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Enhanced chilling tolerance of banana fruit treated with malic acid prior to low-temperature storage
Postharvest Biology and Technology 111 (2016) 209–213
Contents lists available at ScienceDirect
Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Enhanced chilling tolerance of banana fruit treated with malic acid prior to low-temperature storage Hua Huanga,* , Qijie Jiana,b , Yueming Jianga , Xuewu Duana , Hongxia Qua,* a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China b University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 November 2014 Received in revised form 10 September 2015 Accepted 10 September 2015 Available online xxx
The effects of postharvest malic acid (MA) treatment on alleviating the occurrence of chilling injury (CI) symptoms in banana (Musa spp., AAA group, cv. Brazil) fruit under 6 C were evaluated. Application of 80 mM MA alleviated CI symptoms (surface browning), delayed the decrease in chlorophyll fluorescence (Fv/Fm) and chlorophyll content. The activities of peroxidase (POD) and polyphenol oxidase (PPO) were also suppressed by MA. Furthermore, compared with the control group, fruit that were treated with MA showed lower levels of reactive oxygen species, but higher antioxidant activities. The results suggest that the application of MA, as an organic acid, exhibited the potential for alleviating chilling injury symptoms of banana fruit by reducing skin browning and inducing antioxidant activities under low temperature. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Banana Chilling injury Malic acid Antioxidant activity
1. Introduction Banana is highly sensitive to storage temperatures below 13 C that induce chilling injury (CI) of fruit. The CI symptoms are usually observed on the fruit skin, which exhibits delayed yellow color development, browning or blackness spotting of the skin; and finally induce the failure of fruit soften (Jiang et al., 2004). Malic acid (MA) has been found to play pivotal roles not only in affecting starch metabolism and increasing soluble substance and chlorophyll contents during the growth period, but also in regulating maturation and senescence of fruit or flowers (Centeno et al., 2011; Darandeh and Hadavi, 2011). In addition, according to biochemical, molecular or proteomics approaches, the application of exogenous organic acids such as MA, oxalic acid (OA), citric acid (CA) and salicylic acid (SA) has been found to affect fruit quality and induce stress tolerance (Darandeh and Hadavi, 2011; Shoor, 2010; Zheng et al., 2011). These organic acids mainly function in maintaining the ability to inhibit O2 accumulation, delaying H2O2 decrease (Ding et al., 2007; Huang et al., 2013a), enhancing antioxidant enzyme (PPO and POD) activities (Cao et al., 2010) and increasing the expression of senescence-related proteins or defense proteins (Wang et al., 2009) to keep the fruit in good quality during storage. However, little is known about the
* Corresponding authors. E-mail addresses: [email protected] (H. Huang), [email protected] (H. Qu). http://dx.doi.org/10.1016/j.postharvbio.2015.09.008 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
application of malic acid on CI tolerance of banana fruit during storage. The objectives of this study were to investigate the effects of exogenous malic acid in the production of ROS and the antioxidant activities in skin tissues of banana fruit during storage at low temperatures. Knowledge gained from this study will help to understand the role of this organic acid in reducing chilling injury of banana fruit and developing appropriate postharvest technologies accordingly. 2. Materials and methods 2.1. Fruit material and treatments Green mature banana fruit were harvested from a commercial orchard in Guangzhou. Fruit were washed and selected for uniformity of shape, color, and size; then placed into two groups in a completely randomized design: immersion in water only (control) or 80 mM MA for 5 min at 25 C. After treatment, fruit were air dried, then packed in plastic polyethylene bags (200 150 mm, 0.03 mm thickness and 3 fruit per bag), and stored at 6 C, 85–90% relative humidity. Six fruit from each treatment were randomly selected at 0, 2, 4, and 6 d during storage and the chilling index and chlorophyll fluorescence (Fv/Fm) were evaluated. Then the skin tissues were sliced, frozen in liquid N2, and stored at 20 C prior to analyses of enzymatic activity and antioxidant ability.
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2.3. Measurement of chlorophyll fluorescence and chlorophyll content Chlorophyll fluorescence was determined using a chlorophyll fluorometer (FAM 2100, Walz, Germany). Fo and Fm were measured at three equidistant points around the middle position of each fruit after being dark-adapted for 30 min. The maximal variable fluorescence (Fv = Fm–Fo) and PSII quantum yield (Fv/Fm) were calculated. Chlorophyll was extracted by grinding 2 g of peel tissue from six fruit from each treatment in 80% (v/v) cold acetone at 4 C with quartz sand for 30 min. The amount of chlorophyll (a and b) was determined by the absorbance at 663 and 645 nm using a spectrophotometer (UVmini-1240, Shimadzu Corp., Japan), and calculated into mg kg 1 on fresh weight basis. 2.4. Assays for enzyme activities of PPO and POD For analyses of enzyme activities, peel tissue (2.0 g) from six fruit from each treatment were homogenized with 10 mL of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.2 g PVPP (Dingguo, Beijing, China) and then centrifuged at 15,000 g for 20 min (Sigma Laborzenfrifugen, 3K15, Germany) 4 C. The supernatant was collected for enzyme assays. PPO (EC 1.10.3.2) activity was measured by incubating 0.1 mL of enzyme extractionin 2.9 mL of sodium phosphate buffer(0.05 M, pH 7.0) containing 10 mM catechol. One unit of PPO activity was defined as the amount of enzyme causing 0.001 absorbance increase per minute at 398 nm. POD (EC 1.11.1.7) activity was assayed by reaction mixture of 3 mL contained 0.05 mL of enzyme extraction, 0.1 mL of 4.0% guaiacol, 0.1 mL of 0.46% H2O2 and2.75 mL of sodium phosphate buffer (0.05 M, pH 7.0). One unit of POD was defined as the amount of enzyme causing 0.01 absorbance increase per minute at 470 nm. 2.5. Assays of reactive oxygen species (ROS)
Fig. 1. Changes in chilling injury index (A), chlorophyll fluorescence (Fv/Fm) (B), and chlorophyll content of banana fruit treated with malic acid during storage at 6 C. Values are the means of three replicates SE (n = 3). Vertical bars represent the standard errors of the means.
2.2. Determination of chilling injury index Chilling injury was assessed by estimating the extent of browning of the fruit surface using the following scale: 1, almost no browning; 2, 0–1/4 browning of the fruit surface; 3, 1/4–1/ 2 browning area; 4, 1/2–3/4 browning area; and 5, >3/4 browning P area. The chilling injury index was calculated as: (chilling injury scale (corresponding fruit with each class)/number of total fruit (the highest scale)) 100.
Superoxide anion (O2 ) generation rate was measured by the method of Wang and Lou (1990) with modifications. Frozen peel tissues (2.0 g) from six fruit were extracted with 10 mL of extraction buffer (pH 7.8) containing 1 mM EDTA, 2% polyvinylpyrrolidone (PVP, w/v) and 0.3% Triton X-100, centrifuged at 12,000 g for 30 min at 4 C. The 1 mL of supernatant was incubated with 1 mL of 1 m Mhydroxylammoniumchlroride for 30 min at 25 C. Then 1 mL of above solution was mixed with 1 mL of 17 mM 3-amino-benzene-sulfonic acids (Sigma, USA) and 1 mL of 7 mM 1-naphthylamine (Sigma, USA) for 20 min at 25 C. The absorbance of the solution was monitored at 530 nm. The O2 production rate was expressed as nmol s 1 kg 1 on a fresh weight basis. H2O2 content was determined by homogenized frozen peel tissues (2.0 g) from six fruit with 10 mL of cold acetone, centrifuged at 12,000 g for 30 min at 4 C. The supernatant (1 mL) was mixed with 0.1 mL of 5% titanium sulphate and 0.2 mL ammonia, centrifuged at 12,000 g for 10 min at 4 C. The precipitates were dissolved in 3 mL of 10% (v/v) H2SO4, centrifuged at 12,000 g for 10 min. The absorbance of the supernatant was measured at 420 nm and the H2O2 content was expressed on nmol kg 1 on a fresh weight basis. 2.6. Assay of DPPH radical scavenging activity and reducing power Frozen peel tissues (3.0 g) from six fruit were firstly crushed into powder and extracted with 30 mL of methanol for 30 min, centrifuged at 15,000 g for 20 min at 25 C. The supernatants were collected.
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browning during storage (Supplement Fig. 1, S1). After 6 d at 6 C, the MA-treated fruit exhibited a CI index of about 66.7% compared with 93.8% for control fruit (Fig. 1A). Due to the sensitivity to chilling stress of banana fruit, chloroplast integration and ultra-structure change greatly (Kratsch and Wise, 2000), which causes decreases in photosynthesis activity, chlorophyll fluorescence (Fv/Fm), and chlorophyll content (Hashim et al., 2013). Among these parameters, the reduction in Fv/Fm and chlorophyll content of banana fruit were significant slowed (p < 0.05) by MA treatment (Fig. 1B and C). Previous reports showed that application of an organic acid, such as oxalic acid, might alter the pH of fruit peel or fresh tissues (Yoruk et al., 2004; Zheng et al., 2007). However, no significant differences in pH of homogenates of skin tissue treated with MA (pH <3, Supplementary material, S2) were found in our study. These data indicate that MA treatment could delay the rapid disintegration of chloroplast and alleviate the CI symptoms of banana fruit stored at 6 C. 3.2. Effects of malic acid treatment on ROS level and PPO and POD activities Oxidative stress is believed to induce the excess production of ROS including superoxide anion (O2 ) and hydrogen peroxide (H2O2). ROS production causes the oxidation damage in chloroplasts, mitochondria, and apoplast under temperature stresses (Apel and Hirt, 2004; Gill and Tuteja, 2010). In this study, the MAtreated fruit exhibited a significantly (p < 0.05) lower superoxide anion generation rate compared with control fruit. Similarly, the
Fig. 2. Changes in superoxide anion generation rate (A) and contents of hydrogen peroxide (B) in peel tissues of banana fruit treated with malic acid during storage at 6 C. Values are the means of three replicatess SE (n = 3). Vertical bars represent the standard errors of the means.
The DPPH radical scavenging activity was evaluated by the method of Huang et al. (2013a). The above 0.1 mL of supernatant was mixed with 2.9 mL of 0.1 mM DPPH and then measured the absorbance at 517 nm. The DPPH radical scavenging activity (%) of the sample was calculated as: (1 absorbance of sample/absorbance of control) 100. The reducing power was determined by mixing 0.1 mL of the above extraction with 2.5 mL of 0.1 M phosphate buffer (pH7.0) and 2.5 mL of 1% potassium ferricyanide followed by incubation for 20 min at 50 C (Huang et al., 2013b). Then 2.5 mL of 10% TCA, 2.5 mL of distilled water, and 0.5 mL of 0.1% ferric chloride were added for measuring absorbance at 700 nm. 2.7. Statistical analysis The experiments were conducted in a completely randomized design, with three replicates of each treatment. Data are presented as means standard errors. The least significant differences (LSDs) (p = 0.05) were calculated. 3. Results and discussion 3.1. Effects of malic acid on chilling injury and physiological attributes Banana fruit exhibited obvious symptoms of CI when stored at 6 C, discoloration from green skin into browning or black spot, and could not soften normally (Hashim et al., 2011). MA treatment significantly (p < 0.05) delayed the increase of CI-induced
Fig. 3. Changes in the activity of PPO (A) and POD (B) in the peel of banana fruit treated with malic acid during storage at 6 C. Values are the meansof three replicates SE (n = 3). Vertical bars represent the standard errors of the means.
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3.3. Changes in antioxidant activity and reducing power The skin browning of banana fruit induced by CI has been found to involve enzymatic or non-enzymatic oxidation of phenolic substrates and chlorophyll degradation (Nguyen, 2003; Trakulnaleumsai et al., 2006). Phenolic compounds were thought to be important antioxidants, which could inhibit ROS over-production, maintain the radical scavenging activity. This forms the non-enzymatic antioxidant system in plant tissues (Velioglu et al.,1998). The total phenolics content in banana fruit skin tissue decreased gradually with the duration of storage in both control and MA-treated fruit. Compared with the control fruit, a significantly higher content and slower decline of total phenolics were observed in the MA-treated fruit by the end of storage (Supplementary material, S3, A). Meanwhile, after 6 d at 6 C, higher DPPH radical scavenging activity was also detected in MA treatment than that in control fruit peel tissues, decreasing from 68 to 54% and 49%, respectively. The reducing power showed similar trends (Fig. 4). Furthermore, similar to the previous reports (Trakulnaleumsai et al., 2006), the DPPH-radical scavenging activity showed a negative correlation (R2 = 0.83, 95% CI) with chilling injury and a positive correlation (R2 = 0.66, 95% CI) with total phenolic content in the peel tissues (Supplementary material, S4).
4. Conclusion
Fig. 4. Changes in antioxidant activity of the DPPH-radical scavenging activity and reducing power in banana fruit treated with malic acid during storage at 6 C. Values are the meansof three replicates SE (n = 3). Vertical bars represent the standard errors of the means.
accumulation of H2O2 increased to a maximum within the first 2 d and then decreased rapidly (Fig. 2). The MA-treated fruit showed a significantly lower H2O2 content than that in control fruit. Thus, the low temperature induced production and accumulation of H2O2 that is associated with increased generation of superoxide anion at 6 C (Gechev and Hille, 2005; Van Breusegem and Dat, 2006) might contribute to deterioration of chloroplasts and associated decline in Fv/Fm and chlorophyll content (Fig. 2), and the accumulation of membrane lipid oxidation-related MDA byproducts (Supplementary material, S3). Earlier studies have indicated that surface spotting or black browning of banana fruit reflected oxidation and polymerization of phenolics, which were believed to be related to PPO and POD (Choehom et al., 2004; Nguyen et al., 2003). It was suggested that the changes in the membrane system, as indicated by oxidation of membrane lipids, is the primary event resulting in chilling injury, while phenolic substrates oxidized by polyphenol oxidase (PPO) promotes browning or blackening as a secondary injury (Pongprasert et al., 2011; Stewart et al., 2001). In the presentexperiments, both PPO and POD activities increased slowly during the first 2 d of storage and increased rapidly to a significantly higher level in the control fruit than the MA-treated fruit after 4 d (Fig. 3A). The POD activity of the control fruit increased rapidly and was significantly (p < 0.05) higher than that of MA-treated fruit (Fig. 3B). These results correlated well with previous studies of PPO and POD on postharvest fruit (Huang et al., 2013b; Jin et al., 2014; Zheng et al., 2011), in which the inhibition of PPO and POD activities by MA treatment might partially account for inducing resistance to chilling stress.
In the present study, exogenous application of MA, one of the major organic acids present in fruit, exhibited the potential to alleviate CI symptoms to delay skin browning of banana fruit under low-temperature conditions. Suppression of CI by MA treatment was associated with the reduction of MDA accumulation and PPO and POD activities, low levels of superoxide anion generation and H2O2 content, as well as relatively high antioxidant activities. Correlation with previous reports suggested that application of organic acids such as MA, OA, CA, and SA, has the potential for alleviating CI symptoms, extending shelf life, and enhancing pathogen defense on fruit by reducing skin browning and inducing antioxidant activity during storage.
Acknowledgements This work was supported by the National Key Basic Research Program of China (No. 2013CB127102), Guangdong Natural Science Foundation (grant no. S2011020001156), and the National Key Technologies R&D Program (grant no. 2010BAD22B01).
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.2015. 09.008.
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H. Huang et al. / Postharvest Biology and Technology 111 (2016) 209–213 Darandeh, N., Hadavi, E., 2011. Effect of pre-harvest foliar application of citric acid and malic acid on chlorophyll content and post-harvest vase life of Lilium cv. Brunello. Front. Plant Sci. 2. Ding, Z.-S., Tian, S.-P., Zheng, X.-L., Zhou, Z.-W., Xu, Y., 2007. Responses of reactive oxygen metabolism and quality in mango fruit to exogenous oxalic acid or salicylic acid under chilling temperature stress. Physiol. Plant 130, 112–121. Gechev, T.S., Hille, J., 2005. Hydrogen peroxide as a signal controlling plant programmed cell death. J. Cell Biol. 168, 17–20. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Hashim, N., Janius, R.B., Baranyai, L., Rahman, R.A., Osman, A., Zude, M., 2011. KInetic model for colour changes in bananas during the appearance of chilling injury symptoms. Food Bioprocess Technol. 5, 2952–2963. Hashim, N., Pflanz, M., Regen, C., Janius, R.B., Abdul Rahman, R., Osman, A., Shitan, M., Zude, M., 2013. An approach for monitoring the chilling injury appearance in bananas by means of backscattering imaging. J. Food Eng. 116, 28–36. Huang, H., Jing, G., Guo, L., Zhang, D., Yang, B., Duan, X., Ashraf, M., Jiang, Y., 2013a. Effect of oxalic acid on ripening attributes of banana fruit during storage. Postharvest Biol. Technol. 84, 22–27. Huang, H., Zhu, Q., Zhang, Z., Yang, B., Duan, X., Jiang, Y., 2013b. Effect of oxalic acid on antibrowning of banana (Musa spp., AAA group, cv. ‘Brazil’) fruit during storage. Sci. Hortic. 160, 208–212. Jiang, Y., Joyce, D.C., Jiang, W., Lu, W., 2004. Effects of chilling temperatures on ethylene binding by banana fruit. Plant Growth Regul. 43, 109–115. Jin, P., Duan, Y., Wang, L., Wang, J., Zheng, Y., 2014. Reducing chilling injury of loquat fruit by combined treatment with hot air and methyl jasmonate. Food and Biopro.Technol 7 (8), 2259–2266. Kratsch, H., Wise, R., 2000. The ultrastructure of chilling stress. Plant Cell Environ. 23, 337–350. Nguyen, T.B.T., Ketsa, S., van Doorn, W.G., 2003. Relationship between browning and the activities of polyphenoloxidase and phenylalanine ammonia lyase in banana peel during low temperature storage. Postharvest Biol. Technol. 30, 187–193.
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Contents lists available at ScienceDirect
Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Enhanced chilling tolerance of banana fruit treated with malic acid prior to low-temperature storage Hua Huanga,* , Qijie Jiana,b , Yueming Jianga , Xuewu Duana , Hongxia Qua,* a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China b University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 November 2014 Received in revised form 10 September 2015 Accepted 10 September 2015 Available online xxx
The effects of postharvest malic acid (MA) treatment on alleviating the occurrence of chilling injury (CI) symptoms in banana (Musa spp., AAA group, cv. Brazil) fruit under 6 C were evaluated. Application of 80 mM MA alleviated CI symptoms (surface browning), delayed the decrease in chlorophyll fluorescence (Fv/Fm) and chlorophyll content. The activities of peroxidase (POD) and polyphenol oxidase (PPO) were also suppressed by MA. Furthermore, compared with the control group, fruit that were treated with MA showed lower levels of reactive oxygen species, but higher antioxidant activities. The results suggest that the application of MA, as an organic acid, exhibited the potential for alleviating chilling injury symptoms of banana fruit by reducing skin browning and inducing antioxidant activities under low temperature. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Banana Chilling injury Malic acid Antioxidant activity
1. Introduction Banana is highly sensitive to storage temperatures below 13 C that induce chilling injury (CI) of fruit. The CI symptoms are usually observed on the fruit skin, which exhibits delayed yellow color development, browning or blackness spotting of the skin; and finally induce the failure of fruit soften (Jiang et al., 2004). Malic acid (MA) has been found to play pivotal roles not only in affecting starch metabolism and increasing soluble substance and chlorophyll contents during the growth period, but also in regulating maturation and senescence of fruit or flowers (Centeno et al., 2011; Darandeh and Hadavi, 2011). In addition, according to biochemical, molecular or proteomics approaches, the application of exogenous organic acids such as MA, oxalic acid (OA), citric acid (CA) and salicylic acid (SA) has been found to affect fruit quality and induce stress tolerance (Darandeh and Hadavi, 2011; Shoor, 2010; Zheng et al., 2011). These organic acids mainly function in maintaining the ability to inhibit O2 accumulation, delaying H2O2 decrease (Ding et al., 2007; Huang et al., 2013a), enhancing antioxidant enzyme (PPO and POD) activities (Cao et al., 2010) and increasing the expression of senescence-related proteins or defense proteins (Wang et al., 2009) to keep the fruit in good quality during storage. However, little is known about the
* Corresponding authors. E-mail addresses: [email protected] (H. Huang), [email protected] (H. Qu). http://dx.doi.org/10.1016/j.postharvbio.2015.09.008 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
application of malic acid on CI tolerance of banana fruit during storage. The objectives of this study were to investigate the effects of exogenous malic acid in the production of ROS and the antioxidant activities in skin tissues of banana fruit during storage at low temperatures. Knowledge gained from this study will help to understand the role of this organic acid in reducing chilling injury of banana fruit and developing appropriate postharvest technologies accordingly. 2. Materials and methods 2.1. Fruit material and treatments Green mature banana fruit were harvested from a commercial orchard in Guangzhou. Fruit were washed and selected for uniformity of shape, color, and size; then placed into two groups in a completely randomized design: immersion in water only (control) or 80 mM MA for 5 min at 25 C. After treatment, fruit were air dried, then packed in plastic polyethylene bags (200 150 mm, 0.03 mm thickness and 3 fruit per bag), and stored at 6 C, 85–90% relative humidity. Six fruit from each treatment were randomly selected at 0, 2, 4, and 6 d during storage and the chilling index and chlorophyll fluorescence (Fv/Fm) were evaluated. Then the skin tissues were sliced, frozen in liquid N2, and stored at 20 C prior to analyses of enzymatic activity and antioxidant ability.
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2.3. Measurement of chlorophyll fluorescence and chlorophyll content Chlorophyll fluorescence was determined using a chlorophyll fluorometer (FAM 2100, Walz, Germany). Fo and Fm were measured at three equidistant points around the middle position of each fruit after being dark-adapted for 30 min. The maximal variable fluorescence (Fv = Fm–Fo) and PSII quantum yield (Fv/Fm) were calculated. Chlorophyll was extracted by grinding 2 g of peel tissue from six fruit from each treatment in 80% (v/v) cold acetone at 4 C with quartz sand for 30 min. The amount of chlorophyll (a and b) was determined by the absorbance at 663 and 645 nm using a spectrophotometer (UVmini-1240, Shimadzu Corp., Japan), and calculated into mg kg 1 on fresh weight basis. 2.4. Assays for enzyme activities of PPO and POD For analyses of enzyme activities, peel tissue (2.0 g) from six fruit from each treatment were homogenized with 10 mL of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.2 g PVPP (Dingguo, Beijing, China) and then centrifuged at 15,000 g for 20 min (Sigma Laborzenfrifugen, 3K15, Germany) 4 C. The supernatant was collected for enzyme assays. PPO (EC 1.10.3.2) activity was measured by incubating 0.1 mL of enzyme extractionin 2.9 mL of sodium phosphate buffer(0.05 M, pH 7.0) containing 10 mM catechol. One unit of PPO activity was defined as the amount of enzyme causing 0.001 absorbance increase per minute at 398 nm. POD (EC 1.11.1.7) activity was assayed by reaction mixture of 3 mL contained 0.05 mL of enzyme extraction, 0.1 mL of 4.0% guaiacol, 0.1 mL of 0.46% H2O2 and2.75 mL of sodium phosphate buffer (0.05 M, pH 7.0). One unit of POD was defined as the amount of enzyme causing 0.01 absorbance increase per minute at 470 nm. 2.5. Assays of reactive oxygen species (ROS)
Fig. 1. Changes in chilling injury index (A), chlorophyll fluorescence (Fv/Fm) (B), and chlorophyll content of banana fruit treated with malic acid during storage at 6 C. Values are the means of three replicates SE (n = 3). Vertical bars represent the standard errors of the means.
2.2. Determination of chilling injury index Chilling injury was assessed by estimating the extent of browning of the fruit surface using the following scale: 1, almost no browning; 2, 0–1/4 browning of the fruit surface; 3, 1/4–1/ 2 browning area; 4, 1/2–3/4 browning area; and 5, >3/4 browning P area. The chilling injury index was calculated as: (chilling injury scale (corresponding fruit with each class)/number of total fruit (the highest scale)) 100.
Superoxide anion (O2 ) generation rate was measured by the method of Wang and Lou (1990) with modifications. Frozen peel tissues (2.0 g) from six fruit were extracted with 10 mL of extraction buffer (pH 7.8) containing 1 mM EDTA, 2% polyvinylpyrrolidone (PVP, w/v) and 0.3% Triton X-100, centrifuged at 12,000 g for 30 min at 4 C. The 1 mL of supernatant was incubated with 1 mL of 1 m Mhydroxylammoniumchlroride for 30 min at 25 C. Then 1 mL of above solution was mixed with 1 mL of 17 mM 3-amino-benzene-sulfonic acids (Sigma, USA) and 1 mL of 7 mM 1-naphthylamine (Sigma, USA) for 20 min at 25 C. The absorbance of the solution was monitored at 530 nm. The O2 production rate was expressed as nmol s 1 kg 1 on a fresh weight basis. H2O2 content was determined by homogenized frozen peel tissues (2.0 g) from six fruit with 10 mL of cold acetone, centrifuged at 12,000 g for 30 min at 4 C. The supernatant (1 mL) was mixed with 0.1 mL of 5% titanium sulphate and 0.2 mL ammonia, centrifuged at 12,000 g for 10 min at 4 C. The precipitates were dissolved in 3 mL of 10% (v/v) H2SO4, centrifuged at 12,000 g for 10 min. The absorbance of the supernatant was measured at 420 nm and the H2O2 content was expressed on nmol kg 1 on a fresh weight basis. 2.6. Assay of DPPH radical scavenging activity and reducing power Frozen peel tissues (3.0 g) from six fruit were firstly crushed into powder and extracted with 30 mL of methanol for 30 min, centrifuged at 15,000 g for 20 min at 25 C. The supernatants were collected.
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browning during storage (Supplement Fig. 1, S1). After 6 d at 6 C, the MA-treated fruit exhibited a CI index of about 66.7% compared with 93.8% for control fruit (Fig. 1A). Due to the sensitivity to chilling stress of banana fruit, chloroplast integration and ultra-structure change greatly (Kratsch and Wise, 2000), which causes decreases in photosynthesis activity, chlorophyll fluorescence (Fv/Fm), and chlorophyll content (Hashim et al., 2013). Among these parameters, the reduction in Fv/Fm and chlorophyll content of banana fruit were significant slowed (p < 0.05) by MA treatment (Fig. 1B and C). Previous reports showed that application of an organic acid, such as oxalic acid, might alter the pH of fruit peel or fresh tissues (Yoruk et al., 2004; Zheng et al., 2007). However, no significant differences in pH of homogenates of skin tissue treated with MA (pH <3, Supplementary material, S2) were found in our study. These data indicate that MA treatment could delay the rapid disintegration of chloroplast and alleviate the CI symptoms of banana fruit stored at 6 C. 3.2. Effects of malic acid treatment on ROS level and PPO and POD activities Oxidative stress is believed to induce the excess production of ROS including superoxide anion (O2 ) and hydrogen peroxide (H2O2). ROS production causes the oxidation damage in chloroplasts, mitochondria, and apoplast under temperature stresses (Apel and Hirt, 2004; Gill and Tuteja, 2010). In this study, the MAtreated fruit exhibited a significantly (p < 0.05) lower superoxide anion generation rate compared with control fruit. Similarly, the
Fig. 2. Changes in superoxide anion generation rate (A) and contents of hydrogen peroxide (B) in peel tissues of banana fruit treated with malic acid during storage at 6 C. Values are the means of three replicatess SE (n = 3). Vertical bars represent the standard errors of the means.
The DPPH radical scavenging activity was evaluated by the method of Huang et al. (2013a). The above 0.1 mL of supernatant was mixed with 2.9 mL of 0.1 mM DPPH and then measured the absorbance at 517 nm. The DPPH radical scavenging activity (%) of the sample was calculated as: (1 absorbance of sample/absorbance of control) 100. The reducing power was determined by mixing 0.1 mL of the above extraction with 2.5 mL of 0.1 M phosphate buffer (pH7.0) and 2.5 mL of 1% potassium ferricyanide followed by incubation for 20 min at 50 C (Huang et al., 2013b). Then 2.5 mL of 10% TCA, 2.5 mL of distilled water, and 0.5 mL of 0.1% ferric chloride were added for measuring absorbance at 700 nm. 2.7. Statistical analysis The experiments were conducted in a completely randomized design, with three replicates of each treatment. Data are presented as means standard errors. The least significant differences (LSDs) (p = 0.05) were calculated. 3. Results and discussion 3.1. Effects of malic acid on chilling injury and physiological attributes Banana fruit exhibited obvious symptoms of CI when stored at 6 C, discoloration from green skin into browning or black spot, and could not soften normally (Hashim et al., 2011). MA treatment significantly (p < 0.05) delayed the increase of CI-induced
Fig. 3. Changes in the activity of PPO (A) and POD (B) in the peel of banana fruit treated with malic acid during storage at 6 C. Values are the meansof three replicates SE (n = 3). Vertical bars represent the standard errors of the means.
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3.3. Changes in antioxidant activity and reducing power The skin browning of banana fruit induced by CI has been found to involve enzymatic or non-enzymatic oxidation of phenolic substrates and chlorophyll degradation (Nguyen, 2003; Trakulnaleumsai et al., 2006). Phenolic compounds were thought to be important antioxidants, which could inhibit ROS over-production, maintain the radical scavenging activity. This forms the non-enzymatic antioxidant system in plant tissues (Velioglu et al.,1998). The total phenolics content in banana fruit skin tissue decreased gradually with the duration of storage in both control and MA-treated fruit. Compared with the control fruit, a significantly higher content and slower decline of total phenolics were observed in the MA-treated fruit by the end of storage (Supplementary material, S3, A). Meanwhile, after 6 d at 6 C, higher DPPH radical scavenging activity was also detected in MA treatment than that in control fruit peel tissues, decreasing from 68 to 54% and 49%, respectively. The reducing power showed similar trends (Fig. 4). Furthermore, similar to the previous reports (Trakulnaleumsai et al., 2006), the DPPH-radical scavenging activity showed a negative correlation (R2 = 0.83, 95% CI) with chilling injury and a positive correlation (R2 = 0.66, 95% CI) with total phenolic content in the peel tissues (Supplementary material, S4).
4. Conclusion
Fig. 4. Changes in antioxidant activity of the DPPH-radical scavenging activity and reducing power in banana fruit treated with malic acid during storage at 6 C. Values are the meansof three replicates SE (n = 3). Vertical bars represent the standard errors of the means.
accumulation of H2O2 increased to a maximum within the first 2 d and then decreased rapidly (Fig. 2). The MA-treated fruit showed a significantly lower H2O2 content than that in control fruit. Thus, the low temperature induced production and accumulation of H2O2 that is associated with increased generation of superoxide anion at 6 C (Gechev and Hille, 2005; Van Breusegem and Dat, 2006) might contribute to deterioration of chloroplasts and associated decline in Fv/Fm and chlorophyll content (Fig. 2), and the accumulation of membrane lipid oxidation-related MDA byproducts (Supplementary material, S3). Earlier studies have indicated that surface spotting or black browning of banana fruit reflected oxidation and polymerization of phenolics, which were believed to be related to PPO and POD (Choehom et al., 2004; Nguyen et al., 2003). It was suggested that the changes in the membrane system, as indicated by oxidation of membrane lipids, is the primary event resulting in chilling injury, while phenolic substrates oxidized by polyphenol oxidase (PPO) promotes browning or blackening as a secondary injury (Pongprasert et al., 2011; Stewart et al., 2001). In the presentexperiments, both PPO and POD activities increased slowly during the first 2 d of storage and increased rapidly to a significantly higher level in the control fruit than the MA-treated fruit after 4 d (Fig. 3A). The POD activity of the control fruit increased rapidly and was significantly (p < 0.05) higher than that of MA-treated fruit (Fig. 3B). These results correlated well with previous studies of PPO and POD on postharvest fruit (Huang et al., 2013b; Jin et al., 2014; Zheng et al., 2011), in which the inhibition of PPO and POD activities by MA treatment might partially account for inducing resistance to chilling stress.
In the present study, exogenous application of MA, one of the major organic acids present in fruit, exhibited the potential to alleviate CI symptoms to delay skin browning of banana fruit under low-temperature conditions. Suppression of CI by MA treatment was associated with the reduction of MDA accumulation and PPO and POD activities, low levels of superoxide anion generation and H2O2 content, as well as relatively high antioxidant activities. Correlation with previous reports suggested that application of organic acids such as MA, OA, CA, and SA, has the potential for alleviating CI symptoms, extending shelf life, and enhancing pathogen defense on fruit by reducing skin browning and inducing antioxidant activity during storage.
Acknowledgements This work was supported by the National Key Basic Research Program of China (No. 2013CB127102), Guangdong Natural Science Foundation (grant no. S2011020001156), and the National Key Technologies R&D Program (grant no. 2010BAD22B01).
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.2015. 09.008.
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