Active pectin–chitosan films from Spanish lime (Melicoccus bijugatus) shell pectin containing Pimenta racemosa essential oil nanoemulsions for sustainable food packaging

In response to the widespread use and environmental impacts of conventional plastic food packaging, there is growing interest in biodegradable alternatives derived from renewable resources, including agricultural waste. Although pectin is widely investigated for sustainable packaging applications, it is predominantly sourced from citrus and apple by-products, while tropical agro-waste streams remain comparatively underexplored as alternative pectin sources. This study investigated pectin extracted from Spanish lime (Melicoccus bijugatus) shell waste as a polymer source for pectin-chitosan films incorporating nanoemulsified Pimenta racemosa essential oil (BLEO) for potential fresh-produce packaging applications. The films were characterized for structural, mechanical, barrier, antioxidant and antimicrobial properties, and their preliminary applicability was assessed qualitatively using tomatoes. BLEO incorporation (0.5–1.5%) increased antioxidant activity by up to fivefold and enhanced antimicrobial activity against Escherichia coli, Salmonella spp. and Listeria monocytogenes compared with the control film (0% BLEO). FTIR and XRD analyses indicated that the BLEO modified the polymer network, resulting in reductions in tensile strength from 16.20 to 6.88 MPa and Young’s modulus from 597.91 to 133.65 MPa, while elongation at break increased from 12.86% to 20.48%. Oxygen permeability increased from 1.05 to 8.02 cm 3 μm m -2 d -1 kPa -1, whereas water vapor permeability exhibited a non-linear response. In the preliminary tomato storage assessment, coated fruit showed delayed visual ripening and less visible surface deterioration than uncoated controls after 21 days of ambient storage. Unlike many previous pectin-based packaging studies that have relied on commercial or conventional citrus- and apple-derived pectin, this study demonstrates the feasibility of using pectin extracted from M. bijugatus shell waste to develop active biodegradable films with tunable physicochemical and bioactive properties. This approach contributes to the valorization of underutilized tropical agro-waste within a circular bioeconomy framework and highlights its potential for fresh-produce packaging; however, quantitative postharvest studies are required to validate shelf-life extension and assess commercial applicability.

The primary function of food packaging is to protect food products from external environmental factors while maintaining their quality, safety and shelf-life. Petroleum-based plastics have traditionally been the material of choice for food packaging because of their low cost, high mechanical strength, flexibility, processability, and effective moisture and gas barrier properties (Junaid et al., 2025). However, their widespread use has created substantial environmental and sustainability concerns because most conventional plastics are resistant to biodegradation and can persist in landfills, aquatic ecosystems, and terrestrial environments for extended periods. In addition, the degradation and migration of certain plastic-associated chemicals may result in the release of potentially hazardous compounds into food and the environment, with some being associated with endocrine disruption, neurodevelopmental disorders, and other adverse health effects (Eroglu et al., 2026). Consequently, the environmental persistence and potential health concerns associated with conventional food packaging have intensified the need to develop renewable, biodegradable, and safer alternatives that can provide packaging functionality without creating comparable long-term environmental burdens (Madihalli et al., 2025).

Among the alternatives being investigated, edible films and coatings have attracted considerable attention because they can provide a biodegradable protective layer directly on the surface of food products while potentially contributing additional functional benefits (Arshad et al., 2025; Usman et al., 2025; Syarifuddin et al., 2025). However, the development of edible packaging remains challenging because no single biopolymer generally provides all of the mechanical, barrier, structural, and functional properties required for effective food packaging. Consequently, considerable research has focused on exploiting the complementary properties of different biopolymers and incorporating functional additives to overcome their individual limitations.

Edible films and coatings are primarily developed from three major classes of natural biopolymers: lipids, proteins, and polysaccharides. Lipid-based materials, including those prepared from natural waxes, acetylated monoglycerides, and resins, are highly hydrophobic and therefore generally provide effective resistance to moisture transfer. However, their relatively poor mechanical strength can restrict their application when structural integrity is required (Arshad et al., 2025). For example, Devi et al. (2025) demonstrated carnauba wax-, shellac- and carvacrol-based coatings reduced transpiration-related weight loss in mandarin oranges from 51.93% to 19.23%, illustrating the potential of hydrophobic coatings to minimize moisture loss. Protein-based films prepared from zein, gelatin, collagen, and casein, in contrast, generally possess superior mechanical properties and structural integrity but tend to provide weaker moisture barriers, because of the hydrophilic nature of many proteins (Perera et al., 2024). These materials have nevertheless been successfully applied to fresh produce, fish, and meat products to improve quality retention and extend shelf-life (Jiao et al., 2026; Demirtas et al., 2025; Wang et al., 2026). Polysaccharide-based films have received particularly extensive attention because polysaccharides are abundant, renewable, biodegradable, and capable of forming continuous film matrices. They can also be modified or combined with other polymers and can serve as carriers for antimicrobial, antioxidant, and other bioactive compounds, making them attractive platforms for active food packaging (Syarifuddin et al., 2025; Pawase et al., 2025; Junaid et al., 2025; Khan et al., 2025).

Among polysaccharides, pectin has emerged as a particularly promising material because of its excellent film-forming capacity, biodegradability, abundance, and availability from agro-industrial residues (Hlaing et al., 2025; Cotrim et al., 2025; El-Habbasha et al., 2025). Pectin is a complex heteropolysaccharide located predominantly in the cell walls and middle lamella of higher plants, where it contributes to cellular adhesion, structural integrity, and tissue organization (Sharma et al., 2026). The presence of pectin in fruits and vegetables therefore creates an important opportunity to recover this valuable biopolymer from processing residues that would otherwise be discarded. Such an approach provides a dual sustainability benefit by simultaneously valorizing agro-industrial waste and generating renewable raw materials for biodegradable packaging. Despite these advantages, however, films produced from pectin alone frequently exhibit insufficient mechanical strength, water resistance, and barrier performance compared with conventional plastic packaging. These limitations can compromise their structural stability and functional performance during food storage and therefore necessitate polymer blending, crosslinking, or incorporation of suitable additives (Said and Lee, 2025).

Chitosan represents a particularly attractive complementary biopolymer for improving the functionality of pectin-based films. Chitosan is a cationic polysaccharide derived primarily from chitin and is recognized for its biodegradability, film-forming ability, and intrinsic antimicrobial activity (Li and Li, 2025). Because pectin contains negatively charged functional groups whereas chitosan is positively charged, the two polymers can interact electrostatically to form polyelectrolyte complexes. Such interactions can modify the organization of the polymer matrix and potentially improve the mechanical and barrier performance of the resulting films compared with films prepared from pectin alone (Shah et al., 2025). Furthermore, the antimicrobial activity of chitosan, which is associated with interactions between its protonated amino groups and negatively charged components of microbial cell membranes, provides an additional preservation function (Li and Li, 2025). Thus, combining pectin and chitosan provides an opportunity to develop a multifunctional film in which the complementary physicochemical and biological properties of the two polymers are exploited.

The functionality of such biopolymer matrices can be further enhanced through incorporation of essential oils. Essential oils contain numerous volatile bioactive compounds with antioxidant and antimicrobial properties and can therefore provide active protection against oxidative deterioration and microbial spoilage. Their incorporation into edible films and coatings has been reported to improve the preservation of a variety of food products. For example, Cai et al. (2025) demonstrated that orange essential oil incorporated into guar gum-based edible films significantly increased the shelf-life of coated cheese compared with films without the essential oil. Similarly, Çoruhlu et al. (2025), Çoruhlu et al. (2026) reported enhanced antimicrobial activity in chitosan-based films following incorporation of thyme essential oil. Nevertheless, the incorporation of essential oils also presents an important formulation challenge. Since essential oils are hydrophobic and contain numerous chemically active volatile compounds, their incorporation can alter polymer-polymer interactions, matrix organization, film morphology, and consequently mechanical and barrier properties. Excessive concentrations may therefore improve antimicrobial or antioxidant activity while simultaneously compromising film integrity and barrier performance (Bharti et al., 2022). Optimization of essential-oil concentration is consequently essential for achieving an appropriate balance between structural, barrier, and bioactive properties.

Although pectin-chitosan systems and essential-oil-enriched edible films have been increasingly investigated, much of the existing research has relied on commercially available pectin obtained from conventional sources such as apple and citrus processing streams (Sembiring et al., 2025; Dyankova et al., 2026; Ben Dlala et al., 2025). This emphasis has left other regionally abundant and underutilized agro-industrial residues comparatively unexplored as sources of functional film-forming biopolymers. The shell of the Spanish lime fruit (Melicoccus bijugatus), a tropical fruit widely distributed throughout Latin America and the Caribbean, represents one such underutilized resource. The fruit pulp is consumed because of its characteristic flavor and reported gastrointestinal, cardiovascular, and respiratory therapeutic properties (Nwokocha et al., 2019; Nwokocha et al., 2020), whereas the leathery outer shell is generally discarded. The seed and shell together account for approximately 65% of the total fruit mass, highlighting the considerable quantity of residual biomass generated during consumption and processing. Although valorization studies have investigated the seed, comparatively little attention has been given to the shell (Chim-Chi et al., 2025; Ortiz-Fernández, 2023). The shell therefore represents an important but largely untapped source of value-added compounds and biopolymers for sustainable packaging applications.

Our previous work established an optimized extraction process for recovering pectin from Spanish lime shell waste (John and Maharaj, 2025), demonstrating the feasibility of converting this underutilized tropical residue into a functional film-forming biopolymer. However, its application in an active food-packaging system has not yet been investigated. In parallel, the essential oil obtained from West Indian bay leaf (Pimenta racemosa), another plant resource native to the Latin American and Caribbean region, has demonstrated promising antioxidant and antimicrobial properties, yet its incorporation into edible film systems remains unexplored (Eldahshan et al., 2025). Thus, an important research opportunity exists to integrate these two regionally relevant and underutilized resources into a single sustainable packaging platform.

Accordingly, the novelty of the present study lies in the development of a waste-derived pectin-chitosan edible film in which pectin recovered specifically from Spanish lime shell is combined with nanoemulsified P. racemosa essential oil as an active component. To the best of our knowledge, this represents the first investigation of these two underexplored tropical resources within a single active edible packaging system. Beyond providing an alternative source of pectin, this approach simultaneously addresses agro-industrial waste valorization, renewable material utilization, and the development of multifunctional food packaging. Therefore, the present study aimed to investigate the influence of nanoemulsified P. racemosa essential-oil concentration on the structural, mechanical, barrier, antioxidant, and antimicrobial properties of pectin-chitosan edible films. The study was designed to determine whether incorporation of this essential oil could enhance the functional performance of the films while maintaining the physicochemical and mechanical characteristics required for potential food-packaging applications.

Chemicals

Chitosan (medium molecular weight, 75%–85% degree of deacetylation), Tween 80, glycerol, citric acid, acetic acid, ethanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride, 2,4,6-tripyridyl-s-triazine (TPTZ), sodium acetate and all other analytical-grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States).

Preparation of Melicoccus bijugatus shell waste

Shells from harvested M. bijugatus fruits were washed thoroughly to remove residual fruit pulp and subsequently dried in a cabinet dryer (LTE Scientific, Oldham, United Kingdom) at 55 °C for 24 h. The dried shells were milled into a fine powder using a grinding mill (Staub Model 4E, PA, United States).

Pectin extraction from shell waste

Pectin was extracted from the shell powder using a conventional hot acid extraction method as described by Duwee et al. (2022). Based on our previous optimized study, maximum pectin yield was obtained at a solid/liquid ratio of 1:40 (w/v), pH of 2.5 and an extraction time of 3 h (John and Maharaj, 2025). Therefore, for the present study, 5 g of shell powder was dispersed in 200 mL of citric acid solution (pH 2.5), heated at 80 °C for 3 h, filtered, and the pectin precipitated with two volumes of 95% ethanol. After precipitation for 24 h, the pectin was recovered by centrifugation (16,500 × g, 10 min), washed with 70% ethanol, dried at 40 °C for 24 h and milled into shell pectin powder (SPP).

Preparation of nanoemulsions

The essential oil from the West Indian bay leaf plant (P. racemosa) was extracted by steam distillation. Fresh leaves were harvested and thoroughly rinsed with water to remove adhering dirt and other debris. The leaves were then air-dried at ambient temperature for 10 days. The dried leaves were subsequently transferred to a steam distillation apparatus, and distillation was conducted for 3 h. The recovered essential oil hereafter referred to as BLEO was collected and stored in amber glass bottles at 4 °C until further use. Nanoemulsions were prepared following the method described by Norcino et al. (2020) with minor modifications. Bay leaf essential oil (BLEO) was incorporated into distilled water at concentrations of 0.5%, 1.0% or 1.5% (w/w) and stirred at 800 rpm for 5 min. Tween 80 was then added at an oil-to-surfactant ratio of 1:4 (w/w) and the mixture was stirred for a further 5 min to obtain a coarse emulsion. The coarse emulsions were then ultrasonicated using a probe sonicator (Branson, Danbury, CT, United States; 20 kHz, 50 W; 3 mm probe) in an ice bath for 5 min in pulse mode (10 s on, 10 s off) and stored at 4 °C.

Characterization of nanoemulsions

The hydrodynamic diameter (nm), polydispersity index (PDI) and zeta potential (mV) of the three prepared nanoemulsions were determined by dynamic light scattering (DLS) using a Litesizer DLS instrument (Model 701, Anton Paar, Graz, Austria). Prior to analysis, the nanoemulsions were diluted fivefold. Three independently prepared nanoemulsions were analyzed, with each measurement performed in triplicate.

Pectin-chitosan bilayer film formation

The shell pectin-chitosan bilayer films (Figure 1) were prepared using a layer-by-layer casting method adapted from Ivanovska et al. (2025). A 5% (w/v) pectin film-forming solution (FFS) was prepared by dissolving 2 g shell pectin powder (SPP) in 40 mL distilled water under continuous stirring, followed by addition of glycerol (30% w/w, based on pectin). After homogenization and vacuum degassing, the solution was cast (2 mm) onto polymethyl methacrylate plates using an automatic film applicator (Elcometer 4340, United States) and partially dried at room temperature (22 °C) for 24 h. A 2.5% (w/v) chitosan film-forming solution (FFS) containing glycerol (30% w/w based on chitosan) was prepared in 1% (v/v) acetic acid and cast (2 mm) over the partially dried pectin layer to produce the sequentially cast pectin-chitosan bilayer film. Films were dried at 22 °C for 48 h, removed from the casting surface, and conditioned at 50% ± 2% relative humidity before characterization. BLEO-containing films were prepared identically, except that the respective nanoemulsion (0.5%, 1.0% or 1.5%) was incorporated into the pectin FFS at a nanoemulsion:FFS ratio of 1:3 before casting. Film thickness (mm) was measured using a digital micrometer at three random locations on each film, and the mean value was used for subsequent calculations.

Characterization of films

Fourier transform infrared spectroscopy and X-ray diffraction

The prepared films were analyzed using Fourier transform infrared (FTIR) spectroscopy (Shimadzu IRAffinity-1S, Kyoto, Japan) according to the method outlined by Lin et al. (2025), with 32 scans per sample film over a wavenumber range of 450–4000 cm −1 at a spectral resolution of 4 cm −1. The crystalline structure of the pectin-chitosan bilayer films was characterized by X-ray diffraction (Bruker D2 Phaser, Karlsruhe, Germany) according to the method outlined by Boubakeur et al. (2026) with minor modifications. Diffraction patterns were recorded over a 2θ range of 5°–55° at a scan rate of 1° per min. The resulting diffractograms were used to evaluate the crystalline and amorphous regions of the films.

Mechanical properties

The mechanical properties of the films were determined using a universal testing machine (QTest-10, MTS System Corporation, United States) according to the ASTM D882–02 standard (ASTM, 2002a). Prior to testing, samples were conditioned at 50% ± 2% RH for 48 h. Films strips were mounted between the tensile grips and tested using an initial grip separation (gauge length) of 50 mm, and a crosshead speed of 10 mm min −1. Ten film strips were tested for each formulation. Tensile strength (TS), elongation at break (EAB) and Young’s modulus (YM) were determined from the resulting stress-strain curves. TS (MPa) and EAB (%) were calculated using Equations 1,2 while YM (MPa) was obtained from the slope of the initial linear region of the stress–strain curve. where F is the maximum force at break (N), t is the film thickness (mm), w is the film width (mm), d is the extension at break (mm) and d0 is initial gauge length (mm) and Δd = d-d0. 2.7.3 Solubility The solubility of the films was determined according to the method outlined by Zhang et al. (2023) with minor modifications. Film samples (2 cm × 2 cm) were dried at 105 °C to constant weight, m 1 (g) and then immersed in 30 mL of distilled water for 24 h at 25 °C. The undissolved residues were collected by filtration and dried at 40 °C for 24 h to obtain the final weight, m 2 (g). Film solubility (%) was calculated by Equation 3. Measurements were performed in triplicate.

Moisture absorption

The moisture absorption (MA) of the films was determined according to the method outlined by Nwanna et al. (2025) with minor modifications. Film samples (20 mm × 20 mm) were conditioned in a desiccator at 0% RH for 72 h to obtain the initial weight, W 0 (g). The samples were then transferred to a desiccator containing saturated NaCl solution (75% RH) at room temperature (22 °C) and weighed periodically until a constant weight, W t, g was achieved. Moisture absorption (%) was calculated using Equation 4. where W t is the final weight of the film sample (g) at 75% RH and W 0 is the initial dried weight (g) at 0% RH. Measurements were performed in triplicate.

Water vapor permeability

Water vapor permeability (WVP) was determined according to the ASTM E96 standard (ASTM, 2010). Film samples were sealed over test cups (68–3000 EZ-Cup Vapometer, Thwing-Albert Instrument Company United States) containing 20 mL of distilled water. The initial mass (g) of each cup was recorded prior to placement in a desiccator at 0% RH for 5 h. Mass measurements were subsequently recorded at 30 min intervals, and the change in mass over time was plotted to determine the slope (S L). The thickness of the films was taken at 10 randomized points and an average found. Three film specimens were evaluated for each formulation. Water vapor transmission rate (WVT), permeance, and water vapor permeability (g m m −2 s −1 Pa −1) of the films were calculated using Equations 5–7. where A is the test cup area (0.0032 m 2), S is the saturation vapor pressure at the test temperature of 22 °C (20.941 mmHg), R 1 is the relative humidity at the vapor source, (100%) and R 2 is the relative humidity at the vapor sink, (0%) and l is the film thickness (mm).

Oxygen permeability

Oxygen permeability (OP) of the films was determined according to the ASTM D3985–02 standard (ASTM, 2002b). Film samples (6 cm in diameter) were mounted in an oxygen permeation cell (PreSens, Germany) using vacuum grease to ensure an airtight seal. Both cell chambers were purged with nitrogen gas prior to introducing ultrapure oxygen gas into the lower chamber. The increase in oxygen concentration in the upper chamber was recorded by an integrated electrochemical oxygen sensor (PSt 6) and the OP (cm 3 μm m −2 d −1 kPa −1) was calculated using Equation 8. where P O 2 is the rate of increase in oxygen partial pressure in the upper chamber (hPa day −1), V is the volume of the upper chamber (116 cm 3), l is the thickness of the film (mm), P Std is the standard pressure (1013 hPa), A is the permeation area of the film (0.0068 m 2), Δ P O2 is the change in oxygen partial pressure between the upper and lower chambers (0.96 bar), and T Std and T Meas are the standard (273 K) and experimental temperatures (296 K) respectively.

Antibacterial properties

The antibacterial activity of the films was evaluated using the disc diffusion method as described by Norcino et al. (2020). Fresh cultures (24 h) of Escherichia coli (ATCC 35218), Salmonella (ATCC 13076), Listeria monocytogenes (ATCC 7644) and Staphylococcus aureus (ATCC 12600) were inoculated into nutrient broth and incubated at 35 °C for 24 h. The broth cultures were then diluted to 0.5 McFarland standard (approx. 108 CFU mL −1) after which, 0.1 mL was spread onto freshly prepared nutrient agar plates. Film discs (6 mm diameter) were sterilized under UV-C light (254 nm) using a benchtop UV-C chamber equipped with 15W UV-C lamps (Cole-Palmer 15W, Chicago, United States) for 2 min per side and placed onto the inoculated plates. After incubation at 35 °C for 24–48 h, the diameter of the clear inhibition zone (mm), including the 6 mm film disc, was measured using a digital vernier caliper (±0.01 mm). Each assay was performed in triplicate.

Antioxidant capacity

DPPH assay

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined according to Chaichi et al. (2023) with minor modifications. Film samples (25 mg) were dissolved in 1% acetic acid, and 0.1 mL of each solution was mixed with 3.9 mL of 0.1 mM DPPH solution. After incubation in the dark at 30 °C for 30 min, absorbance was measured at 517 nm using a Thermo Scientific UV-visible spectrophotometer (Thermo Fisher Scientific, United States). Measurements were performed in triplicate. Radical scavenging activity (%) was calculated using Equation 9. where A 0 is the absorbance of the DPPH solution (control), A 1 is the absorbance of the sample blank (film solution without DPPH), and A 2 is the absorbance of the reaction mixture containing both the sample and DPPH.

Ferric reducing antioxidant power

Ferric reducing antioxidant power (FRAP) was determined according to Homthawornchoo et al. (2022). Film samples (25 mg) were dissolved in 1% acetic acid, and 0.15 mL of each solution was mixed with 2.85 mL of freshly prepared FRAP reagent. After incubation in the dark at 37 °C for 30 min, absorbance was measured at 593 nm using a Thermo Scientific UV-visible spectrophotometer (Thermo Fisher Scientific, United States). Measurements were performed in triplicate. Results were expressed as µmol Fe (II) equivalents g −1 dry film using a ferric sulphate calibration curve.

Preliminary food storage assessment

To evaluate the practical applicability of the developed bioactive films, a preliminary storage assessment was conducted using mature green tomatoes. Fruits of similar maturity and size, free from visible defects, were obtained from a local market and randomly assigned (ten fruits per treatment) to one of three groups: uncoated control, pectin-chitosan coated or BLEO pectin-chitosan coated. Tomatoes were first surface sanitized by immersion in 100 ppm sodium hypochlorite for 2 min, rinsed with distilled water, and air-dried. Treated fruits were immersed in either the pectin film-forming solution (FFS) or a 0.5% BLEO pectin FFS, partially dried at room temperature (22 °C), then immersed in the chitosan FFS to form a sequentially applied pectin-chitosan film coating before complete drying at ambient conditions. Uncoated fruits served as the control. Individually coated fruits were stored at 22 °C ± 2 °C and approximately 98% relative humidity for 21 days. Representative images were captured on days 0, 4, 7, 14 and 21 to qualitatively monitor ripening progression during storage.

Statistical analysis

All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Statistical analysis was conducted using Minitab software (version 21.4.1). Significant differences between means were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test at a 95% confidence level (p < 0.05).

Nanoemulsion characterization

The prepared nanoemulsions were analyzed by dynamic light scattering (Table 1). All formulations exhibited mean droplet diameters below 50 nm, confirming successful nanoemulsion formation (Mushtaq et al., 2023). Although the 1.5% BLEO formulation exhibited a slightly larger droplet size than the 1.0% formulation, the difference was not significant (p > 0.05). Both formulations had significantly larger droplets than the 0.5% nanoemulsion (p < 0.05) indicating that droplet size increased with BLEO concentration. This trend contrasts with previous reports by Norcino et al. (2020) and Zhang et al. (2021) where increasing essential oil concentration reduced droplet size. One possible explanation is the relatively high density of BLEO (∼1.5 g cm −3), which may reduce the efficiency of ultrasonication at higher oil loadings, thereby producing slightly larger droplets.

The polydispersity index (PDI) reflects the uniformity of droplet size distribution with values below 0.3 indicating a narrow size and homogeneous distribution (Zhao et al., 2022; Pongsumpun et al., 2020). All nanoemulsions exhibited PDI values below this threshold, confirming that the systems were homogeneous with narrow particle size distribution. Zeta potential reflects colloidal stability, with higher absolute values indicating greater electrostatic stabilization (Norcino et al., 2020; Firoozi et al., 2020). Despite the use of Tween 80, a non-ionic surfactant, all nanoemulsions exhibited a net negative charge (Table 1). This phenomenon has been reported previously and is attributed to the presence of ionizable compounds within essential oils (Hasheminya and Dehghannya, 2021). During ultrasonication, mechanical forces may promote the release of functional groups such as hydroxyl and carboxyl moieties, which migrate to the droplet surface and impart a negative charge (Norcino et al., 2020; Hasheminya and Dehghannya, 2021). Among the samples, the 0.5% BLEO nanoemulsion exhibited the highest magnitude of zeta potential (−12.7 mV). In contrast, the 1.5% BLEO nanoemulsion showed the lowest zeta potential (−3.9 mV), suggesting reduced stability. The observed decrease in zeta potentials with increasing BLEO concentration may be related to the higher proportion of non-ionic surfactant used, which can mask surface charge and reduce overall electrostatic repulsion.

Overall, increasing BLEO concentration resulted in slightly larger droplets and a progressive reduction in the magnitude of the zeta potential. This suggests that higher BLEO loadings reduced the relative colloidal stability of the nanoemulsions, potentially due to the greater dispersed-phase content and increased interfacial coverage by Tween 80, which may screen surface charge and reduce electrostatic repulsion. Nevertheless, the PDI remained below 0.3 for all formulations, indicating that relatively homogeneous nanoemulsions were maintained across the BLEO concentrations investigated.

Mechanical properties

Mechanical properties determine film resistance during handling and transportation. Tensile strength (TS) reflects resistance to rupture, whereas Young’s modulus (YM) indicates film stiffness (Baghi et al., 2024; Tafa et al., 2023; Shanbhag et al., 2023). From the results shown in Table 1, the control film (0% BLEO) exhibited the highest tensile strength (TS) and Young’s modulus (YM) values (16.20 MPa and 597.91 MPa, respectively). The incorporation of West Indian bay leaf essential oil (BLEO) nanoemulsion resulted in a significant decrease (p < 0.05) in both the TS and YM. Although differences among BLEO-containing films were not statistically significant (p > 0.05), a decreasing trend was observed with increasing oil concentration. The 0.5% BLEO film exhibited the highest TS and YM among the treated samples, while the 1.5% BLEO film showed the lowest values. At higher BLEO concentrations, the plasticizing effect of the oil could have reduced polymer-polymer interactions and packing density, thereby weakening the mechanical integrity of the films. These results have been reported previously by both Ashraf et al. (2025) and Bharti et al. (2022) as they both showed that the addition of turmeric and caraway essential oil respectively, reduced the TS and YM of chitosan and starch based edible films.

Elongation at break (EAB) reflects the flexibility and extensibility of films representing their ability to deform without rupture (Yin Feng et al., 2025). High EAB values indicate more ductile materials, whereas lower values correspond to more rigid and brittle films. Typically, EAB is inversely related to TS and YM, as rigid and highly ordered polymer networks restrict molecular mobility (Fahrullah et al., 2021; Bhatia et al., 2024a). This inverse relationship was evident in the present study. The control film, which exhibited the highest TS and YM showed the lowest EAB (12.86%), whereas the 1.5% BLEO film, with the lowest TS and YM, exhibited the highest EAB (20.48%). BLEO acted as a plasticizer, reducing intermolecular interactions and increasing polymer chain mobility, thereby producing films with lower TS and YM but greater extensibility (Almasi et al., 2020; Jafari et al., 2023). Further, the progressive increase in EAB with increasing BLEO concentration suggests that the plasticizing effect became more pronounced at higher oil loadings, resulting in more extensible and flexible structures. These results suggest a trade-off between mechanical strength and flexibility, which may be advantageous depending on the intended food packaging application.

Solubility and moisture absorption

Pectin is inherently hydrophilic whereas chitosan exhibits limited solubility in neutral aqueous solutions. Given the hydrophobic nature of BLEO nanoemulsions, their incorporation was expected to reduce film solubility. Film solubility exhibited a non-linear response to BLEO concentration (Table 1). Compared with the control film (33.84%), solubility increased significantly (p < 0.05) to 51.09% at 0.5% BLEO, before decreasing to 36.65% and 29.08% at 1.0% and 1.5% BLEO, respectively.

The increased solubility observed at lower BLEO concentrations is likely due to structural disruption of the polymer matrix by dispersed oil droplets and surfactant molecules (Go and Song, 2020). Although hydrophobic, the dispersed oil phase can act as a plasticizer, reducing intermolecular cohesion and increasing polymer chain mobility, thereby promoting water penetration and dissolution (Schenk et al., 2025). At 1.5% BLEO, the greater proportion of hydrophobic domains likely reduced polymer-water interactions and limited water penetration, resulting in lower film solubility (Bhatia et al., 2024a; Bhatia et al., 2023). Similar trends have been reported in essential oil incorporated films (Jafari et al., 2023; Khalili et al., 2025).

In contrast to the non-linear solubility response, moisture absorption (MA) decreased progressively with increasing BLEO concentration. MA decreased significantly (p < 0.05) from 27.56% in the control to 23.08%, 21.93% and 18.52% at 0.5%, 1.0% and 1.5% BLEO, respectively. This concentration-dependent reduction can be attributed to the increasing hydrophobic character imparted by BLEO and the corresponding reduction in the accessibility of polar sites within the polymer matrix for interaction with water molecules. Thus, although low BLEO concentrations increased film solubility through structural disruption, increasing BLEO consistently reduced the affinity of the films for moisture.

Structural properties (FTIR and XRD)

Fourier transform infrared (FTIR) spectroscopy was used to investigate the functional groups present within the films and to assess the molecular interactions associated with pectin-chitosan complex formation and BLEO nanoemulsion incorporation. The spectra of all formulations as shown in Figure 2 exhibited a broad absorption band at 3343–3353 cm −1, which was assigned to overlapping O–H and N–H stretching vibrations associated with the hydroxyl-rich polysaccharide structure of pectin and the hydroxyl and amino groups of chitosan (Norcino et al., 2020; Xie et al., 2021). The broad nature of this region is indicative of extensive intermolecular hydrogen bonding within the polysaccharide network. The formation of the pectin-chitosan bilayer is expected to involve polyelectrolyte complexation at the interface between the two polymers. Pectin is an anionic polysaccharide containing ionized carboxyl groups (–COO -), whereas under the acidic conditions used to prepare the chitosan film-forming solution, the amino groups of chitosan become protonated (–NH3 +). Electrostatic attraction between these oppositely charged groups can therefore generate an interfacial pectin-chitosan polyelectrolyte complex, while additional hydrogen bonding between hydroxyl, carboxyl and amino groups further stabilizes the polymer network. Strong intermolecular interactions between pectin and chitosan arising from their opposite charges have previously been demonstrated in pectin-chitosan polyelectrolyte complexes (Maciel et al., 2015). In the present films, the bands at 1730–1736 cm −1 and 1604–1620 cm −1 were attributed primarily to esterified carbonyl (C=O) groups and carboxylate-containing environments, respectively. The latter region may also contain contributions associated with chitosan amide and protonated amino-group vibrations. Consequently, the spectral features within this region are consistent with the ionic and hydrogen-bonding interactions expected within the pectin-chitosan system. However, because the films were prepared by sequential casting rather than by homogeneous blending of the polymers, pectin-chitosan complexation would be expected to occur predominantly at the interface between the two layers. Accordingly, substantial shifts in the bulk FTIR spectrum should not necessarily be expected.

The effect of BLEO incorporation was most evident in the aliphatic C–H and polysaccharide fingerprint regions. The BLEO-containing films exhibited a more pronounced C–H stretching band at approximately 2925–2935 cm −1 compared with the control film. This enhancement is consistent with the introduction of aliphatic groups associated with the hydrophobic constituents of the essential oil and therefore provides evidence of BLEO incorporation into the film matrix. Similar changes in C–H stretching regions have been reported following the incorporation of essential oils and essential-oil nanoemulsions into polysaccharide-based films. However, the intensity of this band did not increase progressively with BLEO concentration, suggesting that the spectral response was influenced not only by total oil concentration but also by the distribution of nanoemulsion droplets within the heterogeneous polymer matrix. The C–O–C band at approximately 1096 cm −1 remained comparatively unchanged following BLEO incorporation, whereas the band at approximately 1017–1022 cm −1, associated with C–O vibrations of the polysaccharide pyranose-ring structure, decreased in intensity in the BLEO-containing films. This change suggests modification of the local molecular environment surrounding the polysaccharide chains following nanoemulsion incorporation. Phenolic and other oxygen-containing constituents of essential oils can interact with hydroxyl, carboxyl and amino groups of polysaccharides through hydrogen bonding, while their hydrophobic moieties can associate with less polar regions of the polymer network. Comparable modifications of O–H and C–O-related FTIR bands have been attributed to non-covalent interactions between essential-oil constituents and biopolymer matrices in active packaging films.

Importantly, no new characteristic absorption bands were observed following BLEO incorporation, and the principal pectin–chitosan bands remained present across all formulations. Therefore, the FTIR results do not provide evidence for the formation of new covalent bonds between BLEO constituents and the pectin–chitosan matrix. Rather, the spectral changes support predominantly physical incorporation of BLEO through non-covalent interactions, including hydrogen bonding, hydrophobic association and changes in the local molecular environment of the polysaccharide chains. Nanoemulsification is likely to facilitate these interactions by dispersing the hydrophobic essential oil as nanoscale droplets throughout the hydrophilic film-forming phase, thereby increasing the interfacial contact between BLEO constituents and the polymer matrix.

The crystalline structure of the films was analyzed using X-ray diffraction (XRD), and the resulting diffractograms are shown in Figure 3. Across all films, two main diffraction peaks were observed at 16.5° and 30° (2θ), corresponding to the semi-crystalline domains associated with ordered regions of the pectin-chitosan polymer network. The control film exhibited the highest peak intensities at both positions, indicating a greater degree of structural ordering. In contrast, with increasing BLEO incorporation, the diffraction peaks, particularly at 16.5° became progressively broader and decreased in intensity. This suggests a reduction in crystalline ordering, reflecting disruption of polymer chain packing and intermolecular alignment, by the incorporated essential oil. Similar trends have been reported in essential oil-incorporated edible films (Wu et al., 2025; Abdalla et al., 2023). The observed decrease in crystallinity is consistent with the mechanical properties discussed previously. As BLEO concentrations increased and the polymer network structural order decreased, the films exhibited lower tensile strengths and greater flexibility, consistent with the plasticizing and chain-spacing effects of the essential oil (Jafari et al., 2023).

Barrier properties (OP and WVP)

Oxygen permeability (OP) and water vapor permeability (WVP) are decisive performance indicators for edible films intended for fresh produce applications, as they govern mass transfer processes that dictate oxidative stability, moisture retention, and respiration balance. These parameters are fundamentally controlled by polymer chain packing density, crystallinity and free volume distribution (Saidi et al., 2025; Wu et al., 2024). While films with low OP and WVP can effectively help extend the shelf life of produce through a reduction of oxidative deterioration and moisture loss, films with moderate levels of permeability can also be beneficial in fresh produce systems as they support controlled respiration and transpiration which prevents the formation of anaerobic conditions that would otherwise compromise quality (Jama et al., 2026).

The OP data shown in Figure 4a reveal a statistically significant and concentration-dependent increase (p < 0.05) with BLEO incorporation. Relative to the control film (1.05 cm 3 μm m −2 d −1 ·kPa −1), OP increased to 4.62, 7.15, and 8.02 cm 3 μm m −2 d −1 ·kPa −1 for the 0.5%, 1%, and 1.5% BLEO films respectively, representing nearly an order-of-magnitude increase at the highest loading. Such a pronounced rise in oxygen transmission is consistent with the reduction in crystalline order and changes in mechanical behavior previously evidenced by mechanical testing and XRD analysis. The reduction in crystalline order likely increased polymer free volume, thereby facilitating oxygen diffusion. Additionally, incorporation of the dispersed oil phase may have altered the continuity of the polymer matrix, resulting in less tortuous pathways for oxygen transport. Collectively, these findings suggest that BLEO incorporation reduced the structural order of the polymer matrix, resulting in increased oxygen permeability and a less effective barrier to gas diffusion at higher BLEO concentrations.

The WVP results (Figure 4b) exhibit a non-linear response to BLEO incorporation. While WVP increased significantly (p < 0.05) at 1% (3.36 × 10 −10 g m·m −2 s −1 Pa −1) and 1.5% (3.97 × 10 −10 g m·m −2 s −1 Pa −1) relative to the control (2.70 × 10 −10 g m·m −2 s −1 Pa −1), the 0.5% BLEO film exhibited the lowest WVP value (1.87 × 10 −10 g m·m −2 s −1 Pa −1), indicating an initial improvement in moisture barrier performance. This behavior likely reflects a balance between the hydrophobicity of the dispersed oil phase and the plasticizing effect of BLEO on the polymer matrix. At low BLEO concentrations, hydrophobic domains likely reduced water sorption and diffusion. However, at higher concentrations, increased polymer chain mobility, reduced crystallinity and greater free volume became the dominant factors, facilitating water vapor transport through the films (Go and Song, 2020; Bhatia et al., 2024b). Consequently, the 0.5% BLEO formulation provided the most favorable balance between the hydrophobic contribution of the essential oil and preservation of the structural integrity required for effective resistance to water-vapor transport.

Overall, the permeability behavior of the films indicates that BLEO incorporation progressively altered the structural organization of the polymer matrix, resulting in reduced barrier performance at higher essential oil concentrations. These findings are consistent with the crystallinity reduction observed from XRD and the concomitant decrease in tensile strength, reinforcing the structure-property interdependence within the system. While the precise microstructural changes responsible for these effects were not directly examined, the combined physicochemical data suggest that BLEO incorporation modified polymer chain organization in a manner that facilitated oxygen and water vapor transmission.

Bioactive properties: antioxidant activity

Antioxidant-active edible films can delay oxidative deterioration and extend food shelf life (Muñoz-Tebar et al., 2023). In this study, the antioxidant activity of the films was evaluated using DPPH radical scavenging and ferric reducing antioxidant power (FRAP). In the FRAP assay (Figure 4c), the reducing power more than doubled with the addition of 0.5% BLEO compared to the control film, demonstrating a substantial enhancement in the ferric-reducing capacity even at low essential oil concentrations. At 1.5% BLEO, an approximately five-fold increase was observed indicating a strong concentration-dependent effect (Khan et al., 2023). A similar trend was observed in the DPPH assay (Figure 4d), where the radical scavenging activity increased more than two-fold at 0.5% BLEO and exceeded three-fold at 1.5% BLEO relative to control. These enhancements are attributed to the presence of bioactive compounds in the BLEO, which possesses strong antioxidant properties (Eldahshan et al., 2025). The concentration-dependent increase in both DPPH radical-scavenging activity and FRAP demonstrates that the enhanced antioxidant capacity of the films was directly associated with BLEO incorporation. Increasing BLEO concentration increases the quantity of redox-active essential-oil constituents available within the film matrix. Phenolic and other antioxidant compounds present in essential oils can donate hydrogen atoms or electrons to reactive species, thereby stabilizing free radicals and contributing to ferric-ion reduction. The progressive increase in antioxidant activity with BLEO concentration therefore reflects the greater availability of these bioactive constituents within the films. Similar improvements in oxidative stability have been reported for edible films containing essential oils such as oregano and torch ginger (Matadamas-Ortiz et al., 2022; Marzlan et al., 2022; Macit et al., 2025).

Bioactive properties: antibacterial activity

The antimicrobial activity of the films was evaluated to determine the effect of BLEO incorporation on microbial inhibition. BLEO contains bioactive compounds such as eugenol, chavicol, and other phenolic constituents with well-documented antimicrobial properties. Accordingly, increased antimicrobial activity with higher BLEO concentrations was expected (Eldahshan et al., 2025).

A concentration-dependent increase in antimicrobial activity was observed across the films (Table 2). Films containing higher BLEO concentrations produced larger inhibition zones, indicating enhanced microbial suppression. Additionally, BLEO-incorporated films exhibited antimicrobial activity against three of the four tested strains while the control film showed inhibition only against Salmonella spp. This baseline activity is attributed to the intrinsic antimicrobial properties of chitosan (Abdalla et al., 2023). The results suggest that while only Salmonella spp. Was susceptible to chitosan alone, E. coli and L. monocytogenes required the additional antimicrobial action provided by the BLEO for effective inhibition. Therefore, the antibacterial results demonstrate that BLEO incorporation broadened the antimicrobial activity of the pectin–chitosan films and that the inhibitory effect generally increased with increasing BLEO concentration. This concentration-dependent response can be attributed to the greater availability of antimicrobial constituents from BLEO at higher oil loadings. Hydrophobic essential-oil constituents can interact with microbial cell membranes, disrupting membrane organization and permeability and consequently interfering with essential cellular functions. Nanoemulsification may further promote these interactions by dispersing BLEO as small droplets, thereby increasing contact between the hydrophobic active compounds and microbial cells.

In contrast, no inhibition of S. aureus was observed at any BLEO concentration. This indicates that the antimicrobial effectiveness of the developed films was microorganism-dependent and may reflect differences in strain susceptibility, cellular-envelope characteristics, the concentration of active compounds released from the film, and their diffusion through the agar medium. Therefore, the absence of inhibition against S. aureus highlights a limitation in the antimicrobial spectrum of the present formulation rather than an absence of BLEO-associated antibacterial activity. Although Gram-positive bacteria are often more susceptible to essential oils, strain-dependent variability is well documented and explains this response (Bhatia et al., 2024b; Wang et al., 2020; Galgano et al., 2022; Zhang et al., 2016; Bachir and Benali, 2012). Overall, the results demonstrate that BLEO incorporation significantly enhances the antimicrobial functionality of the films, supporting their potential application as active packaging materials for improving food safety and shelf life.

Tomato storage assessment

The 0.5% BLEO formulation was selected for the preliminary storage assessment because it exhibited the most favorable balance of mechanical properties, barrier performance and bioactivity among the BLEO-containing formulations. From the representative images of the tomatoes (Figure 5), the uncoated control fruit appeared to progress more rapidly through the ripening phases, reaching full red color by day 7 and exhibiting noticeable surface shrivelling by day 21. In contrast, tomatoes from both coated groups appeared to display slower rates of ripening progression, only reaching full red color approximately 7 days later at day 14 and showed little signs of visible surface deterioration at day 21. The delayed visible color development and reduced apparent surface deterioration of the coated tomatoes suggest that the pectin–chitosan coatings modified mass transfer between the fruit and the surrounding environment. The polymeric coating can act as a semi-permeable barrier to gases and moisture, potentially reducing respiration-associated gas exchange and moisture loss and thereby slowing visible processes associated with ripening and senescence. Differences observed between tomatoes coated with the pectin–chitosan formulation and those containing BLEO may additionally reflect the altered permeability of the BLEO-containing films demonstrated in the barrier-property measurements. These observations are consistent with the reported ability of edible coatings to modify the exchange of oxygen, carbon dioxide and water vapor between the fruit and the surrounding environment, thereby slowing the physiological processes associated with ripening (Usman et al., 2025; Syarifuddin et al., 2025). Furthermore, while both coated groups appeared to delay the progression of ripening, it was observed that the non-BLEO coated group retained a more greenish color than the BLEO coated group at day 7. These results may reflect that the non-BLEO film coating led to a slightly slower progression of ripening during the early stages of storage. This observation is in keeping with the earlier barrier characterization of the films in which the pectin-chitosan films exhibited lower oxygen permeability than the BLEO films. This greater gas barrier provided by the pectin-chitosan coating may have therefore reduced the gaseous exchange between the fruit and the surrounding environment more effectively than the BLEO coating, thereby leading to a slower respiration rate and a more delayed initial ripening progression. The present storage assessment was qualitative and did not include quantitative measurements of parameters such as mass loss, firmness, color, respiration rate, soluble solids or microbial load, these findings should be regarded as preliminary evidence of preservation potential rather than definitive confirmation of shelf-life extension. Although these observations were only qualitative and therefore preliminary, they provided initial evidence that the coating formulations, based on the developed films in this study, have the potential to delay ripening and subsequently improve shelf life of fresh produce. However, further comprehensive quantitative postharvest studies would be required to both confirm these effects and determine their extent.

Conclusions

This study demonstrates the successful valorization of M. bijugatus shell waste as a source of pectin for the development of pectin–chitosan bilayer films containing nanoemulsified P. racemosa essential oil (BLEO) for potential active food-packaging applications. BLEO incorporation modified the structural organization of the polymer matrix, resulting in a clear concentration-dependent trade-off between the physicochemical and bioactive properties of the films. Increasing BLEO concentration reduced tensile strength and Young’s modulus while increasing film flexibility, consistent with disruption of polymer-chain packing and increased molecular mobility. BLEO also influenced the barrier properties of the films, with oxygen permeability increasing with essential-oil concentration, while water-vapor permeability initially decreased at 0.5% BLEO before increasing at higher concentrations. In contrast, BLEO substantially enhanced film functionality, producing concentration-dependent improvements in antioxidant activity and increasing antimicrobial activity against E. coli, Salmonella spp. And L. monocytogenes. Collectively, these findings demonstrate that BLEO concentration can be used to tune the balance between structural, barrier and bioactive performance of the developed films.

Among the BLEO-containing formulations, the 0.5% BLEO film provided the most favorable overall balance of mechanical properties, moisture-barrier performance and bioactive functionality and was therefore selected for the preliminary tomato storage assessment. Tomatoes treated with the developed coatings exhibited delayed visible ripening and less apparent surface deterioration over 21 days compared with the uncoated control, providing preliminary evidence of their potential for fresh-produce preservation. However, as the storage assessment was qualitative, further quantitative validation is required before definitive conclusions regarding shelf-life extension can be established. Future research should therefore prioritise quantitative real-food storage trials under controlled and commercial conditions, including measurements of mass loss, firmness, color, respiration, microbial load and other relevant quality indicators. Further investigation of film morphology, BLEO distribution and release behavior would also provide greater insight into the relationships between film structure, active-compound release and packaging performance. Pilot- and industrial-scale production studies should subsequently evaluate processing feasibility, film uniformity and nanoemulsion stability under scalable manufacturing conditions. A comprehensive techno-economic analysis should assess raw-material availability, pectin extraction efficiency, production costs and competitiveness relative to conventional packaging materials, while life-cycle assessment should quantify energy and water requirements, greenhouse-gas emissions, biodegradability and end-of-life performance. Together, these investigations would establish the technical, economic and environmental feasibility of translating M. bijugatus shell-derived active films from laboratory-scale development toward practical and commercially relevant sustainable food-packaging applications.