PVA Combines with oxalic acid treatment to during shelf-life

Scientia Horticulturae 226 (2017) 208–215

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Minimize browning incidence of banana by postharvest active chitosan/PVA Combines with oxalic acid treatment to during shelf-life A.A. Lo’aya, a b

MARK

⁎,1

, H.D. Dawoodb,1

Pomology Department, Faculty of Agriculture, Mansoura University, El-Mansoura, P.O. Box 35516 El-Mansoura, Egypt Chemistry department faculty of agriculture Mansoura University, El-Mansoura, P.O. Box 35336 El-Mansoura, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords: Banana Shelf life Oxalic acid Browning

The effect of anti-browning by exogenous of chitosan/polyvinyl alcohol (CS/PVA) blended with oxalic acid (OA) at different concentration (0, 10, 15 and 20 mM) on ‘Williams’ banana coating to minimize fruit peel browning during shelf life/marketing. Fruits were immersed in different CS/PVA-OA treatments for 10 min and stored at room temperature (24 ± 1 and RH 58 ± 2). Samples were taken every two days for non-destructive and destructive measurements. CS/PVA-OA 20mM presents a significant reduction in cell membrane degrading enzyme activities such as cellulase (CEL), lipoxygenase (LOX) and pectinase (PT). Consequently, it minimizes lipid peroxidation and cell membrane leakage. Then browning enzymes activities polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) minimized by maintaining total phenols and less browning incidence on fruit peel during shelf life. Overall, oxalic acid could minimize the browning enzyme activities to ensure a smooth surface and long term of the shelf life of banana at room temperature.

1. Introduction Banana (Musa spp.) AAA group, CV ‘Williams’ is one of the most popular fruits in worldwide. Traditionally, considered to be functional fruit with a high marketing value, due to of its nutrition, special compounds and good flavor to consumers (Mohapatra et al., 2010). It ripens more rapidly after harvest resulting in a short shelf life. Postharvest precautions such as handling, transportation, ripening, and marketing usually get into the use of advanced logistics chains (Menniti et al., 2004). Fast pre-cooling is the main factor to maintain banana fruit quality at 13 °C by which give a postharvest shelf life of 2–3 weeks (Jiang et al., 2004). However, the most postharvest issues of banana during handling and marketing is susceptible to browning incidence due to banana is more sensitive to the oxidative reaction occurring after harvest or during shelf life (Huang et al., 2013). The major postharvest concern on bananas after harvest is the brown coloration on the fruit peel, which reduces their marketing value and the attractiveness of fruits, as a result of dysfunction of cell membrane integrity. Thus the oxidative reactions are activated enzymatic browning during handling and marketing (Yoruk and Marshall, 2003). Moreover, browning symptoms have a significant effect on fruit quality attributes for consumers that results in a decline of its market value (Zhang and Tian, 2006). Basically, browning phenomena is related to on the oxidation of

⁎

1

flavonoids, mostly ortho-diphenols (O-diphenols, 1,2-diphenols) to semiquinones and quinones. The enzymes involved are polyphenol oxidase (PPO) and peroxidase (POD). Once, formed, the semiquinones and quinones react with each other nun-enzymatically resulting on the brown pigments (Pourcel et al., 2007). Another enzyme is involved, phenylalanine ammonia lyase (PAL), in generation free phenolic compounds that are the base is of browning reactions. Also, many enzymes involved in browning which are located in different cellular compartments. PPO locates in plastids and PAL in the cytoplasm. In addition, cell membrane degrading enzymes is also forced to enhance browning enzymes reacting with substrates (Toivonen, 1992). Therefore, the postharvest technique is necessary to improve or keep banana fruit quality stable during marketing. So, many studies were considered to alleviate fruit browning using different postharvest treatments such as oxalic acid on banana (Huang et al., 2013), or using ascorbic acid on mango ‘Hindi Be-Sennara’ cv (Lo'ay, 2009), ‘Thompson seedless’ grapes (Lo'ay, 2011), and using salicylic acid on longan fruit (Suiubon et al., 2017). Oxalic acid (OA) is the most an organic acid might play important roles in systemic resistance, stress response, programmed cell death and redox homeostasis in the plant (Wu et al., 2011). The exogenous OA treatment could be decreasing the deterioration, extending shelf life, and delaying fruit senescence. Also, reducing the ethylene production rate, repressing fruit reddening,

Corresponding author. E-mail addresses: [email protected] (A.A. Lo’ay), [email protected] (H.D. Dawood). www.mans.edu.eg

http://dx.doi.org/10.1016/j.scienta.2017.08.046 Received 18 May 2017; Received in revised form 25 August 2017; Accepted 29 August 2017 0304-4238/ © 2017 Published by Elsevier B.V.

Scientia Horticulturae 226 (2017) 208–215

A.A. Lo’ay, H.D. Dawood

solution presented the final concentrations/treatments (Najafi et al., 2015).

decreasing alcohol content (Huang et al., 2013). Recently, chitosan application on postharvest of banana fruit has received more attention, as a biopolymer (Pereira et al., 2015). Chitosan is biopolymer which is characterized as non-toxic (Vimala, 2011), and it is often blended with other polymers such as poly-vinyl-alcohol (PVP) which is also a non-toxic and biodegradable polymer. Therefore, considering that CS/PVA can be safely used in fruit coating (Pereira et al., 2015). Possibly, CS/PVA can be incorporated into other compounds such as natural extracts or organic metal as ascorbic acid (Najafi et al., 2015). Using CS/PVA coating as best edible technique and biologically safe preservative coating for different types of fruit which it controls many physiological processes such as lower fruit respiration rates, extended storage period, and firmness retention (Castelló et al., 2010; Wang and Gao, 2013). Other advantages, it extends the postharvest life of fruit by reducing water loss, gas exchange and oxidation reaction stress (Shiekh et al., 2013; Velickova et al., 2013). Therefore, considering that CS/PVA can be safely applied for postharvest of fruit incorporate with antioxidants (Pereira et al., 2015). Exploiting oxalic acid on postharvest fruit as anti-browning to interact with browning enzymes PPO and POD (Zheng et al., 2011). To enlarge the knowledge of chitosan to evaluate the effect of CS/ PVA loaded with oxalic acid at different concentration to alleviate browning incidence on ‘Williams’ banana fruit (Giant Cavendish AAA sub-grope) during handling or marketing. So, the study aims to understand the role of oxalic acid in browning phenomena to present a further technique for extending the shelf life of banana fruit and maintaining fruit quality during commercial postharvest handling or marketing precautions.

2.3. Physical characteristics evaluation Quality elements such as water loss percentage, fruit peel browning index and color hue angle were measured. Water loss% was measured on an initial weight basis and expressed in percentage (Lo'ay and E.LKhateeb, 2017). Fruit peel browning index was recorded and classified into five categories: 1 no browning, 2 slight browning, 3 moderate, 4 severe symptoms and 5 very severe browning symptoms (Lo'ay, 2009). Finally, color measurement was recorded according to the protocol that described (Lo'ay and El Khateeb, 2011) 2.4. Fruit peel total chlorophyll and color Chlorophyll was extracted by immersed 2 g of fruit peel in 20 mL of N, N- dimethylformamide (DMF). Samples were stored in dark at 4 °C for 16 h to allow the DMF to extract the pigment. finally, samples were centrifuged for 5 min at 15,000 rpm, then a clear supernatant sample was determined chlorophyll A at 663 nm and B at 646 nm wavelength using spectrophotometer (UV-vis) and it presented in mg 100g−1 FW. As to carotene was recorded at 452 nm and it presented in mg 100 g−1 FW (Lo'ay, 2005). 2.5. Browning enzymes PPO and PAL and total phenol (TP) Polyphenol oxidase (PPO, EC: 1.14.18.1), one gram of fruit peel was mixed with 20 mM of Tris-HCL buffer, pH 7.0 and homogenized. The mixture was centrifuged at 15,000 rpm for 5 min under cooling at 4 °C. The clear supernatant was stored at −20 °C for assaying PPO. The activity was determined using catechol substrate. The extract 200 μL was rapidly added to 3 mL of 20 mM catechol liquefying in 100 mM sodium phosphate buffer Ph 7.0 (Jiang et al., 2002). The increased activity was recorded at 400 nm on spectrophotometer during 3 min one unit of enzyme activity was defended as the amount of enzymes that causes a change of 0.1 in absorbance min−1. Phenylalanine ammonia-lyase (PAL, EC: 4.3.1.24) was determined (Ke and Salveit, 1986), one gram of fruit peel was mixed with 4 mL of 50 mM of borate buffer (pH 8.5) containing 5 mM of 2-mercaptoethanol and 400 mg PVP. The homogenate was centrifuged at 17,000 rpm for 20 min at 4 °C. The reaction mixture contained 700 μL of 100 mM of Lphenylalanine and 3 mL of 50 mM borate buffer (pH 8.5), which was added to 300 μL of the supernatant. The mixture was incubation at 40 °C for one hour. The reaction was stopped by adding 100 μL of 5 mM of HCL. The activity of PAL was measured at room temperature. PAL has recorded the absorbance of the assay mixture at 290 nm, based on the production of cinnamic acid. As for measuring TP in treated fruits were determined using FolinCiocalteu reagent with gallic acid as slandered. The TP was measured at wavelength 750 nm. The results were reported as mg of gallic acid equivalents (GAE) mg 100 g−1 FW (Hoff and Singleton, 1977).

2. Materials and methods 2.1. Experimental setup The experiment was performed on a banana (Musa spp. L ‘Williams’ CV. Giant Cavendish AAA sub-group). The investigation was conducted during two seasons 2015–2016 in a commercial orchard. To study the effect of biopolymer CS/PVA loaded with different concentration of oxalic acid (OA) on browning incidence during shelf-life. Samples (60 hands) were picked and selected for uniformity in size and color at stage 2 (Soltani et al., 2010). Fruits were cleaned in a solution of 200 μL L−1 chlorine then fruits were dipped for 2 min in 2 g−1 benlate® solution to control fruit rate and were allowed to air dry before treatments. The fruit was immersed in CS/PVA loaded with OA at different concentrations for 10 min ambient air as followed: control fruit, CS/ PVA, CS/PVA- OA 10mM, CS/PVA-OA 15mM and CS/PVA-OA 20mM. Each treatment has 20 hands, 10 hands for non-distractive measurements and 10 hands for distractive assays. Finally, the fruit was stored at room temperature 24 °C ± 1 and RH 58 ± 2 for twelve days. 2.2. Biopolymer chitosan/PVA with OA preparation Chitosan (CS) (MW 71.3 kDa, the degree of deacetylation 94%), Polyvinyl alcohol (PVA) with a degree of polymerization 1700–1800 and 98–99 ° of hydrolysis and Ascorbic acid were purchased from Merck, 64271 Darmstadt Germany. All reagents were of analytical grade. Distilled Water was used to prepare all the solutions. CS/PVA nanoparticle was prepared by dissolving one gram in 100 mL of distilled water and stirring for 12 h at 70 °C until the PVA was completely dissolved. Then, the PVA solution was used for the synthesis of CS/PVA nanoparticle. One gram of CS was dissolved in a 2% (v/v) acetic acid hydrous solution for 12 h under magnetic stirring. The CS/PVA nanoparticle was obtained by polymerization of PVA in CS solution for 12 h under magnetic stirring at 70 °C (De Moura et al., 2008). Oxalic acid (OA) as an organic acid was loaded in polymer CS/PVA at different concentrations into 1000 mL of the solution under magnetic stirring for 4 h at 25 °C. The resulting solution to incorporate organic acid into the

2.6. Cell membrane degrading, cellulase (CEL), lipoxygenase (LOX), and pectinase (PT) Cellulase (EC: 3.2.1.4) activity was measured by assaying the reducing end of carboxymethyl cellulose as substrate (Miller, 1959). One gram of fruit peel was mixed with 20 mM of Tris-HCL buffer, pH 7.0, and homogenized. The mixture was centrifuged at 15,000 rpm for 5 min under cooling at 4 °C. the clear supernatant was stored at −20 °C for measuring CEL at 450 nm. One unit of enzyme activity was expressed as the amount of enzyme. Lipoxygenase (EC: 1.13.11) activity was assayed (Wang et al., 2005), one gram of fruit peel was homogenized with 7 mL of 0.01 mM Tris-HCL (buffer pH 8.0) containing 200 mg PVP. The mixture was 209

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decrease in water loss and fruit peel browning incidence whereas, a high stability of peel hue value and chlorophyll content when fruits immersed in CS/PVA-OA 20mM compared to other treatments during shelf life (10 days). However, the control fruit presented more rapid in water loss (31.38%) at 10th day of shelf life time. Based on the changes to the visible appearance of fruit peel, the report that CS/PVA-OA 20mM is more effective than other treatments. This could be due to the blending CS/PVA with oxalic acid which it is considered as a natural antioxidant and played a role in protecting plant tissue (Wang et al., 2009). Also, the combination of OA with CS/PVA (biopolymer) is more effective than CS or PVA only each (Pereira et al., 2015). It is possible that the immersing fruits in CS/PVA-OA 20mM solution reducing water loss% by minimizing water evaporation fruit peel surface by supporting the natural wax layer on peel surface which it was changed during fruit development (Lo'ay, 2011). In term of, fruit peel browning symptoms in function of browning parameters. Treatment of CS/PVA-OA could effectively minimize browning symptoms on fruit in this investigation, which is consistent with the previous studies on many fruits such as litchi, peach apple, and banana as reported by Huang et al. (2013). The hue angle values show the color change from green to yellow and chlorophyll degradation which indicated that the decline in hue and chlorophyll are attributed to fruit appearance. In this study, hue value and chlorophyll decrease less when fruit was immersed in CS/PVA-OA 20mM compared to other treatments. the result could be due to the effect of oxalic acid to delay chlorophyll breakdown, so, less changes in fruit peel color during shelf life (Wu et al., 2011).

centrifuged at 17,000 rpm for 20 min at 4 °C. the clear supernatant was used for determining the LOX. The substrate contained 4 μL of linoleic acid, 8 mL Of H2O, 2 mL of 0.1 Mm NaOH and 1 μL of Tween 20. The mixture was shocked and diluted with deionized water to 25 mL. The reaction mixture contained 2.85 mL of 0.01 mM sodium phosphate buffer and monitored directly after mixing. One unit of activity was defined as the change in absorbance at 234 nm of 0.01 unit min−1. Pectinase (EC: 3.2.1.15) activity was determined (Collmer et al., 1988), and the extraction used the method (Payasi and Sanwal, 2003). The activity was tested in the mixture of 500 μL of 0.36% (w/v) polygalacturonic acid in 0.05 M Tris-HCL buffer pH 8.5, 300 μL of 4 mM CaCl2, 600 μL enzymes and 600 μL water. The reaction mixture was holed at 37 °C for 3 h. The PT was recorded by the following absorbance at 232 nm. Total soluble protein content in the enzyme extract was determined using the (Bradford, 1976). The specific activity of enzymes was expressed units mg−1 protein. 2.7. Lipid peroxidation (malondialdehyde, MDA) and DPPH% scavenging assay Malondialdehyde (MDA) as terminal product of lipid peroxidation, 2.5 g sample was ground in a mortar and mixed with 25 mL of 5% (w/ v) metaphosphoric acid, 500 μL of 2% (w/v) butylated hydroxytoluene in ethanol, and finally homogenized by a mixer. The calibration curves made by measuring 1,1,3,3-tetraethyoxypropane (Sigma) in the range 0–2 mM (TBARS) which was equivalent to 0–1 mM malondialdehyde (MDA). 1,1,3,3-Tetraethyoxypropane is stoichiometrically converted into MDA during the acid-heating step of the assay (Iturbe-Ormaetxe et al., 1998). The amount of TBARS present is expressed as MDA equivalents. The antioxidant capacity was recorded as the free radical scavenging effect on DPPH%. Initially, three-gram fresh fruit peel from 5 fingers selected randomly from each treatment was mixed with 3o mL methanol and then centrifuged at 10,000 rpm for 15 min. The clear supernatant (100 μL) was added to 3 mL of 0.1 mM DPPH% that dissolved in methanol. The reaction mixture was incubation for 20 min at 25 °C in dark. The control sample containing all reagent without sample and was used as a blank. The DPPH% radical scavenging activity was recorded by determining the absorbance at 517 nm using a spectrophotometer. The DPPH%radical scavenging activity% of the sample was calculated as 1-absorbance of sample/absorbance of control * 100 (Cao et al., 2012).

3.2. Effect of CS/PVA-OA on browning enzyme activities PPO, PAL, and TP Table 2 shows the variation of PPO, PAL, and TP as a function of shelf life time. Clearly, the browning enzymes present a significant interaction at P ≤ 0.001, when the shelf life time and CS/PVA-OA treatments were considered. The changes in PPO activity in control fruit increase gradually during the first 2 days, then the activity increase more rapid up to end of the experiment (0.93 Umg−1 protein). Markedly, the PPO activity is independently according to CS/PVA-OA treatment. Fruits were immersed in CS/PVA-OA 20mM present slightly during 10 days of shelf life which recorded (PPO: 0.274 U mg−1 protein). However, the activity of PAL is more or less of PPO trend even, the PAL activity increases slightly during 4 days, then increase more rapid up to end shelf life period. Treatment CS/PVA-OA 20mM shows more stability of PAL activity during 8 days compared to other CS/PVAOA treatments. In terms of TP, presents less degradation when fruit immersed in CS/PVA-OA 20mM than other treatments. therefore, many reports indicated that oxalic acid has strong anti-browning activity in tissue culture experiments by blocking the copper active receptor in a cell so, PPO and PAL inhibited (Yoruk and Marshall, 2003). Both enzymes have often been found in chloroplast where it is related to internal thylakoid membrane, cytoplasm and vesicles (Obukowicz and Kennedy, 1981), by which, are considered responsible the rapid brown coloration incidence after degreening. These responses suggest that the browning incidence is associated to consume TP amount. TP is mainly substrate for browning enzymes reactions. Decreased both PPO and PAL activities after fruit immersed in CS/PVA-OA 20mM might be indicative of inhibition of PPO and PAL during shelf life so less consuming of TP and less browning symptoms (Promyou et al., 2007).

2.8. Statistical analysis The experiments were arranged in a completely randomized design, and each treatment comprised three replicates. Differences of all the parameters for treatments were tested by two-way ANOVA and least significant difference (LSD), and linear regression analysis at 5% level were conducted. The statistical software package GenStat ver. 11 (Lawes Agricultural Trust, Rothamsted Experimental station, UK) was used. The superscript letters, differ (P < 0.05) according to the LSD and present the significantly between the effect of treatments using Duncan’s test by CoStat software version 6.303 (798 Lighthouse Ave. PMB 320, Monterey, CA, 93940, USA) 3. Results and discussion 3.1. Effect of CS/PVA-oxalic acid on physical quality attributes characteristics fruit weight loss percentage, fruit peel browning index, color profile and total chlorophyll

3.3. Membrane degradation enzymes and permeability Table 3 depicts the differences in cell membrane degradation enzymes such as cellulase (CEL), lipoxygenase (LOX), and pectinase (PT) activities (U mg−1 protein) and cell membrane leakage percentage as a function of shelf life time for ‘Williams’ banana was immersed in different concentration of CS/PVA with oxalic acid. Apparently, the cell membrane degradation enzymes activities present a significant interaction at P ≤ 0.001 when shelf life time and CS/PVA-OA treatments

Physical quality attributes as a function of shelf life (days) for the CS/PVA combined with different oxalic acid (OA) concentrations is shown in Table 1. Chitosan/PVA loaded with oxalic acid, shows a significant effect when it is considered as a factor. Considering the different CS/PVA-OA treatments. It is clear that a significantly greater 210

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Table 1 Effect of postharvest application of Chitosan/PVA film with oxalic acid on water loss percentage, fruit peel color (hue angle), and browning index of banana AAA group, CV ‘Williams’ during ten-day shelf-life at 2015 and 2016 seasons. Treatments

Shelf life time (days)

Water loss% Control CS/PVA-OA 0mM CS/PVA-OA 10mM CS/PVA-OA 15mM CS/PVA-OA 20mM LSD at 5%

D0

D2

0.00 0.00 0.00 0.00 0.00

19.33 17.67 16.53 15.56 14.55 0.569

Fruit peel browning index Control 1.00 1.00 CS/PVA-OA 0mM CS/PVA-OA 10mM 1.00 1.00 CS/PVA-OA 15mM CS/PVA-OA 20mM 1.00 LSD at 5%

± ± ± ± ±

Fruit peel color hue angle (ho) Control 133.66 CS/PVA-OA 0mM 133.66 CS/PVA-OA 10mM 133.66 CS/PVA-OA 15mM 133.66 CS/PVA-OA 20mM 133.66 LSD at 5% Total chlorophyll ab Control CS/PVA-OA 0mM CS/PVA-OA 10mM CS/PVA-OA 15mM CS/PVA-OA 20mM LSD at 5%

0.000 0.000 0.000 0.000 0.000

± ± ± ± ±

7.71 ± 7.71 ± 7.71 ± 7.71 ± 7.71 ± 0.489

1.00 1.00 1.00 1.00 1.00

1.288 1.288 1.288 1.288 1.288

0.201 0.201 0.201 0.201 0.201

D4

± ± ± ± ±

± ± ± ± ±

124.66 129.51 130.63 131.55 133.33 2.688

ab

6.03 7.11 7.45 7.63 7.91

ab ab ab ab

0.420 a 0.365 b 0.294 c 0.216 d 0.11 e

22.27 19.27 18.61 17.81 16.69 0.589

0.000 0.000 0.000 0.000 0.000

± ± ± ± ±

± ± ± ± ±

± ± ± ± ±

1.22 ± 1.00 ± 1.00 ± 1.00 ± 1.00 ± 0.008 c

2.150 0.674 0.169 0.285 0.603

0.075 0.063 0.105 0.036 0.047

D6

107.95 128.18 129.70 130.32 132.75 1.245

b b ab a

j

4.07 5.30 6.58 6.98 7.44

cdef abcd abc a

± ± ± ± ±

0.523 a 0.076 b 0.126 c 0.280 d 0.19 e

0.005 0.000 0.000 0.000 0.000

± ± ± ± ±

D8

25.59 22.41 19.64 18.53 17.82 1.395

a b b b b

± ± ± ± ±

1.37 ± 1.23 ± 1.00 ± 1.00 ± 1.00 ± 0.041 d

1.040 0.746 0.233 0.210 0.612

c b b a

0.24 m 0.379 k 0.043 fghi 0.069 defg 0.221 abcd

0.604 1.109 0.129 0.135 0.241

0.029 0.005 0.000 0.000 0.000

a b c cd d

a b c c c

D10

28.43 23.53 21.26 19.88 19.17 0.728

± ± ± ± ±

2.76 ± 2.35 ± 1.16 ± 1.07 ± 1.00 ± 0.165

0.570 0.623 0.508 0.125 0.266

0.128 0.061 0.027 0.023 0.003

a b c d d

a b c c c

79.05 ± 1.860 d 125.65 ± 0.740 c 128.86 ± 0.123 b 129.71 ± 0.000 ab 131.59 ± 0.866 a 2.540

70.71 ± 3.436 c 122.23 ± 1.534 b 127.62 ± 0.358 a 129.05 ± 0.181 a 130.61 ± 0.550 a 4.340

3.24 ± 0.140 n 4.69 ± 0.301 l 5.99 ± 0.287 j 6.810 ± 0.12 efgh 7.26 ± 0.197 bcde

2.19 3.97 5.27 6.28 6.61

± ± ± ± ±

0.308 o 0.19 m 0.371 k 0.035 hij 0.061 fghi

31.38 27.51 23.18 21.91 20.25 1.071

± ± ± ± ±

4.29 ± 3.22 ± 1.61 ± 1.29 ± 1.16 ± 0.406

1.081 a 0.551 b 0.416 c 0.294 d 0.54 e

0.206 0.210 0.050 0.070 0.029

a b c cd d

65.00 ± 0.690 d 116.00 ± 3.198 c 125.60 ± 0.294 b 127.76 ± 0.550 ab 130.02 ± 0.390 a 4.067 1.32 3.33 5.07 6.10 6.44

0.175 p 0.286 h 0.045 kl 0.069 ij 0.09 ghij

± ± ± ± ±

Means in a column are significantly different at (P < 0.05) according to the LSD. Each value represent mean ± SE (n = 3) during two seasons of 2015 and 2016. The superscript letters, differ (P < 0.05) and represent the significantly between treatments mains using Duncan’s test. Fruit peel browning index categories: (1 = no symptoms healthy; 2 = slightly spots; 3 = moderately; 4 = sever, and 5 = very severe browning incidences).

Table 2 Effect of postharvest application of Chitosan/PVA film with oxalic acid on browning enzyme activities polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) and total phenol (TP) of banana AAA group, CV ‘Williams’ during ten-day shelf-life at 2015 and 2016 seasons. Treatments

Shelf life time (days) D0

PPO activity (Unit mg Control CS/PVA-OA 0mM CS/PVA-OA 10mM CS/PVA-OA 15mM CS/PVA-OA 20mM LSD at 5% PAL activity (Unit mg Control CS/PVA-OA 0mM CS/PVA-OA 10mM CS/PVA-OA 15mM CS/PVA-OA 20mM LSD at 5% TP (mg 100 g‐1 F.wt) Control CS/PVA-OA 0mM CS/PVA-OA 10mM CS/PVA-OA 15mM CS/PVA-OA 20mM LSD at 5%

−1

−1

D2

D4

D6

D8

D10

protein 0.22 ± 0.22 ± 0.22 ± 0.22 ± 0.22 ± 0.009

0.005 n 0.00 n 0.00 n 0.00 n 0.00 n

6.11 ± 6.11 ± 6.11 ± 6.11 ± 6.11 ± 0.887

0.115 0.115 0.115 0.115 0.115

0.25 0.24 0.23 0.23 0.22

± ± ± ± ±

0.003 jk 0.000 lm 0.00 m 0.00 m 0.003 n

7.53 7.07 6.50 6.34 6.07

± ± ± ± ±

0.305 0.033 0.046 0.008 0.033

0.29 0.26 0.25 0.24 0.23

± ± ± ± ±

0.008 h 0.003 j 0.000 kl 0.000 lm 0.00 m

0.56 0.31 0.30 0.26 0.25

± ± ± ± ±

0.005 c 0.00 g 0.005 h 0.003 j 0.003 jk

0.60 0.39 0.37 0.28 0.26

± ± ± ± ±

0.005 b 0.00 e 0.003 f 0.005 i 0.003 j

0.93 0.41 0.39 0.29 0.27

± ± ± ± ±

0.012 a 0.003 d 0.00 e 0.003 h 0.003 i

protein)

117.26 117.26 117.26 117.26 117.26 0.779

± ± ± ± ±

l l l l l

0.263 0.263 0.263 0.263 0.263

abc abc abc abc abc

15.17 16.20 17.11 17.53 17.69

± ± ± ± ±

ij jkl kl l l

0.15 gh 0.083 def 0.157 abcd 0.093 ab 0.012 a

10.89 ± 0.36 g 8.25 ± 0.474 i 7.05 ± 0177 jkl 6.48 ± 0.042 kl 6.25 ± 0.073 l

14.51 16.22 16.67 17.07 17.47

± ± ± ± ±

0.304 0.063 0.163 0.033 0.030

h def bcde abcd ab

16.35 ± 0.738 c 9.40 ± 0.404 h 8.09 ± 0.161 i 7.47 ± 0.083 ijk 6.94 ± 0.048 jkl

10.97 15.48 16.41 16.99 17.11

± ± ± ± ±

0.225 j 0.262fg 0.206 cde 0.008 abcd 0.063 abcd

19.56 ± 0.689 b 12.54 ± 0.848 f 9.21 ± 0.557 h 7.93 ± 0.173 ij 7.45 ± 0.075 ijk

23.07 16.95 14.75 13.59 12.04

9.26 ± 0.627 k 13.59 ± 0.364 i 15.91 ± 0.04 efg 16.59 ± 0.139 bcde 16.96 ± 0.028 abcd

8.64 ± 0.335 k 11.33 ± 0.621 j 14.40 ± 0.281 h 16.19 ± 0.305 def 16.90 ± 0.055 abcd

Means in a column are significantly different at (P < 0.05) according to the LSD. Each value represent mean ± SE (n = 3) during two seasons of 2015 and 2016. The superscript letters, differ (P < 0.05) and represent the significantly between treatments mains using Duncan’s test.

211

± ± ± ± ±

1.062 a 0.413 c 0.357 d 0.13 e 0.358 f

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Table 3 Effect of postharvest application of Chitosan/PVA film with oxalic acid on cell membrane degrading cellulase (CEL), lipoxygenase (LOX), pectinase (PT) and cell membrane ion leakage of banana AAA group, CV ‘Williams’ during ten-day shelf-life at 2015 and 2016 seasons. Treatments

Shelf life time (days) D2

D0

D4

D6

D8

D10

21.00 ± 0.527 c 17.00 ± 0.57 e 11.00 ± 0.577 hi 7.33 ± 0.333 lm 6.33 ± 0.33 mn

29.00 ± 0.577 b 18.33 ± 0.333 d 13.00 ± 0.57 g 9.33 ± 0.333 jk 7.00 ± 0.577 lmn

34.66 20.00 15.66 13.00 10.00

−1

Cellulase activity (Unit mg protein) Control 3.33 ± 0.333 CS/PVA-OA 0mM 3.33 ± 0.333 CS/PVA-OA 10mM 3.33 ± 0.333 CS/PVA-OA 15mM 3.33 ± 0.333 CS/PVA-OA 20mM 3.33 ± 0.333 LSD at 5% 1.261

p

9.33 8.00 6.33 5.66 4.00

± ± ± ± ±

0.333 k 0.577 kl 0.33 mn 0.333 no 0.577 p

14.66 ± 0.881 f 11.33 ± 0.333 h 9.33 ± 0.881 jk 6.33 ± 0.33 mn 4.66 ± 0.333 op

Lipogeneses activity (Unit mg −1 protein) Control 0.65 ± 0.03 efghi CS/PVA-OA 0mM 0.65 ± 0.03 efghi CS/PVA-OA 10mM 0.65 ± 0.03 efghi CS/PVA-OA 15mM 0.65 ± 0.03 efghi CS/PVA-OA 20mM 0.65 ± 0.03 efghi LSD at 5% 0.043

0.70 0.66 0.65 0.64 0.63

± ± ± ± ±

0.017 cd 0.003 cdefgh 0.033 defghi 0.033 fghi 0.03 ghi

0.77 0.70 0.67 0.65 0.64

Pectenase activity (Unit mg −1 protein) Control 0.50 ± 0.05 m CS/PVA-OA 0mM 0.50 ± 0.05 m CS/PVA-OA 10mM 0.50 ± 0.05 m CS/PVA-OA 15mM 0.50 ± 0.05 m CS/PVA-OA 20mM 0.50 ± 0.05 m LSD at 5% 0.096

0.76 0.70 0.63 0.60 0.56

± ± ± ± ±

0.033 0.000 0.033 0.000 0.033

Cell membrane leakage% Control 9.04 ± CS/PVA-OA 0mM 9.04 ± CS/PVA-OA 10mM 9.04 ± CS/PVA-OA 15mM 9.04 ± CS/PVA-OA 20mM 9.04 ± LSD at 5% 0.992

17.31 ± 0.756 k 11.56 ± 0.220 n 10.44 ± 0.133o 10.08 ± 0.040op 9.37 ± 0.133 p

p p p p

0.256 p 0.256 p 0.256 p 0.256 p 0.256 p

j jk kl klm lm

± ± ± ± ±

0.017 b 0.005 cde 0.008 cdefg 0.003 defgh 0.00 ghi

1.06 ± 0.088 fg 0.80 ± 0.000 ij 0.701 ± 0.000 jk 0.70 ± 0.000 jk 0.70 ± 0.000 jk

26.04 23.93 16.51 15.19 13.86

± ± ± ± ±

0.176 f 0.95 g 0.540 k 0.279 l 0.36 m

0.84 0.71 0.69 0.67 0.65

± ± ± ± ±

0.033 0.000 0.003 0.003 0.003

a

1.30 0.93 0.80 0.76 0.70

± ± ± ± ±

0.057 0.033 0.000 0.033 0.000

d

30.19 28.64 22.53 18.54 17.37

± ± ± ± ±

c cdef cdefg defghi

h ij j jk

0.280 d 0.29 e 0.761 h 0.296 j 0.329 k

± ± ± ± ±

0.881 0.577 0.333 0.15 g 0.577

a c f

ij

0.77 0.68 0.66 0.64 0.62

± ± ± ± ±

0.008 b 0.003 cdefg 0.003 cdefgh 0.00 ghi 0.005 hi

0.69 0.68 0.66 0.63 0.60

± ± ± ± ±

0.006 cdef 0.005 cdefg 0.005 cdefgh 0.00 ghi 0.006 i

1.56 1.23 1.13 0.90 0.80

± ± ± ± ±

0.033 b 0.033 de 0.03 ef 0.000 hi 0.000 ij

2.26 1.40 1.23 1.06 0.96

± ± ± ± ±

0.145 a 0.057 c 0.033 de 0.033 fg 0.03 gh

39.51 34.41 25.91 21.41 19.24

± ± ± ± ±

0.416 b 0.650 c 0.261 f 0.427 i 0.584 j

49.13 39.64 30.51 26.52 21.75

± ± ± ± ±

1.455 a 0.866 b 0.500 d 0.748 f 0.317 hi

Means in a column are significantly different at (P < 0.05) according to the LSD. Each value represent mean ± SE (n = 3) during two seasons of 2015 and 2016. The superscript letters, differ (P < 0.05) and represent the significantly between treatments mains using Duncan’s test.

non-enzymatic process and mainly due to LOX. Table 4 depicts that the variation of malondialdehyde (MDA) accumulation as a function of shelf lifetime (days). in fact, the MDA shows a significant interaction at P ≤ 0.001 when shelf life time and CS/PVA-OA treatments were considered. The behavior of immersed fruits in different CS/PVA-OA concentrations and stored at ambient air for 10 days was unexpected for CS/PVA-OA 20mM treatment. There was a slight increase of MDA accumulation were observed. The greatest accumulation of MDA was recorded with control fruit. The effect of OA was observed independently to OA concentrations incorporation with CS/PVA polymer. The variation of MDA accumulation could be explained that OA alleviated of lipid peroxidation process during shelf life (Zhang and Tian, 2006). However, DPPH radical scavenging is usually evaluated as the antioxidant activity of polyphenols in fruit (Table 4). Fruit immersed in CS/PVA-OA20mM showed higher scavenging than other treatments. while control fruit showed the lowest scavenging radicals up to end the experiment. It could be that the oxalic acid attribute to the fruit shelf life due to improving the antioxidant activity (Huang et al., 2013).

were considered. The data on cell membrane enzyme activities increased after harvesting and immersing in CS/PVA-OA treatments up to end of shelf life period. The activity rates are more rapid with control fruits than CS/PVA-OA treatments. in terms of CS/PVA-OA, 20mM decrease the cell membrane derogation enzyme activities compared to control and CS/PVA-OA treatments. This could be due to the effect of CS/PVA biopolymer on cell membrane enzyme activities (Landi et al., 2014). Also, the presence of an oxalic acid with CS/PVA polymer might play important roles in systemic resistance stress response programmed cell death and redox homeostasis in plant cell and an anti-senescence effect in fruit at harvest time (Wu et al., 2011). The changes in CEL activity during shelf life could be related to the change in hemicellulose structure which is responsible for fruit firmness (Pasanphan et al., 2010), while the variation in LOX activity rate is suggested to indicate less membrane disruption (Toivonen, 1992) when fruit immersed in CS/PVA-OA 20mM. However, the variation in PT activity during shelf life experiment could be associated with the changes in fruits firmness that it is related to cell wall disassembly and modification to pectin fraction are a seam of the most apparent changes that take place in the cell wall during ripening (Martin-Cabrejas et al., 2002). Consequently, ion leakage is an indicator of plasma membrane rupture, as ion leak out of the cell. Increase cell membrane enzyme activities rates during shelf life are associated with increasing ion leakage (Kamdee et al., 2009). It is clear that the control fruit presents more rapid cell membrane enzyme activities and ion leakage compared to other treatments (Table 3).

4. Data correlations The breakdown of per-oxidated lipids results in the formation of a multiplicity of low molecular weight volatile compounds such as Ethan methane and ethylene (Cava et al., 2000). Because many of these grasses have low odor and taste thresholds they are responsible for flavor producing either positive or negative consumer reaction (Labuza, 1971). So, malondialdehyde (MDA) and other aldehydes constitute one of the major group of products formed following lipid peroxidation, so, measurement of these compounds as thiobarbituric acid reaction substance is, therefore, a sign of the degree of lipid peroxidation. The shelf life of banana at ambient air caused a more rapid increase

3.4. Lipid peroxidation and DPPH radical scavenging In the present, experiment MDA as an indicator of membrane disruption was used. The level of MDA is indicative of membrane degradation due to lipid peroxidation. MDA accumulation is related to 212

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Table 4 Effect of postharvest application of Chitosan/PVA film with oxalic acid on DPPH% radical and lipid peroxidation (MDA) of banana AAA group, CV ‘Williams’ during ten-day shelf-life at 2015 and 2016 seasons. Treatments

Shelf life time (days) D2

D0 DPPH (mM TE 100 g‐1 F.wt) Control 1.06 ± CS/PVA-OA 0mM 1.06 ± CS/PVA-OA 10mM 1.06 ± CS/PVA-OA 15mM 1.06 ± CS/PVA-OA 20mM 1.06 ± LSD at 5% 0.016

0.003 0.003 0.003 0.003 0.003

D4

D6

a a a a a

0.92 0.94 0.96 0.98 1.03

± ± ± ± ±

0.008 f 0.00 e 0.00 e 0.003 d 0.003 b

0.87 0.89 0.92 0.94 1.01

± ± ± ± ±

0.012 i 0.00 g 0.005 f 0.00 e 0.005 c

Lipid peroxidation (MDA μM g−1 F.wt) Control 0.11 ± 0.005 p 0.11 ± 0.005 p CS/PVA-OA 0mM CS/PVA-OA 10mM 0.11 ± 0.005 p 0.11 ± 0.005 p CS/PVA-OA 15mM CS/PVA-OA 20mM 0.11 ± 0.005 p LSD at 5% 0.02

0.28 0.20 0.18 0.15 0.13

± ± ± ± ±

0.00 gh 0.008 j 0.003 klm 0.005 no 0.005 op

0.32 0.23 0.20 0.17 0.14

± 0.017 f ± 0.012 i ± 0.005 jk ± 0.003 lm ± 0.003 no

D8

D10

0.66 0.85 0.87 0.90 1.00

± ± ± ± ±

0.00 m 0.003 i 0.003 h 0.00 g 0.005 cd

0.50 0.73 0.83 0.85 0.95

± ± ± ± ±

0.00 m 0.020 l 0.003 j 0.006 i 0.00 e

0.42 0.65 0.79 0.87 0.92

± ± ± ± ±

0.40 0.34 0.26 0.23 0.16

± ± ± ± ±

0.012 0.005 0.006 0.008 0.006

0.50 0.37 0.33 0.29 0.19

± ± ± ± ±

0.026 b 0.01 e 0.011 f 0.00 g 0.008 jkl

0.57 0.44 0.37 0.32 0.23

± 0.015 a ± 0.017 c ± 0.01 e ± 0.003 f ± 0.008 i

d f h i nm

0.008 o 0.01 m 0.008 k 0.026 hi 0.005 f

Means in a column are significantly different at (P < 0.05) according to the LSD. Each value represent mean ± SE (n = 3) during two seasons of 2015 and 2016. The superscript letters, differ (P < 0.05) and represent the significantly between treatments mains using Duncan’s test.

Fig. 1. The correlation between cell membrane degradation (CEL, LOX and PT) in function of lipid peroxidation (MDA) of fruit peel of banana stored at (10 days shelf life) after immersing in CS/PVA loaded with oxalic acid at different concentrations. Symbols represent the different treatment of CS/PVA loaded with oxalic acid: −○− control fruit, −□− CS/PVA, −△− CS/PVA- OA 10mM, −◊− CS/PVA-OA 15mM and −X- CS/PVA-OA 20mM. sloid lines represent linear regression at 5%.

Fig. 2. The correlation between cell membrane degradation such as cellulase (CEL), lipoxtgenase (LOX) and pectinase (PT) in function of cell membrane leakage% of fruit peel of banana stored at (10 days shelf life) after immersing in CS/PVA loaded with oxalic acid at different concentrations. Symbols represent the different treatment of CS/PVA loaded with oxalic acid: −○− control fruit, −□− CS/PVA, −△− CS/PVA- OA 10mM, − ◊− CS/PVA-OA 15mM and −X- CS/PVA-OA 20mM. sloid lines represent linear regression at 5%.

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membrane degrading enzymes in function of ion leakage (Fig. 2) shows highly correlation especially with CEL and PT (R2 > 90) compared to LOX (R2 < 50). However, fruit peel browning symptoms in function of parameters such as browning enzyme activities (PAL and PPO unit activity mg−1 protein) and TP content (Fig. 3), shows significantly correlation especially with PAL (R2 < 90) compared to PPO (R2 < 80). However, the relationship TP and fruit peel browning index presents the highest correlation with fruit control and CS/PVA-OA 0mM treatments (R2 < 90), compared to other treatments (R2 < 80). The lowest correlation was observed with CS/PVA-OA 20mM (R2 < 28). It is clear that fruits were coated with CS/PVA-OA 20mM could be imposed a protection for phenolic compounds content and minimized both browning enzyme (PAL and PPO) activities during shelf life. This could be due to the role of CS/PVA to reduce oxidative reaction during fruit ripening (Velickova et al., 2013), or oxalic acid as anti-browning by interacting with browning enzymes (Zheng et al., 2011) In this sense, it is clear that the use of CS/PVA-OA at 20 mM treatment increases cell membrane immunity against the oxidative reaction during shelf life ten days. So, it minimizes cell wall degradation (Table 3) and less accumulation of lipid peroxidation (Table 4), consequently, low cell permeability increasing cell membrane permeability (Table 3). Therefore, the decreases membrane leakage forced browning enzymes to be inactive (Table 2). Then, fruit peel browning index at low index (Table 1). 5. Conclusion The brown color on banana husks is one of the problems that impede the marketing of banana fruits due to the low attractiveness of fruits for the consumer. One of the most important factors affecting the increase of brown color during fruit maturity is the activity of enzymes and cellular cell degeneration, which leads to increased permeability of the plasma membranes of the cells. Thus, browning enzyme activities, which act on phenolic substances, are metabolized with polyphenols. Increasing the activity of these enzymes leads to increase the brown coloration on the peel of the fruit. Therefore, the relationship between the enzymes of decomposition and the termination products of lipid peroxidation (MDA) and the development of the brown color enzymes and the level of brown coloration on the fruits. Finally, we presented that streak browning in ‘Williams’ banana was associated with browning enzyme activities during shelf life by immersing fruit in CS/PVA combined with oxalic acid as anti-browning. We also suggest that the CS/PVA-OA 20mM is very significant impact to minimize the fruit peel browning enzyme activities (PAL and PPO) during shelf life. Also, CS/PVA-OA 20mM treatment decreases the cell wall degradation enzyme activities. So, it protects the phenolic compounds from oxidation.

Fig. 3. correlation between fruit peel browning index in function of the activities of browning enzymes polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and total phenol content (TP) of fruit peel of banana stored at (10 days shelf life) after immersing in CS/PVA loaded with oxalic acid at different concentrations. Symbols represent the different treatment of CS/PVA loaded with oxalic acid: −○− control fruit, −□− CS/PVA, −△− CS/PVA- OA 10mM, −◊− CS/PVA-OA 15mM and −X− CS/PVA-OA 20mM. sloid lines represent linear regression at 5%.

in the MDA content during shelf life (Table 4) which implies membrane oxidation and breakdown. Membrane oxidation alters bilayer stability and increases the membrane permeability (Table 3). The relationship between MDA and cell membrane degrading enzyme activities such as CEL, LOX, and PT, shows that these is an increase in MDA when cell membrane enzyme activities increased during 10 days of shelf life (Fig. 1). Therefore, it seems that the increase of active oxygen species (AOS) formation during shelf life (Hodges et al., 2004) are parallel to increases of cell membrane enzyme activities. These relationships were different depending on the concentration of OA with CS/PVA. All enzymes, CEL, LOX and PT show similar trends during shelf life. While LOX presents less correlation with MDA than CEL and PT more than 90 percent (R2 > 90). Another index of cell injury is ion leakage, which is a commonly used technique to assess cell membrane damage or viability. Ion leakage related to the accumulation of MDA equivalents and as MDA equivalents are formed from cell membrane degrading by increasing enzymes (CEL, LOX, and PT). It could be directly related to increasing ion leakage (Lo’ay, 2005). So, the correlation between cell

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