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Limiting the deterioration of mango fruit during storage at room temperature by oxalate treatment
Food Chemistry 130 (2012) 279–285
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Limiting the deterioration of mango fruit during storage at room temperature by oxalate treatment Xiaolin Zheng ⇑, Libin Ye, Tianjia Jiang, Guoxin Jing, Jianrong Li ⇑ College of Food Science and Biotechnology, Zhejiang Gongshang University, Food Safety Key Lab of Zhejiang Province, Hangzhou 310035, China
a r t i c l e
i n f o
Article history: Received 3 January 2011 Received in revised form 13 May 2011 Accepted 12 July 2011 Available online 23 July 2011 Keywords: Mango fruit Oxalate POD Postharvest deterioration PPO Storage Total phenols
a b s t r a c t Effects of oxalate on the incidence of decay and ripening in mango fruit, and its physiological effects on the peel and flesh of mango were investigated after mango fruit (Mangifera indica L.) were dipped in different oxalate solutions for 10 min and then stored at 25 °C. Oxalate application decreased the incidence of decay and delayed the ripening process in mango fruit during storage. Potassium oxalate treatment resulted in increased activities of peroxidase (POD) in both the peel and the flesh and polyphenol oxidase (PPO) in the peel, without activation of phenylalanine ammonia-lyase activity, and elevated total phenolic content in the peel. The physiological effects of oxalate in increasing activities of POD and PPO and elevating total phenolic level could be involved in induced resistance of mango fruit against postharvest disease. Oxalate application could be a promising method to suppress postharvest deterioration and extend the useful shelf-life of mangoes. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Susceptibility of mango fruit (Mangifera indica L.) to postharvest diseases increases during storage after harvest, as a result of physiological changes and senescence, favouring pathogen development (Prusky & Keen, 1993). Synthetic fungicides, such as benomyl and prochloraz, alone or in combination with other treatments, have shown efficacy in controlling decay incidence in mango fruit during storage at room and lower temperature (Johnson, Sharp, Milne, & Oosthuyse, 1997; Kobiler et al., 2001). However, increasing public concerns with fungicide toxicity, development of fungicide resistance by pathogens and adverse effects on the environment and human health have lead to intensified worldwide research efforts to develop alternative forms of disease control (Droby, Wisniewski, Macarisin, & Wilson, 2009; Wilson et al., 1994). Induction of host resistance is one strategy that holds promise for control of postharvest diseases (Adikaram, 1990). In recent years, inducing resistance by chemical, physical or biological elicitors is becoming a great potential approach for the control of postharvest diseases as an alternative to fungicides (Tian, 2006;
⇑ Corresponding authors. Tel.: +86 571 88071024/7584; fax: +86 571 88053832. E-mail addresses: [email protected] (X. Zheng), [email protected] (J. Li). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.035
Wilson et al., 1994). Previous works have reported that oxalic acid and oxalate induce systemic resistance against diseases caused by fungi, bacteria and viruses in plants (Mucharromah & Kuc, 1991; Toal & Jones, 1999). Oxalic acid treatment inhibits the progress of Alternaria rot in harvested pear fruit associated with an induction of increased defence-related enzyme activities (Tian, Wan, Qin, & Xu, 2006). Wang, Lai, Qin, and Tian (2009) have reported that three proteins related to the defence or stress response are up-regulated by oxalic acid, and contribute to the establishment of systemic resistance induced by oxalic acid in jujube fruits. Moreover, in our previous work, we have found that pre-storage application of oxalic acid can suppress postharvest deterioration and extend the shelf-life of mango fruit, due to a combination of physiological effects associated with delaying the ripening process, and direct effects, including low pH, inhibiting the development of postharvest pathogens such as Colletotrichum gloeosporioides (Zheng, Tian, Gidley, Yue, & Li, 2007; Zheng et al., 2007). In the search for novel treatments to reduce deterioration of mango fruit, as well as to better understand the role of oxalate in improving the limited storage ability of mango fruit, the effects of oxalate on ripening and decay incidence in mango fruit during storage at room temperature, and the effects of potassium oxalate on defence mechanism in association with defence-related enzymes and accumulation of total phenols were further investigated in this study.
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2. Materials and methods
percentage of fruit including class 2–4 without commercial value was taken as fruit decay.
2.1. Material and treatments 2.4. Measurement of SSC and TA
2.1.2. Experiment 2. Postharvest dip treatment with different concentrations of potassium oxalate Mango (M. indica cv. Zill) fruit about 80% matured stage were harvested from a commercial orchard in Panzhihua city, China. Harvested fruit were selected for uniformity of size and appearance. After the selected fruit were cooled for about 2 h in a room at about 25 °C near the orchard, they were dipped in water (as control), 20 or 40 mM potassium oxalate solutions at 25 °C for 10 min. After air drying, each fruit was wrapped with a soft absorbent paper, and then about 15 kg of control and treated fruit were placed in separate cartons. Transit time by plane from harvest to arrival at a Hangzhou laboratory was approximately 24 h. Upon arrival at the laboratory, 20 fruit without injury for control and treatments were placed inside a clean plastic box with fruit touching. Each box was wrapped in a 0.02-mm polyethylene bag and was held in a room at 25 °C (±1 °C). Analysis in triplicate of six fruit each from six plastic boxes was undertaken at 3-day intervals for total soluble solids content (SSC), titratable acidity (TA), enzyme activities of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) and total phenols. Another 60 fruit of each treatment (three replicates, each of 20 fruit) were observed to evaluate firmness index, disease index and fruit decay.
Juice samples were obtained from 12 discs of flesh (about 5 mm deep under peel, 10 mm thickness 13 mm diameter, two discs per fruit on opposite region) from six fruit on the longer transverse axis, and SSC of the fruit juice were determined using a refractometer (Master-a, ATAGO ATC, Japan). Ten grams of flesh tissue (about 5 mm deep under peel) from six fruit on the longer transverse axis (each fruit on opposite region) were homogenised with 25 ml distilled water and filtered, and then TA of the solution was determined by titration to pH 8.1 with 0.1 M NaOH. TA expressed as the percentage of citric acid per 100 g fresh mass. 2.5. Assay for enzymic activities Five-gram peel and ten-gram flesh samples from six fruit in each treatment were ground separately in 30 ml 100 mM sodium phosphate buffer, pH 7.8, containing 0.3 g PVPP (Sigma, St. Louis, MO) for POD and PPO, and in 25 ml of 50 mM sodium borate buffer, pH 8.8, containing 0.3 g PVPP (Sigma) and 5 mM b-mercaptoethanol for PAL analysis using a Kinematica tissue grinder (Kinematica PT2100, Lucerne, Switzerland) and centrifuged at 20,000g for 45 min (Sigma 3–30 K, Osterode am Harz, Germany). The supernatants were used to assay enzymatic activities. All steps in the preparation of extracts were carried out at 4 °C. POD (EC 1.11.1.7) activity was based on the determination of guaiacol oxidation at 470 nm by H2O2. The change in absorbance at 470 nm was followed every 30 s by a spectrophotometer (Shimadzu UV-2550, Kyoto, Japan) (Lacan & Baccou, 1998). One unit of POD was defined as the amount of enzyme causing a 0.01 absorbance increase per min under the conditions of assay. PPO (EC 1.10.3.2) activity was measured by incubating 0.5 ml of enzyme extract to 2.5 ml of buffered substrate (100 mM sodium phosphate, pH 6.4 and 50 mM catechol), and then monitoring the
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2.1.1. Experiment 1. Postharvest dip treatment with prochloraz, potassium oxalate and ammonium oxalate Mango (M. indica cv. Xiaojinhuang) fruit about 80% matured stage were harvested from a commercial orchard in Hainan island of China. Fruit were selected for uniformity of size and colour, and blemished and diseased fruit were discarded. Transit time by train at a temperature of about 25 °C from harvest to arrival at a Zhangjiang City laboratory (Guangdong Province, China) was approximately 12 h. Upon arrival at the laboratory, 20 selected fruit each were placed in a clean plastic box with fruit touching, and then three plastic boxes each were dipped in water (as control), or 0.1% Prochloraz fungicide (Bayer CropScience, China), or 30 mM potassium oxalate or 30 mM ammonium oxalate solutions for 10 min. After air drying, each box was wrapped in a 0.02 mm polyethylene bag to maintain relative humidity and was held in a room at 25 °C (±1 °C). Fruit disease index and fruit decay of each treatment were evaluated at 2-day intervals. Twenty fruit in the same box was considered as a replicate, and three replicates of each treatment were carried out.
2.2. Measurement of fruit firmness index
30 mM potassium oxalate 30 mM ammonium oxalate
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2.3. Measurement of disease index Disease index for fruit was assessed by assessing the extent of total decayed area on each fruit surface using the following scale (Zheng, Tian, Gidley, Yue, & Li, 2007; Zheng et al., 2007): 0 = no visible decay; 1 = <1% decay spots; 2 = 1–20% decayed; 3 = 20–50% decayed; and 4 = >50% decayed. The disease index was calculated using the formula: R (disease scale number of fruit in each class) 100/(number of total fruit highest disease scale). The
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Fruit firmness was assessed by using hand squeeze described by Kobiler et al. (2001) with slight modification, and a firmness index scale from extremely firm to soft ripe (9 = extremely firm, 7 = firm, 5 = sprung, 3 = slight soft and 1 = soft ripe). Firmness index was calculated using the formula: R (firmness scale percentage of fruit within each firmness class).
80 60 40 20 0
Days at 25°C Fig. 1. Changes in disease index (a), and fruit decay (b) in untreated (control), prochloraz and oxalate-treated mango cv. Xiaojinhuang fruit during storage. Data are the means of three replicates ± SD.
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Fig. 2. Appearance of mango cv. Xiaojinhuang fruit treated with 30 mM potassium oxalate (PO), 30 mM ammonium oxalate (AO) and 0.1% prochloraz (P) for 10 min and then held at 25 °C for 1, 10 and 12 days.
change of absorbance at 398 nm (Wang, Tian, Xu, Qin, & Yao, 2004). One unit of activity of PPO was defined as the amount of enzyme that caused 0.01 absorbance increase per min under the conditions of assay. PAL (EC 4.3.1.5) activity was assayed referring to the method of Assis, Maldonado, Munoz, Escribano, and Merodio (2001), with slight modifications. One millilitre of enzyme extract was incubated with 2 ml of borate buffer (50 mM, pH 8.8) and 1 ml of L-phenylalanine (20 mM) for 60 min at 37 °C. The reaction was stopped with 0.5 ml of 6 M HCl. PAL activity was determined by the production of cinnamate, which was measured at 290 nm. The control mixture was stopped by adding 0.5 ml of 6 M HCl immediately after mixing the crude enzyme preparation with L-phenylalanine. Specific enzyme activity was defined in nanomoles cinnamic acid per hour per milligram protein. Protein contents were measured according to the method of Bradford (1976), using bovine serum albumin (BSA) as standard.
2.6. Estimation of total phenols Total phenolic content was determined using the procedures described by Zieslin and Ben-Zaken (1993). Briefly, two-gram peel and five-gram flesh samples from six fruit in each treatment were homogenised separately in 15 ml of 80% methanol and agitated for 15 min at 70 °C, and then 0.5 ml methanolic extract was mixed with 0.5 ml Folin–Ciocalteu reagent by manual shaking for 15–20 s. After 3 min, 1 ml saturated sodium carbonate (75 g/l) and 1 ml of distilled water were added. The reaction mixture was incubated in the dark at room temperature for 2 h and its absorbance was measured at 725 nm against deionised water using a spectrophotometer (Shimadzu UV-2550). The content of the total phenols was calculated based on a standard curve obtained from a Folin–Ciocalteu reaction with phenol and expressed as milligrams of phenol equivalents per gram fresh weight.
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2.7. Statistical analysis
3.2. Effect of potassium oxalate on flesh firmness, SSC and TA
Data represent the means ± SD, and they were analysed by oneway analysis of variance (ANOVA) with SPSS 13.0 (SPSS Inc., Chicago, IL) for comparing the treatments on each day. Significance of difference among means of control and treatments using Duncan’s multiple range tests was at the 5% level. Difference at p < 0.05 was considered significant.
Fruit firmness and TA decreased, while SSC increased in mango fruit during storage. However, potassium oxalate treatment not only resulted in a significantly higher fruit firmness index until Day 6 of storage (Fig. 4a), but also retarded significantly the increase in SSC and decrease in TA compared to the control, particularly from 12 to 15 days after harvest (p < 0.05) (Fig. 4b and c). 3.3. Effect of potassium oxalate on activity of defence-related enzymes
3.1. Effect of oxalate treatments on disease index and fruit decay Oxalate treatments including 30 mM potassium oxalate and 30 mM ammonium oxalate did not show efficacy in slowing the deterioration of mango cv. Xiaojinhuang fruit as well as 0.1% prochloraz treatment during storage at room temperature. However, oxalate treatments apparently reduced the disease index and fruit decay in ‘Xiaojinhuang’ mango fruit after storage of at least 8 days compared to the control. Reductions in disease index and fruit decay were 16% and 35% for the fruit treated with 30 mM potassium oxalate, and 14% and 28% with 30 mM ammonium oxalate, respectively, at the end of storage (12 days) (Fig. 1). The prochloraz, potassium oxalate and ammonium oxalate were also effective for inhibiting diseases such as anthracnose. Among the treatments, the control fruit developed more visible anthracnose, compared to fruit treated with prochloraz and oxalate after storage of at least 10 days, with appreciably less incidence observed in all of three treated fruit (Fig. 2). Mango cv. Zill fruit treated with 20 and 40 mM potassium oxalate also showed decreased deterioration during storage at room temperature, and the disease index and fruit decay for potassium oxalate treated fruit were significantly lower than that of control fruit after storage for both 3 and 6 days (Fig. 3).
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The pattern of change in POD activity in the peel of all fruit was similar during storage, but a significant increase in POD activity in the fruit treated with 20 and 40 mM potassium oxalate was observed from 6 to 12 days and 3 to 9 days in storage, respectively, as compared to the control (Fig. 5a). In the flesh, POD activity in fruit treated with both concentrations of potassium oxalate was also significantly higher than that in the control after 6 days of harvest (p < 0.05) (Fig. 5b). PPO activity in the peel of control fruit increased to a peak on the ninth day, then dropped and remained at a relatively steady level for the remainder of the storage time. For fruit treated with 20 and 40 mM potassium oxalate, PPO activity in the peel also
Firmness index
3. Results
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Days at 25°C Fig. 3. Changes in disease index (a), and fruit decay (b) in untreated (control) and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
Days at 25°C Fig. 4. Changes in flesh firmness index (a), SSC (b) and TA (c) in untreated (control), and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
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Fig. 5. Change in activities of POD, PPO and PAL in the peel (Panel a, Panel c and Panel e) and in the flesh (Panel b, Panel d and Panel f) of untreated (control), and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
increased to a peak on the ninth and the sixth days, respectively, and then decreased steadily during the remaining days. PPO activity in fruit treated with 20 and 40 mM potassium oxalate was significantly higher than that in the control at the beginning of the ninth and sixth days, respectively (p < 0.05) (Fig. 5c). Moreover, PPO activity in the flesh of all fruit increased to a peak on the twelfth day and then decreased, with no differences in the activity between the control and treated fruit (Fig. 5d). PAL activity in the peel of all fruit was maintained at a relatively constant level during the storage period whereas in the flesh it markedly decreased at the beginning of the ninth day and then remained steady during the rest days. However, in the peel and in the flesh, the levels of PAL activity in potassium oxalate-treated fruit were similar to those in the control at all time intervals (Fig. 5e and f). 3.4. Effect of potassium oxalate on total phenolic content Concerning the change of total phenols in mango fruits, the peel of Zill mango fruit contained considerably more total phenolic compounds than the flesh, and this content increased to a maximum on the sixth day after harvest and then declined steadily with duration of storage (Fig. 6). However, the total phenolic content in the peel of 20 and 40 mM potassium oxalate treatments showed
significantly high levels from 6 to 12 days and 6 to 9 days in storage, respectively, relative to the control (p < 0.05) (Fig. 6a). In addition, total phenolic content levels in the flesh varied in the range of 0.3–1.5 mg g 1 during storage, with no significant difference between the control and treated fruit (Fig. 5b).
4. Discussion Mango fruit suffer from a brief shelf-life and postharvest deterioration problems as a result of postharvest disease, insect infestation and over-rapid ripening (Mitra & Baldwin, 1997). To prevent decay development and delay ripening process of mango fruit, a number of strategies have been evaluated instead of fungicides, such as the usage of controlled atmosphere storage (Kim, Brecht, & Talcott, 2007) and application of chemical substances, including salicylic acid (Zainuri, Joyce, Wearing, Coates, & Terry, 2001; Zeng, Cao, & Jiang, 2006), 2,4-dichlorophenoxyacetic acid (Kobiler et al., 2001), 1-methylcyclopropene (Singh & Dwivedi, 2008), potassium phosphate (Zainuri et al., 2001), and oxalic acid (Zheng, Tian, Gidley, Yue, & Li, 2007; Zheng et al., 2007). Data here showed that mango cv. Xiaojinhuang fruit pre-storage treated with 30 mM potassium oxalate and 30 mM ammonium oxalate, and mango cv. Zill fruit treated with 20 and 40 mM potassium oxalate
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successfully decreased postharvest deterioration, as the disease index and fruit decay incidence apparently reduced in treated fruit after storage of several days compared to the control (Figs. 1 and 3). Therefore, these results represented a marked increase in market value for the mango fruit treated with oxalate compared to the control. Softening of the flesh, decreased acidity, increased SSC and total solids, and increased carotenoid pigments are among the recognised parameters of maturity and ripening in mango (Mitra & Baldwin, 1997). Oxalate treatment significantly reduced ripening indices, such as the increase in SSC and decrease in fruit firmness index and TA after storage of 12 or 9 days, respectively, compared with the control (Fig. 4). Therefore, pre-storage application of oxalate delayed the ripening process in mango fruit during storage at room temperature. To our knowledge, this is the first report in which oxalate has been implicated in achieving beneficial effects on decay incidence and ripening in harvested fruit. In addition, a pre-storage dip in a millimolar solution of oxalate did not dramatically raise the price of mango fruit to consumers, as an estimated cost of oxalate treatment was approximately 0.10–0.15 renminbi (RMB) per kilogram. This fact, together with the results that oxalate apparently suppressed postharvest deterioration and extended the shelf-life of mango fruit, suggested that pre-storage oxalate dip might be meaningful in terms of the economics. Generally, defence-related enzymes, including PPO, POD and PAL, are considered potentially important in induced resistance of plants (Sticher, Mauch-Mani, & Métraux, 1997). To date, there are many reports in the literature that increase in activities of PPO, POD and PAL followed by chemical, physical or biological treatments are involved in induced resistance of fruit as well (Chan, Qin, Xu, Li, & Tian, 2007; González-Aguilar, Zavaleta-Gatica, & Tiznado-Hernández, 2007; Tian et al., 2006). Our results indicated that potassium oxalate stimulated significant increase in activities of POD in the fruit peel and flesh, and PPO in the fruit peel, without activation of PAL activity in both the peel and the flesh of mango fruit. Furthermore, phenolic compounds have activity inhibiting pathogens and increase in phenolic content in plants
is correlated with increased resistance to pathogens (Velazhahan & Vidhyasekaran, 1994). Accumulation of phenolic compounds in plant and fruit by abiotic or biotic elicitors has been considered as a defence mechanism. For example, induction of early blight resistance in tomato by Quercus infectoria gall extract is attributed to elevated level of phenolics and enhanced activities of defencerelated enzymes (Yamunarani, Jaganathan, Bhaskaran, Govindaraju, & Velazhahan, 2004). Postharvest UV-C exposition increases the resistance in table grapes (Cantos, Garcia-Viguera, de Pascual-Teresa, & Tomas-Barberan, 2000) and in mango fruit (González-Aguilar et al., 2007) against pathogenic attack by inducing the biosynthesis of large amounts of phenolic compounds. In this experiment, a significant increase in total phenolic content in the peel of mango fruit was also found as a result of the oxalate treatments. Thus, this physiological effect of oxalate treatments in elevating phenolic level and enhancing activities of POD and PPO in mango fruit, particularly marked for the peel, the primary site of potential infestation or infection, might also be involved in induced resistance for mango fruit during storage. This would explain why reduction in fungi infections in whole mango treated with oxalate was observed, and quality of treated mango fruit was consequently maintained for longer periods of time. PAL is the first enzyme of phenylpropanoid metabolism and plays an important role in the regulation of biosynthesis of phenols in plants (Yamunarani et al., 2004), and induction in the level of total phenols in mango fruit by UV-C treatment strongly correlates with the increase in PAL activity (González-Aguilar et al., 2007). Our results were not consistent with this previous report, as total phenols in the peel of mango fruit treated with oxalate were significantly elevated during most of the storage time, without stimulation of PAL enzyme during storage. The different response might be due to the fact of difference in treatment or mango fruit cultivars, and there might be other important enzymes in the phenylpropanoid biosynthetic pathway, such as cinnamate 4-hydroxylase, 4-coumarate CoA ligase and chalcone synthase involved as well. Therefore, for better understanding the possible mode of action of the oxalate treatment, effect of oxalate on those enzymes and specific phytoalexins in mango fruit should be studied. In conclusion, the results obtained in this study showed evidence for the ability of oxalate treatments to limit postharvest deterioration and maintain quality of mango fruit during storage at room temperature. The physiological effects of oxalate that involved enhanced activities of defence-related enzymes such as POD and PPO, and elevated level of total phenols, especially in the fruit peel, collectively contributed to induced resistance of mango Zill fruit against postharvest disease in storage. A pre-storage oxalate dip might be a promising method for suppressing the postharvest deterioration and extending the useful shelf-life of mango fruit.
Acknowledgements The authors acknowledge the financial support provided by National Natural Science Foundation of China (No. 30771509), by the Natural Science Foundation of Guangdong Province, China (No. 7010014), and by the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China (2008).
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Tian, S. P. (2006). Microbial control of postharvest diseases of fruits and vegetables: Current concepts and future outlook. In C. R. Ranesh & P. W. Owen (Eds.), Microbial biotechnology in horticulture (pp. 63–202). Enfield, NH, USA: Science Publishers, Inc. Tian, S. P., Wan, Y. K., Qin, G. Z., & Xu, Y. (2006). Induction of defense responses against Alternaria rot by different elicitors in harvested pear fruit. Applied Microbiology & Biotechnology, 70, 729–734. Toal, E. S., & Jones, P. W. (1999). Induction of systemic resistance to Sclerotinia sclerotiorum by oxalic acid in oilseed rape. Plant Pathology, 48, 759–767. Velazhahan, R., & Vidhyasekaran, P. (1994). Role of phenolic compounds, peroxidase and polyphenol-oxidase in resistance of groundnut to rust. Acta Phytopathologica Et Entomologica Hungarica, 29, 23–29. Wang, Q., Lai, T. F., Qin, G. Z., & Tian, S. P. (2009). Response of jujube fruits to exogenous oxalic acid treatment based on proteomic analysis. Plant Cell Physiology, 50, 230–242. Wang, Y. S., Tian, S. P., Xu, Y., Qin, G. Z., & Yao, H. J. (2004). Changes in the activities of pro-and anti-oxidant enzymes in peach fruit inoculated with Cryptococcus laurentii or Penicillium expansum at 0 or 20 °C. Postharvest Biology and Technology, 34, 21–28. Wilson, C. L., El-Ghaouth, A., Chalutz, E., Droby, S., Steven, C., Lu, J. Y., et al. (1994). Potential of induced resistance to control postharvest diseases of fruit and vegetables. Plant Diseases, 78, 837–844. Yamunarani, K., Jaganathan, R., Bhaskaran, R., Govindaraju, P., & Velazhahan, R. (2004). Induction of early blight resistance in tomato by Quercus infectoria gall extract in association with accumulation of phenolics and defense-related enzymes. Acta Physiologiae Plantarum, 26(3), 281–290. Zainuri Joyce, D. C., Wearing, A. H., Coates, L., & Terry, L. (2001). Effects of phosphonate and salicylic acid treatments on anthracnose disease development and ripening of ‘Kensington Pride’ mango fruit. Australian Journal of Experimental Agriculture, 41, 805–813. Zeng, K. F., Cao, J. K., & Jiang, W. B. (2006). Enhancing disease resistance in harvested mango (Mangifera indica L. cv. ‘Matisu’) fruit by salicylic acid. Journal of the Science of Food and Agriculture, 86, 694–698. Zheng, X. L., Tian, S. P., Gidley, M. J., Yue, H., & Li, B. Q. (2007). Effects of exogenous oxalic acid on ripening and decay incidence in mango fruit during storage at room temperature. Postharvest Biology and Technology, 45, 281–284. Zheng, X. L., Tian, S. P., Gidley, M. J., Yue, H., Li, B. Q., Xu, Y., et al. (2007). Slowing deterioration of mango fruit during cold storage by pre-storage application of oxalic acid. Journal of Horticultural Science and Biotechnology, 82(5), 707–714. Zieslin, N., & Ben-Zaken, R. (1993). Peroxidase activity and presence of phenolic substances in peduncles of rose flowers. Plant Physiology and Biochemistry, 31, 333–339.
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Limiting the deterioration of mango fruit during storage at room temperature by oxalate treatment Xiaolin Zheng ⇑, Libin Ye, Tianjia Jiang, Guoxin Jing, Jianrong Li ⇑ College of Food Science and Biotechnology, Zhejiang Gongshang University, Food Safety Key Lab of Zhejiang Province, Hangzhou 310035, China
a r t i c l e
i n f o
Article history: Received 3 January 2011 Received in revised form 13 May 2011 Accepted 12 July 2011 Available online 23 July 2011 Keywords: Mango fruit Oxalate POD Postharvest deterioration PPO Storage Total phenols
a b s t r a c t Effects of oxalate on the incidence of decay and ripening in mango fruit, and its physiological effects on the peel and flesh of mango were investigated after mango fruit (Mangifera indica L.) were dipped in different oxalate solutions for 10 min and then stored at 25 °C. Oxalate application decreased the incidence of decay and delayed the ripening process in mango fruit during storage. Potassium oxalate treatment resulted in increased activities of peroxidase (POD) in both the peel and the flesh and polyphenol oxidase (PPO) in the peel, without activation of phenylalanine ammonia-lyase activity, and elevated total phenolic content in the peel. The physiological effects of oxalate in increasing activities of POD and PPO and elevating total phenolic level could be involved in induced resistance of mango fruit against postharvest disease. Oxalate application could be a promising method to suppress postharvest deterioration and extend the useful shelf-life of mangoes. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Susceptibility of mango fruit (Mangifera indica L.) to postharvest diseases increases during storage after harvest, as a result of physiological changes and senescence, favouring pathogen development (Prusky & Keen, 1993). Synthetic fungicides, such as benomyl and prochloraz, alone or in combination with other treatments, have shown efficacy in controlling decay incidence in mango fruit during storage at room and lower temperature (Johnson, Sharp, Milne, & Oosthuyse, 1997; Kobiler et al., 2001). However, increasing public concerns with fungicide toxicity, development of fungicide resistance by pathogens and adverse effects on the environment and human health have lead to intensified worldwide research efforts to develop alternative forms of disease control (Droby, Wisniewski, Macarisin, & Wilson, 2009; Wilson et al., 1994). Induction of host resistance is one strategy that holds promise for control of postharvest diseases (Adikaram, 1990). In recent years, inducing resistance by chemical, physical or biological elicitors is becoming a great potential approach for the control of postharvest diseases as an alternative to fungicides (Tian, 2006;
⇑ Corresponding authors. Tel.: +86 571 88071024/7584; fax: +86 571 88053832. E-mail addresses: [email protected] (X. Zheng), [email protected] (J. Li). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.035
Wilson et al., 1994). Previous works have reported that oxalic acid and oxalate induce systemic resistance against diseases caused by fungi, bacteria and viruses in plants (Mucharromah & Kuc, 1991; Toal & Jones, 1999). Oxalic acid treatment inhibits the progress of Alternaria rot in harvested pear fruit associated with an induction of increased defence-related enzyme activities (Tian, Wan, Qin, & Xu, 2006). Wang, Lai, Qin, and Tian (2009) have reported that three proteins related to the defence or stress response are up-regulated by oxalic acid, and contribute to the establishment of systemic resistance induced by oxalic acid in jujube fruits. Moreover, in our previous work, we have found that pre-storage application of oxalic acid can suppress postharvest deterioration and extend the shelf-life of mango fruit, due to a combination of physiological effects associated with delaying the ripening process, and direct effects, including low pH, inhibiting the development of postharvest pathogens such as Colletotrichum gloeosporioides (Zheng, Tian, Gidley, Yue, & Li, 2007; Zheng et al., 2007). In the search for novel treatments to reduce deterioration of mango fruit, as well as to better understand the role of oxalate in improving the limited storage ability of mango fruit, the effects of oxalate on ripening and decay incidence in mango fruit during storage at room temperature, and the effects of potassium oxalate on defence mechanism in association with defence-related enzymes and accumulation of total phenols were further investigated in this study.
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2. Materials and methods
percentage of fruit including class 2–4 without commercial value was taken as fruit decay.
2.1. Material and treatments 2.4. Measurement of SSC and TA
2.1.2. Experiment 2. Postharvest dip treatment with different concentrations of potassium oxalate Mango (M. indica cv. Zill) fruit about 80% matured stage were harvested from a commercial orchard in Panzhihua city, China. Harvested fruit were selected for uniformity of size and appearance. After the selected fruit were cooled for about 2 h in a room at about 25 °C near the orchard, they were dipped in water (as control), 20 or 40 mM potassium oxalate solutions at 25 °C for 10 min. After air drying, each fruit was wrapped with a soft absorbent paper, and then about 15 kg of control and treated fruit were placed in separate cartons. Transit time by plane from harvest to arrival at a Hangzhou laboratory was approximately 24 h. Upon arrival at the laboratory, 20 fruit without injury for control and treatments were placed inside a clean plastic box with fruit touching. Each box was wrapped in a 0.02-mm polyethylene bag and was held in a room at 25 °C (±1 °C). Analysis in triplicate of six fruit each from six plastic boxes was undertaken at 3-day intervals for total soluble solids content (SSC), titratable acidity (TA), enzyme activities of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) and total phenols. Another 60 fruit of each treatment (three replicates, each of 20 fruit) were observed to evaluate firmness index, disease index and fruit decay.
Juice samples were obtained from 12 discs of flesh (about 5 mm deep under peel, 10 mm thickness 13 mm diameter, two discs per fruit on opposite region) from six fruit on the longer transverse axis, and SSC of the fruit juice were determined using a refractometer (Master-a, ATAGO ATC, Japan). Ten grams of flesh tissue (about 5 mm deep under peel) from six fruit on the longer transverse axis (each fruit on opposite region) were homogenised with 25 ml distilled water and filtered, and then TA of the solution was determined by titration to pH 8.1 with 0.1 M NaOH. TA expressed as the percentage of citric acid per 100 g fresh mass. 2.5. Assay for enzymic activities Five-gram peel and ten-gram flesh samples from six fruit in each treatment were ground separately in 30 ml 100 mM sodium phosphate buffer, pH 7.8, containing 0.3 g PVPP (Sigma, St. Louis, MO) for POD and PPO, and in 25 ml of 50 mM sodium borate buffer, pH 8.8, containing 0.3 g PVPP (Sigma) and 5 mM b-mercaptoethanol for PAL analysis using a Kinematica tissue grinder (Kinematica PT2100, Lucerne, Switzerland) and centrifuged at 20,000g for 45 min (Sigma 3–30 K, Osterode am Harz, Germany). The supernatants were used to assay enzymatic activities. All steps in the preparation of extracts were carried out at 4 °C. POD (EC 1.11.1.7) activity was based on the determination of guaiacol oxidation at 470 nm by H2O2. The change in absorbance at 470 nm was followed every 30 s by a spectrophotometer (Shimadzu UV-2550, Kyoto, Japan) (Lacan & Baccou, 1998). One unit of POD was defined as the amount of enzyme causing a 0.01 absorbance increase per min under the conditions of assay. PPO (EC 1.10.3.2) activity was measured by incubating 0.5 ml of enzyme extract to 2.5 ml of buffered substrate (100 mM sodium phosphate, pH 6.4 and 50 mM catechol), and then monitoring the
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2.1.1. Experiment 1. Postharvest dip treatment with prochloraz, potassium oxalate and ammonium oxalate Mango (M. indica cv. Xiaojinhuang) fruit about 80% matured stage were harvested from a commercial orchard in Hainan island of China. Fruit were selected for uniformity of size and colour, and blemished and diseased fruit were discarded. Transit time by train at a temperature of about 25 °C from harvest to arrival at a Zhangjiang City laboratory (Guangdong Province, China) was approximately 12 h. Upon arrival at the laboratory, 20 selected fruit each were placed in a clean plastic box with fruit touching, and then three plastic boxes each were dipped in water (as control), or 0.1% Prochloraz fungicide (Bayer CropScience, China), or 30 mM potassium oxalate or 30 mM ammonium oxalate solutions for 10 min. After air drying, each box was wrapped in a 0.02 mm polyethylene bag to maintain relative humidity and was held in a room at 25 °C (±1 °C). Fruit disease index and fruit decay of each treatment were evaluated at 2-day intervals. Twenty fruit in the same box was considered as a replicate, and three replicates of each treatment were carried out.
2.2. Measurement of fruit firmness index
30 mM potassium oxalate 30 mM ammonium oxalate
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2.3. Measurement of disease index Disease index for fruit was assessed by assessing the extent of total decayed area on each fruit surface using the following scale (Zheng, Tian, Gidley, Yue, & Li, 2007; Zheng et al., 2007): 0 = no visible decay; 1 = <1% decay spots; 2 = 1–20% decayed; 3 = 20–50% decayed; and 4 = >50% decayed. The disease index was calculated using the formula: R (disease scale number of fruit in each class) 100/(number of total fruit highest disease scale). The
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Fruit firmness was assessed by using hand squeeze described by Kobiler et al. (2001) with slight modification, and a firmness index scale from extremely firm to soft ripe (9 = extremely firm, 7 = firm, 5 = sprung, 3 = slight soft and 1 = soft ripe). Firmness index was calculated using the formula: R (firmness scale percentage of fruit within each firmness class).
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Days at 25°C Fig. 1. Changes in disease index (a), and fruit decay (b) in untreated (control), prochloraz and oxalate-treated mango cv. Xiaojinhuang fruit during storage. Data are the means of three replicates ± SD.
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Fig. 2. Appearance of mango cv. Xiaojinhuang fruit treated with 30 mM potassium oxalate (PO), 30 mM ammonium oxalate (AO) and 0.1% prochloraz (P) for 10 min and then held at 25 °C for 1, 10 and 12 days.
change of absorbance at 398 nm (Wang, Tian, Xu, Qin, & Yao, 2004). One unit of activity of PPO was defined as the amount of enzyme that caused 0.01 absorbance increase per min under the conditions of assay. PAL (EC 4.3.1.5) activity was assayed referring to the method of Assis, Maldonado, Munoz, Escribano, and Merodio (2001), with slight modifications. One millilitre of enzyme extract was incubated with 2 ml of borate buffer (50 mM, pH 8.8) and 1 ml of L-phenylalanine (20 mM) for 60 min at 37 °C. The reaction was stopped with 0.5 ml of 6 M HCl. PAL activity was determined by the production of cinnamate, which was measured at 290 nm. The control mixture was stopped by adding 0.5 ml of 6 M HCl immediately after mixing the crude enzyme preparation with L-phenylalanine. Specific enzyme activity was defined in nanomoles cinnamic acid per hour per milligram protein. Protein contents were measured according to the method of Bradford (1976), using bovine serum albumin (BSA) as standard.
2.6. Estimation of total phenols Total phenolic content was determined using the procedures described by Zieslin and Ben-Zaken (1993). Briefly, two-gram peel and five-gram flesh samples from six fruit in each treatment were homogenised separately in 15 ml of 80% methanol and agitated for 15 min at 70 °C, and then 0.5 ml methanolic extract was mixed with 0.5 ml Folin–Ciocalteu reagent by manual shaking for 15–20 s. After 3 min, 1 ml saturated sodium carbonate (75 g/l) and 1 ml of distilled water were added. The reaction mixture was incubated in the dark at room temperature for 2 h and its absorbance was measured at 725 nm against deionised water using a spectrophotometer (Shimadzu UV-2550). The content of the total phenols was calculated based on a standard curve obtained from a Folin–Ciocalteu reaction with phenol and expressed as milligrams of phenol equivalents per gram fresh weight.
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2.7. Statistical analysis
3.2. Effect of potassium oxalate on flesh firmness, SSC and TA
Data represent the means ± SD, and they were analysed by oneway analysis of variance (ANOVA) with SPSS 13.0 (SPSS Inc., Chicago, IL) for comparing the treatments on each day. Significance of difference among means of control and treatments using Duncan’s multiple range tests was at the 5% level. Difference at p < 0.05 was considered significant.
Fruit firmness and TA decreased, while SSC increased in mango fruit during storage. However, potassium oxalate treatment not only resulted in a significantly higher fruit firmness index until Day 6 of storage (Fig. 4a), but also retarded significantly the increase in SSC and decrease in TA compared to the control, particularly from 12 to 15 days after harvest (p < 0.05) (Fig. 4b and c). 3.3. Effect of potassium oxalate on activity of defence-related enzymes
3.1. Effect of oxalate treatments on disease index and fruit decay Oxalate treatments including 30 mM potassium oxalate and 30 mM ammonium oxalate did not show efficacy in slowing the deterioration of mango cv. Xiaojinhuang fruit as well as 0.1% prochloraz treatment during storage at room temperature. However, oxalate treatments apparently reduced the disease index and fruit decay in ‘Xiaojinhuang’ mango fruit after storage of at least 8 days compared to the control. Reductions in disease index and fruit decay were 16% and 35% for the fruit treated with 30 mM potassium oxalate, and 14% and 28% with 30 mM ammonium oxalate, respectively, at the end of storage (12 days) (Fig. 1). The prochloraz, potassium oxalate and ammonium oxalate were also effective for inhibiting diseases such as anthracnose. Among the treatments, the control fruit developed more visible anthracnose, compared to fruit treated with prochloraz and oxalate after storage of at least 10 days, with appreciably less incidence observed in all of three treated fruit (Fig. 2). Mango cv. Zill fruit treated with 20 and 40 mM potassium oxalate also showed decreased deterioration during storage at room temperature, and the disease index and fruit decay for potassium oxalate treated fruit were significantly lower than that of control fruit after storage for both 3 and 6 days (Fig. 3).
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The pattern of change in POD activity in the peel of all fruit was similar during storage, but a significant increase in POD activity in the fruit treated with 20 and 40 mM potassium oxalate was observed from 6 to 12 days and 3 to 9 days in storage, respectively, as compared to the control (Fig. 5a). In the flesh, POD activity in fruit treated with both concentrations of potassium oxalate was also significantly higher than that in the control after 6 days of harvest (p < 0.05) (Fig. 5b). PPO activity in the peel of control fruit increased to a peak on the ninth day, then dropped and remained at a relatively steady level for the remainder of the storage time. For fruit treated with 20 and 40 mM potassium oxalate, PPO activity in the peel also
Firmness index
3. Results
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Days at 25°C Fig. 3. Changes in disease index (a), and fruit decay (b) in untreated (control) and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
Days at 25°C Fig. 4. Changes in flesh firmness index (a), SSC (b) and TA (c) in untreated (control), and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
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Fig. 5. Change in activities of POD, PPO and PAL in the peel (Panel a, Panel c and Panel e) and in the flesh (Panel b, Panel d and Panel f) of untreated (control), and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
increased to a peak on the ninth and the sixth days, respectively, and then decreased steadily during the remaining days. PPO activity in fruit treated with 20 and 40 mM potassium oxalate was significantly higher than that in the control at the beginning of the ninth and sixth days, respectively (p < 0.05) (Fig. 5c). Moreover, PPO activity in the flesh of all fruit increased to a peak on the twelfth day and then decreased, with no differences in the activity between the control and treated fruit (Fig. 5d). PAL activity in the peel of all fruit was maintained at a relatively constant level during the storage period whereas in the flesh it markedly decreased at the beginning of the ninth day and then remained steady during the rest days. However, in the peel and in the flesh, the levels of PAL activity in potassium oxalate-treated fruit were similar to those in the control at all time intervals (Fig. 5e and f). 3.4. Effect of potassium oxalate on total phenolic content Concerning the change of total phenols in mango fruits, the peel of Zill mango fruit contained considerably more total phenolic compounds than the flesh, and this content increased to a maximum on the sixth day after harvest and then declined steadily with duration of storage (Fig. 6). However, the total phenolic content in the peel of 20 and 40 mM potassium oxalate treatments showed
significantly high levels from 6 to 12 days and 6 to 9 days in storage, respectively, relative to the control (p < 0.05) (Fig. 6a). In addition, total phenolic content levels in the flesh varied in the range of 0.3–1.5 mg g 1 during storage, with no significant difference between the control and treated fruit (Fig. 5b).
4. Discussion Mango fruit suffer from a brief shelf-life and postharvest deterioration problems as a result of postharvest disease, insect infestation and over-rapid ripening (Mitra & Baldwin, 1997). To prevent decay development and delay ripening process of mango fruit, a number of strategies have been evaluated instead of fungicides, such as the usage of controlled atmosphere storage (Kim, Brecht, & Talcott, 2007) and application of chemical substances, including salicylic acid (Zainuri, Joyce, Wearing, Coates, & Terry, 2001; Zeng, Cao, & Jiang, 2006), 2,4-dichlorophenoxyacetic acid (Kobiler et al., 2001), 1-methylcyclopropene (Singh & Dwivedi, 2008), potassium phosphate (Zainuri et al., 2001), and oxalic acid (Zheng, Tian, Gidley, Yue, & Li, 2007; Zheng et al., 2007). Data here showed that mango cv. Xiaojinhuang fruit pre-storage treated with 30 mM potassium oxalate and 30 mM ammonium oxalate, and mango cv. Zill fruit treated with 20 and 40 mM potassium oxalate
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Days at 25°C Fig. 6. Change in total phenolic content in the peel (a), and in the flesh (b) of untreated (control), and potassium oxalate-treated mango cv. Zill fruit during storage. Data are the means of three replicates ± SD.
successfully decreased postharvest deterioration, as the disease index and fruit decay incidence apparently reduced in treated fruit after storage of several days compared to the control (Figs. 1 and 3). Therefore, these results represented a marked increase in market value for the mango fruit treated with oxalate compared to the control. Softening of the flesh, decreased acidity, increased SSC and total solids, and increased carotenoid pigments are among the recognised parameters of maturity and ripening in mango (Mitra & Baldwin, 1997). Oxalate treatment significantly reduced ripening indices, such as the increase in SSC and decrease in fruit firmness index and TA after storage of 12 or 9 days, respectively, compared with the control (Fig. 4). Therefore, pre-storage application of oxalate delayed the ripening process in mango fruit during storage at room temperature. To our knowledge, this is the first report in which oxalate has been implicated in achieving beneficial effects on decay incidence and ripening in harvested fruit. In addition, a pre-storage dip in a millimolar solution of oxalate did not dramatically raise the price of mango fruit to consumers, as an estimated cost of oxalate treatment was approximately 0.10–0.15 renminbi (RMB) per kilogram. This fact, together with the results that oxalate apparently suppressed postharvest deterioration and extended the shelf-life of mango fruit, suggested that pre-storage oxalate dip might be meaningful in terms of the economics. Generally, defence-related enzymes, including PPO, POD and PAL, are considered potentially important in induced resistance of plants (Sticher, Mauch-Mani, & Métraux, 1997). To date, there are many reports in the literature that increase in activities of PPO, POD and PAL followed by chemical, physical or biological treatments are involved in induced resistance of fruit as well (Chan, Qin, Xu, Li, & Tian, 2007; González-Aguilar, Zavaleta-Gatica, & Tiznado-Hernández, 2007; Tian et al., 2006). Our results indicated that potassium oxalate stimulated significant increase in activities of POD in the fruit peel and flesh, and PPO in the fruit peel, without activation of PAL activity in both the peel and the flesh of mango fruit. Furthermore, phenolic compounds have activity inhibiting pathogens and increase in phenolic content in plants
is correlated with increased resistance to pathogens (Velazhahan & Vidhyasekaran, 1994). Accumulation of phenolic compounds in plant and fruit by abiotic or biotic elicitors has been considered as a defence mechanism. For example, induction of early blight resistance in tomato by Quercus infectoria gall extract is attributed to elevated level of phenolics and enhanced activities of defencerelated enzymes (Yamunarani, Jaganathan, Bhaskaran, Govindaraju, & Velazhahan, 2004). Postharvest UV-C exposition increases the resistance in table grapes (Cantos, Garcia-Viguera, de Pascual-Teresa, & Tomas-Barberan, 2000) and in mango fruit (González-Aguilar et al., 2007) against pathogenic attack by inducing the biosynthesis of large amounts of phenolic compounds. In this experiment, a significant increase in total phenolic content in the peel of mango fruit was also found as a result of the oxalate treatments. Thus, this physiological effect of oxalate treatments in elevating phenolic level and enhancing activities of POD and PPO in mango fruit, particularly marked for the peel, the primary site of potential infestation or infection, might also be involved in induced resistance for mango fruit during storage. This would explain why reduction in fungi infections in whole mango treated with oxalate was observed, and quality of treated mango fruit was consequently maintained for longer periods of time. PAL is the first enzyme of phenylpropanoid metabolism and plays an important role in the regulation of biosynthesis of phenols in plants (Yamunarani et al., 2004), and induction in the level of total phenols in mango fruit by UV-C treatment strongly correlates with the increase in PAL activity (González-Aguilar et al., 2007). Our results were not consistent with this previous report, as total phenols in the peel of mango fruit treated with oxalate were significantly elevated during most of the storage time, without stimulation of PAL enzyme during storage. The different response might be due to the fact of difference in treatment or mango fruit cultivars, and there might be other important enzymes in the phenylpropanoid biosynthetic pathway, such as cinnamate 4-hydroxylase, 4-coumarate CoA ligase and chalcone synthase involved as well. Therefore, for better understanding the possible mode of action of the oxalate treatment, effect of oxalate on those enzymes and specific phytoalexins in mango fruit should be studied. In conclusion, the results obtained in this study showed evidence for the ability of oxalate treatments to limit postharvest deterioration and maintain quality of mango fruit during storage at room temperature. The physiological effects of oxalate that involved enhanced activities of defence-related enzymes such as POD and PPO, and elevated level of total phenols, especially in the fruit peel, collectively contributed to induced resistance of mango Zill fruit against postharvest disease in storage. A pre-storage oxalate dip might be a promising method for suppressing the postharvest deterioration and extending the useful shelf-life of mango fruit.
Acknowledgements The authors acknowledge the financial support provided by National Natural Science Foundation of China (No. 30771509), by the Natural Science Foundation of Guangdong Province, China (No. 7010014), and by the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China (2008).
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