# Eco-friendly films from lemon peel pectin including essential oils for the sustainable tomato preservation

Sirine Ben Dlala, Zeineb Mzoughi, Mariem Itaimi Dammak, Khaoula Khwaldia, Didier Le Cerf, Hamdi Ben Halima, Nicole Jaffrezic-Renault, Hafsa Korri-Youssoufi, Hatem Majdoub

► To cite this version:

Sirine Ben Dlala, Zeineb Mzoughi, Mariem Itaimi Dammak, Khaoula Khwaldia, Didier Le Cerf, et al. Eco-friendly films from lemon peel pectin including essential oils for the sustainable tomato preservation. Process Biochemistry, 2025, 157, pp.56-64. 10.1016/j.procbio.2025.06.018. hal-05184489

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Submitted on 24 Jul 2025

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# Eco-friendly films from lemon peel pectin including essential oils for the sustainable tomato preservation

Sirine Ben Dlala<sup>a</sup>, Zeineb Mzoughi<sup>a</sup>, Mariem Itaimi Dammak<sup>a</sup>, Khaoula Khwaldia<sup>b</sup>, Didier Le Cerf<sup>c</sup>, Hamdi Ben Halima<sup>d</sup>, Nicole Jaffrezic-Renault<sup>d,∗</sup>, Hafsa Korri-Youssoufi<sup>e</sup>, Hatem Majdoub<sup>f,∗</sup>

## ARTICLE INFO

### Keywords:

Pectin films

Citrus limon

Lemon essential oil

Laurel essential oil

Antimicrobial activity

Packaging films

Tomato preservation

Biodegradability

## ABSTRACT

Several fruits and vegetables deteriorate within a few days, which poses a major challenge for their preservation and commercialization. To address this issue, this study reports on developing eco-friendly and safe films based on pectin extracted from lemon peel, enriched with essential oils, for a sustainable preservation of tomatoes. Pectin was extracted from *Citrus limon* peels using an innovative high-pressure treatment, achieving higher yields than conventional methods. The extracted pectin was characterized using advanced analytical techniques, FT-IR, Nuclear Magnetic Resonance (NMR), Size Exclusion Chromatography (SEC), and Gas Chromatography-Mass Spectrometry (GC-MS). Additionally, essential oils extracted from lemon and laurel were characterized by GC-MS to identify their active components. Pectin-chitosan-based films, with or without laurel and lemon essential oils, were prepared and evaluated for their mechanical properties, antimicrobial activity, moisture resistance, solubility, and biodegradability. These films were then tested for tomato preservation, demonstrating a significant extension of their shelf life beyond one month. Acting as a protective second skin, these films extend the freshness of fruits and vegetables by preventing microbial growth and reducing moisture loss. The results highlight the potential of these safe and eco-friendly films as a promising alternative to conventional plastic films.

## 1. Introduction

The preservation of fruits and vegetables, particularly tomatoes, remains a major challenge in the agro-food industry due to their high-water content and susceptibility to microbial degradation. Traditionally, chemical treatments have been used to extend their shelf life. However, growing concerns about chemicals' impact on health and the environment have driven the search for natural and sustainable food preservation solutions.

This study introduces an innovative biofilm formulation using pectin extracted from lemon peels, using for the first time a high-pressure treatment, and chitosan derived from shrimp exoskeletons. Both by-products (pectin and chitosan) are abundant on an industrial scale. By

valorizing these materials, we developed eco-friendly and biodegradable films, hence contributing to waste reduction. To enhance the biofilms' effectiveness, the formulation incorporates lemon and bay laurel essential oils. Bay laurel essential oil, applied for the first time in preservation films, provides antibacterial and antifungal benefits.

The methodology used in this work includes several key steps:

First, pectin was extracted from *Citrus limon* peels using an innovative high-pressure treatment, achieving higher yields than conventional extraction methods. The extracted pectin was then characterized using advanced analytical techniques, such as (FT-IR) spectroscopy, Nuclear Magnetic Resonance (NMR), Size Exclusion Chromatography (SEC), and Gas Chromatography-Mass Spectrometry (GC-MS). Essential oils from lemon and bay laurel were also extracted and characterized by GC-MS to

<footnote><sup>a</sup> University of Monastir, Laboratory of Interfaces and Advanced Materials, Faculty of Sciences of Monastir, Monastir 5000, Tunisia</footnote>

<footnote><sup>b</sup> Laboratory of Natural Substances (LSN), National Institute of Research and Physicochemical Analysis (INRAP), BiotechPole, Sidi Thabet 2020, Tunisia</footnote>

<footnote><sup>c</sup> Normandie University, INSA Rouen, CNRS, PBS Laboratory, Rouen 76000, France</footnote>

<footnote><sup>d</sup> University of Franche-Comté, UTINAM Institute, UMR CNRS 6213, 16 Route de Gray, Besançon 25030, France</footnote>

<footnote><sup>e</sup> University Paris-Saclay, Laboratory of Bioorganic and Bioinorganic Chemistry, ICMMO, ECBB, 17 Avenue des Sciences, Site Henri Moissan, Orsay 91400, France</footnote>

<footnote><sup>f</sup> Department of Chemistry, College of Science, Qassim University, Buraidah 51452, Saudi Arabia</footnote>

<footnote>\* Corresponding authors.</footnote>

<footnote>E-mail addresses: nicole.jaffrezic-renault@univ-fcomte.fr (N. Jaffrezic-Renault), h.majdoub@qu.edu.sa (H. Majdoub).</footnote>
identify their active components. Next, pectin-chitosan-based films were prepared, with or without the addition of lemon and bay laurel essential oils. These films were evaluated for their mechanical properties, antimicrobial activity, moisture resistance, solubility, and biodegradability. Finally, the films were tested for their effectiveness in tomato preservation.

While the study by Jelena Jovanović et al. [1] demonstrates effective preservation for up to 8 days using chitosan-pectin films with lemon essential oil, our formulation, with bay laurel essential oil, extended tomato shelf life to 32 days. Additionally, our formulation improved mechanical properties, reduced moisture content, and enhanced biodegradability. Furthermore, our films outperformed curcumin-loaded films from Qingtong Xie et al. [2], especially in terms of water resistance and mechanical strength, providing a more sustainable solution for food preservation.

Beyond protecting against microbial spoilage and moisture loss, these biofilms offer a sustainable alternative to conventional plastic films. Utilizing natural resources and industrial by-products helps reduce environmental impact and promotes responsible resource management.

This study highlights the potential of agro-food waste valorization in developing innovative and eco-friendly solutions for tomato preservation, addressing key challenges in sustainability and food security.

## 2. Materials and methods

### 2.1. Extraction of Citrus lemon polysaccharide (CLP)

#### 2.1.1. Preparation of raw materials

The *Citrus lemon* fruits were acquired from a fruit orchard located in Mahdia, Tunisia. After washing, the lemons were peeled, and their peels were chopped into tiny pieces and dried for four days in a laboratory oven at 50°C, being turned three times a day until they reached a constant weight. CLP was extracted by preparing an alcohol-insoluble substance (AIS) to remove low molecular weight compounds, proteins, and partially polar substances. The AIS was then treated with 95 % ethanol for 24 hrs using a Soxhlet.

Chitosan derived from shrimp exoskeletons (100–300 kDa) was purchased from Sigma-Aldrich. All used chemicals were of analytical grade. They were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo).

#### 2.1.2. CLP extraction

the polysaccharide was extracted using the same technique as Wang et al. [3] with a slight modification. 30 g of AIS were blended with 2400 g of deionized water and the mixture underwent high-pressure treatment at 121°C for 1 hr.

The resulting mixtures were filtered, centrifuged for 15 min at 3500 rpm, and precipitated with three volumes of ethanol (96 %) for 12 hrs at 4°C. The formed precipitates were dissolved in distilled water and deproteinized three times using Savag's method [4]. After that, the aqueous phase was dialyzed using a dialysis membrane (cut-off 14 kDa) against deionized water for 3 days at 4°C. To extract the CLP fraction, the dialysate was finally lyophilized. The extract yield percentage (%) was determined using Eq. (1):

$$ \text{Yield}(\%) = \frac{\text{mass of extracted CLP}}{\text{mass of citrus peel}} \times 100 \quad (1) $$

#### 2.1.3. Physicochemical characterization of CLP

- Colorimetric assays

CLP's carbohydrate content was determined using the phenolsulfuric acid method and galactose as the standard [5]. Galacturonic acid was used as the benchmark in the carbazole technique to calculate the uronic acid rate [6]. The protein rate was determined using the Lowry method [7].

- FT-IR and NMR Analysis

The CLP infrared spectra were acquired using a spectrophotometer (PerkinElmer, Waltham, MA, USA) equipped with an Attenuated Total Reflectance (ATR-Germanium crystal). The spectra were scanned in wavelength between 400 and 4000 cm⁻¹ at a resolution of 4 cm⁻¹. The degree of esterification (DE) of CLP was determined by analyzing the FTIR spectra [8]. DE was calculated using the band areas at 1700–1750 cm⁻¹ (methyl esterified uronic acids) and at 1600–1630 cm⁻¹ (free uronic acids). DE was computed using Eq. (2):

$$ DE = [(A1736 / (A1763 + A1613)) + 0.107] \times 100 \quad (2) $$

NMR experiments were conducted at 600 MHz using a Bruker Avance II spectrometer. Before NMR analysis, the polysaccharide solution (30 mg/mL) was lyophilized and re-dissolved in 0.5 mL of deuterium oxide, and tetramethylsilane (TMS) was used as an internal standard.

- Monosaccharide Composition

CLP sugar analysis was performed using a gas chromatograph-mass spectrometer (GC-MS) (Varian CP 3800) with a DB-5 capillary column and a detector (Varian Saturn 2000) after hydrolysis and derivation using the same method as [9] with some optimization. In brief, CPL (0.45 mg) was hydrolyzed for 2 h at 70 °C under a nitrogen atmosphere with 1 mL of 2 M TFA solution and converted into its trimethylsilyl derivative. The aqueous solution of D-myo-inositol (100 µL, 1800 µg/mL) was added to the mixture as an internal standard. The hydrolyzed product was then derivatized into its trimethylsilyl (TMS) form by combining the dried sample with dry pyridine and N₂O-bis (trimethylsilyl) trifluoroacetamide. Monosaccharide standards were prepared utilizing the same method. These analyses were carried simultaneously.

Size exclusion chromatography

CLP analysis was performed using a size exclusion chromatograph (SEC) outfitted with triple detection: a viscometer detector (VD) (Viscostar II, Wyatt Technology, Ca, USA), a multi-angle light scattering (MALS) detector (HELEOS II, Wyatt Technology, Ca, USA), and a differential refractive index (DRI) (RID 10 A Shimadzu, Japan) to establish the average molecular weights (Mn and Mw), the dispersity (D = Mw/Mn). The polysaccharides were decomposed in the eluent LiNO₃ (0.1 mol/L) at a concentration of 2 g/L before being filtered through a 0.45 mm membrane and injected (100 mL) using an automatic injector (SIL-20A, Shimadzu, Japan). Separation was achieved using two columns in series, OHPAC 804HQ and 806HQ columns (Shodex, Japan). The analysis was carried out using a Zimm "order 1" data processing system, with angles ranging from 34.8° to 142.8°. dn/dc was 0.15 mL/g.

### 2.2. Essential oil extraction

*Citrus limon* powder and dry leaves of *Laurus nobilis* were separately subjected to hydrodistillation for 3 hrs, employing a Clevenger-type apparatus. The obtained essential oils were separated through decantation, dehydrated using anhydrous sodium sulfate (Na₂SO₄), filtered, transferred into small opaque bottles, and stored in a light-protected environment at a temperature of 4°C.

#### 2.2.1. GC/MS analysis of essential oils

Essential oil biochemical composition was determined according to an earlier procedure [10]. GC-MS analysis was conducted using a Varian CP-3800 gas chromatograph. The experimental conditions were as follows: the injector temperature and transfer line temperature were set to 220 and 240 °C, respectively. The oven temperature was increased from 60 to 240 °C at a rate of 3 °C/min. A hexane solution of 10 % essential oils (0.2 µL) was injected with a split ratio of 1:30. Compounds were identified by comparing retention times to standards and involved computer matching against commercial libraries (NIST 98 and ADAMS 95), along with a custom library of mass spectra derived from pure
substances, known essential oil components, and MS literature data. Linear retention indices were calculated using the Van den Dool and Kratz formula based on the n-alkanes series (C8–C23).

## 2.3. CLP-CH films preparation

The film, composed of CLP and chitosan (CH), was prepared following the published procedure [11], with certain adjustments. It was found that excess chitosan reduced flexibility, while too much pectin weakened mechanical strength. The chosen balance (50:50) ensures adequate flexibility while maintaining good mechanical strength and optimal biodegradability. CH (2 % w/v) was dispersed in 1 % (v/v) glacial acetic acid, under continuous stirring at 500 rpm for 24 hrs. Simultaneously, the 2 % (w/v) pectin solution was dispersed in water and heated on a hot plate at 40°C for 1 hr, under continuous stirring at 1500 rpm. Subsequently, the dispersions were cooled to 25°C and filtered to eliminate non-dissolved polymer. Glycerol (0.2 g/g CLP-CH) was added during stirring at 500 rpm for 15 min.

Essential oils were incorporated in accordance with previous studies exploring the use of eucalyptus essential oil in biopolymer films [12]. These studies show that a 3 % oil concentration is optimal for enhancing antimicrobial properties while preserving the films’ structural integrity. Based on these results, a 3 % concentration of essential oil in our formulation was adopted. Tween 80 (0.2 % v/v) was incorporated into the essential oil of *Laurus nobilis* leaves (EOLA) or the essential oil of *Citrus limon* peels (EOLE) as an emulsifier to aid its dispersion in the film solution. After 15 min of stirring, the essential oil (3 %) was added to the dispersion. The mixtures were then homogenized for 10 min using a high-speed mixer (Ultra-Turrax) at 5000 rpm, and air bubbles were eliminated through ultrasound. Finally, 50 mL of the solution was poured into a Petri dish and left to dry at room temperature.

### 2.3.1. Characterizations of the film

Scanning electron microscope (SEM) micrographs were recorded using a ZEISS Sigma HD [13].

The pectin films’ infrared (IR) spectra were registered using a Fourier-transform infrared spectrometer (BX FTIR system spectrometer, PerkinElmer, MA, USA) equipped with ATR germanium. The spectra were collected in the 4000 cm⁻¹- 400 cm⁻¹ regions.

The thermal properties of the films are relevant for applications that require processing at high temperatures. The thermal stability of the films was evaluated using ThermoGravimetric Analysis (TGA) with a DuPont Instrument.

The film moisture content (MC) was assessed by determining the reduction in weight during the drying process in an oven set at 110 °C until achieving a constant weight (the weight of the dry sample).

The film solubility in water (SW) was determined by immersing the film pieces (1 cm×3 cm) in 50 mL of distilled water at 25°C under constant agitation for 24 hrs. After immersion, the remaining film pieces were dried at 110°C until reaching a constant weight (the final dry weight W2). The initial dry weight (W1) was determined when a constant weight was reached after heating at 110°C. The measurements were performed in triplicate, where all data were expressed as the mean ± standard deviation. The solubility in water (%) was calculated according to Eq. 3.

$$ \text{Film solubility \%} = ((W1 - W2)/ W1) \times 100 \quad (3) $$

A ProGage thickness tester from Thwing-Albert Instrument Company (Philadelphia, PA, USA) was used to assess the film thickness. Ten distinct positions on each film were measured with eight replicates conducted for each film formulation.

The mechanical properties of the films: tensile strength (TS), elongation at break (EB), and Young’s Modulus were evaluated at 23°C under controlled humidity conditions (50 % RH) using an INSTRON 3345 universal testing machine (High Wycombe, UK). The testing

followed the ASTM standard method D882 (ASTM, 2001). Six rectangular film strips (15 mm × 60 mm) were tested for each formulation at a head speed of 100 mm/min with a clamp separation of 50 mm. The tensile strength (TS), elongation at break (EB), and Young’s modulus (YM) were directly calculated by the machine’s software.

The film specimens’ color was assessed employing a color difference meter (Spectraflash 600 plus, Datacolor International, USA) based on the CIE Lab scale chromaticity parameters. The recorded CIE color values included L* for lightness (0 = black, 100 = white), a* representing greenness (-a* = green, +a* = red), and b* indicating blueness (-b* = blue, +b* = yellow).

Descriptive analyses were used to analyze data from SEM, FTIR, and TGA. FTIR spectra and TGA curves were analyzed using the software Origin® 8.0.

## 2.4. Biodegradability test

The biodegradation of the films in indoor soil was assessed following the method reported in [14]: Film Section (1 cm×1 cm) were weighed ($m_0$) after being dried at 70°C for 1 day and buried 5 cm deep in aerated and moist soil collected from INRAP (Tunisia). To maintain moisture, 1 mL of water was added daily. Each week, the films were unburied, cleaned, and weighed ($m_t$) after being dried at 70°C for 1 day. It was necessary to weigh continuously until a constant weight was achieved. The experiments were carried out in triplicate. The weight loss was calculated as follows:

$$ \text{Weight loss (\%)} = \left( \frac{m_0 - m_t}{m_0} \right) \times 100 \quad (4) $$

where $m_t$ is the dry weight at time t and $m_0$ is the initial weight.

## 2.5. Antimicrobial activity

The antimicrobial properties of the films were tested against a range of bacteria, including *Salmonella typhimurium* ATCC 14028 G (-), *Escherichia coli* ATCC 8739 G (-), *Staphylococcus aureus* ATCC 6538 G (+), and *Enterococcus faecium* ATCC 19434 G (+), and the fungus *Candida albicans* ATCC 10231.

The antimicrobial activity of the films and their components was determined using the disk diffusion method on Gram (+) and Gram (-) bacteria, and fungi. The method [15], involved spreading 100 µL of each bacterial suspension (10⁸ CFU/mL) onto Müller-Hinton Agar plates. Gentamicin (10 µg per disc) was used as the positive control. Discs, 6 mm in diameter, were cut from each film, placed on the agar plates, and incubated at 37°C for 24 hrs. The contact area between the films and the agar surface, as well as the inhibition zones surrounding the film samples, were observed, and the diameter of the visible inhibition zones was measured (three assays).

## 2.6. Coating application

The film-forming dispersions were used to coat tomatoes via immersion, following the technique reported in [16,17], with some adjustments. Before coating, both the tomatoes and the materials were sanitized using a chlorinated water solution followed by rinsing with water. Each tomato was individually immersed in the film-forming dispersions (50 mL) at 4°C for 1 min, ensuring a single coating layer was formed. Control tomatoes were immersed in water instead. Subsequently, the tomatoes were positioned on plastic shelves, sepals and stems facing downwards, and allowed to dry for 4 h at room temperature (25°C). Once dry, the tomatoes were transferred to Petri dishes inside rectangular plastic containers, sealed, and stored at 4°C for 32 days.
<table><caption>**Table 1**
Physicochemical data, monosaccharide compositions, and macromolecular characteristics for *Citrus limon* peels.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Yield (%)</th><th rowspan="2">Total sugars (%)</th><th rowspan="2">Uronic acid (%)</th><th rowspan="2">Protein (%)</th><th colspan="6">Monosaccharide composition (%)</th><th rowspan="2">M<sub>n</sub> (kDa)</th><th rowspan="2">M<sub>w</sub> (kDa)</th><th rowspan="2">D</th></tr><tr><th>Glc</th><th>Man 2.53</th><th>Ara</th><th>Gal</th><th>Xyl</th><th>Rha</th></tr></thead><tbody><tr><td>CLP</td><td>8.53</td><td>43.77</td><td>56.23</td><td>-</td><td>1.31</td><td>57.84</td><td>21.67</td><td>12.09</td><td>4.56</td><td>16.6</td><td>157 ± 140</td><td>9.5</td></tr></tbody></table>

Ara = arabinose, Rha = rhamnose, Xyl = xylose, Man = mannose, Fu = fructose, Gal = galactose, Glc = glucose, M<sub>w</sub>: weight average molecular mass; M<sub>n</sub>: number average molecular weights

# 3. Results and discussion

## 3.1. Physicochemical properties of CLP

Lemon peel is an abundant agro-industrial waste, hence, the necessity to valorize it. Furthermore, pectin is one of the most versatile bio-materials and is considered an effective biopolymer for the production of edible films due to its biocompatibility, biodegradability, and non-toxicity.

Several studies have demonstrated the feasibility of using pectin as a biopolymer to formulate edible films due to its ability to form gels [18].

Pectin films and coatings exhibit crystalline or amorphous areas, making them suitable for integrating additives and immobilizing water molecules within the film structure, thereby facilitating the retention of hydrophilic compounds. Additionally, pectin can carry functional substances, as it is well-suited for the incorporation of phytochemical compounds, allowing the development of nutritionally fortified edible films [19].

Chemical analysis of CLP is shown in Table 1. The FTIR spectrum of CLP, discussed in SM, was very similar to that of commercial citrus pectin (CCP) (Figure S1A). NMR spectra (Figure S2) are presented and discussed in SM.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Scanning electron microscopy images illustrate the surface characteristics of the pectin-chitosan control film (A), the film incorporating lemon essential oil (B), and the film containing laurel essential oil (C).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 2. Thermal characteristics of (A) CLP-CH, (B) EOLE and (C) EOLA using TGA/DTGA.</figcaption></figure>

The monosaccharide content of CLP was determined using GC-MS after hydrolysis and derivatization to their volatile states. As shown in Table 1, the total monosaccharide content in CLP was 43.77 %, consisting primarily of arabinose (57.84 %), followed by galactose (21.67 %), xylose (12.09 %), rhamnose (4.56 %), mannose (2.53 %) and glucose (1.31 %). Moreover, as the polysaccharide is rich in galactose and rhamnose and contains an important amount of uronic acid (56.23 %), CLP is a pectin-type polysaccharide, specifically Rhamnogalacturonan I [20].

The results SEC analysis (Fig. S3) revealed that CLP possesses a molecular weight of 157 ± 140 kDa with a large heterogeneity, as evidenced by a high dispersity value (D = 9.5) (Table 1). The molecular weight values of the acid-extracted pectins from citrus are between 123 and 386 [20], which frames the obtained value for CLP.

## 3.2. Chemical composition of essential oils

The laurel essential oil was obtained with a yield of 0.77 %, which is very close to the yields reported in the literature (0.71 % for oil isolated from laurel [21]). The major constituents of laurel essential oil were identified through GC-MS analysis, and the corresponding analytical data are shown in Table S1.

Indeed, 17 compounds were identified, representing 83.17 % of the total essential oil. The most abundant constituents were 1,8-cineole (33.15 %), alpha-terpinenyl acetate (12.29 %), methyl eugenol (11.07 %), and linalool (10.12 %). The essential oil composition from *Laurus nobilis* showed a 1,8-cineole pattern as a major constituent, which is similar to those reported for oils collected from other geographical regions in Morocco [22], Egypt [23] and Turkey [24].

The *Citrus limon* essential oil (EOLE) was obtained with a yield of 0.91 % and the different components of the essential oil were determined using the same method GC-MS as previously, and displayed in Table S1. The data revealed the identification of 17 compounds, which made up 96.69 % of the total essential oil. The most abundant constituents were limonene (57.67 %), alpha-terpineol (9.37 %), and beta-pinene (6.14 %). Similar findings have been reported by [25,26] which showed that lemon essential oil consists mainly of limonene.

## 3.3. Characterization of the CLP-CH film

### 3.3.1. Scanning electron microscopy

To investigate the microstructural changes and homogeneity of the films after EO enrichment, the film samples were visualized by SEM, and the results are shown in Fig. 1. The surface morphology of CLP-CH films showed an intermediate texture. This may be due to the network disorganization caused by the presence of chitosan in the pectin matrix, inducing some interactions. Pectin is recognized as a representative anionic linear polysaccharide, while chitosan is identified as a cationic linear polysaccharide. Additionally, through electrostatic attraction, chitosan can create a three-dimensional network structure in conjunction with negatively charged pectin [27]. The control film exhibited an intermediate texture on its surface. However, upon the addition of essential oil to the pectin-chitosan film, its microstructure changed significantly, resulting in a heterogeneous structure. The films incorporating essential oils displayed a rough surface, a looser texture, and a sponge-like structure with micro-cavities. According to Fernandez-Pan et al. [28], this disparity in appearance between the control and the enriched films could be attributed to the introduction of essential oils, which induced the emergence of discontinuities associated with the presence of the two immiscible phases in the matrix. Surface irregularities are caused by the migration of aggregates or droplets to the film surface during film drying. Flocculation and creaming of oil droplets also occur during film drying [29].

### 3.3.2. Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of CLP-CH films incorporating essential oils (Fig. S4) showed an absorption band at 3263 cm⁻¹ attributed to N-H and O-H stretching, while absorption at 2920 cm⁻¹ represents the C-H stretching of the alkyl chain, and the absorption at 1743 cm⁻¹ represents the C=O stretching vibration [30]. Furthermore, the absorption at 1591 cm⁻¹ might be attributed to N-H bending, whereas the absorption at 1415 cm⁻¹ may be related to C-N and C-O stretching [27]. The spectrum discrepancies between different film samples are mostly due to changes in chain conformation and orientation caused by EO inclusion. When EO was added to the CLP-CH film, no substantial difference (no additional
absorption peaks) was observed. In general, films with and without EO displayed identical major peaks, but the amplitude of the peaks varied.

### 3.3.3. Thermal analysis

The thermal behavior of the membranes was studied by thermogravimetric analysis (TGA) over a temperature range from 20 to 800 °C (Fig. 2). The broad initial minimum observed on the thermogravimetric derivative (DTGA) curves at a Tpeak lower than 150 °C is attributed to water loss due to the hydrophilicity of the membranes [31]. A significant weight loss occurred between 235 °C and 375 °C for the pectin-chitosan blend, indicating the degradation of the polymer. The incorporation of EOLE and EOLA modified the thermal behavior of the films, causing shifts in degradation temperatures and DTGA peaks. This suggests strong interactions between the polymer chains and the added components. Notably, the first degradation phase in the presence of laurel oil shifted to a higher temperature, probably due to attractive interactions between the polymer complex and the acidic features of laurel oil.

### 3.3.4. Moisture content (MC) and film solubility in water (SW)

Table S2 displays the impact of the incorporated essential oils on the physical properties of CLP-CH films. Interactions between the CLP-CH film matrix and EO molecules resulted in a decrease in the affinity of the CLP-CH molecules for water molecules, producing films with lower MC (%) and lower SW (%), which is valuable for the integrity and water resistance of food products. The incorporation of EOLE into the film matrix reduced the MC (%) of the films from 19.43 % to 15.66 % and film solubility from 25.85 % to 19.33 %. Similarly, the incorporation of EOLA into the film matrix lowered MC (%) of the films from 19.43 % to 11.38 % and film solubility from 25.37 % to 13.38 %. These outcomes may be attributed to the reduction in the hydrophilic properties of the edible films, as well as the interaction of EO components with the hydroxyl groups of the film matrix, which could limit the interaction of these hydroxyl groups with water, consequently leading to the formation of more water-resistant films. These observations are also in agreement with the data reported by Hafsa et al. [12] and by Nisar et al. [32], who observed that in the presence of EO, chitosan films significantly present a lower water content.

### 3.3.5. Mechanical properties

The mechanical properties of CLP-CH films are important for ensuring their durability and stability during handling, shipping, and storage [33]. These properties are intricately linked to the physicochemical characteristics of the used polymer, the types and quantities of additives, the intermolecular interactions, the water content, and the resulting microstructure. Table S2 presents the mechanical properties, including tensile strength (TS), elongation at break (EAB), and Young's Modulus.

A comparison of the different film formulations revealed that the film made from CLP-CH-EOLE was more fragile (TS: 1.56 MPa) and less flexible (EAB: 17.01 %) than those of the CLP-CH film (TS: 3.96 MPa, EAB: 28.06 %). Interestingly, the addition of EOLA to CLP-CH films led to an unexpected increase in TS from 3.96 MPa to 4.16 MPa. This result is surprising because many previous studies have reported that the inclusion of essential oils typically reduces the TS of polymeric films [34]. This notable increase in TS with EOLA addition can be attributed to the strong interactions between EOLA and CLP-CH chains, as supported by the results of the thermogravimetric analysis. Similar findings have been reported by Jie Liu for films based on soybean polysaccharides and chitosan, where essential oil addition led to covalent bond formation between phenolic compounds and polysaccharide functional groups, enhancing TS [33]. Moreover, the film (CLP-CH-EOLA) with the highest TS also exhibited the highest EAB, indicating that incorporating CH and EOLA can simultaneously enhance both the strength and flexibility of the coating films.

This significant difference in mechanical behavior between CLP-CH-EOLA and CLP-CH-EOLE can be explained by the distinct chemical

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 3. Biodegradation of films containing different essential oils.</figcaption></figure>

compositions of the used essential oils and their molecular interactions with the polymer matrix. In CLP-CH-EOLA, laurel essential oil, which is rich in 1,8-cineole, induced the formation of hydrogen and covalent bonds with the chitosan-pectin polymer matrix due to its oxygenated groups (ethers and alcohols). These interactions strengthen the film structure and improve its mechanical properties.

In contrast, in CLP-CH-EOLE, lemon essential oil, mainly composed of limonene (57.67 %), is a non-polar hydrocarbon that interacts weakly with the polymer matrix. This weak interaction disrupts the cohesion of the polymer network, leading to structural discontinuities and a plasticizing effect, thereby reducing TS and EAB. This result is consistent with
<figure><img src="image_9.png" /><figcaption>Fig. 4. Antibacterial efficiency of the various constituent (A) and (B) films against four bacteria (E. coli, S. typhimurium, S. aureus, E. faecium) and fungi (C. albicans).</figcaption></figure>

the findings of Gabriela Abdalla et al.[16], who reports that the incor-
poration of lemon essential oil reduces the mechanical properties of
chitosan-pectin films.

3.3.6. *Film color*

The color and transparency of the edible film are important in-
dicators of their overall aesthetic appearance and consumer acceptance.
Table S2 shows the effect of EO addition on the color coordinates (L*, a*,
and b*) of CLP-CH films. Edible films without EO seemed clearer and
more transparent, while the presence of oil altered the hue and trans-
parency of the films. Control films exhibited lighter colors, as indicated
by higher L* values. The L* a* and b* values of the film specimens
decreased from 79.98 to 76.01, from 4.66 to 1.32 and, from 60.93 to
45.01, respectively, with the addition of EOLE. Similarly, for EOLA, the

values of L*, a*, and b* decreased compared to the control film, as
indicated in Table S2.

3.3.7. *Biodegradability test*

The biodegradability of pectin-chitosan films was assessed using the
soil burial method, tracking weight loss (%) over time. As shown in
Fig. 3, all film formulations exhibited progressive weight loss
throughout 70 days, confirming their biodegradation. Films incorpo-
rating laurel essential oil demonstrated the highest biodegradation, with
weight loss increasing from 56.75 % at 10 days to 73.75 % by day 70. In
comparison, films containing lemon essential oil showed slower initial
degradation (45.71 % at 10 days), but steadily increased to 70.05 % at
the end of the test. Control films (CPL-CH) followed a more moderate
trend, with weight loss rising from 53.81 % at 10 days to 67.32 % by day
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Appearance changes of uncoated (control) tomato and tomatoes coated with representative pectin-based coating materials (CLP-CH, CLP-CH-EOLE and CLP-CH-EOLA) stored at 4°C for 32 days.</figcaption></figure>

70. These findings indicate that the incorporation of essential oils enhances the biodegradability of CPL-CH films, with laurel essential oil leading to a faster rate of degradation.

### 3.3.8. Antimicrobial activity

The antimicrobial effects of CPL-CH films incorporating different essential oils (EOLA and EOLE) against four bacteria and fungi are shown in Fig. 4. Neat CPL-CH films were ineffective against any of the tested microorganisms, which is consistent with the findings of Hafsa et al. (2016)[32], who reported that antimicrobial properties are negligible when chitosan is in the form of insoluble films. As shown in Fig. 4, no significant antibacterial efficiency was observed for the pure CPL-CH film against either Gram-positive or Gram-negative bacteria.

The antimicrobial efficacy of chitosan relies on the presence of its positively charged amino groups, which can interact with the anionic charges of pectin, inhibiting chitosan's active sites and inducing a subsequent loss of antimicrobial activity. However, incorporating EOLE into the CPL-CH films resulted in clear inhibitory zones; with a noticeable absence of bacterial growth around the film discs, particularly against *E. coli*, *S. aureus*, *E. faecium*, and *C. albicans*.

### 3.3.9. Application to tomato preservation

The CPL-CH, CPL-CH-EOLE, and CPL-CH-EOLA coatings were applied to evaluate their effectiveness as tomato preservatives (Fig. 5). At the beginning of the experiment, all tomatoes had bright red skin with no signs of mold. After 25 days, mold spots appeared on the control sample as well as on tomatoes coated with CLP-CH, while those coated with CLP-CH-EOLE and CLP-CH-EOLA showed no visible signs of mold growth.

On the 32nd day, tomatoes coated with CLP-CH-EOLE showed slight alterations, whereas those coated with CLP-CH-EOLA remained intact. These results suggest that biofilms made from pectin extracted from lemon peels and chitosan, enriched with lemon and bay laurel essential oils, provide an eco-friendly and sustainable solution for tomato

preservation. In particular, the addition of bay laurel essential oil effectively extended the shelf life of tomatoes up to 32 days. Compared to the study by Jovanović et al. [1] which demonstrates effective preservation for up to 8 days using chitosan-pectin films with lemon essential oil, our results show a better protection. Our findings are consistent with the study conducted by Roshandel-Hesari et al. [17], who developed an edible coating based on chitosan and casein containing *Origanum vulgare* L. essential oil, thus successfully preserving cherry tomatoes for 32 days.

## 4. Conclusion

In this study, pectin was successfully extracted from *Citrus limon* peels using an innovative high-pressure treatment, achieving a higher yield (8.53 %) than conventional methods. Additionally, essential oils from lemon and laurel were effectively extracted and characterized, highlighting their potential as bioactive compounds.

Pectin (2 %)-chitosan (2 %)-based films enriched with 3 % essential oils were formulated, optimizing their composition to enhance their functional properties. The optimized films demonstrated excellent performance in preserving tomatoes, extending their shelf life for up to 32 days. These films act as a protective barrier, preventing microbial growth and reducing moisture loss, providing a sustainable alternative to conventional plastic films. This innovation holds potential for reducing post-harvest losses, improving the quality of exported products, and offering a sustainable solution for food preservation and waste reduction.

## Funding

S. Ben Dlala thanks the University of Monastir for the traineeship grant. Centre National de la Recherche Scientifique is acknowledged for the IRP NARES. Campus France is acknowledged for its financial support through PHC Maghreb EMBISALIM.
CRediT authorship contribution statement

**Hatem Majdoub:** Writing – review & editing. **Hafsa Korri-Yous-soufi:** Methodology. **Sirine Ben Dlala:** Investigation. **Zeineb Mboughi:** Conceptualization. **Dammak Mariem:** Conceptualization. **Khaoula Khwaldia:** Methodology. **Didier Le Cerf:** Methodology. **Hamdi Ben Halima:** Writing – original draft. **jaffrezic-renault nicole:** Writing – review & editing.

**Declaration of Competing Interest**

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nicole JAFFREZIC-RENAULT reports equipment, drugs, or supplies was provided by University of Franche-Comté. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgments**

The authors express their gratitude to Dr. Christophe Rihouey for his technical assistance with SEC analysis, and to Dr. Imed Chraief, Chief Technical Assistant, for his support with GC-MS analyses. S. Ben Dlala thanks the University of Monastir for the traineeship grant. CNRS is acknowledged for the IRP NARES. Campus France is acknowledged for its financial support through PHC Maghreb EMBISALIM.

**Data availability**

Data will be made available on request.

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