Effect of ultrasound-assisted emulsification on cinnamon essential oil dispersion: a strategy to improve biopolymer-based active films for sustainable food packaging

This study investigated how the emulsification method of cinnamon essential oil (CEO) influences its dispersion characteristics and, consequently, the physicochemical and biological properties of active films based on sodium alginate and carboxymethylcellulose. Two systems were evaluated: a pre-emulsion obtained by Ultra-Turrax and an ultrasound-assisted emulsion produced by combining Ultra-Turrax and sonication. Although both dispersions presented droplet sizes within the nanometric range, the ultrasound-treated system exhibited significantly lower polydispersity index (0.2 ± 0.0) and smaller droplet size (40.2 ± 3.66 nm) compared to the pre-emulsion (67.9 ± 4.2 nm and 0.9 ± 0.0, respectively), indicating improved dispersion uniformity. This enhanced homogeneity resulted in films with a more compact and uniform microstructure, as well as improved thermal stability (endothermic peak at 175.2 °C ± 4.7 °C). In addition, films containing the ultrasound-assisted emulsion exhibited greater flexibility and extensibility, with higher elongation at break (4.88% ± 0.48%) and lower tensile strength (30.64 ± 0.77 MPa), suggesting a more effective plasticizing effect. CEO incorporation also provided UV-blocking capacity and reduced WVP regardless of the emulsification method. Both active films inhibited the growth of Colletotrichum gloeosporioides in the contact area, with no significant differences between systems. These results indicate that, under the evaluated conditions, dispersion uniformity, rather than nominal droplet size classification, plays a key role in governing film structure and performance, highlighting the relevance of ultrasound-assisted emulsification for improving functional properties of active biopolymer films.

Food packaging is essential in the supply chain, protecting products from processing to transportation. However, conventional materials such as plastic and paper have environmental limitations. For example, petroleum-based plastics are single-use and pollute oceans and landfills, in addition to contributing to greenhouse gas emissions and global warming (Kumar et al., 2021; Sundqvist-Andberg and Åkerman, 2021). In response to environmental concerns and changing consumer behavior, there is a movement toward sustainable packaging materials, such as those made with biodegradable materials. Such packaging can extend the shelf life of food, especially when it contains active ingredients (Mohamed et al., 2020; de Oliveira Júnior et al., 2023).

Films are thin layers, usually up to 0.3 mm thick, that can cover the surface of the food, functioning as primary packaging (Ribeiro et al., 2021). Polysaccharides such as sodium alginate and carboxymethylcellulose have been highlighted in the production of films. Extracted from brown algae, sodium alginate (SA) is a polymer formed by two base monomers, β-D-mannuronyl and α-L-guluronyl. Carboxymethylcellulose (CMC), which is a polymer derived from cellulose, has high mechanical strength, adjustable hydrophilicity, good viscosity and is biocompatible (Kocira et al., 2021). These polysaccharides can be used as a base to form solutions and, after drying, usually by the casting method, can form films (Han, 2014; Khan et al., 2024b; Ullah et al., 2025).

In recent years, essential oils have been widely explored as natural antimicrobials and can be incorporated into polymeric solutions to form active films (de Oliveira et al., 2023). Cinnamon essential oil (CEO), for example, is considered a natural preservative due to its antioxidant and antimicrobial properties. The main active component of CEO is cinnamaldehyde, a phenolic compound capable of neutralizing free radicals and slowing the growth of microorganisms, thus contributing to food preservation (Han et al., 2018). However, the direct incorporation of CEO into film-forming solutions containing biopolymers and water is a challenge, since essential oils are highly volatile and insoluble, causing phase separation during film preparation (Bolouri et al., 2022). Furthermore, essential oils, when incorporated directly into polymeric solutions, can cause color and flavor changes in foods, and their bioactive compounds can undergo degradation when exposed to environmental factors (temperature, pH, oxygen, light, and moisture) (Atarés and Chiralt, 2016). Thus, the production of nanoemulsions has been suggested as an alternative to enable the incorporation of essential oils into polymeric solutions to form films.

Nanoemulsions are systems in which two immiscible liquids are homogenized to form droplets measuring 20–200 nm in size (Zhang et al., 2022). In this system, emulsion is more resistant to phase separation compared to conventional emulsions (Lago et al., 2019). Furthermore, the reduction in droplet size implies an increase in the contact surface area between the active agents and the food, improving the biological capacity of essential oils, which allows the use of smaller doses (Kumar et al., 2021). Nanoemulsions are obtained by high or low energy methods. Among high energy methods, ultrasound and that obtained by rotor-stator devices. Emulsification obtained with a rotor-stator device is popular and generally forms coarse emulsions. Emulsification obtained by ultrasound has been successfully applied to obtain finer and more uniform emulsions (Espitia et al., 2019; Mushtaq et al., 2023; Khan et al., 2024a).

Although these methods are often used interchangeably to produce emulsions in the nanometric range, differences in droplet size distribution and dispersion uniformity may lead to distinct interactions within polymeric matrices. However, it remains unclear whether improvements achieved at the emulsion level, such as droplet size reduction via ultrasound, effectively translate into functional advantages in film-based systems.

Film properties are strongly governed by polymer–polymer interactions and by how active compounds are dispersed within the matrix, which directly affects structural, physicochemical, and biological performance. This study is part of a broader research effort in which CEO emulsions have been previously developed and characterized, including their physicochemical stability and biological properties (de Oliveira et al., 2024; Oliveira et al., 2025). Building on these findings, the present work focuses on evaluating the impact of CEO incorporation into film-forming systems in different dispersion forms (pre-emulsion obtained by Ultra-Turrax and nanoemulsion produced by Ultra-Turrax combined with ultrasound). Thus, this study investigates whether differences in CEO dispersion translate into measurable changes in the structural, functional, and antifungal performance of SA/CMC films, with a view toward their application as active packaging materials.

Material

CEO (Cinnamomum cassia) was purchased from Ferquima Indústria e Comércio Ltda (Vargem Grande Paulista, SP, Brazil). Sodium alginate, Tween 80, dimethyl sulfoxide (DMSO), and calcium chloride were obtained from Sigma-Aldrich Brasil Ltda (São Paulo, SP, Brazil). Sodium carboxymethylcellulose was acquired from Êxodo Científica (Sumaré, Brazil), and glycerol from Neon Comercial (Suzano, Brazil). The synthetic fungicide Graduate A+ was kindly provided by Syngenta Proteção de Cultivos Ltda (São Paulo, Brazil). The fungal isolate Colletotrichum gloeosporioides used in this study was obtained from typical anthracnose lesions on mango fruits and confirmed via Koch’s postulates. Fungal identification was performed by extracting genomic DNA from the mycelium, followed by amplification of the internal transcribed spacer (ITS) regions of the rDNA using the standard primer set ITS4 (5′-ATT​AAC​CCT​CAC​TAA​AGT​CCT​CCG​CTT​ATT​GAT​ATG​C-3′) and ITS5 (5′-TAA​TAC​GAC​TCA​CTA​TAG​GGG​GAA​GTA​GAA​GTC​GTA​ACA​AGG-3′) (Bruns et al., 2014). Preliminary identification was conducted using the online platform “UNITE–rDNA ITS-based identification of Eukaryotes and their communication via DOIs” (Kõljalg et al., 2020). The isolates were cultured on potato dextrose agar (PDA; HiMedia, Mumbai, India) at 25 °C ± 0.5 °C for 7 days.

Film preparation

SA and CMC were separately dissolved in a water bath at 50 °C and homogenized using an Ultra-Turrax T25 homogenizer (IKA-Werke GmbH & Co., Germany) at 10,000 rpm for 5 min. The final film-forming solution contained SA and CMC at a ratio of 2:1 (w/v). A calcium chloride solution was added to the SA solution at 0.75% (w/w) relative to the SA mass. The SA and CMC solutions were subsequently combined and homogenized again under the same conditions. Glycerol was incorporated as a plasticizer at 20% (w/w) relative to the total polymer mass, followed by homogenization using the Ultra-Turrax T25 under the same conditions.

The polymeric film-forming solution was prepared based on a previous study conducted by our research group (de Oliveira et al., 2024; Oliveira et al., 2025). The proportions of CEO during formulation were carefully controlled to ensure that the final film-forming solution presented a concentration of 0.25% (w/v). CEO and Tween 80 (1:1 w/w) were dispersed in distilled water and homogenized at 10,000 rpm for 5 min using the Ultra-Turrax T25 to form a pre-emulsion. A portion of this pre-emulsion was reserved, while the remaining part underwent ultrasonic treatment using a UIP1000hdT ultrasonic processor (Hielscher, Germany) equipped with a BS4d34 sonotrode (34 mm diameter), operating at a nominal power of 150 W for 3 min in continuous mode (no pulse applied), to generate a nanoemulsion. The amplitude varied around 70% during processing, while the treated sample volume was 200 mL, resulting in a specific energy input ranging from 7,465 to 7,568 kWh. The probe was immersed approximately 10 mm into the sample during processing. Temperature was controlled using an ice bath during sonication and monitored with a thermometer, maintaining an average temperature of approximately 30.1 °C ± 3 °C. Both the pre-emulsion and the nanoemulsion were separately incorporated into the polymer solution under magnetic stirring for 10 min at room temperature. A formulation without CEO was used as the control. An aliquot of 50 g of each film-forming solution was poured into 150 mm diameter Petri dishes and dried in a ventilated oven with air circulation at 35 °C for 18 h to allow film formation (Corrêa-Filho et al., 2024).

Characterization of emulsions regarding droplet size distribution and zeta potential

Twenty-four hours after the preparation of the pre-emulsion and nanoemulsion, the average droplet size (Z-average), polydispersity index (PDI), and zeta potential were measured. These parameters were determined by dynamic light scattering (DLS) using a Zetasizer Nano Series instrument (Malvern Instruments Inc., Worcestershire, United Kingdom). Prior to analysis, the emulsions were diluted in ultrapure water at a 1:5 ratio and evaluated at 25 °C (Chu et al., 2020).

Film characterization

Analysis of the surface morphology of the films by scanning electron microscopy (SEM)

A scanning electron microscope (TESCAN® VEGA 3) was used to evaluate the surface microstructure of the incorporated films. To increase conductivity and image quality, the films were coated with platinum and finally examined at 10 kV with a magnification of 15000x.

Film thickness, swelling and water solubility

Film thickness was measured using a digital micrometer (IP 54, Fowler, Newton, United States of America), with each measurement performed at least five times. To evaluate water solubility and swelling, the films were cut into circular samples (25 mm diameter) using a manual round cutter. The samples were initially dried in a ventilated oven at 105 °C for 24 h to determine their initial dry mass (M 0).

Subsequently, the samples were immersed in 50 mL of distilled water and subjected to continuous shaking at 65 rpm at room temperature in a shaker incubator (SL-222/E, Solab, Piracicaba, Brazil) for 24 h. After immersion, the wet mass (M 1) was recorded by gently blotting the surface moisture with absorbent paper. The samples were then re-dried in a ventilated oven at 105 °C for an additional 24 h to obtain the final dry mass (M 2). All measurements were conducted in quintuplicate. Swelling and solubility were calculated using Equations 1, 2, respectively (Mei et al., 2013). where M 0 is the initial film mass and M 1 represents the film mass after the swelling time. where M 0 represents the initial film mass and M 2 is the final dry mass.

Moisture content

Moisture content was determined by drying 25 mm film samples in a ventilated oven (Model MA 035/3, Marconi, São Paulo, Brazil) at 105 °C for 24 h. Moisture content was calculated as the proportion of weight lost relative to the initial mass of the film. Each measurement was carried out in at least five replicates.

Analysis of the optical properties of the films

The films (10 mm × 40 mm) were evaluated for their barrier properties against ultraviolet (UV) and visible light. Each sample was affixed to one side of a colorimetric cuvette and analyzed over a wavelength range of 200–800 nm using a UV-Vis spectrophotometer (UV M-51, BEL, Monza, Italy). Measurements were performed in triplicate, with an empty cuvette used as the reference. Film opacity was calculated as the ratio of absorbance at 600 nm to film thickness (mm).

X-ray diffraction (XRD) and differential scanning calorimetry (DSC)

X-ray diffraction analysis of the films was conducted using a Bruker 224 AXS D2 Phaser diffractometer (Bruker, Germany). CuKα radiation (λ = 1.5418 Å) was used, with a voltage of 30 kV and a current of 10 mA. The film scans were performed between 5° and 60° (2θ), at a speed of 2°/min, at room temperature (Wu et al., 2019).

In order to measure the amount of energy thermally absorbed or released by the films, DSC analysis was performed. The curves were recorded on a Q200 differential scanning calorimeter (TA Instruments, New Castle, United States of America), according to procedures established by Ghiasi et al. (2020). Approximately 2 mg of each film sample, with previously determined moisture content, were placed in hermetically sealed aluminum pans and subjected to a heating rate of 10 °C·min -1. The temperature profile ranged from −90 °C–250 °C, in an inert nitrogen atmosphere at 50 mL min -1.

Mechanical properties

Tensile strength (TS) represents the maximum stress the film can endure before breaking, while Young’s modulus (YM) indicates the film’s stiffness. Elongation at break (EB) reflects the extent to which the film can stretch prior to rupture. Mechanical properties were assessed using a texture analyzer (TA.XT Plus, Stable Microsystems, Surrey, United Kingdom) equipped with a 30 kg load cell. Film specimens (100 mm × 30 mm) were clamped with an initial grip separation of 50 mm, and a test speed of 6 mm/min was applied. For each sample, EB, TS, and YM were calculated using Equations 3–5, respectively. A minimum of ten replicates was used for each film formulation (Vieira et al., 2021).

L 0 is the initial film length and L 1 is the film break length.

F max is the maximum tension during the stretching process. L is the width of the films, and W is the thickness.

σ is slope of the tensile curve and γ is deformation.

Puncture tests were performed to determine the puncture force (FP) and puncture deformation (DP) using a TA. XT Plus texture analyzer (Stable Micro Systems, Surrey, United Kingdom). A cylindrical probe with a 5 mm diameter was applied perpendicularly to the center of film samples (25 mm × 25 mm) at a constant speed of 1 mm/s until film rupture occurred. The samples were positioned over an orifice with a 10 mm diameter. For each film type, a minimum of ten specimens was tested. FP and DP values were obtained from the resulting force–deformation curves. Puncture force was defined as the maximum force recorded before rupture, while puncture deformation corresponded to the displacement at the point of rupture (Cazón et al., 2019).

Water vapor permeability (WVP)

Water vapor permeability (WVP) of the films was determined gravimetrically using a modified version of the ASTM E96-92 standard method, as described by Rocha et al. (2014). Circular film samples (40 mm in diameter) were sealed onto permeation cells containing distilled water (100% RH; water vapor pressure = 3.167 kPa at 25 °C). The cells were then placed in desiccators containing blue silica gel (0% RH; water vapor pressure = 0 Pa at 25 °C) to establish a water vapor pressure gradient across the film.

The permeation cells were weighed every 24 h over a period of 5 days. The amount of water vapor transmitted through the film and absorbed by the desiccant was determined from the weight loss of the cells. The water vapor transmission rate (WVTR) was calculated from the slope of the linear regression of weight loss versus time (g/h). Finally, the WVP was determined using Equation 6. where WVP is the water vapor permeability (gmm·h -1 ·m -2 ·kPa -1), w is the weight loss of the permeation system (g), t is the time (h), x is the film thickness (mm), A is the exposed film area (m 2), and Δ p is the water vapor pressure difference across the film (kPa) at 25 °C. For each type of film, four independent WVP measurements were performed.

Fourier transform infrared spectroscopic (FTIR)

Fourier-transform infrared (FTIR) spectra were obtained using a Vertex 70 spectrometer (Bruker Instruments) equipped with a diamond attenuated total reflectance (ATR) accessory. Measurements were performed in ATR mode over a spectral range of 4,000 to 400 cm -1, with samples analyzed directly without additional preparation.

Evaluation of the antifungal activity of the films against Colletotrichum gloeosporioides

A spore suspension was prepared using saline solution (0.9% NaCl) added to plates previously inoculated with Colletotrichum gloeosporioides and cultured for 7 days. The surface of the colonies was gently rubbed with a sterile inoculating loop, promoting the release of spores. The suspension, containing fungal fragments, was then filtered through sterile gauze and collected in Falcon tubes. The spore concentration was adjusted to 10 5 spores·mL -1 by counting under a microscope. Aliquots of 20 μL of the suspension were spread onto plates containing PDA medium using a Drigalski loop. Immediately afterward, films (25 mm) were applied to three distinct points on each plate. The plates were incubated at 25 °C (±1 °C) for 48 h, and fungal growth was subsequently evaluated using a digital caliper (Power Fix Professional). Colony diameter was measured in two perpendicular directions (horizontal and vertical), and the results were expressed as the mean ± standard deviation of these measurements. A total of five plates were prepared, each containing three film samples (Muñoz-Tébar et al., 2022).

Statistical analysis

All experiments were performed in triplicate, except for FTIR analysis, which was conducted in duplicate. Data are expressed as mean ± standard deviation. Statistical analysis was carried out using analysis of variance (ANOVA) with Statistica software, version 10.0 (StatSoft Inc., Tulsa, OK, United States of America). When significant differences were detected, Tukey’s post hoc test was applied for multiple comparisons. The level of significance was set at p < 0.05. The statistical results are presented in the tables, where different lowercase letters within the same column indicate significant differences between samples.

Characterization of emulsions

The emulsification method directly influenced droplet characteristics, which are known to affect the final properties of films. The pre-emulsion droplets presented a size of 67.9 ± 4.2 nm, a PDI of 0.9 ± 0.0, and a Zeta potential of −26.6 ± 2.7 mV. In contrast, the nanoemulsion exhibited significantly smaller droplets (40.2 ± 3.66 nm) and a markedly lower PDI (0.2 ± 0.0), with a Zeta potential of −21.9 ± 1.9 mV (p < 0.05). Although both systems were within the nanometric range, the high PDI of the pre-emulsion indicates a heterogeneous size distribution. This is commonly associated with reduced stability and a greater tendency toward destabilization phenomena such as coalescence and flocculation (Mutlu, 2023). This behavior may be associated with previous findings for coarse CEO emulsions (de Oliveira et al., 2024).

The reduction observed in both droplet size and PDI in the nanoemulsion may be attributed to the application of ultrasonic treatment. These data suggest the effectiveness of sonication in reducing droplet size and homogenizing the particle distribution. Several researchers have already reported the ability of high-energy methods, such as ultrasound, to produce nanometric-sized emulsions for Pereskia aculeata Miller and cinnamon oils, characterized by lower PDI and enhanced kinetic stability (Lago et al., 2019; Chu et al., 2020). For instance, the effectiveness of sonication in obtaining nanometric emulsions was further demonstrated by Fattahi et al. (2020). The authors reported that macroemulsions, produced by Ultra-Turrax, had droplet diameters in the range of 242–362 nm. However, after sonication, the resulting nanoemulsions showed a drastic reduction in size, reaching 59–80 nm. This transition was associated with an improvement in emulsion quality, with sonication being responsible for reducing not only the size but also the PDI (from 0.5 in emulsions produced by Ultra-Turrax to 0.3 in emulsions produced by ultrasound).

The Zeta potential of the emulsions presented a negative value that may be related to the surface charge of the CEO droplets. This behavior is mainly attributed to the adsorption of hydroxide ions (OH-) on the oil droplet surface, as well as the presence of hydroxyl (R-O-) and carboxyl (R-COO-) groups of compounds present in the CEO (Ojagh et al., 2010). This behavior may be explained by the physicochemical effects of ultrasound on the interfacial structure. The application of ultrasonic energy promotes droplet disruption and increases the interfacial area, which enhances the adsorption and rearrangement of surfactant molecules at the oil–water interface. At the same time, cavitation and intense shear forces may expose or redistribute functional groups, leading to partial neutralization of surface charges and, consequently, a reduction in the absolute zeta potential (Yang et al., 2022; de Oliveira et al., 2024).

Physicochemical properties of the films

SEM analysis

Figure 1, which presents SEM micrographs of the surface of the polymeric films, shows significant changes in structural organization as a result of the addition of the pre-emulsion and nanoemulsion of CEO. The control film, containing only SA crosslinked with Ca 2+, glycerol, and CMC, exhibited a heterogeneous morphology characterized by regions of varying density and surface texture. This heterogeneity may be attributed to the non-uniform ionic crosslinking of SA by Ca 2+, resulting from the uneven diffusion of ions within the polymeric matrix. The competition between SA and CMC for the available carboxylate sites. The formation of more densely crosslinked zones interspersed with partially crosslinked regions, along with the differential redistribution of glycerol, may have led to the occurrence of microdomains, as also reported in similar calcium-crosslinked alginate systems (Azeredo et al., 2012; Morozkina et al., 2022).

The incorporation of the essential oil pre-emulsion appears to intensify surface irregularity, resulting in a more heterogeneous and discontinuous structure. During the drying process, the removal of water from between molecular structures can promote flocculation and coalescence of oil droplets. Common in systems with broad droplet size distributions, phenomena such as flocculation and coalescence drive phase separation. This, in turn, creates disordered domains within the film matrix (Vargas et al., 2009; Espitia et al., 2019; Avramescu et al., 2020).

Thickness, swelling and moisture content

The moisture content in polymeric films is a fundamental parameter, as it directly influences the stability, functionality, and durability of the material. In general, lower moisture values are desirable, since they contribute to reducing the risk of microbial growth and preserving the structural integrity of the polymeric matrix. Typically, the addition of an oily phase results in a decrease in moisture content, an effect attributed to the hydrophobic nature of the oil which, when concentrated during drying, forms a lipid barrier capable of hindering water retention in the polymer network (Sharma et al., 2021; Lopes et al., 2025).

The literature on SA films enriched with oils demonstrates that the incorporation of hydrophobic components tends to increase surface hydrophobicity and, consequently, reduce water absorption. Table 1 shows that the samples containing CEO presented similar moisture values (p < 0.05), regardless of the form of oil incorporation into the polymer solution. This behavior may be related to the efficiency with which the oily phase was dispersed in the polymer solution. Both emulsions presented droplets on a nanometric scale, which favors a more homogeneous distribution of the essential oil in the matrix. This efficiency may be enhanced by the structure of the nanoemulsions. After drying, the hydrophilic portion of the surfactant interacts with the polymer chains through hydrogen bonds, instead of interacting with the evaporated water. This interaction is more intense in nanoemulsions with smaller particles, due to the larger surface area and better dispersion, resulting in a more consistent and uniform reinforcement of the hydrophobic barrier (Pires and Moura, 2016).

On the other hand, an interesting paradox is observed. The lower uniformity in droplet size (higher PDI) in the pre-emulsion may not have been sufficient to significantly increase the moisture content compared to the control. However, it seems to have contributed to the greater swelling of the films containing the CEO pre-emulsion. The heterogeneity of the droplets may reduce the stability of the system and favors agglomeration during drying. The formation of these agglomerations or lipid domains may generate imperfections, pores, and discontinuities in the polymeric matrix. These structural defects, in turn, facilitate the penetration and retention of water, promoting greater swelling in this type of film. The use of nano-scale emulsions, with a low polydispersity index, has proven more effective in producing films with more regular surfaces and less porous internal structures (How et al., 2024). The micrographs presented in Figure 1 confirm this behavior. The films containing the nanoemulsion exhibited a more regular surface compared to the film containing the pre-emulsion, which corroborates the explanation above.

Thickness is a key characteristic of polymeric films, as it directly affects their mechanical strength, transparency, and barrier properties (Odila Pereira et al., 2019). Therefore, precise control of the solid’s concentration and emulsion stability in the film-forming solution is essential to optimize the final performance of the material. In this study, films containing CEO in both pre-emulsion and nanoemulsion forms exhibited similar thickness values, however, both were significantly thicker than the control film (p < 0.05) (Table 1). A similar trend was reported by Bhatia et al. (2023) in alginate–gum arabic films enriched with CEO. The increase in thickness following the incorporation of essential oils is frequently attributed to the higher viscosity of the film-forming solution. The introduction of a hydrophobic phase into a hydrophilic polymeric matrix promotes molecular interactions that increase viscosity, ultimately leading to the formation of thicker films after drying (Fattahi et al., 2020).

Moreover, the final thickness of a film is strongly influenced by the solids content in the initial formulation (Fattahi et al., 2020). Other studies have confirmed this pattern, for example, Zhou et al. (2021) demonstrated that the thickening of cassava starch-based films was directly related to higher CEO concentrations. Similarly, Valizadeh et al. (2019) observed an increase in the thickness of carboxymethylcellulose–chitosan films after the incorporation of cinnamon essential oils and other hydrophobic additives.

Water solubility and WVP

The results in Table 1 indicate that there was no significant difference (p < 0.05) in water solubility values between the control film and those incorporated with CEO. This similarity suggests that the concentration of added CEO was not sufficient to drastically alter the intrinsically hydrophilic nature of the base polymer matrix. The control film exhibits high solubility due to the hydrophilic nature of SA and CMC, which contain numerous hydroxyl groups (Kowalonek et al., 2023). Although the hydrophobic nature of CEO tends to decrease solubility, the absence of a statistical difference between the samples reinforces that the primary impact of SA crosslinking in the formation of the gel network structure was the dominant factor in controlling solubility, as reported in the literature (Ghasemlou et al., 2013; Odila Pereira et al., 2019).

WVP is a critical indicator of a film’s ability to limit moisture transfer between the product and the environment, and is influenced by factors such as hydrophobicity, thickness, and structural integrity (Kanmani and Lim, 2013; Lan et al., 2018). In our study, the control film exhibited the highest WVP, which is expected given the high concentration of hydrophilic hydroxyl groups of SA and CMC, which are highly hygroscopic. The crosslinking of SA attenuates this effect, but does not eliminate it. The incorporation of CEO in both emulsified forms demonstrated an impact on WVP compared to the control, although the difference between the films containing oil was not statistically significant. The trend of reduced WVP is attributed to the hydrophobicity of the oil phase, which creates a barrier in the diffusion path of water vapor through the polymer matrix (Liu et al., 2017; Lan et al., 2018). The statistical similarity between the WVP values of the films containing CEO can be explained by the total amount of oil added. This parameter appears to be the main factor conferring hydrophobicity to the film, overriding small structural variations. The addition of oil, in addition to being hydrophobic, can also act as a plasticizer in the polysaccharide network. This effect can influence chain mobility and, consequently, WVP, highlighting the complexity of interactions in the composite film matrix (Noshirvani et al., 2017).

Appearance and optical properties

Visual evaluation of the films is a relevant aspect, as the uniformity of the matrix can be directly associated with the functionality of the material, especially in applications intended for food packaging. In the present study, all the films produced showed an intact and continuous surface (Figure 2). Regarding transparency, it was observed that, in general, the films exhibited a high degree of transparency. However, those incorporated with CEO were slightly more turbid compared to the control film. This slight increase in turbidity was also evidenced in the opacity results (Table 1), in which the control film presented significantly lower values (p < 0.05). The difference observed in both the visual analysis and the opacity data can be attributed to the presence and characteristic coloration of the CEO. Opacity is an essential parameter for evaluating the transparency of a film, with higher values indicating lower transparency. CEO, depending on the extraction method, can present colors ranging from orange to reddish (Freche et al., 2022). The incorporation of this intrinsic coloration into the polymer matrix results in a visually less translucent appearance compared to the control. Similarly, Bhatia et al. (2023), when analyzing sodium alginate and acacia gum hydrogel films containing CEO, reported that the presence of the oil reduced the transparency of the films.

The films containing the pre-emulsion showed the highest opacity values (Table 1). This behavior can be explained by the lower uniformity of the oil droplets in the pre-emulsion, which possibly underwent flocculation during the drying process, leading to the formation of droplet agglomerates. The presence of these agglomerates in the polymer matrix acts as a barrier to the passage of light, increasing light scattering. The intensity of light scattering is related to the concentration and distribution of oil droplets in the polymer matrix; therefore, the greater the quantity or heterogeneity of these droplets, the more pronounced the light scattering will be, resulting in higher opacity values (Hosseini et al., 2015).

Light transmittance is a fundamental optical property of polymeric films. On the one hand, low transmittance values in the ultraviolet (UV) region are highly desirable, as they contribute to increasing the shelf life of packaged foods, protecting them against photo-induced deterioration processes, such as lipid oxidation (Simona et al., 2021). On the other hand, high transparency in the visible spectrum range is necessary to allow visual inspection of the product by the consumer (Hu et al., 2016). The analysis of Figure 3 shows that the control film presented the highest transmittance values across the entire spectral range, reaching levels above 90% in the visible region (≥750 nm). In contrast, the films incorporated with CEO, both in pre-emulsion and nanoemulsion form, exhibited a significant reduction in transmittance compared to the control. This decrease appears to be more pronounced in the UV region and at the beginning of the visible spectrum. The film containing the pre-emulsion showed the most intense barrier effect, with transmittance varying between 10% and 15% in the visible region, while the film containing the nanoemulsion presented intermediate values (approximately 60%–70%).

These results confirm that the films containing CEO are opaquer, which is in full agreement with the visual analysis and the opacity results obtained. The reduction in transmittance is likely associated with the presence of oil droplets in the polymer matrix, which promote light scattering and refraction (Hu et al., 2016; Rincón et al., 2019). It is also observed that the transmittance of the films with CEO undergoes a sharp change in the region near the UV-visible (below 400 nm), indicating that the incorporation of the essential oil confers excellent UV radiation blocking capacity, protecting the packaging material against photo-oxidative degradation and, consequently, prolonging the stability of the packaged food. The significant difference between the control film and the films containing oil reinforces the fundamental role of the CEO in modifying the optical properties, without completely compromising transparency in the visible region in the case of the film prepared with the nanoemulsion (Nowicki et al., 2003; Simona et al., 2021).

X-ray diffraction (XRD)

XRD analysis was performed to assess the structural organization of the SA/CMC films and to determine the effects of incorporating CEO on the crystalline and amorphous regions of the polymeric matrix. The diffraction patterns shown in Figure 4 exhibited broad halos and the absence of well-defined crystalline peaks for all samples, indicating a predominantly amorphous nature. This behavior is typical of hydrophilic biopolymer-based films containing plasticizers, where molecular disorder prevails due to hydrogen bonding and the presence of ionic crosslinking (Hamsan et al., 2021).

The control sample, composed of calcium-crosslinked alginate and CMC, presented a halo centered at 2θ = 26.27°, with a crystalline fraction of 21% and an amorphous fraction of 79%. This low crystallinity is attributed to the semicrystalline character of SA and the plasticizing effect of glycerol, which enhances chain mobility and favors the formation of a more disordered structure. Moreover, the presence of CMC, a highly amorphous polymer, further reduces matrix ordering, resulting in a predominantly disorganized material (Bhatia et al., 2023; Kandil and El Desouky, 2025).

Upon incorporation of the CEO, a decrease in apparent crystallinity was observed, reaching 18% in films incorporated with the pre-emulsion and 16.5% in films incorporated with the nanoemulsion. This reduction suggests that the oil phase interferes with the ordered arrangement of the polymer, thereby increasing the amorphous fraction. The CEO droplets may act as physical barriers that hinder chain packing and limit the formation of crystalline regions. Similar findings were reported by Azadi et al. (2023) who developed chitosan, tragacanth gum, and polyvinyl alcohol films incorporated with CEO nanoemulsion.

Furthermore, the use of ultrasonication in the nanoemulsion formulation promoted smaller and more homogeneously distributed droplets, increasing the interfacial area between the oil and polymer phases. This enhanced interfacial interaction may intensify molecular rearrangements, contributing to a higher amorphous content and explaining the lower crystallinity observed in films incorporated with the nanoemulsion (Chu et al., 2020). In this context, the reduced droplet size and lower PDI observed for the nanoemulsion can be directly associated with a more effective disruption of polymer chain packing, establishing a cause–effect relationship in which improved dispersion leads to increased interfacial interactions and, consequently, reduced molecular ordering. The reduction in crystallinity and the increase in molecular disorder within the polymer matrix are functionally relevant, as more amorphous films generally exhibit greater flexibility, a desirable property for biodegradable and active packaging materials. These structural changes may also be reflected in the mechanical properties of the films.

Differential scanning calorimetry (DSC)

DSC analysis was performed to investigate the thermal transitions and energetic stability of the films. Thermal stability is a critical parameter for the performance and functionality of edible films, as it directly affects their shelf life and suitability for various food packaging applications. The corresponding thermal parameters are listed in Table 2.

The glass transition temperature (Tg) could not be detected for SA-based films. According to Chiumarelli and Hubinger (2014), determining Tg in plasticized polymeric films using DSC is often difficult because the change in heat capacity during this transition is very subtle. All films exhibited two main thermal events: a strong endothermic peak between 155 °C and 180 °C, followed by a moderate exothermic peak between 219 °C and 221 °C. The first event is associated with the removal of strongly bound water and/or the disruption of amorphous regions, while the second corresponds to the onset of thermal degradation of the polymeric matrix.

The control film showed an endothermic peak at 157.7 °C ± 3.1 °C with an enthalpy of 370.4 ± 26.8 J g -1, followed by an exothermic peak at 220.7 °C ± 0.2 °C with an enthalpy of 72.7 ± 1.6 J g -1. After the incorporation of the CEO, the endothermic enthalpy decreased to 247.3 ± 32.4 J g -1 in films incorporated with the pre-emulsion and 230.7 ± 7.2 J g -1 in films incorporated with the nanoemulsion, while the corresponding peaks shifted slightly toward higher temperatures, reaching up to 175.2 °C ± 4.7 °C in films with the nanoemulsion.

This behavior suggests that the incorporation of CEO nanoemulsions reduces the amount of water strongly bound to the polymer chains and promotes internal structural reorganization, resulting in lower energy required to break intra- and intermolecular interactions. Similar findings were reported by Mazlan et al. (2025), who observed a reduction in endothermic enthalpy in pectin–pineapple juice films incorporated with ginger essential oil nanoemulsion.

The decrease in endothermic enthalpy is directly correlated with the XRD results, which showed a reduction in the crystalline fraction in films incorporated with the nanoemulsion. More amorphous materials tend to exhibit endothermic transitions of lower magnitude since the absence of ordered domains reduces the energy involved in local melting processes. Moreover, the shift of the endothermic peak to higher temperatures in films with the nanoemulsion suggests enhanced local thermal stability, likely due to stronger polymer–oil interfacial interactions and improved dispersion of the oil phase (Bhatia et al., 2023). In this context, the smaller droplet size and lower PDI achieved through ultrasonication increase the interfacial area between the CEO and the polymer matrix, promoting more effective interactions and structural reorganization. This relationship suggests a possible cause–effect pathway in which improved droplet dispersion leads to reduced crystallinity and modified intermolecular interactions, which in turn influence the thermal transitions observed. Therefore, the DSC results are consistent with the structural findings (XRD) and molecular interactions (FT-IR), reinforcing that droplet size distribution plays a key role in defining the thermal behavior of the films.

FT-IR spectroscopy

FT-IR spectroscopy in ATR mode was performed to identify the main functional groups present in the SA/CMC films and to assess possible chemical interactions between the components of the polymeric matrix and the different CEO emulsions. The spectra, presented in Figure 5, cover the range of 4,000–400 cm -1 and were obtained in ATR mode.

All films exhibited a broad band between 3,300 and 3,200 cm -1, attributed to the O–H stretching vibrations of hydroxyl groups from SA, CMC, and glycerol, as well as contributions from adsorbed water. This band reflects the hydrophilic character of the matrix and indicates the presence of a hydrogen-bonded network among polysaccharides, the plasticizer, and the oil phase (Mostaghimi et al., 2023). In films containing CEO, an increase in intensity and a slight shift toward lower wavenumbers were observed, suggesting strengthened hydrogen bonding and reorganization of the polymeric network after CEO incorporation (Qayum et al., 2024). Similar results were reported by Wang et al. (2025) for carboxymethylcellulose-based films containing CEO.

In the 2,920–2,850 cm -1 region, C–H stretching bands characteristic of methylene and methyl groups, originating from glycerol and the lipophilic fraction of the essential oil, showed higher intensity in films containing CEO, confirming the presence of the oil phase in the films. Between 1,260 and 1,000 cm -1, the C–O–C and C–O stretching bands associated with glycosidic linkages displayed slight broadening and intensification, suggesting changes in the chemical environment of C–O bonds due to interactions between the polymer hydroxyl groups and oxygenated compounds from the essential oil, such as cinnamaldehyde and eugenol (Mostaghimi et al., 2023; Wang et al., 2025).

The vibrations corresponding to the carboxylate groups of SA (1,593 cm -1 for the asymmetric mode and ≈1,408 cm -1 for the symmetric mode) became more intense in films incorporated with CEO in the form of pre-emulsion and nanoemulsion., indicating electrostatic interactions and possible modifications in Ca 2+ ionic coordination promoted by phenolic and aldehydic compounds from the essential oil (Han et al., 2018; Bacha et al., 2025).

These interactions may reduce the rigidity and crystallinity of the polymer, consistent with the lower crystalline fraction observed by XRD and the thermal changes detected by DSC, such as reduced water removal enthalpy and peak shifts. In addition, the more pronounced interactions observed in films containing the nanoemulsion can be associated with the smaller droplet size and lower PDI, which increase the interfacial area and favor stronger interactions between CEO compounds and polymer chains. Therefore, the improved dispersion achieved through ultrasonication contributes to the modification of intermolecular forces, reinforcing the increase in amorphous character and helping to explain the structural and thermal behavior of the films. The results suggest that the incorporation of the CEO, regardless of the preparation method, did not alter the polymer’s chemical structure but modified its intermolecular interactions, particularly hydrogen bonding, leading to a more amorphous and homogeneous network, in agreement with the XRD and DSC analyses.

Mechanical properties

Films with good mechanical strength and extensibility are essential for food packaging applications, as they must withstand external stresses during processing, transportation, handling, and storage (Aloui et al., 2021). The incorporation of the CEO nanoemulsion significantly affected the mechanical properties of SA/CMC films, as shown in Table 3.

The control film exhibited the highest tensile strength (40.66 ± 2.96 MPa) and Young’s modulus (28.95 ± 2.32). However, the lowest elongation at break (2.86% ± 0.22%) and puncture deformation (3.73% ± 0.34%) was observed. Generally, an increase in tensile strength is accompanied by a decrease in elongation at break (Li et al., 2022). After the addition of nanoemulsion, a decrease in strength and modulus was observed, along with an increase in deformability, particularly in the films containing the nanoemulsion, which showed the lowest tensile strength (30.64 ± 0.77 MPa) but the highest puncture deformation (7.62% ± 0.62%). These results suggest that the nanoemulsion acted as a plasticizing agent, modifying the polymeric network and promoting a less rigid, more elastic structure capable of withstanding greater deformation before failure.

These findings are consistent with those reported by Han et al. (2018), who incorporated CEO into sodium SA/CMC films. According to the authors, the CEO may reduce the cohesive forces within the polymeric network, enhances SA/CMC chain mobility, and improves the films’ extensibility and flexibility. The improvement in puncture deformation observed in the films containing the nanoemulsion reflects the influence of the ultrasonication process on the film structure. The combined use of Ultra-Turrax and ultrasound promoted a more homogeneous dispersion and smaller oil droplets, as shown in the surface structure of the films (Figure 1), resulting in better integration between the nanoemulsion and the polymer matrix.

The decrease in tensile strength and Young’s modulus in films containing CEO may be associated with a reduction in crystallinity, as evidenced by the XRD results, and the lower enthalpy of water removal observed by DSC. The larger amorphous fraction and greater segmental mobility reduce stiffness and facilitate the movement of polymer chains under tension, leading to greater flexibility and deformability. In this context, the smaller droplet size and lower PDI of the nanoemulsion promote a more homogeneous dispersion of the oil phase, increasing interfacial interactions and enhancing its plasticizing effect. This improved dispersion contributes to a more uniform stress distribution within the matrix and reinforces the reduction of intermolecular forces, resulting in films that are less rigid and more deformable. Thus, the mechanical behavior is likely a direct consequence of the reduction in droplet size and PDI. This reduction promotes improved dispersion and stronger interfacial interactions between the CEO and the polymer matrix. These effects may contribute to a decrease in crystallinity and an increase in molecular mobility, ultimately resulting in enhanced flexibility and deformability of the films.

Antifungal activity of the films

The use of natural preservatives, such as plant essential oils, in films is an effective strategy to extend the shelf life of food. Encapsulating or entrapping these oils in the polymer matrix ensures the maintenance of antimicrobial efficacy. It also minimizes possible unfavorable effects on sensory properties that would occur if they were applied directly to the product (de Oliveira et al., 2023). Figure 6 demonstrates the action of the active films against the fungus C. gloeosporioides. It was verified that the films containing the CEO promoted the inhibition of fungal growth in the area of direct contact. However, it is crucial to note the absence of inhibition halo formation (Table 4). This result suggests that the release of the bioactive compounds from the CEO did not occur significantly by diffusion into the culture medium, with the inhibition restricted to the film interface (Bacha et al., 2025). Under these conditions, the inhibited area corresponded exclusively to the contact region defined by the film disc, with no radial inhibition beyond this zone, indicating that the antifungal effect was localized and directly dependent on film–fungus interaction.

The potent antifungal activities of CEO are widely attributed to its main phenolic and aldehydic components, such as cinnamaldehyde. This compound acts by the reaction of its highly electrophilic carbonyl groups with nucleophilic compounds in the microorganism’s structure (Fattahi et al., 2020). A central point of discussion is that the method of preparing the emulsions did not generate differences in antifungal activity against C. gloeosporioides in this study. In the literature, it is frequently reported that reducing the oil droplet size and uniformity tends to improve antifungal properties by increasing the bioavailability and penetration of the active compounds into microbial cell membrane (Chen and Liu, 2016). Hashemi Gahruie et al. (2017) also observed that reducing the size of essential oil droplets by sonication resulted in improved bacterial inhibition. However, the equivalence of results observed in the present study suggests that, for the contact inhibition of C. gloeosporioides with CEO, the sustained release rate and the total amount of oil present in the contact area may have been more decisive than the droplet size. This observation implies that the uniformity of oil droplet dispersion may not be the limiting factor for antifungal efficacy in polymeric films, differing from the behavior observed in other studies (Muñoz-Tébar et al., 2022), which highlights the need for further investigations into the specific interaction between CEO and the SA/CMC matrix polysaccharide.

This study demonstrates that the emulsification method of CEO directly influences the structural and functional properties of SA/CMC films, primarily through changes in dispersion uniformity. Ultrasound-assisted nanoemulsification improved droplet distribution, which translated into more homogeneous microstructures, reduced crystallinity, enhanced thermal stability, and increased film flexibility. However, no significant differences were observed in antifungal performance, indicating that, under contact-driven conditions, factors such as CEO concentration and local availability at the film interface are more decisive than droplet size.

These findings highlight that improving dispersion uniformity does not necessarily translate into enhanced biological activity, revealing a key limitation in the current understanding of structure–function relationships in active films. In this context, the antifungal effect was restricted to direct contact, suggesting limited diffusion of active compounds into the surrounding medium. From an application perspective, this behavior is particularly relevant for the control of surface-associated pathogens such as C. gloeosporioides, the causal agent of anthracnose (a major postharvest disease affecting tropical fruits). The ability of the films to inhibit fungal growth at the contact interface indicates their potential as antifungal active packaging materials for fresh produce, where contamination predominantly occurs at the surface.

Nevertheless, further studies are required to better understand the interactions between CEO and the polymeric matrix, as well as to optimize release mechanisms and evaluate performance under real storage conditions.