life at 25 °C, 2 mmol/L PUT treated fruit had the highest total antioxidant capacity, while control fruit had the lowest. The total antioxidant content using FRAP assay had a similar trend as DPPH radical assay (Fig. 3B). The total antioxidant increased gradually until day 9 + 6, but on day 9 + 9, it exhibited a slight reduction. It was noticed that the 2 mmol/L PUT treated fruit showed the highest total antioxidant content when compared to other treatments. The determination
3.3. Effect of exogenous putrescine treatment on hydrogen peroxide Levels of H2O2 content in all treatment tended to increase gradually during ripening. The H2O2 content in 2 mmol/L PUT treated fruit was 27
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of antioxidant capacity is one of the approaches that expresses the nonenzymatic antioxidant system and biological properties of fruit which plays a vital role in maintaining fruit quality during storage period. In this work, the application of 2 mmol/L PUT was effective in maintaining the total antioxidant contents during postharvest storage both assayed by the FRAP and DPPH methods. These antioxidants were well maintained by putrescine, which are basically anti-senescence agents (Seiler and Rual, 2005). In addition, 2 mmol/L PUT also maintained total antioxidant property by increasing the SOD, CAT, GPOX, APX, and GR as shown in our data. Previous works describe polyamines as being associated with maintaining the total antioxidant in several fruits such as pomegranate (Mirdehghan et al., 2007), plum (Davarynejad et al., 2015), kiwi (Jhalegar et al., 2012), and zucchini (Palma et al., 2015).
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4. Conclusion Exogenous application of 2 mmol/L PUT was the most effective treatment to improve the postharvest quality and antioxidant activities of ‘Nam Dok Mai No.4′ mango during storage. Positive effects of PUT treatment were evidently confirmed from its capability to maintain high SOD, CAT, GPOX, APX, and GR enzyme activities and non-enzymatic antioxidant contents while reduce hydrogen peroxide content. These finding suggested that the delaying of mango fruit ripening after the PUT treatment is promising for manufacturers and can be economically beneficial as mango can potential reach a wider market. Future studies may focus on PUT induction of antioxidant activity and internal polyamines for reducing chilling injury during the low temperature storage of mango fruit. Acknowledgements Financial support for this research was provided by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and the Overseas Research Experience Scholarship (ORES) from the Graduate School and Faculty of Science. The author was a recipient of a Science Achievement Scholarship of Thailand. References AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed. AOAC, Washington, DC. Abbott, J.A., Conway, S.W., Sams, E.C., 1989. Postharvest calcium chloride infiltration affects textural attributes of apples. J. Am. Soc. Hortic. Sci. 114, 932–936. Barman, K., Asrey, R., 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. Beers, R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Benzie, I.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 91, 179–194. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248–254. Chancharoenrit, J., 2002. Effects of Hot Water Dips on Physiological Changes and Chilling Injury After Storage of ‘Hom Thong’ Variety of Banana. Master’s Thesis. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. Choi, Y., Lee, S.M., Chun, J., Lee, H.B., Lee, J., 2006. Influence of heat treatment on the antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom. Food Chem. 99, 381–387. Davarynejad, G., Zarei, M., Ardakani, E., Nasrabadi, M.E., 2013. Influence of putrescine application on storability: postharvest quality and antioxidant activity of two Iranian apricot (Prunus armeniaca L.) cultivars. Notulae Sci. Biol. 5, 212–219. Davarynejad, G.H., Zarei, M., Nasrabadi, M.E., Ardakani, E., 2015. Effects of salicylic acid and putrescine on storability: quality attributes and antioxidant activity of plum cv.‘Santa Rosa’. J. Food Sci. Technol. 52, 2053–2062. Ding, Z.S., Tian, S.P., Zheng, X.L., Zhou, Z.W., Xu, Y., 2007. Responses of reactive oxygen metabolism and quality in mango fruit to exogenous oxalic acid or salicylic acid under chilling temperature stress. Physiol. Plant. 130, 112–121. Duan, J., Li, J., Guo, S., Kang, Y., 2008. Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances short-term salinity
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