# Double-layered starch-based films containing citronella oil prepared by solution casting

Nandi Sukri, Fransisca Fiendra Natasha, Yana Cahyana, Herlina Marta, Bambang Nurhadi, Sandi Darniadi, Sergio Torres-Giner, Zuhaib Bhat, Abderrahmane Aït-Kaddour

► To cite this version:

Nandi Sukri, Fransisca Fiendra Natasha, Yana Cahyana, Herlina Marta, Bambang Nurhadi, et al. Double-layered starch-based films containing citronella oil prepared by solution casting. Carbohydrate Polymer Technologies and Applications, 2025, 11, pp.100894. 10.1016/j.carpta.2025.100894. hal-05251243

HAL Id: hal-05251243

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Submitted on 12 Sep 2025

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# Double-layered starch-based films containing citronella oil prepared by solution casting

Nandi Sukri<sup>a,e</sup>, Fransisca Fiendra Natasha<sup>a</sup>, Yana Cahyana<sup>a</sup>, Herlina Marta<sup>a</sup>, Bambang Nurhadi<sup>a</sup>, Sandi Darniadi<sup>b</sup>, Sergio Torres-Giner<sup>c</sup>, Zuhaib F. Bhat<sup>d</sup>, Abderrahmane Aït-Kaddour<sup>a,e,w</sup>

## ARTICLE INFO

### Keywords:

Starch-based films

Double-layered casting

Active packaging

Citronella oil

Antimicrobial

Antioxidant

## ABSTRACT

The study aimed to determine the effect of drying time on the quality characteristics of the double-layer starch-based film and to evaluate the efficacy of citronella oil as a bioactive agent. The double-layered starch-based composite film was developed using a layer-by-layer solution casting method and dried for 15, 20, or 25 h. The citronella oil as an emulsion was added as a bioactive agent to one layer (food contact surface) at different levels (0, 1, 3, and 5 %, w/w). The film was evaluated for various physico-mechanical characteristics, thermal stability and bioactive properties. The results showed a significant impact of the drying time on the physical characteristics of the film and the most optimum results were found for the film dried for 20 h. The addition of citronella oil increased the thickness, water solubility, tensile strength, Young's modulus and heat absorption of the film and decreased the WVTR and elongation at break. An effect of the concentration was found and the film containing the highest concentration (5 %) of citronella oil showed bigger-sized inhibitory halos and lower IC₅₀ values. Overall, this film proved to be effective in releasing citronella oil specifically on its inner active layer to inhibit microbial growth.

## 1. Introduction

During the last decade, there has been a revolution in food packaging technology targeted to enhance food safety and quality. Packaging is not only required to fulfil basic functions (contain, protect, and market the product) but also to actively protect the product from spoilage and quality deterioration (active packaging) and to have a lower environmental impact. Active packaging is used to increase shelf life, improve quality, and prevent damage to food ingredients (Pavelková & Flimelová, 2012). The principle behind this is to combine an active ingredient with the packaging system, such as essential oils, which can enhance its bioactive properties such as antimicrobial and antioxidant activities (Beltrán, Roncalés & Bellés, 2019).

Essential oils with strong antioxidant and antimicrobial properties are generally used as bioactive agents to develop active packaging systems (Sharma, Barkauskaite, Jaiswal & Jaiswal, 2021). The most

popular essential oils exported for their active properties include citronella, clove, nutmeg, patchouli, and vetiver oil (Rastuti et al., 2020). Citronella essential oil has proven antimicrobial activities against various microbes with reported inhibitory halos higher than 20 mm against *Escherichia coli* and *S. aureus* (Sagala, Kambira, Gunawan & Pollin, 2022). These properties make it a potential candidate for developing active and biodegradable packaging material for food and can lead to innovations in food packaging technology. Developing bioactive packaging using citronella essential oil can provide a way to preserve foods without adding preservatives and can avoid the negative effects of essential oils on the sensory quality of foods.

While the art of developing bioactive films for food preservation has been known for decades, developing double, triple or multilayer films using layer-by-layer solution casting methods is a relatively recent development. This method allows the development of films with more than one layer which are more effective in preserving foods. The

<footnote><sup>a</sup> Department of Food Industrial Technology, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, West Java, Indonesia</footnote>

<footnote><sup>b</sup> Research Center for Appropriate Technology, National Research and Innovation Agency (BRIN), Subang 41243, Indonesia</footnote>

<footnote><sup>c</sup> University Institute of Food Engineering-FoodUPV, Universitat Politécnica de València (UPV), Camino de Vera s/n, Valencia, 46022, Spain</footnote>

<footnote><sup>d</sup> Division of Livestock Products Technology, SKUAST-Jammu, J&K, India</footnote>

<footnote><sup>e</sup> Université Clermont Auvergne, INRAE, VetAgro Sup, UMRF, Lempdes F-63370, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: abderrahmane.aitkaddour@vetagro-sup.fr (A. Aït-Kaddour).</footnote>
solution casting method is widely used as it can effectively produce thin bioplastic films (Gustafsson et al., 2019; Kurabetta et al., 2024).

The present study aimed at developing a starch-based double-layered bioactive film using a solution casting method, where the first layer was developed using starch and the second active layer (food contact surface) was developed using starch and emulsified citronella oil. Starch is recognized as a promising matrix for developing food packaging materials (Sivapragasam, Maqsood & Rupasinghe, 2024). The bioactive compounds in citronella oil are volatile and get released into the food in contact with the inner active layer (Ribeiro-Santos, Andrade, de Melo & Sanches-Silva, 2017).

However, despite growing interest in multilayer film production, no research has been reported concerning the optimization of the drying time of the first layer before the second layer is cast. This step is critical because it introduces two distinct drying processes, and the drying condition of the first layer can significantly affect the adhesion, structure, and performance of the final double-layered film. Therefore, the present study also evaluates the effect of first-layer drying time and citronella oil concentration on the quality characteristics of starch-based double-layered films.

## 2. Materials and methods

### 2.1. Materials

The material used in this study were cassava starch with an amylose content of 14 % wt. and amylopectin content of 86 % wt. (Rose Brand with a moisture content of 14.15 % total weight) purchased from PT. Budi Acid Jaya Tbk Indonesia, stems and leaves of citronella (Cymbo-pogon nardus) plant, glycerol, carboxymethyl cellulose (CMC), silica gel, tween 80 surfactant, Mueller Hinton Agar (MHA), paper disc, amoxicillin antibiotics, DPPH (2,2-diphenyl-1-picrylhydrazyl) solution, and methanol. All the chemicals were of analytical or food grade.

### 2.2. Citronella oil (CO) extraction

The oil was obtained from the stems and leaves of the citronella plant using an extraction method described by Verma and Goyal (Verma & Goyal, 2017). Briefly, the stems and leaves were washed, cut into pieces and dried for 14 h at 50 °C inside an oven with forced air. The dried stems and leaves were placed in a portable distiller and distillation was carried out for 6 h at a temperature of 95–105 °C. Citronella oil was

separated using a separating funnel, weighed, stored in dark vials, and stored away from light.

### 2.3. Citronella oil emulsification

The emulsification of citronella oil was done following the method elaborated by Agrawal et al. (Agrawal, Maddikeri & Pandit, 2017). Different levels of citronella oil (0, 1, 3, and 5 % w/w) and tween 80 (mass ratio of 2:1) were added to distilled water (emulsion medium) and the solution was homogenized using an ultrasonic homogenizer (60 % power for 5 min).

### 2.4. Preparation of the bioactive film

This bioactive film was developed following the method of Arifin et al. (Arifin et al., 2022) with minor modifications. A layer-by-layer solvent casting method was used and a filmogenic solution was prepared by mixing 3 % (w/v) cassava starch and 0.5 % (w/v) CMC in distilled water (Fig. 1). This mixture was homogenized using a hotplate stirrer (Infitek HPS-380-2) until it reached the initial gelatinization temperature (65 °C). Thereafter, 1 % (v/v) glycerol was added and the solution mixture was again homogenized until it reached the final gelatinization temperature (73.90–75.0 °C). The solution was cooled to a temperature of 50 °C and degassed for 20 min using an ultrasonicator (Branson 1510). This filmogenic solution was used to develop the first layer of the double-layered film by pouring it on a glass plate (20 × 20 cm) and drying it in an oven at 50 °C for 15, 20, or 25 h to standardize the optimum drying time. Another filmogenic solution was prepared for the second layer of the film using citronella oil emulsion as a bioactive agent at different levels (0, 1, 3, and 5 % w/w) using the same procedure. The range of citronella oil concentration values used in this research refers to research conducted by Zhao et al. (Zhao et al., 2023). This filmogenic solution was poured over the first dried layer of the film and dried in an oven at 50 °C for the same amount of time (15, 20, and 25 h) as used initially. The dried films were cooled, removed from the glass plate and stored at room temperature for characterization. The first stage of this research is to find the best drying time between 15, 20 and 25 h using 3 % citronella oil concentration in accordance with research conducted by Zhao et al. (Zhao et al., 2023). The second stage of the research is to observe the effect of different citronella oil concentrations by using the best drying time obtained from the first stage.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Schematic diagram for preparation of the double-layered bioactive film.</figcaption></figure>
## 2.5. Physico-mechanical characteristics

The mechanical characteristics of the film were studied by determining tensile strength (MPa), elastic modulus and elongation at break using a texture analyzer (Hazirah et al.) and Eq. (1). The films were equilibrated at 23 °C at 50 % RH for 40 h before analysis. The films were clamped (6 cm x 2 cm) on the texture analyzer (stable micro systems TA XT Express) with an initial distance of 5 cm between the clamps. The initial thickness and length of the sample were recorded and the samples were tested until they broke in the center.

$$ \sigma = \frac{F_{max}}{A} \varepsilon = \frac{\Delta L}{Lo} \times 100\%E = \frac{\sigma}{\varepsilon} = \frac{F \cdot Lo}{A \cdot \Delta L} \quad (1) $$

where $\sigma$ = tensile strength (MPa), $F$ = Force applied to the material (N, lb), $A$ = Area (cm², in²), $\varepsilon$ = Stretch/Elongation (%), $\Delta L$ = Change in elongation (cm), $L$ = Initial length (cm), and $E$ = modulus of elasticity (MPa).

## 2.6. Thickness

Thickness was measured using a screw micrometre at five measurement points (four angles and the center of the film). The average measurement of all the five points of each sample was taken (Sultan et al., 2024). The thickness is expressed in mm and the screw micrometer used had an accuracy of 0.01 mm.

## 2.7. Water vapour transmission rate (WVTR)

The water vapor transmission rate was characterized according to Slavutsky and Bertuzzi (Slavutsky & Bertuzzi, 2014). The film samples were cut to a size equivalent to the diameter of the cup mouth. A glass jar with a volume of 8 liters was filled with 40 % (w/v) NaCl to become a 75 % RH condition. The film sample was placed on the mouth of the cup containing silica gel and glued with double tape. The initial weight of the cup was weighed using an analytical balance. The cup's weight was weighed every hour for 8 h and the weight gain was calculated. WVTR (Water Vapor Transmission Rate) value was determined using the formula (2).

$$ WVTR = \frac{\text{Slope}}{A} \quad (2) $$

where WVTR = water vapor transmission rate (g/m²h), slope = absorption of moisture every hour (g/h), A = film area (m²).

## 2.8. Water solubility (WS)

Film samples were cut into pieces (2 × 2 cm) and dried in an oven (105 °C) for 24 h (Arifin et al., 2022). Samples were weighed as initial dry weight (a), then immersed in 40 mL of distilled water in a centrifuge tube for 24 h. The samples were filtered using filter paper (Whatman Number 1) that had been weighed beforehand (b) and dried in an oven (105 °C) for 24 h. The sample was allowed to stand in a desiccator for 10 min and weighed as the final weight (c). The percentage solubility of the sample in water was calculated by the Eq. (3).

$$ Solubility (\%) = \frac{a - (c - b)}{a} \times 100\% \quad (3) $$

## 2.9. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA)

The thermal behaviour (thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of the bioactive films were performed using a thermogravimeter (DTG-60, Schimadzu). Briefly, 5 mg of the samples were accurately measured and heated from 25 to 400 °C at a heating rate of 10 °C/min and their weight changes were recorded. The

gas nitrogen flow rate was 100 mL/min.

## 2.10. Fourier transform infrared spectroscopy analysis (FTIR)

The characterization of the developed packaging was done using an FTIR spectrometer (Bruker VERTEX70, Germany). The spectra were recorded at room temperature in the 4000 to 600 cm⁻¹. For each sample spectrum, 24 scans were recorded in order to increase the signal-to-noise ratio. Before acquisition, a background spectrum was recorded under the same conditions. Before spectral comparison, the spectra were subjected to scattering and pathlength corrections with the SNV (Standard Normal variate) technique by using the Matlab® software (R2012b, The Mathworks Inc., Natick, Mass., U.S.A) associated with the PLS toolbox 852 (Eigenvector Research, WA, U.S.A).

## 2.11. Antimicrobial activity

The antimicrobial activity of the double-layered film was evaluated by determining the inhibitory halos following the agar diffusion method described by Fahrullah et al. (Fahrullah & Rosyidi, 2021). For this purpose, the bacterial suspensions of E. coli and Staphylococcus aureus cultures equivalent to 1.5 × 10⁸ CFU/mL were prepared. MHA (Mueller Hinton Agar) media poured (25 mL) into the sterile petri dish were inoculated aseptically and spread in streaks with a cotton swab. Paper discs were immersed into citronella oil samples, distilled water (negative control), and amoxicillin antibiotic (positive control) and placed aseptically onto the Petri dish. Film samples with different levels of citronella oil (0, 1, 3, and 5 %) were cut into round disks of 7 mm diameter and placed directly on the agar inside the Petri dish (carried out in duplicate). The Petri dishes were covered with clingwrap and incubated for 24 h at 37 °C.

## 2.12. Antioxidant activity

The antioxidant activity of the films was determined following the previously reported method by Byun et al. (Byun, Kim & Whiteside, 2010) with a slight modification. A 0.5 g film sample was mixed with 50 mL of distilled water and stirred for 2 min. The suspension was filtered, 50 mL of distilled water was added, and five dilution series were prepared using methanol. The suspension sample was mixed with 0.5 mL of DPPH solution, vortexed until a homogeneous mixture was obtained and incubated in a dark room for 30 min. The absorbance of the sample was measured using a UV-Vis spectrophotometer at a wavelength of 517 nm. The inhibition value was calculated using the following formula (4).

$$ \%Inhibition = 1 - \frac{A_{sample}}{A_{control}} \times 100\% \quad (4) $$

The $IC_{50}$ value was calculated using the linear regression equation (5).

$$ Y = aX + b \quad (5) $$

where $A$ = absorbance, $Y$ = 50, $a$ = slope, $b$ = intercept, $X$ = the value of $IC_{50}$ (µg/mL).

## 2.13. Statistical analysis

The research data collected during the experiments was analyzed using one-way ANOVA using SPSS 22.0 statistical software. Data analysis was continued with Duncan's test to determine the significance between individual pairs of means at a 5 % level of significance ($p < 0.05$).
# 3. Results and discussion

## 3.1. Effect of drying time on quality of the double-layered film

The first objective of the study was to determine the optimum time to develop a double-layered starch-based film with the best quality characteristics. The degree of dryness of the first layer with varying drying times is the determining parameter in the addition of the second layer. The film was dried for 15, 20 or 25 h and the samples were evaluated for various physico-mechanical characteristics such as tensile strength (MPa), elongation at break ( %), Young's modulus (MPa), water vapour transmission rate (WVTR) and solubility in water (%).

### 3.1.1. Mechanical characteristics

The mechanical characteristics studied included tensile strength (MPa), elongation at break ( %), and modulus of elasticity (Young's modulus, MPa). Effect of drying time on physical and mechanical characteristics of double-layered film containing 3 % citronella oil presented on Table 1. Tensile strength is the maximum tensile force a film can sustain until it breaks. A lower tensile strength indicates that the film is weak, brittle and less strong, making it less suitable for packaging. A significant (*p* < 0.05) effect of drying time was observed on the tensile strength of the film. The highest value was found for the film dried for 20 h (1.98 ± 0.01 MPa) and the lowest for the film dried for 25 h (1.39 ± 0.03 MPa). While an increase was observed in the tensile strength of the film with increasing drying time (15 to 20 h), a decline was observed after 20 h of drying. Increasing drying time and temperature can drive particles to form a strong and homogeneous structure in film matrices. The addition of ingredients, such as cellulose, has been reported to enhance the tensile strength and gas barrier properties without affecting the thermal stability and optical properties of the film (Hernández-García, Chiralt, Vargas & Torres-Giner, 2023). Starch and cellulose during longer drying time form more hydrogen bonds that lead to a strong homogeneous compact structure of the film matrix, however, a further higher temperature and longer drying time may reduce the tensile strength due to excessive moisture loss (evaporation) and reduced inter- and intra-molecular contacts between the polymers, reducing the compact film structure (Pulungan, Kapita & Dewi, 2020).

A significant effect of drying time was also recorded on the elongation at break (%) with an increasing trend (56 to 66 %) from 15 to 20 h, however, a decline (52 %) was observed when the films were dried for 25 h, indicating a negative effect of excessive drying time. The elongation at break value of a film is its ability to stretch under force and the higher value indicates the higher ductility and flexible nature of the film. Longer drying times can make the films brittle and affect their flexibility. A decrease in elongation at break can be caused by an increase in drying time which causes further evaporation of water, thereby compacting and homogenizing the particle structure of the film matrix (Pulungan, Kapita & Dewi, 2020). In addition, the crystallization rate can be accelerated by the retraction ratio of the film, so that during stretching, there is a restriction of hydrogen bonds with the weakening of hydrogen bonds, which plays a role in the movement of molecular chains and stimulates

<caption>**Table 1**</caption>

Effect of drying time on physical and mechanical characteristics of double-layered film containing 3 % citronella oil.

<table><thead><tr><th rowspan="2">Parameters</th><th colspan="3">Drying time (hours)</th></tr><tr><th>15</th><th>20</th><th>25</th></tr></thead><tbody><tr><td>Tensile strength (MPa)</td><td>1.79 ± 0.04<sup>b</sup></td><td>1.98 ± 0.01<sup>c</sup></td><td>1.39 ± 0.03<sup>a</sup></td></tr><tr><td>Elongation at break (%)</td><td>56.75 ± 2.59<sup>a</sup></td><td>66.13 ± 2.54<sup>b</sup></td><td>52.25 ± 3.54<sup>a</sup></td></tr><tr><td>Young's modulus (MPa)</td><td>3.31 ± 0.01<sup>a</sup></td><td>3.00 ± 0.10<sup>a</sup></td><td>2.68 ± 0.25<sup>a</sup></td></tr><tr><td>WVTR (g/m<sup>2</sup>.h)</td><td>7.08 ± 0.41<sup>a</sup></td><td>9.20 ± 0.22<sup>b</sup></td><td>9.90 ± 0.28<sup>b</sup></td></tr><tr><td>Water Solubility (%)</td><td>35.97 ± 1.11<sup>c</sup></td><td>33.29 ± 0.01<sup>b</sup></td><td>29.76 ± 0.53<sup>a</sup></td></tr></tbody></table>

Means ± SD with different superscripts (a, b, c) in a row differ significantly (*p* < 0.05).

crystallization caused by stretching when the film is pulled (Hernández-García et al., 2023).

No significant (*p* > 0.05) effect of drying time was observed on the value of Young's modulus of the film although a decreasing trend was found with increasing drying time. The value of Young's modulus (elasticity) indicates the stiffness of the film and a higher value means the film is stiffer and changes its shape only slightly under load.

### 3.1.2. Water vapour transmission rate (WVTR)

The water vapour transmission rate (WVTR) is determined to evaluate the permeability of the film to water vapours. A significant impact of drying time was found on the WVTR of the film. A significant increasing (*p* < 0.05) trend was observed with increasing drying time and the highest WVTR was recorded for the film dried for 25 h (Table 1). In general, a lower WVTR is considered as a good quality characteristic for the bioactive film, however, most of the biodegradable films are not efficient in retaining the gases and water vapours. In contrast to our results, Pérez-Gago & Krochta (Pérez-Gago & Krochta, 2001) reported a decrease in WVTR of the film with the increase in temperature and drying time. Increased temperature and drying time can increase the cohesive force between polymer chains and increased heat will cause polymer chain movement leading to more extensive interactions. This discrepancy between our results and the literature may have been caused due to the differences in the film thickness between treatments as the WVTR of a film is also influenced by its thickness, which acts as a barrier to water vapour from the environment into packaged products (Siskawardani, Warkoyo, Hidayat & Sukardi, 2020). In addition, molecules in amorphous substances have an unstable arrangement, where the bonds between amorphous substance molecules are not identical and tend to vary at certain points, the density is uneven and can change back at any time on temperature changes (Benz & Bonten, 2021).

### 3.1.3. Water solubility

The water solubility was determined to evaluate the level of solubility of the film in water. The solubility value is expressed as a percent of the dry weight of the sample that has been soaked in distilled water for 24 h (Atef, Rezaei & Behrooz, 2014). The lower solubility value indicates better water resistance. A significant decreasing trend was observed with increasing drying time and the lowest solubility (%) was recorded for the film dried for 25 h. The drying temperature and time can affect the hydrophobic properties of biopolymers, where the higher temperature and longer drying time result in a tighter and more compact film structure (Pérez-Gago & Krochta, 2001). Longer and intermediate drying times allow the formation of films with low solubility and promote hydrophobic interactions between ingredients, such as starch, oil and cellulose, in the film matrix (Tapia-Blácido, do Amaral Sobral & Menegalli, 2013).

### 3.1.4. Optimization of the drying time

The determination of the best drying time is based on two stages of the drying process, namely the drying of the first layer and the drying after casting the second layer. The drying time optimization consisted of three drying times, namely 15, 20 and 25 h and the second layer used contained 3 % citronella oil. This step is very important, as the drying conditions of the first layer can significantly affect the adhesion, structure and performance of the final double-layer film. The double-layered film dried for 20 h showed the highest values for both the tensile strength and elongation at break compared to the films dried for 15 and 25 h (Fig. 2). The values of tensile strength and elongation at break were used as parameters in selecting the optimum drying time because the results of the analysis for both parameters showed significant differences in values with respect to drying time (Table 1). Further, the WVTR of the film dried for 20 h was higher than 15 h films and comparable with 25 h films. The films dried for 15 h were too sticky and the films dried for 25 h were too dry and brittle whereas the films dried for 20 h were of optimum quality. Therefore, based on these quality characteristics the
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Effect of drying time on tensile strength and elongation at break of bioactive film. Means with different superscripts (a, b, c) in a row differ significantly (p < 0.05).</figcaption></figure>

drying time of 20 h was considered as optimum and chosen for further studies.

## 3.2. Effect of citronella oil on quality characteristics of the double-layered bioactive film

The study's second objective was to evaluate the effect of the addition of citronella oil (0, 1, 3, and 5%) emulsion on the quality characteristics of the double-layered starch-based film dried for 20 h. The film was evaluated for various quality parameters viz. tensile strength (MPa), elongation at break (%), Young's modulus (MPa), solubility (%), WVTR (g/m²·h), thickness (mm), antimicrobial activity (inhibitory halos), and antioxidant activity [IC₅₀ (µg/mL)].

### 3.2.1. Mechanical characteristics

A significant effect of CO emulsion was observed on all the physico-mechanical characteristics of the double-layered starch-based film (Table 2). The addition of CO showed a significant (p < 0.05) increase in the tensile strength of the film in comparison to the control film containing 0 % CO. The highest values were found for the film containing 1 % CO (2.54 ± 0.13 MPa) and thereafter a decreasing trend was observed with increasing concentrations of CO. No significant difference (p > 0.05) was observed in the tensile strength of control samples and the film containing 5 % CO. A similar pattern was also found for the values of Young's modulus and highest values were recorded for the film

<table><caption>**Table 2**
Effect of addition of citronella oil on physical and mechanical characteristics of double-layered bioactive film.</caption><thead><tr><th rowspan="2">Parameters</th><th colspan="4">Level of citronella oil (%)</th></tr><tr><th>0</th><th>1</th><th>3</th><th>5</th></tr></thead><tbody><tr><td>Tensile strength (MPa)</td><td>1.09 ± 0.013<sup>a</sup></td><td>2.54 ± 0.13<sup>c</sup></td><td>1.98 ± 0.01<sup>b</sup></td><td>1.23 ± 0.12<sup>a</sup></td></tr><tr><td>Young's modulus (MPa)</td><td>1.25 ± 0.02<sup>a</sup></td><td>4.10 ± 0.24<sup>c</sup></td><td>2.88 ± 0.07<sup>b</sup></td><td>1.50 ± 0.19<sup>a</sup></td></tr><tr><td>Elongation at break (%)</td><td>87.42 ± 3.54<sup>c</sup></td><td>62.00 ± 0.71<sup>a</sup></td><td>69.58 ± 2.36<sup>b</sup></td><td>81.79 ± 3.00<sup>c</sup></td></tr><tr><td>Thickness (mm)</td><td>0.145 ± 0.005<sup>a</sup></td><td>0.156 ± 0.001<sup>b</sup></td><td>0.163 ± 0.001<sup>c</sup></td><td>0.167 ± 0.001<sup>c</sup></td></tr><tr><td>WVTR (g/m²·h)</td><td>11.94 ± 0.42<sup>c</sup></td><td>10.71 ± 0.33<sup>b</sup></td><td>9.64 ± 0.18<sup>a</sup></td><td>9.79 ± 0.05<sup>a</sup></td></tr><tr><td>Water Solubility (%)</td><td>30.24 ± 1.18<sup>a</sup></td><td>31.86 ± 0.48<sup>ab</sup></td><td>34.59 ± 1.44<sup>b</sup></td><td>40.87 ± 0.79<sup>c</sup></td></tr></tbody></table>

Means ± SD with different superscripts (a, b, c) in a row differ significantly (p < 0.05).

containing 1 % CO and thereafter a decreasing trend was observed with increasing concentrations of CO. No significant difference (p > 0.05) was observed in the mean values for Young's modulus of control samples and the film containing 5 % CO. A positive relationship was found between the tensile strength and Young's modulus as the former was directly proportional to the later (Sun, Kothari & Sun, 2018).

These results agree with the findings of Amni and Supardi (Amni & Supardi, 2021) who observed a significant increase in the tensile strength of banana starch-based edible film incorporated with lemongrass oil and the lowest values were found for the film containing the highest concentration of lemongrass oil. Another study reported a similar effect of the addition of lemongrass oil on the tensile strength of cassava starch-based edible film, where the addition of lemongrass oil increased the tensile strength compared to the control film but a decrease was observed on further increase in oil concentration (Resianingrum et al., 2016). While evaluating the impact of the addition of nanoemulsions of Thymus daenensis essential oil on Young's moduli of cellulose-based edible films, Moghimi et al. (Moghimi, Aliahmadi & Rafati, 2017) reported a similar increase in the value of Young's moduli and a similar declining trend with increasing concentration of the nanoemulsions. The addition of higher levels of CO may have caused the weakening of polymer bonds in the film matrix in our study, thereby affecting the film elasticity.

The addition of CO significantly (p < 0.05) decreased the values of elongation at break of the film in comparison to the control film. An increasing trend was observed with increasing concentrations of CO and the highest values were found for the film containing 5 % CO which was comparable with that of the control film. The elongation values decreased with the addition of citronella oil indicating that it allows fewer interconnections to occur in the film structure, thereby limiting the flexibility of the film. Further increase in the concentration of CO, which is hydrophobic in nature, will increase intermolecular interactions in the film matrix, thereby increasing its flexibility value. An inverse relationship has been reported between the tensile strength and the elongation at break for edible films (Sharma et al., 2020). The stronger the film material, the lower the flexibility. Resianingrum et al. (Resianingrum et al., 2016) reported that solid components in citronella oil can reduce the intercellular voids in edible films, form a more compact matrix and affect the tensile strength of cassava starch-based film. However, within the film network, some weak polymer bonds of citronella oil lead to the formation of gaps between polymer chain bonds and decrease the tensile strength values as the concentration of oil increases (Santos, Ramos, Luís & Amaral, 2023). In addition, solute components in citronella oil that enter the film matrix network can
disrupt and weaken the bonds between starch polymers and glycerol when present at higher levels (Suput et al., 2016). As the concentration of CO increases, it increases the number of OH groups in the film matrix which plays a role in increasing the mobility of the polymer chain and affects the flexibility of the film (Benavides, Villalobos-Carvajal & Reyes, 2012). There were some minor differences in the colour of the film due to the presence or absence of CO as shown in Fig. 3. The side of the film that contained CO was doffed and the other side was glossy.

### 3.2.2. Thickness

The addition of CO significantly (p < 0.05) increased the thickness of the film in comparison to the control film. An increasing trend was observed with increasing concentrations of CO and the highest values were found for the film containing 5 % CO which were comparable with the film containing 3 % CO. These results are in line with the findings of Ganapathy and Venkatasami (Ganapathy & Venkatasami, 2019) who reported a similar increase in the thickness of rice flour-based films incorporated with increasing levels of pomelo essential oil. The increase in film thickness indicates the interaction, and homogenization of CO with starch biopolymers (Valizadeh et al., 2019).

### 3.2.3. Water vapour transmission rate (WVTR)

The addition of CO significantly (p < 0.05) decreased the WVTR of the film in comparison to the control film. A decreasing trend was observed with increasing concentrations of CO and the lowest values were found for the film containing 3 % CO which were comparable with the film containing 5 % CO. This is in agreement with the findings of Yanwong and Threepopnatkul (Yanwong & Threepopnatkul, 2015) who observed a similar decrease in WVTR of fish skin-based edible film incorporated with increasing levels of CO. A similar decreasing pattern in WVTR of starch-based edible film has been reported by Resianingrum et al. (Resianingrum et al., 2016) on the incorporation of increasing concentrations of lemongrass oil. A decrease in the WVTR of the film with increasing levels of CO may also be attributed to increasing thickness with increasing levels of CO. In general, the thicker the film, the lower the WVTR (Jung et al., 2019). This decrease in WVTR with increasing concentration of CO may also be attributed to the hydrophobic nature of the CO (Resianingrum et al., 2016). The interaction between compounds in cassava starch and polyphenolic compounds in CO through hydrogen bonds may also be responsible for reduced WVTR (Han Lyn & Nur Hanani, 2020).

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Pictorial presentation of the two layers of the film: layer without citronella oil (a) and layer with citronella oil (b).</figcaption></figure>

### 3.2.4. Water solubility

The addition of CO significantly (p < 0.05) increased the solubility of the film in comparison to the control film. An increasing trend was observed with increasing concentrations of CO and the highest values were found for the film containing 5 % CO. CO contains many polyphenolic bioactive compounds which contain hydrophilic hydroxyl (-OH) and carbonyl functional groups (Fitri et al., 2022). In addition, cassava starch also contains hydroxyl groups (-OH) and increases the hydrophilic nature of the film and its water solubility (Yoshida, Antunes & Antunes, 2002). Go and Song (Go & Song, 2020) reported a similar increase in the solubility of a composite film incorporated with increasing concentrations of citronella essential oil. CO can loosen the film network, which causes water molecules to diffuse into the film network and causes the film to dissolve in water (Hosseini, Razavi & Mousavi, 2009). In addition, molecules in amorphous substances have an unstable arrangement, where the bonds between molecules are not identical and tend to vary at certain points, the density is uneven and can also affect the solubility value of the film (Benz & Bonten, 2021).

### 3.2.5. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA)

Thermogravimetric analysis of double-layered bioactive films containing different levels of CO is presented in Fig. 4. The TGA analysis of the film revealed two phases of mass losses in the film samples. The first phase occurred between 70–170 °C. In this temperature range, the films showed a mass loss in a range of 0.57–2.44 %. This weight loss is generally related to water evaporation (Morales et al., 2018). The second phase of mass loss (38.70–51.54 %) was recorded between 239–251 °C and is generally related to the evaporation of the plasticizers. Afterwards, the film underwent a high weight loss due to the complete degradation of starch at 400 °C.

The DTA of the double-layered bioactive film containing different levels of CO is presented in Fig. 5. DTA indicates heat absorption or emission due to phase transitions and reactions of substances upon external heat absorption. In this case, there were differences in the content of volatile material (CO) which can cause differences in heat absorption by the film. Fig. 5 reveals the heat absorption phase in the temperature range of 291–322 °C when the phase transition occurred and indicates a significant difference in the amount of heat absorbed by the four types of films. The amount of heat absorbed was 121.53, 180.09, 255.70, and 292.56 mcal for CO 0 % (K0), CO 1 % (K1), CO 3 %
<figure><img src="image_5.png" /><figcaption>Fig. 4. Thermogravimetric analysis of the double-layered bioactive film for every concentration of citronella oil (K0 = 0 % CO, K1 = 1 % CO, K3 = 3 % CO and K5 = 5 % CO w/w).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 5. Differential thermal analysis of the double-layered bioactive film for every concentration of citronella oil (K0 = 0 % CO, K1 = 1 % CO, K3 = 3 % CO and K5 = 5 % CO w/w).</figcaption></figure>

(K3), and CO 5 % (K5), respectively.

### 3.2.6. Fourier transform infrared spectroscopy analysis (FTIR)

Fig. 6 presents the FTIR spectra of the double-layered bioactive films containing different levels of CO. All the spectra exhibited bands in the 3000–3650 cm⁻¹, 2880–2925 cm⁻¹, 1650–1200 cm⁻¹ and 800–1200 cm⁻¹ regions. The 3000–3650 cm⁻¹ region exhibited a prominent peak around 3290 cm⁻¹ assigned to the vibration of O—H groups (Ayala Valencia et al., 2015). The band around 1643 cm⁻¹ is also associated with O—H vibrations of water molecules (Valencia, Henao & Zapata, 2012). Peaks observed between 2880 and 2925 cm⁻¹ are related to the vibration of C—H groups (Malki, Wijesinghe, Ratnayake & Thilakarathna, 2023). The peaks in the 800 to 1200 cm⁻¹ region are associated with the stretching of the C—O-C and C—O-O groups and the C—O-H group (Malki, Wijesinghe, Ratnayake & Thilakarathna, 2023). The spectra of the films were all similar. It was found that adding CO did not induce the apparition of new bands. This is certainly associated with the small

differences in the concentration of CO added into the packaging formulation (1 to 5 % w/w). This observation is consistent with the study of Zhao et al. (Zhao et al., 2023) who added CO to soy protein isolate-based packaging.

### 3.2.7. Antimicrobial activity

The antimicrobial properties of the double-layered film were evaluated by enumerating inhibitory halos (Table 3) against two types of pathogenic bacteria, *E. coli* and *Staphylococcus aureus*, which often contaminate food products. The present study evaluated the effectiveness of the antimicrobial and antioxidant capacity of the film and use the film without citronella oil as a control (0 % CO). A concentration-dependent effect was found and the inhibitory halos of significantly greater sizes were observed for the films containing increasing levels of CO against *E. coli*. While a significant effect of CO was found and significantly greater sizes were observed for all the films containing CO against *S. aureus* compared to the control film, no significant difference
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Fourier transform infrared spectroscopy analysis (FTIR) of the double-layered bioactive film for every concentration of citronella oil (K0 = 0 % CO, K1 = 1 % CO, K3 = 3 % CO and K5 = 5 % CO w/w).</figcaption></figure>

<table><caption>**Table 3**
The antimicrobial and antioxidant properties of the double-layered bioactive film.</caption><thead><tr><th rowspan="2">Treatments</th><th colspan="2">Zone of Inhibition (mm)</th><th rowspan="2">IC<sub>50</sub> (µg/mL)</th></tr><tr><th><i>E. coli</i></th><th><i>S. aureus</i></th></tr></thead><tbody><tr><td>0 % CO</td><td>0.00 ± 0.00<sup>a</sup></td><td>0.00 ± 0.00<sup>a</sup></td><td>(inactive)<sup>a</sup></td></tr><tr><td>1 % CO</td><td>4.88 ± 0.18<sup>b</sup></td><td>7.88 ± 0.88<sup>b</sup></td><td>364.51 ± 4.01<sup>c</sup></td></tr><tr><td>3 % CO</td><td>5.13 ± 0.18<sup>b</sup></td><td>7.75 ± 1.06<sup>b</sup></td><td>364.52 ± 6.58<sup>c</sup></td></tr><tr><td>5 % CO</td><td>6.25 ± 0.35<sup>c</sup></td><td>9.13 ± 0.53<sup>b</sup></td><td>325.82 ± 3.12<sup>b</sup></td></tr></tbody></table>

Means±SD with different superscripts (a, b, c) in a column differ significantly (p < 0.05).

was recorded between the three treatments. In addition, the inhibition properties of the layer of the film without CO were evaluated (Fig. 7) and no antimicrobial activities (no inhibition zone was formed) were found, indicating that only one layer (or one side) of the double-layered film (containing CO) showed antimicrobial activities. This indicates that CO did not diffuse from one layer to the other layer of the film possibly due to the hydrophobic nature of CO emulsion. Similar findings were reported by Maizura et al. (Maizura, Fazilah, Norziah & Karim, 2007) who found the inhibitory properties of sago starch alginate-based film containing lemongrass oil against *E. coli* at all the concentrations (0.1–0.4 %, v/v). In a similar study, the size of inhibitory halos against *Pseudomonas fluorescens* tended to increase with the increasing concentration of lemongrass oil in cassava starch-based film (Resianingrum et al., 2016).

Fahrullah et al. (Fahrullah & Rosyidi, 2021) also found a significant increase in the size of inhibitory halos with increasing concentration of clove essential oil in the edible film. The size of inhibitory halos is also affected by various other factors, such as incubation temperature, incubation time, homogeneity, microbial density, the turbidity of the bacterial suspension, drying time/impregnation into the agar medium, and the thickness of the agar medium (Bubonja-Šonje, Knezević & Abram, 2020). The inhibition zones formed against *S. aureus* were larger than *E. coli* and might be attributed to the cell walls of gram-positive bacteria which are hydrophobic allowing essential oils to penetrate and damage the cells (Nazzaro et al., 2013). Its hydrophobic nature allows better binding of non-polar compounds such as citronella oil. Further, the non-polar nature of the oil can inhibit the interaction of OH-ions of catechins with peptidoglycans, which ultimately causes leakage of gram-positive bacterial cells.

### 3.2.8. Antioxidant activity

The antioxidant activity of the double-layered film was evaluated by determining IC₅₀ (µg/mL, DPPH method) values (Table 3). A significant effect of CO was observed and the films containing CO showed significantly (p < 0.05) higher antioxidant activities compared to the control film (which showed no activity). Significantly lower IC₅₀ values of the film containing 5 % CO showed an effect of the concentration of CO on the antioxidant activity of the film. The IC₅₀ value is inversely proportional to the antioxidant activity of a compound. Similar concentration-dependent antioxidant activity has been reported by Mehdizadeh et al.

<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Zone of inhibition: (a) = layer without citronella oil and (b) = layer with citronella oil (K0 = 0 % CO, K1 = 1 % CO, K3 = 3 % CO and K5 = 5 % CO w/w).</figcaption></figure>
(Mehdizadeh, Tajik, Mehdi Razavi Rohani & Rassol Oromiehie, 2012) for starch and chitosan-based films containing Thymus kotschyanus essential oil. Another study has documented a similar effect of the concentration of citronella essential oil on the antioxidant activity of film composites (Go & Song, 2020). Antioxidants are chemical compounds that can donate electrons to unpaired free radicals, thereby reducing the oxidizing effects of free radicals that can cause carcinogenic and cardiovascular diseases and premature ageing (Schiavano et al., 2015). The citronella oil contains several antioxidant compounds such as terpenoids and secondary metabolites such as phenolic groups. These phenolic compounds can scavenge a free radical by donating hydrogen atoms through an electron transfer process (Mathew, Abraham & Zakaria, 2015).

## 4. Conclusions

The results showed a significant impact of drying time on tensile strength, elongation at break, solubility, and WVTR of the double-layered bioactive film and based on the quality characteristics, a drying period of 20 h was considered optimum. Where, in the treatment with drying for 20 h, the tensile strength value is 1.98 ± 0.01 MPa, elongation at break value is 66.13 ± 2.54 %, young's modulus value is 3.00 ± 0.10 MPa, water vapor transmission rate (WVTR) value is 9.20 ± 0.22 g/m2.h and water solubility value is 33.29 ± 0.01 %. The addition of citronella oil significantly affected the physicochemical characteristics of the film. It increased thickness, water solubility, tensile strength, and Young's modulus and decreased the WVTR and elongation at break. It also imparted antimicrobial and antioxidant activities to the double-layered film. The film containing the highest concentration (5 %) of citronella oil showed a larger circle of inhibition and a lower IC50 value. Overall, the double-layered film containing citronella essential oil on the active side proved to be effective in inhibiting microbial growth.

### CRediT authorship contribution statement

**Nandi Sukri:** Writing – original draft, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Fransisca Fiendra Natasha:** Writing – original draft, Validation, Investigation, Formal analysis, Data curation. **Yana Cahyana:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Herlina Marta:** Writing – review & editing, Methodology, Conceptualization. **Bambang Nurhadi:** Writing – review & editing, Methodology, Conceptualization. **Sandi Darniadi:** Writing – review & editing, Methodology, Conceptualization. **Sergio Torres-Giner:** Writing – review & editing, Validation, Data curation. **Zuhaib F. Bhat:** Writing – review & editing, Validation, Data curation. **Abderrahmane Aït-Kaddour:** Writing – original draft, Validation, Resources, Methodology, Data curation.

### Declaration of competing interest

The 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 are thankful to Universitas Padjadjaran for this research's facilities.

### Data availability

The authors do not have permission to share data.

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