Quality and biochemical changes of ‘Hindi-Besennara’ mangoes during shelf life as affected by chitosan, trans-resveratrol and glycine betaine postharvest dipping

Scientia Horticulturae 217 (2017) 156–163

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Quality and biochemical changes of ‘Hindi-Besennara’ mangoes during shelf life as affected by chitosan, trans-resveratrol and glycine betaine postharvest dipping Mohamed A. Awad a,b,∗ , Adel D. Al-Qurashi a , El-Refaey F.A. El-Dengawy c , Mohamed I. Elsayed a a Department of Arid land Agriculture, Faculty of Meteorology, Environment and Arid land Agriculture, King Abdulaziz University, 80208, Jeddah, Saudi Arabia b Pomology Department, Faculty of Agriculture, Mansoura University, El-Mansoura, Egypt c Pomology Department, Faculty of Agriculture, Damietta University 34517, Damietta, Egypt

a r t i c l e

i n f o

Article history: Received 21 October 2016 Received in revised form 24 January 2017 Accepted 25 January 2017 Keywords: Mango Chitosan Antioxidants Resveratrol Glycine betaine Enzymes

a b s t r a c t Effects of chitosan (1%), trans-resveratrol (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 ‘Hindi-Besennara’ mangoes during ripening at shelf life (SL) (18± 2 ◦ C, 60–70% RH) were studied. Resveratrol, especially at low rate followed by GB, especially at high rate, decreased decay percentage after one and two weeks of SL compared to other treatments. Both compounds at all rates retained higher fruit firmness during SL and higher titratable acidity (TA) (only after one week of SL). Both GB and resveratrol at all rates retained higher vitamin C level than control with no effect on total soluble solids (TSS) after two weeks of SL. These compounds had no effect on weight loss after one week, but increased it after two weeks of SL compared to control. Chitosan showed higher weight loss during SL but, retained higher TA and vitamin C, and lower pH with no significant impact on firmness, TSS and decay compared to control. Chitosan, low and medium resveratrol rates, and low and high GB rates maintained higher membrane stability of peel after two weeks of SL compared to control. All treatments showed lower ␣-amylase but, higher peroxidase (POD) activities in peel than control after two weeks of SL. High resveratrol rate retained higher total phenols level than control, in contrast to chitosan after two weeks of SL while, total flavonoids was not affected. Compared to initial, peel free radical scavenging capacity (FRSC) decreased after one week followed by a sharp increase after two weeks of SL. Chitosan, medium and high resveratrol rates, and medium GB rate showed higher FRSC than control after one week of SL but, with no differences after two weeks of SL. In conclusion, both trans-resveratrol and GB treatments retained quality of ‘Hindi-Besennara’ mangoes during SL and being suggested as natural alternatives to synthetic chemicals. © 2017 Elsevier B.V. All rights reserved.

1. Introduction As climacteric fruit, mangoes (Mangifera indica L.) possess a relatively short shelf life at ambient conditions due to rapid softening and the development of several physiological and pathological disorders (Sivakumar et al., 2011; López-Mora et al., 2013). Also, mangoes are highly sensitive to chilling injuries when stored at

∗ Corresponding author at: Department of Arid land Agriculture, Faculty of Meteorology, Environment and Arid land Agriculture, King Abdulaziz University, 80208, Jeddah, Saudi Arabia. E-mail addresses: [email protected], [email protected] (M.A. Awad). http://dx.doi.org/10.1016/j.scienta.2017.01.043 0304-4238/© 2017 Elsevier B.V. All rights reserved.

a temperature below 13 ◦ C (Sivakumar et al., 2011). 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 mangoes 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; Al-Qurashi and Awad, 2015). Postharvest dipping of ‘Tainong’ mangoes in 2% chitosan decreased respiration rate, and the loss of firmness, color change, acidity, ascorbic acid and fruit weight as well as inhibited diseases progress during storage at 15 ◦ C (Zhu et al., 2008). Also dipping ‘Nam Dok Mai’ mangoes, previously inoculated with C. gloeosporioides, in chitosan (from 0.5 to 2.0%) delayed ripening and reduced respiration rate, ethylene production, and

M.A. Awad et al. / Scientia Horticulturae 217 (2017) 156–163

weight loss, ascorbic acid, and acidity and reduced diseases progression (Jitareerat et al., 2007). However, ‘Tommy Atkins’ mangoes dipped in 1% chitosan had no effects on fruit ripening, weight loss and black spot incidence, but inhibited the extension of this disease during storage at 12 ◦ C and 25 ◦ C (López-Mora et al., 2013). 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 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 trans-resveratrol dipping at 1.6 × 10−5 M, 1.6 × 10−4 M and 1.6 × 10−3 M reduced decay, increased antioxidant compounds, POD and polyphenoloxidase (PPO) and decreased polygalacturonase 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, 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 during cold storage (Fu-gui et al., 2013). Postharvest GB dipping at 10, 15 and 20 mM 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 ‘Hindi-Besennara’ mangoes to postharvest dipping in chitosan, resveratrol and GB treatment as an attempt to maintain quality during SL. 2. Materials and methods 2.1. Plant materials and experimental procedure This experiment was performed on mature hard-green ‘HindiBesennara’ mangoes collected from a commercial orchard located in Jizan region (17.4751◦ N, 42.7076◦ E), Kingdom of Saudi Arabia. Fruit were packed in perforated cardbox (12 fruit of each box, about 3.0–3.5 kg) and transported to the postharvest laboratory at King Abdulaziz University in Jeddah within about 8 h at 15 ◦ C. Fruit of uniform size, weight (250–300 g/fruit) and appearance and free of visual defects were selected for this experiment. 2.2. Fruit treatment A completely randomized experimental design with three replicates (20 fruit of each) was established. Fruit of each treatment/replicate were soaked either into water (control), 1% acetic acid, 1% chitosan (100,000–300,000 MW) (Acros Organic, New Jersey, USA) dissolved in 1% acetic acid, trans-resveratrol (Baoji

157

Guokang Bio-Technology 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 2 weeks. Before applying the treatments, additional three samples (5 fruit of each) were randomly collected for initial quality and biochemical analyses as described below. After one and two weeks of shelf life, weight loss and decay incidence were recorded for each treatment/replicate as described below. Also, samples (5 fruit 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 total phenols, flavonoids, enzymes and antioxidant activity analysis. Pulp firmness was measured in each sample directly following peeling then, the pulp tissue was sliced and mixed. Random portion of this pulp tissue was directly used for TSS, TA, 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. Decay incidence Decay incidence, due to skin browning, shriveling and diseases, was recorded and calculated on initial fruit number basis for each samples and expressed in percentage. 2.5. Firmness, TSS, TA, pH and vitamin C measurements in fruit pulp Fruit pulp firmness was measured independently in 5 fruit (two opposite measurements in the middle of each fruit) 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 5 fruit per replicate for measuring TSS content, TA, pH and vitamin C concentration. TSS content was measured in fruit pulp juice with a digital refractometer (Pocket Refractometer PAL 3, ATAGO, Japan) and expressed in percentage. TA was determined in distilled water diluted fruit juice (1: 2) by titrating with 0.1N sodium hydroxide up to pH 8.2, using automatic titrator (HI 902, HANNA Instrument, USA) and the results expressed as a percentage of citric 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-dichlorophenol endophenol dye and the results expressed as g Kg−1 on a fresh weight (FW) basis (Ranganna, 1979). 2.6. Leakage of ions from fruit peel 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 conductivity digital meter (Orion 150A+, Thermo Electron Corporation, USA). The same disks were kept in a boiling water bath (100 ◦ C)

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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 5 fruit/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 ␮L 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 capacity (FRSC) 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:

Table 1 Decay and weight loss percentage of ‘Hindi-Besennara’ mangoes during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (1%) Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM/L) 10 15 20 F-test LSD (0.05) Shelf life (SL) (week) 1 2 F-test T × SL F-test

Decay (%)

Weight loss (%)

0.0

0.0

23.6a 24.9a 24.6a

9.4f 14.0a 11.5c

5.3d 9.4c 7.2cd

9.8ef 9.6f 10.1ed

14.2b 14.8b 10.4c *** 3.2

10.6c 12.4b 11.2c *** 0.48

10.9 19.0 ***

7.8 14.2 ***

*

***

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

Table 2 The interaction effect between treatment and shelf life on decay and weight loss percentage of ‘Hindi-Besennara’ mangoes as affected by postharvest chitosan, transresveratrol and glycine betaine dipping. Treatment

Shelf life (week) Decay (%)

Weight loss (%)

1

2

1

2

Control Acetic acid (1%) Chitosan (1%)

17.7b 19.5b 18.9b

29.4a 30.3a 30.3a

7.3ij 7.4ij 8.7h

11.6f 20.6a 14.3cd

Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3

2.3f 7.8de 5.9ef

8.2de 11.1cd 8.6de

7.2j 6.9j 7.4ij

12.5e 12.2ef 12.8e

Glycine betaine (mM/L) 10 15 20

9.2de 11.5cd 5.2ef

19.2b 18.0b 15.5bc

7.5ij 9.4g 7.9i

13.7d 15.3b 14.4c

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

1959). The reaction mixture (0.5 ml) containing 5 mg substrate, DPPHradicalscavenging% = [(Abscontrol − Abssample)/Abscontrol] × 100 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 The inhibition concentration (IC50 ) was defined as ␮g phenolics dinitrosalicylic acid reagent was added to each tube and heated in of the test sample that decreases 50% of initial radical. The IC50 a boiling water bath for 10 min. After cooling to room temperavalues were calculated from the dose responses curves. ture, the absorbance was measured at 560 nm. Starch was used as a substrate. One unit of enzyme activity was defined as the amount 2.8. Enzymes measurements of fruit peel of enzyme which liberated 1 ␮M of reducing sugar per min under standard assay conditions. 2.8.1. Crude extract preparation One gram of fruit peel (randomly collected from 5 fruit/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 both ␣-amylase and peroxidase assay. 2.8.2. ˛-amylase assay ␣-amylase (EC 3.2.1.1) activity was assayed by determining the liberated reducing end products using maltose as a standard (Miller,

2.8.3. Peroxidase assay Peroxidase (EC 1.11.1.7) activity 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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Table 3 Quality characteristics and peel membrane stability index (MSI) of ‘Hindi-Besennara’ mangoes during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (1%) Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM/L) 10 15 20 F-test LSD (0.05) Shelf life (SL) (week) 1 2 F-test T × SL F-test

Firmness (N)

TSS (%)

Acidity (%)

pH

Vitamin C (g kg-1 )

MSI (Index)

6.90

12.8

1.35

3.42

0.45

20.0

3.85ed 3.41e 4.14d

17.3bcd 16.0d 16.7bcd

0.23c 0.28bc 0.42ab

5.3a 4.9bc 4.8bc

0.31c 0.46a 0.45a

8.8e 11.4cd 19.3a

5.44ab 5.48ab 4.74c

16.0d 17.6abc 19.0a

0.37ab 0.47a 0.47a

4.8bc 4.9b 4.9b

0.38b 0.43ab 0.46a

17.9a 15.1b 9.7de

5.87a 5.55ab 5.11bc *** 0.45

17.2bcd 16.5cd 18.1ab *** 1.48

0.42ab 0.42ab 0.50a *** 0.14

4.8bc 4.7bc 4.7c *** 0.24

0.45a 0.47a 0.45a *** 6.1

12.8c 9.2de 8.8e *** 2.2

5.15a 4.54b ***

15.5b 18.2a ***

0.56a 0.24b ***

4.5b 5.2a ***

0.42a 0.44a NS

15.3a 11.2b ***

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. Table 4 The interaction effect between treatment and shelf life on acidity (%), pH, vitamin C (g Kg−1 ) and peel membrane stability index (MSI) of ‘Hindi-Besennara’ mangoes as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping. Treatment

Shelf life (week) Acidity (%)

pH

Vitamin C

MSI

1

2

1

2

1

2

1

2

Control Acetic acid (1%) Chitosan (1%)

0.20bc 0.38bc 0.63a

0.25bc 0.19c 0.22bc

5.4ab 4.4f 4.3f

5.2abc 5.4ab 5.4a

0.33fg 0.50ab 0.37defg

0.30g 0.42bcde 0.53a

9.4efg 13.0d 21.6a

8.2fg 9.7efg 16.9b

Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3

0.39b 0.73a 0.70a

0.35bc 0.22bc 0.24bc

4.9cd 4.7de 4.4ef

4.7de 5.1bcd 5.4ab

0.36efg 0.39cdef 0.50ab

0.41cdef 0.46abc 0.43bcde

23.9a 16.6bc 10.9def

11.8de 13.5d 8.4fg

Glycine betaine (mM/L) 10 15 20

0.64a 0.62a 0.75a

0.19c 0.23bc 0.25bc

4.3f 4.2f 4.2f

5.2abc 5.2abc 5.2abc

0.40cdef 0.45abcd 0.46abc

0.50ab 0.50ab 0.45abc

13.7cd 10.8def 17.2b

11.9de 7.5g 13.0d

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

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 F-test and the least significant differences (LSD) at P ≤ 5%. 3. Results There were significant interaction effects between treatment and SL on decay and weight loss percentage (Tables 1 and 2). Decay percentage increased during SL in all treatments, except for the medium and high rates of resveratrol. In this respect, after one and two weeks of shelf life, decay percentage was lower in resveratrol and GB treatments at all rates than other treatments (Table 2). After one and two weeks of SL, the low rate of resveratrol and the high rate of GB showed the lowest decay percentage compared to other rates. Weight loss percentage increased during SL in all treatments. After one week of SL, there were no significant differences among the applied treatments, except for chitosan and the medium rate

of GB that showed higher weight loss percentage than other treatments (Table 2). However, after two weeks of SL, all the applied treatments showed higher weight loss percentage than control, except for the medium rate of resveratrol. In this respect, resveratrol at all rates gave lower weight loss percentage than GB at all rates. Firmness decreased during SL, showed lower values than initial, and was significantly higher at resveratrol and GB at all rates than other treatments (Table 3). In this respect, the low and medium rates of resveratrol showed higher firmness than the high rate. Also, the low rate of GB gave higher firmness than the high rate. However, there were no significant differences in firmness among chitosan, acetic acid and control treatments. TSS content increased during SL, showed higher values than initial, and was higher at the high rate of resveratrol than control (Table 3). There were no significant interaction effect between treatment and SL on fruit firmness and TSS content (Table 3). There were significant interaction effects between treatment and shelf life on TA, pH, vitamin C and MSI (Tables 3 and 4). TA concentration showed much lower values than initial and decreased during SL in all treatments, except for control, acetic acid and the low rate of resveratrol (Tables 3 and 4). In this respect, after one week of SL chitosan, resveratrol at medium

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and high rates and GB at all rates showed higher TA concentration than acetic acid and control treatments (Table 4). However, after two weeks of shelf life there were no significant differences among treatments. pH showed much higher values than initial and increased during SL in all treatments, except for control and the low and medium rates of resveratrol (Tables 3 and 4). After one week of SL, pH was lower in all treatments than control while, after two weeks of SL, pH was lower at the low rate of resveratrol than other treatments, except for the medium rate of resveratrol (Table 4). Vitamin C concentration showed similar values to initial and increased during SL only in chitosan and the low rate of GB treatments (Tables 3 and 4). After one week of SL, vitamin C concentration was higher in acetic acid, high rate of resveratrol, and medium and high rates of GB than other treatments (Table 4). However, after two weeks of shelf life, all treatments gave higher vitamin C concentration than control. In this respect, there were no significant differences in vitamin C concentration among the different rates of resveratrol and GB. MSI showed lower values than initial and decreased during SL in all treatments, except for control and the low rate of GB (Tables 3 and 4). After one week of SL, the applied treatments maintained higher MSI than control, except for the high rate of resveratrol and the medium rate of GB (Table 4). However, after two weeks of SL, chitosan, the low and medium rates of resveratrol and the low and high rates of GB showed higher MSI than other treatments including control. There were significant interaction effect between treatment and shelf life on total phenols and flavonoids concentrations and FRSC values (Tables 5 and 6). Total phenols concentration decreased during SL in acetic acid, chitosan, low rate of resveratrol, and low and medium rates of GB treatments, while it did not change in the other treatments (Table 6). After one week of SL, acetic acid, and the low and medium rates of GB showed higher total phenols concentration than other treatments. However, after two weeks of SL, the high rate of resveratrol gave higher total phenols concentration than control, in contrast to chitosan treatment. Total flavonoids concentration increased during SL only in acetic acid and the low rate of GB while, it did not significantly change in the other treatments (Table 6). After one week of SL, total flavonoids concentration was lower in acetic acid, and the low rate of resveratrol and GB than control. However, after two weeks of SL, there were no significant differences in total flavonoids concentration among the treatments. Compared to initial, FRSC of peel greatly decreased (higher DPPH IC50 values) after one week followed by a sharp increase (lower DPPH IC50 values) after two weeks of SL (Tables 5 and 6). After one week of SL, chitosan, the medium and high rates of resveratrol and the medium rate of GB showed higher FRSC than other treatments (Table 6). However, after two weeks of

Table 5 Total phenols and flavonoids concentration and free radical scavenging capacity (FRSC) of peel of ‘Hindi-Besennara’ mangoes during shelf life as affected by postharvest chitosan, trans- resveratrol and glycine betaine dipping. Phenols (g kg-1 )

Flavonoids (g kg-1 )

FRSC (DPPH IC50 value)

20.5

1.21

9.5

22.4cd 26.5abc 17.7e

2.00ab 1.51de 1.72bcde

17.0bc 20.6ab 13.1cd

Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3

24.6bcd 21.1de 22.7cd

1.44e 1.85abcd 2.12a

17.4b 11.7d 9.9d

Glycine betaine (mM/L) 10 15 20 F-test LSD (0.05)

29.3a 27.7ab 23.7bcd *** 4.4

1.65cde 1.89abc 1.88abc *** 0.35

17.0bc 11.8d 22.7a *** 4.0

26.5a 21.4b ***

1.66b 1.91a ***

27.2a 4.2b ***

***

**

***

Initial Treatment (T) Control Acetic acid (1%) Chitosan (1%)

Shelf life (SL) (week) 1 2 F-test T × SL F-test

Means within each column followed by the same letter are not significantly different at level P ≤ 0.05. (**) and (***) significant at P ≤ 0.01 and 0.001, respectively.

SL there were no significant differences in FRSC among treatments. There were significant interaction effect between treatment and shelf life on ␣-amylase and peroxidase activities (Tables 7 and 8). ␣-amylase activity increased during SL and showed higher values than initial (Tables 7 and 8). After one week of SL, both the low rate of resveratrol and control showed higher ␣-amylase activity than other treatments (Table 8). However, after two weeks of SL, all the treatments showed lower ␣-amylase activity than control. POD activity increased during SL in all treatments (Table 8). After one week of SL, POD activity was lower in control, medium rate of resveratrol, and low and medium rates of GB than other treatments (Table 8). However, after two weeks of SL, all the treatments showed higher peroxidase activity than control, in contrast to chitosan treatment. After both one and two weeks of shelf life, the high rate of GB treatment showed the highest POD activity.

Table 6 The interaction effect between treatment and shelf life on total phenols and flavonoids concentration (g kg−1 ) and free radical scavenging capacity (FRSC) (DPPH IC50 value) in peel of ‘Hindi- Besennara’ mangoes as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping. Treatment

Shelf life (week) Phenols

Flavonoids

FRSC

1

2

1

2

1

2

Control Acetic acid (1%) Chitosan (1%)

24.5cde 34.2ab 21.7def

20.2def 18.8efg 13.6g

2.1a 1.0e 1.8abc

1.8abc 2.0ab 1.6bcd

31.0bc 35.8b 22.7d

2.9f 5.2f 3.4f

Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3

30.4abc 21.6def 16.1fg

18.8efg 20.6def 29.3bc

1.4cde 1.7abcd 2.1ab

1.4cde 2.0ab 2.1a

31.3bc 19.7de 14.5e

3.4f 3.7f 5.3f

Glycine betaine (mM/L) 10 15 20

33.3ab 35.8a 21.4def

25.2cd 19.7defg 25.9cd

1.2de 1.6abcd 1.8abc

2.0ab 2.1ab 1.9ab

28.9c 18.9de 41.9a

5.1f 4.7f 3.5f

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

M.A. Awad et al. / Scientia Horticulturae 217 (2017) 156–163 Table 7 ␣-amylase and peroxidase activity of ‘Hindi-Besennara’ mangoes peel during shelf life as affected by postharvest chitosan, trans-resveratrol and glycine betaine dipping.

Initial Treatment (T) Control Acetic acid (1%) Chitosan (1%) Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3 Glycine betaine (mM/L) 10 15 20 F-test LSD (0.05) Shelf life (SL) (week) 1 2 F-test T × SL F-test

␣-amylase (U min g FW)

Peroxidase (U min g FW)

0.45

0.90

0.78a 0.58e 0.62d

1.13f 1.58bc 1.19f

0.73b 0.68c 0.62d

1.43de 1.33e 1.64b

0.68c 0.68c 0.70c *** 0.025

0.99g 1.50cd 2.11a *** 0.10

0.56b 0.79a ***

1.01b 1.85a ***

***

***

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

Table 8 The interaction effect between treatment and shelf life on ␣-amylase and peroxidase activity (U min g FW) of ‘Hindi-Besennara’ mangoes peel as affected by postharvest chitosan, trans- resveratrol and glycine betaine dipping. Treatment

Shelf life (week) ␣-amylase

Peroxidase

1

2

1

2

Control Acetic acid (1%) Chitosan (1%)

0.66e 0.46i 0.49hi

0.89a 0.70e 0.75d

0.81k 1.3gh 1.1i

1.4fg 1.8c 1.3h

Resveratrol (M) 1.6 × 10−5 1.6 × 10−4 1.6 × 10−3

0.68e 0.53g 0.46i

0.78cd 0.82b 0.77d

0.97ij 0.90jk 1.0i

1.9c 1.7cd 2.2b

Glycine betaine (mM/L) 10 15 20

0.61f 0.53gh 0.58f

0.75d 0.83b 0.82bc

0.43l 0.90jk 1.6de

1.5ef 2.1b 2.6a

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

4. Discussion As a climacteric fruit, mango fruit show a relatively high rate of physiological activity including high respiration rate and ethylene production following harvest that shorten the SL. Due to raising consumers concerns against the use of synthetic chemicals, several natural compounds are currently evaluated for their effectiveness on delaying ripening and maintaining fruit quality during SL. In this regard, the current study evaluated the response of ‘Hindi-Besennara’ mangoes to postharvest dipping in chitosan, as a natural edible 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. Our results showed that resveratrol, especially at low rate followed by GB, especially at high rate treatments, significantly decreased decay percentage after one and two weeks of SL compared to control and other treatments (Tables 1 and 2). Moreover, both compounds at all rates retained

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higher fruit firmness during SL and higher TA (only after one week of SL). Also, after two weeks of SL, both GB and resveratrol at all rates retained higher level of vitamin C than control with no negative impact on TSS content (Tables 3 and 4). However, these compounds showed no significant effect on weight loss after one week, but increased it after two weeks of SL compared to control. These results partially confirm those of Jimenez et al. (2005) in which trans-resveratrol especially at 1.6 × 10−4 M reduced water loss, maintained turgidity and firmness of apples, grapes and tomatoes during storage compared to control with no negative impact on other fruit quality. Such effects were attributed to two distinct mechanisms namely the fungicidal and the impermeabilizer properties of trans-resveratrol, as suggested by Jimenez et al. (2005). Accordingly, trans-resveratrol may form a coat on fruit surface that decrease water loss and retain higher fruit firmness than control. trans-resveratrol dipping of ‘Crimson’ table grapes controlled decay, but showed no synergistic effect when applied in combination with chitosan. Trans-resveratrol proved broad antifungal activity especially against Botrytis cinerea (Gonzalez Urena et al., 2003; Jimenez et al., 2005). Also, Awad et al. (2015) reported that both trans-resveratrol and GB treatments retained higher firmness but had no effect on weight loss, TSS, TA and pH after cold storage and SL of ‘El-Bayadi’ table grapes. The induction of natural defense system that include trans-resveratrol accumulation in ‘Cardinal’ grapes tissue by pre-storage carbon dioxide treatment was critical in controlling fungus decay during storage and shelf life. The observed positive effects of GB on decay and other quality parameters of ‘Hindi-Besennara’ mangoes (Tables 1–4) might be related 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; Ashraf and Foolad, 2007; Mansour, 2000; Yang et al., 2003; 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 of endogenous non-enzymatic antioxidant defense such as increasing the levels of S-adenosylmethionine 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. It is well known that weight loss during SL is mainly due to loss of water by transpiration through fruit peel and also to the respiration process (Jitareerat et al., 2007; Zhu et al., 2008; Jagadeesh et al., 2015). Chitosan as an edible coating is expected to reduce transpiration and thus decrease weight loss during SL. However, in our experiment, chitosan showed higher weight loss after one and two weeks of SL but, retained higher TA and vitamin C, and lower pH with no significant impact on firmness, TSS and decay of fruit compared to control (Tables 1–4). These results partially confirm those of López-Mora et al. (2013) in which 1% chitosan dipping of ‘Tommy Atkins’ mangoes had no effects on fruit ripening, weight loss and black spot incidence, but inhibited the extension of this disease during storage at 12 ◦ C and 25 ◦ C. However, our results partially contradict with those of Jitareerat et al. (2007), Zhu et al. (2008) and Jagadeesh et al. (2015) on mangoes where edible coatings such as chitosan and gum Arabic delayed ripening and reduced the loss in weight, vitamin C and TA. Such

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contradictions are 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. Mangoes treated with either 100 ppm laboratory-produced chtiosan or 500 ppm Sigmaproduced chitosan delayed fruit ripening and effectively decreased the incidence of postharvest stem-end rot disease (caused by D. natalensis) (Baclayon and Calibo, 2013). Our results showed that chitosan, resveratrol at low and medium rates, and GB at low and high rates significantly maintained higher membrane stability index (MSI) of peel tissues after two weeks of SL (Table 4) compared to control. Fruit ripening and senescence is possibly an oxidative process in which the transition from mature stage into ripening/senescence stage is accompanied by a progressive shift toward an oxidative state (Goulao and Oliveira 2008). Accordingly, excessive ROS production could participate in the oxidation of lipids and proteins of cell membrane that are involved in mango ripening. Indeed a steady decrease in membrane stability index, as measured by the leakage of ions, was observed upon the progression of fruit ripening (Tables 3 and 4), indicates a gradual loss of membrane’s stability due to changes occurring in the biochemical and biophysical properties of cell membranes. During ripening, the burst in ROS was combined with a surge in the expression of genes that encode enzymes involved in the generation of antioxidant systems in fruit skin and flesh such as POD, PPO and catalase to enhance resistance against pathogens were reported (Ali et al., 2011; Zhang et al., 2013). Our results showed that all the applied treatments maintained higher vitamin C concentration than control after two weeks of SL. Also, all treatments showed lower activity of the hydrolytic enzyme ␣-amylase but, higher the antioxidant enzyme peroxidase in peel tissues than control after two weeks of SL (Table 8). These results might indicate that these compounds, especially resveratrol and GB, enhanced the antioxidant network of fruit, providing more efficient control of metabolic free radicals level, thus maintain cell membranes integrity of peel and retain higher flesh firmness. However, after two weeks of SL, high resveratrol rate retained higher total phenols concentration than control, in contrast to chitosan while, total flavonoids concentration was not affected (Table 6). After one week of SL, chitosan, the medium and high rates of resveratrol and the medium rate of GB showed higher FRSC than control (Table 6). However, there were no differences in FRSC among all treatments after two weeks of SL. These results partially confirm those of Cherukuri, (2007) in which trans-resveratrol dipping 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. Also, our results are in partial agreement with those of Fu-gui et al. (2013) in which 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. Moreover, postharvest GB dipping at 10, 15 and 20 mM increased antioxidant compounds, POD and PPO activities of ‘El-Bayadi’ table grapes after cold storage and shelf life (Awad et al., 2015). Compared to initial, FRSC of fruit peel greatly decreased (higher DPPH IC50 values) after one week followed by a sharp increase (lower DPPH IC50 values) after two weeks of SL (Tables 5 and 6). The increase in the antioxidant capacity (lower IC50 values) during shelf life confirm those of Kondo et al. (2005) where DPPH-radical scavenging activity of ‘Choke anan’ mangoes peel increased during 10 days of storage at 6 and 12 ◦ C. The decrease

in total phenols and the relative stability of total flavonoids levels with the increase in FRSC (lower DPPH IC50 values) of peel during SL (Tables 5 and 6) might suggest qualitative changes in phenolic classes toward higher antioxidant potential. In other fleshy climacteric fruit, Fernando et al. (2014) found no significant correlation between the total phenols concentration and antioxidant activity measured by DPPH and FRAP, except for vitamin C level and FRAP in ‘Khai’ banana pulp. However, Sulaiman et al. (2011) obtained only minor to moderate correlation between phenolic concentration and antioxidant activities in nine Malaysian banana cultivars. These results imply that antioxidant compounds other than phenolics and vitamin C such as carotenoids and xanthophylls might also be involved. It was reported that the antioxidant capacity of phenols possibly has a concentration saturation limit above which the activity could not increase further with the concentration (Dani et al., 2012). Also, parallel different assays should be used to investigate the principles of antioxidant/oxidation activity of a certain horticultural commodity (Ciz et al., 2010). In conclusion, both postharvest trans-resveratrol and GB treatments retained quality of ‘Hindi-Besennara’ mangoes during ripening at shelf life conditions 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-37-1551437. The authors, therefore, acknowledge with thanks DSR for technical and financial support. Also, we would like to thank Nageeb Al-Masoudi, MSc., and Nour Gamal, BSc. 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. Al-Qurashi, A.D., Awad, M.A., 2015. Postharvest chitosan treatment affects quality, antioxidant capacity, antioxidant compounds and enzymes activities of ‘El-Bayadi’ table grapes after storage. Sci. Hortic. 197, 392–398. Ali, M.B., Howard, S., Chen, S., Wang, Y., Yu, O., Kovacs, L.G., Qiu, W., 2011. Berry skin development in Norton grape: distinct patterns of transcriptional regulation and flavonoid biosynthesis. BMC Plant Biol. 11, 7–29. 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. Baclayon, D.P., Calibo, C.L., 2013. Effects of chitosan isolated from crab exoskeleton on postharvest stem-end rot disease and on the quality of mango fruit. Ann. Tropic. Res. 35, 23–34. 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, e02210–15. Cherukuri, K., 2007. Effect of Trans-Resveratrol on Shelf Life and Bioactive Compounds in Satsuma Mandarin MSc. Thesis. Auburn, Alabama, USA, 96 pages. 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. Fernando, H.R.P., Srilaong, V., Pongprasert, N., Boonyaritthongchai, P., Jitareerat, P., 2014. Changes in antioxidant properties and chemical composition during ripening in banana variety ‘Hom Thong’ (AAA group) and ‘Khai’ (AA group). Int. Food Res. J. 21, 749–754.

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