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Influence of chitosan coating combined with spermidine on anthracnose disease and qualities of ‘Nam Dok Mai’ mango after harvest
Scientia Horticulturae 224 (2017) 180–187
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Influence of chitosan coating combined with spermidine on anthracnose disease and qualities of ‘Nam Dok Mai’ mango after harvest Pornchan Jongsria, Pranee Rojsitthisakb, Teerada Wangsomboondeea, Kanogwan Seraypheapa, a b
MARK ⁎
Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Metallurgy and Materials Science Research Institute, Chulalongkorn University, Bangkok 10330, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords: Fruit coating Postharvest storage Plant defense mechanism Fruit softening
Chitosan (CTS) combined with spermidine (SPD) was applied as fruit coating for ‘Nam Dok Mai’ mango (Mangifera indica L.) compared with CTS and SPD treatments after harvest and stored at 25 ± 2 °C for 9 days. Influence of all treatments on anthracnose disease and qualities of mango fruit was investigated after inoculation fruit with Colletotrichum gloeosporioides. Inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD exhibited the smallest area of lesion development (0–1 cm); while non-coating inoculated fruit presented the most severe fungal decay (4–5 cm). Furthermore, inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD showed higher plant defense mechanisms than control and other treated fruits. These phenomena were represented by the production of high levels of H2O2 and phenolic compounds during storage and the induction of defense enzyme activities including chitinase, β-1,3-glucanase and peroxidase whereas fruit treated with only CMS or SPD expressed lower effect on induction of plant defense mechanisms. Therefore, synergistic effect of chitosan and spermidine combination can increase the ability to inhibit anthracnose disease development on ‘Nam Dok Mai’ mango fruit. Delayed mango fruit softening by 1% CTS combined with 0.1 ppm SPD was in correlation with reduced soluble pectin content during ripening stage. The results obtained suggested that 1% CTS combined with 0.1 ppm SPD had potential to improve firmness and delay deterioration processes of ‘Nam Dok Mai’ mango fruit after harvest.
1. Introduction Around the world, mango (Mangifera indica L.) is a popular tropical fruit with its high qualities and nutritional properties. Mango is an important export fruit of Thailand to which the national total production reached 33,000 tons in 2015 (Office of Agricultural Economics (OAE), 2016: online). However, mango is a climacteric fruit, making it perishable after harvest during the ripening process. Furthermore, a major disease of mango fruit that causes reduction of fruit qualities in both preharvest and postharvest is anthracnose stemming from Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Akem, 2006). This disease reduces shelf life of mango tremendously. Therefore, treatments used to control the pathogen development are essential for maintaining fruit qualities. Chitosan, a deacetylated form of chitin, is a natural carbohydrate polymer (Rinaudo, 2006). Chitosan has been eagerly used in agriculture because of its advantageous effects on plant growth and its biodegradable property (Pichyangkura and Chadchawan, 2015). Postharvest coating is well recognized as an effective application on fruits to extend
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Corresponding author. E-mail address: [email protected] (K. Seraypheap).
http://dx.doi.org/10.1016/j.scienta.2017.06.011 Received 2 February 2017; Received in revised form 24 May 2017; Accepted 10 June 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
shelf life and maintain fruit quality during transportation and storage (Olivas et al., 2008; Mattiuz et al., 2015; Munhuweyi et al., 2017). Chitosan could create modified atmosphere when used as fruit coating which could lead to an enhanced shelf life and maintain qualities of fruit during storage. Generally, pathogenesis-related (PR) proteins are defense enzymes that are induced in plants by pathogen infection (Ohashi and Oshima, 1992). PR proteins can be separated into 17 families by protein property (Sels et al., 2008). Chitinase, β-1,3-glucanase and peroxidase (POD) are major PR proteins that gene expressions are also increased when plant is attacked by pathogens. Phenylalanine ammonia-lyase (PAL) is one of the major enzymes in plant defense mechanism that activates after pathogen infection (Passardi et al., 2004). Previous studies indicated that chitosan coating had the potential to prolong shelf life by inducing antioxidant properties, reducing respiration rate, ethylene production, and transpiration and controlling decay of many fruits and vegetables (Hong et al., 2012; Dhall, 2013; Zhang et al., 2013; Jongsri et al., 2016a,b). Chitosan was reported as an exogenous elicitor that could induce plant defense mechanism by increasing activity of defense enzymes such as chitinase, β-1,3-glucanase
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2.2. Fungal isolation and culture
and POD in chitosan coated grapevine leaves, mango and pear fruits (Trotel-Aziz et al., 2006; Jitareerat et al., 2007; Meng et al., 2010). Preharvest chitosan spray and postharvest chitosan coating significantly induced the activities of PAL and POD of table grape fruit while activities decreased in control fruit. Thus, chitosan promoted protection in grape fruit against latent infection of pathogens (Meng et al., 2008). In addition, levels of H2O2, lignin, phytoalexin and phenolic compounds were increased in many plants treated with chitosan (Bhaskara-Reddy et al., 1999; Agrawal et al., 2002). However, the physiological differences of fruit ripening phenomenon in response to chitosan treatment could be due to the molecular weight of the applied chitosan. Jongsri et al. (2016b) reported that among the chitosan coatings tested on ‘Nam Dok Mai’ mango fruit, 1% high molecular weight chitosan (Mw. 360,000 Da) could prolong shelf life of ‘Nam Dok Mai’ mango fruit by delaying respiration rate and ethylene production and inhibiting disease occurrence. Polyamines (PAs), mainly diamine putrescine (Put), triamine spermidine (Spd) and tetraamine spermine (Spm), are organic compounds that are present in all living organisms (Handa and Mattoo, 2010). PAs also play a role in the developmental processes in plant, such as morphogenesis, fruit ripening and responses to biotic and abiotic stresses (Galston and Sawhney, 1990; Ziosi et al., 2006). Changes in PAs content have been correlated with fruit growth during the cell division stage of several annual and woody crops, suggesting that biosynthesis is associated with post-fertilization growth and development of ovary tissues (Slocum and Galston, 1985). During climacteric fruit ripening, pectin is the major component of the primary cell wall and middle lamella which are degraded by pectin degrading enzymes leading to fruit softening (Posé et al., 2015). Polygacturonase (PG) and pectin methyl esterase (PME) are mainly cell wall degrading enzymes that cause the reduction of cell wall structure (Sozzi, 2004). In 1992, Charney et al. studied the effect of three PAs (Put, Spd and Spm) on PME activity in soybean and orange. They presented that PAs could inhibit PME activity by interacting with negative charges of pectic substrate that condition the binding of PME. This report indicated that PAs could control the esterification of pectin within cell wall. Therefore, PAs might have a role in all regulatory mechanisms in which cell-wall enzymes were involved. Furthermore, exogenous PAs could induce levels of endogenous PAs in litchi fruits so it could delay browning, peroxide level and ethylene production (Jiang and Chen, 1995). Previous study by Santivipanond et al. (2012) presented that 0.1 ppm spermidine was the best treatment for maintaining firmness and delaying chemical changes of ‘Hom Thong’ banana fruit. Jongsri et al. (2016a) used a combination of 1% chitosan and 0.1 ppm spermidine to coat ‘Nam Dok Mai’ mango and resulted in higher firmness than other treatments. However, there are still only few reports about the effect of polyamine on postharvest response of mango fruit. Although chitosan and polyamines have been known to control decay and prolong storage life in fruit and vegetable, no reports have been published regarding the use of the combination of chitosan and spermidine to control postharvest disease in mango. The coating application as antimicrobial compounds, elicitor or protective of decay needs to be further explored. We hypothesized that combining spermidine at 0.1 ppm and 1% chitosan coating could present a greater result in the defense mechanisms and delay deterioration processes on ‘Nam Dok Mai’ mango.
The phytopathogenic fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. was isolated from diseased tissues of symptomatic mango fruit and identified by morphological technique (Barnett and Hunter, 1998) and molecular technique (White et al., 1990; Bunyard et al., 1994) by DNA sequencing performed by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Koch’s postulate was used to confirm the causal agent of the disease (Juangbhanich, 1988). Pure culture was grown on potato dextrose agar (PDA) at room temperature for 7 days before usage. 2.3. Experimental treatments Fruit was washed under running water and left to air dry in the laboratory. Mango fruit was surface sterilized by immersion in 70% ethanol for 1 min and prepared for inoculation by inflicting one 1-mmdeep wound in the middle of each fruit with a sterile needle. Each wound was then inoculated with 10 μL conidial suspension (106 conidia/mL) of C. gloeosporioides. The inoculated fruit was incubated overnight at room temperature before dipping treatments (Yenjit et al., 2010). The experiment comprised of 6 treatments: (1) control treatment (inoculated with distilled water) (2) inoculated with C. gloeosporioides then dipped in distilled water (3) inoculated with C. gloeosporioides then dipped in 0.5% acetic acid solution (4) inoculated with C. gloeosporioides then dipped in 1% chitosan (CTS) solution (A.N. Lab Thailand: Mw. 360,000 Da, %DD = 84.90 ± 0.72) combined with 0.1 ppm spermidine (SPD) (Sigma) (5) inoculated with C. gloeosporioides then dipped in 1% CTS solution (6) inoculated with C. gloeosporioides then dipped in 0.1 ppm SPD solution. After treatments, fruit was stored at 25 ± 2 °C for 9 days. There were 3 replications in each treatment and each replication consisting of 2 samples. The result was measured every 3 days during storage. 2.4. Disease severity The diameter of lesion (cm) was measured and calculated after inoculation every 3 days (Jitateerat et al., 2007). 2.5. Physico-chemical analysis 2.5.1. H2O2 content One gram of mango pulp was grinded in liquid nitrogen then cold phosphate buffer (pH 6.5) containing hydroxylamine was added. Tube content was mixed and centrifuged at 8000 rpm for 25 min at 4 °C. Supernatant was added with 0.3% titanium sulphate in 20% H2SO4 (v/ v) and centrifuged at 8000 rpm for 15 min at 4 °C. H2O2 content was measured at 410 nm by spectrophotometrically assay following Jana and Choudhuri (1982). According to standard curve, H2O2 content was shown as μmol/g FW. 2.5.2. Total phenolic content The Folin-Ciocalteu assay, adapted from Ramful et al. (2010), was used for determining total phenolic present in the fruit extracts. One gram of mango pulp was added to 80% methanol. Tube content was centrifuged at 9000 rpm for 20 min at 4 °C. Plant extract and distilled water were mixed with Folin-Ciocalteu reagent (Merck) and incubated at room temperature for 3 min. After incubation, 20% sodium carbonate was added to tube and incubated for 40 min in a water bath at 40 °C. The absorbance of the blue coloration formed was read at 685 nm. Results were expressed in mg of gallic acid/g FW.
2. Materials and methods 2.1. Plant material Mature green mango fruit (Mangifera indica L. cv. Nam Dok Mai) was harvested from a commercial orchard in Nakornratchasrima province (90–100 days after fruit set). Fruit was selected for uniformity in size, color, shape and without any blemishes and disease symptoms and transferred to the Laboratory within 4 h.
2.5.3. Phenylalanine ammonia-lyase activity One gram of mango pulp was used for analyzing following the method of D'Cunha et al. (1996). Mango pulp was homogenized with extraction buffer (Tris-HCl pH 7.0) then centrifuged at 13,000 rpm at 181
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(Hardness tester FHM-1, Takemura, Japan) at 3 regions on fruit (blossom end, middle and stem end of the fruit). Fruit firmness value of each fruit was computed as mean of 3 measurements and was recorded as kg-force in Newtons (N) (Chancharoenrit, 2002).
4 °C for 15 min. The resulting supernatant was used as crude enzymes. Tris-HCl (pH 8.5) containing 2-mercaptoethnaol and L-phenylalanine were used as substrate. The reaction was started when adding crude enzymes into the substrate and incubated at 40 °C for 60 min. Then the reaction was stopped by adding 6 N HCl. The sample was determined by spectrophotometer at 290 nm. Unit of phenylalanine ammonia-lyase activity was defined as 1 μg of cinnamic/h/mg protein.
2.6. Titratable acidity (TA) Method was adjusted from AOAC (1984). Ten grams of sliced mango pulp was grinded and mixed with 100 mL of distilled water by vortex for 1 min. The macerate was filtered by Whatman filter paper No.4 into a 125 mL volumetric flask. Ten mL of filtrate was titrated with NaOH. Phenolphthalein was used as indicator. Then titration was performed until reaching its end point of pinkish hue. The percentage of citric acid was calculated.
2.5.4. Chitinase activity Chitinase activity was measured according to the modified method of Somogyi (1952). One gram of mango pulp was extracted with sodium acetate buffer (pH 5.0) and centrifuged at 13,000 rpm for 20 min at 4 °C. The reaction was started when mixed crude enzyme with colloidal chitin (in sodium acetate buffer pH 5.0) then incubated at 37 °C for 60 min. The reaction was stopped by adding 3,5-dinitrosalicylic acid and boiled samples in water bath at 100 °C for 5 min. The absorbance of N-acetyl-D-glucosamine (GlcNAc) forms was read at 550 nm. One unit of enzyme is defined as the amount of enzyme which produces 1 μg of GlcNAc/h/mg protein.
%TA = NaOH(mL) × Conc. NaOH(N) × 0.07 × 100 10g 2.7. Total soluble solids (TSS) content Five grams of sliced mango pulp was grinded and mixed with 20 mL of distilled water by vortex for 1 min. Then macerate was filtered by Whatman filter paper No. 4 into a 50 mL volumetric flask. One mL of filtrate was used for measuring total soluble solids with a hand refractometer (N-1E, Japan) and TSS was expressed as °Brix.
2.5.5. β-1,3-glucanase activity β-1,3-glucanase activity was done as described by Somogyi (1952). Crude enzyme was extracted by adding sodium acetate buffer (pH 5.0) into one gram of mango pulp and centrifuged at 13,000 rpm for 20 min at 4 °C. Crude enzyme was mixed with laminarin (in sodium acetate buffer pH 5.0) then incubated at 37 °C for 60 min. The reaction was stopped by adding 3,5-dinitrosalicylic acid and boiled samples in water at 100 °C for 5 min. The absorbance of D-glucose was read at 550 nm. One unit of enzyme is defined as the amount of enzyme which produces 1 μg of D-glucose/h/mg protein.
2.8. Statistical analysis Data were analyzed using IBM SPSS V.20 software (SPSS Inc., USA). Statistical comparisons were made by one-way analysis of variance (ANOVA). Mean differences were regarded as significant when the pvalues were less than 0.05. Mean separations were performed by employing Duncan’s Multiple Range Test comparison procedure.
2.5.6. Peroxidase activity Peroxidase activity was measured by method of MacAdam et al. (1992). One gram of mango pulp was used for extraction. Crude enzyme was extracted by adding potassium phosphate buffer (pH 6.0) and centrifuged at 13,000 rpm for 20 min at 4 °C. The substrate solution containing potassium phosphate buffer (pH 6.0), H2O2 (0.2 mM) and guaiacol (3.0 mM) were prepared then the reaction was started by adding crude enzyme into substrate solution. The kinetic of peroxidase activity was measured by monitoring changes in absorbance at 470 nm for 5 min. One unit of peroxidase activity represented 1 μmol of tetraguaiacol/min/mg protein.
3. Results 3.1. Antifungal effect of chitosan coating combined with spermidine on disease severity and 3.1.1. Lesion diameter of mango fruit inoculated with C. gloeosporioides The postharvest disease of ‘Nam Dok Mai’ mango, anthracnose, caused by C. gloeosporioides, resulted in fruit decay with disease lesions (1–3 cm) 3 days after inoculation on inoculated fruit dipped in distilled water, 0.5% acetic acid and 0.1 ppm SPD treatments. Whereas, control fruit and inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD or 1% CTS alone did not show fungal decay (Fig. 1). Disease lesion development measured every 3 days after inoculation revealed that inoculated fruit dipped in distilled water, 0.5% acetic acid and 0.1 ppm SPD treatments significantly presented more disease severity during storage (4–5 cm at day 9) than control fruit and inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD or 1% CTS alone (Figs. 1 and 2). Therefore, fungal development in infected ‘Nam Dok Mai’ mango was inhibited in this experiment.
2.5.7. Total protein assay Total protein assay as described by Bradford (1976) was conducted. Fifty μL Bradford dye reagent (BioRad) was added to test tubes containing 50 μL enzyme extract samples and 100 μL distilled water and the tubes were incubated at room temperature for 5 min. The samples were then thoroughly mixed and read at a wavelength of 595 nm in a spectrophotometer. Bovine serum albumin (BSA) was used as the standard protein. 2.5.8. Soluble pectin content Soluble pectin content was analyzed based on the methods of Robertson (1979). One gram of mango pulp was used for soluble pectin extraction. Extraction was diluted with distilled water and added with sodium tetraborate decahydrate (in conc. HCl). After mixing, tube content was boiled in waterbath at 100 °C for 10 min, then added with 0.1 mL mhydroxydiphenyl (in 0.5%NaOH) and incubated at room temperature for 15 min. Soluble pectin content was read at 410 nm by spectrophotometer. Values were calculated by standard curve of D-galacturonic acid (μg/g fresh weight (FW)).
3.2. Effect of chitosan coating combined with spermidine on physicochemical changes of ‘Nam Dok Mai’ mango fruit 3.2.1. H2O2, total phenolic contents and PAL activity H2O2 level in fruit of all treatments increased during 6 days after inoculation then decreased until the end of storage. High level of H2O2 content was promoted in inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD and 1% CTS treatments on day 6 with significant differences from control treatment (Fig. 3A). Total phenolic content was also increased on day 6 after inoculation until the end of storage. Inoculated fruit coated with 1% chitosan combined with 0.1 ppm SPD significantly showed the highest value of phenolic content during storage compared to control treatment (Fig. 3B). Mango fruit represented
2.5.9. Fruit firmness Pulp firmness was measured by using handheld penetrometer 182
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Fig. 1. Disease severity of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days.
3.2.3. Soluble pectin content and firmness Soluble pectin content highly increased on day 3 then gradually decreased until the end of storage. Fruit coated with 1% CTS combined with 0.1 ppm SPD significantly showed lower soluble pectin content than control treatment. Inoculated fruit and control fruit presented the highest soluble pectin content on day 3 and 6 during storage, respectively. After that, soluble pectin content slightly reduced until the end of storage (Table 1). Fruit firmness rapidly decreased on day 3 after storage then gradually decreased until the last day of storage. Only 1% CTS combined with 0.1 ppm SPD and 1% CTS coating alone had significant effect to inhibit fruit softening by maintaining fruit firmness throughout the experiment. The other 3 treatments had no differences in firmness when compared to the control treatment (Table 1).
the highest PAL activity on day 6 whereas control fruit presented the lowest PAL activity. However, there were no significant differences in PAL activity among treatments (Fig. 3C).
3.2.2. Chitinase, β-1,3-glucanase and peroxidase activities Activities of defense enzymes, chitinase and β-1,3-glucanase, decreased in the first 3 days after treatments then increased until day 6 in all treatments except for control and inoculated fruit dipped in 0.1 ppm SPD treatments. Inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD significantly presented the highest chitinase and β-1,3glucanase activities until the end of storage (Fig. 4A and B). All treatments except for control and inoculated fruit dipped in 0.1 ppm SPD significantly presented high level of peroxidase activities on day 3 after inoculation (Fig. 4C) then enzyme activities was sharply decreased. Overall, it was evident that 1% CTS combined with 0.1 ppm SPD showed strong effect to induce chitinase, β-1,3-glucanase and peroxidase activities.
3.2.4. Titratable acidity (TA) and total soluble solids (TSS) contents The changes of TA and TSS contents were observed after 3 days of storage. TA instantaneously decreased on day 3 (from 2% to 0.3%) in all treatments. After that, TA slightly decreased until the end of storage Fig. 2. Disease lesion diameter (cm.) of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Vertical bars represent ± SE.
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Fig. 3. H2O2 (A), total phenolic (B) and PAL activity (C) of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Each data point was calculated from three replications. Vertical bars represent ± SE.
combined with 0.1 ppm SPD significantly presented the highest H2O2 content. In addition, phenolic compounds are well-known antimicrobial compounds in plants (Sivaprakasan and Vidhyasekaran, 1993; Schlösser, 1994). According to Matern and Kneusel (1988), plant expressed rapid accumulation of phenols at the first step of the defense mechanism after infection by a pathogen which limited or slowed down pathogen growth. Our result showed that phenolic content slightly increased on day 6 after inoculation until the end of storage. Fruit coated with 1% CTS combined with 0.1 ppm SPD exhibited higher value of total phenolic content than other treatments. The accumulation of H2O2, phenolic, lignin and phytoalexin were increased in many plants treated with chitosan such as wheat seed, pea pods and rice (BhaskaraReddy et al., 1999; Agrawal et al., 2002). Moreover, previous experiment by Yamakawa et al. (1998) indicated that spermine functions as a signal molecule to transduce defense responses. According to the study by Takahashi et al. (2004), spermine acted as an endogenous inducer of pathogenesis-related (PR) proteins during TMV-induced HR resulting in the production of H2O2 into the cytosol. The exogenous spermine specifically activated the expression of four HR marker genes in tobacco leaves. Activation of these HR marker genes may provide to defense resistance against pathogen attack to host plant and lead to programmed cell. Therefore, combining chitosan and spermidine in our experiment completely presented the best effect in increasing the accumulation of H2O2 and phenolic production leading to the induction of plant defense mechanisms for protecting mango fruit from pathogen infection. Our research found that the treatment could enhance activities of chitinase, β-1,3-glucanase and peroxidase resulting in suppress
time. Fruit coated with 1% CTS combined with 0.1 ppm SPD significantly presented higher TA content during storage than control and 1% CTS treatments (Table 1). In this study, TSS swiftly increased on day 3 and day 6 then gradually decreased. TSS values were differences between 1% CTS combined with 0.1 ppm SPD treatment and other treatments on day 6. The lowest TSS presented in 1% CTS combined with 0.1 ppm SPD conferred delayed ripening process of ‘Nam Dok Mai’ mango fruit (Table 1). 4. Discussions In Thailand, anthracnose is a major constraint in ‘Nam Dok Mai’ mango fruit production and export. Chemical fungicides are routinely applied to control the pathogen which causes environmental toxicity and residual effect. Combining chitosan with spermidine as fruit coating had a potential to control disease severity of inoculated mango fruit by inhibiting fungal decay and reduce fruit softening during storage. The present investigation clearly indicated that the combination of 1% CTS combined with 0.1 ppm SPD provided better effect on inhibiting fungal development than solely chitosan usage. Inducing plant defense mechanisms such as H2O2, total phenolic content, chitinase, β1,3-glucanase and peroxidase activities were involved. Plant promoted H2O2 as hypersensitive response in program cell death after plant was infected by pathogens (Levine et al., 1994; Tenhaken et al., 1995). H2O2 had the ability to inhibit the growth and viability of diverse microbial pathogens (Kiraly et al., 1993; Wu et al., 1995). Our study showed that ‘Nam Dok Mai’ coated with 1% CTS 184
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Fig. 4. Chitinase (A), β-1,3-glucanase (B) and peroxidase (C) activities of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Each data point was calculated from three replications. Vertical bars represent ± SE.
Table 1 Influence of chitosan coating combined with spermidine on soluble pectin content, firmness, % titratable acidity (TA) and total soluble solid (TSS) of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Storage (days)
Treatment
Soluble pectin content (μg/g FW)
Firmness (N)
TA (%)
Day 0
Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD
1.57 1.57 1.57 1.57 1.57 1.57 2.97 3.06 2.25 1.97 2.59 2.72 2.72 1.82 1.18 1.88 2.04 1.13 0.63 0.95 0.80 1.72 1.87 0.65
7.54 7.54 7.54 7.54 7.54 7.54 5.11 4.97 5.11 5.63 5.40 5.16 4.83 4.65 4.83 5.44 5.39 4.74 4.58 4.26 4.39 5.40 5.22 4.73
1.91 1.91 1.91 1.91 1.91 1.91 0.26 0.29 0.32 0.39 0.21 0.30 0.25 0.21 0.27 0.25 0.16 0.26 0.12 0.11 0.10 0.23 0.09 0.11
Day 3
Day 6
Day 9 a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.30a 0.30a 0.30a 0.30a 0.30a 0.30a 0.35c 0.14c 0.08ab 0.25a 0.07abc 0.21bc 0.32b 0.36ab 0.41a 0.29ab 0.17ab 0.22a 0.14a 0.19ab 0.14a 0.34bc 0.33c 0.08a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.24a 0.24a 0.24a 0.24a 0.24a 0.24a 0.16b 0.16b 0.11b 0.20a 0.07ab 0.12b 0.16b 0.12b 0.10b 0.25a 0.09a 0.20b 0.13b 0.17b 0.18b 0.11a 0.19a 0.13b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
TSS (°Brix) 0.01a 0.01a 0.01a 0.01a 0.01a 0.01a 0.01bc 0.02bc 0.02ab 0.06a 0.03c 0.01ab 0.24ab 0.31ab 0.14a 0.47ab 0.15b 0.35ab 0.01b 0.01b 0.01b 0.02a 0.01b 0.01b
Values followed by the same letter in the column on the same day were not significantly different according to Duncan’s Multiple Range Test (P < 0.05).
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16.00 16.00 16.00 16.00 16.00 16.00 22.50 22.75 20.75 19.88 20.00 20.63 24.00 24.75 25.00 20.00 23.00 23.00 20.75 24.50 22.75 19.50 21.90 20.75
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.74a 0.74a 0.74a 0.74a 0.74a 0.74a 1.50a 0.98a 0.25a 0.72a 1.48a 0.94a 1.10b 1.33b 1.32b 0.63a 0.63ab 1.00ab 1.12ab 1.26b 1.66ab 1.34a 1.87ab 1.05ab
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fungal development in mango fruit. This finding is in agreement with the previous observation in strawberry and raspberry fruits (Zhang and Quantick, 1997). They reported that strawberries and raspberries coated with chitosan showed high level of chitinase and β-1,3-glucanase activities. Activity of peroxidase in cucumber plants was also induced after treated with chitosan (Ben-Shalom et al., 2003; Lin et al., 2005). During ripening, fruit softening is an important aspect which is associated with extensive structural alterations within pectin matrix leading to loss of cell wall structure (Seymour et al., 1990 Posé et al., 2015). Pectin is degraded by pectin degrading enzymes becoming soluble pectin which is always increased during ripening period (Redgwell et al., 1997a,b). The combination of 1% CTS combined with 0.1 ppm SPD significantly inhibited the increase in soluble pectin content and retained fruit firmness. The results are in agreement with the findings of Liu et al. (2016) where chitosan coating delayed softening of cherimoya (Annona cherimola Mill.) fruit and significantly affected the expression of several cell wall-related genes. Similarly, banana fruit showed a remarkable increase of soluble pectin level during ripening (Duan et al., 2008) and the amount of soluble pectin in strawberry also increased from the small green to the white stage (Rosli et al., 2004). In addition, previous study in citrus fruit showed that polyamines mainly interacted with negative charges of pectic substance which condition the binding of PME thus it could inhibit PME activity leading to maintain fruit firmness (Charney et al., 1992). Similarly, spermine or spermidine treatment could retard softening of apple and strawberry fruits (Kramer et al., 1991; Ponappa et al., 1993). The maximum ‘Summer Bahisht Chaunsa’ mango firmness was reported in fruit coated with crab chitosan (Abbasi et al., 2009). Chitosan had ability to avoid shrinkage and reduction in cell wall loosening which in turn maintained cell integrity (Salunkhe and Desai, 1984; Medlicott et al., 1986). Chitosan coatings have been used for controlling the internal gas atmosphere of fruits and vegetables. It acted as a barrier to water vapor for reducing moisture loss and delaying fruit dehydration that could maintain fruit firmness and protect weight loss (Srinivasa et al., 2002; Ribeiro et al., 2007; Duan et al., 2011). In 2016a, Jongsri et al. reported that 1% chitosan combined with 0.1 ppm spermidine significantly induced endogenous polyamines including free putresine, spermidine and spermine in ‘Nam Dok Mai’ mango when compared with control fruit and other treatments. Higher endogenous polyamines in fruit resulted in higher fruit firmness in these fruits. Moreover, coating with 1% chitosan combined with 0.1 ppm spermidine also maintained fresh weight and retarded peel color changes. However, our results found that fruit treated with only chitosan or spermidine presented lower properties for prolong shelf life of mango fruit. Therefore, the combination of 1% chitosan and 0.1 ppm spermidine has synergistic effect on fruit qualities. Using a suitable coating could maintain fruit firmness, reduction of respiration and water loss (Tasdelen and Bayindirli, 1998). Besides fruit softening, TSS and TA are very important quality factors used to estimate the storage longevity of mango. TSS refer to sugar within fruit that always increases during ripening stage and TA always decreases during ripening stage of the fruit, which are major characteristic for climacteric fruit ripening (Mahto and Das, 2013). During storage, we found that TA swiftly decreased on day 3 in all treatments and TSS instantaneously increased until the end of storage period. Fruit coated with 1% CTS combined with 0.1 ppm SPD presented the highest TA content and the lowest TSS content during storage. This same result was observed with the use of chitosan-lactoperoxidase coating in “Kent” mango. Uncoated mangoes showed the maximum of TSS content and pH and the minimum of TA acidity (Cissé et al., 2015). In addition, dipping papaya fruits in spermine could prolong shelf life by delaying fruit softening and the increase of TSS and peel color changes (Purwoko et al., 1998). Previous study by Santivipanond et al. (2012) reported that spermidine at 0.1 ppm could improve firmness and delayed TSS in ‘Hom Thong’ bananas. During postharvest storage, acid metabolism
converted starch and acid to sugar resulting in the decrease of TA and the increase of TSS (Duan et al., 2011). Apricot, plum, kiwi and lemon fruits treated with putresine showed higher fruit firmness and titratable acidity, lower soluble solid content, ethylene production and respiration rate, reduced weight loss and delayed color changes leading to extend storage life of fruit. Moreover, chitosan coating could delay respiration rate and ethylene production leading to retain qualities in citrus and mango fruits (Chien et al., 2007; Jitareerat et al., 2007; Jongsri et al., 2016b). Therefore, combining 1% CTS and 0.1 ppm SPD could inhibit deterioration processes of mango fruit resulting in the delay chemical changes in fruit during ripening stage. 5. Conclusions One percent CTS combined with 0.1 ppm SPD could prolong shelf life and maintain qualities of ‘Nam Dok Mai’ mango fruit after harvest and was the appropriate coating for postharvest ‘Nam Dok Mai’ mango. Furthermore, 1% CTS combined with 0.1 ppm SPD had potential to control anthracnose disease caused by C. gloeosporioides and induce defense mechanisms in ‘Nam Dok Mai’ mango fruit. Therefore, this formulated coating can be beneficial coating for commercial use in postharvest storage of mango fruit. Acknowledgements The first author was recipient of a Postdoctoral Fellowship, Rachadapisek Sompote Fund, Chulalongkorn University. The authors would like to thank National Research Project Management (NRPM) for their financial support. References AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed. AOAC, Washington, DC. Abbasi, N.A., Iqbal, Z., Maqbool, M., Hafiz, I.A., 2009. Postharvest quality of mango (Mangifera indica L.) fruit as affected by chitosan coating. Pak. J Bot. 41 (1), 343–357. Agrawal, G.K., Rakwal, R., Tamogami, S., Yonekura, M., Kubo, A., Saji, H., 2002. Chitosan activates defense/stress response(s) in the leaves of Oryza saliva seedlings. Plant Physiol. Biochem. 40, 1061–1069. Akem, C.N., 2006. Mango anthracnose disease: present status and future research priorities. Plant Pathol. J. 5, 266–273. Barnett, H.L., Hunter, B.B., 1998. Illustrated Genera of Imperfect Fungi, 4thed. APS press, Saint Paul, Minnnesota, America. Ben-Shalom, N., Ardi, R., Pinto, R., Aki, C., Fallik, E., 2003. Controlling gray mold caused By Botrytis cinerea in cucumber plants by means of chitosan. Crop Prot. 22, 285–290. Bhaskara-Reddy, M.V., Arul, J., Angers, P., Couture, L., 1999. Chitosan treatment of wheat seed induces resistance to Fusarium gramninearum and improves seed quality. J. Agric. Food Chem. 47, 1208–1216. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bunyard, B.A., Nicholson, M.S., Royse, D.J., 1994. A systematic assessment of Morchella with RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86, 762–772. Chancharoenrit, J., 2002. Effects of Hot Water Dips on Physiological Changes and Chilling Injury After Storage of ‘Hom Thong’ Variety of Banana. Master’s Thesis. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. Charney, D., Nari, J., Noat, G., 1992. Regulation of plant cell-wall pectin methyl esterase by polyamines-Interactions with the effects of metal ions. Eur. J. Biochem. 205, 711–714. Chien, P.J., Sheu, F., Lin, H.R., 2007. Coating citrus (Murcott tangor) fruit with low molecular weight chitosan increases postharvest quality and shelf life. Food Chem. 100, 1120–1164. Cissé, M., Polidori, J., Montet, D., Loiseau, G., Ducamp-Collin, M.N., 2015. Preservation of mango quality by using functional chitosan lactoperoxidase systems coatings. Postharvest Biol. Technol. 101, 10–14. D’Cunha, G.B., Satyanaraan, V., Nair, P.M., 1996. Stabilization of phenylalanine ammonia- lyase containing Rhodotorula glutatinis cells for the continuous synthesis of phenylalanine methyl ester. Enzyme Microbial. Technol. 19, 421–427. Dhall, R.K., 2013. Advances in edible coatings for fresh fruits and vegetables: a review. Crit. Rev. Food Sci. Nutr. 53 (5), 435–450. Duan, X., Cheng, G., Yang, E., Yi, C., Ruenroengklin, N., Lu, W., Luo, Y., Jiang, Y., 2008. Modification of pectin polysaccharides during ripening of postharvest banana fruit. Food Chem. 111, 144–149. Duan, J., Wu, R., Strik, B.C., Zhao, Y., 2011. Effect of edible coatings on the quality of fresh blueberries (Duke and Elliott) under commercial storage conditions. Postharvest Biol. Technol. 59, 71–79.
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Influence of chitosan coating combined with spermidine on anthracnose disease and qualities of ‘Nam Dok Mai’ mango after harvest Pornchan Jongsria, Pranee Rojsitthisakb, Teerada Wangsomboondeea, Kanogwan Seraypheapa, a b
MARK ⁎
Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Metallurgy and Materials Science Research Institute, Chulalongkorn University, Bangkok 10330, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords: Fruit coating Postharvest storage Plant defense mechanism Fruit softening
Chitosan (CTS) combined with spermidine (SPD) was applied as fruit coating for ‘Nam Dok Mai’ mango (Mangifera indica L.) compared with CTS and SPD treatments after harvest and stored at 25 ± 2 °C for 9 days. Influence of all treatments on anthracnose disease and qualities of mango fruit was investigated after inoculation fruit with Colletotrichum gloeosporioides. Inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD exhibited the smallest area of lesion development (0–1 cm); while non-coating inoculated fruit presented the most severe fungal decay (4–5 cm). Furthermore, inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD showed higher plant defense mechanisms than control and other treated fruits. These phenomena were represented by the production of high levels of H2O2 and phenolic compounds during storage and the induction of defense enzyme activities including chitinase, β-1,3-glucanase and peroxidase whereas fruit treated with only CMS or SPD expressed lower effect on induction of plant defense mechanisms. Therefore, synergistic effect of chitosan and spermidine combination can increase the ability to inhibit anthracnose disease development on ‘Nam Dok Mai’ mango fruit. Delayed mango fruit softening by 1% CTS combined with 0.1 ppm SPD was in correlation with reduced soluble pectin content during ripening stage. The results obtained suggested that 1% CTS combined with 0.1 ppm SPD had potential to improve firmness and delay deterioration processes of ‘Nam Dok Mai’ mango fruit after harvest.
1. Introduction Around the world, mango (Mangifera indica L.) is a popular tropical fruit with its high qualities and nutritional properties. Mango is an important export fruit of Thailand to which the national total production reached 33,000 tons in 2015 (Office of Agricultural Economics (OAE), 2016: online). However, mango is a climacteric fruit, making it perishable after harvest during the ripening process. Furthermore, a major disease of mango fruit that causes reduction of fruit qualities in both preharvest and postharvest is anthracnose stemming from Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Akem, 2006). This disease reduces shelf life of mango tremendously. Therefore, treatments used to control the pathogen development are essential for maintaining fruit qualities. Chitosan, a deacetylated form of chitin, is a natural carbohydrate polymer (Rinaudo, 2006). Chitosan has been eagerly used in agriculture because of its advantageous effects on plant growth and its biodegradable property (Pichyangkura and Chadchawan, 2015). Postharvest coating is well recognized as an effective application on fruits to extend
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Corresponding author. E-mail address: [email protected] (K. Seraypheap).
http://dx.doi.org/10.1016/j.scienta.2017.06.011 Received 2 February 2017; Received in revised form 24 May 2017; Accepted 10 June 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
shelf life and maintain fruit quality during transportation and storage (Olivas et al., 2008; Mattiuz et al., 2015; Munhuweyi et al., 2017). Chitosan could create modified atmosphere when used as fruit coating which could lead to an enhanced shelf life and maintain qualities of fruit during storage. Generally, pathogenesis-related (PR) proteins are defense enzymes that are induced in plants by pathogen infection (Ohashi and Oshima, 1992). PR proteins can be separated into 17 families by protein property (Sels et al., 2008). Chitinase, β-1,3-glucanase and peroxidase (POD) are major PR proteins that gene expressions are also increased when plant is attacked by pathogens. Phenylalanine ammonia-lyase (PAL) is one of the major enzymes in plant defense mechanism that activates after pathogen infection (Passardi et al., 2004). Previous studies indicated that chitosan coating had the potential to prolong shelf life by inducing antioxidant properties, reducing respiration rate, ethylene production, and transpiration and controlling decay of many fruits and vegetables (Hong et al., 2012; Dhall, 2013; Zhang et al., 2013; Jongsri et al., 2016a,b). Chitosan was reported as an exogenous elicitor that could induce plant defense mechanism by increasing activity of defense enzymes such as chitinase, β-1,3-glucanase
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2.2. Fungal isolation and culture
and POD in chitosan coated grapevine leaves, mango and pear fruits (Trotel-Aziz et al., 2006; Jitareerat et al., 2007; Meng et al., 2010). Preharvest chitosan spray and postharvest chitosan coating significantly induced the activities of PAL and POD of table grape fruit while activities decreased in control fruit. Thus, chitosan promoted protection in grape fruit against latent infection of pathogens (Meng et al., 2008). In addition, levels of H2O2, lignin, phytoalexin and phenolic compounds were increased in many plants treated with chitosan (Bhaskara-Reddy et al., 1999; Agrawal et al., 2002). However, the physiological differences of fruit ripening phenomenon in response to chitosan treatment could be due to the molecular weight of the applied chitosan. Jongsri et al. (2016b) reported that among the chitosan coatings tested on ‘Nam Dok Mai’ mango fruit, 1% high molecular weight chitosan (Mw. 360,000 Da) could prolong shelf life of ‘Nam Dok Mai’ mango fruit by delaying respiration rate and ethylene production and inhibiting disease occurrence. Polyamines (PAs), mainly diamine putrescine (Put), triamine spermidine (Spd) and tetraamine spermine (Spm), are organic compounds that are present in all living organisms (Handa and Mattoo, 2010). PAs also play a role in the developmental processes in plant, such as morphogenesis, fruit ripening and responses to biotic and abiotic stresses (Galston and Sawhney, 1990; Ziosi et al., 2006). Changes in PAs content have been correlated with fruit growth during the cell division stage of several annual and woody crops, suggesting that biosynthesis is associated with post-fertilization growth and development of ovary tissues (Slocum and Galston, 1985). During climacteric fruit ripening, pectin is the major component of the primary cell wall and middle lamella which are degraded by pectin degrading enzymes leading to fruit softening (Posé et al., 2015). Polygacturonase (PG) and pectin methyl esterase (PME) are mainly cell wall degrading enzymes that cause the reduction of cell wall structure (Sozzi, 2004). In 1992, Charney et al. studied the effect of three PAs (Put, Spd and Spm) on PME activity in soybean and orange. They presented that PAs could inhibit PME activity by interacting with negative charges of pectic substrate that condition the binding of PME. This report indicated that PAs could control the esterification of pectin within cell wall. Therefore, PAs might have a role in all regulatory mechanisms in which cell-wall enzymes were involved. Furthermore, exogenous PAs could induce levels of endogenous PAs in litchi fruits so it could delay browning, peroxide level and ethylene production (Jiang and Chen, 1995). Previous study by Santivipanond et al. (2012) presented that 0.1 ppm spermidine was the best treatment for maintaining firmness and delaying chemical changes of ‘Hom Thong’ banana fruit. Jongsri et al. (2016a) used a combination of 1% chitosan and 0.1 ppm spermidine to coat ‘Nam Dok Mai’ mango and resulted in higher firmness than other treatments. However, there are still only few reports about the effect of polyamine on postharvest response of mango fruit. Although chitosan and polyamines have been known to control decay and prolong storage life in fruit and vegetable, no reports have been published regarding the use of the combination of chitosan and spermidine to control postharvest disease in mango. The coating application as antimicrobial compounds, elicitor or protective of decay needs to be further explored. We hypothesized that combining spermidine at 0.1 ppm and 1% chitosan coating could present a greater result in the defense mechanisms and delay deterioration processes on ‘Nam Dok Mai’ mango.
The phytopathogenic fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. was isolated from diseased tissues of symptomatic mango fruit and identified by morphological technique (Barnett and Hunter, 1998) and molecular technique (White et al., 1990; Bunyard et al., 1994) by DNA sequencing performed by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. Koch’s postulate was used to confirm the causal agent of the disease (Juangbhanich, 1988). Pure culture was grown on potato dextrose agar (PDA) at room temperature for 7 days before usage. 2.3. Experimental treatments Fruit was washed under running water and left to air dry in the laboratory. Mango fruit was surface sterilized by immersion in 70% ethanol for 1 min and prepared for inoculation by inflicting one 1-mmdeep wound in the middle of each fruit with a sterile needle. Each wound was then inoculated with 10 μL conidial suspension (106 conidia/mL) of C. gloeosporioides. The inoculated fruit was incubated overnight at room temperature before dipping treatments (Yenjit et al., 2010). The experiment comprised of 6 treatments: (1) control treatment (inoculated with distilled water) (2) inoculated with C. gloeosporioides then dipped in distilled water (3) inoculated with C. gloeosporioides then dipped in 0.5% acetic acid solution (4) inoculated with C. gloeosporioides then dipped in 1% chitosan (CTS) solution (A.N. Lab Thailand: Mw. 360,000 Da, %DD = 84.90 ± 0.72) combined with 0.1 ppm spermidine (SPD) (Sigma) (5) inoculated with C. gloeosporioides then dipped in 1% CTS solution (6) inoculated with C. gloeosporioides then dipped in 0.1 ppm SPD solution. After treatments, fruit was stored at 25 ± 2 °C for 9 days. There were 3 replications in each treatment and each replication consisting of 2 samples. The result was measured every 3 days during storage. 2.4. Disease severity The diameter of lesion (cm) was measured and calculated after inoculation every 3 days (Jitateerat et al., 2007). 2.5. Physico-chemical analysis 2.5.1. H2O2 content One gram of mango pulp was grinded in liquid nitrogen then cold phosphate buffer (pH 6.5) containing hydroxylamine was added. Tube content was mixed and centrifuged at 8000 rpm for 25 min at 4 °C. Supernatant was added with 0.3% titanium sulphate in 20% H2SO4 (v/ v) and centrifuged at 8000 rpm for 15 min at 4 °C. H2O2 content was measured at 410 nm by spectrophotometrically assay following Jana and Choudhuri (1982). According to standard curve, H2O2 content was shown as μmol/g FW. 2.5.2. Total phenolic content The Folin-Ciocalteu assay, adapted from Ramful et al. (2010), was used for determining total phenolic present in the fruit extracts. One gram of mango pulp was added to 80% methanol. Tube content was centrifuged at 9000 rpm for 20 min at 4 °C. Plant extract and distilled water were mixed with Folin-Ciocalteu reagent (Merck) and incubated at room temperature for 3 min. After incubation, 20% sodium carbonate was added to tube and incubated for 40 min in a water bath at 40 °C. The absorbance of the blue coloration formed was read at 685 nm. Results were expressed in mg of gallic acid/g FW.
2. Materials and methods 2.1. Plant material Mature green mango fruit (Mangifera indica L. cv. Nam Dok Mai) was harvested from a commercial orchard in Nakornratchasrima province (90–100 days after fruit set). Fruit was selected for uniformity in size, color, shape and without any blemishes and disease symptoms and transferred to the Laboratory within 4 h.
2.5.3. Phenylalanine ammonia-lyase activity One gram of mango pulp was used for analyzing following the method of D'Cunha et al. (1996). Mango pulp was homogenized with extraction buffer (Tris-HCl pH 7.0) then centrifuged at 13,000 rpm at 181
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(Hardness tester FHM-1, Takemura, Japan) at 3 regions on fruit (blossom end, middle and stem end of the fruit). Fruit firmness value of each fruit was computed as mean of 3 measurements and was recorded as kg-force in Newtons (N) (Chancharoenrit, 2002).
4 °C for 15 min. The resulting supernatant was used as crude enzymes. Tris-HCl (pH 8.5) containing 2-mercaptoethnaol and L-phenylalanine were used as substrate. The reaction was started when adding crude enzymes into the substrate and incubated at 40 °C for 60 min. Then the reaction was stopped by adding 6 N HCl. The sample was determined by spectrophotometer at 290 nm. Unit of phenylalanine ammonia-lyase activity was defined as 1 μg of cinnamic/h/mg protein.
2.6. Titratable acidity (TA) Method was adjusted from AOAC (1984). Ten grams of sliced mango pulp was grinded and mixed with 100 mL of distilled water by vortex for 1 min. The macerate was filtered by Whatman filter paper No.4 into a 125 mL volumetric flask. Ten mL of filtrate was titrated with NaOH. Phenolphthalein was used as indicator. Then titration was performed until reaching its end point of pinkish hue. The percentage of citric acid was calculated.
2.5.4. Chitinase activity Chitinase activity was measured according to the modified method of Somogyi (1952). One gram of mango pulp was extracted with sodium acetate buffer (pH 5.0) and centrifuged at 13,000 rpm for 20 min at 4 °C. The reaction was started when mixed crude enzyme with colloidal chitin (in sodium acetate buffer pH 5.0) then incubated at 37 °C for 60 min. The reaction was stopped by adding 3,5-dinitrosalicylic acid and boiled samples in water bath at 100 °C for 5 min. The absorbance of N-acetyl-D-glucosamine (GlcNAc) forms was read at 550 nm. One unit of enzyme is defined as the amount of enzyme which produces 1 μg of GlcNAc/h/mg protein.
%TA = NaOH(mL) × Conc. NaOH(N) × 0.07 × 100 10g 2.7. Total soluble solids (TSS) content Five grams of sliced mango pulp was grinded and mixed with 20 mL of distilled water by vortex for 1 min. Then macerate was filtered by Whatman filter paper No. 4 into a 50 mL volumetric flask. One mL of filtrate was used for measuring total soluble solids with a hand refractometer (N-1E, Japan) and TSS was expressed as °Brix.
2.5.5. β-1,3-glucanase activity β-1,3-glucanase activity was done as described by Somogyi (1952). Crude enzyme was extracted by adding sodium acetate buffer (pH 5.0) into one gram of mango pulp and centrifuged at 13,000 rpm for 20 min at 4 °C. Crude enzyme was mixed with laminarin (in sodium acetate buffer pH 5.0) then incubated at 37 °C for 60 min. The reaction was stopped by adding 3,5-dinitrosalicylic acid and boiled samples in water at 100 °C for 5 min. The absorbance of D-glucose was read at 550 nm. One unit of enzyme is defined as the amount of enzyme which produces 1 μg of D-glucose/h/mg protein.
2.8. Statistical analysis Data were analyzed using IBM SPSS V.20 software (SPSS Inc., USA). Statistical comparisons were made by one-way analysis of variance (ANOVA). Mean differences were regarded as significant when the pvalues were less than 0.05. Mean separations were performed by employing Duncan’s Multiple Range Test comparison procedure.
2.5.6. Peroxidase activity Peroxidase activity was measured by method of MacAdam et al. (1992). One gram of mango pulp was used for extraction. Crude enzyme was extracted by adding potassium phosphate buffer (pH 6.0) and centrifuged at 13,000 rpm for 20 min at 4 °C. The substrate solution containing potassium phosphate buffer (pH 6.0), H2O2 (0.2 mM) and guaiacol (3.0 mM) were prepared then the reaction was started by adding crude enzyme into substrate solution. The kinetic of peroxidase activity was measured by monitoring changes in absorbance at 470 nm for 5 min. One unit of peroxidase activity represented 1 μmol of tetraguaiacol/min/mg protein.
3. Results 3.1. Antifungal effect of chitosan coating combined with spermidine on disease severity and 3.1.1. Lesion diameter of mango fruit inoculated with C. gloeosporioides The postharvest disease of ‘Nam Dok Mai’ mango, anthracnose, caused by C. gloeosporioides, resulted in fruit decay with disease lesions (1–3 cm) 3 days after inoculation on inoculated fruit dipped in distilled water, 0.5% acetic acid and 0.1 ppm SPD treatments. Whereas, control fruit and inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD or 1% CTS alone did not show fungal decay (Fig. 1). Disease lesion development measured every 3 days after inoculation revealed that inoculated fruit dipped in distilled water, 0.5% acetic acid and 0.1 ppm SPD treatments significantly presented more disease severity during storage (4–5 cm at day 9) than control fruit and inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD or 1% CTS alone (Figs. 1 and 2). Therefore, fungal development in infected ‘Nam Dok Mai’ mango was inhibited in this experiment.
2.5.7. Total protein assay Total protein assay as described by Bradford (1976) was conducted. Fifty μL Bradford dye reagent (BioRad) was added to test tubes containing 50 μL enzyme extract samples and 100 μL distilled water and the tubes were incubated at room temperature for 5 min. The samples were then thoroughly mixed and read at a wavelength of 595 nm in a spectrophotometer. Bovine serum albumin (BSA) was used as the standard protein. 2.5.8. Soluble pectin content Soluble pectin content was analyzed based on the methods of Robertson (1979). One gram of mango pulp was used for soluble pectin extraction. Extraction was diluted with distilled water and added with sodium tetraborate decahydrate (in conc. HCl). After mixing, tube content was boiled in waterbath at 100 °C for 10 min, then added with 0.1 mL mhydroxydiphenyl (in 0.5%NaOH) and incubated at room temperature for 15 min. Soluble pectin content was read at 410 nm by spectrophotometer. Values were calculated by standard curve of D-galacturonic acid (μg/g fresh weight (FW)).
3.2. Effect of chitosan coating combined with spermidine on physicochemical changes of ‘Nam Dok Mai’ mango fruit 3.2.1. H2O2, total phenolic contents and PAL activity H2O2 level in fruit of all treatments increased during 6 days after inoculation then decreased until the end of storage. High level of H2O2 content was promoted in inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD and 1% CTS treatments on day 6 with significant differences from control treatment (Fig. 3A). Total phenolic content was also increased on day 6 after inoculation until the end of storage. Inoculated fruit coated with 1% chitosan combined with 0.1 ppm SPD significantly showed the highest value of phenolic content during storage compared to control treatment (Fig. 3B). Mango fruit represented
2.5.9. Fruit firmness Pulp firmness was measured by using handheld penetrometer 182
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Fig. 1. Disease severity of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days.
3.2.3. Soluble pectin content and firmness Soluble pectin content highly increased on day 3 then gradually decreased until the end of storage. Fruit coated with 1% CTS combined with 0.1 ppm SPD significantly showed lower soluble pectin content than control treatment. Inoculated fruit and control fruit presented the highest soluble pectin content on day 3 and 6 during storage, respectively. After that, soluble pectin content slightly reduced until the end of storage (Table 1). Fruit firmness rapidly decreased on day 3 after storage then gradually decreased until the last day of storage. Only 1% CTS combined with 0.1 ppm SPD and 1% CTS coating alone had significant effect to inhibit fruit softening by maintaining fruit firmness throughout the experiment. The other 3 treatments had no differences in firmness when compared to the control treatment (Table 1).
the highest PAL activity on day 6 whereas control fruit presented the lowest PAL activity. However, there were no significant differences in PAL activity among treatments (Fig. 3C).
3.2.2. Chitinase, β-1,3-glucanase and peroxidase activities Activities of defense enzymes, chitinase and β-1,3-glucanase, decreased in the first 3 days after treatments then increased until day 6 in all treatments except for control and inoculated fruit dipped in 0.1 ppm SPD treatments. Inoculated fruit coated with 1% CTS combined with 0.1 ppm SPD significantly presented the highest chitinase and β-1,3glucanase activities until the end of storage (Fig. 4A and B). All treatments except for control and inoculated fruit dipped in 0.1 ppm SPD significantly presented high level of peroxidase activities on day 3 after inoculation (Fig. 4C) then enzyme activities was sharply decreased. Overall, it was evident that 1% CTS combined with 0.1 ppm SPD showed strong effect to induce chitinase, β-1,3-glucanase and peroxidase activities.
3.2.4. Titratable acidity (TA) and total soluble solids (TSS) contents The changes of TA and TSS contents were observed after 3 days of storage. TA instantaneously decreased on day 3 (from 2% to 0.3%) in all treatments. After that, TA slightly decreased until the end of storage Fig. 2. Disease lesion diameter (cm.) of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Vertical bars represent ± SE.
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Fig. 3. H2O2 (A), total phenolic (B) and PAL activity (C) of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Each data point was calculated from three replications. Vertical bars represent ± SE.
combined with 0.1 ppm SPD significantly presented the highest H2O2 content. In addition, phenolic compounds are well-known antimicrobial compounds in plants (Sivaprakasan and Vidhyasekaran, 1993; Schlösser, 1994). According to Matern and Kneusel (1988), plant expressed rapid accumulation of phenols at the first step of the defense mechanism after infection by a pathogen which limited or slowed down pathogen growth. Our result showed that phenolic content slightly increased on day 6 after inoculation until the end of storage. Fruit coated with 1% CTS combined with 0.1 ppm SPD exhibited higher value of total phenolic content than other treatments. The accumulation of H2O2, phenolic, lignin and phytoalexin were increased in many plants treated with chitosan such as wheat seed, pea pods and rice (BhaskaraReddy et al., 1999; Agrawal et al., 2002). Moreover, previous experiment by Yamakawa et al. (1998) indicated that spermine functions as a signal molecule to transduce defense responses. According to the study by Takahashi et al. (2004), spermine acted as an endogenous inducer of pathogenesis-related (PR) proteins during TMV-induced HR resulting in the production of H2O2 into the cytosol. The exogenous spermine specifically activated the expression of four HR marker genes in tobacco leaves. Activation of these HR marker genes may provide to defense resistance against pathogen attack to host plant and lead to programmed cell. Therefore, combining chitosan and spermidine in our experiment completely presented the best effect in increasing the accumulation of H2O2 and phenolic production leading to the induction of plant defense mechanisms for protecting mango fruit from pathogen infection. Our research found that the treatment could enhance activities of chitinase, β-1,3-glucanase and peroxidase resulting in suppress
time. Fruit coated with 1% CTS combined with 0.1 ppm SPD significantly presented higher TA content during storage than control and 1% CTS treatments (Table 1). In this study, TSS swiftly increased on day 3 and day 6 then gradually decreased. TSS values were differences between 1% CTS combined with 0.1 ppm SPD treatment and other treatments on day 6. The lowest TSS presented in 1% CTS combined with 0.1 ppm SPD conferred delayed ripening process of ‘Nam Dok Mai’ mango fruit (Table 1). 4. Discussions In Thailand, anthracnose is a major constraint in ‘Nam Dok Mai’ mango fruit production and export. Chemical fungicides are routinely applied to control the pathogen which causes environmental toxicity and residual effect. Combining chitosan with spermidine as fruit coating had a potential to control disease severity of inoculated mango fruit by inhibiting fungal decay and reduce fruit softening during storage. The present investigation clearly indicated that the combination of 1% CTS combined with 0.1 ppm SPD provided better effect on inhibiting fungal development than solely chitosan usage. Inducing plant defense mechanisms such as H2O2, total phenolic content, chitinase, β1,3-glucanase and peroxidase activities were involved. Plant promoted H2O2 as hypersensitive response in program cell death after plant was infected by pathogens (Levine et al., 1994; Tenhaken et al., 1995). H2O2 had the ability to inhibit the growth and viability of diverse microbial pathogens (Kiraly et al., 1993; Wu et al., 1995). Our study showed that ‘Nam Dok Mai’ coated with 1% CTS 184
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Fig. 4. Chitinase (A), β-1,3-glucanase (B) and peroxidase (C) activities of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Each data point was calculated from three replications. Vertical bars represent ± SE.
Table 1 Influence of chitosan coating combined with spermidine on soluble pectin content, firmness, % titratable acidity (TA) and total soluble solid (TSS) of inoculated fruit during storage at room temperature (25 ± 2 °C) for 9 days. Storage (days)
Treatment
Soluble pectin content (μg/g FW)
Firmness (N)
TA (%)
Day 0
Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD Control Inoc + H2O Inoc + Acetic acid Inoc + CTS + SPD Inoc + CTS Inoc + SPD
1.57 1.57 1.57 1.57 1.57 1.57 2.97 3.06 2.25 1.97 2.59 2.72 2.72 1.82 1.18 1.88 2.04 1.13 0.63 0.95 0.80 1.72 1.87 0.65
7.54 7.54 7.54 7.54 7.54 7.54 5.11 4.97 5.11 5.63 5.40 5.16 4.83 4.65 4.83 5.44 5.39 4.74 4.58 4.26 4.39 5.40 5.22 4.73
1.91 1.91 1.91 1.91 1.91 1.91 0.26 0.29 0.32 0.39 0.21 0.30 0.25 0.21 0.27 0.25 0.16 0.26 0.12 0.11 0.10 0.23 0.09 0.11
Day 3
Day 6
Day 9 a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.30a 0.30a 0.30a 0.30a 0.30a 0.30a 0.35c 0.14c 0.08ab 0.25a 0.07abc 0.21bc 0.32b 0.36ab 0.41a 0.29ab 0.17ab 0.22a 0.14a 0.19ab 0.14a 0.34bc 0.33c 0.08a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.24a 0.24a 0.24a 0.24a 0.24a 0.24a 0.16b 0.16b 0.11b 0.20a 0.07ab 0.12b 0.16b 0.12b 0.10b 0.25a 0.09a 0.20b 0.13b 0.17b 0.18b 0.11a 0.19a 0.13b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
TSS (°Brix) 0.01a 0.01a 0.01a 0.01a 0.01a 0.01a 0.01bc 0.02bc 0.02ab 0.06a 0.03c 0.01ab 0.24ab 0.31ab 0.14a 0.47ab 0.15b 0.35ab 0.01b 0.01b 0.01b 0.02a 0.01b 0.01b
Values followed by the same letter in the column on the same day were not significantly different according to Duncan’s Multiple Range Test (P < 0.05).
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16.00 16.00 16.00 16.00 16.00 16.00 22.50 22.75 20.75 19.88 20.00 20.63 24.00 24.75 25.00 20.00 23.00 23.00 20.75 24.50 22.75 19.50 21.90 20.75
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.74a 0.74a 0.74a 0.74a 0.74a 0.74a 1.50a 0.98a 0.25a 0.72a 1.48a 0.94a 1.10b 1.33b 1.32b 0.63a 0.63ab 1.00ab 1.12ab 1.26b 1.66ab 1.34a 1.87ab 1.05ab
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fungal development in mango fruit. This finding is in agreement with the previous observation in strawberry and raspberry fruits (Zhang and Quantick, 1997). They reported that strawberries and raspberries coated with chitosan showed high level of chitinase and β-1,3-glucanase activities. Activity of peroxidase in cucumber plants was also induced after treated with chitosan (Ben-Shalom et al., 2003; Lin et al., 2005). During ripening, fruit softening is an important aspect which is associated with extensive structural alterations within pectin matrix leading to loss of cell wall structure (Seymour et al., 1990 Posé et al., 2015). Pectin is degraded by pectin degrading enzymes becoming soluble pectin which is always increased during ripening period (Redgwell et al., 1997a,b). The combination of 1% CTS combined with 0.1 ppm SPD significantly inhibited the increase in soluble pectin content and retained fruit firmness. The results are in agreement with the findings of Liu et al. (2016) where chitosan coating delayed softening of cherimoya (Annona cherimola Mill.) fruit and significantly affected the expression of several cell wall-related genes. Similarly, banana fruit showed a remarkable increase of soluble pectin level during ripening (Duan et al., 2008) and the amount of soluble pectin in strawberry also increased from the small green to the white stage (Rosli et al., 2004). In addition, previous study in citrus fruit showed that polyamines mainly interacted with negative charges of pectic substance which condition the binding of PME thus it could inhibit PME activity leading to maintain fruit firmness (Charney et al., 1992). Similarly, spermine or spermidine treatment could retard softening of apple and strawberry fruits (Kramer et al., 1991; Ponappa et al., 1993). The maximum ‘Summer Bahisht Chaunsa’ mango firmness was reported in fruit coated with crab chitosan (Abbasi et al., 2009). Chitosan had ability to avoid shrinkage and reduction in cell wall loosening which in turn maintained cell integrity (Salunkhe and Desai, 1984; Medlicott et al., 1986). Chitosan coatings have been used for controlling the internal gas atmosphere of fruits and vegetables. It acted as a barrier to water vapor for reducing moisture loss and delaying fruit dehydration that could maintain fruit firmness and protect weight loss (Srinivasa et al., 2002; Ribeiro et al., 2007; Duan et al., 2011). In 2016a, Jongsri et al. reported that 1% chitosan combined with 0.1 ppm spermidine significantly induced endogenous polyamines including free putresine, spermidine and spermine in ‘Nam Dok Mai’ mango when compared with control fruit and other treatments. Higher endogenous polyamines in fruit resulted in higher fruit firmness in these fruits. Moreover, coating with 1% chitosan combined with 0.1 ppm spermidine also maintained fresh weight and retarded peel color changes. However, our results found that fruit treated with only chitosan or spermidine presented lower properties for prolong shelf life of mango fruit. Therefore, the combination of 1% chitosan and 0.1 ppm spermidine has synergistic effect on fruit qualities. Using a suitable coating could maintain fruit firmness, reduction of respiration and water loss (Tasdelen and Bayindirli, 1998). Besides fruit softening, TSS and TA are very important quality factors used to estimate the storage longevity of mango. TSS refer to sugar within fruit that always increases during ripening stage and TA always decreases during ripening stage of the fruit, which are major characteristic for climacteric fruit ripening (Mahto and Das, 2013). During storage, we found that TA swiftly decreased on day 3 in all treatments and TSS instantaneously increased until the end of storage period. Fruit coated with 1% CTS combined with 0.1 ppm SPD presented the highest TA content and the lowest TSS content during storage. This same result was observed with the use of chitosan-lactoperoxidase coating in “Kent” mango. Uncoated mangoes showed the maximum of TSS content and pH and the minimum of TA acidity (Cissé et al., 2015). In addition, dipping papaya fruits in spermine could prolong shelf life by delaying fruit softening and the increase of TSS and peel color changes (Purwoko et al., 1998). Previous study by Santivipanond et al. (2012) reported that spermidine at 0.1 ppm could improve firmness and delayed TSS in ‘Hom Thong’ bananas. During postharvest storage, acid metabolism
converted starch and acid to sugar resulting in the decrease of TA and the increase of TSS (Duan et al., 2011). Apricot, plum, kiwi and lemon fruits treated with putresine showed higher fruit firmness and titratable acidity, lower soluble solid content, ethylene production and respiration rate, reduced weight loss and delayed color changes leading to extend storage life of fruit. Moreover, chitosan coating could delay respiration rate and ethylene production leading to retain qualities in citrus and mango fruits (Chien et al., 2007; Jitareerat et al., 2007; Jongsri et al., 2016b). Therefore, combining 1% CTS and 0.1 ppm SPD could inhibit deterioration processes of mango fruit resulting in the delay chemical changes in fruit during ripening stage. 5. Conclusions One percent CTS combined with 0.1 ppm SPD could prolong shelf life and maintain qualities of ‘Nam Dok Mai’ mango fruit after harvest and was the appropriate coating for postharvest ‘Nam Dok Mai’ mango. Furthermore, 1% CTS combined with 0.1 ppm SPD had potential to control anthracnose disease caused by C. gloeosporioides and induce defense mechanisms in ‘Nam Dok Mai’ mango fruit. Therefore, this formulated coating can be beneficial coating for commercial use in postharvest storage of mango fruit. Acknowledgements The first author was recipient of a Postdoctoral Fellowship, Rachadapisek Sompote Fund, Chulalongkorn University. The authors would like to thank National Research Project Management (NRPM) for their financial support. References AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed. AOAC, Washington, DC. Abbasi, N.A., Iqbal, Z., Maqbool, M., Hafiz, I.A., 2009. Postharvest quality of mango (Mangifera indica L.) fruit as affected by chitosan coating. Pak. J Bot. 41 (1), 343–357. Agrawal, G.K., Rakwal, R., Tamogami, S., Yonekura, M., Kubo, A., Saji, H., 2002. Chitosan activates defense/stress response(s) in the leaves of Oryza saliva seedlings. Plant Physiol. Biochem. 40, 1061–1069. Akem, C.N., 2006. Mango anthracnose disease: present status and future research priorities. Plant Pathol. J. 5, 266–273. Barnett, H.L., Hunter, B.B., 1998. Illustrated Genera of Imperfect Fungi, 4thed. APS press, Saint Paul, Minnnesota, America. Ben-Shalom, N., Ardi, R., Pinto, R., Aki, C., Fallik, E., 2003. Controlling gray mold caused By Botrytis cinerea in cucumber plants by means of chitosan. Crop Prot. 22, 285–290. Bhaskara-Reddy, M.V., Arul, J., Angers, P., Couture, L., 1999. Chitosan treatment of wheat seed induces resistance to Fusarium gramninearum and improves seed quality. J. Agric. Food Chem. 47, 1208–1216. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bunyard, B.A., Nicholson, M.S., Royse, D.J., 1994. A systematic assessment of Morchella with RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86, 762–772. Chancharoenrit, J., 2002. Effects of Hot Water Dips on Physiological Changes and Chilling Injury After Storage of ‘Hom Thong’ Variety of Banana. Master’s Thesis. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. Charney, D., Nari, J., Noat, G., 1992. Regulation of plant cell-wall pectin methyl esterase by polyamines-Interactions with the effects of metal ions. Eur. J. Biochem. 205, 711–714. Chien, P.J., Sheu, F., Lin, H.R., 2007. Coating citrus (Murcott tangor) fruit with low molecular weight chitosan increases postharvest quality and shelf life. Food Chem. 100, 1120–1164. Cissé, M., Polidori, J., Montet, D., Loiseau, G., Ducamp-Collin, M.N., 2015. Preservation of mango quality by using functional chitosan lactoperoxidase systems coatings. Postharvest Biol. Technol. 101, 10–14. D’Cunha, G.B., Satyanaraan, V., Nair, P.M., 1996. Stabilization of phenylalanine ammonia- lyase containing Rhodotorula glutatinis cells for the continuous synthesis of phenylalanine methyl ester. Enzyme Microbial. Technol. 19, 421–427. Dhall, R.K., 2013. Advances in edible coatings for fresh fruits and vegetables: a review. Crit. Rev. Food Sci. Nutr. 53 (5), 435–450. Duan, X., Cheng, G., Yang, E., Yi, C., Ruenroengklin, N., Lu, W., Luo, Y., Jiang, Y., 2008. Modification of pectin polysaccharides during ripening of postharvest banana fruit. Food Chem. 111, 144–149. Duan, J., Wu, R., Strik, B.C., Zhao, Y., 2011. Effect of edible coatings on the quality of fresh blueberries (Duke and Elliott) under commercial storage conditions. Postharvest Biol. Technol. 59, 71–79.
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