Effect of molecular weights of chitosan coating on postharvest quality and physicochemical characteristics of mango fruit

LWT - Food Science and Technology 73 (2016) 28e36

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Effect of molecular weights of chitosan coating on postharvest quality and physicochemical characteristics of mango fruit Pornchan Jongsri a, Teerada Wangsomboondee a, Pranee Rojsitthisak b, Kanogwan Seraypheap a, * a b

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

Article history: Received 5 February 2016 Received in revised form 19 May 2016 Accepted 20 May 2016 Available online 21 May 2016

’Nam Dok Mai’ mango is an important export fruit of Thailand. However, the quality of fruit is reduced after harvest. Therefore, it is necessary to develop a postharvest treatment to maintain the quality of ’Nam Dok Mai’ mango after harvest. Chitosan solutions (high molecular weight (HM-CTS), medium molecular weight (MM-CTS), and low molecular weight (LM-CTS)) were applied as fruit coating for ‘Nam Dok Mai’ mango (Mangifera indica L.) and stored at 25
C for 16 days. The film forming properties of chitosan were influenced by molecular weight and significantly impacted postharvest quality of mango fruit during storage. HM-CTS could delay mango fruit ripening and thus maintaining the highest value of titratable acidity, fruit firmness, and also resulting in a reduction of weight loss, ethylene production, and respiration rate of mango fruit. Moreover, HM-CTS coated fruit exhibited no incidences of disease throughout storage. DPPH inhibition and ascorbic acid content were maintained in coated fruit during storage. H2O2 content was inhibited by catalase and ascorbate peroxidase activities in HM-CTS coated fruit. These data indicated that the application of HM-CTS could be used to reduce deteriorative processes, maintain quality, and increase the shelf life of ‘Nam Dok Mai’ mango during postharvest storage. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Disease Antioxidant activity Scanning electron microscopy Ethylene Softening

1. Introduction Mango (Mangifera indica L.) is a popular tropical fruit that is in high demand around the world (Kim, Brecht, & Talcott, 2007) because of its nutritional properties, attractive fragrance, aesthetic color, and excellent exotic flavour (Ulloa, Guatemala, Arriola, Escalona, & Díaz, 2009). However, mango is a climacteric fruit which quickly ripens and softens after harvest because of high ethylene synthesis, and in addition to accelerated ethylene synthesis, chilling injury can occur if mango is stored at temperatures under 13
C for several days (Acosta et al., 2000). Moreover, anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is a major postharvest disease in mango where the pathogen can attack immature fruit (Dodd, Prusky, & Jeffries, 1997). These severe problems of postharvest mango cause reduction in the quality of fruit during storage, transport, and marketing period (Mitra & Baldwin, 1997). Fungicides are used to resolve the problem

* Corresponding author. E-mail address: [email protected] (K. Seraypheap). http://dx.doi.org/10.1016/j.lwt.2016.05.038 0023-6438/© 2016 Elsevier Ltd. All rights reserved.

caused by anthracnose despite many countries having concerns over toxic residues and human health (Bautista-Banos et al., 2006). Thus, it is necessary to develop alternative postharvest technologies to better maintain overall mango fruit qualities during the export chain or market period. Fruit coating is becoming one of the most popular methods to extend the commercial shelf-life of fruits by delaying ripening, water loss, and decay (Baldwin, Nisperos, Hagenmaier, & Baker, 1997). Coatings can lead to a change in the composition of the atmosphere surrounding the fruit which results in creating a modified atmosphere (MA) that can act as a barrier to gas exchange, especially for O2, CO2, and ethylene. Storage under MA allows for some controls of fruit softening and senescence. In addition, fruit coating can maintain fruit quality by slowing fungal development and improving appearance during transportation and storage (Amarante & Banks, 2001; Baldwin, 1994). Chitosan (poly-b-(1,4)-D-glucosamine), a deacetylated form of chitin (poly-N-acetylglycosamine), has been widely used as fruit coating (Djioua et al., 2010; Ippolito, El-Ghaouth, Wisniewski, & Wilson, 2000; Jiang & Li, 2001; Rinaudo, 2006) due to its filmforming property, biocompatibility, and biodegradability (Shahidi,

P. Jongsri et al. / LWT - Food Science and Technology 73 (2016) 28e36

Arachchi, & Jeon, 1999). Chitosan coating can create MA (Baldwin, Nisperos, Shaw, & Burns, 1995) that decrease the response of fruit to environmental conditions by reducing gas exchange leading to retardant ripening, water loss, respiration rate, and ethylene production (Baldwin et al., 1997; Jitareerat, Paumchai, Kanlayanarat, & Sangchote, 2007). Chitosan can act as an exogenous elicitor inducing activities of several defense-related enzymes and accumulating special substances in some plants (Cabrera, Messiaen, Cambier, & Van, 2006; Trotel-Aziz, Couderchet, Vernet, & Aziz, 2006), which are known to participate in defense mechanisms and to prevent pathogen infections. The effects of chitosan coatings on the extension of storage life of many fruits were reported (Ampaichaichok, Rojsitthisak, & Seraypheap, 2014; Chien, Sheu, & Lin, 2007; Zhang & Quantick, 1998). It was found that coating ‘Nam Dok Mai’ mango fruit with 1% chitosan (Mw 350 kDa) could maintain ascorbic acid content, weight loss, peel color change, and total titratable acidity while reducing respiration rate and ethylene production. In addition, the treated fruit showed higher chitinase and b-l,3-glucanase activities than control fruit (Jitareerat et al., 2007). To date, there are few reports on the effect of different molecular weights of chitosan coatings on fruit storage, especially in ‘Nam Dok Mai’ mango fruit. Therefore, the aims of this study are to develop a fruit coating for mango that improves fruit shelf life and quality, and to further investigate the effect of different molecular weights of chitosan on postharvest qualities and physicochemical characteristics of mango fruit during storage. 2. Materials and methods 2.1. Chitosan materials Chitosan flakes prepared from shrimp shells were obtained from A.N. Lab, Thailand. Chitosan properties were analyzed for molecular weight (Gel Permeation Chromatography (GPC, Water 600E, Waters Corp., USA)), solubility (Robert, 1992), moisture content (AOAC International, 2000), deacetylation degree (Muzzarelli & Rocchetti, 1985), and viscosity (Brookfield Viscometer, model DV-IIþ, Japan). Chitosan materials were divided into three different molecular weight groups: high molecular weight chitosan (HM-CTS: 360 kDa), medium molecular weight chitosan (MM-CTS: 270 kDa), and low molecular weight chitosan (LM-CTS: 40 kDa). One percent (w/v) chitosan solution was prepared in 0.5% (v/v) acetic acid; the solution was then stirred at 25
C overnight. After stirring, the solution was amended with 0.1% (w/v) tween® 80 and stirred at room temperature for 30 min before treatment. 2.2. Plant materials and treatments Mature green mango (Mangifera indica L. cv Nam Dok Mai) fruit were harvested 90e100 days after fruit set from a commercial orchard in Nakornratchasrima province, Thailand. Afterwards, fruit were selected for their uniformity in size, color, shape, and as well as lack of blemishes and disease symptoms. The 160 fruits were separated into 4 replications for each treatment. Fruit were dipped for 1 min into a solution of 1% (w/v) HM-CTS, MM-CTS, and LM-CTS. The control fruit were dipped in 0.5% acetic acid containing 0.1% tween® 80. After treatment, fruit were air-dried for 30 min and stored at room temperature (25 ± 2
C). Every 4 days, 32 fruits were randomly sampled until day 16. 2.3. Scanning electron microscopy (SEM) analysis Thickness and surface properties of chitosan coating film were measured by using a scanning electron microscope (FEI Quanta 250

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ESEM, Netherlands) with voltage set at 10 kV. 2.4. Respiration rate and ethylene production Fruit from each treatment were weighed and placed in 2.4 L jars fitted with a rubber septum for 1 h at 25
C. One mL sample of the internal atmosphere was extracted and injected into a gas chromatograph equipped with a thermal conductivity detector (GC-RIA, Shimadzu, Kyoto, Japan) for carbon dioxide. The column temperature for carbon dioxide was at 50
C and helium was used as the carrier gas. The respiration rate measured by CO2 production was expressed in mg CO2 kg1 h1. For ethylene, one mL sample was analyzed using a gas chromatograph (GC-14, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector held at 80
C using nitrogen as the carrier gas. Ethylene levels were determined and expressed as mL kg1 h1. 2.5. Peel color Mango peel color was determined by using colorimeter (Color Reader CR-10, Konica Minolta Sensing, Inc., Japan). Lightness (L) and hue angle value were measured for peel color change. The measurement was taken from three equatorial regions of the fruit peel (blossom end, middle, and stem end). 2.6. Fruit firmness Pulp firmness was evaluated by using a handheld penetrometer (Hardness tester FHM-1, Takemura, Japan) on the same three regions as for the peel color measurement. Firmness was recorded as kg-force in Newtons (N). 2.7. Percentage of weight loss Weight loss was calculated as percentage loss from initial weight. Fruit were weighed regularly to determine weight loss using formula as described by AOAC (1984). 2.8. Titratable acidity (TA) The titratable acidity analysis method was applied from AOAC (1984). One hundred mL of distilled water was mixed with 10 g of sliced mango pulp by vortex for 1 min and filtered. Ten mL of filtrate was titrated with 0.1 N NaOH. Filtrate was added with 1% phenolphthalein indicator; then titrated until the end point. The TA (%) was calculated as follows:

TAð%Þ ¼

NaOHðmLÞ  0:1NaOHðNÞ  0:07  100 10g

2.9. Total soluble solids content (TSS) Mango juice was used to measure total soluble solids with a hand refractometer (N-1E, Japan), and TSS was expressed as ºBrix. 2.10. H2O2 content One gram of mango pulp was ground with liquid nitrogen then 10 mL of 50 mM phosphate buffer (pH 6.5) containing 1 mM hydroxylamine at 0
C was added. The mixture was centrifuged at 8000  g for 25 min; 1 mL supernatant was added with 1 mL of 0.3% titanium sulphate in 20% H2SO4 (v/v) and then centrifuged again at 8000  g for 15 min. The H2O2 content was measured at 410 nm by

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Table 1 Chitosan properties.

a

Chitosan

Molecular weight (kDa)

Solubility (%) (in 0.5% acetic acid)

Moisture (%)

Deacetylation (%)

Viscosity (cps)

HM-CTS MM-CTS LM-CTS

360 270 40

99.21 ± 0.63a 99.71 ± 0.36a 98.68 ± 0.40a

12.26 ± 0.25b 10.62 ± 0.11ab 12.93 ± 0.22b

84.90 ± 0.72 90.50 ± 0.99 91.30 ± 0.15

34.07 ± 0.55 29.77 ± 0.60 6.30 ± 0.05

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).

Fig. 1. Cross-section image of mango peel coated with high (HM-CTS), medium (MM-CTS), and low molecular weight chitosan (LM-CTS) by scanning electron microscope. The arrows indicate the chitosan film on mango peel after coating.

spectrophotometrically assay following Jana and Choudhuri (1982). According to standard curve, the H2O2 content was shown as mmol/ g fresh weight (FW).

DPPH inhibitionð%Þ ¼

ð1  AbsorbanceðsampleÞÞ  100 AbsorbanceðcontrolÞ

2.11. DPPH inhibition The method to determine the percentage of a, a-diphenyl-bpicrylhydrazyl (DPPH) inhibition was adapted from Choi, Lee, Chun, Lee, and Lee (2006). The color of reaction was measured by absorbance (Ab) at 520 nm. Water used in control and blank was 80% methanol. The percentage of DPPH inhibition was calculated as follows:

P

PDI for severity ¼

2.12. Ascorbic acid content Ascorbic acid (AA) content was determined according to the method of Shin, Liu, Nock, Holliday, and Watkins (2007). Total AA was measured by Ab at 540 nm using a standard curve. The concentrations were expressed as mg/g FW.

ðnumerical ratingsÞ  100 Total number of observations  maximum disease score

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Fig. 2. Surface image of mango peel coated with high (HM-CTS), medium (MM-CTS), and low molecular weight chitosan (LM-CTS) by scanning electron microscope. The arrow indicate stomatal aperture on mango peel.

2.13. Free flavonoid content Flavonoid contents in pulp were determined by a colorimeter method (Djeridane et al., 2006). The color of supernatant was monitored by the decrease in Ab at 433 nm. The flavonoid contents were expressed as mg rutin (RE)/g FW. 2.14. Catalase and ascorbate peroxidase activities Catalase (CAT) and ascorbate peroxidase (APX) activities were assayed by the method of Nakano and Asada (1987). The CAT activity was measured by the decline in Ab at 240 nm. One unit of CAT activity was a decomposition of 1 mmole of H2O2 per min. The APX reaction rate was monitored by the decrease in Ab at 290 nm. One unit of APX activity was defined as that which is oxidized 1 mmole of ascorbate per min. The specific activity was expressed as U/mg protein. Protein content was measured according to the method of Bradford (1976) using bovine serum albumin as the standard. 2.15. Disease incidence and severity Disease incidence was measured by counting diseased fruit in each timing and the percentage of disease incidence (Baldwin et al., 1999) was calculated as follows:

Disease incidenceð%Þ ¼

No: of diseased fruits  100 No: of total fruits

Disease severity was measured by using score 0e7 as: 0 ¼ no disease, 1 ¼ 1e2% disease, 2 ¼ 5% disease, 3 ¼ 10% disease, 4 ¼ 20%

disease, 5 ¼ 40% disease, 6 ¼ 60% disease, and 7 ¼ more than 80% disease (Pedroso, Lage, Henz, & Cafe-Filho, 2011). Then the percent of disease index (PDI) for severity was calculated as reported by Hossain, Hossain, Bakr, Rahman, and Hossain (2010):

2.16. Fungal identification The phytopathogenic fungus was isolated from diseased tissues of symptomatic mango fruit and identified by both morphological technique (Barnett & Hunter, 1998) and molecular technique by DNA sequencing (National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand). 2.17. Statistic analysis Statistical comparisons were made by one-way analysis of variance (ANOVA). Differences were regarded as significant when the p-values were less than 0.05. Mean separations were performed by employing Duncan’s Multiple Range Test comparison procedure. 3. Results and discussion 3.1. Evaluation of chitosan characteristics and chitosan coating film The bioactivity of chitosan is a function of its physicochemical properties which have an effect on film forming characteristics (No, Meyers, Prinyawiwatkul, & Xu, 2007). Different physicochemical properties of chitosan solutions which were classified as high (HM-

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Fig. 4. Peel color change of mango in control and after coating with chitosan during storage at room temperature (25 ± 2
C) for 16 days. Fig. 3. Ethylene production (A) and respiration rate (B) of control and chitosan coated fruit during storage at room temperature (25 ± 2
C) for 16 days. Each data point represents the mean of eight fruit. Vertical bars represent ± SE. (A) Control; ( ) HMCTS; (:) MM-CTS; (✕) LM-CTS.

▫

CTS), medium (MM-CTS), and low (LM-CTS) molecular weight chitosan are shown in Table 1. The respective SEM images are shown in Fig. 1. It was found that more than 98% of chitosan materials of three different molecular weights could dissolve in 0.5% acetic acid (Table 1). All chitosan materials showed percentages of moisture content between 10 and 12%. Rabea et al. (2003) suggested that the

viscosity of chitosan solutions is affected by the molecular weight of chitosan, the concentration of solution, the ionic strength, the pH, and the temperature. HM-CTS showed the highest viscosity, and LM-CTS showed the lowest viscosity which related to the molecular weight of the chitosan samples. In addition, the samples presented a degree of deacetylation (DD) between 84 and 91% which had no significant differences. Therefore, the expected differences in response of coated mango fruit should be mainly due to the molecular weight of the chitosan applied. The thickness and film forming characteristics of chitosan film were also affected by molecular weight of chitosan and viscosity.

Table 2 Effects of chitosan coating on L value, hue angle, firmness, weight loss (%), TA (%) and TSS of fruit during storage at room temperature (25 ± 2
C) for 16 days. Storage time (Days)

Treatment

L value

0

Control HM MM LM Control HM-CTS MM-CTS LM-CTS Control HM-CTS MM-CTS LM-CTS Control HM-CTS MM-CTS LM-CTS Control HM-CTS MM-CTS LM-CTS

62.87 62.87 62.87 62.87 63.98 62.91 63.02 63.89 66.07 63.88 64.46 65.33 68.38 65.08 65.92 67.84 71.44 66.85 67.84 69.07

4

8

12

16

a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Hue angle 0.59a 0.59a 0.59a 0.59a 0.37a 0.59a 0.62a 0.38a 0.26c 0.51a 0.51ab 0.48bc 0.26b 0.59a 0.59a 0.39b 0.44c 0.52a 0.28ab 0.52b

96.84 96.84 96.84 96.84 93.62 95.86 95.57 95.72 84.26 95.18 90.91 85.44 77.35 93.11 84.27 78.46 73.82 91.00 79.79 73.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.30a 0.30a 0.30a 0.30a 1.88a 0.59a 0.43a 0.55a 1.97c 0.71a 1.13b 1.16c 1.53c 1.32a 1.48b 0.98c 0.97c 1.83a 1.65b 0.63c

Fruit firmness (N) 0.96 0.96 0.96 0.96 0.94 0.95 0.92 0.94 0.76 0.78 0.83 0.76 0.62 0.74 0.67 0.65 0.64 0.68 0.62 0.65

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01a 0.01a 0.01a 0.01a 0.01a 0.01a 0.02a 0.00a 0.01b 0.03ab 0.01a 0.01b 0.03b 0.03a 0.01b 0.02b 0.02ab 0.00a 0.02b 0.01ab

Weight loss (%)

TA (%)

e e e e 5.22 ± 0.25a 4.77 ± 0.25a 5.21 ± 0.24a 5.47 ± 0.25a 10.39 ± 0.36a 9.39 ± 0.44a 10.30 ± 0.44a 10.49 ± 0.34a 13.63 ± 0.47b 11.61 ± 0.28a 13.33 ± 0.57b 13.81 ± 0.46b 15.15 ± 0.52ab 13.59 ± 0.59a 14.97 ± 0.64ab 15.54 ± 0.54b

1.65 1.65 1.65 1.65 1.44 1.51 1.40 1.31 0.53 1.49 1.36 0.79 0.12 0.28 0.20 0.16 0.08 0.22 0.09 0.08

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

TSS (ºBrix) 0.14a 0.14a 0.14a 0.14a 0.03a 0.11a 0.09a 0.07a 0 .005b 0.14a 0.03a 0.15b 0.01b 0.09a 0.03ab 0.03ab 0.00b 0.01a 0.02b 0.00b

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).

9.63 ± 0.31a 9.63 ± 0.31a 9.63 ± 0.31a 9.63 ± 0.31a 14.00 ± 0.23b 11.63 ± 0.84a 14.25 ± 0.94b 15.38 ± 0.25b 17.63 ± 0.47b 15.75 ± 0.49a 17.25 ± 0.49b 18.00 ± 0.49b 18.94 ± 0.29b 16.88 ± 0.47a 18.44 ± 0.91ab 19.19 ± 0.68b 19.94 ± 0.38a 19.88 ± 0.47a 19.75 ± 0.34a 19.88 ± 0.52a

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Fig. 5. H2O2 content of control and chitosan coated fruit during storage at room temperature (25 ± 2
C) for 16 days and sampling every four days. Each value is the mean of eight fruit. Vertical bars indicate ± SE. (A) Control; ( ) HM-CTS; (:) MMCTS; (✕) LM-CTS.

▫

From the cross-section SEM images (Fig. 1), the arrows indicate the chitosan film on coated mango peel. Mango fruit coated with HMCTS exhibited the thickest of the chitosan film coating on mango peel (3.47 ± 0.50 mm) while MM-CTS coated mango resulted in thinner chitosan film coating (1.29 ± 0.45 mm). LM-CTS showed very thin chitosan film, and consequently, the thickness of film on the coated fruit could not be measured. The arrows in Fig. 2 indicate stomatal aperture on mango peel in control and LM-CTS coated fruit. Both HM-CTS and MM-CTS film coating thoroughly sealed and completely covered stomata on mango peel. However, HM-CTS was tightly compacted while MMCTS exhibited a thin, semi-transparent film. LM-CTS coated film was highly porous and did not thoroughly seal mango peel resulting in exposed stomatal aperture. Thus, the dissimilar filmforming properties of chitosan which are influenced by molecular weight significantly impact postharvest qualities and physicochemical characteristics of ‘Nam Dok Mai’ mango fruit during storage.

3.2. Effects of chitosan coating on ethylene production and respiration The changes in the rate of ethylene production and respiration of mango fruit during storage at 25 ± 2
C were observed for 16 days (Fig. 3A and B) to investigate the effects of chitosan coating on fruit physiology. The rate of ethylene production was significantly lower in mango fruit coated with HM-CTS and MM-CTS than LM-CTS coated fruit on day 4, 8, and 12 during storage. CO2 suppression in HM-CTS and MM-CTS coated fruit exhibited a significant difference from control, whereas, LM-CTS coated fruit was only affected on day 8. These results indicated that HM-CTS and MM-CTS coatings affected the ability to reduce ethylene production and respiration rate of the mango fruit and thus retarding the ripening process. This is in accordance with research where fruits such as mango and papaya have been reported to show an increase in the internal CO2 concentrations when coated with chitosan (Abbasi, Iqbal, Maqbool, & Hafiz, 2009; Ali, Muhammad, Sijam, & Siddiqui, 2011; Jitareerat et al., 2007). Previous studies have revealed that chitosan can act as a barrier film that creates a modified internal atmosphere and selectively permeates C2H4, CO2, and O2 inside and out of the fruit, leading to a reduced rate of respiration, transpiration, and production of ethylene (Ali et al., 2011; Jitareerat et al., 2007).

Fig. 6. Percentage of a, a-diphenyl-b-picrylhydrazyl (DPPH) inhibition (A), ascorbic acid (B) and free flavonoids (C) of control and chitosan coated fruit during storage at room temperature (25 ± 2
C) for 16 days and sampling every four days. Each value is the mean of eight fruit. Vertical bars indicate ± SE. (A) Control; ( ) HM-CTS; (:) MM-CTS; (✕) LM-CTS.

▫

3.3. Effects of chitosan coating on postharvest qualities and physicochemical characteristics 3.3.1. Peel color Peel lightness (L value) and hue angle have been used as ideal indicators of peel color change. L values increase and hue angles decrease when fruit reaches ripening stage in conjunction with peel color changes from green to yellow as mango ripens. HM-CTS and MM-CTS coatings could delay peel color change during storage.

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Fig. 7. Catalase (A) and ascorbate peroxidase (B) activities of control and chitosan coated fruit during storage at room temperature (25 ± 2
C) for 16 days and sampling every four days. Each data point represents the mean of eight fruit. Vertical bars represent ± SE. (A) Control; ( ) HM-CTS; (:) MM-CTS; (✕) LM-CTS.

▫

L value gradually increased in uncoated fruit while mango fruit coated with HM-CTS and MM-CTS resulted in a significant delay of peel color change on day 8, 12, and 16 (Table 2) (Fig. 4). Hong, Xie, Zhang, Sun, and Gong (2012) reported that chitosan coating was significant in maintaining chlorophyll content in guava fruit. Moreover, chitosan had an effect on the reduction of chlorophyll content in sweet pepper (Xing et al., 2011). The maintenance of ‘Nam Dok Mai’ mango peel color may be attributed to the modification of the internal atmosphere in the fruit by chitosan coating, leading to a delay in chlorophyll degradation process.

3.3.2. Fruit firmness and weight loss Fruit firmness was decreased when fruit began to ripen and drastically decreased in uncoated fruit (Table 2). There was a consistent increase in the percentage of weight loss during storage leading to fruit wilting. Mango fruit coated with HM-CTS showed the lowest percentage of weight loss on day 12 and day 16. Uncoated fruit and fruit coated with LM-CTS showed a drastic increased in the percentage of weight loss on day 12 and day 16 (Table 2). This study agreed with a previous report where weight loss of litchi fruit coated with chitosan was slower than the control during storage time (Lin et al., 2011). Correspondingly, fresh cut strawberries (Fragaria ananassa Duchesne) cv. 329 dipped in 1.0% chitosan solution resulted in a delayed percentage of weight loss compared with 1.0% carboxymethyl cellulose (CMC) when stored at 2
C for 10 days (Inkha, Kongtong, & Rattanapanone, 2005). Furthermore, chitosan coating effectively delayed weight loss and prolonged shelf life of sliced mango fruit (Chien et al., 2007). Chitosan was reported to be associated with stomatal response. The stomatal aperture of tomato and Commelina communis were

Fig. 8. Disease incidence (A) and disease severity (B) of control and chitosan coated fruit during storage at room temperature (25 ± 2
C) for 16 days and sampling every four days. Each data point was calculated from four replications. (A) Control; ( ) HMCTS; (:) MM-CTS; (✕) LM-CTS.

▫

reduced when the epidermis was treated with chitosan (Lee et al., 1999). Our study showed that stomatal aperture on mango peel was covered by chitosan film especially in HM-CTS. Thus, HM-CTS could maintain fresh weight of mango fruit by decreasing fruit transpiration and respiration rate. 3.3.3. Titratable acidity (TA) and total soluble solids (TSS) The results showed that fruit coated with HM-CTS represented the highest of TA on day 8, 12, and 16 whereas uncoated fruit showed the lowest TA during storage (Table 2). TSS content gradually increased during ripening. TSS was the lowest in fruit coated with HM-CTS on day 4, 8, and 12 (Table 2) which is a significant difference between coated and uncoated fruit. The higher TA and the lower of TSS values of HM-CTS coated fruit are evidently linked to the reduced rate of ethylene production and respiration. 3.4. Effects of chitosan coating on H2O2 and antioxidant contents During fruit ripening, the production of reactive oxygen species (ROS) increased while the antioxidant defense system decreased (Kim et al., 2007). H2O2, an oxidative parameter, significantly increased in uncoated mango during ripening while lowered in coated mango (Fig. 5). HM-CTS demonstrated the lowest H2O2 content among all treatments. These results suggested that HMCTS chitosan coating resulted in a significant reduction in the H2O2 content of mango fruit during the ripening stage. Chitosan solution was reported to induce reactive oxygen species scavenging capacity and to have an increase in phenolic compounds, flavonoid, and lignin contents in many fruits (Liu, Tian, Meng, & Xu, 2007;

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Meng, Li, Liu, & Tian, 2008; Gonzalez-Aguilar, Villa-Rodriguez, Ayala-Zavala, & Yahia, 2010). The antioxidant and reactive oxygen species scavenging potential status of ‘Nam Dok Mai’ mango tissue were evaluated for bioactive compound contents and activities. Our results showed that HM-CTS coated fruit had higher ascorbic acid content, DPPH inhibition, free flavonoids, and enzyme activities (CAT and APX) than uncoated fruit during storage. DPPH inhibition of mango extract gradually decreased during ripening period. HM-CTS treatment showed the highest DPPH inhibition (Fig. 6A) whereas uncoated fruit showed the lowest DPPH inhibition on day 12 and 16. Many bioactive compounds including ascorbic acid and flavonoids contributed to the antioxidant activity. The ascorbic acid content rapidly decreased during storage time; however, the ascorbic acid content of coated fruit was significantly higher than uncoated fruit on day 4 (Fig. 6B). Coating could delay the reduction of ascorbic acid content of mango fruit during storage at 25 ± 2
C. Although some fluctuations in free flavonoids were observed during storage, the content of free flavonoids was higher in coated fruit when compared to uncoated fruit. A sharp reduction of free flavonoids values was observed during the first 4 days of storage (Fig. 6C) then free flavonoids was marginally increased after 8 days of storage. Both CAT and APX are important antioxidant enzymes in the scavenging of hydrogen peroxide which is a reactive oxygen species that can damage plant cells (Asada, 1992). HM-CTS treatment showed the highest CAT and APX activity on day 4 (Fig. 7A and B). HM-CTS treatment significantly delayed the reduction of CAT and APX activities. This is in accordance with Zeng, Deng, Ming, and Deng (2010) who reported that the activity of peroxidase (POD) in navel orange fruit was significantly enhanced by chitosan coating, which could protect tissues from injury caused by excessively high levels of reactive oxygen species. Therefore, chitosan coating, resulting of high antioxidant activities leading to a protection of mango from oxidative stress induced ripening and senescence, could prolong shelf life of ‘Nam Dok Mai’ mango. 3.5. Effects of chitosan coating on disease incidence and disease severity Anthracnose started to develop in uncoated fruit on day 8 and sharply increased until the end of storage life (Fig. 8). Uncoated fruit showed the highest disease incidence and disease severity whereas mango fruit coated with HM-CTS did not have any disease symptoms. These defense responses induced by HM-CTS coating might be the result of lower internal oxygen levels created by a barrier film of chitosan (Amarante & Banks, 2001). Low internal oxygen levels and the slow ripening of fruit were the limiting factor for fungal growth and fungal metabolism. Significant reduction of disease incidence and disease severity in mango fruit treated with chitosan could also be involved with the elicitation of phenylalanine ammonia-lyase, chitinase, and b-1,3-glucanase activities. After treated with chitosan, these antimicrobial activities have been induced in several fruits, including grape berries (Romanazzi, Nigro, Ippolito, Di Venere, & Salerno, 2002) and mango (Jitareerat et al., 2007). Therefore, the antimicrobial effect of HM-CTS coating on mango fruit in this study could be attributed to a combination of its antimicrobial activity and the defense responses induced by chitosan. 4. Conclusions Among the chitosan coatings tested on ‘Nam Dok Mai’ mango fruit, HM-CTS (360 kDa) effectively delayed ripening and maintained postharvest quality of mango. HM-CTS film coating thoroughly sealed and completely covered stomata on mango peel thus

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