Nanoencapsulation of Zataria multi ﬂ ora essential oil preparation and characterization with enhanced antifungal activity for controlling Botrytis , the causal agent of gray mould disease Innovative Food Science and Emerging Technologies

Thisstudy wasundertaken to investigatethe nanoencapsulation of Zatariamulti ﬂ ora essentialoil(ZEO) inchito- san nanoparticles (CSNPs) in order to enhance antifungal activity and stability of the oils against one isolate of Botrytiscinerea Pers.,thecausalagentofgraymoulddisease.ZEOwasencapsulatedbyanionicgelationtechnique into CSNPs with an average size of 125 – 175 nm as observed by transmission electron microscopy (TEM). From UV-vis spectrophotometry results, the drug encapsulation and loading ef ﬁ ciency of ZEO decreased from 45.24% to 3.26% and from 9.05% to 5.22%, respectively, upon increasing initial ZEO content from 0.25 to 1 g/g chitosan. Invitro releasestudiesalsodemonstratedacontrolledandsustainedreleaseofZEOfor40days.Thesuperiorper- formance of ZEOwhen encapsulatedbyCSNPs under both invitro and invivo conditionsin comparison with unmodi ﬁ ed ZEO against B. cinerea was revealed. The in vivo experiment also showed that the encapsulated oils at 1500ppmconcentrationsigni ﬁ cantlydecreasedbothdiseaseseverityandincidenceof Botrytis -inoculatedstraw-berriesduring7daysofstorageat4°Cfollowedby2 – 3moredaysat20°C.These ﬁ ndingsrevealedthepromising role of CSNPs as a controlled release system for EOs in order to enhance antifungal activities. Industrial relevance: Application of plant essential oil (EOs) treatment at pre- or postharvest stage has been considered as an alternative treatment to the use of synthetic fungicides to prevent fruit postharvest decay and to extend the storage life while retaining the overall quality of different fresh commodities. Although EOs have proved to be good antimicrobial agents, their use for maintaining fruit quality and reducing fungal decay is often limited due to their volatile compounds which can easily suffer degradation under the action of heat, pressure, light and oxygen. Furthermore, they are insoluble inwater, and for certainapplications a controlledre- leaseisrequired.Inthis regard, nano-sizecarriers providemoresurfaceareaand canpossiblyupgrade solubility, enhance bioavailability and improve controlled release and targeting of the encapsulated food ingredients, in comparison to micro-size carriers. These ﬁ ndings revealed the promising role of CSNPs as a controlled release system for EOs in order to enhance their antimicrobial activities.


Introduction
The pathogen Botrytis cinerea Pers. Fr. (known as "gray mould fungus") causes serious losses in more than 200 plant species worldwide (Jarvis, 1977), including important crops and harvested commodities, such as grapevine, tomato, strawberry, raspberry, blackberry, cucumber, cut flowers and ornamental plants (Droby & Lichter, 2007). With increasing international trade in cold-stored produce, this fungus has attained great importance because it can grow effectively over long periods at just above freezing temperatures in products such as kiwifruit, apples, pears and strawberries (Williamson, Tudzynski, Tudzynski, & van Kan, 2007). The synthetic fungicides provide the primary means for controlling postharvest decay of fruit (Soylu, Kurt, & Soylu, 2010). Although the synthetic fungicides are effective, their continued or repeated application has significant drawbacks, including cost, handling hazards, contamination of fruits and vegetables with fungicide residues, and threats to human health and the environment (Paster & Bullerman, 1988). These adverse effects have led to intensified worldwide research efforts to develop safe and biodegradable alternatives as natural fungicides to replace synthetic chemicals (Costa et al., 2000).
The application of plant essential oil (EOs) treatment at pre-or postharvest stage has been considered as an alternative treatment to the use of synthetic fungicides to prevent fruit postharvest decay and to extend the storage life while retaining the overall quality of different fresh commodities (Aloui et al., 2014). Among a wide variety of EOs, Zataria multiflora Boiss EOs (ZEO) appear as promising natural compounds for controlling postharvest decay in fruits. Quantitatively, the most abundant components in hydro-distilled ZEO are oxygenated monoterpenes (approximately 70%) followed by monoterpene hydrocarbons, sesquiterpenes hydrocarbons and oxygenated sesquiterpenes (Sajed, Sahebkar, & Iranshahi, 2013). They have been proven to have antifungal properties against several postharvest phytopathogens, including B. cinerea and species of Aspergillus, Rhizopus and Penicillium (Abdollahi, Hamzehzarghani, & Saharkhiz, 2011;Mohammadifar et al., 2012).
Although EOs have proved to be good antimicrobial agents, their use for maintaining fruit quality and reducing fungal decay is often limited due to their volatile compounds which can easily suffer degradation under the action of heat, pressure, light and oxygen. Furthermore, they are insoluble in water, and for certain applications a controlled release is required (Martín, Varona, Navarrete, & Cocero, 2010). Hence, controlled or sustained delivery is crucial to obtain maximum benefits of using EOs as antimicrobial agents. Nano-/micro-encapsulation technology of these compounds can be a practical and efficient approach to solve some of these problems such as the physical instability and enhance their bioactivity, while at the same time, controlling fruit postharvest decay, by lowering the diffusion processes and maintaining high concentrations of active molecules on the surface of the fruit (Aloui et al., 2014). In this regard, nano-size carriers provide more surface area and can possibly upgrade solubility, enhance bioavailability and improve controlled release and targeting of the encapsulated food ingredients, in comparison to micro-size carriers (Mozafari et al., 2006).
Recently, chitosan (CS) has attracted a great attention in the encapsulation of bioactive compounds because of its general recognition as safe (GRAS) and its advantageous biological properties, such as biodegradability, biocompatibility and nontoxicity, as well as ability to form films, membranes, gels, beads, fibers and particles (Keawchaoon & Yoksan, 2011). Several studies on antimicrobial activities of CS alone or in combination with other natural agents have been recently carried out (Ajun, Yan, Li, & Huili, 2009;Aloui et al., 2014). In particular, in our previous study, CS in combination with ZEO showed strong in vitro and in vivo antimicrobial activity against some pathogenic bacteria and fungi (under publication). The CS nanoparticles (CSNPs) has shown its capacity for the loading and delivery of sensitive bioactive compounds such as lipophilic drugs (Ajun et al., 2009), polyphenolic compounds (Keawchaoon & Yoksan, 2011), proteins (Avadi et al., 2010), genes (Csaba, Köping-Höggård, & Alonso, 2009) and vitamins (Luo, Zhang, Whent, Yu, & Wang, 2011). However, to our knowledge, preparations of ZEO combined in CSNPs with in vitro release have not been studied. The present study was set to encapsulate hydrophobic ZEO in CSNPs using ionic gelation technique in order to enhance antifungal activity and stability of the oils against B. cinerea.

Materials
Chitosan derived from crab shell with a molecular weight of 684 kDa and deacetylation degree of ∼ 85% was purchased from Sigma (Germany). ZEO (98%) was purchased from Magnolia Co (IRAN). Pentasodium triphosphate (TPP), acetic acid glacial and sodium hydroxide were purchased from Merck (Germany). All chemicals used were of analytical grade and used as received. B. cinerea (IRAN 1304C) was obtained from the Iranian Research Institute of Plant Protection (IRIPP).

Preparation of ZEO-loaded CSNPs
ZEO-loaded CSNPs (ZEO@CSNPs) were prepared by an ionic gelation technique as described by Keawchaoon and Yoksan (2011) with slight variation. Briefly, CS (0.3% w/v, 50 mL) was dispersed in an aqueous solution of glacial acetic acid (1%, v/v) at ambient temperature overnight, followed by ultrasonication (MISONIX Inc. S-4000, USA) for 4 min at 60 W. ZEO was gradually dropped into the stirring mixture of CS solution for 30 min to obtain an oil-in-water emulsion. Various contents of ZEO, i.e., 0, 375, 750, 1500 and 3000 ppm, were used to obtain different weight ratios of CS to ZEO of 1:0, 1:0.25, 1:0.50, 1:0.75 and 1:1.00, respectively. A TPP solution (0.3% w/v, 20 mL) was prepared in water and its pH was adjusted to 5.6 using 1 N NaOH. ZEO@CSNPs were spontaneously obtained by the addition drop-wise of TPP solution into an o/w emulsion under constant stirring at room temperature for 60 min. The final pH of mixture solution was ∼5.0. The resultant suspensions were subjected to particle-size analysis. Further, the formed particles were collected by centrifugation (Tomy Kongyo Co. LTD. Suprema 25, JAPAN) at 27000 ×g for 14 min at 25°C and washed with distilled water to remove free ZEO. The obtained particles were dispersed in distilled water and kept at 4°C.

Characterization of ZEO-loaded CSNPs
Morphological characteristics of CSNPs and ZEO@CSNPs were examined by high-resolution transmission electron microscope (TEM) (Hitachi, H-600) at an accelerating voltage of 100 kV. Samples were immobilized on copper grids. They were dried under reduced pressure at an ambient temperature overnight prior to TEM observation and then were examined using a TEM without being stained. Particle size was determined by dynamic light scattering (DLS) using a photon correlation spectroscopy (PCS) assembly (Zetasizer 3000 HS, Malvern Instruments, UK). Zeta potential was measured by laser Doppler anemometry on the same instrument. Results are reported as the mean of three measurements ± SD (standard deviation). UV-vis absorption spectra were recorded over a wavelength ranging from 220 to 400 nm by a TECAN spectrometer (infinite M200 Pro, Switzerland).

Determination of encapsulation efficiency and loading capacity
The percentage of ZEO encapsulated was determined after lysis of the prepared NPs with hydrochloric acid solution and alcohol, as described previously (Keawchaoon & Yoksan, 2011). The concentration of ZEO in absolute alcohol was determined spectrophotometrically at 230 nm (maximum absorption wavelength of ZEO) using a UV-visible spectrophotometer in triplicate. Encapsulation efficiency (EE) was calculated through the following relationship: Loading capacity (LC) was articulated as

Nanoparticle yield determination
Each nanoparticle sample was centrifuged as described in Section 2.2, and the residue was lyophilized. Nanoparticle yield was calculated from the weight of the lyophilized nanoparticles (W 1 ) and the sum of the initial dry weight of starting materials (W 2 ) as using the following formula: Results are reported as the mean of three measurements ± SD.

In vitro release kinetics
The release kinetics of ZEO@CSNPs was investigated using a spectrophotometer during an 8-week period. The release of ZEO was monitored daily during the first week of the experiment followed by weekly monitoring afterwards.

Effects 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, 188, 375, 750 and 1500 ppm for ZEO, CSNPs and ZEO@CSNPs. After inoculating the mycelia of fungus onto the center of agar, the dishes were sealed with parafilm and incubated at 25°C for 5-7 days, until the growth in the control plates (without the treatments) reaches the edge of the plates. Then, the antifungal index of treatment was calculated as follows: where C and T are the radial growth (mm) of fungus in the control and treated plates, respectively.

In vivo antifungal assay: strawberry decay
To enable the comparison with the results obtained in the in vitro studies, the in vivo study conducted on strawberries (Fragaria × ananassa). The fruits of uniform size, shape and color and without any signs of mechanical damage or fungal decay were selected and disinfected with sodium hypochlorite solution (2.5%) prior to coating. After abundant washing with distilled water (× 3), the fruits were dipped in the respective solutions for 1 min. Strawberries were inoculated with suspension of 5 × 10 5 conidia per ml of B. cinerea and dried in air for 1 h. Thirty non-coated inoculated strawberries were used as control. Fruits were then placed in covered plastic boxes and stored for 7 days at 4°C, 95-98% RH, and next exposed to 3 days shelf life at 20°C, 95-98% RH. Five replicates of 30 strawberries were used for each of the treatments. During the storage, the percentage of decayed strawberries was recorded. Disease severity was also recorded according to an empirical scale with six degrees: 0, healthy fruit; 1, 1-20% fruit surface infected; 2, 21-40% fruit surface infected; 3, 41-60% fruit surface infected; 4, 61-80% fruit surface infected; 5, more than 81% of the strawberry surface infected and showing sporulation (Romanazzi, Nigro, & Ippolito, 2000).

Successful loading of ZEO into CS particles
ZEO-loaded CSNPs were prepared through the formation of oil-inwater emulsion droplets followed by the solidification of the ZEO droplets via ionic gelation of the surrounding CS with TPP. CS in acidic media (pK a 6.5) has amino groups that can undergo protonation and interact with the negatively charged TPP, forming inter-and intramolecular cross-linkages, yielding ionically cross-linked CSNPs (De Campos, Sánchez, & Alonso, 2001;Xu & Du, 2003). This method results in spontaneous formation of NPs of smaller size with positive charge (De Campos et al., 2001;Pan et al., 2002) without using any organic solvent or surfactants (Dudhani & Kosaraju, 2010;Hu et al., 2002). In this regard, the concentrations of the CS and TPP solutions and the ratio of CS to TPP have important effects on nanochitosan preparation (Calvo, Remunan-Lopez, Vila-Jato, & Alonso, 1997). In our study, 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 (poly dispersity index b1) as shown in Table 1. This finding is in agreement with Huang, Sheu, and Chao (2009), who suggested that the cross-linking was best when the CS/TPP mass ratio was approximately 5:2. The recovery yield of CSNPs was 88.6 ± 4.64%, while those of ZEO-loaded CSNPs prepared using weight ratios of CS to ZEO of 1:0.25, 1:0.50, 1:0.75 and 1:1.00 were 60.90 ± 2.22%, 63.98 ± 5.98%, 71.99 ± 4.93% and 66.65 ± 7.94%, respectively.
The successful loading of ZEO into CSNPs was optimized by varying the concentrations of ZEO (350 to 3000 ppm) and was further confirmed by UV-visible spectrophotometry. CSNPs and ZEO@CSNPs were digested in HCL solution (2 M) at 95°C to break up the NPs and allow the release of encapsulated ZEO. ZEO-ethanol solution gives a maximum absorption peak at 230 nm, whereas the supernatant of CSNPs in ethanol after 1 h, exhibited no absorption peak at any wavelength ranging from 220 to 350 nm. However, the medium obtained from the immersion of ZEO@CSNPs prepared using weight ratios of CS to ZEO of 1:0.25, 1:0.5, 1:0.75 and 1:1 gave a maximum absorption peak at 230 nm, which corresponded to the max of ZEO. This result indicated the successful loading of ZEO into CSNPs.

Encapsulation efficiency and loading capacity
Following the synthesis of ZEO@CSNP, their loading capacity (LC) and encapsulation efficiency (EE) were calculated using Eqs. (1) and (2), respectively. As shown in Fig. 1, the EE% of ZEO was in a range of 3.26-45.24% when the initial ZEO varied from 25% to 100% (w/w) of CS. This result could indicate that the EE and LE decreased upon increasing initial ZEO concentration with a decrease in particle size. However, maximum EE and LC were obtained for the sample prepared using the weight ratio of CS to ZEO of 1:0.25 (45.24% and 9.05%, respectively). The result indicated that 100 g of particles contained ZEO content of 9.05 g. The possible reason for the decrease of EE could be that the excess ZEO gets loosely adsorbed onto the surface of the CS and finally gets detached during the separation of the CSNPs by centrifugation.  This result was in agreement with the findings regarding the loading of oregano EOs or ellagic acid into CSNPs, which have been reported by Hosseini, Zandi, Rezaei, and Farahmandghavi (2013) and Arulmozhi, Pandian, and Mirunalini (2013).

Shape, size and surface charge of ZEO-loaded CS particles
Particle size and surface charge are the major criteria to be focused in order to have an effective drug delivery (Arulmozhi et al., 2013). Mean particle size of CSNPs and ZEO-loaded CSNPs are shown in Table 1.Size of CSNPs was 96.93 ± 1.00 nm. The hydrodynamic diameter of the particles became larger when ZEO was loaded: i.e., 124.67-174.03 nm. Indeed, the particle diameter tended to slightly increase with increasing initial content of ZEO. In addition, surface charge and thereby the stability of the prepared nanoparticle systems was determined by zeta potential measurements. As shown in Table 1, the CSNPs have a high zeta potential (+53.2 mV), implying a positively charged surface and good stability of the particles. The zeta potential increased to values ranging from + 58 to + 67.8 mV for ZEO-loaded nanoparticles. The ZEO in media converts to positively charged group that could be responsible to increase in the zeta potential. The interactions between phenolic groups of ZEO and amino groups of CS (over phosphate group of TPP) may lead to decrease in the cross-linking density. A similar phenomenon were also found in other published works who showed lowered zeta potential value on CSNPs compared to drug-loaded CSNPs . In this regard, Dudhani and Kosaraju (2010) noted that the zeta potential increased for catechin-loaded CSNPs by 5.8 mV in comparison with CSNPs, which strongly supports our findings. All these data suggested that CSNPs and CSNP-loaded ZEO prepared here were stable.
The obtained morphological studies by TEM showed CSNPs with or without ZEO were nearly spherical in shape with smooth surfaces (Fig. 2a, b). The result indicates loading of ZEO in CSNPs increased the particle size, and it is in agreement with quercetin-loaded CS NPs (Zhang, Yang, Tang, Hu, & Zou, 2008).
It should be pointed out that the larger diameter of particles observed by DLS compared to that by TEM might be a result of swelling and aggregation of the nanoparticles during dispersion in an aqueous medium (Woranuch & Yoksan, 2013). Indeed, image analysis on the TEM micrographs gives the "true radius" of the particles (though determined on a statistically small sample), and DLS provides the hydrodynamic radius on an ensemble average (Berne & Pecora, 2000). As a result, the size determined by laser light scattering was larger than the size measured by TEM. This seems to be consistent with the findings of Woranuch and Yoksan (2013), who noted that the particle size results from TEM studies are smaller than the sizes obtained from the DLS for Eugenol-loaded CSNPs and CSNPs.

In vitro release characteristics of ZEO from CS particles
The in vitro release profiles of ZEO from ZEO-loaded CSNPs, prepared using a weight ratio of CS to ZEO of 1:0.25, were investigated for 40 days in buffer solutions with pH of 3, 5 and 7 at ambient condition. Fig. 3 illustrates that the release of ZEO is divided into two stages based on the release rate (the slope of the release profile). For the initial stage, the release rate was rapid, especially in the case of citrate buffer. ZEO was released up to 34.3%, 28% and 14.8% in buffer solutions with pH 3 and 5 (citrate buffer) and pH 7 (phosphate buffer), respectively (Fig. 3). The mechanism of ZEO release at this stage was attributed to the ZEO molecules adsorbed on the surface of the particles and oil entrapped near the surface, as the dissolution rate of the polymer near the surface is high, the amount of drug released will be also high (Anitha et al., 2011).
In the second stage, the release rate was relatively slow, i.e., almost zero (Fig. 3). At this stage, the rate of release fell as the dominant release Table 2 Diffusion exponential (n), Pearson coefficient (r 2 ) and diffusion constant (k) of ZEO released from CSNPs in different pH media. mechanism was changed to ZEO diffusion through CS matrix as previously reported (Papadimitriou, Bikiaris, Avgoustakis, Karavas, & Georgarakis, 2008). This might be due to the inability of buffer solutions to break or destroy the NPs, resulting in no additional release of ZEO at this stage. Agnihotri, Mallikarjuna, and Aminabhavi (2004) reported that the release of a drug by diffusion involved the penetration of medium into the particulate system, which caused swelling of the matrix. The concentrations of ZEO released on day 40 were 49.2%, 37.4% and 20.3% in buffer solutions with pH ∼3, 5 and 7, respectively. Recently, Arulmozhi et al. (2013) have mentioned that drugs loaded in CSNPs generally show two-step release pattern with an initial rapid release followed by sustained release. In addition, similar release pattern was reported for other CS-encapsulated polyphenolic compounds (Liang et al., 2011), which strongly supports our findings. Thus, our results proved that the release of ZEO in a controlled manner from ZEO@CSNP is an essential requirement for an effective ZEO delivery. However, the release profile of ZEO was affected by the pH of the medium, as shown in Fig. 3. At low pH, i.e., 3 and 5, ZEO was released from CSNPs very quickly, and the released ZEO content was relatively high as compared to the release at high pH, i.e., 7. The greater release of ZEO in acidic medium might be explained by the swelling and partial dissolution of NPs caused by the ionic repulsion of protonated free amino groups on one CS chain with its neighboring chains (Zhang, Mardyani, Chan, & Kumacheva, 2006).
The release mechanism and release kinetics of ZEO from CSNPs were also investigated using Korsmeyer-Peppas model (Eq. (5)): Here, M t /M ∞ is the fractional ZEO release at time t, k is a constant characteristic of the drug-polymer interaction and n is the characteristic power for the release. The value of n is used to indicate the Fickian and non-Fickian (anomalous) behaviors. In this context, n ≤ 0.43 indicates a Fickian release (case I transport), while n ≥ 0.85 indicates a case II transport (a purely relaxation-controlled delivery). Intermediate values of n, i.e., 0.43 b n b 0.85, indicate a non-Fickian release (anomalous transport) (Keawchaoon & Yoksan, 2011;Stulzer, Tagliari, Parize, Silva, & Laranjeira, 2009). The linear plot of ln (M t /M ∞ ) versus ln (t) yielded the diffusion exponential (n), the Pearson coefficient (r 2 ) and the diffusion constant (k) (Stulzer et al., 2009). Table 2 shows that n values were in the range of 0.14-0.27 for ZEO released in buffer solutions with pH of 3, 5 and 7. This implied that under acid and neutral conditions, the mechanism involved in the ZEO release displayed Fickian kinetics in which the release was mainly caused by diffusion and tiny swelling (Ritger & Peppas, 1987). The k values were related to the ZEO release kinetics: i.e., a higher k value indicates a faster release. The value of k was highest for the release in citrate buffer solution with pH of 3 (0.0468), followed by citrate buffer with pH of 5 (0.0237) and phosphate buffer solution with pH of 7 (0.0162), as shown in Table 2. The results suggest that the diffusion rate, or the release of ZEO from CSNPs, is faster in acidic solutions (pH ∼ 3 and 5) than in neutral media (pH ∼ 7). A similar result on in vitro release study of carvacrol-loaded CSNPs obtained by Keawchaoon and Yoksan (2011). This might be a result of the weakening of electrostatic interaction between the cationic material and anionic TPP, which caused both faster release and shorter release periods, as mentioned above (Yoksan, Jirawutthiwongchai, & Arpo, 2010). However, the amount of ZEO released in acidic solutions (pH ∼3 and 5) was higher than that in neutral media (pH ∼7), which might be due to the superior solubility of ZEO in acidic media.

In vitro antifungal effects
The antifungal activities of tested materials were first examined at various concentrations, and the results are shown in Fig. 4. The rate of growth reduction was directly proportional to the concentration of the tested material in the medium, i.e., as the concentration increases the radial growth decreases. ANOVA results demonstrated that only one concentration of ZEO and CSNPs out of four reduced in vitro mycelial growth of B. cinerea more than 50% (p b 0.05). While from the four concentrations tested of ZEO@CSNPs, three were able to reduce mycelial growth of fungus more than 50% (Fig. 4). The results obtained showed that ZEO in in vitro condition was not able to suppress mycelial growth even at concentrations as high as 1500 ppm. On the contrary, when encapsulated by CSNPs, the oils completely inhibited fungal growth at 1500 ppm. In fact, these new compounds exhibited an in vitro antifungal activity greater than unmodified ZEO against B. cinerea. This finding was in agreement with two studies of Russo et al. (2014) and Beyki et al. (2014) that resulted in better performance of EOs at lower amount. As demonstrated in those studies, the superior performance of EO-loaded CSNPs was due to 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 CSNPs itself. On the basis of the results from the in vitro inhibition of radial growth assay, only 1500 ppm for ZEO, CSNPs and ZEO@CSNPs were screened for the in vivo assay in mould-inoculated strawberries.

Antifungal effects on fruits
Figs. 5 and 6 present the results of the in vivo study of the ZEO@ CSNPs using strawberry fruit infected by B. cinerea. The statistical analysis revealed that the lowest percentage of infected strawberries was recorded for 1500 ppm ZEO@CSNPs (16.67%), followed by 1500 ppm CSNPs (66.67%) (p b 0.05) at day 9 (Fig. 5a). Meaning, the most effective treatments to reduce the disease incidence were those based on ZEO-loaded CSNPs. This treatment were not only effective in controlling strawberry decay but also in delaying the onset of disease symptoms and slowing down B. cinerea growth during the storage period (p b 0.05). Uncoated strawberries started to decay from the fourth day of storage at 4°C (Fig. 5a). The decay process was delayed to 7 and 9 days at strawberries coated with CSNPs and ZEO@CSNPs treatments, respectively.
Similarly, strawberries immersed in CSNPs or ZEO@CSNPs showed significant reduction of gray mold disease severity, when compared to the control after 2 days shelf life at 20 ± 1°C (Fig. 5b). They able to 1500 ppm ZEO@CSNPs 1500 ppm CSNPs Control Fig. 6. Appearance of strawberries coated with CSNPs (1500 ppm) and ZEO@CSNPs (1500 ppm) during storage. Fruits were treated, inoculated with fungus and stored for 7 days at 4°C followed by 2 days at 20°C. reduce disease severity to less than 1.5 and 2.6 in ZEO@CSNPs and CSNPs treated fruit, respectively, compared to 4.86 units (based on a 0-6 empirical scale) in the control strawberry. Furthermore, the ZEO@ CSNPs treatments did not cause any apparent phytotoxicity on strawberries (Fig. 6).
Results obtained in this study indicated that ZEO-loaded CSNPs significantly decreased both disease severity and incidence of infected strawberries by B. cinerea, after 1 or 2 days shelf life at 20°C following removal from storage at 4°C. Indeed, the in vivo trials confirmed the strong efficacy shown in vitro by ZEO@CSNPs treatment. Nonetheless, interactions between the coating components and the fruit surface can lead to divergent behavior with respect to that observed in the in vitro studies (Perdones, Sánchez-González, Chiralt, & Vargas, 2012). Numerous factors can affect the biological activity of certain compounds when in contact with fruit tissue. In this sense, Vu, Hollingsworth, Leroux, Salmieri, and Lacroix (2011) reported a higher antifungal effect of limonene than peppermint essential oil, encapsulated in coatings of modified CS, when applied to cold-stored strawberries, although the in vitro antifungal effect of the free (non-encapsulated) oils showed the opposite trend.

Conclusions
CSNPs, a prospective carrier for sustained release of ZEO, were prepared by a modified ionotropic gelation method and characterized by UV-vis spectrophotometry, DLS and TEM.
The average particle size and encapsulation efficiency could be controlled through the mass ratios of CS to ZEO. The release of ZEO from CSNPs in buffer solutions with pH of 3, 5 and 7 followed a Fickian behavior. ZEO was released relatively quickly in an acidic solution, followed by neutral media. The superior performance of ZEO when encapsulated by CSNPs under both in vitro and in vivo conditions in comparison with free oils was revealed. Having considered the remarkable antifungal activity of ZEO@CSNPs, further investigations on nanoencapsulation as drug carriers are also suggested.