Chitosan nanoparticles loaded with Cinnamomum zeylanicum essential oil enhance the shelf life of cucumber during cold storage Postharvest Biology and Technology

This study aimed to improve the antimicrobial activity and stability of Cinnamomum zeylanicum essential oil (CEO) against Phytophthora drechsleri , the causal agent of cucumber ( Cucumis sativus ) fruit rot in laboratory media and when applied as a coating on cucumbers. Additionally, the in ﬂ uence of chitosan nanoparticles (CSN)-based coatings on several physicochemical characteristics of the fruit during storage was assessed. CEO was encapsulated by an ionic gelation technique into CSN with an average size of 100 – 190 nm. The encapsulation ef ﬁ ciency (EE) and loading capacity (LC) of CEO-loaded CSNs (CEO-CSN) were about 2 – 17% and 3 – 4%, respectively, with the initial CEO content of 0.25 – 1 kg/kg chitosan. In vitro release studies showed a controlled and sustained release of CEO for 40 d. The superior performance of CEO when encapsulated by CSNs under both in vitro and in vivo conditions in comparison with unmodi ﬁ ed CEO against P. drechsleri was studied. The in vivo experiment also showed that the encapsulated oils at 1.5 g/L concentration signi ﬁ cantly decreased both disease severity and incidence of Phytophthora -inoculated cucumbers during 7 d of storage at 4 (cid:1) C followed by 2 – 3 more days at 20 (cid:1) C. Furthermore, the CEO-CSN coating extended the shelf life of cucumbers up to 21 d at 10 (cid:3) 1 (cid:1) C while uncoated fruit were unmarketable in less than 15 d. CEO-CSN coating improved the microbiological and physicochemical quality of cucumbers. Coated fruit were ﬁ rmer, maintained color, water content, and showed lower microbial counts ( P < 0.05) throughout storage. According to these results, CEO-CSN coatings can be an effective method to extend cucumber shelf life. ã 2015 Elsevier B.V. All rights reserved.


Introduction
Cucumber (Cucumis sativus L.), which belongs to the gourd family Cucurbitaceae, is an economically important vegetable crop worldwide (Li et al., 2013). They have a short shelf life (<14 d) that is mainly related to firmness loss, discoloration, desiccation and fungal rot (Martin-Belloso and Fortuny, 2011). Among postharvest fungal pathogens, the filamentous oomycete pathogen Phytophthora drechsleri is a common fruit-damaging agent (water mold). This oomycete causes damping off, gummosis and fruit rot in many vegetable crops such as Cucurbitaceae and Solanaceae families (Saberi-Riseh et al., 2004). In Iran, where these fruit are produced on >250,000 ha of irrigated land, melons and cucumbers are frequently devastated by a severe wilt disease known locally as 'green death', caused by P. drechsleri (Alavi et al., 1982). Oomycetes such as P. drechsleri, have a distinct physiology that causes most fungicides to be ineffective against them (Thakur and Mathur, 2002). In this regard, some studies have exhibited an interesting inhibitory effect of essential oils (EOs) and their constituents on food-related spoiling and pathogenic microorganisms (De Azeredo et al., 2011;de Sousa et al., 2012). Within a great variety of EOs, Cinnamomum zeylanicum (cinnamon) essential oil (CEO) has emerged as a growth inhibitor of different fungal species, including postharvest pathogens (Carmo et al., 2008). The antimicrobial activities of CEO are mainly because the cinnamaldehyde (major component of CEO), eugenol, and the monoterpenes hydrocarbons (Miyazawa et al., 2001;Simi c et al., 2004). However, like other EOs, CEO is a volatile compound that can easily suffer degradation under the action of heat, pressure, light, and oxygen. Furthermore, CEO is insoluble in water, and for certain applications a controlled release is required (Martín et al., 2010). Accordinaly, encapsulation represents a viable and efficient approach to increase the physical stability of antimicrobial compounds and enhance their bioactivity during food processing and storage. Additionally, encapsulation could improve water solubility, control the delivery, improve absorption, and reduce the toxicity and cost of bioactive compounds due to the reduction of the required quantity (Fang and Bhandari, 2010).
While microcapsules may guarantee protection of antimicrobial compounds against evaporation or degradation, nanoencapsulation systems, due to high surface area to volume ratios, have versatile advantages for targeted site-specific delivery and efficient absorption through cells that could lead to higher antimicrobial activity (Ezhilarasi et al., 2013). In this regard, the encapsulation of eugenol and carvacrol into nanometric surfactant micelles resulted in enhanced antimicrobial activity (Gaysinsky et al., 2005), although the addition of micelle-encapsulated eugenol to milk was found to be less or only as inhibitory as unencapsulated eugenol (Gaysinsky et al., 2007).
In recent years, chitosan (CS) has received a lot of interest in the encapsulation of bioactive compounds owing to its status as generally recognition as safe (GRAS), its biocompatibility, and its excellent biodegradability (Keawchaoon and Yoksan, 2011). CS has shown its capacity for the loading and delivery of sensitive bioactive compounds such as lipophilic drugs (Ajun et al., 2009;Arya et al., 2009), polyphenolic compounds (Hu et al., 2008;Keawchaoon and Yoksan, 2011), and vitamins (Luo et al., 2011). However, the encapsulation of CEO using CS particles as an outer shell at a nanoscale range apparently has not been studied.
This study was performed with the following objectives: (i) to optimize the encapsulation of CEO in CS nanoparticles; (ii) to characterize the physicochemical properties of the obtained nanoparticles by ultraviolet and visible (UV-Vis) spectrophotometry and dynamic light scattering (DLS); (iii) to evaluate the efficacy of the CSNs obtained under optimized conditions to inhibit P.drechsleri, the causal agent of cucumber fruit rot, in laboratory media and as a coating on the cucumbers; as well as (iv) to assess the effect of the CEO-CSN coating on the physicochemical and microbial characteristics of the cucumbers during storage.
Cucumbers obtained at commercial maturity from an organic farm, were transported at low temperature to the laboratory and coated on the same day.
The fungal strain, P. drechsleri (IRAN 1156C), was used as the test microorganism. The strain was grown for 7 d on potato dextrose agar (PDA; 1 L of an infusion from potatoes containing 20 g/L glucose and 15 g/L agar) at 25 C. Spores from a seven-day-old culture were suspended in sterile saline solution (8.5 g/L NaCl) and adjusted to 10 5 spores/mL by using a hemocytometer (Shao et al., 2015).

Preparation of CEO-loaded CSNs
CEO-CSNs were prepared by an ionic gelation, according to a previous report (Mohammadi et al., 2015). Briefly, CS solution (0.3% w/v, 50 mL, pH = 4.6) was prepared by dissolving CS in an aqueous acetic acid solution (1% v/v) at an ambient temperature overnight. Tween 80 (CAS 9005-65-6) was then added as a surfactant to the solution and stirred at 45 C for 2 h to obtain a homogeneous mixture. CEO was gradually dropped into the stirring mixture of CS solution, and agitation was carried out for 30 min to obtain an oilin-water emulsion. Various contents of CEO were used to obtain different weight ratios of CS to CEO of 1:0, 1:0.25, 1:0.50, 1:0.75 and 1:1.00, respectively. CEO-loaded CSNs were spontaneously obtained by the addition drop-wise of TPP solution (0.3% w/v, 20 mL, pH = 5.6) into o/w emulsion (for CEO-CSN) or CS solution (for CSN) under constant stirring at room temperature for 60 min. The resultant nanosuspensions were subjected to particle-size analysis. Further, nanoparticles were centrifuged at 27,000Âg for 14 min, resuspended in distilled water and freeze-dried used for further analyses. Two coating solutions of CSN and CEO-CSN (1:0.25) with final concentration of 1.5 g/L were prepared separately by dispersing 0.15% (w/v) of each one in distilled water and kept at 4 C.

Characterization of CSNs loaded with and without CEO
UV-Vis absorption spectra were recorded over a wavelength ranging from 250 to 400 nm by a Helios Gamma Thermo Spectronic spectrometer (England). The particle size of the nanoparticles were determined using quasi-elastic laser light scattering with a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) at 25 AE 1 C.

Determination of encapsulation efficiency and loading capacity
The percentage of CEO encapsulated was determined after lysis of the prepared nanoparticles (NPs) with hydrochloric acid solution and alcohol, as described previously (Keawchaoon and Yoksan, 2011). The concentration of CEO in absolute alcohol was determined spectrophotometrically at 290 nm (maximum absorption wavelength of CEO) (Shekar et al., 2012) using a UV-Vis spectrophotometer in triplicate. The encapsulation efficiency (EE) and loading capacity (LC) of CEO were calculated using Eqs. (1) and (2), respectively:

In vitro CEO release experiment
The release kinetics of the CSN-encapsulated CEO oils at ambient temperature were investigated using a spectrophotometer during a six-week period. The release of the EOs was monitored daily during the first week of the experiment followed by weekly monitoring afterwards.

Effects of CSNs and CEO-CSNs on fungal mycelial growth
Antifungal assays were performed with the pour-plate method as described by Askarne et al. (2012). In this method, solutions of serial concentrations of each treatment were mixed with sterilized potato dextrose agar (PDA) in petri dish (9 cm diameter) containing 15 mL of agar to obtain final concentrations as follows: 0-3 g/L (based on preliminary work) for CEO, CSNs and CEO-CSNs. After inoculating the mycelia of fungus onto the center of the agar, the dishes were sealed with parafilm and incubated at 25 C for 5-7 d, until the growth in the control plates (without the treatments) reached the edge of the plates. Then, the antifungal index of each treatment was calculated as follows: Where C and T are the radial growth (mm) of the fungus in the control and treated plates, respectively. Based on this assay, a concentration of 1.5 g/L was selected for the further screening.

Effects of CSN and CEO-CSN coating on fungus infection of cucumbers
Cucumbers were selected for uniform size, shape and color, and without any signs of mechanical damage or deterioration. Then, the fruit were washed in a sodium hypochlorite solution (1% v/v) to decontaminate their surface, and left for 2 h in a safety cabinet to dry.
To verify the results obtained from the in vitro studies, 60 cucumbers were first wounded (1.2 cm deep and 1.5 cm diameter-three wounds per fruit) with a cork borer, and 30 mL of CSN and/or CEO-CSN (1.5 g/L) coating formula were pipetted into each wound, allowing the droplet to be absorbed into the fruit. After two hours incubation at room temperature, each wound was inoculated with 10 mL of an inoculum solution (approximately 10 4 conidia/mL). Thirty non-coated inoculated cucumbers were used as control. Then, the inoculated fruit were kept for 7 d at cold temperature (4 C) followed by 2 d at room temperature (20 C) to observe the decay development. Five fruit pieces constituted a single replicate; each treatment was performed in triplicate, and the entire experiment repeated twice. Decay incidence and lesion diameter were measured. Decay incidence was expressed as the percentage of infected wounds, and lesion diameter was expressed as the means of the width and length of the decay area (Askarne et al., 2012).

Cucumber coating
Selected cucumbers were dipped in one of two coating solutions (CEO-CSN or CSNs) for 2 min. A total of 400 cucumbers were coated and dried on a nylon filter to drain the excess liquid. Cucumbers immersed in distilled water for 2 min served as untreated controls. Next, the fruit were placed into polypropylene trays ($5 cucumbers per tray) and stored at 10 AE 1 C with 90-95% RH (Gross et al., 2004;Martin-Belloso and Fortuny, 2011) for 21 d. Samples were withdrawn every 3 d for physicochemical and microbiological evaluation. In addition, fruit were examined for the presence of visible fungal infection, and the results were expressed as the percentage of fruit infected at different times (Feng and Zheng, 2007). Five fruit constituted a single replicate. Each treatment was performed in triplicate, and the entire experiment repeated twice.

Effects of coating on physical and physicochemical quality of cucumber
The fruit were evaluated for weight loss and general quality aspects such as color, respiration rate and firmness at the same observation intervals as the examination for fungal infection.
The weight loss of fresh cucumbers in each treatment during storage was measured by monitoring the weight of the 40 fruit at different intervals. The weight loss was calculated as a percentage of the initial weight (Meng et al., 2008).
The skin color was assessed by a Labscan XE (16,437) colorimeter (HunterLab, Inc., VA) with the CIELAB system with measuring aperture diameter of 36 mm, and illuminant/viewing geometry of D65/10 . The unit was calibrated using standard white and black plates. Means of 15 replications were used to determine the color coordinates, L*(lightness), a*(greenness), and b*(yellowness).
Firmness was evaluated using a texture analyzer (Hounsfield Test Equipment, UK) with a 500 N load cell. At least fourteen fruit per control and coating samples were punctured with a 7 mm diameter ball probe at a speed of 12 mm/s at their geometric center, and the results were expressed as Newtons (N) (Chien et al., 2007).
For respiration rate, three replicates of five fruit per treatment at each time point were placed in an airtight chest, and a CO 2 sensor (Testo AG-435-2, Germany) was used to monitor CO 2 concentration. The sensor was programmed to collect CO 2 concentration at 1-min intervals for 60 min. Respiration rate was calaulated from the regression analysis slant of CO 2 concentration versus time and expressed as mg kg À1 s À1 (Eshghi et al., 2014).

Microbiological analysis
The microbiological characteristics of a 10 g sample were obtained following homogenization in 90 ml 0.1% peptone water. Other decimal dilutions were prepared from a 10 À1 dilution and each was spread-plated (0.1 ml) as follows: plate count agar (PCA) for total aerobic bacteria (incubated at 35 C for 2 d); and sabouraud dextrose agar (SDA) with chloramphenicol for yeasts and molds (incubated at 25 C for 5 d). Quadruplicate counts were averaged for graphic presentation.

Statistical analysis
The results were analysed by a multifactor analysis of variance (ANOVA) and Tukey's test with a 95% significance level using GraphPad Prism, version 5.02 (GraphPad Software, Inc., San Diego, CA, USA).

Loading of CEO into chitosan nanoparticles
The preparation of CSN and CEO-CSN is based on an ionic gelation interaction between positively charged CS and negatively charged TPP at room temperature (De Campos et al., 2001;Qi et al., 2004). Among the variety of methods developed to load bioactive compounds into CSNs, ionotropic gelation has attracted considerable attention due to this process is non-toxic, organic solvent free, convenient and controllable (Agnihotri et al., 2004). During the preparation process, TPP electrostatically attracted to the NH3 + groups in CS to produce ionically cross-linked CSNs (Aydin and Pulat, 2012;Yang and Hon, 2009). Calvo et al. (Calvo et al., 1997) pointed out that the concentrations of the CS and TPP solutions and the ratio of CS to TPP have important effects on nanochitosan preparation. It is evident from our findings, that the concentration of 0.3% (w/w) CS and TPP with weight ratios of 5:2 (CS: TPP) resulted in formation of NPs sized below 200 nm (polydispersity index <1) as shown in Table 1. This finding is in agreement with Huang et al. (2009) (Huang et al., 2009), who suggested that the cross-linking was best when the CS/TPP mass ratio was approximately 5:2. CEO-CSNs showed an average diameter in the range of 97-182 nm. The initial CEO content had no influence on the size of the NPs (Table 1). The successful loading of CEO into CSNs particles was confirmed by UV-Vis spectrophotometry. CEO-ethanol solution gives a maximum absorption peak at 290 nm, which is very close to the wavelength reported by Shekar et al. (Shekar et al., 2012). After immersion of CSNs in ethanol for 1 h, the supernatant exhibited no absorption peak at wavelengths ranging from 250 to 350 nm. However, the medium obtained from the immersion of CEO-CSNs gave a maximum absorption peak at 290 nm, which corresponded to the max of CEO. This result indicated the successful loading of CEO into CSNs.

Encapsulation efficiency and loading capacity
From UV-Vis spectrophotometry results, the LC% of CEO was in the range of 3.18-3.78%, when the initial CEO content ranged from 0.25 to 1 kg/kg CS (Table 1). In other word, the LC% showed no significant change (p > 0.05) as a function of initial CEO content. While our findings in this work are in agreement with those of some researchers (Papadimitriou et al., 2012), they are contrary to Hosseini et al. (Hosseini et al., 2013), who reported that the loading of oregano EO (OEO) into CSNs increased as a function of initial OEO content.
In addition, based on our results the EE% of CEO ranged from 1.99 to 16.91%. We also observed that the EE tended to decrease with an increase of the initial CEO content. However, the maximum EE value was obtained for the sample prepared using the weight ratio of CS to CEO of 1:0.25 (16.91%). The decrease of EE for the samples prepared using higher initial CEO content might be explained due to the saturation of CEO loading into CSNs. This finding was in accordance with the previous findings that showed reduced EE with increased initial drug content (Ajun et al., 2009;Hosseini et al., 2013;Yoksan et al., 2010).

In vitro release characteristics of CEO from CEO-CSNs
The in vitro release profile of CEO from CEO-CSNs, prepared using a weight ratio of CS to CEO of 1:0.25, has been presented in  Results were reported as mean AE SD, n = 3. Fig. 1. As shown, the overall release process can be characterized as a biphasic procedure, i.e., an initial burst release followed by subsequent slower release. The initial burst release of CEO was observed for the first 9 d, which released up to 28.4% (Fig. 1). This burst effect may be attributed to the desorption of the CEO close to the surface during preparation of the NPs, which then diffused rapidly when the NPs came into contact with the release medium, as previously reported (Zhang et al., 2008).
After the burst release period, the release rate was relatively slow, i.e., almost zero that the release of CEO reached plateau. The concentration of CEO released on day 40 was 31.65%. At this stage, the rate of release fell as the dominant release mechanism was changed to CEO diffusion through CS matrix (Papadimitriou et al., 2008). This might be due to the inability of buffer solution to break or destroy the NPs, resulting in no additional release of CEO at this stage. In this regard, Agnihotri et al. (2004) stated that the release of a drug by diffusion involved the penetration of medium into the particulate system, which caused swelling of the matrix.

In vitro antifungal effects
The antifungal activities of tested materials were first examined at various concentrations, and the results are shown in Fig. 2. The rate of growth reduction was directly proportional to the concentration of the tested material in the medium. CSNs led to complete fungal inhibition at 3 g/L concentration. ANOVA results demonstrated that only two concentrations of CEO out of five reduced mycelial growth of P. drechsleri more than 50% (P < 0.05) and the complete inhibition was obtained at concentration higher than 1.5 g/L (3 g/L). On the contrary, when encapsulated by CSNs, the oils completely inhibited fungal growth at !0.75 g/L. In fact, these new compounds exhibited an in vitro antifungal activity greater than unmodified CEO against P. drechsleri. This finding was in agreement with two studies of Russo et al. (2014) (Russo et al., 2014) and Beyki et al. (2014) (Beyki et al., 2014) that resulted in better performance of EOs at lower amount. As demonstrated in those studies, the superior performance of EOs-loaded CSNs was due to the volatile nature of the EOs and that the nanoencapsulation well preserved the oils in the media. Furthermore, the slow-release of the preserved volatile compounds from the NPs during the assay leading to better inhibitory effect as well as the inhibitory effect of the CSNs itself. Although 3 g/L of the CEO were necessary for the complete inhibition of fungal growth, the use of such high amounts for controlling postharvest decay in fruits can greatly affect their taste or exceed acceptable flavor thresholds (Hsieh et al., 2001). For this reason, a concentration of 1.5 g/L for CEO, CSNs and CEO-CSNs resulting in 67.15, 75.5 and 100% radial growth inhibition respectively, was screened for the assays on artificially infected cucumbers. Fig. 3 presents the results of the in vivo study of the treatments using cucumber fruit infected by P. drechsleri. The statistical analysis revealed that the lowest percentage of infected cucumbers was recorded for 1.5 g/L CEO-CSNs (0%), followed by 1.5 g/L CSNs (38.66%) and CEO (75.84%) (P < 0.05) at day 9 (Fig. 3A). Meaning, the most effective treatment to reduce the disease incidence were those based on CEO loaded CSNs. This treatment was not only effective in controlling cucumber decay, but also in delaying the onset of disease symptoms and slowing down P. drechsleri growth during the storage period (P < 0.05). Uncoated cucumbers started to decay from the fourth day of storage (Fig. 3A). The decay process was delayed to the fifth and sixth days for cucumbers coated with CEO and CSN treatments, respectively. Moreover, fruit coated with CEO-CSNs and contaminated with P. drechsleri displayed no visible fungal growth throughout the storage period (9 d).

Antifungal effects of treatments on artificially inoculated fruit
Similarly, the treatments exhibited significant reduction of disease severity compared with the control. According to Fig 3B, CEO-CSNs reduced disease severity for 100% compared with control cucumbers, at day 7 for 70% in CEO and 97% in CSN, while at day 9 for 26% in CEO and 70% in CSN, respectively. Furthermore, no visible symptoms of phytotoxicity were detected on cucumbers treated with CEO-CSNs. However, fruit treated with pure CSNs and CEO exhibited symptoms of phytotoxicity manifested as drying and browning around the treated wounds.
Results obtained in the in vivo trials confirmed the strong efficacy shown in vitro by CEO-CSNs treatment. Numerous factors can affect the biological activity of certain compounds when in contact with fruit tissue. In this sense, Vu et al. (2011) reported a higher antifungal effect of limonene than peppermint Eos encapsulated in coatings of modified CS, when applied to coldstored strawberries, although the in vitro antifungal effect of the free (non-encapsulated) oils showed the opposite trend.
On the basis of the results from the in vitro and in vivo inhibition of fungal growth assay, only the CEO-CSNs treatment was screened for the shelf life and quality assay of cucumber.
3.6. Efficacy of CSN and CEO-CSN coatings on cucumber 3.6.1. Cucumber decay The effect of coatings on the decay percentage of cucumbers, during 21 d of storage at 10 AE 1 C, is presented in Figs. 4-5. The infection level increased gradually throughout the storage time and was significantly higher for control fruit, followed by CSN and CEO-CSN treated cucumbers (P 0.05) (Fig. 4). According to Fig. 4, the decay level of cucumbers immersed in CEO-CSN (3.2%) was significantly less than that of CSNs treated (10.95%) and control (97.73%) samples after 15 d shelf life at 10 AE 1 C. At day 21, the decay levels of cucumbers treated with CEO-CSN (26.1%), were still significantly less than that of control (100%) and CSNs treatment (44.87%) (P 0.05). The fruit are presented in Fig. 5. It can be easily observed that uncoated cucumbers are dehydrated and are largely contaminated by mold at day 15 whereas coated cucumbers maintained a good hydrated, green-colored appearance even at day 21.

Cucumber physicochemical quality
Treated as well as control cucumbers differed (P 0.05) in weight during storage time, although the weight loss of both cucumber groups increased gradually during storage (Fig. 6A), the control (untreated) had higher loss of weight over all the storage period compared with coated treatments. The maximum weight loss was observed in the control was about 12% compared to CEO-CSNs and CSNs treatments which lost only 8.55% and 9.82% at 15 d of storage, respectively. This result means the CSN treatments (with and without CEO) succeed in reducing the weight loss percentage in treated cucumbers by 29% and 18% respectively, compared with untreated samples. As shown in Fig. 6A, incorporation of CEO into the CSNs did not have any significant effect on weight loss reduction of cucumbers until the fifteenth day of storage. After 18 and 21 d of storage, CEO-CSNs treatment showed greater effectiveness for maintaining fruit weight during storage than that obtained for CSNs treatment (P 0.05). This finding is in contrast to the results of the study by Eshghi et al. (2014), who reported that both nanochitosan and copper-loaded nanochitosan coatings provided a similar control in reducing weight of strawberries during 3 weeks (Eshghi et al., 2014).
According to the experimental results in this study, coated cucumbers exhibited higher flesh firmness values than uncoated fruit (Fig. 6B). After 15 d of storage, the loss of firmness in control fruit was around 30%, while the coated samples were significantly firmer (P < 0.05) throughout storage and only 13-16% firmness loss was recorded for the treated fruit with CEO-CSNs and CSNs at the 15th day. Control fruit softened faster and were fully ripe after approximately 15 d at 10 AE 1 C. Firmness is one of the components of texture which is a complex sensory attribute that also includes crispiness and juiciness (Konopacka and Plocharski, 2004) and is critical in determining the acceptability of horticultural commodities (Gross et al., 2004). In agreement with results in this study, other studies have reported that the application of nanochitosan and EOs alone or in combination as a coating material improved the firmness of fruits (Abdolahi et al., 2010;Eshghi et al., 2014).
Application of two coating solutions helped preserve (P < 0.05) the color attributes of cucumber, whereas there was no significant difference between CEO-CSNs and CSNs coated cucumbers (Fig. 6C, D and E). Lightness (L*) of all samples decreased (P 0.05) throughout time; however, the coated samples showed higher lightness values (P 0.05) than the controls (darker fruits) by the end of shelf life (Fig. 6C). Loss of water and browning on the surface was more noteworthy in the uncoated fruit as found in different studies (Brasil et al., 2012;Rattanapanone et al., 2001). The a * - values increased throughout storage, with all fruit showing a greenish color (negative a*-values) (Fig. 6D). The b*-values of coated samples varied less and were significantly (P 0.05) lower than uncoated controls (yellower) by day 15 (Fig. 6E). The coating maintained the green color of cucumber by preventing oxidative or enzymatic browning. The small changes in a* and b*-values are good indicators of the absence of oxidative browning of cucumber (Rocha and Moraes, 2000).
The respiration rate is a good index for the quality of fruit during storage. Edible coatings lead to high carbon dioxide and low oxygen internal gas concentrations in coated fruit so as to lower respiration rates (Özden and Bayindirli, 2002). Zhang et al. (2014) reported that the respiration rate of cucumber was reduced with chitosan-g-salicylic acid coating under cold and ambient temperature storage (Zhang et al., 2014). In this study, the respiration rates of cucumber coated with CEO-CSNs and CSNs were significantly reduced (P 0.05) compared to the control samples (Fig. 6F). CEO-CSNs coating was also more effective in reducing the respiration rates of cucumber than CSNs (Fig. 6F), although the difference between the two coating treatments was significant only at day 6 and 12 (P 0.05). In this context, some researchers have reported that EOs and their monoterpenoid components due to their lipophilic character, destroy cellular integrity and inhibit respiration in different micro-organisms (Andrews et al., 1980;Cox et al., 2000). The better performance of CEO-CSN coatings relative to reducing respiration, is due to inhibition activity of CEO; it induced damage to the surface and structure of fungal cell wall accompanied the decline in respiratipon rate.

Microbiological quality of Cucumber
The coating with CSNs and CEO-CSNs were very effective for inhibiting total aerobic bacteria, yeasts and mold growth (Fig. 7). Coated fruit had significantly lower microbial counts (P < 0.05) throughout storage. There was also significant difference between the microbial counts of two coated cucumbers. The CFU values of CEO-CSNs treated cucumbers were significantly (P < 0.05) lower than that with CSNs alone at !6 d of storage. The total aerobic bacteria count of uncoated samples increased from 2.05 to 11.67 log CFU/mL at the end of the storage, while coated samples with CEO-CSNs maintained below 5 log, as shown in Fig. 7A.
In both coated and uncoated fruit, the yeast and molds counts increased (P < 0.05) with time. However, counts in coated samples were lower (P < 0.05) throughout storage (Fig. 7B). If 5 log CFU/mL of aerobic plate counts or yeast and molds counts is considered as the critical limit (Pao and Petracek, 1997), only the CEO-CSN coating extended the microbial shelf life of cucumbers. Similarly, Brasil et al. (2012) showed that the application of polysaccharide-based multilayered antimicrobial edible coating preserved the microbiological quality of fresh-cut papaya (Brasil et al., 2012).

Conclusions
In this study, chitosan nanoparticles loaded with CEO were prepared with size range of 100-190 nm, loading capacity of 3-4%, and encapsulation efficiency of 2-17%. The superior performance of CEO when encapsulated by CSNs in comparison with free oils and CSNs was demonstrated. The in vivo experiment also showed that the encapsulated oils at 1.5 g/L concentration significantly decreased both disease severity and incidence of infected cucumbers by P. drechsleri, during 7 d of storage at 4 C followed by 1-2 more days at 20 C. Furthermore, the CEO-CSNs was more effective than pure CSNs coating at reducing respiration rates, improving the microbiological quality, preserving the fruit weight and quality of cucumbers during the storage period. These results demonstrate the potential of CSNs containing cinnamon oil for coating cucumbers to extended the postharvest shelf life.