Impact of Electrostatic Atomized Water Particles Treatment on Chlorophyll Degradation and Delay Ripening in a Thai Banana (Musa × paradisiaca, cv. ‘Namwa’ Banana) during Storage

Abstract

Electrostatic atomized water particles (EAWPs) treatment was applied to investigate its effect on chlorophyll (Chl) degradation and ripening delay in ‘Namwa’ bananas. Banana fruits were pretreated with EAWPs generated from a device (Panasonic F-GMK01) for 0 (control), 0.5, 1.0, 1.5, and 3.0 h in a closed 50 L container, and then kept in perforated polypropylene plastic bags and stored at ambient temperature (25 ± 2°C) under dark conditions. The results showed that 1.0 h-EAWPs treatment best retained peel greenness with a significantly higher hue angle and lower L* value than other treatments on day 6. Also, the 1.0 h-EAWPs treatment maintained the total Chl content, firmness, total soluble solids (TSS), and delayed the ripening index (RI) of fruit accompanied by a delayed climacteric rise in ethylene and respiration rate compared to the control. It was found that the 1.0 h-EAWP treatment induced accumulations of nitric oxide (NO) in peel tissues and suppressed the activities of Chl-degrading enzymes (chlorophyllase, Mg-dechelatase, Chl-degrading peroxidase, and pheophytinase) in the peel. Furthermore, Chl derivatives levels (chlorophyllide a, pheophobide a, 13²-hydroxychlorophyll a, and pheophytin a) were higher in fruits treated with EAWPs than the control fruits. The results suggest that EAWPs technology could be an alternative approach to delay Chl degradation and ripening in ‘Namwa’ bananas.

Introduction

The ‘Namwa’ banana (Musa × paradisiaca) is an important commercial crop in Thailand and is very popular, being consumed locally and internationally (Ploetz et al., 2007; Youryon and Supapvanich, 2016). Recently, the demand for ‘Namwa’ bananas has increased in global markets, including China, Hong Kong, and Japan (Suvittawat et al., 2014), owing to its abundance of vitamin, minerals, and phenolics (Bennett et al., 2010). Banana is a climacteric fruit that is able to produce ethylene after harvest to trigger and accelerate the ripening process (Reid, 1992; Siriboon and Banlusilp, 2004). During postharvest storage, the green color of ‘Namwa’ banana peel turns to yellow quickly, resulting in a short storage-life of less than two days (Salaemae et al., 2021; Seymour et al., 1993). This loss of greenness in horticultural crops occurs with the progress of senescence, resulting from chlorophyll (Chl) degradation (Jitareerat et al., 2015). This problem is a major concern as agricultural produce may ripen before reaching distributors and consumers. Therefore, postharvest techniques are needed to extend the storage life and maintain the quality of agricultural produce. Previous techniques relied on chemical methods to improve the shelf life of banana fruits. However, concern about these chemicals harming human health has encouraged researchers to look into non-chemical treatments as an alternative.

One of the non-chemical treatments is to induce stress in the produce. There are several postharvest stress treatments to suppress senescence and delay Chl degradation in horticultural crops, including heat treatment, irradiation, and others (Lurie, 1998; Sivakumar and Fallik, 2013; Yamauchi, 2015). Ummarat et al. (2011) reported that hot water treatment at 50°C for 10 min efficiently delayed ripening, retarded Chl a and b, total Chl breakdown, and increased antioxidant activity of banana fruit. Similarly, UV-C irradiation delayed yellowing and Chl degradation due to the inhibition of chlorophyllase and Chl-degrading peroxidase activities in banana fruit (Pongprasert et al., 2011). With advancements in technology, electrostatic atomized water particles (EAWPs) have been introduced for agricultural application. EAWPs is a technology that can produce reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, hydrogen peroxide (H₂O₂), nitric oxide (NO), nitrate, and nitrite ions by electrostatic atomization of moisture (Shimokage et al., 2005; Yamauchi et al., 2014). This technology has been applied to preserve the greenness of citrus fruit cultivars such as green Nagato-yuzukichi and green yuzu (Yamauchi et al., 2014). Nomura et al. (2017) reported that EAWPs treatment delayed de-greening of broccoli florets by inhibiting chlorophyll degradation. Similarly, EAWPs treatment of mangosteen fruit suppressed the de-greening of calyx by reducing Chl-degrading enzyme activities and reducing the levels of Chl derivatives (Kaewsuksaeng et al., 2019). Recent research has also found that EAWPs treatment was able to improve the antioxidative system and protect cells from oxidative stress damage resulting in delayed senescence and peel blackening in ‘Namwa’ banana (Salaemae et al., 2021). In addition, EAWPs treatment not only maintained postharvest quality of agricultural produce, but also the accumulation of NO, which effective in controlling postharvest diseases such as gray mold in tomato (Imada et al., 2015). NO acts as an important signaling molecule in plants and plays a role in plant responses to stresses (Lamattina et al., 2003; Salgado et al., 2013); for example, the accumulation of NO increased the Chl content in pea leaves and retarded the Chl degradation in phytophthora-infected potato leaves. (Laxalt et al., 1997). These previous studies led us to hypothesize that EAWPs treatment may induce NO accumulation and consequently reduce Chl degradation in ‘Namwa’ bananas. Moreover, EAWPs treatment may also delay the ripening and maintain the quality of ‘Namwa’ bananas during postharvest storage.

Materials and Methods

Banana fruit samples

Banana fruit (Musa × paradisiaca cv. ‘Namwa’ banana) were harvested at the commercial mature green stage (70% maturity). The fruit were selected based on their uniformity of weight, peel color, and absence of physical damage or any defects due to plant pests. Each banana hand was cut into individual fingers.

Electrostatic atomized water particles (EAWPs) treatment

The banana fruit were divided into five treatment groups and pre-treated with EAWPs at 0.5, 1.0, 1.5, and 3.0 h. Non-treated EAWPs fruits were used as a control. EAWPs treatment was performed according to a method previously described by Salaemae et al. (2021). The fruit were separated from the banana hand into individual fruits. Thirty fruits were placed in a closed container of 50 L and treated with EAWPs. The control fruits were enclosed in the same type of containers without the device. All fruit samples were kept in polypropylene plastic bags (0.03 mm thick, size 200 × 300 mm, with two 5 mm holes) and stored at room temperature (25 ± 2°C) with 80–90% RH under a dark condition for 8 d. During storage, the samples were evaluated for peel color changes (L* value and hue angle) and the optimal EAWPs treatment condition was chosen. From the preliminary study, EAWPs treatment for 1.0 h was selected for further study to be compared with the untreated control. The banana samples were randomly collected to examine any changes in postharvest physiology every other day throughout the storage period. Each treatment consisted of five replications and each replicate contained four fruits.

Peel color and total chlorophyll content

The peel color of banana fruit was measured using a colorimeter (Model NF 777; Nippon Denshoku, Japan) to determine the L* and hue angle values. Three locations were taken from each fruit: the top, middle, and bottom of the banana.

Total chlorophyll content was analyzed according to the method of Moran (1982). Banana peel weighing 0.5 g was extracted in 10 mL of N,N-dimethylformamide and incubated at 4°C in the dark overnight. The extracts were filtered with Whatman No. 1 filter paper. Then, the filtrate was measured at absorbance of 664 and 647 nm using a spectrophotometer (U-2000; Hitachi, Japan) and the results were recorded in terms of g·kg⁻¹FW.

Evaluation of the ripening index (RI)

The ripening stages were determined using a scale of 1 to 7 according to the method of Kader (2005), where stage 1 = hard and green; stage 2 = green with a trace of yellow; stage 3 = more green than yellow; stage 4 = yellow with a green hint; stage 5 = all yellow with a green tip on the crown; stage 6 = all yellow; stage 7 = yellow with brown spots. The ripening index (RI) was calculated using the formula below:

  

$$\mathrm{RI}=\frac{\mathrm{Ʃ}(\text{number of fruit at the corresponding stage}\times \text{ripening stage 1–7})}{\mathrm{Ʃ}\left(\text{number of total fruit}\right)}$$

Determination of fruit firmness and total soluble solids

Fruit firmness was determined using a texture analyzer (TA-XT Plus; Stable Micro Systems, UK). The measurements were taken at the top, middle, and bottom points from each banana fruit with a conical probe (0.45 cm). The mean values were expressed as maximum force (N).

For total soluble solids (TSS), the banana pulp was homogenized with 10 mL of deionized water for 2 min. The homogenate was filtered through a filter cloth. Then, TSS was determined using a digital refractometer (Model PAL-1; Atago, Japan).

Determination of respiration rate and ethylene production

Respiration rates and ethylene production were measured by gas chromatography (GC 2014; Shimadzu, Japan) using a Porapack-Q column (60/80 mesh) equipped with a flame ionization detector (FID). The fruit sample was kept in a 700 mL sealed plastic container and incubated at 25°C for 3 h. Then, a 1 mL sample was withdrawn from the gas headspace and used for carbon dioxide (CO₂) and ethylene determination. The results were calculated and expressed as mg CO₂ kg⁻¹FW·h⁻¹ and μL C₂H₄ kg⁻¹FW·h⁻¹, respectively.

Detection of endogenous nitric oxide (NO)

NO accumulation was analyzed using the NO-sensitive dye Diaminofluorescein-2-diacetate (DAF-2DA) according to the method of Imada et al. (2015). The peels of banana fruit were cut into 1 × 1 mm pieces. The samples were immersed with 25 μM DAF-2DA in 20 mM sodium phosphate buffer (pH 7.2) and kept in a desiccator for 5 min, followed by immediate incubation for 30 min in the dark at room temperature. Then, the banana tissues were observed for fluorescence from diaminotriazolo-fluorescein using a fluorescence microscope (BZ-9000, BIOREVO; Keyence, Japan).

Protein extraction to analyze Chl-degrading enzyme activities

Acetone powder from the peel tissue of banana fruit was prepared according to the method described by Aiamla-or et al. (2014). Chlorophyllase extraction was prepared by suspending acetone powder in 15 mL of 10 mM phosphate buffer (pH 7.0) containing 0.6% CHAPS (3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate). For Mg-dechelatase and Chl-degrading peroxidase extraction, 500 mg acetone powder was suspended in 15 mL of 50 mM phosphate buffer (pH 7.0) containing 50 mM KCl and 0.24% Triton-X 100. For pheophytinase extraction, the acetone powder (500 mg) was suspended in 15 mL of 50 mM Tris–HCl buffer (pH 8.0). Afterwards, the mixture was stirred at 4°C in ice for 1 h and then filtered with two layers of Miracloth. Then, the crude enzyme was centrifuged at 13,000 × g at 4°C for 20 min. The supernatants from each extraction were used as the crude enzyme extract for analysis of Chl-degrading enzyme activities. Also, each extract was used to analyze protein contents according to the method of Bradford (1976).

Analysis of Chl-degrading enzyme activities

Chlorophyllase activity

Chlorophyllase activity was conducted following the method of Aiamla-or et al. (2010). The reaction mixture contained 0.5 mL of 0.1 mM phosphate buffer (pH 7.5), 0.2 mL of 500 μg·mL⁻¹ Chl a acetone solution (100 μg·mL⁻¹), and 0.5 mL crude enzyme. The reaction mixture was incubated at 25°C for 40 min and 4 mL of acetone was added to stop the reaction. Chlorophyllide (Chlide) a was separated by adding 4 mL of hexane. The upper phase contained the remaining Chl a, while the lower phase contained the Chlide a. The enzymes activity was determined by Chlide a formation at 667 nm. The results were calculated and reported as U mg⁻¹ protein.

Mg-dechelatase activity

Mg-dechelatase activity was assessed using the method of Kaewsuksaeng et al. (2006). The reaction mixture contained 0.2 mL of Chlide a, 0.5 mL of 50 mM Tris-HCl buffer (pH 8.0), and 0.2 mL of crude enzyme. The activity was analyzed using a spectrophotometer based on pheophorbide a formation by following the increase in absorbance at 535 nm per min and the result was expressed in terms of U·mg⁻¹ protein.

Chl-degrading peroxidase activity

Chl-degrading peroxidase activity was determined according to the method of Yamauchi et al. (1997) with modifications. The reaction mixture included 0.2 mL of 500 μg·mL⁻¹ Chl a acetone solution, 0.1 mL of 1.0% Triton-X 100, 0.4 mL of enzyme solution, 1.5 mL of 0.1 mM phosphate buffer (pH 5.5), 0.1 mL of 50 mM p-coumaric acid, and 0.1 mL of 0.3% hydrogen peroxide. The Chl-degrading peroxidase activity was determined by the decrease in 1.0 μg Chl a in absorbance at 686 nm per min per mg protein at 25°C.

Pheophytinase activity

Pheophytinase activity was determined according to the method of Schelbert et al. (2009). The reaction mixture contained 0.5 mL of enzyme solution, 0.5 mL of 50 mM Tris-HCl buffer (pH 8.0), and 0.2 mL of pheophytin a. The resultant reaction mixture was incubated in darkness at 25°C for 40 min, and the enzyme reaction was stopped by adding 2 mL of acetone. The sample was analyzed with a high performance liquid chromatography (HPLC) system using a pump (L-7100; Hitachi) with a UV–Visible spectrophotometer (L-7420; Hitachi). The pheophorbide a formation was monitored at 665 nm. The pheophytinase activity was estimated with the standard of pheophorbide a formation (μg) based on the peak area per min per mg protein.

Analyses of Chl and derivatives in banana peel

Preparation of Chl a and resultant derivatives

Standards of Chl derivatives such as Chlide a, pheophytin (Phein) a, and pheophorbide (Pheide) a were obtained from Tama Biochemical (Japan) and Wako Pure Chemical Industries (Japan), respectively. The standard of Phein a was prepared according to the method of Holm-Hansen et al. (1965). Two drops of 0.1 N HCl were added to the Chl a acetone solution (100 μg·mL⁻¹) to obtain Phein a. After that, two drops of 0.1 N NaOH were added to neutralize the solution. Meanwhile, 13²-hydroxychlorophyll a (OHChl a) analysis was conducted as previously described by Kaewsuksaeng et al. (2007). OHChl a was obtained by adding peroxidase (horseradish, Sigma-Aldrich, St. Louis, MO, USA) to a Chl a solution.

Measurement of Chl and derivatives

The peel (2.5 g) was homogenized in 22.5 mL of acetone-HEPES buffer (AHB) solution. The preparation of AHB solution followed the method previously described by Kaewsuksaeng et al. (2019). Subsequently, the homogenate was kept under dark conditions on ice for 5 min, and filtrated through Whatman No. 2 filter paper and a DISMIC filter (0.45 μm, ADVANTEC, Japan). Chl and derivatives were analyzed by using HPLC (L-700 pump + automatic gradient controller; Hitachi) followed by a diode array detector (L-2450; Hitachi) or a UV–vis spectrophotometer (L-7240; Hitachi) at A665 nm. The Chl and derivatives were recorded based on the peak area.

Statistical analysis

The experiments were conducted using a completely randomized experimental design. The data were analyzed using analysis of variance (ANOVA) and performed using SAS software (SAS Institute Inc., Cary, NC, USA). The data are reported as the difference between means, and the means were compared using least significant difference (LSD) at P < 0.05.

Results

Effects of EAWPs on peel color, total Chl content, and fruit ripening

The changes in banana peel color are shown in Table 1. Hue angle and L* values decreased rapidly on day 6 during storage at 25°C. The banana fruit treated with EAWPs for 1.0 h remained green, exhibiting higher hue angle and lower L* value than other treatments. The results showed that treating the banana fruit with EAWPs for 1 h was sufficient to delay peel discoloration compared to other treatments. Thus, EAWPs treatment for 1.0 h was selected for further experiments.

Table 1

Effect of Electrostatic atomized water particles (EAWPs) treatment for 0, (control), 0.5, 1.0, 1.5, and 3.0 h. on the hue angle and L* value in ‘Namwa’ banana storage at 25°C. The average values were calculated by the F-test one-way ANOVA with SE (n = 5). Different superscript letters (a–c) within the same column indicate significant differences between treatments. The asterisks (**) indicate that the value is significantly different from the corresponding control (P < 0.01).

Total Chl content in the peel of banana fruit with or without EAWPs declined during storage. As shown in Figure 1a, the control fruit showed a rapid decrease in total chlorophyll content after two days. However, the total Chl content in fruit treated with EAWPs for 1.0 h remained significantly higher than the control fruit. In addition, the amount of total Chl was positively correlated with the change in peel color.

Fig. 1

Total chlorophyll content (a), ripening index (b), and the appearance of postharvest disease (c) in the peel of ‘Namwa’ bananas treated with EAWPs for 1 h compared with the control during storage at 25°C. The error bar indicates ± SE (n = 5). The statistical significance was determined using the Student’s t-test (**P ≤ 0.01).

Control fruit showed rapid ripening and the peel turned yellow quickly within two days of storage (Fig. 1b, c). However, banana fruit treated with EAWPs for 1.0 h turned yellow gradually and significantly delayed the ripening until day 4 during storage. These results indicated that EAWPs can delay ripening and color changes in banana fruit. Moreover, the bananas treated with EAWPs for 1.0 h did not show any postharvest disease symptoms from the initial day to the end of storage. On the contrary, the control fruit showed symptoms of postharvest disease on day 4 of storage (Fig. 1c). Therefore, the results also suggested that EAWPs treatment for 1.0 h can reduce the occurrence of postharvest disease in ‘Namwa’ bananas.

Effect of EAWPs on ethylene production and respiration rate during ripening

Figure 2a, b show that the untreated fruit had a marked elevation in both ethylene production and respiration rate after harvest, which peaked on day 4 of storage. These elevations are normal in banana as they are categorized as climacteric fruit. However, with EAWPs treatments, ethylene production was inhibited and the respiration rate reduced by 92% (0.007 μL C₂H₄·kg⁻¹·h⁻¹) and 36% (28.55 mg CO₂·kg⁻¹·h⁻¹) compared to the control (0.09 μL C₂H₄·kg⁻¹·h⁻¹ and 44.53 mg CO₂·kg⁻¹·h⁻¹), respectively, during the first two days of storage (Fig. 2a). On day 4 of storage, EAWPs treatment significantly delayed the climacteric increase in ethylene and respiration rate (peaking on day 6 of storage) of ‘Namwa’ banana fruits compared to controls.

Fig. 2

Changes in ethylene production (a) and respiration rate (b) of ‘Namwa’ bananas treated with EAWPs for 1 h compared with the control during storage at 25°C. The error bar indicates ± SE (n = 5). The statistical significance was determined using the Student’s t-test (*P ≤ 0.05; **P ≤ 0.01).

Effect of EAWPs on firmness and total soluble solids (TSS) during ripening

EAWPs treatment had an obvious positive effect on maintaining fruit firmness. The 1.0 h-EAWPs treatment markedly delayed fruit softening; the fruit firmness dropped slightly within six days, and decreased quickly thereafter (Fig. 3a). On the other hand, control fruit softened rapidly with 6-fold lower fruit firmness than EAWPs-treated fruit measured on the 6^th day of storage. Banana fruit softening was correlated with the change in TSS. The TSS concentration of fruit in both control and treated fruits increased during storage (Fig. 3b). Nonetheless, the EAWPs-treated fruits recorded significantly lower TSS than control fruits. In fact, the TSS concentration in control fruit increased markedly after two days of storage, but the fruit treated with EAWPs increased gradually after four days of storage. This indicated that the TSS concentrations increased with fruit ripening and softening.

Fig. 3

Changes in firmness (a) and total soluble solids (a) in ‘Namwa’ bananas treated with EAWPs for 1 h compared with the control during storage at 25°C. The error bar indicates ± SE (n = 5). The statistical significance was determined using the Student’s t-test (*P ≤ 0.05; **P ≤ 0.01).

Effect of EAWP on the accumulation of NO in peel tissues of banana fruit

The NO-sensitive fluorescence dye DAF-2DA was used to detect NO that had accumulated in the untreated and EAWPs-treated banana peel. The intensity of green fluorescence is indicative of the amount of NO accumulation. A more intense green fluorescence in the EAWPs-treated banana peel than in the control (Fig. 4) was found on the initial day (day 0). The results suggested that EAWPs treatment induced NO accumulation in banana fruit during the initial storage day. However, the intensity of green fluorescence of the EAWPs-treated fruit decreased after that. In control fruit, the green fluorescence intensity of banana peel increased from two days after storage, indicating a higher NO accumulation than EAWPs during storage.

Fig. 4

Accumulation of nitric oxide (denoted by arrows) in ‘Namwa’ banana peel with diamino fluorescein diacetate (DAF-2DA) after treatment with EAWPs for 1 h compared with untreated fruit (control) during storage at 25°C.

Effects of EAWPs on chlorophyll-degrading enzyme activities in peel of banana fruit

The high amount of total Chl content in the fruit treated with EAWPs was correlated with a low activity of chlorophyll-degrading enzymes, as shown in Figure 5. In control fruit, Chl-degrading enzymes such as chlorophyllase, Mg-dechelatase, Chl-degrading peroxidase, and pheophytinase increased with storage period (Fig. 5). Nevertheless, the activities of these enzymes were always significantly lower in the fruit treated with 1.0 h-EAWPs than the control fruit throughout the storage days at 25°C.

Fig. 5

Changes in enzyme activities of Chlorophyllase (a), Mg-dechelatase (b), Chl-degrading peroxidase (c), and pheophytinase (d) of ‘Namwa’ bananas with or without treatment with EAWPs for 1 h during storage at 25°C. The error bar indicates ± SE (n = 5). The statistical significance was determined using the Student’s t-test (**P ≤ 0.01).

Effect of EAWPs on Chl derivatives in peel of banana fruit

Figure 6 shows the changes in Chl derivative levels in untreated and EAWPs-treated banana fruit during storage at 25°C. All Chl derivative levels in control fruit disappeared completely after four days of storage in control fruits (not detectable on day 6 and 8). A similar trend was observed for Chlide a, OHChl a, and Phein a, which showed a marked reduction on day 2 and 4, while fruits treated with EAWPs recorded higher levels than control fruit (Fig. 6a, c, d). As for Pheide a, the level increased on day 2 and decreased after that (Fig. 6b). The highest level was on day 2 in fruit treated with EAWPs and was always significantly higher than in control fruit. In the control, Pheide a decreased after four days of storage.

Fig. 6

Changes in chlorophyll derivatives levels of chlorophyllide a (a), pheophobide a (b), 13²-Hydroxy chlorophyll a (c), and pheophytin a (d) of ‘Namwa’ bananas with or without treatment with EAWPs for 1 h during storage at 25°C. The error bar indicates ± SE (n = 5). The statistical significance was determined using the Student’s t-test (**P ≤ 0.01).

Discussion

Banana fruits are subjected to continuous changes after harvesting and during transportation. There are several major problems affecting banana quality, including physical damage, decay, and ripening with loss of green color, especially during long-distance transportation. Due to these problems, stress treatments are used to maintain fruit quality and extending shelf life. Treatments using physical techniques such as hot water treatment, UV-C irradiation treatment, and precooling treatment have been evaluated to delayed ripening and inhibition chlorophyll degradation in banana fruit (Pongprasert et al., 2011; Ummarat et al., 2011; Zhang et al., 2010). Although physical stress treatments have been used to overcome these problems, there are newer technologies (i.e., EAWPs) which not only address the ripening problem, but also postharvest diseases.

EAWPs technology is known to be an effective treatment to suppress de-greening and delay senescence in stored horticultural crops (Kaewsuksaeng et al., 2019; Nomura et al., 2017; Salaemae et al., 2021; Yamauchi et al., 2014). Our study showed that 1.0 of EAWPs treatment was the best in terms of maintaining the peel color by delaying changes in hue angle and L* values in banana peel (Table 1) compared with those of the control. However, a previous report showed that 1.0 h-EAWPs treatment generated H₂O₂ at an average of 0.13 mg·L⁻¹, which is the optimum concentration to regulate the antioxidation system that led to delayed senescence in ‘Namwa’ banana (Salaemae et al., 2021). Also, H₂O₂ formed by 1.0 h-EAWPs treatment induced catalase (CAT), which could be related to the delay in the degreening of broccoli (Nomura et al., 2017). In this study, the effectiveness of 1.0 h-EAWPs treatment to inhibit postharvest ripening was supported by a reduction in chlorophyll degradation as shown in Figure 1a. Chlorophyll breakdown is commonly a visual indication of ripening in bananas. The high chlorophyll content in EAWPs treatment corresponded with the delay in ripening in ‘Namwa’ bananas (Fig. 1b, c). Our results agreed with previous studies which reported that the degree of fruit ripening was related to the loss of chlorophyll content in banana, mango, and papaya (Wang et al., 2009; Wu et al., 2018). Therefore, retarding chlorophyll breakdown is one of the most important factors to slow down the ripening of ‘Namwa’ bananas during transportation.

Normally, fruit ripening is associated with an increase in the respiration rate and ethylene production (Johnson et al., 1997). The action of ethylene results in softening of the fruit and conversion of starch to sugars, acceleration of deterioration, and shortening of postharvest shelf life (Saltveit, 1999). Our results indicated that EAWPs treatments suppressed ethylene production and the respiration rate in banana fruits during storage at 25°C as compared to the control (Fig. 2a, b). Furthermore, we observed that EAWPs treatment for 1 h maintained the firmness and TSS of ‘Namwa’ bananas during storage (Fig. 3a, b). This suggests that EAWPs delay the ripening process by mediating reductions in respiration and ethylene production. Previous studies have reported that the EAWPs effectively suppressed ethylene formation and the respiratory rate, and delayed color changes in broccoli florets after treatment with EAWPs (Kaewsuksaeng et al., 2015; Ma et al., 2012). In addition, EAWPs-mediated reductions in respiration and ethylene production resulted from the role of ROS, especially H₂O₂, which acts as a signaling molecule in cell metabolism as reported in broccoli (Kaewsuksaeng et al., 2015; Ma et al., 2012; Nomura et al., 2017).

EAWPs treatment also works by inducing the antioxidant system of banana fruits. Previous research has shown that EAWPs suppressed senescence and minimized the degradation of chlorophyll in broccoli and citrus (Nomura et al., 2017; Yamauchi et al., 2014). The reduction in chlorophyll degradation in broccoli could be explained by the H₂O₂ signaling of molecules in cell metabolism that controls the de-greening process. Interestingly, this study found NO accumulation in EAWPs-treated ‘Namwa’ bananas just after treatment, and this decreased gradually with increased storage days (Fig. 4). This finding indicated that the EAWPs increased the NO content, which may enhance antioxidant enzyme activities and reduce the oxidative damage to the biological membranes in banana fruits (Wang et al., 2015). EAWPs treatment was reported to induce catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD), which are important enzymes that are involved in the antioxidant system (Nomura et al., 2017). Therefore, it is possible that the role of NO in chlorophyll degradation may act partially through its effect on enhancing the whole antioxidant system in banana fruits. NO has been noted as an antisenescence signal that can extend the postharvest life of fruits and vegetables (Leshem et al., 2001). Besides that, NO treatment delayed the yellowing process and retarded the onset of chlorophyll degradation in broccoli florets during storage at 20°C (Eum et al., 2009). Consequently, EAWPs treatment could suppress the activities of chlorophyllase, Mg-dechelatase, pheophytinase, and Chl-degrading peroxidase, which can be seen in the lower activity in the peel of the treated fruit than in the control fruit (Fig. 5). Thus, the delay in peel-yellowing in ‘Namwa’ bananas may be due to the inhibition of Chl-degrading enzymes by EAWPs. A similar result was reported in EAWPs-treated mangosteen, with observed reductions in Chl-degrading enzyme activity and delayed fruit ripening (Kaewsuksaeng et al., 2019). It has been reported that Chl a and b contents in horticultural crops decreased with senescence during storage (Gross, 1991). Here, high derivative levels of Chlide a, Pheide a, OHChl a, and Phein a were detected in the peel of EAWPs treated ‘Namwa’ banana fruits during storage (Fig. 6). On the other hand, the control fruit had low Chl derivatives levels during the storage period, suggesting that the activities of Chl-degrading enzymes amy be effectively suppressed by EAWPs treatment.

In addition, NO production induced stomata closure by way of abscisic acid action (Murata et al., 2015). The stomata closure resulted in a reduction in respiration, transpiration, and water loss (Shinozaki and Yamaguchi-Shinozaki, 2007), thus maintaining the stored quality of the banana fruits. NO is known as an essential signaling molecule that regulates diverse defense responses of plants to biotic stress. Accumulating evidence demonstrates that NO-mediated disease resistance involves protein S-nitrosylation and the activation of the salicyclic acid signaling pathway (Hong et al., 2008). Our results showed that postharvest disease in ‘Namwa’ bananas was reduced by EAWPs for 1 h (Fig. 1c), suggesting the action of NO in enhancing resistance to senescence and diseases. These results agreed with Imada et al. (2015) who found that EAWPs-induced NO plays a role in the defense responses and against gray mold disease in tomato plants.

Conclusions

The present study results showed that 1.0 h-EAWPs could delay changes in hue angle and L* values, and total chlorophyll content in ‘Namwa’ bananas. The treatment with 1.0 h-EAWPs suppressed the ripening process by reducing ethylene production and respiration rates, and maintaining firmness and TSS. Also, the EAWPs-induced accumulation of NO in cells immediately after the treatment may enhance the capacity of the antioxidative system to delay chlorophyl degradation by suppressing chlorophyll-degrading enzyme activities and maintaining the level of several chlorophyll derivatives.

Acknowledgements

We also thank the Postharvest Technology Innovation Center, Ministry of Higher Education, Science, Research and Innovation, Thailand, and Graduate School of Science and Technology for Innovation, Yamaguchi University, Japan for the facilities provided to conduct our study.

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