Natural polymer nanocapsules containing eugenol for nectarine coating to control brown rot caused by Monilinia fructicola

Natural polymer nanocapsules containing eugenol for nectarine coating to control brown rot caused by Monilinia fructicola | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Natural polymer nanocapsules containing eugenol for nectarine coating to control brown rot caused by Monilinia fructicola Joslaine Jacumazo, Gabriela Pereira Parchen, Meira Janete Ballesteros-Garcia, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1654686/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The fruit infection by fungi reduces the quantity and quality of food for human consumption and causes economic damage. Thus, this study aimed to address the effects of nanocapsules (NCs) based on chitosan and carboxymethylcellulose containing eugenol in protecting nectarines against Monilinia fructicola , a brown rot agent, a worldwide important disease. NCs were prepared by Layer-by-Layer self-assembly starting from an anionic template and deposition with up to two polymeric layers. The sizes ranged from 158 nm (nanoemulsion) to 398 nm (two polymeric layers). In vitro release showed an initial burst followed by a gradual release. In addition to showing a good adherence to nectarine surface, the NCs coated with chitosan proved to be the best formulation for the control of brown rot in the antifungal test. Therefore, this study demonstrated that chitosan NCs containing eugenol could be an alternative to preserve fruit for longer periods in the post-harvest. natural polymers layer-by-layer adherence eugenol Monilinia fructicola disease control Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Fruits are an essential part of a healthy diet for humans due to their composition based on vitamins, minerals, and fibers (Slavin & Lloyd, 2012 ). In 2018, according to the United Nations Food and Agriculture Organization (FAO), they are perishable products, and post-harvest losses are between 30–50% of total world production. Nectarine ( Prunus persica var. nucipersica ) is a popular fruit and highly nutrient, belonging to the Rosaceae Family (Aubert et al., 2014 ). The nectarine and peach production in 2019 reached 22.3 million tons (FAO, 2022 ). However, due to its typical climacteric properties, the fruit ripens quickly under environmental conditions, and after harvesting, there is an increase in the production of plant hormone ethylene which is responsible for its ripening and, therefore, limits the storage time in the post-harvest (Aubert et al., 2014 ). This condition affects the fruit characteristics such as the decrease in firmness, in addition to the loss of color and flavor (Aubert et al., 2014 ; Lurie & Crisosto, 2005 ; Xi et al., 2017 ). Furthermore, under these situations, infection by microorganisms such as fungi may occur. Brown rot is one of the main diseases that affect nectarine (Oliveira-Lino et al., 2016). This disease is caused by the fungi Monilinia fructicola (Wint) Honey. This pathogen is responsible for infecting the plant during the blossom time and also during the fruit grown stage (Oliveira-Lino et al., 2016; Keske, Amorin & Mio, 2011; May-De Mio, Luo & Michailides, 2011 ). and it can remain in the fruit latently, showing symptoms only when the environment becomes favorable, for example, in the post-harvest. Chemical control methods are still predominantly using fungicides, reducing the loss to 5–10% in the post-harvest. However, this microorganism has been selected for resistance to fungicides (Oliveira-Lino et al., 2016; May-De Mio, Luo & Michailides 2011 ; Garcia-Benitez & Melgarejo, 2017; Hong et al., 1997 ; Pereira et al., 2018 ). Thus, in recent years, some alternatives have been proposed to minimize losses in the post-harvest period and increase the fruit’s shelf life, among them, the use of low temperature (Zhao et al., 2019 ), coating with polysaccharides (Amon et al., 2015), immersion in hot water (Akgun et al, 2010 ) and use of essential oils (Amiri et al., 2008 ). One of the essential oils that we can highlight is the clove essential oil, composed mainly of eugenol (45–90%) (Bakkali et al., 2008 ; Khalil et al., 2017 ). Eugenol (4-allyl-2-methoxy phenol) is a phenolic compound widely used in the pharmaceutical, cosmetic, and food industries due to its antimicrobial and antioxidant properties (Marchese et al, 2017 ; Shao et al., 2018 ; Jin et al. 2019 ). Eugenol has also effects to control plant pathogens, presenting a fungitoxic mode of action (Rozwaka et al., 2020). In previous and non-published studies carried out by our research group, eugenol proved to be efficient in controlling conidia germination and mycelium growth of Monilinia fructicola . However, its application to control agricultural diseases becomes limited because it is volatile and can be degraded by light and oxygen (Turek & Stintzing, 2013 ). Low solubility in water is another limiting fator (Turek & Stintzing, 2013 ; Li et al., 2015 ). To overcome these difficulties, nanoencapsulation has become an interesting and promising alternative. Polymeric nanocapsules (NCs) can be used as a protective barrier for active substances, decreasing their losses and increasing their efficiencies by promoting a more controlled release (Lima et al., 2022 ). They can be obtained by various methods, including nanoprecipitation, emulsion-diffusion, coacervation, and layer-by-layer (LbL) (Mora-Huertas, Fessi & Elaissari, 2010 ; Rao & Geckeler, 2011 ). The mechanism of NCs formation by the LbL self-assembly method, from a colloidal template, is based on irreversible ionic interactions that lead to the adsorption of polyelectrolytes. Among the cationic and anionic polyelectrolytes, chitosan and carboxymethylcellulose, respectively, can be mentioned (Mora-Huertas, Fessi & Elaissari, 2010 ) Chitosan is a linear copolymer of (1–4) linked 2-acetamido-2-deoxy-β- D -glucopyranose and 2-amino-2-deoxy-β- D -glucopyranose, soluble in dilute acidic solutions below pH 6.5 due to the quaternization of the amine groups that have a pK a value of 6.3–6.5 (Sahariah & Másson, 2017 ; Wang, Qian & Ding, 2018 ). It has excellent biocompatibility and biodegradability, in addition to low toxicity, making it an ideal alternative for interaction with other polymers and the development of NCs (Wang, Qian & Ding, 2018 ; Dash et al., 2011 ). A polymer of opposite charge, biodegradable and non-toxic that can interact ionically with chitosan is carboxymethylcellulose (Zhang et al., 2013 ), a molecule formed by the partial carboxymethylation of β- D -glucopyranose cellulose. The anionic character is due to the formation of carboxylate groups through the deprotonation that occurs at pH above its pK a (2.0–4.0) (Heinze & Koschella et al., 2005). In this sense, this work aimed to use the LbL technique to prepare NCs containing eugenol using polysaccharides derived from renewable sources such as chitosan and carboxymethylcellulose, and to develop a modified release fruit coating system to evaluate the control of pathogenic fungi. Our hypothesis is that coating fruit, such as nectarine, using eugenol nanocapsules coated with biopolymers, could be a new strategy to reduce the incidence of brown rot caused by Monilinia fructicola . This strategy offers new perspectives to increase fruit quality and reduce post-harvest losses. 2. Materials And Methods 2.1. Chemicals Chitosan (C 6 H 11 NO 4 ) n was purchased from Shangyu Biotech Co, Ltda (Shangyu, China) and submitted to a purification procedure before use as described in previous work (Jacumazo et al, 2020 ). The chitosan weight average molar mass (M w ) was 1.9x10 5 g moL − 1 and deacetylation degree of 76%. Sodium dodecyl sulphate (99.0 wt% purity), eugenol (99.0% V V − 1 purity), formamide (99.5% V V − 1 purity) and 2,2-diphenyl-1-picryl-hydrazil (DPPH) were purchased from Sigma-Aldrich (St. Louis, USA). Diiodomethane (99.0% V V − 1 purity) was purchased from Neon (Suzano, Brazil). Carboxymethylcellulose of M w of 6.7x10 5 g moL − 1 and DS 0.84 was purchased from Acros Organics (Geel, Belgium). All solutions were prepared with purified water obtained by a reverse osmose system. 2.2. Nanoemulsion of eugenol The nanoemulsions of eugenol (Ne) were obtained from the addition of eugenol in a sodium dodecyl sulfate solution (5.0 mmol L − 1 ), the ratio eugenol:sodium dodecyl sulfate was 0.78:1 wt. V − 1 . This mixture was sonicated (Ultrasonics Sonicator, USA) at 30% amplitude with a nominal power of 750 W and frequency of 20 kHz. Then, the sonication time was varied from 30, 120, 240, 360, 480 to 600 s at 0 ºC. At the end of the sonication process, the Ne were left under magnetic stirring at 900 rpm (Magnital Agitator MAG15, Marte Scientific, Brazil) for 15 min. 2.3. Layer-by-Layer of eugenol-loaded nanocapsules The LbL technique was used for the self-assembly of polymer layers. Polymeric dispersions of chitosan (2 mg mL − 1 ), carboxymethylcellulose (2 mg mL − 1 ), and Ne were performed in sodium acetate buffer (0.01 mol L − 1 , pH 4.6). The self-assembly step was performed in two different situations: condition 1, the polymer dispersion was added into the Ne; and condition 2, the Ne was added into the polymer dispersion. From the Ne, the NCs were formed by alternating deposition of the polymer: cationic (Ne-LbL 1 NCs) and anionic (Ne-LbL 2 NCs). After each polymer deposition, the nanocapsules were centrifuged (5 x 10 4 g, 30 min) at 25 ºC, washed with water, and kept under continuous magnetic stirring at 900 rpm, 25°C for 1 h after resuspension. Using the same protocol, free eugenol particles were produced as controls for antimicrobial assays and named LbL 1 NCs and LbL 2 NCs. 2.3.1. Eugenol-loaded nanocapsules characterization The average apparent hydrodynamic diameter (D apph ) was determined using a dynamic light scattering (DLS) on a NANO DLS Particle Size Analyzer apparatus of Brookhaven Instruments (Holtsville, New York, USA) in water at 20 ºC. All experiments were conducted using a 15 mW solid-state He–Ne laser, operating at 90° and wavelength at 632.8 nm. The samples containing the NCs were diluted in water 1:50 (V V − 1 ). For zeta potential (ζ-potential) analyses were carried out in a Particle Charge Mapping Stabino apparatus (Germany). The samples were diluted in water 1:50 (V V − 1 ) and analyzed for 100 s at 20 ºC. A 10 mL measuring cell coupled to a 400 µm piston was used. Thermogravimetric analysis (TGA) of eugenol (6.1 mg), chitosan and carboxymethylcellulose mixture (5.2 mg), Ne-LbL 1 NCs (4.9 mg), Ne-LbL 2 NCs (4.6 mg) were performed using 0.065 mL alumina crucibles with a Netzsch analyzer (STA 449 F3 series EP), following a heating rate at 10°C min − 1 from 25 to 600 ºC under nitrogen atmosphere of 50 mL min − 1 . The NCs with eugenol were previously lyophilized at -50 ºC (MicroModulyo, Thermo Electron Corporation, USA) prior to the TG analysis. The antioxidant activity of eugenol, intending to confirm the release of eugenol from NCs, was evaluated using DPPH radical tests (Thaipong et al., 2006 ). For more details, please access the Supporting Information. 2.4. Incorporation efficiency The incorporation efficiency (IE%) of eugenol was determined according to the method described by Jacumazo et al. ( 2020 ). The solubility of eugenol in water and chloroform was determined using spectroscopy in the UV-vis region (281 nm) and the values were 2.41 g L − 1 e 8.18 g L − 1 , respectively. The samples (2 mL) were centrifuged (4.000 g , 15 min) at 5 ºC. The supernatant was removed, and chloroform (2 mL) was added to the sedimented material, mixed, and centrifuged (4.000 g , 20 min) at 25 ºC. The supernatant was collected, and the absorbance was measured at 281 nm using UV–vis spectrophotometry. The eugenol IE% was estimated following Eq. 1. The experiments were run in triplicate. \(\text{IE(\%)=}\left(\frac{{\text{NCs}}_{\text{Eugenol}}}{{\text{Initial}}_{\text{Eugenol}}}\right)\text{x 100}\) ( Eq. 1 ) where NCs Eugenol is the mass of eugenol determined in the NCs and Initial Eugenol is the mass of eugenol added in the experiment. 2.5. In vitro eugenol release from nanocapsules The eugenol release experiments were measured using a dialyzes procedure. Samples were added in a cellulose dialysis bag (cut-off of 12 kg mol − 1 , D0530-100FT, Sigma-Aldrich, Germany, USA) and then placed in the receptor system containing water. The saturation concentration of eugenol in water was 69.8 mg L − 1 , and all the experiments were maintained under sink conditions. The experiment was performed at pH 6.8, 25 ºC, and continuous magnetic stirring (900 rpm) for 96 h. At defined time intervals (0, 0.25, 0.5, 1, 2, 3, 4, 5, 24, 48, 72, and 96 h), an aliquot of 2 mL of the receptor medium was collected and analyzed using a UV-vis spectrophotometer in the wavelength of 281 nm. The medium receptor was immediately replenished with equal volumes of water. The experiments were run in triplicate. The eugenol quantification was estimated using an equation of the analytical curve, with R 2 = 0.998, \(Eugenol mg {L}^{-1}=\left(\frac{absorbance-0.011}{0.015 L {mg}^{-1}}\right)\) . The limit of detection and quantification was 0.40 mg L −1 and 1.34 mg L − 1 , respectively. 2.6. Nectarine coating from nanocapsules 2.6.1. Preparation The nectarine coating was carried out with the Ne-LbL 1 NCs and Ne-LbL 2 NCs, and nectarine without coating was used for the control. The nectarines were placed in contact with the dispersions (Ne-LbL 1 − 2 NCs) or distilled water (control) for 30 s and then dried at 25 ºC for 24 h. After drying, the nectarine peel was withdrawn, and the contact angle analysis was performed in duplicate with six drops per sample. 2.6.2. Characterization: contact angle, surface free energy, and adhesion work The contact angle measurements were performed using three liquids: water, formamide, and diiodomethane, and measured at 20 ºC using a DATAPHYSICS Instruments GmbH Contact Angle System OCA15 + tensiometer (Germany). The contact angle at the nectarine surface was measured by the sessile drop method, 5 µL of each liquid was dripped on the samples. The right and left side of the drop was measured to average the contact angle. The contact angles were automatically calculated by fitting the captured drop shape (software SCA20). The calculation of the surface energy of the nectarine coating was based on the method described by Owens and Wendt ( 1969 ) and Kaelble ( 1970 ), using the Owens, Wendt, Rabel, and Kaelble (OWRK) method to calculate the components of the solid surface energy. The calculation was performed using Eq. 2. \(\frac{{\gamma }_{L}(cos{\theta }_{c}+1)}{2\left(\sqrt{{\gamma }_{L}^{D}}\right)}= \sqrt{{\gamma }_{S}^{P}}\left(\frac{\sqrt{{\gamma }_{L}^{P}}}{\sqrt{{\gamma }_{L}^{D}}}\right)+ \sqrt{{\gamma }_{S}^{D}}\) ( Eq. 2) where \({\theta }_{c}\) is the experimental contact angle, \({\gamma }_{L}\) is the total liquid surface tension, \({\gamma }^{D}\) is the dispersive component, \({\gamma }^{P}\) is the polar component. The subscripts S and L stand for solid and liquid, respectively. The NCs work of adhesion of LbL 1 NCs and Ne-LbL 1 NCs with the nectarine surface, as well as the LbL 2 NCs with the chitosan layer (LbL 1 ), was calculated using Equations 3, 4 and 5. \({W}_{a}= {{W}_{a}}^{P}+ {{W}_{a}}^{D}\) (Eq. 3) \({{W}_{a}}^{P}=2\sqrt{{\gamma }_{x}^{P}{\gamma }_{y}^{P}}\) (Eq. 4) \({{W}_{a}}^{D}=2\sqrt{{\gamma }_{x}^{D}{\gamma }_{y}^{D}}\) (Eq. 5) where \({W}_{a} x,y\) is the total work of adhesion between layers x and y, and \({\gamma }_{x} and {\gamma }_{y}\) are the free energy of the two different interfaces. 2.7. Microbial assays for brown rot control The fungus, isolated PpMfSP15/575 from Monilinia fructicola , belongs to the LEMID-UFPR collection. The conidial suspension was prepared to collect the spores on the surface of pre-inoculated canned peach and disperse into a sterile Tween 20 (0.5% V V − 1 ) aqueous solution. Nectarines ( Prunus persica var. nucipersica ) of Sungold cultivar (Santa Catarina – Brazil) were used in the experiment. These fruits were previously sanitized by immersion (1 min) in ethanol/water (70% V V − 1 ), an aqueous solution of sodium hypochlorite (1% V V − 1 ), and distilled water (3 x) and then let dry at ambient temperature (25°C). The nectarine fruits were treated with sterile water (control), an aqueous solution of eugenol using dimethyl sulfoxide and Adivex® as co-solvents (formulation control), NCs without eugenol, Ne-LbL 1− 2 NCs, and fungicide (iprodione – Rovral SC® 500 g L − 1 Basf, France) as the positive control, efficient to control brown rot (Kaelble, 1970 ). The nectarines were dripped in each treatment for 30 s and dried for 24 h. Then, each nectarine was inoculated with 40 µL of the Monilinia fructicola conidial suspension (10 5 conidial mL − 1 ) and the samples were placed in the humid chamber. Seven repetitions were performed for each treatment and symptoms evaluations were performed every 24 h for 7 days. The experiment was conducted twice in a completely randomized design. 2.8. Data analysis The statistical analysis was performed with GraphPad Prism 8 software, using a one-way analysis of variance (ANOVA), followed by Tuckey’s Post Hoc test (p ≤ 0.05). Survival analysis was estimated by the Kaplan-Meier method and the comparison of curves between treatments was estimated by the non-parametric log-rank test. In addition, Cox's proportional hazards model was also used for all tested samples. For all statistical inferences, p < 0.05 was considered a nominal significance level. The statistical software R (version 3.4.3) was used for data analysis and graphical representation. 3. Results And Discussion 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. 1 A. The mean value of D apph 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. 1 B. 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 (pK a < 6.5), and was added over the Ne, making the dispersion immediately milky-like (Fig. 1 C - C1). For this situation, the D apph (Fig. 1 C) 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. 1 C – C2). In this situation, the droplet coating with chitosan molecules occurs immediately, and the D apph (Fig. 1 C) and PDI were 360 ± 30 nm and 0.390, respectively, lower than C1 (p 0.05), and the values obtained were D apph : 473 ± 164 nm and PDI: 0.440 for C1 and D apph : 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. 1 D, 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-LbL 1 NCs and 5.1 ± 0.3% for Ne-LbL 2 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-LbL 1 − 2 the rapid release phase occurs up to 5 h, followed by a slow and gradual release. The decrease in the release, comparing Ne-LbL 2 NCs with Ne-LbL 1 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-LbL 1 NCs and Ne-LbL 2 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-LbL 1 − 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-LbL 1 − 2 NCs) and NCs in the absence of eugenol (LbL 1 − 2 NCs), were performed. The contact angles were obtained using three liquids of different polarities (water, formamide, and diiodomethane) as shown in Fig. 3 A. 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 (𝛾 total ), dispersive and polar components using the OWRK model. The total surface tension (𝛾 total ), dispersive (𝛾 D ), and polar (𝛾 P ) 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. 3 B), on the other hand, the nectarines with Ne-LbL 1 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 LbL 1 and LbL 2 and the values of total surface-free energy were different for the untreated and treated samples with Ne-LbL 1 NCs (p < 0.05). In the nectarines coated with chitosan with Ne-LbL 1 NCs and LbL 1 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 LbL 1 , the surface polarity increased, however maintaining equivalent dispersive composition (Fernández et al., 2011 ). The nectarines coated with the anionic polymer, Ne-LbL 2 NCs, and LbL 2 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-LbL 2 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 (W a ) (Fig. 3 C). As observed, the W a determined to LbL 1 on nectarines (W a 0,1), or for LbL 2 on LbL 1 (W a 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 W a (W a 0,1 or W a 0,2). However, the LbL 1 of chitosan turns the surface much more polar than LbL 2 , 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-LbL 1-2 NCs), aqueous solution of eugenol, NCs in absence of eugenol (LbL 1 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, LbL 1 NCs and aqueous solution of eugenol expressed disease symptoms more rapidly than those treated with Ne-LbL 1-2 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 - - - LbL 1 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-LbL 1 NCs > 7 0.2351 0.1480 0.3736 Ne-LbL 2 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-LbL 1 NCs to nectarines, as shown in the work of adhesion data. In this sense, although treatments with LbL 1 NCs, NCs with the outer layer of carboxymethylcellulose (Ne-LbL 1-2 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-LbL 1 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-LbL 1 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-LbL 1 ) promoted better adherence and showed the best antimicrobial control. However, the Ne-LbL 2 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. ABBREVIATIONS AND NOMENCLATURE United Nations Food and Agriculture Organization (FAO); nanocapsules (NCs); layer-by-layer (LbL); nanoemulsions of eugenol (Ne); NCs with deposition of the cationic polymer containing eugenol (Ne-LbL 1 NCs); NCs with deposition of the anionic polymer containing eugenol (Ne-LbL 2 NCs); NCs with deposition of the cationic polymer (LbL 1 NCs), NCs with deposition of the anionic polymer (NCs with deposition of the anionic polymer), apparent hydrodynamic diameter (D apph ); dynamic light scattering (DLS); zeta potential (ζ-potential); thermogravimetric analysis (TGA); incorporation efficiency (IE%); work of adhesion (Wa), polydispersity index (PDI), confidence interval (CI) Declarations AUTHOR’S CONTRIBUTION J.J., M.J.B.G, N.C.S. and G.P.P performed the experiments and wrote the main manuscript; F.A.M. reviewed the manuscript and supervised the chemical characterization of eugenol; L.L.M.D.M. reviewed the manuscript and supervised the nectarine experiments with Monilinia fructicola; R.A.F. reviewed the manuscript and supervised the chemical and physical chemical experiments of the manuscript. ACKNOWLEDGMENTS The authors would like to thank Universidade Federal do Paraná (UFPR) for the scientific and technical assistance they provided. The authors would like to thank MSc. Rafaele R. Moreira for survival analysis. FUNDING DECLARATION J.J., M.J.B.G, N.C.S. and G.P.P are grateful for the doctoral scholarships received from CAPES (Finance Code 001). R.A.F. (nº 303312/2019-0) and L.L.M.D.M. (306907/2017-8) are Research Member of the National Research Council of Brazil (CNPq). This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES), coordinated by R.A.F CAPESPRINT 41/2017 (n o 88887.311748/2018-00) and CNPq (nº 400117/2016-9 and 430451/2018-0). DATA AVAILABILITY STATEMENT All data generated or analyzed during this study are included in this published article and it supplementary information files. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1654686","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":106667868,"identity":"282ea6b2-8c38-4c08-820b-4b15ebefa9dc","order_by":0,"name":"Joslaine Jacumazo","email":"","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Joslaine","middleName":"","lastName":"Jacumazo","suffix":""},{"id":106667871,"identity":"42b38aaf-00ce-46ed-b371-ebdb3c251979","order_by":1,"name":"Gabriela Pereira Parchen","email":"","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Pereira","lastName":"Parchen","suffix":""},{"id":106667873,"identity":"c8503d9b-4579-40cb-be56-46050fa6f789","order_by":2,"name":"Meira Janete Ballesteros-Garcia","email":"","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meira","middleName":"Janete","lastName":"Ballesteros-Garcia","suffix":""},{"id":106667874,"identity":"5eaa3374-b536-4be3-bc2b-b6fcbc6fa9f3","order_by":3,"name":"Nayana Cristina da Silva","email":"","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nayana","middleName":"Cristina da","lastName":"Silva","suffix":""},{"id":106667875,"identity":"37c06f7e-379d-4ee0-bcee-93899373bab8","order_by":4,"name":"Louise Larissa May de Mio","email":"","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Louise","middleName":"Larissa May","lastName":"de Mio","suffix":""},{"id":106667876,"identity":"9ab564ef-6c5d-451c-8930-ee4158831a6e","order_by":5,"name":"Francisco de Asssis Marques","email":"","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"de Asssis","lastName":"Marques","suffix":""},{"id":106667877,"identity":"ceeb3ac6-61b0-4728-8af1-d2eeab634921","order_by":6,"name":"Rilton Alves de Freitas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYDACZuYGIGnDwA6kmInUwtgIVJzGwHOAaC0MYC2HSdBi3s7Y/uDjjvOJPdK9D5gL9xChReYwY2PjzDO3E3tkjhswz3hGhBYJoF+aedtuJ+6XSGNgBrmOWC3nEntI1XKARC0zZ7YlG/fIHGM4PIMoLfyHD3z42GYn2yPdxvi4gBgtSJoZGEjTANYyCkbBKBgFowArAADSfDN1190/HAAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Paraná","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rilton","middleName":"Alves","lastName":"de Freitas","suffix":""}],"badges":[],"createdAt":"2022-05-13 22:44:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1654686/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1654686/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21832960,"identity":"42f6fc52-4eda-4723-a855-db712936b96c","added_by":"auto","created_at":"2022-05-24 16:08:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":510413,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/6fc17cfc5b72683ad35850ea.jpg"},{"id":21833536,"identity":"afaa3881-a385-4af3-82e1-20007851be10","added_by":"auto","created_at":"2022-05-24 16:13:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":328509,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/706541b69d6b10bbb6b4c492.jpg"},{"id":21832959,"identity":"bb11f823-a7b0-4579-bd56-28484c3b66d8","added_by":"auto","created_at":"2022-05-24 16:08:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101862,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Average values and standard deviation (n = 6) of contact angle of liquids water, formamide and diiodomethane for nectarines not treated (control) and treated with NCs in the presence of eugenol (Ne-LbL\u003csub\u003e1-2\u003c/sub\u003e NCs) and absence of eugenol (LbL\u003csub\u003e1-2\u003c/sub\u003e NCs); (B) Surface-free energy (𝛾\u003csup\u003etotal\u003c/sup\u003e), dispersive (𝛾\u003csup\u003eD\u003c/sup\u003e) and polar (𝛾\u003csup\u003eP\u003c/sup\u003e) components calculate by the OWRK model; (C) work of adhesion (W\u003csub\u003ea\u003c/sub\u003e) of NCs in the absence and presence of eugenol over nectarines were subscript 0 means the nectarine surface, 1 the film formed with particles LbL\u003csub\u003e1\u003c/sub\u003e and 2 the film formed with particles LbL\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/eb44a5bf594b450d0dfd62d0.jpg"},{"id":21833535,"identity":"c0bd65ef-cbb7-4f69-be92-4807c78a9382","added_by":"auto","created_at":"2022-05-24 16:13:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":167468,"visible":true,"origin":"","legend":"\u003cp\u003eEstimation of Kaplan-Meier curves by survival analysis, indicating the probability over time of nectarines to remain without the occurrence of symptoms of \u003cem\u003eMonilinia fructicola\u003c/em\u003e with different treatments (-) control, (-) LbL\u003csub\u003e1\u003c/sub\u003e NCs, (-) iprodione, (-) aqueous solution of eugenol, (-) Ne-LbL\u003csub\u003e1\u003c/sub\u003e and (-) Ne-LbL\u003csub\u003e2 \u003c/sub\u003eNCs.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/19ba44128023d8fa64ab93f7.jpg"},{"id":21833538,"identity":"72185bfa-5394-43fd-bfd0-0958efc7fba6","added_by":"auto","created_at":"2022-05-24 16:13:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":742796,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/374ece40-4cec-4449-9c83-aa2f71f3a80d.pdf"},{"id":21833537,"identity":"9dbcb726-1ef3-420a-acea-f15e9f86a284","added_by":"auto","created_at":"2022-05-24 16:13:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":546351,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/2d8cb96f-ff0f-44fc-a727-87d9686ad520.pdf"},{"id":21832963,"identity":"3e9c0c4a-3259-4ea2-a7d0-63a180e7676f","added_by":"auto","created_at":"2022-05-24 16:08:40","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":9711584,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationM.fructicola13052022.docx","url":"https://assets-eu.researchsquare.com/files/rs-1654686/v1/dee3461b1cfdc645fc552546.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNatural polymer nanocapsules containing eugenol for nectarine coating to control brown rot caused by \u003cem\u003eMonilinia fructicola\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFruits are an essential part of a healthy diet for humans due to their composition based on vitamins, minerals, and fibers (Slavin \u0026amp; Lloyd, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In 2018, according to the United Nations Food and Agriculture Organization (FAO), they are perishable products, and post-harvest losses are between 30\u0026ndash;50% of total world production. Nectarine (\u003cem\u003ePrunus persica\u003c/em\u003e var. \u003cem\u003enucipersica\u003c/em\u003e) is a popular fruit and highly nutrient, belonging to the Rosaceae Family (Aubert et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The nectarine and peach production in 2019 reached 22.3\u0026nbsp;million tons (FAO, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, due to its typical climacteric properties, the fruit ripens quickly under environmental conditions, and after harvesting, there is an increase in the production of plant hormone ethylene which is responsible for its ripening and, therefore, limits the storage time in the post-harvest (Aubert et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This condition affects the fruit characteristics such as the decrease in firmness, in addition to the loss of color and flavor (Aubert et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lurie \u0026amp; Crisosto, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Xi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, under these situations, infection by microorganisms such as fungi may occur.\u003c/p\u003e \u003cp\u003eBrown rot is one of the main diseases that affect nectarine (Oliveira-Lino et al., 2016). This disease is caused by the fungi \u003cem\u003eMonilinia fructicola\u003c/em\u003e (Wint) Honey. This pathogen is responsible for infecting the plant during the blossom time and also during the fruit grown stage (Oliveira-Lino et al., 2016; Keske, Amorin \u0026amp; Mio, 2011; May-De Mio, Luo \u0026amp; Michailides, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). and it can remain in the fruit latently, showing symptoms only when the environment becomes favorable, for example, in the post-harvest. Chemical control methods are still predominantly using fungicides, reducing the loss to 5\u0026ndash;10% in the post-harvest. However, this microorganism has been selected for resistance to fungicides (Oliveira-Lino et al., 2016; May-De Mio, Luo \u0026amp; Michailides \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Garcia-Benitez \u0026amp; Melgarejo, 2017; Hong et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Pereira et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, in recent years, some alternatives have been proposed to minimize losses in the post-harvest period and increase the fruit\u0026rsquo;s shelf life, among them, the use of low temperature (Zhao et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), coating with polysaccharides (Amon et al., 2015), immersion in hot water (Akgun et al, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and use of essential oils (Amiri et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the essential oils that we can highlight is the clove essential oil, composed mainly of eugenol (45\u0026ndash;90%) (Bakkali et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Khalil et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Eugenol (4-allyl-2-methoxy phenol) is a phenolic compound widely used in the pharmaceutical, cosmetic, and food industries due to its antimicrobial and antioxidant properties (Marchese et al, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Eugenol has also effects to control plant pathogens, presenting a fungitoxic mode of action (Rozwaka et al., 2020). In previous and non-published studies carried out by our research group, eugenol proved to be efficient in controlling conidia germination and mycelium growth of \u003cem\u003eMonilinia fructicola\u003c/em\u003e. However, its application to control agricultural diseases becomes limited because it is volatile and can be degraded by light and oxygen (Turek \u0026amp; Stintzing, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Low solubility in water is another limiting fator (Turek \u0026amp; Stintzing, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To overcome these difficulties, nanoencapsulation has become an interesting and promising alternative.\u003c/p\u003e \u003cp\u003ePolymeric nanocapsules (NCs) can be used as a protective barrier for active substances, decreasing their losses and increasing their efficiencies by promoting a more controlled release (Lima et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). They can be obtained by various methods, including nanoprecipitation, emulsion-diffusion, coacervation, and layer-by-layer (LbL) (Mora-Huertas, Fessi \u0026amp; Elaissari, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rao \u0026amp; Geckeler, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The mechanism of NCs formation by the LbL self-assembly method, from a colloidal template, is based on irreversible ionic interactions that lead to the adsorption of polyelectrolytes. Among the cationic and anionic polyelectrolytes, chitosan and carboxymethylcellulose, respectively, can be mentioned (Mora-Huertas, Fessi \u0026amp; Elaissari, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eChitosan is a linear copolymer of (1\u0026ndash;4) linked 2-acetamido-2-deoxy-β-\u003cem\u003eD\u003c/em\u003e-glucopyranose and 2-amino-2-deoxy-β-\u003cem\u003eD\u003c/em\u003e-glucopyranose, soluble in dilute acidic solutions below pH 6.5 due to the quaternization of the amine groups that have a pK\u003csub\u003ea\u003c/sub\u003e value of 6.3\u0026ndash;6.5 (Sahariah \u0026amp; M\u0026aacute;sson, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang, Qian \u0026amp; Ding, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It has excellent biocompatibility and biodegradability, in addition to low toxicity, making it an ideal alternative for interaction with other polymers and the development of NCs (Wang, Qian \u0026amp; Ding, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Dash et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A polymer of opposite charge, biodegradable and non-toxic that can interact ionically with chitosan is carboxymethylcellulose (Zhang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), a molecule formed by the partial carboxymethylation of β-\u003cem\u003eD\u003c/em\u003e-glucopyranose cellulose. The anionic character is due to the formation of carboxylate groups through the deprotonation that occurs at pH above its pK\u003csub\u003ea\u003c/sub\u003e (2.0\u0026ndash;4.0) (Heinze \u0026amp; Koschella et al., 2005).\u003c/p\u003e \u003cp\u003eIn this sense, this work aimed to use the LbL technique to prepare NCs containing eugenol using polysaccharides derived from renewable sources such as chitosan and carboxymethylcellulose, and to develop a modified release fruit coating system to evaluate the control of pathogenic fungi. Our hypothesis is that coating fruit, such as nectarine, using eugenol nanocapsules coated with biopolymers, could be a new strategy to reduce the incidence of brown rot caused by \u003cem\u003eMonilinia fructicola\u003c/em\u003e. This strategy offers new perspectives to increase fruit quality and reduce post-harvest losses.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eChitosan (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eNO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003en\u003c/sub\u003e was purchased from Shangyu Biotech Co, Ltda (Shangyu, China) and submitted to a purification procedure before use as described in previous work (Jacumazo et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The chitosan weight average molar mass (M\u003csub\u003ew\u003c/sub\u003e) was 1.9x10\u003csup\u003e5\u003c/sup\u003e g moL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and deacetylation degree of 76%. Sodium dodecyl sulphate (99.0 wt% purity), eugenol (99.0% V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e purity), formamide (99.5% V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e purity) and 2,2-diphenyl-1-picryl-hydrazil (DPPH) were purchased from Sigma-Aldrich (St. Louis, USA). Diiodomethane (99.0% V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e purity) was purchased from Neon (Suzano, Brazil). Carboxymethylcellulose of M\u003csub\u003ew\u003c/sub\u003e of 6.7x10\u003csup\u003e5\u003c/sup\u003e g moL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and DS 0.84 was purchased from Acros Organics (Geel, Belgium). All solutions were prepared with purified water obtained by a reverse osmose system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Nanoemulsion of eugenol\u003c/h2\u003e \u003cp\u003eThe nanoemulsions of eugenol (Ne) were obtained from the addition of eugenol in a sodium dodecyl sulfate solution (5.0 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the ratio eugenol:sodium dodecyl sulfate was 0.78:1 wt. V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This mixture was sonicated (Ultrasonics Sonicator, USA) at 30% amplitude with a nominal power of 750 W and frequency of 20 kHz. Then, the sonication time was varied from 30, 120, 240, 360, 480 to 600 s at 0 \u0026ordm;C. At the end of the sonication process, the Ne were left under magnetic stirring at 900 rpm (Magnital Agitator MAG15, Marte Scientific, Brazil) for 15 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Layer-by-Layer of eugenol-loaded nanocapsules\u003c/h2\u003e \u003cp\u003eThe LbL technique was used for the self-assembly of polymer layers. Polymeric dispersions of chitosan (2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), carboxymethylcellulose (2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and Ne were performed in sodium acetate buffer (0.01 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, pH 4.6). The self-assembly step was performed in two different situations: condition 1, the polymer dispersion was added into the Ne; and condition 2, the Ne was added into the polymer dispersion. From the Ne, the NCs were formed by alternating deposition of the polymer: cationic (Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs) and anionic (Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs). After each polymer deposition, the nanocapsules were centrifuged (5 x 10\u003csup\u003e4\u003c/sup\u003e g, 30 min) at 25 \u0026ordm;C, washed with water, and kept under continuous magnetic stirring at 900 rpm, 25\u0026deg;C for 1 h after resuspension.\u003c/p\u003e \u003cp\u003eUsing the same protocol, free eugenol particles were produced as controls for antimicrobial assays and named LbL\u003csub\u003e1\u003c/sub\u003e NCs and LbL\u003csub\u003e2\u003c/sub\u003e NCs.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Eugenol-loaded nanocapsules characterization\u003c/h2\u003e \u003cp\u003eThe average apparent hydrodynamic diameter (D\u003csub\u003eapph\u003c/sub\u003e) was determined using a dynamic light scattering (DLS) on a NANO DLS Particle Size Analyzer apparatus of Brookhaven Instruments (Holtsville, New York, USA) in water at 20 \u0026ordm;C. All experiments were conducted using a 15 mW solid-state He\u0026ndash;Ne laser, operating at 90\u0026deg; and wavelength at 632.8 nm. The samples containing the NCs were diluted in water 1:50 (V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eFor zeta potential (ζ-potential) analyses were carried out in a Particle Charge Mapping Stabino apparatus (Germany). The samples were diluted in water 1:50 (V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and analyzed for 100 s at 20 \u0026ordm;C. A 10 mL measuring cell coupled to a 400 \u0026micro;m piston was used.\u003c/p\u003e \u003cp\u003eThermogravimetric analysis (TGA) of eugenol (6.1 mg), chitosan and carboxymethylcellulose mixture (5.2 mg), Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs (4.9 mg), Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs (4.6 mg) were performed using 0.065 mL alumina crucibles with a Netzsch analyzer (STA 449 F3 series EP), following a heating rate at 10\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from 25 to 600 \u0026ordm;C under nitrogen atmosphere of 50 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The NCs with eugenol were previously lyophilized at -50 \u0026ordm;C (MicroModulyo, Thermo Electron Corporation, USA) prior to the TG analysis.\u003c/p\u003e \u003cp\u003eThe antioxidant activity of eugenol, intending to confirm the release of eugenol from NCs, was evaluated using DPPH radical tests (Thaipong et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For more details, please access the Supporting Information.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Incorporation efficiency\u003c/h2\u003e \u003cp\u003eThe incorporation efficiency (IE%) of eugenol was determined according to the method described by Jacumazo et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The solubility of eugenol in water and chloroform was determined using spectroscopy in the UV-vis region (281 nm) and the values were 2.41 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e e 8.18 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The samples (2 mL) were centrifuged (4.000 \u003cem\u003eg\u003c/em\u003e, 15 min) at 5 \u0026ordm;C. The supernatant was removed, and chloroform (2 mL) was added to the sedimented material, mixed, and centrifuged (4.000 \u003cem\u003eg\u003c/em\u003e, 20 min) at 25 \u0026ordm;C. The supernatant was collected, and the absorbance was measured at 281 nm using UV\u0026ndash;vis spectrophotometry. The eugenol IE% was estimated following Eq.\u0026nbsp;1. The experiments were run in triplicate.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{IE(\\%)=}\\left(\\frac{{\\text{NCs}}_{\\text{Eugenol}}}{{\\text{Initial}}_{\\text{Eugenol}}}\\right)\\text{x 100}\\)\u003c/span\u003e \u003c/span\u003e (\u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e)\u003c/p\u003e \u003cp\u003ewhere NCs\u003csub\u003eEugenol\u003c/sub\u003e is the mass of eugenol determined in the NCs and Initial\u003csub\u003eEugenol\u003c/sub\u003e is the mass of eugenol added in the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eIn vitro\u003c/em\u003e eugenol release from nanocapsules\u003c/h2\u003e \u003cp\u003eThe eugenol release experiments were measured using a dialyzes procedure. Samples were added in a cellulose dialysis bag (cut-off of 12 kg mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, D0530-100FT, Sigma-Aldrich, Germany, USA) and then placed in the receptor system containing water. The saturation concentration of eugenol in water was 69.8 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and all the experiments were maintained under sink conditions. The experiment was performed at pH 6.8, 25 \u0026ordm;C, and continuous magnetic stirring (900 rpm) for 96 h. At defined time intervals (0, 0.25, 0.5, 1, 2, 3, 4, 5, 24, 48, 72, and 96 h), an aliquot of 2 mL of the receptor medium was collected and analyzed using a UV-vis spectrophotometer in the wavelength of 281 nm. The medium receptor was immediately replenished with equal volumes of water. The experiments were run in triplicate. The eugenol quantification was estimated using an equation of the analytical curve, with R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.998, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(Eugenol mg {L}^{-1}=\\left(\\frac{absorbance-0.011}{0.015 L {mg}^{-1}}\\right)\\)\u003c/span\u003e\u003c/span\u003e. The limit of detection and quantification was 0.40 mg L\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 1.34 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Nectarine coating from nanocapsules\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Preparation\u003c/h2\u003e \u003cp\u003eThe nectarine coating was carried out with the Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs and Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs, and nectarine without coating was used for the control. The nectarines were placed in contact with the dispersions (Ne-LbL\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e NCs) or distilled water (control) for 30 s and then dried at 25 \u0026ordm;C for 24 h. After drying, the nectarine peel was withdrawn, and the contact angle analysis was performed in duplicate with six drops per sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Characterization: contact angle, surface free energy, and adhesion work\u003c/h2\u003e \u003cp\u003eThe contact angle measurements were performed using three liquids: water, formamide, and diiodomethane, and measured at 20 \u0026ordm;C using a DATAPHYSICS Instruments GmbH Contact Angle System OCA15\u0026thinsp;+\u0026thinsp;tensiometer (Germany). The contact angle at the nectarine surface was measured by the sessile drop method, 5 \u0026micro;L of each liquid was dripped on the samples. The right and left side of the drop was measured to average the contact angle. The contact angles were automatically calculated by fitting the captured drop shape (software SCA20).\u003c/p\u003e \u003cp\u003eThe calculation of the surface energy of the nectarine coating was based on the method described by Owens and Wendt (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1969\u003c/span\u003e) and Kaelble (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1970\u003c/span\u003e), using the Owens, Wendt, Rabel, and Kaelble (OWRK) method to calculate the components of the solid surface energy. The calculation was performed using Eq.\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\frac{{\\gamma }_{L}(cos{\\theta }_{c}+1)}{2\\left(\\sqrt{{\\gamma }_{L}^{D}}\\right)}= \\sqrt{{\\gamma }_{S}^{P}}\\left(\\frac{\\sqrt{{\\gamma }_{L}^{P}}}{\\sqrt{{\\gamma }_{L}^{D}}}\\right)+ \\sqrt{{\\gamma }_{S}^{D}}\\)\u003c/span\u003e \u003c/span\u003e (\u003cb\u003eEq.\u0026nbsp;2)\u003c/b\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }_{c}\\)\u003c/span\u003e\u003c/span\u003e is the experimental contact angle, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }_{L}\\)\u003c/span\u003e\u003c/span\u003e is the total liquid surface tension, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }^{D}\\)\u003c/span\u003e\u003c/span\u003e is the dispersive component, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }^{P}\\)\u003c/span\u003e\u003c/span\u003e is the polar component. The subscripts S and L stand for solid and liquid, respectively.\u003c/p\u003e \u003cp\u003eThe NCs work of adhesion of LbL\u003csub\u003e1\u003c/sub\u003e NCs and Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs with the nectarine surface, as well as the LbL\u003csub\u003e2\u003c/sub\u003e NCs with the chitosan layer (LbL\u003csub\u003e1\u003c/sub\u003e), was calculated using Equations 3, 4 and 5.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({W}_{a}= {{W}_{a}}^{P}+ {{W}_{a}}^{D}\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003e(Eq.\u0026nbsp;3)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({{W}_{a}}^{P}=2\\sqrt{{\\gamma }_{x}^{P}{\\gamma }_{y}^{P}}\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003e(Eq.\u0026nbsp;4)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({{W}_{a}}^{D}=2\\sqrt{{\\gamma }_{x}^{D}{\\gamma }_{y}^{D}}\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003e(Eq.\u0026nbsp;5)\u003c/b\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({W}_{a} x,y\\)\u003c/span\u003e\u003c/span\u003e is the total work of adhesion between layers x and y, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }_{x} and {\\gamma }_{y}\\)\u003c/span\u003e\u003c/span\u003e are the free energy of the two different interfaces.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Microbial assays for brown rot control\u003c/h2\u003e \u003cp\u003eThe fungus, isolated PpMfSP15/575 from \u003cem\u003eMonilinia fructicola\u003c/em\u003e, belongs to the LEMID-UFPR collection. The conidial suspension was prepared to collect the spores on the surface of pre-inoculated canned peach and disperse into a sterile Tween 20 (0.5% V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) aqueous solution.\u003c/p\u003e \u003cp\u003eNectarines (\u003cem\u003ePrunus persica\u003c/em\u003e var. \u003cem\u003enucipersica\u003c/em\u003e) of Sungold cultivar (Santa Catarina \u0026ndash; Brazil) were used in the experiment. These fruits were previously sanitized by immersion (1 min) in ethanol/water (70% V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), an aqueous solution of sodium hypochlorite (1% V V\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and distilled water (3 x) and then let dry at ambient temperature (25\u0026deg;C).\u003c/p\u003e \u003cp\u003eThe nectarine fruits were treated with sterile water (control), an aqueous solution of eugenol using dimethyl sulfoxide and Adivex\u0026reg; as co-solvents (formulation control), NCs without eugenol, Ne-LbL\u003csub\u003e1\u0026minus;\u0026thinsp;2\u003c/sub\u003e NCs, and fungicide (iprodione \u0026ndash; Rovral SC\u0026reg; 500 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Basf, France) as the positive control, efficient to control brown rot (Kaelble, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). The nectarines were dripped in each treatment for 30 s and dried for 24 h. Then, each nectarine was inoculated with 40 \u0026micro;L of the \u003cem\u003eMonilinia fructicola\u003c/em\u003e conidial suspension (10\u003csup\u003e5\u003c/sup\u003e conidial mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the samples were placed in the humid chamber. Seven repetitions were performed for each treatment and symptoms evaluations were performed every 24 h for 7 days. The experiment was conducted twice in a completely randomized design.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Data analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed with GraphPad Prism 8 software, using a one-way analysis of variance (ANOVA), followed by Tuckey\u0026rsquo;s Post Hoc test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Survival analysis was estimated by the Kaplan-Meier method and the comparison of curves between treatments was estimated by the non-parametric log-rank test. In addition, Cox's proportional hazards model was also used for all tested samples. For all statistical inferences, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a nominal significance level. The statistical software R (version 3.4.3) was used for data analysis and graphical representation.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. Layer-by-Layer of eugenol-loaded nanocapsules\u003c/h2\u003e\n\u003cp\u003eThe Ne formation is an important step to form nanocapsules, since it was used as a template for the LbL process (Abbas et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. The mean value of D\u003csub\u003eapph\u003c/sub\u003e 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Between 30 s (PDI\u0026thinsp;=\u0026thinsp;0.494) and 600 s (PDI\u0026thinsp;=\u0026thinsp;0.230), there is a considerable difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the dispersion due to the greater amount of energy supplied to the system Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. The PDI\u0026thinsp;\u0026lt;\u0026thinsp;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.\u003c/p\u003e\n\u003cp\u003eAfter 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., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). 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.\u003c/p\u003e\n\u003cp\u003eIn condition C1, the dispersion of chitosan has a positive charge in acid medium (pK\u003csub\u003ea\u003c/sub\u003e \u0026lt; 6.5), and was added over the Ne, making the dispersion immediately milky-like (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC - C1). For this situation, the D\u003csub\u003eapph\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) and the PDI were 368\u0026thinsp;\u0026plusmn;\u0026thinsp;113 nm and 0.546, respectively. The higher PDI value indicated a non-uniform formation of NCs.\u003c/p\u003e\n\u003cp\u003eFor C2, the Ne was added to chitosan dispersion, and the original light-yellow dispersion color gradually became light-milky (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC \u0026ndash; C2). In this situation, the droplet coating with chitosan molecules occurs immediately, and the D\u003csub\u003eapph\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) and PDI were 360\u0026thinsp;\u0026plusmn;\u0026thinsp;30 nm and 0.390, respectively, lower than C1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The size and PDI variation were monitored for the other self-assembly layers. However, no significant differences were observed in D\u003csub\u003eapph\u003c/sub\u003e and PDI for adding carboxymethylcellulose (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and the values obtained were D\u003csub\u003eapph\u003c/sub\u003e: 473\u0026thinsp;\u0026plusmn;\u0026thinsp;164 nm and PDI: 0.440 for C1 and D\u003csub\u003eapph\u003c/sub\u003e: 398\u0026thinsp;\u0026plusmn;\u0026thinsp;106 nm and PDI: 0.396 for C2.\u003c/p\u003e\n\u003cp\u003eIn addition to the NCs size, stability by \u0026zeta;-potential was observed after each coating for C1 and C2. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2. Incorporation efficiency and \u003cem\u003ein vitro\u003c/em\u003e eugenol release from nanocapsules\u003c/h2\u003e\n\u003cp\u003eThe IE (%) of eugenol was determined as 8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% for Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs and 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% for Ne-LbL\u003csub\u003e2\u003c/sub\u003e 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 \u003cstrong\u003eFig. S1\u003c/strong\u003e and \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe eugenol release profile was investigated to understand the mechanism of release from NCs. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e was possible to observe for free eugenol a rapid release (burst release) in the first 15 min (34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7%), reaching to 100% ~ 4 h. In contrast, eugenol encapsulated in Ne-LbL\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e the rapid release phase occurs up to 5 h, followed by a slow and gradual release.\u003c/p\u003e\n\u003cp\u003eThe decrease in the release, comparing Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs with Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs was directly related to the number of polymer layers added as observed in previous studies (Jacumazo et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). A first-order equation was used to adjust the release of eugenol. In Eq.\u0026nbsp;6, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{m}}_{\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e represents the fraction released at time t, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{m}}_{{\\infty }}\\)\u003c/span\u003e\u003c/span\u003e is the amount of the active in the formulation, and \u003cem\u003ek\u003c/em\u003e is the first-order constant.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{ln}\\left(\\frac{{m}_{t}}{{m}_{\\infty }}\\right)=kt\\)\u003c/span\u003e \u003c/span\u003e \u003cstrong\u003e(Eq.\u0026nbsp;6)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe values of \u003cem\u003ek\u003c/em\u003e using a first-order model for free eugenol, Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs and Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs were 0.057, 0.033, and 0.031 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 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 (\u003cstrong\u003eFig. S2\u003c/strong\u003e), that Ne-LbL\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e NCs make eugenol release and DPPH inhibition slower.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Surface properties, contact angle, surface free energy, and work of adhesion\u003c/h2\u003e\n\u003cp\u003eNCs 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 \u003cstrong\u003eFig. S3\u003c/strong\u003e. 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.\u003c/p\u003e\n\u003cp\u003eTo obtain more information about these coatings, contact angle measurements of untreated nectarines (control), treated with NCs in the presence of eugenol (Ne-LbL\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e NCs) and NCs in the absence of eugenol (LbL\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e NCs), were performed. The contact angles were obtained using three liquids of different polarities (water, formamide, and diiodomethane) as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA.\u003c/p\u003e\n\u003cp\u003eWhen 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 \u0026amp; Algecira, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eOn the other hand, the contact angles of the samples treated with the NCs decreased, compared to the control, increasing the wettability (p\u0026thinsp;\u0026lt;\u0026thinsp;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 \u003cstrong\u003eFig S2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWith the obtained contact angle values for all liquids, it was possible to calculate the values of total surface-free energy (𝛾\u003csup\u003etotal\u003c/sup\u003e), dispersive and polar components using the OWRK model. The total surface tension (𝛾\u003csup\u003etotal\u003c/sup\u003e), dispersive (𝛾\u003csup\u003eD\u003c/sup\u003e), and polar (𝛾\u003csup\u003eP\u003c/sup\u003e) components values for the three liquids used can be seen in \u003cstrong\u003eTable S2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe control sample, nectarine peel treated with water, had a lower surface free energy value (26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), on the other hand, the nectarines with Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs coating showed a higher total surface-free energy value (36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). Comparatively, the control sample has a less polar surface than LbL\u003csub\u003e1\u003c/sub\u003e and LbL\u003csub\u003e2\u003c/sub\u003e and the values of total surface-free energy were different for the untreated and treated samples with Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eIn the nectarines coated with chitosan with Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs and LbL\u003csub\u003e1\u003c/sub\u003e NCs, values of 18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e were observed respectively for the dispersive components and 17.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for the polar components, where the dispersive and polar components do not differ (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After coating with LbL\u003csub\u003e1\u003c/sub\u003e, the surface polarity increased, however maintaining equivalent dispersive composition (Fern\u0026aacute;ndez et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe nectarines coated with the anionic polymer, Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs, and LbL\u003csub\u003e2\u003c/sub\u003e NCs, the values of 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 and 17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e were observed respectively for the dispersive components and 4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 and 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 mJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for the polar components, and the dispersive and polar components do not differ (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs have a larger dispersive component, possibly due to the strong ionic interaction between chitosan and carboxymethylcellulose and exposing less polar sites of cellulose.\u003c/p\u003e\n\u003cp\u003eWith the calculated data of total surface-free energy and dispersive and polar components, it was possible to obtain the coating work of adhesion (W\u003csub\u003ea\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). As observed, the W\u003csub\u003ea\u003c/sub\u003e determined to LbL\u003csub\u003e1\u003c/sub\u003e on nectarines (W\u003csub\u003ea\u003c/sub\u003e 0,1), or for LbL\u003csub\u003e2\u003c/sub\u003e on LbL\u003csub\u003e1\u003c/sub\u003e (W\u003csub\u003ea\u003c/sub\u003e 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 W\u003csub\u003ea\u003c/sub\u003e (W\u003csub\u003ea\u003c/sub\u003e 0,1 or W\u003csub\u003ea\u003c/sub\u003e 0,2). However, the LbL\u003csub\u003e1\u003c/sub\u003e of chitosan turns the surface much more polar than LbL\u003csub\u003e2\u003c/sub\u003e, and this could promote interesting biological properties.\u003c/p\u003e\n\u003cp\u003eAccording to Vel\u0026aacute;squez, Skurtys, Enrione, \u0026amp; Osorio (\u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e), 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. Effect of nanocapsules on the brown rot\u003c/h2\u003e\n\u003cp\u003eIn the present study, antimicrobial activity was evaluated using NCs with up to two layers of polymers containing eugenol (Ne-LbL\u003csub\u003e1-2\u003c/sub\u003e NCs), aqueous solution of eugenol, NCs in absence of eugenol (LbL\u003csub\u003e1\u003c/sub\u003e NCs) and aqueous solution of the commonly used fungicide iprodione for the control of brown rot (Dutra, Pereira, May de Mio et al., 2019).\u003c/p\u003e\n\u003cp\u003eIn this sense, the estimate of the relative risk for the expression of symptoms of \u003cem\u003eMonilinia fructicola\u003c/em\u003e was analyzed using the Cox semiparametric model (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) 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.\u003c/p\u003e\n\u003cp\u003eThe survival analysis of the healthy fruits is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e 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, LbL\u003csub\u003e1\u003c/sub\u003e NCs and aqueous solution of eugenol expressed disease symptoms more rapidly than those treated with Ne-LbL\u003csub\u003e1-2\u003c/sub\u003e 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.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEstimates of relative risk for the expression of symptoms of \u003cem\u003eMonilinia fructicola\u003c/em\u003e estimated by the Cox semi-parametric model, followed by 95% confidence intervals for nectarine\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTreatment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIncubation\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eRelative risk\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCI (95%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eperiod (days)\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eLL\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eUS\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLbL\u003csub\u003e1\u003c/sub\u003e NCs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.7218\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.0194\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIprodione\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.9272\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.6724\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.2786\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEugenol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5651\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3918\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.8150\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNe-LbL\u003csub\u003e1\u003c/sub\u003e NCs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.2351\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.1480\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3736\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNe-LbL\u003csub\u003e2\u003c/sub\u003e NCs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4959\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.3423\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.7184\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eCI \u0026ndash; confidence interval (95%), LL: lower limits, US: upper limit.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003e* 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).\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, 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-LbL\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;NCs to nectarines, as shown in the work of adhesion data. In this sense, although treatments with LbL\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;NCs, NCs with the outer layer of carboxymethylcellulose (Ne-LbL\u003csub\u003e1-2\u003c/sub\u003e\u0026nbsp;NCs) and aqueous solution of eugenol enable the efficient control of the pathogen\u0026nbsp;\u003cem\u003eMonilinia fructicola\u003c/em\u003e\u0026nbsp;when compared to treatment with iprodione, only the fruits treated with NCs with the outer layer of chitosan (Ne-LbL\u003csub\u003e1\u003c/sub\u003e\u0026nbsp;NCs) showed above 70% probability that the fruits remain in the absence of symptoms until the seventh day of the study.\u003c/p\u003e\n\u003cp\u003eThe images of the experiments containing all treatments during the 7 days can be seen in \u003cstrong\u003eFig. S4\u003c/strong\u003e, as some fruits showed more accentuated symptoms, they were removed from the experiment to minimize possible contamination in other fruits.\u003c/p\u003e\n\u003cp\u003eThe 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 \u0026amp; Stintzing, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). 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-LbL\u003csub\u003e1\u003c/sub\u003e NCs had the best fungicidal activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eIn 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 \u0026amp; Abdou et al., 2013).\u003c/p\u003e\n\u003cp\u003eAnother factor that may be associated with the better performance of chitosan-coated NCs is their positive surface charge (\u0026zeta;-potential: 32\u0026thinsp;\u0026plusmn;\u0026thinsp;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., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn 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-LbL\u003csub\u003e1\u003c/sub\u003e) promoted better adherence and showed the best antimicrobial control. However, the Ne-LbL\u003csub\u003e2\u003c/sub\u003e 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 \u003cem\u003eMonilinia fructicola\u003c/em\u003e, to increase fruit storage or shelf life, in addition to using much less toxic natural substances as fungicides.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"ABBREVIATIONS AND NOMENCLATURE","content":"\u003cp\u003eUnited Nations Food and Agriculture Organization (FAO); nanocapsules (NCs); layer-by-layer (LbL); nanoemulsions of eugenol (Ne); NCs with deposition of the cationic polymer containing eugenol (Ne-LbL\u003csub\u003e1\u003c/sub\u003e NCs); NCs with deposition of the anionic polymer containing eugenol (Ne-LbL\u003csub\u003e2\u003c/sub\u003e NCs); NCs with deposition of the cationic polymer (LbL\u003csub\u003e1\u003c/sub\u003e NCs), NCs with deposition of the anionic polymer (NCs with deposition of the anionic polymer), apparent hydrodynamic diameter (D\u003csub\u003eapph\u003c/sub\u003e); dynamic light scattering (DLS); zeta potential (\u0026zeta;-potential); thermogravimetric analysis (TGA); incorporation efficiency (IE%); work of adhesion (Wa), polydispersity index (PDI), confidence interval (CI)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR\u0026rsquo;S CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.J., M.J.B.G, N.C.S. and G.P.P performed the experiments and wrote the main manuscript;\u003c/p\u003e\n\u003cp\u003eF.A.M. reviewed the manuscript and supervised the chemical characterization of eugenol;\u003c/p\u003e\n\u003cp\u003eL.L.M.D.M. reviewed the manuscript and supervised the nectarine experiments with \u003cem\u003eMonilinia fructicola;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eR.A.F. reviewed the manuscript and supervised the chemical and physical chemical experiments of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Universidade Federal do Paran\u0026aacute; (UFPR) for the scientific and technical assistance they provided. The authors would like to thank MSc. Rafaele R. Moreira for survival analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING DECLARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.J., M.J.B.G, N.C.S. and G.P.P are grateful for the doctoral scholarships received from CAPES (Finance Code 001). R.A.F. (n\u0026ordm; 303312/2019-0) and L.L.M.D.M. (306907/2017-8) are Research Member of the National Research Council of Brazil (CNPq). This study was financed by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES), coordinated by R.A.F CAPESPRINT 41/2017 (n\u003csup\u003eo\u003c/sup\u003e 88887.311748/2018-00) and CNPq (n\u0026ordm; 400117/2016-9 and 430451/2018-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and it supplementary information files. However, extra information, data set, and raw data generated and/or analyzed during the current study are available from the corresponding authors of reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLIC OF INTEREST STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSlavin, J. \u0026amp; Lloyd, B. (2012). 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Biomater.\u003c/em\u003e, \u003cem\u003e2012\u003c/em\u003e, 1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2012/632698\u003c/span\u003e\u003cspan address=\"10.1155/2012/632698\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"natural polymers, layer-by-layer, adherence, eugenol, Monilinia fructicola, disease control","lastPublishedDoi":"10.21203/rs.3.rs-1654686/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1654686/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe fruit infection by fungi reduces the quantity and quality of food for human consumption and causes economic damage. Thus, this study aimed to address the effects of nanocapsules (NCs) based on chitosan and carboxymethylcellulose containing eugenol in protecting nectarines against \u003cem\u003eMonilinia fructicola\u003c/em\u003e, a brown rot agent, a worldwide important disease. NCs were prepared by Layer-by-Layer self-assembly starting from an anionic template and deposition with up to two polymeric layers. The sizes ranged from 158 nm (nanoemulsion) to 398 nm (two polymeric layers). In vitro release showed an initial burst followed by a gradual release. In addition to showing a good adherence to nectarine surface, the NCs coated with chitosan proved to be the best formulation for the control of brown rot in the antifungal test. Therefore, this study demonstrated that chitosan NCs containing eugenol could be an alternative to preserve fruit for longer periods in the post-harvest.\u003c/p\u003e","manuscriptTitle":"Natural polymer nanocapsules containing eugenol for nectarine coating to control brown rot caused by Monilinia fructicola","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-24 16:08:36","doi":"10.21203/rs.3.rs-1654686/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"34edb4c9-da85-4faa-bf10-2a720e8829dd","owner":[],"postedDate":"May 24th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-30T07:14:10+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-24 16:08:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1654686","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1654686","identity":"rs-1654686","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}