Postharvest chitosan, trans-resveratrol and glycine betaine dipping affect quality, antioxidant compounds, free radical scavenging capacity and enzymes activities of ‘Sukkari’ bananas during shelf life

Scientia Horticulturae 219 (2017) 173–181

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Postharvest chitosan, trans-resveratrol and glycine betaine dipping affect quality, antioxidant compounds, free radical scavenging capacity and enzymes activities of ‘Sukkari’ bananas during shelf life Adel D. Al-Qurashi a , Mohamed A. Awad a,b,∗ , Saleh A. Mohamed c,d , Mohamed I. Elsayed a a Department of Arid Land Agriculture, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, P.O.Box. 80208, Jeddah, Saudi Arabia b Pomology Department, Faculty of Agriculture, Mansoura University, El-Mansoura, Egypt c Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, P.O.Box 80208, Jeddah, Saudi Arabia d Molecular Biology Department, National Research Centre, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 10 October 2016 Received in revised form 26 February 2017 Accepted 28 February 2017 Keywords: Banana Chitosan Antioxidants Resveratrol Glycine betaine Enzymes

a b s t r a c t Effects of chitosan (0.5 and 1%), trans-resveratrol (1.6 × 10−5 M, 1.6 × 10−4 M and 1.6 × 10−3 M) and glycine betaine (GB) (10, 15 and 20 mM) dipping on quality and biochemical changes of ‘Sukkari’ bananas were studied during ripening at shelf life (SL) conditions for 13 days. Weight loss increased during shelf life but was not affected by treatments. Peel color index increased during SL and was lower at both resveratrol and GB treatments than control. Membrane stability index (MSI) of peel decreased during SL and was higher, especially after 6 days, at resveratrol and GB treatments than control. Firmness decreased during SL and was higher, especially after 6 days, at resveratrol and low and moderate rates of GB than control. TSS increased during SL and was lower at all treatments than control. Acidity concentration decreased during SL and was higher, especially after 6 days, at low rate of chitosan, resveratrol and high rate of GB than control. pH increased during SL and was higher at low rate of GB and lower at low and moderate rates of resveratrol than control. Vitamin C concentration decreased during SL but was not affected by treatments. Total phenols concentration decreased during SL and was higher at GB and moderate rate of resveratrol than control. Total flavonoids concentration decreased during SL and was lower at resveratrol and low and high rates of GB than control. FRSC (DPPH IC50 ) increased during SL and was lower at GB and low and moderate rates of resveratrol than other treatments, especially after 6 days. The relations of such biochemical changes with ␣-amylase, xylanase, polygalacturonase, peroxidase and polyphenoloxidase activities were discussed. In conclusion, both postharvest trans-resveratrol and GB treatments retained quality of ‘Sukkari’ bananas during SL and being suggested as natural alternatives to synthetic chemicals. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Banana (Musa spp.) is one of the most important fruit that consumed worldwide due to its high functional nutritional value (Pereira and Maraschin, 2015). As a climacteric fruit, banana possess a relatively short shelf life (a few days) at ambient conditions due to rapid softening, oxidation damages, peel spotting and fungal decay (Duan et al., 2007; Bhande et al., 2008; Hailu et al., 2013). Also, as tropical fruit, bananas are highly sensitive to low

∗ Corresponding author at: Department of Arid land Agriculture, Faculty of Meteorology, Environment and Arid land Agriculture, King Abdulaziz University, P.O.Box. 80208, Jeddah, Saudi Arabia. Tel.: +966 566123090; fax: +966 26952364. E-mail addresses: [email protected], [email protected] (M.A. Awad). http://dx.doi.org/10.1016/j.scienta.2017.02.046 0304-4238/© 2017 Elsevier B.V. All rights reserved.

storage temperature (below 10 ◦ C) and pathogens attack (Cano et al., 1997). Synthetic chemical preservatives application at pre or postharvest stages is restricted due to rising consumers concerns on both human health and the environment. Thus, alternative tools to maintain bananas quality during shelf life are critically required. Chitosan, a bioactive natural edible coat, is widely considered as a promising alternative to chemical preservatives (Romanazzi et al., 2013). Postharvest dipping of ‘Embul’ bananas in 1% chitosan (Jinasena et al., 2011) or ‘Cavendish’ bananas in 2% chitosan (Suseno et al., 2014) reduced weight loss, delayed ripening and maintained quality during shelf life compared to control. Resveratrol, its 3-glucopyranoside piceid, and their cis isomers are natural antioxidant plant phenolics, representing the major active compound of stilbene phytoalexins that mainly occur in grapes, berries, and other dietary constituents and are presumed to be involved in

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defense system against plant pathogens and metabolic diseases in human (Adrian et al., 1997; Gonzalez Urena et al., 2003; Jimenez et al., 2005; Gülc¸in, 2010; Chen et al., 2016). Postharvest dipping in trans-resveratrol at 1.6 × 10−4 M maintained firmness, sensory and nutritional value and reduced water loss during storage of apples, grapes and tomatoes compared to control (Jimenez et al., 2005). Cherukuri (2007) reported that exogenous trans-resveratrol treatment at 1.6 × 10−3 M, 1.6 × 10−4 M and 1.6 × 10−5 M increased total phenolics, vitamin C and total carotenoids concentration and antioxidant capacity of Satsuma mandarins. Postharvest transresveratrol dipping at 1.6 × 10−5 M, 1.6 × 10−4 M and 1.6 × 10−3 M reduced decay, increased antioxidant compounds, peroxidase (POD) and polyphenoloxidase (PPO) and decreased polygalacturonase (PG) activities of ‘El-Bayadi’ table grapes after cold storage and shelf life (Awad et al., 2015). Glycine betaine (GB) is a naturally occurring compatible solute that function as an effective osmotic stress protectant and stabilizing photosynthetic pigments and cell membranes in plants (Robinson and Jones, 1986; Genard et al., 1991; Ashraf and Foolad, 2007; Mansour, 2000; Yang et al., 2003; Chen and Murata, 2011). Foliar application of GB at 50 and 100 mM/l increased antioxidant enzymes activities and enhanced photosynthesis of maize under salinity stress (Nawaz and Ashraf, 2009). Postharvest GB dipping of Shredded Iceberg lettuce, at especially 0.2 mM/l, maintained sensory quality during cold storage (Hurme et al., 1999). Also, dipping of ‘Zhongnong 8’ cucumbers in GB at 5, 10, and 15 mM decreased lipoxygenase (LOX) activity but increased POD and catalase (CAT), restrained malondialdehyde (MDA) and hydrogen peroxide (H2 O2 ) accumulations, especially at 10 mM/l during cold storage (Fu-gui et al., 2013). Postharvest GB dipping at 10, 15 and 20 mM/l increased antioxidant compounds, POD and PPO activities of ‘El-Bayadi’ table grapes after cold storage and shelf life (Awad et al., 2015). This study aim to evaluate the response of ‘Sukkari’ bananas to postharvest dipping in chitosan, resveratrol and GB treatment as an attempt to maintain quality during shelf life. In this study, the effects of chitosan, trans-resveratrol (resveratrol) and GB on antioxidant compounds, antioxidant and hydrolytic enzymes activities, free radical scavenging capacity (FRSC measured by DPPH assay) of ‘Sukkari’ bananas were evaluated. 2. Materials and methods 2.1. Plant materials and experimental procedure This experiment was performed on bananas (cv. ‘Sukkari’) imported from Yemen, and purchased in a local commercial company in Jeddah, Kingdom of Saudi Arabia (KSA). Fruit were harvested, packed as hands in polyethylene film in perforated cardbox (about 30 kg) and transported from Yemen to Jeddah within 48 h at 15 ◦ C. Bananas (at the ripening stage 1, according to a banana ripening chart, were directly pre-treated with ethylene gas (about 0.01% by volume in air) at 18 ◦ C and 85% RH for 24 h for ripening induction at a commercial airtight ground warehouses with a great deal of bananas. Then, uniform hands (at the ripening stage 2) were randomly selected at the warehouse and rapidly transported to the horticulture laboratory of King Abdulaziz University in Jeddah.

dissolved in 1% acetic acid, trans-resveratrol (Baoji Guokang BioTechnology Co., Ltd., China) solution (1.6 × 10−5 M, 1.6 × 10−4 M and 1.6 × 10−3 M; referring to 0.00365, 0.0365 and 0.365 g/l, respectively) or glycine betaine (Danisco, Finland) solution (10, 15, 20 mM; refereeing to 1.172, 1.757 and 2.343 g/l, respectively) for 1 min. A surfactant (Tween 20 at 0.5 ml/l) was added to all treatments. Following air draying of about 1 h, all treatments/replicates were weighted and stored at 18 ± 2 ◦ C and 60–70% (RH) in perforated cardboard cartons for 13 days. Before applying the treatments, additional three samples (10 fingers of each) were randomly collected for initial quality and biochemical analyses. After 3, 6, and 13 days of shelf life, weight loss and peel color stage were recorded for each treatment/replicate. After 6 and 13 days of shelf life, samples (10 fingers of each) from each treatment/replicate were randomly collected for quality and biochemical analyses. Then, these fruit samples were peeled and the peel tissue was sliced and mixed. Random part of this peel was used for electrolyte leakage measurement and the remaining peel was kept at −80 ◦ C for later enzyme, total flavonoids and phenols and antioxidant activity analysis. Pulp firmness was measured in each sample directly following peeling. The pulp tissue was later sliced, mixed and a random portion was used for total soluble solids (TSS), titratable acidity, pH, and vitamin C determinations. 2.3. Weight loss determination The total fruit weight loss was calculated on initial weight basis and expressed in percentage. 2.4. Peel color change estimation Peel color change score was recorded for each sample (10 individual fingers of each) with the help of a banana ripening chart (1–7 scale; 1 – green, 2 – green with trace of yellow, 3 – more green than yellow, 4 – more yellow than green, 5 – yellow with trace of green, 6 – full yellow and 7 – yellow with brown spots. 2.5. Firmness, TSS, acidity, pH and vitamin C measurements in fruit pulp Fruit pulp firmness was measured independently in 10 fingers (in the middle of each finger) per replicate by a digital basic force gauge, model BFG 50N (Mecmesin, Sterling, Virginia, USA) supplemented with a probe of 11 mm diameter and the results were expressed as Newton. A homogeneous sample was prepared from these 10 fingers per replicate for measuring TSS, acidity, pH and vitamin C. TSS concentration was measured as a percentage in fruit pulp juice with a digital refractometer (Pocket Refractometer PAL 3, ATAGO, Japan). Titratable acidity was determined in fruit juice diluted in water at a ratio 1: 2 by titrating with 0.1 N sodium hydroxide up to pH 8.2, using automatic titrator (HI 902, HANNA Instrument, USA) and the results expressed as a percentage of malic acid. Fruit juice pH was measured by a pH meter (WTW 82382, Weilheim, Germany). Vitamin C was measured by the oxidation of ascorbic acid with 2,6-dichlorophenolindophenol dye and the results expressed as g kg−1 on a fresh weight (FW) basis (Ranganna, 1979).

2.2. Fruit treatment 2.6. Leakage of ions from fruit peel Bananas (at the color stage 2) carefully prepared in small uniform hands (about 5 fingers each, free of visual defects and with similar weight and size) were selected. A completely randomized experimental design with three replicates (six hands each) was established. Fruit of each treatment/replicate were soaked either into water (control), 1% acetic acid, 0.5 or 1% chitosan (100,000–300,000 MW) (Acros Organic, New Jersey, USA)

Leakage of ions was measured in peel disks according to Sairam et al. (1997) with some modifications and was expressed as membrane stability index percentage (MSI %). Three grams of peel disks per replicate/treatment was randomly taken and placed in 30 ml of deionized water at ambient temperature for 4 h in a shaker. Conductivity before boiling (C1 ) was measured with an electrical

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Table 1 Fruit weight loss, color and membrane stability index (MSI) of peel of ‘Sukkari’ bananas during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (%) 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20 F-test LSD (0.05) Shelf life (SL) (d) 3 6 13 F-test LSD (0.05) T × SL F-test

Weight loss (%)

Peel color (Index)

MSI (Index)

0.0

2.0

69.8

4.6 4.9

6.0a 5.7ab

14.1f 15.2ef

4.2 4.9

5.8ab 5.7ab

15.0ef 15.2ef

4.3 4.5 4.5

5.3bc 5.3bc 5.3bc

20.0cd 23.3bc 37.3a

4.2 4.6 4.3 NS –

5.0c 5.1c 5.1c *** 0.45

23.0bc 24.2b 18.1ed *** 3.47

1.6c 3.0b 9.2a *** 0.25

3.6c 6.0b 6.7a *** 0.34

– 30.3a 10.7b *** –

NS

NS

***

Means within each column followed by the same letter are not significantly different at level P ≤ 0.05. (***), significant at P ≤ 0.001; (NS), not significant; (–), not calculated.

conductivity digital meter (Orion 150A+, Thermo Electron Corporation, USA). The same disks were kept in a boiling water bath (100 ◦ C) for 30 min to release all electrolytes, cooled to 22 ± 2 ◦ C with running water, and conductivity after boiling was recorded (C2 ). MSI was expressed in percentage using the formula: [1 − (C1 /C2 )] × 100. 2.7. Preparation of methanol extract of fruit peel Two grams of fruit peel (randomly collected from 10 fingers/replicate) were extracted by shaking at 150 rpm for 12 h with 20 mL methanol (80%) and filtered through filter paper No. 1. The filtrate designated as methanol extract that will be used for total phenols, total flavonoids and antioxidant activity estimations. 2.7.1. Estimation of total phenols Total phenols concentration was measured according to Hoff and Singleton (1977). Fifty microliters of the methanol extract was mixed with 100 ␮l Folin-Ciocalteu reagent, 850 ␮l of methanol and allowed to stand for 5 min at ambient temperature. A 500 ␮l of 20% sodium carbonate was added and allowed to react for 30 min. Absorbance was measured at 750 nm. Total phenols was quantified from a calibration curve obtained by measuring the absorbance of known concentrations of gallic acid and the results expressed as g kg−1 FW gallic acid equivalent. 2.7.2. Estimation of total flavonoids Total flavonoids concentration was determined using a modified colorimetric method described previously by Zhishen et al. (1999). Methanol extract or standard solution (250 ␮l) was mixed with distilled water (1.25 ml) and 5% NaNO2 solution (75 ␮l). After standing for 6 min, the mixture was combined with 10% AlCl3 solution (150 ␮l), 1 M NaOH (0.5 ml) and distilled water (275 ␮l) were added to the mixture 5 min later. The absorbance of the solutions at 510 nm was then measured. Total flavonoids was quantified from a calibration curve obtained by measuring the absorbance of known concentrations of catechin and the results expressed as g kg−1 FW catechin equivalent.

2.7.3. Evaluation of DPPH radical scavenging assay of fruit peel Free radical scavenging activity of methanol extract of fruit peel was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method (Ao et al., 2008). A methanol extract (0.1 ml) was added to 0.9 ml of freshly prepared DPPH methanol solution (0.1 mM). An equal amount of methanol was used as a control. After incubation for 30 min at room temperature in the dark, the absorbance (Abs) was measured at 517 nm using a spectrophotometer. Activity of scavenging (%) was calculated using the following formula:

 DPPH radical scavenging %=



(Abs control − Abs sample) × 100 Abs control

The inhibition concentration (IC50 ) was defined as ␮g phenolics of the test sample that decreases 50% of initial radical. The IC50 values were calculated from the dose responses curves.

2.8. Enzymes measurements of fruit peel 2.8.1. Crude extract preparation One gram of fruit peel (randomly collected from 10 fingers/replicate) was homogenized with 20 mM Tris–HCl buffer, pH 7.2 using homogenizer. The homogenate was centrifuged at 10,000 rpm for 10 min at 4 ◦ C. The supernatant was designed as crude extract and stored at −20 ◦ C for peroxidase, polyphenoloxidase, polygalacturonase, xylanase and ˛-amylase assay.

2.8.2. Peroxidase assay Peroxidase (EC 1.11.1.7) activity (POD) was assayed according to Miranda et al. (1995). The reaction mixture containing in one ml: 0.008 ml of 0.97 M H2 O2 , 0.08 ml of 0.5 M guaiacol, 0.25 ml of 0.2 M sodium acetate buffer, pH 5.5 and least amount of enzyme preparation. The change in absorbance at 470 nm due to guaiacol oxidation was followed for 1 min using a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that increases the O.D. 1.0 per min under standard assay conditions.

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2.8.3. Polyphenoloxidase assay Polyphenoloxidase (EC 1.14.18.1) (PPO) activity was assayed with catechol as a substrate according to the spectrophotometric procedure of Jiang et al. (2002). The extract (0.2 ml) was rapidly added to 2.8 ml of 20 mM catechol solution prepared in 0.01 M sodium phosphate buffer (pH 6.8). The increase in absorbance at 400 nm was recorded for 3 min using a spectrophotometer. One unit of enzyme activity was defined as the amount of the enzyme that causes a change of 0.1 in absorbance per min. 2.8.4. Polygalacturonase, ˛-amylase and xylanase assays Polygalacturonase (EC 3.2.1.15) (PG), ␣-amylase (EC 3.2.1.1) and xylanase (EC 3.2.1.8) activities were assayed by determining the liberated reducing end products using galacturonic acid, maltose and xylose, respectively as standards (Miller, 1959). The reaction mixture (0.5 ml) containing 5 mg substrate, 0.25 ml of 0.2 M sodium acetate buffer pH 5.5 and a suitable amount of crude extract. Assays were carried out at 37 ◦ C for 1 h. Then 0.5 ml dinitrosalicylic acid reagent was added to each tube and heated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance was measured at 560 nm. Substrates used were polygalacturonic acid, starch and xylane for polygalacturonase, ␣-amylase and xylanase, respectively. One unit of enzyme activity was defined as the amount of enzyme which liberated 1 ␮M of reducing sugar per min under standard assay conditions. 2.9. Statistical analysis The data were statistically analyzed as a completely randomized design with three replicates by analysis of variance (ANOVA) using the statistical package software SAS (SAS Institute Inc., 2000, Cary, NC, USA). Comparisons between means were made by the least significant differences (LSD) at P ≤ 5%. 3. Results Fruit weight loss gradually increased during SL reaching 9.2% after 13 days but was not significantly affected by the applied treatments (Table 1). There were no significant interaction effects between treatment and SL on fruit weight loss. Peel color index

Table 2 The interaction effect between treatment and shelf life on membrane stability index (MSI) of ‘Sukkari’ banana fruit peel during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping. Treatment

Shelf life (d) MSI (Index) 6

Control Acetic acid 1% Chitosan (%) 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20

13

22.4d 22.1d

5.9g 8.3fg

19.1d 22.1d

10.9fe 8.3fg

30.6c 32.7bc 51.8a

9.6feg 13.8e 22.7d

36.9b 36.7b 28.9c

9.0feg 11.6fe 7.2fg

Means within and between columns followed by the same letter are not significantly different at level P ≤ 0.05.

increased (more yellow and less green) during shelf life reaching a value of 6.7 after 13 days and was significantly lower at both resveratrol and GB treatments than control (Table 1). However, chitosan treatment at both rates showed no significant effect on peel color index. There were no significant interaction effects between treatment and SL on peel color index (Table 1). MSI of peel tissue sharply decreased during SL and was higher at both resveratrol and GB treatments than control. However, chitosan treatment at both rates showed no significant effect on MSI (Table 1). The significant interaction effects between treatment and SL revealed that, after 13 days of SL, chitosan at low rate gave higher MSI than control (Table 2). At the same time, the low rate of resveratrol and the low and high rates of GB showed similar MSI to control (Table 2). Firmness sharply decreased during SL and was significantly higher at all resveratrol rates and the low and moderate rates of GB compared to control (Table 3). The significant interaction effects between treatment and SL on firmness revealed that, after 13 days of SL, there were no significant differences among all treatments (Table 4). TSS significantly increased during SL and was

Table 3 Firmness, TSS, titratable acidity, pH and vitamin C of ‘Sukkari’ bananas during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (%) 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20 F-test LSD (0.05) Shelf life (SL) (d) 6 13 F-test T × SL F-test

Firmness (N)

TSS (%)

Acidity (%)

pH

Vitamin C (g kg−1 )

41.5

3.3

0.52

5.0

0.095

7.1d 7.7cd

16.1a 15.1ab

0.61cd 0.68bc

5.1bc 5.2ab

0.058 0.062

6.9d 7.7cd

14.6bc 14.1bcd

0.75b 0.68bc

4.9bc 5.2ab

0.060 0.065

9.2bc 9.4bc 10.2ab

13.1d 14.3bcd 11.6e

0.94a 0.75b 0.74b

4.2e 4.4de 4.9bc

0.065 0.065 0.063

11.3a 10.2ab 8.3cd *** 1.47

11.3e 11.5e 13.4cd *** 1.43

0.62cd 0.54d 0.95a *** 0.075

5.5a 4.9bc 4.7cd *** 0.32

0.067 0.065 0.062 NS 0.0089

11.0a 6.2b ***

11.9b 15.0a ***

1.12a 0.34b ***

4.7b 5.2a ***

0.073a 0.054b ***

***

NS

***

NS

NS

Means within each column followed by the same letter are not significantly different at level P ≤ 0.05. (***), significant at P ≤ 0.001; (NS), not significant; (–), not calculated.

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Table 4 The interaction effect between treatment and shelf life on firmness and acidity of ‘Sukkari’ bananas during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping. Treatment

Shelf life (d) Firmness (N) 6

Control Acetic acid 1% Chitosan 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20

Acidity (%) 13

6

13

8.0cde 9.3c

6.2def 6.2def

0.86fe 1.03d

0.36gh 0.33gh

8.1cd 9.3c

5.8f 6.2def

1.17c 1.03d

0.33gh 0.33gh

12.4b 12.5b 14.1b

5.9fe 6.3def 6.2def

1.45b 1.11cd 1.55c

0.42g 0.38gh 0.32hg

16.5a 13.8b 10.0c

6.2def 6.7def 6.5def

0.91e 0.77f 1.58a

0.33gh 0.31h 0.33gh

For each parameter, means within and between columns followed by the same letter are not significantly different at level P ≤ 0.05. Table 5 Total phenols and total flavonoids concentrations and free radical scavenging capacity (FRSC) of ‘Sukkari’ banana fruit peel during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (%) 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20 F-test LSD (0.05) Shelf life (SL) (d) 6 13 F-test T × SL F-test

Phenols (g kg−1 )

Flavonoids (g kg−1 )

0.33

0.23

2.52

0.38cd 0.36cd

0.16a 0.15ab

2.67e 3.30de

0.38cd 0.36cd

0.13bc 0.15ab

3.52d 3.30de

0.40bc 0.43ab 0.34d

0.13c 0.13c 0.12c

3.88cd 4.31bc 2.68e

0.47a 0.45a 0.46a *** 0.043

0.13c 0.15ab 0.13c *** 0.022

5.15a 4.37bc 4.79ab *** 0.67

0.48a 0.30b ***

0.17a 0.11b ***

6.45a 1.15b ***

NS

NS

***

FRSC (DPPH IC50 value)

Means within each column followed by the same letter are not significantly different at level P ≤ 0.05. (***), significant at P ≤ 0.001; (NS), not significant.

lower at all treatments than control, except for acetic acid treatment. Acidity concentration significantly decreased during SL and was higher at the low rate of chitosan, all rates of resveratrol and the high rate of GB than control (Table 3). The significant interaction effects between treatment and SL on acidity concentration revealed that, after 13 days of SL, there were no significant differences among all treatments (Table 4). pH significantly increased during SL and was higher at low rate of GB and lower at low and moderate rates of resveratrol than control. Vitamin C concentration decreased during SL and was not significantly affected by the applied treatments (Table 3). There were no significant interaction effects between treatment and SL on TSS, pH and vitamin C concentrations (Table 3). Total phenols concentration significantly decreased during SL and was higher at all rates of GB and the moderate rate of resveratrol than control (Table 5). Chitosan and acetic acid treatments gave similar phenols concentration to control. Total flavonoids concentration significantly decreased during SL and was lower at all rates of resveratrol and the low and high rate of GB than control (Table 5). Chitosan and acetic acid treatments gave simi-

lar flavonoids concentration to control. There were no significant interaction effects between treatment and SL on both total phenols and flavonoids concentrations (Table 5). FRSC of peel extract measured by the DPPH assay increased during SL (lower IC50 value) (Table 5). FRSC (DPPH IC50 ) values ranged from 1.15 to 6.45 ␮g phenolics concentration among the treatments. However, resveratrol at low and moderate rates and GB at all rates showed significantly lower FRSC (higher IC50 values) than other treatments including control (Table 5). The significant interaction effects between treatment and SL on FRSC revealed that, after 13 days of SL, there were no significant differences in FRSC among all treatments (Table 6). Both ␣-amylase and xylanase activities showed higher values than initial and were not significantly affected by both treatments and SL period (Table 7). PG activity significantly decreased during SL and was lower at all treatments than control. The significant interaction effects between treatment and SL on PG activity revealed that, after 6 days of SL, acetic acid, chitosan at both rates and GB at low rate showed similar values as control (Table 8). POD activity increased during SL and showed higher values than initial, but was not

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Table 6 The interaction effect between treatments and shelf life on free radical scavenging capacity (FRSC) of ‘Sukkari’ banana fruit peel during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping. Treatment

Control Acetic acid 1% Chitosan 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20

6

13

4.23f 5.16fe

1.13g 1.44g

significantly affected by treatments. There were no significant interaction effects between treatment and SL on ␣-amylase, xylanase and POD activities (Table 7). PPO activity decreased during SL and was significantly higher at low and moderate rates of both resveratrol and GB than other treatments including control. The significant interaction effects between treatment and SL on PPO activity revealed that, after 6 days of SL, both resveratrol and GB treatments gave similar values to control, while acetic acid and chitosan at high rate showed lower PPO activity than other treatments (Table 8).

5.79de 5.16fe

1.26g 1.44g

4. Discussion

6.69cd 7.48bc 4.45f

1.01g 1.13g 0.92g

9.40a 7.75b 8.43b

0.91g 1.00g 0.92g

Shelf life (d) FRSC (DPPH IC50 value)

Means within and between columns followed by the same letter are not significantly different at level P ≤ 0.05.

Banana is a typical climacteric fruit with a relatively high rate of physiological activity including high respiration rate and ethylene production following harvest. Due to raising consumers concerns against the use of synthetic chemicals, several natural compounds are currently evaluated for their effectiveness on maintaining and improving fruit quality during shelf life. In this regard, the current study investigated the effects of chitosan, as a natural edible

Table 7 Hydrolytic and antioxidant enzymes activities (U min g FW) of ‘Sukkari’ banana peel during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (%) 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20 F-test LSD (0.05) Shelf life (SL) (d) 6 13 F-test T × SL F-test

␣-amylase

Xylanase

PG

POD

14.0

8.0

81

11.2

394

16.5 17.5

12.0 13.2

102a 72b

29.0 27.0

597cd 496e

17.7 17.3

12.0 13.2

72b 68bc

24.2 27.2

559d 496e

14.5 15.2 13.2

12.0 11.0 12.5

54de 56de 46e

24.7 24.2 25.0

14.6 15.8 15.0 NS -

15.0 14.7 13.5 NS -

63bcd 57cde 57cde *** 12.4

24.2 28.0 28.7 NS -

711a 712a 641bc *** 60

14.4 11.4 NS

15.8 15.6 NS

88a 41b ***

14.9b 36.6a ***

728a 513b ***

NS

NS

***

NS

PPO

678ab 669ab 654abc

***

PG, POD and PPO refereeing to polygalacturornase, peroxidase and polyphenoloxidase, respectively. Means within each column followed by the same letter are not significantly different at level P ≤ 0.05. (***), significant at P ≤ 0.001; (NS), not significant; (–), not calculated. Table 8 The interaction effect between treatments and shelf life on polygalacturornase (PG) and polyphenoloxidase (PPO) activities (U min g FW) of ‘Sukkari’ banana peel during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping. Treatment

Shelf life (d) PG

PPO

6 Control Acetic acid 1% Chitosan (%) 0.5 1 Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM) 10 15 20

13

6

13

104a 88abcd

100abc 55fg

755abc 550efg

439hi 441hi

103ab 88abcd

42ghi 48gh

729bc 550efg

388i 441hi

81de 77de 69fe

26ij 34hij 23j

782ab 816a 791ab

574efg 521efg 516gh

101abc 86bcde 84cde

25ij 28ij 29ij

819a 813ab 681cd

603def 610ed 602def

For each parameter, means within and between columns followed by the same letter are not significantly different at level P ≤ 0.05.

A.D. Al-Qurashi et al. / Scientia Horticulturae 219 (2017) 173–181

coating, resveratrol, as a natural antioxidant compound, and GB, as a natural compatible solute that function as an osmotic stress protectant and stabilizing photosynthetic pigments and cell membrane in plants on quality, antioxidant activity, antioxidant compounds and enzymes activities of ‘Sukkari’ bananas during SL. It is well known that weight loss occurs during SL due to mainly loss of water by transpiration through fruit peel and to the respiration process (Ayranci and Tunc, 2003; Bhande et al., 2008; Maqbool et al., 2011). Chitosan as an edible coating is expected to reduce transpiration and thus decrease weight loss during SL. However, in the current experiment none of the applied treatments including chitosan affected weight loss (Table 1). These results regarding chitosan contradict with those of Suseno et al. (2014) on ‘Cavendish’ bananas and Jinasena et al. (2011) on ‘Embul’ bananas. This is possibly attributed to the degree of deacetylation (DD), molecular weight and concentration of the used chitosan that affect the quality and homogeneity of the resulting coating. The weight loss of bananas coated with 70% DD chitosan was higher than those coated with 80% DD chitosan (Suseno et al., 2014). They also found that increasing chitosan concentration from 1 to 2% (w/w) increased the barrier to the moisture loss and reduced weight loss during SL, but that was not true for vitamin C. Our results showed that chitosan retained higher acidity and lower TSS concentration but had no significant effect on peel color index, firmness, pH and MSI of ‘Sukkari’ bananas during SL (Tables 1–4). In another study, 1% chitosan treatment of ‘Embul’ bananas had no effect on TSS, acidity and pH but slightly retained higher firmness than control during storage at 13.5 ◦ C and 95% RH for 14 days (Jinasena et al., 2011). In the current study, resveratrol and GB dipping delayed ripening of ‘Sukkari’ bananas as reflected by higher peel color index (more green), higher MSI and firmness (only after 6 days of SL), and acidity and lower TSS concentration during 13 days of SL than control (Table 1). However, the effects of these two compounds on fruit weight loss were insignificant (Table 1). These results partially confirm those of Jimenez et al. (2005) in which the exogenous trans-resveratrol treatment (1.6 × 10−4 M) reduced water loss, maintained turgidity, firmness, sensory and nutritional value during storage with no negative impact on other parameters of apples, grapes and tomatoes compared to control. Also, Awad et al. (2015) reported that both trans-resveratrol and GB treatments retained higher firmness but had no effect on weight loss, TSS, acidity and pH after cold storage and SL of ‘El-Bayadi’ table grapes. trans-resveratrol dipping of ‘Crimson’ table grapes controlled decay, but showed no synergistic effect when applied in combination with chitosan (Freitas et al., 2015). The positive effects of trans-resveratrol on fruit quality might be attributed to its general antioxidant properties (Jimenez et al., 2005). Accordingly, trans-resveratrol treatment may form a coat on fruit surface that decrease water loss and retain higher firmness than control. The observed positive effects of GB on quality parameters of ‘Sukkari’ bananas during SL (Tables 1–4) might be attributed to its well known general properties as a natural compatible solute that function as a photosynthetic pigment and membrane stabilizing agent and osmoregulator in many plant species (Robinson and Jones, 1986; Genard et al., 1991; Mansour, 2000; Yang et al., 2003; Ashraf and Foolad, 2007; Chen and Murata, 2011). However, the exact mechanism of especially GB on fruit physiology needs further investigations. Zhang et al. (2016) reported that betaine was not able to scavenge free radicals (as measured by classical chemical assays such as DPPH, ABTS, FRAP) or enhance the enzymatic antioxidant system in human hepatocellular carcinoma (HepG2) cells. However, such classical assays did not always reflect the antioxidant activity, since a compound without free radical scavenging ability may still possess antioxidant activity in an organism. Accordingly, Zhang et al (2016) suggested that betaine exert its antioxidant activity via two mechanisms. One mechanism involves scavenging reactive oxygen species (ROS) in cells via up-regulation

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of endogenous non-enzymatic antioxidant defense such as increasing the levels of S-adenosylmethionine (SAM) and methionine via the methionine-homocysteine cycle. The other inhibits ROS generation by forming a protective membrane with an electronegative outer surface around cells, thereby preventing the contact of free radicals with the cytomembrane. The observed decrease in total phenols and flavonoids concentration during SL (Tables 5 and 6) are in accordance with those of Kondo et al. (2005) who found that total phenols decreased ‘Namwa’ bananas peel stored at 6 o C but slightly changed in fruit stored at 12 ◦ C for 6 d. Also, Wang et al. (2014) found that total phenols in ‘Brazil’ banana peel slightly increased during the first 10 days of storage at 7 ◦ C then sharply decreased. The decrease of phenols concentration in fruit during ripening might be due to breakdown of cell structure because of the senescence phenomena during storage (Macheix et al., 1990). Our results showed that FRSC of fruit peel increased (lower IC50 values) during SL. However, Kondo et al. (2005) found that IC50 values of superoxide (O2 –) and DPPH-radical scavenging capacity of ‘Namwa’ bananas peel stored at 6 and 12 ◦ C decreased (higher antioxidant capacity) during the first 2 d and then gradually increased (lower antioxidant capacity) during the following 8 d of storage. Also, Fernando et al. (2014) reported that total antioxidant activities (mmol TE/100 g fw) measured by DPPH and FRAP of ‘Hom Thong’ and ‘Khai’ bananas flesh during ripening at 25 ◦ C for 10 d increased with ripening but rapidly decreased with senescence. The decrease in phenols concentration with the increase in FRSC during ripening might suggest qualitative changes in phenolic classes toward higher antioxidant potential. It was reported that the antioxidant capacity of phenolics possibly has a concentration saturation limit above which the activity could not increase further with the concentration (Dani et al., 2012). Also, phenolics are possibly not the only factor that contributes to FRSC of fruit but it might work synergistically with several vitamins and minerals (Dani et al., 2012). Accordingly, parallel several assays should be used to investigate the principles of antioxidant/oxidation activity of a certain horticultural commodity. Both GB at all rates and resveratrol at the moderate rate retained higher total phenols concentration of fruit peel than control during SL (Table 5). While, total flavonoids concentration was lower at all resveratrol and GB rates than control, except for the moderate rate of GB. However, vitamin C concentration was not affected by any of the treatments. Also, FRSC (DPPH IC50 ) of peel of both resveratrol and GB treatments showed lower FRSC (higher IC50 values), only after 6 days of SL, than control (Table 5). These results partially contradict with those of Cherukuri (2007) in which trans-resveratrol application at different concentration (1.6 × 10−3 M, 1.6 × 10−4 M and 1.6 × 10−5 M) on ‘Satsuma’ mandarin retained higher total phenols, vitamin C and total carotenoids concentration and antioxidant capacity during 12 weeks of cold storage but, with no effect on total flavonoids concentration. However, in other study, antioxidant activity of ‘El-Bayadi’ table grapes peel measured by ABTS method showed that trans-resveratrol only at the high rate (1.6 × 10−3 M) gave higher antioxidant activity (lower IC50 values) than other GB rates and control treatments (Awad et al., 2015). The differences between the antioxidant assays might be attributed to differences in sensitivity/potential among antioxidant compounds such as phenols and flavonoids classes and vitamin C toward a specific assay (Ou et al., 2002; Ciz et al., 2010). The significant decrease in the hydrolytic PG enzyme activity by chitosan, resveratrol and GB treatments accompanied by higher MSI of fruit peel in both resveratrol and GB treatments compared to control might explain the observed retention of fruit firmness by these treatments (Tables 1–3, 7 and 8). PPO, as a defense enzyme (Campos-Vargas and Saltveit, 2002), showed much higher activity during SL in the control treatment compared to initial. While both resveratrol and glycine betaine treatments at low and moderate rates retained both higher PPO activity and total phenols concentration than control (Tables 5, 7

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and 8), although phenols, especially (+)-catechin, gallic acid, chlorogenic acid, and ellagic acid as the most important PPO substrates, could be oxidized to quinones by the action of both PPO and POD (Campos-Vargas and Saltveit, 2002). Quinones polymerizations produce brown-colored compounds that are highly toxic to several pathogens and cause tissue browning (Campos-Vargas and Saltveit, 2002). In Mangoes, PPO activity was 3-folds higher in ripe than that of unripe fruit and its activity was positively correlated with total phenols during 12 days of storage at 30 ◦ C (Hossain et al., 2014). Both PPO and total phenols were involved in anthracnose resistance of mangoes and suggested as indicators for cultivars resistant to postharvest diseases (Gong et al., 2013). Thus, the regulation of phenols metabolism is, however, more likely determined not only by PPO but also by several other phenolic-biosynthetic enzymes such as phenylalanine ammonia-lyas (Liu et al., 2007). In conclusion, both postharvest trans-resveratrol (especially at 1.6 × 10−3 M) and GB (especially at 10 mM) treatments retained quality of ‘Sukkari’ bananas during shelf life and being suggested as natural alternatives to synthetic chemicals. Acknowledgments This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant G-53-1551437. The authors, therefore, acknowledge with thanks DSR for technical and financial support. Also, we would like to thank Nageeb Al-Masoudi, M.Sc., and Nour Gamal, B.Sc. at the Arid land Agriculture Department, Faculty of Meteorology, Environment and Arid land Agriculture, King Abdulaziz University, for their indispensable technical support. References Adrian, M., Jeandet, P., Veneau, J., Weston, L.A., Bessis, R., 1997. Biological activity of resveratrol, a stilbenic compound from grapevines, against Botrytis cinerea, the causal agent for gray mold. J. Chem. Ecol. 23, 1689–1702. Ao, C., Li, A., Elzaawely, A.A., Xuan, T.D., Tawata, S., 2008. Evaluation of antioxidant and antibacterial activities of Ficus microcarpa L. fil. extract. Food Control 19, 940–948. Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216. Awad, M.A., Al-Qurashi, A.D., Mohamed, S.A., 2015. Postharvest trans-resveratrol and glycine betaine treatments affect quality, antioxidant capacity, antioxidant compounds and enzymesactivities of ‘El-Bayadi’ table grapes after storage and shelf life. Sci. Hortic. 197, 350–356. Ayranci, E., Tunc, S., 2003. A method for the measurement of the oxygen permeability and the development of edible films to reduce the rate of oxidative reactions in fresh foods. Food Chem. 80, 423–431. Bhande, S.D., Ravindra, M.R., Goswami, T.K., 2008. Respiration rate of banana fruit under aerobic conditions at different storage temperatures. J. Food Eng. 87, 116–123. Campos-Vargas, R., Saltveit, M.E., 2002. Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce. Physiol. Plant. 114, 73–84. Cano, M.P., De Ancos, B., Matallana, C., Camara, M., Reglero, G., Tabera, J., 1997. Difference among Spanish and latin-american banana cultivars: morphological, chemical and sensory characteristics. Food Chem. 59, 411–419. Chen, T.H., Murata, N., 2011. Glycine betaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ. 34, 1–20. Chen, M.-L., Yi, L., Zhang, Y., Zhou, X., Ran, L., Yang, J., Zhu, J.-D., Zhang, Q.-Y., Mi, M.-T., 2016. Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota. mBio 7, e02210–e2215. Cherukuri, K., 2007. Effect of trans-resveratrol on shelf life and bioactive compounds in Satsuma mandarin. In: MSc Thesis. Auburn, Alabama, USA, pp. 96. Ciz, M., Cizova, H., Denev, P., Kratchanova, M., Slavov, A., Lojek, A., 2010. Different methods for control and comparison of the antioxidant properties of vegetables. Food Control 21, 518–523. Dani, C., Oliboni, L.S., Pra, D., Bonatto, D., Santos, C.E., Yoneama, M.L., Dias, J.F., Salvador, M., Henriques, J.A.P., 2012. Mineral content is related to antioxidant and antimutagenic properties of grape juice. Genet. Mol. Res. 11, 3154–3163. Duan, X.W., Joyce, D.C., Jiang, Y.M., 2007. Postharvest biology and handling of banana fruit: review. Fresh Produce 1, 140–152. Fernando, H.R.P., Srilaong, V., Pongprasert, N., Boonyaritthongchai, P., Jitareerat, P., 2014. Changes in antioxidant properties and chemical composition during

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