Postharvest Biology and Technology 84 (2013) 2227

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Postharvest Biology and Technology

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Effect of oxalic acid on ripening attributes of banana fruit during storage

Hua Huang a,b , Guoxing Jing c , Lifang Guo d , Dandan Zhang a , Bao Yang a , Xuewu Duan a , Muhammad Ashraf e , Yueming Jiang a,
a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, Peoples Republic of China b Graduate University of Chinese Academy of Sciences, Beijing 100039, Peoples Republic of China c College of Food Science and Biotechnology, Zhejiang Gongshang University, Food Safety Key Laboratory of Zhejiang Province, Hangzhou 310035, Peoples Republic of China d Department of Bioengineering, Jinan University, Guangzhou 510632, Peoples Republic of China e Department of Botany, Faculty of Sciences, University of Agriculture, Faisalabad 38040, Pakistan

a r t i c l e

i n f o

a b s t r a c t
The effect of exogenous oxalic acid treatment on ripening attributes of banana fruit during storage was investigated. Banana fruit were dipped into solutions of 0 (control) or 20 mM oxalic acid for 10 min and then stored at room temperature (23 2 C) and 7590% relative humidity. The application of oxalic acid reduced fruit deterioration during storage. The oxalic acid treatment also reduced the rates of respiration and ethylene production, and delayed the decreases in rmness, hue angle, and maximal chlorophyll uorescence (Fv/Fm) of banana fruit during storage. Furthermore, fruit treated with oxalic acid exhibited higher superoxide dismutase activity and antioxidant capability with a lower production of reactive oxygen species at the late storage period compared with non-oxalic acid-treated fruit. Overall, the oxalic acid treatment was effective in inhibiting postharvest ripening of banana fruit and exhibited the potential for commercial application to store the bananas at room temperature. It can be concluded that the delay in banana fruit ripening associated with oxalic acid treatment could be due to inhibition of respiration and ethylene production rates, and reduction of oxidative injury caused by reactive oxygen species through increased antioxidant activity. 2013 Elsevier B.V. All rights reserved.

Article history: Received 17 August 2012 Accepted 4 April 2013 Keywords: Banana Fruit Oxalic acid Ripening Antioxidant activity

1. Introduction Banana fruit after harvest undergo a rapid softening progress, resulting in a short shelf life (Menniti et al., 2004). Banana handling, transportation, ripening and marketing typically involve the use of sophisticated technologies and facilities (Jiang et al., 1999). Technologies such as fast cooling and temperature control at approximately 13 C give a postharvest shelf life of 23 weeks. Though storage at low temperature (<13 C) delays fruit ripening and extends the postharvest life of banana fruit, the benecial effects are limited by the development of chilling injury-associated disorders, including skin browning and failure of the fruit to soften (Jiang et al., 2004). Especially in developing countries, there is a need for alternative non-sophisticated technologies for green life extension at ambient temperature so subsistence producers can transport green banana fruit long distances without refrigeration. Investigations have documented that some fruits experience postharvest deterioration due to the imbalance between the

Corresponding author. Tel.: +86 20 37252525; fax: +86 20 37252831. E-mail addresses: ymjiang@scbg.ac.cn, ymjiang@scib.ac.cn (Y. Jiang). 0925-5214/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.04.002

amount of reactive oxygen species (ROS) produced and the scavenging ROS capacity, which resulted in enhanced lipid peroxidation and membrane injury and reduced resistance of these fruits to pathogens (Macarisin et al., 2010). Oxidation injury caused by ROS is an important factor leading to quality deterioration by affecting cell wall disassembly in pulp tissues of banana fruit (Cheng et al., 2008). Excessive production of ROS might result in loss of compartmentalization of litchi fruit between enzymes and their substrates, which was responsible for enzymatic browning (Sun et al., 2012). Therefore, developing postharvest handling to inhibit ROS production and decrease lipid peroxidation could be important for reducing quality deterioration and extending the storage life of banana fruit. Recently, the application of oxalic acid (OA) to harvested fruit has received much attention. As an organic acid present in various organisms (Kostman et al., 2001; Munir et al., 2001), oxalic acid has shown some antioxidant activities (Zheng et al., 2007) and, thus, could play an important role in systemic resistance, programmed cell death, redox homeostasis in plants, and an anti-senescence effect in harvested fruits (Ding et al., 2007; Wu et al., 2011). Zheng et al. (2011) reported that postharvest oxalic acid treatment could be a promising method to suppress quality deterioration and

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extend the storage shelf-life of mango fruit. In addition, the organic acid can delay jujube fruit senescence by reducing the ethylene production rate, repressing fruit reddening, and decreasing alcohol content (Wang et al., 2009). Unfortunately, little information on the effect of oxalic acid on ripening and storage life of harvested banana fruit has been available. The objective of the present study was to investigate the role of oxalic acid in postharvest ripening of banana fruit and to examine its effects on physiological activity and antioxidant systems during storage. The study should be helpful for further development of commercial postharvest technology for quality maintenance and shelf life extension of banana fruit. 2. Materials and methods 2.1. Fruit materials and treatments Fruit of banana (Musa spp., AAA group, cv. Brazil) were harvested at 8090% maturity from a commercial orchard in Guangzhou and then cut into ngers. Fruit ngers were dipped in water to clean the surfaces. They were then air-dried until visible moisture on fruit surfaces disappeared completely. The fruit were selected for uniformity of weight and shape and for lack of visual defects. In a small-scale experiment, the optimum concentration of oxalic acid that affected postharvest ripening of banana fruit was assessed. Oxalic acid at 0 (control), 5, 10 or 20 mM was applied to evaluate the green life of harvested banana fruit at room temperature (23 2 C). During storage, the simple green life was evaluated rapidly based on the changes in fruit rmness and skin color. Results showed that immersion into 20 mM oxalic acid for 10 min was the best (supplementary Fig. 1) in terms of the lowest changes in fruit rmness (softening) (supplementary Fig. 2) and skin color (supplementary Fig. 3 Fig. 3). Thus, this treatment was used for further experiments. In the experiment to examine the effect of oxalic acid on ripening attributes of banana fruit during storage, the selected fruit were divided into two groups. One group was immersed in distilled water for 10 min as control while the other group was dipped into 20 mM oxalic acid for 10 min. The control and treated fruit were air-dried, then packed into plastic polyethylene bags (0.03 mm), and stored at room temperature (23 2 C) and 7590% relative humidity (RH). Ten ngers selected randomly from each treatment every 4 d were used for measurements of ethylene production and respiration rates, followed by evaluations of fruit rmness, hue angle, and photochemical efciency of photosystem II (PSII and Fv/Fm). Another ten ngers from each treatment every 4 d were peeled and the peel tissues were collected, sliced, frozen in liquid N2 and stored at 20 C, prior to measurements of superoxide dismutase (SOD) activity, ROS level, and antioxidant capability. 2.2. Determination of fruit rmness Fruit rmness was determined using a penetrometer (Model GY-3, Zhejiang Scientic Instruments, Zhejiang, China) that measured the force required to penetrate into the fruit. Ten fruit ngers were withdrawn randomly from each treatment. Each nger was measured at three equidistant points around the middle position of the fruit with a at probe. The results were expressed as N. 2.3. Measurements of color and chlorophyll uorescence Fruit color was determined using a Minolta Chroma Meter CR400 (Minolta Camera Co. Ltd., Osaka, Japan) by the method of Chen et al. (2008), with a few modications. Ten fruit ngers withdrawn randomly from each treatment were used for determination of peel color. Each fruit nger was measured at three equidistant points

around the middle position of the fruit surface. Color was recorded using the CIE L*, a* and b* scales. L denotes the lightness or darkness and a* indicates green to red color while b* shows blue to yellow color. Numerical values of L*, a* and b* were converted into the hue angle [h0 = tan1 (b*/a*)]. Chlorophyll uorescence was determined using a portable chlorophyll uorometer (FAM 2100, Walz, Germany) (Wu et al., 2011). Ten fruit ngers withdrawn randomly from each treatment were dark-adapted for at least 30 min prior to measurement. The photochemical efciency of PSII was determined and expressed as the ratio of the maximum variable uorescence to the maximum yield of uorescence (Fv/Fm). 2.4. Measurements of ethylene production and respiration rates Fruit ngers withdrawn randomly from each treatment and three ngers were sealed in a 2.6 L plastic container and then held for 2 h at 25 C. The fruit in each treatment were divided into three replicate groups to measure the ethylene production rate. One milliliter headspace gas was withdrawn from the container and then injected into a gas chromatograph (GC-2010; Shimadzu, Kyoto, Japan) equipped with a 30 m HP-PLOT Q capillary column (Agilent Technologies, USA) and a ame ionization detector (FID). Ethylene production rate was expressed on a fresh weight basis as g kg1 s1 . Respiration rate was determined by the methods with a LI-6262 CO2 /H2 O Analyzer (LI-6262, LI COR, America) (Huang and Jiang, 2012). Fruit ngers withdrawn randomly from each treatment and then three ngers were sealed into a box that connects the LI6262 CO2 /H2 O Analyzer before the amount of CO2 was recorded for 4 min. The fruit in each treatment were divided into three replicate groups to measure the respiration. Respiration rate was expressed on a fresh weight basis as rate of CO2 production on a fresh weight basis as mg kg1 s1 . 2.5. Assay for superoxide dismutase activity Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by the method of Duan et al. (2011). Fresh peel tissues (2.0 g) from ten ngers withdrawn randomly from each treatment were homogenized with 10 mL of 50 mM sodium phosphate buffer (pH 7.0) containing 0.3 g insoluble polyvinylpolypyrrolidone (Sigma, St. Louis, MO) and then centrifuged at 15,000 g for 45 min (Sigma 3K15, Instrument Co. Ltd., Germany). The supernatant was collected to assay enzymatic activity. All steps in the preparation of enzymatic extract were carried out at 4 C. Enzyme extract (50 L) was mixed with 3 mL of reaction buffer solution consisting of 1.3 M riboavin, 13 mM methionine, 63 M nitro blue tetrazolium (NBT) and 0.66 mM EDTA-Na2 in 0.05 M sodium phosphate buffer (pH 7.0). The reaction was started under 4000 lx irradiance at 25 C for 30 min. A non-irradiated reaction mixture that did not develop color was used as the control. One unit (U) of SOD activity was dened as the amount of enzyme required to cause a 50% inhibition of the reduction of NBT as monitored at 560 nm. 2.6. Determinations of superoxide anion generation rate and H2 O2 content Superoxide anion generation (O2 ) rate was measured according to the method of Song et al. (2009), with a few modications. Fresh peel tissues (2.0 g) from ten ngers withdrawn randomly from each treatment were homogenized with 10 mL of extract solution consisting of 1 mM EDTA, 0.3% Triton X-100 and 2% insoluble polyvinylpolypyrrolidone, and then centrifuged at 12,000 g for 30 min at 4 C. The extract solution (1 mL) was mixed with 1 mL of 1 mM hydroxylammonium chlroride, and then incubated for

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Flesh firmness (N)

30 min at 25 C. One milliliter of the incubated solution was added to 1 mL of 17 mM 3-aminobenzenesulfonic acid (Sigma, USA) and 1 mL of 7 mM 1-naphthylamine (Sigma, USA) for further incubation for 20 min at 25 C. The absorbance of the solution was monitored at 530 nm. A standard curve with NO2 was used to calculate the O2 production rate from the reaction equation of O2 with hydroxylamine. Superoxide anion generation rate was expressed on a fresh weight basis as nmol kg1 s1 . H2 O2 content was determined by the previous method of Cheng et al. (2007). Fresh peel tissues (2.0 g) from ten ngers withdrawn randomly from each treatment was homogenized with 10 mL of cold acetone, and then centrifuged at 12,000 g for 30 min at 4 C. One milliliter of the extract solution was mixed with 0.1 mL of 5% titanium sulphate and 0.2 mL ammonia, and then centrifuged at 12,000 g for 10 min at 4 C. The pellets were dissolved into 3 mL of 10% (v/v) sulfuric acid solution, and then centrifuged at 12,000 g for 10 min. The absorbance of the supernatant was measured at 420 nm. The known concentrations of H2 O2 were used to make a standard curve. The H2 O2 content was expressed on a fresh weight basis as mmol kg1 . 2.7. Measurement of malondialdehyde Malondialdehyde (MDA) measurement was carried out by the method of Heath and Packer (1968), with a slight modication. Fresh peel tissue (2.0 g) from ten ngers was homogenized in 10 mL of 10% trichloroacetic acid, and centrifuged at 10,000 g for 20 min. The supernatant was collected, and 1 mL of it was mixed with 3 mL of 0.5% thiobarbituric acid. The mixture was boiled for 15 min, then quickly cooled in an ice bath and centrifuged at 12,000 g for 15 min. The supernatant was collected and used to measure absorbance at 450, 532, and 600 nm. The MDA concentration was calculated according to the formula: 6.453 (A532 A600) 0.563 A450. The concentration of MDA on a fresh weight basis was calculated mmol kg1 . 2.8. DPPH radical scavenging activity The DPPH radical scavenging activity was evaluated by the method of Gorinstein et al. (2011). Initially, fresh peel tissue (3.0 g) from ten ngers withdrawn randomly from each treatment was mixed with 30 mL methanol and then centrifuged at 10,000 g for 15 min. The supernatant (0.1 mL) was mixed with 2.9 mL of 0.1 mM DPPH that dissolved in methanol. The reaction mixture was incubated for 20 min at 25 C in the dark. The control containing all reagents without the sample and was used as blank. The DPPH radical scavenging activity was determined by measuring 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. 2.9. Determination of superoxide anion radical scavenging activity Superoxide anion radical scavenging activity was analyzed by the method described previously (Duan et al., 2007), with a slight modication. Fresh peel tissue (2.0 g) from ten ngers withdrawn randomly from each treatment was mixed with 20 mL methanol, then centrifuged at 10,000 g for 15 min and nally, the supernatant was collected. The reaction mixture contained 0.5 mL of 0.05 M phosphate buffer (pH 7.0), 0.3 mL of 50 M riboavin, 0.3 mL of 20 mM methionine, and 0.3 mL of 0.51 mM nitro blue tetrazolium, prior to addition of 1 mL of sample solution. The reaction was started by illuminating the reaction mixture with a uorescent lamp. After 20 min of incubation, the absorbance was measured at 560 nm with a spectrophotometer. The reaction mixture without

sample was used as the control. The superoxide anion radical scavenging activity (%) was calculated as (1 absorbance of sample/absorbance of control) 100. 2.10. Determination of hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was assayed by the method of of Siddhuraju and Becker (2007). Fresh peel tissue (2.0 g) from ten ngers withdrawn randomly from each treatment was mixed with 20 mL methanol and then centrifuged at 10,000 g for 15 min. The collected supernatant (1 mL) was mixed with 1 mL reaction buffer containing 100 M ferric chloride, 104 M EDTA, 2.5 mM H2 O2 , 2.5 mM 2-deoxyribose and 100 M ascorbic acid. The reaction mixture was incubated for 1 h at 37 C. A solution of thibabituric

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Fig. 1. Changes in esh rmness (A), hue angle (B) and chlorophyll uorescence (Fv/Fm) (C) of banana fruit treated with oxalic acid at 0 ( , control) and 20 mM () for 10 min during storage at 25 C and 75 90% relative humidity. Data were presented as the means standard errors (n = 10). The least signicant difference (LSD) (P = 0.05) was calculated for mean separation.

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acid (TBA) in 1 mL (0.5%; w/v) of 25 mM NaOH solution and 1 mL 2.8% (w/v) trichloroacetic acid (TCA) was added. The mixture was heated for 30 min at 80 C and then cooled rapidly. The absorbance was measured at 532 nm with a spectrophotometer. The methanol instead of the sample was used as the control. The hydroxyl radical scavenging activity (%) was calculated as (1 absorbance of sample/absorbance of control) 100. 2.11. Statistical analysis These experiments were arranged in completely randomized design. Data were present as the means and standard errors (SE). The least signicant differences (LSDs) (P = 0.05) were calculated for mean separation. 3. Results and discussion

Chlorophyll uorescence is an indirect measurement of the color in chlorophyll-containing plant tissues. When proteinchlorophyll complexes are degraded with fruit ripening and senescence, a marked change in the chlorophyll uorescence will occur. In the present study, the fruit treated with oxalic acid had a slower change in the hue angle compared to control fruit (Fig. 1B). The chlorophyll uorescence parameter Fv/Fm exhibited a similar trend (Fig. 1C). Furthermore, as fruit rmness was less than 38.26 1.36 N, the hue angle and Fv/Fm values declined rapidly. After 24 d of storage, the hue angle and Fv/Fm ratio of the control fruit decreased from 115.44 0.95 to 88.62 2.33 and from 0.76 0.01 to 0.29 0.07, respectively, which might imply reduced chloroplast activity due to the partial senescence, while the oxalic acid-treated fruit

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3.1. Effect of oxalic acid on fruit rmness, color and chlorophyll uorescence It has been reported that the formation of oxalate-soluble pectin was related to fruit rmness of tomato and banana during storage (Guillon et al., 2008; Emaga et al., 2008). In the present study, banana fruit began to soften after 12 d of storage and the oxalic acid treatment markedly slowed down fruit softening, as shown in Fig. 1A. Color and chlorophyll uorescence could be expressed as the hue angle (Alkarkhi et al., 2011) and Fv/Fm ratio (Wu et al., 2011).

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-1 -1 g kg s )

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Fig. 3. Changes in superoxide anion generation rate (A) and contents of hydrogen peroxide (B) and MDA (C) in peel tissues of banana fruit treated with oxalic acid at 0 ( , control) and 20 mM () for 10 min during storage at 25 C and 7590% relative humidity. Data presented are the means standard errors (n = 3). The least signicant difference (LSD) (P = 0.05) was calculated for mean separation.

Days of storage
Fig. 2. Changes in rates of ethylene production (A) and respiration (B) of banana fruit treated with oxalic acid at 0 ( , control) and 20 mM () for 10 min during storage at 25 C and 7590% relative humidity. Data are the means standard errors (n = 3). The least signicant difference (LSD) (P = 0.05) was calculated for mean separation.

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Superoxide anion savenging activity (%)

exhibited hue angle of 104.23 2.49 and the Fv/Fm ratio of 0.44 0.04. Some studies have shown that the decline in chlorophyll uorescence during fruit ripening of apple, banana, mango, jujube, and papaya was related to loss of chlorophyll content and chloroplast competence (Wang et al., 2009; Wu et al., 2011). The results further indicated that the oxalic acid treatment could delay a decline of chlorophyll uorescence and reduce the change in skin color of banana fruit during storage. 3.2. Effect of ethylene production and respiration rates Oxalic acid treatment not only inhibited the ethylene production rate but also delayed the time to the peak ethylene production rate of banana fruit during storage (Fig. 2A). In the control fruit, a peak of 0.075 g kg1 s1 occurred after 16 d of storage, while no obvious peak of ethylene production rate appeared in the oxalic acid-treated fruit by the end of storage. Similarly, the respiration rate of banana fruit during storage was inhibited by oxalic acid treatment (Fig. 2B). Furthermore, the oxalic acid treatment delayed the time to the respiration peak of banana fruit during storage. Banana fruit shows a typical climacteric characteristic during storage. The peaks of ethylene production and respiration rates almost appeared at the same time (Jiang et al., 1999). The previous study found that oxalic acid treatment reduced the respiration rate of peach fruit and inhibited the ethylene production rate in mango and plum fruits (Ding et al., 2007; Wu et al., 2011; Zheng et al., 2007). The mechanism might be due to the inhibition of 1aminocyclopropane-1-carboxylic acid synthase activity by oxalic acid (Wang et al., 2009) as the enzyme is a key regulatory enzyme of ethylene biosynthesis. 3.3. Effects of oxalic acid on ROS level and lipid peroxidation Overall, the superoxide anion generation rate in control banana fruit increased during storage. The oxalic acid-treated fruit exhibited a signicantly (P = 0.05) lower level of superoxide anion generation rate in peel tissues of banana fruit by the end of storage compared with the control fruit (Fig. 3A). Similarly, the oxalic acid treatment markedly reduced H2 O2 content of banana fruit at the later storage (Fig. 3B). Fig. 3C presents the inhibition of lipid peroxidation of banana fruit by exogenous oxalic acid treatment, which was associated with lower superoxide generation rate and H2 O2 content. As ROS can induce the formation of several oxidative

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Fig. 5. Changes in scavenging activities of superoxide anion radical (A), hydroxyl radical (B) and DPPH radical (C) in peel tissues of banana fruit treated with oxalic acid at 0 ( , control) and 20 mM () for 10 min during storage at 25 C and 7590% relative humidity. Data presented are the means standard errors (n = 3). The least signicant difference (LSD) (P = 0.05) was calculated for mean separation.

3 -1 SOD activity (x10 U kg )

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byproducts such as MDA (Duan et al., 2011), imbalance between ROS production and the ROS scavenging capacity enhanced lipid peroxidation and membrane injury, which could account for accelerated fruit ripening and senescence (Marangoni et al., 1996; Wang et al., 2004).
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Days after storage

Fig. 4. Change in SOD activity in peel tissues of banana fruit treated with oxalic acid at 0 ( , control) and 20 mM () for 10 min during storage at 25 C and 7590% relative humidity. Data presented are the means standard errors (n = 3). The least signicant difference (LSD) (P = 0.05) was calculated for mean separation.

3.4. Superoxide dismutase activity and radical scavenging capability Superoxide anion is involved in the formation of free radicals (Apel and Hirt, 2004) while superoxide dismutase as a major antioxidant enzyme in cells plays a protective role against superoxide

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anion generation (Mittler, 2002). The enzyme can break superoxide anion into molecular oxygen and hydrogen peroxide (Lushchak, 2011). Thus, the antioxidant capability includes SOD activity and radical scavenging activity. Figs. 4 and 5 present SOD activity and radical scavenging activities in banana fruit during storage. Generally, the SOD activity and scavenging activities of superoxide anion and hydroxyl radicals in peel tissue of the oxalic acid-treated banana fruit at the later storage stage was at a higher level than in the control fruit. ROS, such as superoxide anion and H2 O2 , are mainly generated in the chloroplasts, mitochondria, peroxisomes, and apoplast during photosynthesis, respiration, and other physiological processes involving electron transfer (Tovar-Mendez et al., 2011). It has been found that oxalic acid can induce the programmed cell death response in plant tissues by increasing ROS level (Kim et al., 2008). Some evidence also shows that oxalic acid has a potential induction effect on antioxidant systems, and the resistance against pathogen (Wang et al., 2009). Thus, the low superoxide anion generation rate and H2 O2 content and high radical scavenging activity of the oxalic acid-treated banana fruit during the later storage suggested that the oxalic acid treatment reduced the injury caused by ROS and delayed fruit ripening and senescence. In conclusion, the results obtained in this study provide evidence of the ability of oxalic acid to maintain peel appearance and to extend the green shelf life of banana fruit during storage at room temperature and exhibited the potential for commercial application to store the bananas at ambient temperature. Together with other studies (Whangchai et al., 2006; Zheng and Tian, 2006), it is suggested that the oxalic acid treatment is a safe and promising postharvest handling technology for maintaining quality and prolonging storage life of harvested banana fruit. Further work is needed to understand better the mechanism by which oxalic acid delays fruit ripening at the molecular level. Acknowledgments This work was supported by the National Key Basic Research Program of China (No. 2013CB127100), the National Key Technologies R&D Program (grant no. 2010BAD22B01), Science and Technology Planning Project of Guangdong Province, China (grant no. 2009B040600004) and the National Natural Science Foundation of China (grant no. 30972071). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.postharvbio.2013.04.002. References
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