3.1. Layer-by-Layer of eugenol-loaded nanocapsules
The Ne formation is an important step to form nanocapsules, since it was used as a template for the LbL process (Abbas et al., 2015) and in this work, the variation of the sonication time to obtain the Ne was evaluated. The droplet size and the polydispersity index (PDI) of the Ne as a function of sonication time are shown in Fig. 1A. The mean value of Dapph was calculated from up to 20 repetitions of each measurement. After 240 s of sonication, there is a decrease in the droplets size and sample dispersion (p < 0.05). Between 30 s (PDI = 0.494) and 600 s (PDI = 0.230), there is a considerable difference (p < 0.05) in the dispersion due to the greater amount of energy supplied to the system Fig. 1B. The PDI < 0.250 suggested a more homogeneous distribution, reducing the Ostwald ripening effect and contributing to stability. In this context, the best condition to form homogeneous Ne droplets was 600 s of sonication.
After selecting the best condition to obtain Ne, NCs were obtained by LbL. As already noted in the literature, the order of addition of the precursors can alter some final properties of the NCs, for example, the average diameter (Liu et al., 2012). Two different ways of adding the precursor materials were followed to identify the best protocol to obtain NCs. In condition 1 (C1), the polymeric dispersion was added over the Ne and in condition 2 (C2) the Ne was added over the polymeric dispersion.
In condition C1, the dispersion of chitosan has a positive charge in acid medium (pKa < 6.5), and was added over the Ne, making the dispersion immediately milky-like (Fig. 1C - C1). For this situation, the Dapph (Fig. 1C) and the PDI were 368 ± 113 nm and 0.546, respectively. The higher PDI value indicated a non-uniform formation of NCs.
For C2, the Ne was added to chitosan dispersion, and the original light-yellow dispersion color gradually became light-milky (Fig. 1C – C2). In this situation, the droplet coating with chitosan molecules occurs immediately, and the Dapph (Fig. 1C) and PDI were 360 ± 30 nm and 0.390, respectively, lower than C1 (p < 0.05). The size and PDI variation were monitored for the other self-assembly layers. However, no significant differences were observed in Dapph and PDI for adding carboxymethylcellulose (p > 0.05), and the values obtained were Dapph: 473 ± 164 nm and PDI: 0.440 for C1 and Dapph: 398 ± 106 nm and PDI: 0.396 for C2.
In addition to the NCs size, stability by ζ-potential was observed after each coating for C1 and C2. In Fig. 1D, it is possible to observe the zeta potential inversion for each polymeric deposition and to infer that regardless of the order of addition of the polysaccharides, the NCs are close to the stability region (~|30| mV). Considering the lower PDI, further experiments were performed using the condition C2.
3.2. Incorporation efficiency and in vitro eugenol release from nanocapsules
The IE (%) of eugenol was determined as 8.3 ± 0.1% for Ne-LbL1 NCs and 5.1 ± 0.3% for Ne-LbL2 NCs. The differences were probably associated to the dragging of eugenol molecules associated to centrifugation steps during NCs preparation. The presence of eugenol in the NCs was also confirmed by thermogravimetric analysis present in Fig. S1 and Table S1.
The eugenol release profile was investigated to understand the mechanism of release from NCs. In Fig. 2 was possible to observe for free eugenol a rapid release (burst release) in the first 15 min (34.4 ± 3.7%), reaching to 100% ~ 4 h. In contrast, eugenol encapsulated in Ne-LbL1 − 2 the rapid release phase occurs up to 5 h, followed by a slow and gradual release.
The decrease in the release, comparing Ne-LbL2 NCs with Ne-LbL1 NCs was directly related to the number of polymer layers added as observed in previous studies (Jacumazo et al., 2020). A first-order equation was used to adjust the release of eugenol. In Eq. 6, \({\text{m}}_{\text{t}}\) represents the fraction released at time t, \({\text{m}}_{{\infty }}\) is the amount of the active in the formulation, and k is the first-order constant.
\(\text{ln}\left(\frac{{m}_{t}}{{m}_{\infty }}\right)=kt\) (Eq. 6)
The values of k using a first-order model for free eugenol, Ne-LbL1 NCs and Ne-LbL2 NCs were 0.057, 0.033, and 0.031 min− 1, respectively. The first-order kinetic model refers to the process where eugenol release was concentration-dependent. Also, the presence of polymeric layers decreased k, due to the formation of porous layers that limit the diffusion process, but apparently is the same for one or two polymeric layers, suggesting only partial coating using carboxymethylcellulose. It can be inferred that the polymer layers reduced the eugenol diffusibility and such results corroborates with the antioxidant activity evaluation of the NCs (Fig. S2), that Ne-LbL1 − 2 NCs make eugenol release and DPPH inhibition slower.
3.3. Surface properties, contact angle, surface free energy, and work of adhesion
NCs can be used to coat products such as fruits and vegetables by increasing their shelf life (Zambrano-Zaraboza et al., 2018), providing, in addition, the release of actives for protection against pathogens. In this sense, nectarine coatings were made with NCs containing eugenol. The macroscopic aspects of the coatings can be seen in Fig. S3. It is notable that after 24 h the coatings are homogeneous and transparent, and it is not possible to observe macroscopic differences concerning the control sample.
To obtain more information about these coatings, contact angle measurements of untreated nectarines (control), treated with NCs in the presence of eugenol (Ne-LbL1 − 2 NCs) and NCs in the absence of eugenol (LbL1 − 2 NCs), were performed. The contact angles were obtained using three liquids of different polarities (water, formamide, and diiodomethane) as shown in Fig. 3A.
When the liquid drop encounters the nectarine surface, intermolecular interactions are established between the epicarp surface or film surface and the specific liquid drop which can be attractive or repulsive forces (Moncayo, Buitrago & Algecira, 2013). Thus, considering a polar liquid, the greater the contact angle, the lower the affinity of the surface in question to the liquid, that is, the more hydrophobic this surface is and the less wettable. This can be observed for the control sample when in contact with water and formamide liquids (p < 0.05).
On the other hand, the contact angles of the samples treated with the NCs decreased, compared to the control, increasing the wettability (p < 0.05). In samples with the chitosan layer, there was a greater increase in wettability compared to the other samples, possibly a better nectarine coating. This may be related to the better interaction of the chitosan acetyl groups with the nectarine, thus making the hydrophilic groups of chitosan more exposed. In the case of coating with the second layer of polymer (carboxymethylcellulose), this decrease is not so marked, this may be related to an incomplete coating as seen in Fig S2.
With the obtained contact angle values for all liquids, it was possible to calculate the values of total surface-free energy (𝛾total), dispersive and polar components using the OWRK model. The total surface tension (𝛾total), dispersive (𝛾D), and polar (𝛾P) components values for the three liquids used can be seen in Table S2.
The control sample, nectarine peel treated with water, had a lower surface free energy value (26.9 ± 2.9 mJ m− 2) (Fig. 3B), on the other hand, the nectarines with Ne-LbL1 NCs coating showed a higher total surface-free energy value (36.1 ± 3.7 mJ m− 2). Comparatively, the control sample has a less polar surface than LbL1 and LbL2 and the values of total surface-free energy were different for the untreated and treated samples with Ne-LbL1 NCs (p < 0.05).
In the nectarines coated with chitosan with Ne-LbL1 NCs and LbL1 NCs, values of 18.2 ± 3.9 mJ m− 2 and 14.3 ± 3.1 mJ m− 2 were observed respectively for the dispersive components and 17.0 ± 6 mJ m− 2 and 13.3 ± 4.1 mJ m− 2 for the polar components, where the dispersive and polar components do not differ (p > 0.05). After coating with LbL1, the surface polarity increased, however maintaining equivalent dispersive composition (Fernández et al., 2011).
The nectarines coated with the anionic polymer, Ne-LbL2 NCs, and LbL2 NCs, the values of 23.6 ± 1.9 and 17.7 ± 4.1 mJ m− 2 were observed respectively for the dispersive components and 4.9 ± 1.9 and 9.3 ± 4.4 mJ m− 2 for the polar components, and the dispersive and polar components do not differ (p > 0.05). The Ne-LbL2 NCs have a larger dispersive component, possibly due to the strong ionic interaction between chitosan and carboxymethylcellulose and exposing less polar sites of cellulose.
With the calculated data of total surface-free energy and dispersive and polar components, it was possible to obtain the coating work of adhesion (Wa) (Fig. 3C). As observed, the Wa determined to LbL1 on nectarines (Wa 0,1), or for LbL2 on LbL1 (Wa 1,2) was almost of the same order of magnitude. It is important to highlight that both polysaccharides could be useful to coat nectarines, with almost the same Wa (Wa 0,1 or Wa 0,2). However, the LbL1 of chitosan turns the surface much more polar than LbL2, and this could promote interesting biological properties.
According to Velásquez, Skurtys, Enrione, & Osorio (2011), the chemical composition of the wax is a mixture of long-chain compounds, including hydrocarbons, ketones, alcohols, aldehydes, and free and esterified fatty acids, the percentage of the composition varies from fruit to fruit. Thus, the components of the nectarine epicuticular wax may be promoting intermolecular interactions with the components of the NCs, chitosan and carboxymethylcellulose.
3.4. Effect of nanocapsules on the brown rot
In the present study, antimicrobial activity was evaluated using NCs with up to two layers of polymers containing eugenol (Ne-LbL1-2 NCs), aqueous solution of eugenol, NCs in absence of eugenol (LbL1 NCs) and aqueous solution of the commonly used fungicide iprodione for the control of brown rot (Dutra, Pereira, May de Mio et al., 2019).
In this sense, the estimate of the relative risk for the expression of symptoms of Monilinia fructicola was analyzed using the Cox semiparametric model (Table 1) using nectarine in the absence of treatment as a standard. It is possible to observe that the nectarines coated with the NCs in the presence of eugenol, followed by the aqueous solution of eugenol were the ones that presented the lowest relative risk, therefore, the lowest probability of the fruit becoming ill. On the other hand, fruits treated with the fungicide were more susceptible to the onset of disease symptoms. Regarding the confidence interval (CI, 95%) the NCs containing eugenol and the aqueous solution of eugenol are the samples that differ from the control sample, confirming the lower risk of contamination of the fruits.
The survival analysis of the healthy fruits is shown in Fig. 4 with a study time of 7 days. It can be observed that over time there is a decrease in the probability of the fruits remaining without symptoms of brown rot for all treatments. Untreated (control) and fungicide treated nectarines, LbL1 NCs and aqueous solution of eugenol expressed disease symptoms more rapidly than those treated with Ne-LbL1-2 NCs. For the control fruits and those treated with iprodione, on the fifth day, there was less than 50% probability that the fruits remained without disease symptoms.
Table 1
Estimates of relative risk for the expression of symptoms of Monilinia fructicola estimated by the Cox semi-parametric model, followed by 95% confidence intervals for nectarine
Treatment
|
Incubation
|
Relative risk
|
CI (95%)
|
period (days)
|
LL
|
US
|
Control
|
3
|
-
|
-
|
-
|
LbL1 NCs
|
4
|
0.7218
|
0.5111
|
1.0194
|
Iprodione
|
2
|
0.9272
|
0.6724
|
1.2786
|
Eugenol
|
5
|
0.5651
|
0.3918
|
0.8150
|
Ne-LbL1 NCs
|
> 7
|
0.2351
|
0.1480
|
0.3736
|
Ne-LbL2 NCs
|
5
|
0.4959
|
0.3423
|
0.7184
|
CI – confidence interval (95%), LL: lower limits, US: upper limit. |
* Incubation period is the number of days between the inoculation (contact of the pathogen with the nectarine fruit) and the symptoms expression on at least 50% of the sample (inoculated fruit). |
Furthermore, the fruits treated with iprodione were the ones that showed the most symptoms of the disease, with a probability below 20% of the fruits remaining without the disease until the end of the study (7 days). In the case of fruits treated with NCs with the outer layer of chitosan, there was a higher probability of survival compared to NCs with the outer layer of carboxymethylcellulose, which corroborates the better adherence of Ne-LbL1 NCs to nectarines, as shown in the work of adhesion data. In this sense, although treatments with LbL1 NCs, NCs with the outer layer of carboxymethylcellulose (Ne-LbL1-2 NCs) and aqueous solution of eugenol enable the efficient control of the pathogen Monilinia fructicola when compared to treatment with iprodione, only the fruits treated with NCs with the outer layer of chitosan (Ne-LbL1 NCs) showed above 70% probability that the fruits remain in the absence of symptoms until the seventh day of the study.
The images of the experiments containing all treatments during the 7 days can be seen in Fig. S4, as some fruits showed more accentuated symptoms, they were removed from the experiment to minimize possible contamination in other fruits.
The lower brown rot control efficiency promoted by the aqueous solution of eugenol may be associated with its rapid volatilization and low stability when exposed to light, temperature, or humidity, as documented in the literature (Turek & Stintzing, 2013). On the other hand, its encapsulation process enabled the formation of a protective barrier to the factors mentioned above, enabling the control of the pathogen for a longer period and with a smaller amount of active, since the IE% of eugenol in NCs with the first polymeric coating was close at 8.3%. As a second layer of the polymer was added, there was a decrease in pathogen inhibition. This may be related to the fact that systems with lower release rates may take longer to efficiently reach the fungus, for this reason, Ne-LbL1 NCs had the best fungicidal activity (p < 0.05).
In addition, the effective control of treatments containing NCs may be related to the coating that NCs promote on the surface of nectarines, this coating provides a barrier against external elements, in addition to protecting against moisture loss (Elsabee & Abdou et al., 2013).
Another factor that may be associated with the better performance of chitosan-coated NCs is their positive surface charge (ζ-potential: 32 ± 5 mV), which can ionically interact with the negative charge of the fungal membrane phospholipids. This interaction increases membrane permeability causing loss of cell content and leading to fungus death (Devlieghere, Vermeulen, Debevere et al., 2004). Furthermore, the size of the NCs may also be related to better antifungal activity, as by reducing the size, the contact surface area increases, promoting a better affinity with fungal cells (Ing et al., 2012).
In this way, considering the factors that influence the surface properties of nectarine adherence and antimicrobial activity, the NCs containing eugenol with the outer layer of chitosan (Ne-LbL1) promoted better adherence and showed the best antimicrobial control. However, the Ne-LbL2 NCs also showed control of the pathogen, in lesser intensity, but higher or at the level of the commercial fungicide. Therefore, these polymeric systems proved to be promising for fruit coating in the protection against brown rot caused by Monilinia fructicola, to increase fruit storage or shelf life, in addition to using much less toxic natural substances as fungicides.