Effects of chitosan coatings on physicochemical and nutritional quality of clementine mandarins cv. ‘Oronules’

Abstract

A commercial solution of chitosan was applied on mandarins ‘Oronules’ at different solid content (0.6%, 1.2% and 1.8%). Additionally, one group of mandarins was coated with a polyethylene-shellac commercial wax, and another group remained uncoated (control). Mandarins were stored at 5 °C up to 28 days followed by 7 days at 20 °C simulating retail conditions. One group of mandarins was stored at 20 °C for 9 days simulating direct retail conditions. The commercial wax decreased weight and firmness loss of mandarins compared to uncoated samples, whereas the chitosan coating did not effectively decrease weight loss of mandarins. Chitosan-coated mandarins at the highest solid content retained firmness after cold storage and contained more phenolics than uncoated ones. Although all the coatings restricted gas exchange and modified the internal atmosphere of the mandarins, with a greater effect at higher chitosan concentration, sensory quality was not affected. In general, the internal quality and the health-related properties of mandarins were not negatively affected by coating application. However, there is a need to further improve the water barrier properties of the chitosan coating.

Keywords

Citrus wax ethanol weight loss vitamin C

Introduction

Spain is the main exporter of clementine citrus mandarins, including in this group Oronules ‘clementine’. This clementine cultivar is highly appreciated for its excellent organoleptic quality (Ortiz et al., 1988). Citrus fruit are coated in the citrus packing houses to improve their appearance and extend their shelf-life (Petracek et al., 1999). In general, commercial waxes used by the citrus industry enhance shine, reduce water loss and act as a vehicle for fungicides. However, it has also been reported by many authors that waxing of citrus can adversely affect fruit flavor (Baldwin et al., 1995; Hagenmaier, 2002; Hagenmaier and Baker, 1993; Hagenmaier and Shaw, 2002; Porat et al., 2005), due to the overproduction of volatiles associated with anaerobic conditions when coatings offer a high barrier to gases. The success of coatings for citrus mainly depends on the selection of appropriate formulations that not only reduced fruit weight and firmness loss, but also give a desirable internal gas composition. In addition, for a specific coating formulation, the solid content (SC) of the formulation can affect the gas barrier of the coating (Cisneros-Zevallos and Krochta, 2003).

Commercial coatings used for citrus fruit include waxes such as carnauba, petroleum-based waxes such as polyethylene wax, acetoglycerides, oleic acid, and resins such as shellac to improve fruit gloss (Baldwin et al., 1997). In recent years, concerns about environmental protection and food safety, has led to an increased interest in the development of ‘edible coatings’ to replace synthetic ingredients, like polyethylene, by natural ingredients.

Edible coatings incorporate proteins, polysaccharides and lipids (Baldwin, 1994). Polysaccharides have been widely used because of their ability to form films and their selective permeabilities to O₂ and CO₂ (Nisperos-Carriedo, 1994). Among polysaccharides, chitosan (poly β-(1 → 4)N-acetyl-D-glucosamine) has been used as coating of some fruits and vegetables, due to its antimicrobial and biostimulant activities, as well as film forming properties (Zeng et al., 2010; Zhang et al., 2011). Films and coatings based on chitosan present a selective permeability to O₂ and CO₂, and good mechanical properties. However, its hydrophilic nature makes it poor moisture barrier (Vargas et al., 2008). Many works have shown the benefits of chitosan in fruits, such as strawberry, mango, peach, grapes, by providing a modified atmosphere and controlling decay (Bautista-Baños et al., 2006; Li and Yu, 2000; Sanchez-Gonzalez et al., 2011; Srinivasa et al., 2002; Vargas et al., 2006). In citrus fruit, significant reduction of postharvest Penicillium decay and delay of fruit senescence during long-term cold storage of different citrus species and cultivars have been observed after the application of certain chitosan formulations (Abbas et al., 2008; Chien and Chou, 2006; Chien et al., 2007; El Ghaouth et al., 2000; Fornes et al., 2005; Galed et al., 2004). However, the antimicrobial activity seems to depend on its molecular weight and the degree of deacetylation and of chemical degradation.

Fruits and vegetables, especially citrus fruit, constitute an important nutritional source of health related compounds, mainly vitamin C, as well as polyphenolic compounds such as flavonoids. The variety and abundance of antioxidant compounds in citrus make synergy between these compounds possible, contributing to the high total antioxidant capacity of the fruit (Sanchez-Moreno et al., 2003). The functional and nutritional quality of fruits should be maintained during postharvest until these reach the consumer. Despite this interest for maintaining the health-related quality of fruit, scarce works have studied the influence of citrus postharvest treatments on their bioactive compounds (Biolatto et al., 2005; Contreras-Oliva et al., 2011; Del Caro et al., 2004; Girennavar et al., 2008; Patil et al., 2004; Perez et al., 2005; Rapisarda et al., 2008; Vanamala et al., 2005, 2007), with no works related to the effect of fruit coating. Therefore, there is a necessity for determining possible effects of usual postharvest practices applied on citrus fruit such as waxing, especially when the coating formulation includes bioactive substances like chitosan. Although it is well known that coatings induced important changes in the fruit internal atmosphere, no information can be found about their effect on health-related compounds of clementine mandarins. Therefore, the objective of this work was to study the effect of a commercial chitosan formulation applied at different SC on the physiology, sensory, and health-related quality of mandarins cv. ‘Oronules’ and compare its effect with a commercial coating.

Materials and methods

Materials

Crab-shell chitosan, Biorend®, was supplied by Idebio, S.L. This product is manufactured as a water solution (pH = 4.8) of chitosan at 1.8 g/100 mL. Polysorbate 80 (Tween 80) was from Panreac Química, S.A. (Barcelona, Spain). 2,2-diphenyl-1-picrylhydrazyl (DPPH^•), potassium dihydrogen phosphate (KH₂PO₄), meta-phosphoric acid (MPA), phosphoric acid (H₃PO₄), Folin-Ciocalteu's phenol reagent, sodium carbonate (Na₂CO₃), gallic acid and standard L-ascorbic acid (AA) were purchased from Sigma (Sigma-Aldrich Chemie, Steinhein, Germany). Acetic acid glacial and dimethyl sulfoxide (DMSO) were from Scharlau (Sentmenat, Spain). Methanol was from BDH Prolabo (Poole, UK). 1,4-dithio-DL-threitol (DTT) and hesperidin (hesperitin-7-0-rutinoside) were obtained from Fluka (Sigma Co., Barcelona, Spain). Narirutin (naringenin-7-rutinoside) and didymin (isosakuranetin-7-rutinoside) were purchased from Extrasynthese (Genay, France). All solvents used were of HPLC-grade and ultrapure water (Milli-Q) was used for the analysis.

Methods

Sample preparation and coating application

Mandarins cv. ‘Oronules’ were harvested from a local grove in Valencia (Spain) and transported the same day to the research laboratory. Fruit were selected for size, color and absence of physical damage, and then dipped 1 min in 500 ppm imazalil solution to avoid decay during storage, following air drying.

Mandarins were coated on the commercial chitosan solution prepared at three soluble contents (SC): 0.6%, 1.2% or 1.8% (0.6% Ch, 1.2% Ch and 1.8% Ch). To improve wetting of the coating to the citrus surface, 0.1% (v/v) of Tween 80 was added to the chitosan solutions. Fruit were dip-coated by immersion in the chitosan solutions for 20 s, drained of excess coating and dried in a tunnel at 55 °C for 2 min. With the aim of comparing these chitosan coatings with commercial procedures, a commercial wax coating (CW): Citrashine conservación 10% (Cerexagri Ibérica, S.A., Spain) was applied by spraying with nozzles in the citrus processing line placed at the Instituto Valenciano de Investigaciones Agrarias (IVIA), followed by drying in a tunnel at the same conditions used with the chitosan coatings. Control fruit consisted on uncoated mandarins that were initially washed and dried in the tunnel under similar conditions to coated fruit. CW consisted of a polyethylene/shellac mixture at total SC of 10%. After coating, fruit were stored for 28 days at 5 °C and 90–95% relative humidity (RH), followed by 6 days at 20 °C simulating retail handling conditions. Additionally, one group of fruit from each treatment was stored directly in retail conditions at 20 °C and 75% RH for 8 days.

Weight loss

Three lots of 30 fruit per treatment were used to measure weight loss. The same fruit were weighted at the beginning of the experiment and at the end of each storage period. The results were expressed as the percentage loss of initial weight.

Fruit firmness

Firmness of 20 fruit per treatment was determined at the end of each storage period using an Instron Universal Testing Machine (Model 4301, Instron Corp., Canton, MA). Each fruit was placed between two flat surfaces and compressed at a constant speed of 5 mm/min, by closing together the upper surface, which consists on a probe that ends in a flat area of 2.5 cm in size. The machine gave the deformation (mm) after application of a load of 10 N to the equatorial region of the fruit. Results were expressed as percentage of deformation related to initial diameter.

Internal gas composition

Ten fruit per treatment were used to calculate internal gas concentrations. Internal CO₂ and O₂ concentrations of each sample were obtained by withdrawing 1 mL internal gas sample from the mandarin central cavity with a syringe while the fruit was immersed under water. The gas sample was then injected into a gas chromatograph (Thermo Fisher Scientific, Inc., Waltham, MA) fitted with a Porapak QS 80/100 (1.2 m × 0.32 cm) column, followed by a molecular sieve 5A 45/60 (1.2 m × 0.32 cm²) column. Temperatures were 35 °C, 125 °C and 180 °C, respectively, for the oven, injector and thermal conductivity detector. Helium was used as carrier gas at 22 mL/min flow rate. Peak areas obtained from standard gas mixtures were determined before and after analysis of samples and results were expressed as percentage.

Ethanol and acetaldehyde content

Ethanol and acetaldehyde concentration in juice were determined by head-space gas chromatography (Davis and Chace, 1969). Three juices of 10 fruit each, per treatment, were prepared for the analysis. Five milliliters of juice were transferred to 10 mL vials with crimp-top caps and TFE/silicone septa seal. Volatiles were analyzed using a gas chromatograph (Thermo Fisher Scientific, Inc., Waltham, MA) with a flame ionization detector and a 1.2 m × 0.32 cm Porapack QS 80/100 column. Temperatures of the oven, injector, and detector were 150 °C, 175 °C, and 200 °C, respectively. Helium was used as the carrier gas at a flow rate of 28 mL/min. A 1 mL sample of the headspace was withdrawn from each vial previously equilibrated in the autosampler incubation chamber for 10 min at 40 °C. Ethanol and acetaldehyde were identified by comparison of retention times with standards. Results were expressed as milligram per liter juice.

Sensory evaluation

The effect of coatings on sensory quality of the samples, initially and after each storage period, was conducted by 10 to 15 trained judges. Panelists rated flavor on a 9-point scale, where 1 to 3 represented a range of non-acceptable quality with the presence of off-flavor, 4 to 6 represented a range of acceptable quality, and 7 to 9 represented a range of excellent quality. Off-flavor presence was evaluated using a 6-point scale, in which 0 represented absence of off-flavor and 5 high presence of off-flavor.

One sample consisted of segments taken from about 4 individual fruits. Samples were presented to panelists in trays labeled with 3-digit random codes and served at room temperature (25 ± 1 °C). The judges had to taste several segments of each sample in order to compensate, as far as possible, for biological variation of the material. Mineral spring water was provided for rinsing between samples.

Fruit internal quality

Soluble solids content (SSC) was measured with a digital refractometer (Atago, Model PR1) and titratable acidity (TA) was determined by titration with 0.1 N NaOH to pH 8.1 and expressed as g citric acid per L of juice. The maturity index (MI) was calculated as SSC/TA ratio. Three juices of 10 fruit each, per treatment, were prepared to determine the above parameters.

Bioactive compounds

The total antioxidant capacity was analyzed by the DPPH^• assay (Brand-Williams et al., 1995). One-mL of juice diluted with 2 mL of methanol was centrifuged at 14,000 rpm and 4 °C for 5 min. Five methanolic dilutions from the supernatant (7.5 µL) were mixed with 392.5 µL of DPPH^• (24 mg/L) and kept in darkness for 30 min. Afterwards, the change in absorbance at 515 nm was measured in a Multiskan spectrum microplate reader (Thermo Labsystem, USA). For each dilution, the percentage of remaining DPPH^• was determined on the basis of the DPPH^• standard curve. The amount of juice in each dilution was plotted against the amount of DPPH^• radical remaining. Using this curve, the EC₅₀ value was calculated, which expresses the amount of mandarin juice needed to reduce 1 kg of DPPH^• by 50% (L juice/kg of DPPH^•); thus, lower EC₅₀ values mean higher antioxidant capacity.

Total ascorbic acid (TAA) was determined by the sum of ascorbic acid (AA) plus dehydroascorbic acid (DAA), by using the reducing agent DTT (Dhuique-Mayer et al., 2005). One-mL of sample was diluted to 10 mL with 2.5% (w/v) meta-phosphoric. Two-ml of this solution were mixed with 0.4 mL of DTT (0.02 g de DTT in 1 mL ultrapure water) for 2 h in darkness. Afterwards, the extracts were filtered through a 0.45 mm Millipore filter and analyzed by HPLC. The HPLC analyses were performed on a Lachrom Elite HPLC (Merck Hitachi, Germany) equipped with a L-2200 autosampler, L-2130 quaternary pump, L-2300 column oven and L-2450 diode array detector. System conditions were: injection volume 20 µL, oven 25 °C, detector wavelength 245 nm, flow rate 1 mL/min, column Lichospher 100 RP-18 of 25 cm × 0.4 cm with 5 µm particle size (Merck, Darmstadt, Germany). The mobile phase was 2% KH₂PO4 adjusted to pH 2.3 with H₃PO₄. Results were expressed as milligrams of ascorbic acid per litre of juice.

Flavanone glycosides, hesperidin, narirutin and didymin (mg/L) were determined by the method described by Cano et al. (2008) slightly modified. Two-mL of juice were homogenized with 2 mL of DMSO:MeOH (1:1 v/v) and centrifuged for 30 min at 12,000 rpm and 4 °C. The supernatant was filtered through a 0.45 µm nylon filter and analyzed by HPLC-DAD using the HPLC equipment described above. System conditions were: injection volume 10 µL, oven 25 °C, detector wavelength 280 nm, flow rate 1 mL/min, column Lichospher 100 RP-18 of 25 cm × 0.4 cm preceded by a precolumn (4 mm × 4 mm) with 5 µm particle size (Merck, Darmstadt, Germany). The mobile phase was acetonitrile (phase A):0.6% acetic acid (phase B) with initial condition of 10% A for 2 min, reaching 75% A in the following 28 min, then back to the initial condition in 1 min and held for 5 min prior to the next sample injection. The main flavanone glycosides were identified by matching their respective spectra and retention times with those of commercially obtained standards. Narirutin, hesperidin and didymin contents were calculated by comparing the integrated peak areas of each individual compound to that of its pure standards.

Mandarin juice was analyzed for total phenolic concentration by the Folin-Ciocalteu (FC) colorimetric method (Singleton and Rossi, 1965): 0.3 mL of mandarin juice was diluted with 1.7 mL of 80% aqueous methanol. Appropriately diluted extract (0.4 mL) was mixed with 2 mL of FC commercial reagent (previously diluted with water 1:10, v:v) and incubated for 1 min before 1.6 mL sodium carbonate (7.5% w/v) was added. The mixture was incubated for 1 h at room temperature. The absorbance of the resulting blue solution was measured spectrophotometrically at 765 nm (Thermo UV1, Thermo Electron Corporation, UK) and the concentration of total phenolics was expressed as gallic equivalents (mg/L).

Antioxidant capacity, total vitamin C, flavanone glycosides and total phenolics were determined in 3 juices of 10 fruit each, per treatment.

Statistical analysis

A complete randomized design was used to perform the analysis of the samples. Two-way ANOVA was performed to determine the effect of treatment and storage time on the quality attributes. Because of significant interactions, individual one-way ANOVA was also performed for each level of each factor. Significant differences between means were determined by least significant difference (LSD) at p ≤ 0.05. Data were analyzed using STATGRAPHICS Plus 2.1 (Manugistics, Inc., Rockville, Maryland, USA).

Results and discussion

Weight loss

As expected, weight loss of coated and uncoated ‘Oronules’ mandarins increased as storage time increased, reaching values around 10% (Figure 1). For all storage periods, the commercial wax was the most effective treatment reducing weight loss of the mandarins; whereas, chitosan only reduced weight loss compared to the control in mandarins coated with 1.2% and 1.8% Ch after 9 days of storage at 20 °C, and mandarins coated with 1.2% Ch after 1 week of storage at 5 °C plus 1 week at 20 °C.

Figure 1.

Weight loss (%) of coated and uncoated ‘Oronules’ mandarins during storage. Means within each storage time with the same letter are not different (p ≤ 0.05).

Commercial waxes used by the citrus industry are made of natural or synthetic waxes (e.g., beeswax, carnauba, polyethylene), fatty acids, oils, shellac, emulsifier, plasticizers, anti-foam agents, and surfactants (Hagenmaier, 1998). The hydrophobic nature of these ingredients contributes to significantly reduce weight loss of coated fruit. However, the hydrophilic nature of polysaccharides makes them to be poor moisture barrier at high RH, which is the case of fruit application. In this sense, at a RH gradient 100/50 (%/%) the value of water vapor permeability (WVP) of stand-alone chitosan films is 360 × 10⁻¹¹ g/m.s.Pa (Park et al., 2001); whereas, for lipids and resins, such as shellac and carnauba, WVPs are 0.5 × 10⁻¹¹ and 0.033 × 10⁻¹¹ g/m.s.Pa, respectively (Martín-Polo et al., 1992; Vargas et al., 2008) at a RH gradient 0/100 (%/%).

In spite of the low moisture barrier offered by chitosan films, some works have shown an effect of chitosan reducing weight loss of citrus fruit (Chien et al., 2007; Galed et al., 2004; Salvador et al., 2003), pepper and cucumbers (El Ghaouth et al., 1991), and strawberries (Hernández-Muñoz et al., 2006). Differences with our results might be due to differences in the fruit type and cultivar, storage conditions and the chitosan nature. Previous works reported that similar hydroxypropyl methylcellulose-lipid edible coatings were effective reducing weight loss of ‘Ortanique’ mandarins, whereas they did not reduce weight loss of coated ‘Valencia’ oranges (Valencia-Chamorro, 2009; Valencia-Chamorro et al., 2009). Many works have also shown that the effectiveness of chitosan coatings depend, among others factors, on the molecular weight (Mw) and the degree of deacetylation (Chien et al., 2007; Bautista-Baños et al., 2006). For instance, coating ‘Murcott’ mandarins with low Mw chitosan (15 kDa) was effective controlling fruit weight loss, whereas no differences were found between mandarins coated with high Mw chitosan (357 kDa) and control samples (Chien et al., 2007).

Fruit firmness

Firmness of uncoated mandarins after storage was lower than firmness at harvest (6.7% deformation), as can be seen by the higher percentage deformation of the fruit, whereas mandarins coated with the commercial wax maintained mandarin firmness during cold storage followed by 1 week storage at 20 °C (Figure 2). After cold storage at 5 °C followed by one week at 20 °C, mandarins coated with 1.8% Ch were firmer (lower deformation) than uncoated mandarins. However, after 9 days of storage at 20 °C firmness of 1.8% Ch-coated mandarins did not differ from control samples, and only 1.2% Ch showed a slight effect maintaining firmness.

Figure 2.

Firmness (% of deformation) of coated and uncoated ‘Oronules’ mandarins during storage. Means within each storage time with the same letter are not different (p ≤ 0.05). Firmness at harvest was 6.7% deformation.

Rodov et al. (2000) reported that firmness of citrus fruit depends primarily on turgidity and weight loss rate. However, other authors have reported that a correlation between citrus fruit weight loss and firmness is not always observed (Hagenmaier, 2000; Navarro-Tarazaga et al., 2008; Pérez-Gago et al, 2002). In this work, the lower weight loss observed on mandarins coated with the commercial wax correlates with a higher firmness of these mandarins. However, such a correlation between firmness and weight loss was not observed on mandarins coated with chitosan. Although the chitosan coating had little or no effect controlling mandarin weight loss, mandarins coated with 1.8% Ch showed higher firmness than those coated with the chitosan at lower SC. The slight positive effect of the 1.8% Ch coating slowing down firmness loss of mandarins could be due to the biostimulating properties of chitosan, through an improvement of the rind of ‘Oronules’ mandarins. This cultivar, although very appreciated by its excellent flavor quality, shows a tendency to puffing after storage. Puffing is a physiological disorder characterized by the separation of the pulp from the rind (Burdon et al., 2007). Even though this defect did not become visible in this experiment, early stages of puffing could accelerate firmness loss of mandarins during storage. The beneficial effect of the high chitosan content (1.8% Ch) or commercial coating reducing firmness loss of ‘Oronules’ mandarins could help to slow down or minimize the appearance of puffing.

Internal gas composition

The application of the coatings modified the internal atmosphere in the fruit, increasing the CO₂ level and reducing the O₂ level compared to the control (Figure 3). Mandarins coated with 0.6% Ch presented the lowest internal CO₂ and the highest O₂ values among coated samples. As SC of the chitosan coating was increased, internal CO₂ level of mandarins increased and the O₂ level decreased. Many works have described a direct relation between the internal gas modification of coated fruit and coating thickness, which depends on SC, viscosity, and density of the coating formulation (Banks et al., 1993; Cisneros-Zevallos and Krochta, 2003; Navarro-Tarazaga and Pérez-Gago, 2006; Park et al., 2007). In our case, as SC of the chitosan coating increased the internal CO₂ content increased by nearly a 30%, reaching values similar to mandarins coated with the commercial wax. Similarly, Salvador et al. (2003) also found no differences in the internal CO₂ content of ‘Fortune’ mandarins coated with a commercial wax and with a chitosan coating at 1.25% SC.

Figure 3.

Internal CO₂ and O₂ contents of coated and uncoated ‘Oronules’ mandarins during storage. Means within each storage time with the same letter are not different (p ≤ 0.05). At harvest, internal CO₂ and O₂ were 2.83 and 16.2 kPa, respectively.

Among the different ingredients incorporated into coating formulations, shellac has been known to reduce the O₂ gas exchange to a greater extent than waxes, creating in many cases an anaerobic/fermentative environment within the fruit when internal O₂ concentration becomes too low (Baldwin et al., 1995; Hagenmaier, 2000). Therefore, coatings having low O₂ permeabilities were rated by a sensory panel as markedly less fresh than fruit with higher permeability coatings (Hagenmaier, 2002).

At the end of the cold storage period the concentration of internal CO₂ and O₂ levels on mandarins coated with the commercial coating reached values around 6% and 15%, respectively. In general, these levels of internal O₂ could be considered not low enough to create anaerobic conditions inside the fruit (Baldwin et al., 1997).

Ethanol and acetaldehyde contents

Fruit coating application induces an increase in the amount of internal volatiles associated with anaerobic conditions due to the gas barrier offered by coatings. In this work, mandarins coated with 0.6% Ch showed no differences on volatile content with uncoated mandarins (Figure 4). As soluble contents of the chitosan coating increased, ethanol and acetaldehyde content of mandarins increased, which confirmed the creation of a modified atmosphere within the fruit. At the end of the storage period, the concentrations of ethanol and acetaldehyde of mandarins coated with 1.8% Ch and the commercial coating were the highest. These results correlate with changes in the internal gas composition (Figure 3).

Figure 4.

Acetaldehyde and ethanol contents of coated and uncoated ‘Oronules’ mandarins during storage. Means within each storage time with the same letter are not different (p ≤ 0.05). At harvest, acetaldehyde and ethanol contents were 1.07 and 50.8 mg/L, respectively.

In general, the concentration of ethanol in the juice of coated mandarins after 4 weeks of storage at 5 °C followed by 1 week at 20 °C was in the range of 100–700 mg/L. Different works have reported higher ethanol content within coated citrus fruit after prolonged cold storage, with values that depended on citrus cultivar, coating type, and storage conditions (Baldwin et al., 1995; Hagenmaier and Baker, 1993; Hagenmaier and Shaw, 2002; Navarro-Tarazaga et al., 2008; Rojas-Argudo et al., 2009). In this work, the internal atmosphere caused by the coatings did not create anaerobic conditions inside the fruit, which translated in a moderated increase in the content of volatile components.

Sensory evaluation

Sensory quality of mandarins was evaluated within the range of acceptability after 4 weeks of storage at 5 °C plus 1 week at 20 °C, with values from 5.3 to 5.9 and no differences among treatments (data not shown). Several works showed that the contribution to off-flavor of volatile content depends on citrus cultivar. Ke and Kader (1990) established the minimum ethanol content associated with off-flavor in ‘Valencia’ oranges to be 2000 mg/L; whereas, Pérez-Gago et al. (2002) found flavor degradation in ‘Fortune’ mandarin at an ethanol content above 3000 mg/L and Navarro-Tarazaga and Pérez-Gago (2006) found that ethanol content of 1000 mg/L reduced flavor quality of ‘Clemenules’ mandarins. In our work, the highest ethanol content reached for ‘Oronules’ mandarins was 710 mg/L after 4 weeks of storage at 5 °C (Figure 4), which was not enough to induce flavor degradation in this citrus cultivar.

In an informal visual evaluation, coating was not perceived on the fruit surface other than for the higher gloss than all coated mandarins had compared to the control. When comparing fruit gloss, all the coated mandarins had higher gloss than the control and it increased as soluble contents of the chitosan formulation increased, observing little differences between the commercial wax and the 1.8% Ch coating.

Fruit internal quality

Coating application did not affect TA, SSC, or MI of ‘Oronules’ mandarins. SSC ranged from 11.5 at harvest to 12.8 at the end of storage. Acidity ranged from 10.9 g/L at harvest to 9.2 g/L at the end of storage. MI ranged from 10.6 at harvest to 12.0 at the end of storage. No differences among treatments were observed for these parameters. The effect of coating application on internal quality parameters has been shown to depend on coating type, fruit cultivar and storage conditions. Some authors have found no differences in these parameters after coating application on different citrus cultivars (Baldwin et al., 1995; Obenland et al., 2008); whereas others have found a decrease in SSC and TA losses compared to uncoated fruits, which was always related to a decrease in weight loss and respiration rate (Togrul and Arslan, 2004). Chien et al. (2007) observed that a low molecular weight chitosan coating beneficially influenced internal quality of ‘Murcott’ mandarins and coated fruit had higher acidity and lower SSC than uncoated fruit; whereas, a high molecular weight chitosan coating did not influence these parameters.

Bioactive compounds

Table 1 shows the content of the bioactive compounds of coated and uncoated ‘Oronules’ mandarins. The TAA of mandarins was not affected by coating application or the storage length. Togrul and Arslan (2004), however, reported that ascorbic acid loss after storage was delayed when mandarins citrus were coated with carboxymethyl cellulose. This result was explained by the gas barrier of the coatings which decreased the potential autoxidation of ascorbic acid in the presence of oxygen. In our work, although commercial and chitosan coatings reduced the level of internal O₂ (Figure 3), these levels could be not low enough to affect the TAA of the mandarin.

Table 1.

Total phenolics, flavonoids, ascorbic acid contents and antioxidant activity of coated and uncoated ‘Oronules’ mandarins after storage

		Total ascorbic acid (mg/L)	Hesperidin (mg/L)	Narirutin (mg/L)	Dydimin (mg/L)	Total phenolics (mg/L)	EC₅₀ (L juice/kg DPPH)
At harvest		583.6 ± 24.7	220.1 ± 14.6	16.6 ± 1.8	3.8 ± 0.1	640.4 ± 17.6	187.83 ± 11.32
9 d 20 °C	Control	626.1 ± 31.2 a	255.7 ± 6.9 b	20.2 ± 1.0 c	4.5 ± 0.3 b	655.0 ± 12.2 a	187.96 ± 5.32 a
	Commercial wax	602.0 ± 21.7 a	193.5 ± 14.2 a	16.9 ± 1.7 b	3.4 ± 0.3 a	662.6 ± 24.0 a	179.98 ± 12.14 a
	0.6% Chitosan	580.3 ± 55.9 a	193.3 ± 12.2 a	16.1 ± 1.0 ab	3.3 ± 0.2 a	647.3 ± 31.8 a	193.91 ± 6.51 a
	1.2% Chitosan	598.6 ± 12.3 a	201.2 ± 2.6 a	14.4 ± 1.8 a	3.1 ± 0.1 a	655.2 ± 10.5 a	179.17 ± 6.78 a
	1.8% Chitosan	597.7 ± 4.3 a	188.5 ± 15.9 a	14.0 ± 0.6 a	3.4 ± 0.3 a	630.1 ± 22.0 a	179.37 ± 2.76 a
1 wk 5 °C+ +1 wk 20 °C	Control	561.9 ± 35.3 a	204.6 ± 7.4 a	16.4 ± 1.9 a	4.0 ± 0.2 c	653.5 ± 0.8 b	–
	Commercial wax	528.8 ± 52.6 a	214.9 ± 19.4 a	16.7 ± 1.2 a	3.7 ± 0.2 bc	626.7 ± 8.0 ab
	0.6% Chitosan	586.2 ± 52.2 a	211.2 ± 12.1 a	13.5 ± 1.7 a	3.4 ± 0.1 ab	652.7 ± 38.5 b
	1.2% Chitosan	602.6 ± 55.3 a	202.5 ± 16.9 a	13.9 ± 2.7 a	3.3 ± 0.2 a	656.7 ± 38.1 b
	1.8% Chitosan	527.1 ± 36.6 a	206.6 ± 5.7 a	16.2 ± 1.8 a	3.4 ± 0.1 ab	586.5 ± 1.1 a
2 wk 5 °C+ +1 wk 20 °C	Control	622.3 ± 45.9 a	179.6 ± 11.2 a	12.2 ± 1.5 a	3.1 ± 0.3 a	659.4 ± 29.1 a	–
	Commercial wax	623.4 ± 12.1 a	206.1 ± 6.8 b	14.4 ± 0.6 a	3.4 ± 0.1 a	669.2 ± 45.2 a
	0.6% Chitosan	585.5 ± 11.3 a	174.6 ± 10.9 a	12.8 ± 1.3 a	3.1 ± 0.2 a	641.6 ± 23.1 a
	1.2% Chitosan	629.2 ± 88.0 a	188.4 ± 10.0 a	12.6 ± 1.2 a	3.2 ± 0.4 a	674.3 ± 13.5 a
	1.8% Chitosan	555.8 ± 43.9 a	189.9 ± 4.2 ab	13.0 ± 0.4 a	3.2 ± 0.1 a	686.2 ± 15.5 a
3 wk 5 °C+ +1 wk 20 °C	Control	566.9 ± 71.3 a	205.8 ± 8.0 a	14.8 ± 1.0 b	3.6 ± 0.1 b	643.0 ± 22.9 a	185.37 ± 3.16 b
	Commercial wax	543.3 ± 15.1 a	203.4 ± 9.1 a	13.5 ± 0.4 ab	3.1 ± 0.1 a	613.6 ± 11.6 a	189.79 ± 1.89 bc
	0.6% Chitosan	572.5 ± 53.7 a	193.3 ± 9.4 a	12.3 ± 0.8 a	3.0 ± 0.1 a	615.9 ± 26.5 a	159.33 ± 7.89 a
	1.2% Chitosan	565.8 ± 6.4 a	214.4 ± 3.7 a	14.7 ± 1.0 b	3.3 ± 0.0 a	616.7 ± 35.4 a	155.15 ± 6.38 a
	1.8% Chitosan	548.2 ± 37.4 a	225.2 ± 22.3 a	15.3 ± 1.5 b	3.2 ± 0.2 a	630.1 ± 30.3 a	196.39 ± 6.79 c
4 wk 5 °C+ +1 wk 20 °C	Control	649.9 ± 67.8 a	221.3 ± 2.1 a	16.7 ± 1.2 a	3.3 ± 0.1 a	654.5 ± 6.4 b	158.12 ± 15.60 a
	Commercial wax	593.8 ± 19.4 a	236.5 ± 26.2 a	18.3 ± 2.3 a	3.1 ± 0.3 a	682.8 ± 9.2 bc	178.92 ± 3.29 b
	0.6% Chitosan	580.6 ± 61.7 a	203.3 ± 11.0 a	13.9 ± 1.2 a	2.8 ± 0.2 a	584.3 ± 27.8 a	218.72 ± 7.84 c
	1.2% Chitosan	529.7 ± 15.1 a	214.2 ± 21.3 a	14.6 ± 2.1 a	2.9 ± 0.2 a	653.2 ± 23.2 b	206.18 ± 4.63 c
	1.8% Chitosan	643.6 ± 63.0 a	225.7 ± 15.4 a	15.2 ± 2.3 a	3.0 ± 0.4 a	719.2 ± 41.3 c	180.20 ± 8.94 b

Means within each storage time followed by the same letter are not different (p ≤ 0.05).

Hesperidin was the more abundant flavonoid in mandarins ‘Oronules’ followed by narirutin and didymin (Table 1). The contents of the different flavonoids were not affected by storage. In general coating application did not have an important effect on flavonoid levels, although some significant differences were found among treatments. Uncoated mandarins stored at 20 °C for 9 days contained higher content of hesperidine, narirutin and didymin than coated mandarins, which could be related to the higher weight loss of uncoated mandarins under this storage condition.

In addition to flavanones, citrus fruit also contain other phenolic compounds, such as flavones and hydroxycinnamic acids (represented by ferulic, caffeic, synapic, and p-coumaric acids) that, although present in a lower concentration, contribute to the total phenolic concentration (Gil-Izquierdo et al., 2002; Rapisarda et al., 1999). Several authors have reported that chitosan act as an exogenous elicitor in plant tissue inducing different responses, such as the de novo biosynthesis of phenolic compounds (Bautista-Baños et al., 2006; Lafontaine and Benhamou, 1996; Meng et al., 2008). In this work, some significant differences were found among treatments depending on storage time, which makes it difficult to draw any conclusion regarding the effect of coating composition (Table 1). Interestingly, at the end of the storage period, total phenolic content increased in mandarins coated with 1.8% Ch, which could be an indication of the chitosan activity as an exogenous elicitor of plant tissue.

The antioxidant capacity was expressed as EC₅₀ or juice quantity necessary to reduce by 50% the DPPH^., thus the lower the value the higher the antioxidant capacity of the citrus fruit. Table 1 shows the antioxidant capacity of the mandarins stored 9 days at 20 °C and stored 3 or 4 wk at 5 °C plus 1 week at 20 °C. Coating application did not affect the total antioxidant capacity of samples stored 9 days at 20 °C. After cold storage, some significant differences were found among treatments depending on storage time, which makes it difficult to draw any conclusion regarding the effect of coating composition. After 4 weeks of cold storage plus 1 week at 20 °C, mandarins coated with 0.6% Ch and 1.2% Ch presented the lowest antioxidant capacity (i.e. highest EC₅₀). These results could be related to the lowest total phenolic content of 0.6% Ch-coated mandarins (p ≤ 0.05) or slightly lower TAA content of 1.2% Ch-coated mandarins (p = 0.08). Previous works have found correlations between vitamin C and antioxidant capacity (Pretel et al., 2005) or phenolic content and antioxidant capacity of citrus (Togrul and Arslan, 2004).

Conclusion

In general, the internal quality and the health-related properties of mandarins were not negatively affected by coating application, although the chitosan formulation should be improved by the incorporation of hydrophobic components to reduce weight loss. Ch-coated mandarins at the highest SC retained firmness after cold storage and contained more phenolics than uncoated ones, which could be related to the biostimulating properties of chitosan. Both commercial wax and the chitosan coating modified the internal atmosphere of the mandarins, with a greater effect at higher chitosan concentration, without affecting the sensory quality.

Footnotes

Acknowledgments

The authors thank Idebio S.L. and Fontestad S.A. for supplying the chitosan and fruit, respectively. Adriana Contreras was also funded by a scholarship from the Consejo Nacional de Ciencias y Tecnología (CONACyT).

Funding

This work was funded by the Consellería de Educación de la Generalitat Valenciana through the project GV/2007/187 and the European Social Fund.

References

Abbas

Abbasi

Maqbool

Yaseen

(2008). Influence of irradiated chitosan coating on post harvest quality of Kinnow (Citrus reticulata Blanco). Asian Journal of Chemistry 20(8): 6217–6227.

Baldwin

(1994). Edible coatings for fresh fruits and vegetables, past, present, and future. In: Krochta

Baldwin

Nisperos-Carriedo

(eds). Edible Coatings and Films to Improve Food Quality. Lancaster: Technomic Publishing Co., 25–64.

Baldwin

Nisperos Carriedo

Shaw

Burns

(1995). Effect of coatings and prolonged storage-conditions on fresh orange flavor volatiles, degrees Brix, and ascorbic-acid levels. Journal of Agricultural and Food Chemistry 43(5): 1321–1331.

Baldwin

Nisperos

Hagenmaier

Baker

(1997). Use of lipids in coatings for food products. Food Technology 51(6): 56–62.

Banks

Dadzie

Cleland

(1993). Reducing gas exchange of fruits with surface coatings. Postharvest Biology and Technology 3(3): 269–284.

Bautista-Baños

Hernández-Lauzardo

Velázquez-Del Valle

Hernández-López

Ait Barka

Bosquez-Molina

(2006). Chitosan as potential natural compounds to control pre and postharvest diseases of horticultural commodities. Crop Protection 25(2): 108–118.

Biolatto

Salitto

Cantet

RJC

Pensel

(2005). Inﬂuence of different postharvest treatments on nutritional quality of grapefruits. Lebensmittel Wissenschaft und Technologie 38(2): 131–134.

Brand-Williams

Cuvelier

Berset

(1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie 28(1): 25–30.

Burdon

Lallu

Yearsley

Osman

Billing

Boldingh

(2007). Postharvest conditioning of Satsuma mandarins for reduction of acidity and skin puffiness. Postharvest Biology and Technology 43(1): 102–114.

10.

Cano

Medina

Bermejo

(2008). Bioactive compounds in different citrus varieties. Discrimination among cultivars. Journal of Food Composition and Analysis 21(5): 377–381.

11.

Chien

Chou

(2006). Antifungal activity of chitosan and its application to control post-harvest quality and fungal rotting of Tankan citrus fruit (Citrus tankan Hayata). Journal of the Science of Food and Agriculture 86(12): 1964–1969.

12.

Chien

Sheu

Lin

(2007). Coating citrus (Murcott tangor) fruit with low molecular weight chitosan increases postharvest quality and shelf life. Food Chemistry 100(3): 1160–1164.

13.

Cisneros-Zevallos

Krochta

(2003). Dependence of coating thickness on viscosity of coating solution applied to fruits and vegetables by dipping method. Journal of Food Science 68(2): 503–510.

14.

Contreras-Oliva

Pérez-Gago

Palou

Rojas-Argudo

(2011). Effect of insecticidal atmosphere and low dose X-ray irradiation in combination with cold quarantine storage on bioactive compounds of clementine mandarins cv. ‘Clemenules'. International Journal of Food Science and Technology 46(3): 612–619.

15.

Davis

Chace

(1969). Determination of alcohol in citrus juice by gas chromatographic analysis head space. HortScience 4(2): 117–119.

16.

Del Caro

Piga

Vacca

Agabbio

(2004). Changes of ﬂavonoids, vitamin C and antioxidant capacity in minimally processed citrus segments and juices during storage. Food Chemistry 84(1): 99–105.

17.

Dhuique-Mayer

Caris-Veyrat

Ollitrault

Curk

Amiot

(2005). Varietal and interspecific influence on micronutrient content in citrus from the Mediterranean area. Journal of Agricultural and Food Chemistry 53(6): 2140–2145.

18.

El Ghaouth

Arul

Ponnanmpalam

(1991). Use of chitosan coating to reduce weight loss and maintain quality of cucumbers and bell pepper fruits. Journal of Food Processing and Preservation 15(5): 359–368.

19.

El Ghaouth

Smilanick

Wilson

(2000). Enhancement of the performance of Candida saitoana by the addition of glycolchitosan for the control of postharvest decay of apple and citrus fruit. Postharvest Biology and Technology 19(1): 103–110.

20.

Fornes

Almela

Abad

Agustí

(2005). Low concentrations of chitosan coating reduce water spot incidence and delay peel pigmentation of Clementine mandarine fruit. Journal of the Science of Food and Agriculture 85(7): 1105–1112.

21.

Galed

Fernández-Valle

Martínez

Heras

(2004). Application of MRI to monitor the process of ripening and decay in citrus treated with chitosan solutions. Magnetic Resonance Imaging 22(1): 127–137.

22.

Gil-Izquierdo

Gil

Ferreres

(2002). Effect of processing techniques at industrial scale on orange juice antioxidant and beneficial health compounds. Journal of Agricultural and Food Chemistry 50(18): 5107–5114.

23.

Girennavar

Jayaprakasha

Mclin

Maxim

Yoo

Patil

(2008). Influence of electron-beam irradiation on bioactive compounds in grapefruits (Citrus paradisi Macf.). Journal of Agricultural and Food Chemistry 56(22): 10941–10946.

24.

Hagenmaier

(1998). Wax microemulsion formulations used as fruit coatings. Proceedings of the Florida State Horticultural Society 111: 251–255.

25.

Hagenmaier

(2000). Evaluation of a polyethylene-candelilla coating for ‘Valencia’ oranges. Postharvest Biology and Technology 19(2): 147–154.

26.

Hagenmaier

(2002). The flavor of mandarin hybrids with different coatings. Postharvest Biology and Technology 24(1): 79–87.

27.

Hagenmaier

Baker

(1993). Reduction in gas-exchange of citrus-fruit by wax coatings. Journal of Agricultural and Food Chemistry 41(2): 283–287.

28.

Hagenmaier

Shaw

(2002). Changes in volatile components of stored tangerines and other specialty citrus fruits with different coatings. Journal of Food Science 67(5): 1742–1745.

29.

Hernández-Muñoz

Almenar

Ocio

Gavara

(2006). Effect of calcium dips and chitosan coatings on postharvest life of strawberries (Fragaria x ananassa). Postharvest Biology and Technology 39(3): 247–253.

30.

Kader

(1990). Tolerance of ‘Valencia’ oranges to controlled atmospheres as determined by physiological responses and quality attributes. Journal of the American Society for Horticultural Science 115(5): 779–783.

31.

Lafontaine

Benhamou

(1996). Chitosan treatment: an emerging strategy for enhancing resistance of greenhouse tomato plants to infection by Fusarium oxysporum f. sp. radicis-lycopersici. Biocontrol Science and Technology 6(1): 111–124.

32.

(2000). Effect of chitosan on incidence of brown rot, quality and physiological attributes of postharvest peach fruit. Journal of the Science of Food and Agriculture 81(2): 269–274.

33.

Martín-Polo

Voilley

Blond

Colas

Mesnier

Ploquet

(1992). Hydrophobic films and their efficiency against moisture transfer. 2 Influence of the physical state. Journal of Agricultural and Food Chemistry 40(3): 413–418.

34.

Meng

Liu

Tian

(2008). Physiological responses and quality attributes of table grape fruit to chitosan preharvest spray and postharvest coating during storage. Food Chemistry 106(2): 501–508.

35.

Navarro Tarazaga

MLL

Pérez-Gago

(2006). Effect of edible coatings on quality of mandarins cv. Clemenules. Proceedings of the Florida State Horticultural Society 119: 350–352.

36.

Navarro-Tarazaga

MLL

Del Río

Krochta

Pérez-Gago

(2008). Fatty acid effect on hydroxypropyl methylcellulose-beeswax edible film properties and postharvest quality of coated ‘Ortanique’ mandarins. Journal of Agricultural and Food Chemistry 56(2): 10689–10696.

37.

Nisperos-Carriedo

(1994). Edible coatings and films based on polysaccharides. In: Krochta

Baldwin

Nisperos-Carriedo

(eds). Edible Coatings and Films to Improve Food Quality. Lancaster: Technomic Publishing Co., 305–335.

38.

Obenland

Collin

Sievert

Fjeld

Doctor

Arpaia

(2008). Commercial packing and storage of navel oranges alters aroma volatiles and reduces flavor quality. Postharvest Biology and Technology 47(2): 159–167.

39.

Ortiz

Zaragoza

Bono

(1988). The major citrus cultivars in Spain. HortScience 23(4): 691–693.

40.

Park

Bunn

Vergano

Testin

(2007). Gas permeation and thickness of the sucrose polyester Semperfresh coatings on apples. Journal of Food Processing and Preservation 18(5): 349–358.

41.

Park

Lee

Jung

Park

(2001). Biopolymer composite films based on k-carrageenan and chitosan. Materials Bulletin Research 36(3-4): 511–519.

42.

Patil

Vanamala

Hallman

(2004). Irradiation and storage influence on bioactive components and quality of early and late season ‘Rio Red’ grapefruit (Citrus paradisi Macf.). Postharvest Biology and Technology 34(1): 53–64.

43.

Perez

Luaces

Oliva

Rios

Sanz

(2005). Changes in vitamin C and flavour components of mandarin juice due to curing of fruits. Food Chemistry 91(1): 19–24.

44.

Pérez-Gago

Rojas

Del Río

(2002). Effect of lipid type and amount of edible hydroxypropyl methylcellulose-lipid composite coatings used to protect postharvest quality of mandarins cv. Fortune. Journal of Food Science 67(8): 2903–2909.

45.

Petracek

Hagenmaier

Dou

(1999). Waxing effects on citrus fruit physiology. In: Schirra

(ed.) Advances in Postharvest Diseases and Disorders Control of Citrus Fruit. Trivandrum: Research Signpost, 71–92.

46.

Porat

Weiss

Cohen

Daus

Biton

(2005). Effects of polyethylene wax content and composition on taste, quality, and emission of off-flavor volatiles in 'Mor' mandarins. Postharvest Biology and Technology 38(3): 262–268.

47.

Pretel

Botella

Zapata

Amorós

Serrano

(2005). Antioxidative activity and general fruit characteristics in different traditional orange (Citrus sinensis (L.) Osbeck) varieties. European Food Research and Technology 219(5): 474–478.

48.

Rapisarda

Tomaino

Lo Cascio

Bonina

De Pasquale

Saija

(1999). Antioxidant effectiveness as influenced by phenolic content of fresh orange juices. Journal of Agricultural and Food Chemistry 47(11): 4718–4723.

49.

Rapisarda

Lo Bianco

Pannuzzo

Timpanaro

(2008). Effect of cold storage on vitamin C, phenolics and antioxidant activity of five orange genotypes [Citrus sinensis (L.) Osbeck]. Postharvest Biology and Technology 49(3): 348–354.

50.

Rodov

Agar

Peretz

Nafussi

Kim

Ben-Yehoshua

(2000). Effect of combined application of heat treatments and plastic packaging on keeping quality of ‘Oroblanco’ fruit (Citrus grandis L. x C. paradisi Macf.). Postharvest Biology and Technology 20(3): 287–294.

51.

Rojas-Argudo

Del Río

Pérez-Gago

(2009). Development and optimization of locust bean gum (LBG)-based edible coatings for postharvest storage of ‘Fortune' mandarins. Postharvest Biology and Technology 52(2): 227–234.

52.

Salvador

Cuquerella

Monterde

(2003). Efecto del quitosano aplicado como recubrimiento en mandarinas ‘Fortune’. Revista Iberoamericana de Tecnología Poscosecha 5(2): 122–127.

53.

Sanchez-Gonzalez

Pastor

Vargas

Chiralt

Gonzalez-Martinez

Chafer

(2011). Effect of hydroxypropylmethylcellulose and chitosan coatings with and without bergamot essential oil on quality and safety of cold-stored grapes. Postharvest Biology and Technology 60(1): 57–63.

54.

Sanchez-Moreno

Plaza

De Ancos

Cano

(2003). Quantitative bioactive compounds assessment and their relative contribution to the antioxidant capacity of commercial orange juice. Journal of the Science of Food and Agriculture 83(5): 430–439.

55.

Singleton

Rossi

Jr (1965). Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. American Journal of Enology and Viticulture 16(3): 144–158.

56.

Srinivasa

Baskaran

Ramesh

Prashanth

KVH

Tharanathan

(2002). Storage studies of mango packed using biodegradable chitosan film. European Food Research Technology 215(6): 504–508.

57.

Togrul

Arslan

(2004). Carboxymethyl cellulose from sugar beet pulp cellulose as a hydrophilic polymer in coating of mandarin. Journal of Food Engineering 62(3): 271–279.

58.

Valencia-Chamorro SA. (2009). Desarrollo de recubrimientos comestibles con actividad antifúngica en frutos cítricos. PhD Dissertation, Departamento de Tecnología de Alimentos, Universitat Politécnica de València, Spain, 263 pp.

59.

Valencia-Chamorro

Pérez-Gago

Del Río

Palou

(2009). Effect of antifungal hydroxypropyl methylcellulose (HPMC)-lipid edible composite coatings on postharvest decay development and quality attributes of cold-stored ‘Valencia’ oranges. Postharvest Biology and Technology 54(2): 72–79.

60.

Vanamala

Cobb

Turner

Lupton

Yoo

Pike

(2005). Bioactive compounds of grapefruit (Citrus paradisi cv. Rio red) respond differently to postharvest irradiation, storage, and freeze drying. Journal of Agricultural and Food Chemistry 53(10): 3980–3985.

61.

Vanamala

Cobb

Loaiza

Yoo

Pike

Patil

(2007). Ionizing radiation and marketing simulation on bioactive compounds and quality of grapefruit (Citrus paradisi cv. Rio Red). Food Chemistry 105(4): 1404–1411.

62.

Vargas

Albors

Chiralt

González-Martínez

(2006). Quality of cold-stored strawberries as affected by chitosan–oleic acid edible coatings. Postharvest Biology and Technology 41(2): 164–171.

63.

Vargas

Pastor

Chiralt

McClements

González-Martínez

(2008). Recent advances in edible coatings for fresh and minimally-processed fruits. Critical Reviews in Food Science and Nutrition 48(6): 496–511.

64.

Zeng

Deng

Ming

Deng

(2010). Induction of disease resistance and ROS metabolism in navel oranges by chitosan. Scientia Horticulturae 126(2): 223–228.

65.

Zhang

Liu

(2011). Effects of chitin and its derivative chitosan on postharvest decay of fruits: a review. International Journal of Molecular Sciences 12(2): 917–934.