# Carvacrol encapsulation in different carriers for PHBV-based active films: A comparative study of release profiles

Aynura Rzayeva, Mina Hariri, Nathalie Gontard, Valérie Guillard, Fanny Coffigniez

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Aynura Rzayeva, Mina Hariri, Nathalie Gontard, Valérie Guillard, Fanny Coffigniez. Carvacrol en-capsulation in different carriers for PHBV-based active films: A comparative study of release profiles. Food Packaging and Shelf Life, 2025, 50, pp.101565. 10.1016/j.fpsl.2025.101565. hal-05493246

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# Carvacrol encapsulation in different carriers for PHBV-based active films: A comparative study of release profiles

Aynura Rzayeva<sup>a,b</sup>, Mina Hariri<sup>a</sup>, Nathalie Gontard<sup>a</sup>, Valérie Guillard<sup>a</sup>, Fanny Coffigniez<sup>a,w</sup>

## ARTICLE INFO

Keywords:

Carvacrol encapsulation

Coating

Release

Headspace

Food simulant

## ABSTRACT

This study investigated the encapsulation of carvacrol, a natural phenolic compound with antimicrobial properties, into three different carriers: PHBV nanoparticles, β-cyclodextrin, and halloysite nanotubes. The encapsulated carvacrol was then integrated as a coating on PHBV film, and the release of the molecule in the headspace and food simulants was compared to the carvacrol release from the impregnated PHBV film. The results showed a high encapsulation efficiency (66 ± 5 %) but a slow release in the headspace (around 45 % in 30 days) for β-cyclodextrin, a moderate encapsulation efficiency (31 ± 2 %) but a faster release (more than 90 % in 10 days) for PHBV nanoparticles, and a very low encapsulation efficiency (less than 2 %) for halloysite. For all cases, the production of the film generated a 30 % loss of carvacrol, but the remaining concentration was homogeneous in all active films. The carvacrol release from the film in the headspace and food simulant demonstrated a faster release for coated films than impregnated ones, a higher affinity of carvacrol for ethanol than water (food simulant D1 with 50 % ethanol than A with 10 % ethanol), and a higher affinity for β-cyclodextrin than PHBV nanoparticles (coefficient partition higher for PHBV nanoparticles). These results highlighted that coated film with encapsulated carvacrol (PHBV nanoparticles and β-cyclodextrin) could be used for short shelf-life products (one week), while impregnated carvacrol film could be used for longer shelf-life products (a few months), the minimal inhibitory concentration against microorganisms being reached in headspace after this period for all cases.

## 1. Introduction

One of today's major challenges is the development of sustainable packaging that can all at once reduce environmental impact, limit food loss, maintain food quality, and assure food safety (Coffigniez et al., 2021; Guillard et al., 2018). To maximize food preservation and prolong food shelf life, active packaging is of interest in the food domain. This type of packaging is based on the integration of active substances, such as antioxidant or antimicrobial compounds, that could interact with food or the food environment (Bruni et al., 2024; Di Giuseppe et al., 2022; Rzayeva et al., 2023).

In this context, essential oils seem to be good candidates as active molecules thanks to their natural intrinsic antimicrobial, antifungal, and antioxidant properties, but also to their consumer-friendly status, the molecules from essential oils being generally recognised as safe (GRAS) (Atarés & Chiralt, 2016; Carpena et al., 2021). Essential oils are secondary metabolites of aromatic plants that possess bioactive substances

such as phenolic compounds, terpenes, and terpenoids (Nostro & Papalia, 2012; Varghese et al., 2020). Many studies demonstrated the effectiveness of essential oils, and more specifically of carvacrol, a natural phenolic compound derived from oregano oil, against a broad spectrum of food-borne pathogens (Mascheroni et al., 2011; Requena et al., 2017; Requena et al., 2019; Shi et al., 2022; Tampau et al., 2018; Yang et al., 2020). However, these kinds of active molecules (carvacrol and essential oils) tend to degrade during material processing owing to their sensitivity to environmental factors such as light, oxygen, and temperature, and they can quickly evaporate due to their volatile nature. Therefore, encapsulation is a crucial solution to prevent the loss of carvacrol by creating a protective environment around the volatile molecule, but also to control the active molecule release during storage, thus preserving their efficacy over time (Corrado et al., 2022; Hosseini et al., 2013).

Different carriers, such as cyclic oligosaccharides (cyclodextrins), polymeric nanoparticles, clay-based carriers (halloysite,

<footnote><sup>a</sup> IATE, Agro Polymers Engineering & Emerging Technology, Univ Montpellier, INRAE, Institut Agro, Montpellier & CIRAD, Montpellier, France</footnote>

<footnote><sup>b</sup> Department of Chemical Engineering, School of Engineering and Applied Science, Khazar University, 41 Mahsati Str., Baku AZ1096, Azerbaijan</footnote>

<footnote>* Correspondence to: IATE, 2 place Pierre Viala, Bat 31, Montpellier 34060, France.</footnote>

<footnote>E-mail address: fanny.coffigniez@umontpellier.fr (F. Coffigniez).</footnote>
montmorillonite), chitosan, etc., have been studied in the last decade to encapsulate carvacrol, enhance its stability and provide a controlled release (Alkan Tas et al., 2019; Mascheroni et al., 2010; Santos et al., 2015; Shakeri et al., 2014). Beta-cyclodextrin (β-cyclodextrin) is a cyclic oligosaccharide composed of seven glucopyranose units linked by alpha-(1-4) bonds, which is capable of forming an inclusion complex (active-carrier complex) through molecular complexation with a wide range of compounds (Del Valle, 2004). Its lipophilic inner cavity allows it to trap apolar molecules such as essential oils (Fang et al., 2013). When complexation occurs in an aqueous solution, water molecules within the β-cyclodextrin cavity are replaced by the more hydrophobic molecules in the solution. Upon drying, the complex remains stable, however, when rehydrated, it disintegrates, releasing the trapped volatile molecules (Del Valle, 2004). Encapsulation within β-cyclodextrin has been extensively studied and serves as a reference for comparing other carriers. From inorganic carriers, while montmorillonite has received significant attention and investigation for its ability to encapsulate volatile compounds (Abdullayev & Lvov, 2011; Alu'datt et al., 2022; Mascheroni et al., 2010, 2011; Rehman et al., 2020), halloysite is a less explored clay that shows promise for essential oil encapsulation. Indeed, the halloysite possesses a high surface area and unique tubular structure, which lets expect a high essential oil loading capacity (Abdullayev & Lvov, 2011; Santos et al., 2018). However, its potential as a bioactive carrier is still being explored and needs to be confirmed. Similarly, the use of biopolymers, such as Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), to nanoencapsulate carvacrol or essential oils have been employed thanks to their advantageous physical and chemical properties, as well as, their safe, bio-benign and eco-friendly characteristics (Rehman et al., 2020; Rzayeva et al., 2024), but still needs to be explored to confirm their interest regarding more usual carrier as β-cyclodextrin.

In addition to the encapsulation strategy, the incorporation method of the encapsulated molecule into the final packaging material can influence the release of the molecule and consequently affect the efficiency of the packaging against microbial growth (Kurek et al., 2014). Indeed, active molecules such as carvacrol could be incorporated directly into the polymer matrix or coated on the surface of the packaging material in an encapsulated (Cui et al., 2023; López-Gómez et al., 2023; Rivera et al., 2024; Shi et al., 2022; Tampau et al., 2018; Wicochea-Rodríguez et al., 2019) or non-encapsulated form (Chalier et al., 2009; Kurek et al., 2014; Mascheroni et al., 2011; Requena et al., 2016), using casting, electrospinning, spraying, compression molding or extrusion methods.

Several authors have studied the release of carvacrol into a headspace and/or food simulant, obtaining different results, probably due to the different incorporation and encapsulation strategies. For example, Wicochea-Rodríguez et al. (2019) showed almost 80 % release of carvacrol from soy protein isolate coated paper when the molecule was encapsulated in β-cyclodextrin after 50 days at 30 °C, while Kurek et al. (2014) showed a total release of carvacrol from chitosan including carvacrol, after 7 days at 20 °C. However, most of the work studied the impact of environmental conditions (temperature and relative humidity), the amount of active compound, or the strategy of integration of the molecule (in the whole mass of the film or coating on a surface of the film) on the release of the molecules (Chalier et al., 2009; Kurek et al., 2014; López-Gómez et al., 2023; Mascheroni et al., 2011; Shi et al., 2022). To the best of our knowledge, a comparison of carvacrol release using different encapsulation and incorporation strategies has not been conducted, although this information could be helpful in selecting a strategy that will be as close as possible to the needs of the food.

In this context, the aim of the present study was to compare different strategies of carvacrol encapsulation: β-cyclodextrin, PHBV nanoparticles and halloysite and their impact on the release of the molecules into a headspace and different food simulants from an active coated PHBV film. PHBV was chosen as a polymer for encapsulation, coating and film support for their sustainable characteristics: this latest one

being bio-based, biodegradable and non-toxic; but also for their strong mechanical and barrier properties (Chea et al., 2016; Di Giuseppe et al., 2022). An impregnated PHBV film with carvacrol was produced as a reference for a non-encapsulation strategy and integration throughout the entire film mass.

To the best of our knowledge, this work (1) represents the first systematic comparison of encapsulation carriers and integration methods for carvacrol in PHBV films, bridging critical gaps in optimizing controlled-release packaging to specific food needs, (2) develops a dual-pathway analysis providing actionable insights for both vapor-phase and direct-contact applications, (3) advances sustainable packaging by eliminating microplastic persistence risks associated with traditional carriers thanks to the use of PHBV, a biodegradable polymer.

## 2. Materials and methods

### 2.1. Materials

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was used in pellet form (PHI002) or powder (PHI003) form and was purchased from NaturePlast (Ifs, France). PHI002 (pellet) is a commercially formulated grade that contains 3 % valerate and 1 % boron nitride (nucleating agent) and presents a molecular weight (Mw) of 244 ± 15 kDa. PHI003 (powder) is a commercial non-formulated grade that contains 3 % valerate and presents a molecular weight (Mw) >1100 kDa.

All compounds: boron nitride (98 %), carvacrol (98 %), β-cyclodextrin (≥ 97 %), halloysite, sodium dodecyl sulfate (SDS, ≥ 99 %) and solvents: dichloromethane (≥ 99.9 %), chloroform (≥ 99.8 %), ethanol (≥ 99.8 % and 96 %), hexane (≥ 95 %), hydrochloric acid (37 %) were purchased from Sigma Aldrich (France).

### 2.2. Carvacrol encapsulation

#### 2.2.1. The different encapsulation methods

##### 2.2.1.1. Encapsulation of carvacrol in PHBV nanoparticles.
Carvacrol encapsulation in PHBV nanoparticles was done using the emulsification method, adapted from Corrado et al. (2022) and Rzayeva et al. (2024). The first step is to decrease the molecular weight of the PHBV, which was achieved by a thermo-mechanical process. After drying PHBV (PHI002) at 60 °C, pellets were transformed into films (size of 12 × 12 cm and 300 µm of thickness) using a hydraulic press (CFM 20 T, Pinette Emidecau Industries, Chalon sur Saone Cedex, France) at 180 °C with the following time-pressure conditions: 1 min 30 at 5 bar, then 30 s at 25 bar, 30 s at 50 bar, 30 s at 75 bar, 30 sec at 100 bar and 1 min 30 sec at 150 bar. The PHBV film was then cut into fragments of 1 cm × 1 cm and dissolved, under stirring, in a dichloromethane solution (5 mg/mL) at 40 °C for 2 h in a closed bottle. After cooling the solution to room temperature (22 °C), 25 mg of carvacrol was added to the stirred solution for an additional hour. This solution was poured into a SDS solution at 5 mg/mL, and the mixture was homogenized at 7000 rpm for 10 min using a shear mixer (L4RT, High, Silverson, England) in an ice bath. The dichloromethane was evaporated by heating the mixture at 40 °C under agitation, and the nanoparticles were recovered after three centrifugations at 2795 g (5000 rpm) for 10 min using a Megafuge centrifuge (16 R, ThermoScientific, Germany). The nanoparticles were then dried in a desiccator for 24 h. The nanoparticles were prepared in triplicate and stored in a closed container at -20 °C for a maximum of one month before use in the release assay or film production.

##### 2.2.1.2. Encapsulation of carvacrol in β-cyclodextrin.
Carvacrol encapsulation in β-cyclodextrin was done using the freeze-drying method, adapted from Santos et al. (2015). Carvacrol and β-cyclodextrin were mixed in distilled water at room temperature (22°C) for 48 h under
stirring at 1000 rpm, using a 1:1 molar mass ratio at a solution con- centration of 10 g/L (preparation in closed bottle). After freezing at – 20 °C, the solution was lyophilized at – 50 °C under a vacuum of 0.01 mbar for 48 h using a freeze dryer (Cryotec Cosmos 20 K, France). The samples were prepared in triplicate and stored in a closed container at – 20 °C for a maximum of one month before use in the release assay or film production.

2.2.1.3. Encapsulation of carvacrol in halloysite. Preparation of halloy- site: Carvacrol encapsulation in halloysite was done after acidification of the encapsulant. The acidification of the halloysite was performed as described by Alkan Tas et al. (2019). Halloysites were dispersed in 0.1 M hydrochloric acid at a concentration of 1 g/L and then mixed for 8 h at 60 °C. The solution was centrifuged at 2795 g (5000 rpm) for 10 min using a Megafuge centrifuge (16R, ThermoScientific, Germany). The recovered halloysites were washed with ultrapure water until a neutral pH was obtained, then dried at 50 °C for 24 h. Finally, the samples were crushed and filtered through using a 100 µm sieve (Retsch, Germany) for carvacrol encapsulation.

Encapsulation of carvacrol: Carvacrol and halloysite were mixed using a 17:1 molar mass ratio, ultrasonicated for 30 min using an ultrasonication bath (Digital Ultrasonic Cleaner), then put two times under vacuum cycle (Buchi V-600) of 1 mbar for 30 min twice, by pausing for 10 min between cycles. Afterwards, carvacrol-loaded hal- loysite were rinsed with ethanol, centrifuged at 2795 g (5000 rpm) for 5 min using a Megafuge centrifuge (16R, ThermoScientific, Germany) and dried in a desiccator for 24 h. The samples were prepared in trip- licate and stored in a closed container at – 20 °C for a maximum of one month before being used in the release assay or film production.

2.2.2. Carvacrol extraction from encapsulants

2.2.2.1. Carvacrol extraction from PHBV nanoparticles. To proceed to carvacrol extraction, 10 mg of nanoparticles were mixed and stirred in 5 mL of hexane: ethanol (1:1) solution at room temperature for 72 h, then the solution was filtered through 0.22 µm (ait, France) as described by Rzayeva et al. (2024). The carvacrol was then quantified by absor- bance measurement at 275 nm using a spectrophotometer (Evolution 300 UV-Vis Spectroscopy, Thermo Scientific). The analysis was done in triplicate.

The quantity of encapsulated carvacrol in the nanoparticles (Carv$_{t}$) (mg) was calculated using the following equation:

$$
\text{Carv}_{\text{t}} = \frac{(\text{Abs} - \text{Abs}_{0}) \times \text{Dil} \times \text{V}_{\text{tot}} \times \text{W}_{\text{t}}}{k \times l \times \text{W}_{\text{te}}} \quad (1)
$$

where Abs is the sample absorbance at 275 nm; Abs₀ is the blank absorbance, corresponding to the negative control going through the same encapsulation and extraction protocol procedures as the sample but without carvacrol; k is the extinction coefficient of carvacrol at 275 nm (Rzayeva et al., 2024), l is the width of the spectrophotometer cell, Dil is the dilution factor used before measurement to spectropho- tometer if necessary, Vtot is the total volume of the assay, Wt is the quantity of encapsulated carvacrol obtained after encapsulation (mg), and Wte is the quantity of encapsulated carvacrol used for carvacrol extraction.

2.2.2.2. Carvacrol extraction from β-cyclodextrin. Carvacrol extraction from β-cyclodextrin was performed by stirring 30 mg of carvacrol- encapsulated β-cyclodextrin complexes in 5 mL of ultrapure water at room temperature for 10 min (López-Gómez et al., 2023). Then, the mixture was ultrasonicated twice for 5 min, using an ultrasonication bath (Digital Ultrasonic Cleaner). 10 mL of hexane was added to the mixture, and samples were let to decant for 30 min. The hexane and water phases were then separated by centrifugation at 2795 g (5000 rpm) for 10 min using a Megafuge centrifuge (16R,

ThermoScientific, Germany). The carvacrol was quantified by measuring the absorbance of the hexane phase at 275 nm using a spectrophotometer (Evolution 300 UV-Vis Spectroscopy, Thermo Sci- entific). The analysis was done in triplicate and the quantity of encap- sulated carvacrol in β-cyclodextrin (Carv$_{t}$) (mg) was calculated using the Eq. (1).

2.2.2.3. Carvacrol extraction from halloysite. The extraction of the carvacrol from halloysite was performed by stirring 20 mg of encapsu- lated carvacrol in 10 mL of hexane at room temperature for 10 min (Zheng et al., 2022). Then the mixture was ultrasonicated for 30 min, using an ultrasonication bath (Digital Ultrasonic Cleaner). Samples were centrifuged at 2795 g (5000 rpm) for 10 min, using a Megafuge centri- fuge (16R, ThermoScientific, Germany). The carvacrol was quantified by measuring the absorbance of the hexane at 275 nm using a spectro- photometer (Evolution 300 UV-Vis Spectroscopy, Thermo Scientific). The analysis was done in triplicate, and the quantity of encapsulated carvacrol in halloysite (Carv$_{t}$) (mg) was calculated using the Eq. (1).

2.2.3. Determination of efficiency of the encapsulation method

For all types of encapsulants (PHBV, β-cyclodextrin and halloysite), process efficiency, carvacrol recovery, encapsulant recovery and loading capacity were determined as described by Rzayeva et al. (2024).

The process efficiency was the quantity of material recovered after encapsulation over the total quantity of initial material involved in the formulation and was calculated using the following equation:

$$
\text{Process Efficiency} = \frac{W_t}{\text{Carv}_t + E_i} \times 100 \qquad (2)
$$

where Wt is the quantity of material obtained after encapsulation (mg), Carv$_{t}$ and E$_{i}$ are the quantity of carvacrol and encapsulants, respectively, initially introduced in the preparation (mg).

The carvacrol recovery was the quantity of carvacrol recovered after encapsulation over the initial quantity used to make the formulation and was calculated using the following equation:

$$
\text{Carvacrol recovery} = \frac{\text{Carv}_t}{\text{Carv}_t} \times 100 \qquad (3)
$$

where Carv$_{t}$ is the quantity of carvacrol detected in the material after encapsulation (mg) and Carv$_{i}$ is the quantity of carvacrol initially introduced in the preparation (mg).

The encapsulant recovery was the quantity of encapsulant material (PHBV, β-cyclodextrin, or halloysite) obtained after encapsulation (mg) over the initial quantity used to make the formulation and was calcu- lated using the following equation:

$$
\text{Encapsulant recovery} = \frac{W_t - \text{Carv}_t}{E_i} \times 100 \qquad (4)
$$

Where Wt is the quantity of material obtained after encapsulation (mg), Carv$_{t}$ is the quantity of carvacrol detected in the material after encap- sulation (mg) and E$_{i}$ is the quantity of encapsulant initially introduced in the preparation (mg).

The loading capacity was the quantity of carvacrol recovered after encapsulation over the quantity of encapsulant (PHBV, β-cyclodextrin, or halloysite) present in the material after encapsulation and was calculated using the following equation:

$$
LC = \frac{Carv_t}{W_t - Carv_t} \times 100 \qquad (5)
$$

where, Carv$_{t}$ is the quantity of carvacrol detected in material after encapsulation (mg), and W$_{t}$ is the quantity of material obtained after encapsulation (mg).
### 2.2.4. Evaluation of carvacrol release from encapsulants into the headspace

15 mg of carvacrol encapsulated in PHBV or 30 mg of carvacrol encapsulated in β-cyclodextrin (powder form) were stored in a 0.5 L glass cells (Le Parfait, France), hermetically closed with steel lid. A saturated humidity was obtained by adding 50 mL of distilled water in a separate container at the bottom of the glass cell. Then, glass cells were placed in a climatic chamber (Memmert, Germany) at 30 °C for 80 days. Periodically, the remaining carvacrol in the material was extracted and quantified as described in Section 2.2.2. The experiments were done in triplicate. The released carvacrol in the headspace was determined as follows:

$$ \%Carv_r = \frac{m_{Carv0} - m_{Carvt}}{m_{Carv0}} \times 100 \qquad (6) $$

where %Carv_r is the percentage of the released carvacrol into headspace (%), mCarv0 and mCarvt are the amount of the carvacrol extracted at “0” and t times, respectively (mg).

The concentration of carvacrol in the headspace was calculated using the following equation:

$$ Carv_h = \frac{m_{Carv0} - m_{Carvt}}{V_h} \times 10^3 \qquad (7) $$

where Carv_h is the quantity of the released carvacrol into headspace (ug/mL), mCarvt is the amount of the carvacrol extracted at t times (mg) and V_h is the volume of headspace in the glass cell (mL or cm³).

## 2.3. Active PHBV films containing carvacrol

### 2.3.1. Preparation of the active film

#### 2.3.1.1. Production of PHBV film.
PHBV powder (PHI003) was mixed with boron nitride (0.5 % w/w) in a powder blender (Turbula T10B, Laboservices, Montpellier, France) and dried at 60 °C for 48 h. Then, the mixture (2 g for one film) was melt and transformed into film between two Teflon-coated plates (Taconic, France) using a hydraulic thermo-press (CFM 20 T, Pinette Emidecau Industries, Chalon sur Saone Cedex, France) at 178°C and the following time/pressure conditions: 5 min at 0 Pa (just contact), 1 min 30 sec at 5 bar, then 30 s at 25 bar, 30 s at 50 bar, 30 s at 75 bar, 30 s at 100 bar, and 1 min 30 s at 150 bar. The obtained films were cooled to room temperature by placing a cold-water bath on the surface of the metal plates for 5 min. The films, measuring 6 cm × 6 cm (1.8 g) and approximately 300 µm in thickness, were stored in a desiccator at room temperature until use.

#### 2.3.1.2. Coating of PHBV film.
The coating solution was prepared by solving and mixing 4 % w/v of PHBV powder (PHI003) in chloroform at 60 °C for an hour. Then encapsulated carvacrol (in β-cyclodextrin or PHBV) was added and stirred to 1 mL of this solution so as to obtain a coated film with 0.05 % of carvacrol (for example, 0.9 mg of carvacrol was necessary for a film of 1.8 g, meaning 6.3 mg of carvacrol encapsulated in PHBV or 10.2 mg of carvacrol encapsulated in β-cyclodextrin, using the loading capacity). The 1 mL of the active solution was deposited on the surface of the PHBV (produced as described before in this present section) film using a Terumo syringe (Luer slip tip, Thermo Scientific). Then, the chloroform was dried at room temperature for a few minutes. Coating with blank encapsulants (see Section 2.2.2) was also prepared. The weight and thickness of each film were measured before and after coating using a balance LX 220 A (BALCO, Switzerland) and a micrometer MDC-25SX (Mitutoyo, Japan). The samples were stored in a closed container at – 20 °C for a maximum of one month before release evaluation.

#### 2.3.1.3. Impregnation of PHBV film.
PHBV powder (PHI003) was mixed with boron nitride (0.5 % w/w) in a powder blender (Turbula T10B, Laboservices, Montpellier, France) and dried at 60 °C for 48 h. A

solution of 20 mg/mL of carvacrol was prepared in ethanol and added to the PHBV mixed in closed bottle in order to obtain a final material with 0.05 % w/w of carvacrol. The impregnation was done at 40 °C for 7 days using a Reax 2 rotator (Heidolph, Germany). The mixture was then transformed into film using a hydraulic thermopress, as described before in this present section. The samples were stored in a closed container at – 20 °C for a maximum of one month before release evaluation.

### 2.3.2. Evaluation of carvacrol recovery and distribution in/on the film after production

#### 2.3.2.1. From a macroscopic point of view.
Active films (impregnated and coated) of 36 cm² were cut into four equal parts from which carvacrol was separately extracted at room temperature for 72 h using 20 mL of hexane/ethanol mixture as solvent (1:1). Samples were then ultrasonicated for 30 min, and filtered through 0.22 µm (ait, France). The carvacrol was quantified by absorbance measurement at 275 nm using a spectrophotometer (Evolution 300 UV-Vis Spectroscopy, Thermo Scientific). The analysis was done in triplicate.

#### 2.3.2.2. From a microscopic point of view.
Active films of 0.5 cm width were deposited on carbon adhesive tape and metallized with a 4 nm layer of Au/Pd sputter coating (Quorum Sputter Coater SC7620) at room temperature. The structure of the films was observed using a benchtop Phenom Pro X scanning electron microscope (Phenom World, Eindhoven, The Netherlands) with a backscattered electron detector and an acceleration voltage of 10 kV.

### 2.3.3. Evaluation of carvacrol release from an active film into the headspace

One active film of 1.8 g containing 0.05 % w/w (6 cm×6 cm) of carvacrol was stored in 0.5 L glass cells (Le Parfait, France), hermetically closed with a steel lid. The release of carvacrol into headspace was made in the same conditions as described in Section 2.2.4, and the remaining carvacrol was extracted from the film and quantified, as described in Section 2.3.2. Experiments were done in triplicate. The percentage of released carvacrol into headspace is calculated using Eq. (6).

### 2.3.4. Evaluation of carvacrol migration from active film into food simulants

Two food simulants were selected to study the evaluation of the carvacrol migration: food simulant A (10 % (v/v) ethanol) corresponding to hydrophilic food and simulant D1 (50 % (v/v) ethanol) corresponding to lipophilic food (European Standard No 10/, 2011). 60 cm² of films (6 discs of 10 cm²) were immersed in 100 mL of each food simulant (area-to-volume ratio around 6 dm²/L) into a closed glass cell of 150 mL at 30 °C in a climatic chamber (Memmert, Germany) for 1–10 days, depending on incorporation strategies and food simulant type. Then, the remaining carvacrol in/on the film was extracted and quantified using the procedure described in Section 2.3.2. Additionally, the carvacrol that migrated into the food simulant was quantified by measuring the absorbance of the food simulant at 275 nm using a spectrophotometer (Evolution 300 UV-Vis Spectroscopy, Thermo Scientific). The experiments were conducted in triplicate. The percentage of the total released carvacrol from the film is calculated using Eq. (6). The percentage of carvacrol in food simulant and headspace, respectively, were calculated as follows:

$$ \%Carv_f = \frac{m_{Carv\_food}}{m_{Carv0}} \times 100 \qquad (8) $$

$$ \%Carv_h = \%Carv_r - \%Carv_f \qquad (9) $$

where %Carv_f and %Carv_h are the percentage of the carvacrol into food and headspace respectively (%), %Carv_r is the percentage of the total released carvacrol from the film (%), mCarv,food is the amount of carvacrol
crol present in food simulant at “t” time (mg), and $m_{carv0}$ is the amount of carvacrol extracted from the film at “0” time (mg).

## 2.4. Evaluation of partition coefficient and apparent diffusivities

### 2.4.1. Partition coefficient

The partition coefficient is defined as the ratio of the carvacrol concentration between two phases, as following:

$$K = \frac{C_{g,orL}}{C_s} \qquad (10)$$

With

$$C_g = \frac{(m_{carv0} - m_{carvt.eq} - m_{Carvt.food.eq})}{V_h} \times 10^3 \qquad (11)$$

$$C_L = \frac{m_{Carvt.food.eq}}{V_L} \times 10^3 \qquad (12)$$

$$C_s = \frac{m_{Carvt.eq} \times d_s}{m_s} \times 10^3 \qquad (13)$$

where $C_{g,orL}$ is the carvacrol concentration in the headspace (gas) or food simulant (liquid) (µg/mL), and $C_s$ is the carvacrol concentration in the solid media: powder or film (µg/mL), $m_{carvt.eq}$ and $m_{Carvt.food.eq}$ are the amount of the carvacrol extracted from powder or film and the amount of carvacrol present in food simulant at equilibrium (mg), $m_{carv0}$ is the amount of carvacrol extracted from the film at “0” time (mg), $V_h$ is the volume of headspace in the glass cell (mL or cm³), $V_L$ is the volume of food simulant (mL), $m_s$ is the mass of powder, film, or coated layer used or obtained during experiments (g), $d_s$ is the density of PHBV (1.25 g/mL) or β-cyclodextrin (1.44 g/mL).

### 2.4.2. Apparent diffusivities

The apparent diffusion of carvacrol from film into headspace was modeled using the Fick's second law (Eq. (14)), assuming that (i) the film is a one dimensional infinite plane sheet (ii) the thickness of the film is homogeneous and unchanged during the experiments, and (iii) carvacrol is homogeneously distributed in the film and in headspace (Di Giuseppe et al., 2022; Bruni et al., 2024).

$$\frac{M_t}{M_\infty} = 1 - \sum_{n=0}^{\infty} \frac{2\alpha(1+\alpha)}{1+\alpha+\alpha^2 q_n^2} \exp\left\{-\frac{D q_n^2}{L^2} t\right\} \qquad (14)$$

$$With \alpha = \frac{1}{K_{P,HS}} \frac{V_{HS}}{V_P} \qquad (15)$$

Where, Mt is the total amount of carvacrol in headspace at time t and M∞ is the total amount of carvacrol in headspace at the steady state, VP is the polymer volume and VHS the headspace volume, (qn)n, the positive roots of the equation tanq= -αq and KP,HS, the partition coefficient of the additive in the polymer/headspace system.

The numerical simulations were carried out using Matlab® software and the lsqnonlin function to estimate the apparent diffusivity. For each model fitting, the quality of fit was estimated through the percentage of Root Mean Square Error (RMSE):

$$RMSE = \frac{1}{M_0} \sqrt{\frac{1}{N} \sum_{i=1}^{N} ((M_t)_{experimental} - (M_t)_{predicted})^2} \times 100 \qquad (16)$$

where M0 is the initial mass of carvacrol in the film and Mt is the mass of carvacrol into headspace at time t.

## 2.5. Statistical analysis

All experiments were performed in triplicate (n = 3) and data were expressed as mean ± standard deviation. A student test was conducted

on data in order to determine differences with a significance level of 5 % (p < 0.05).

# 3. Results and discussion

## 3.1. Carvacrol encapsulation

### 3.1.1. Efficiency of the encapsulation method

Fig. 1 presents the recovery of carvacrol and encapsulant, as well as the loading capacity and process efficiency using PHBV, β-cyclodextrin and halloysite to encapsulate carvacrol. Results showed that β-cyclodextrin was the encapsulant allowing to obtain the highest carvacrol recovery and process efficiency after encapsulation (65.5 ± 5.4 % and 86.2 ± 1.1 % respectively), following by PHBV (30.7 ± 2.0 % and 71.0 ± 0.0 % respectively), then halloysite (2.0 ± 0.0 % and 9.3 ± 0.8 % respectively), while encapsulant recovery was high and quite similar for all encapsulants (between 80.3 % and 91.2 %). On the contrary, the loading capacity was higher for halloysite (23.9 ± 0.3 %) and PHBV (16.9 ± 1.3 %) than β-cyclodextrin (9.7 ± 0.8 %). It means that less encapsulant material could be used to encapsulate carvacrol in halloysite and PHBV, which could reduce the total cost of encapsulation. However, using the molecular mass of each encapsulant (258 g/mol; 244 g/mol and 1135 g/mol for halloysite, PHBV and β-cyclodextrin respectively), this loading capacity corresponded to the encapsulation of 0.41; 0.27 and 0.73 mol of carvacrol for one mole of encapsulant (halloysite, PHBV and β-cyclodextrin respectively). Therefore, in the light of these results, β-cyclodextrin remains the better encapsulant for carvacrol (higher carvacrol recovery), PHBV is a promising solution (high loading capacity), while halloysite is a strategy to exclude (carvacrol recovery and process efficiency are both too low).

These results are similar to the ones found in the literature (Phunpee et al., 2016; Trindade et al., 2019). For example, Phunpee et al. (2016), Shakeri et al. (2014) and Trindade et al. (2019) obtained a carvacrol recovery of 81 % and 21 % using β-cyclodextrin and PHBV, respectively, while Lu et al. (2022) and Shakeri et al. (2014) obtained a loading capacity of carvacrol of 12.5 % and 24.2 % using PHBV and halloysite, respectively.

The difference of encapsulation efficiency between the three complexes could be due to a difference of morphology and structural properties. Indeed, Fig. 2 displayed spherical PHBV nanoparticles; crystalline complexes for β-cyclodextrin and fibrous aspect for halloysites, as already observed by Krishnaswamy et al. (2012), Levis and Deasy (2002); and Rzayeva et al. (2024). Based on geometrical consideration, Lisuzzo et al. (2019) showed that halloysite had a maximal encapsulation efficiency of 10 % due to lumen capacity.

The stability of complexes was observed on short term (one month),

<figure><img src="image_4.png" /><figcaption>Fig. 1. Encapsulation efficiency of carvacrol (carvacrol recovery, encapsulant recovery, loading capacity and process efficiency) using different encapsulants: PHBV, β-cyclodextrin and halloysite. Bars with same letters were not significantly different (p < 0.05).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Scanning electron microscopy images of the complexes obtained after encapsulation of carvacrol in (A) nanoparticles of PHBV, (B) β-cyclodextrin and (C) halloysite. The scale bar represented 10 µm.</figcaption></figure>

but still need to be validated on long term (6 months) under different environmental conditions (temperature, humidity).

### 3.1.2. Carvacrol release from encapsulants into the headspace

Fig. 3 shows the release of carvacrol from PHBV nanoparticles and β-cyclodextrin encapsulants into the headspace at 30 °C and 100 % relative humidity. From PHBV nanoparticles, the release of carvacrol increased with time until reaching a plateau of 95 % after 10 days, whereas from β-cyclodextrin, a plateau of 45 % was reached after 30 days. This release allowed to obtain a carvacrol concentration in the headspace at equilibrium of 3.7 µg/mL from PHBV nanoparticles and 2.8 µg/mL from β-cyclodextrin complexes (Fig. 3), that was enough to overpass or be closed to the minimal inhibitory concentration, that is known to be between $4.62 \times 10^{-2}$ µg/mL (Kurek et al., 2013) and 5 µg/mL (Arfa et al., 2007). Similar release observations have been reported in the literature (Alinaqi et al., 2021; Hosseini et al., 2013; Oliveira et al., 2023; Shakeri et al., 2014; Yang et al., 2020). For example, Yang et al. (2020) showed a close to 100 % release of cinnamaldehyde encapsulated in polyhydroxyalkanoate after 7 days at room temperature, while Li et al. (2010) showed a 93 % release of cinnamon encapsulated in β-cyclodextrin after 30 days at 40 °C (98 % relative humidity).

The faster and higher release of carvacrol from PHBV nanoparticles than β-cyclodextrin could be due to the presence of carvacrol molecules on the external surface instead of trapped molecules in the core of the PHBV nanoparticles and to lower H-bonding and hydrophobic interaction between carvacrol and PHBV than carvacrol and β-cyclodextrin (Cheng et al., 2024; Szente & Szemán, 2013); but also to a difference in complexes morphology (Fig. 2). The higher affinity of carvacrol for β-cyclodextrin than PHBV was also confirmed by the partition

coefficient estimation (Fig. 4). Indeed, the carvacrol partition coefficient between headspace and powder was evaluated at $9.1 \times 10^{-4}$ (log K = -3.0) and $4.4 \times 10^{-5}$ (log K = -4.4) for PHBV nanoparticles and β-cyclodextrin respectively, indicated in both cases a higher affinity for solid polymeric phases than air, and a higher affinity for PHBV than β-cyclodextrin (around 20 times more).

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Logarithm of partition coefficient K of carvacrol between headspace or food simulant and the solid media (film or powder) at 30 °C (and 100 % HR for headspace) when the carvacrol was impregnated into the film or encapsulated in nanoparticles or β-cyclodextrin and coated on PHBV films. Bars with same letters were not significantly different (p < 0.05).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Carvacrol release from PHBV nanoparticles (A) and β-cyclodextrin complexes (B) into headspace at 30 °C and 100 % RH in a closed system.</figcaption></figure>
## 3.2. Active PHBV films containing carvacrol

### 3.2.1. Impact of the process production on carvacrol stability and distribution into the PHBV film

The PHBV/carvacrol sheets were divided into four equal parts, and the carvacrol was extracted and quantified from each part separately. The results showed a similar quantity of carvacrol in each part of the film, with a value of 0.033 ± 0.003 g/100 g for impregnated carvacrol in PHBV film, 0.031 ± 0.002 g/100 g for carvacrol encapsulated in nanoparticles and coated on a PHBV film, and 0.030 ± 0.003 g/100 g for carvacrol encapsulated in β-cyclodextrin and coated on a PHBV film. Although the film production allowed a good homogeneity of carvacrol in films, the total amount of carvacrol extracted from film was only 65 ± 6 % of the initial amount of carvacrol introduced in impregnated film; 62 ± 3 % of the initial amount of carvacrol encapsulated in nanoparticles and coated on PHBV film; and 61 ± 5 % of the initial amount of carvacrol encapsulated in β-cyclodextrin and coated on PHBV film. Therefore, the impregnation (40 °C), compounding and thermopressing (180 °C) processes lead to 35 % loss of carvacrol, essentially due to the high volatility of the molecule and its increasing release to increasing temperature (Chalier et al., 2009). Moreover, with the thermal degradation of carvacrol being close to 200 °C, partial degradation of the molecule could occur during compounding and thermopressing processes (Li et al., 2022). The coating processes generated almost 40 % of carvacrol loss, probably occurring during the evaporation of the chloroform phase. Indeed, some of the carvacrol molecules could be non-entrapped and fixed on the surface of the film due to modification of the structure, environment or bounding between carvacrol and encapsulant generated by the solvent presence (chloroform) and were volatilized during the solvent evaporation (Alinaqi et al., 2021; Astray et al., 2009; Loftsson & Duchène, 2007)

Fig. 5 shows the surface and cross-section of PHBV (A and E); PHBV impregnated with carvacrol (B and F); PHBV coated with carvacrol encapsulated in nanoparticles (C and G), and PHBV coated with carvacrol encapsulated in β-cyclodextrin (D and H). The surface and cross-section of PHBV films impregnated with carvacrol were smooth and homogeneous (A, B, E, F). However, the surfaces of the coated films with encapsulated carvacrol (in nanoparticle or β-cyclodextrin) were non-

homogeneous, with the presence of some pores (C, D), probably generated by the solvent evaporation, as already observed by Bruni et al. (2024). Moreover, PHBV films coated with carvacrol encapsulated in β-cyclodextrin (D) presented some heaps on the surface, unlike the PHBV films coated with carvacrol encapsulated in nanoparticles (C), proving that dispersion of PHBV nanoparticles in chloroform was easier than dispersion of β-cyclodextrin, this latest one being more soluble in water than organic solvent (Allan Hedges, 2009). However, the cross-sectional views of the coated PHBV films (G and H) showed a homogeneous coating layer that was measured to be 24 ± 6 µm and 21 ± 6 µm for the PHBV nanoparticles and β-cyclodextrin layers, respectively, corroborating the macroscopic results.

### 3.2.2. Carvacrol release from PHBV films into the headspace

Fig. 6 shows the carvacrol release from PHBV film (i) impregnated with carvacrol, (ii) coated with carvacrol encapsulated in PHBV nanoparticles, and (iii) coated with carvacrol encapsulated in β-cyclodextrin into the headspace at 30 °C and 100 % relative humidity.

From impregnated PHBV films, the carvacrol release increased with time, reaching a maximum of 90 % of the initial carvacrol after 80 days (no plateau reached), corresponding to a carvacrol headspace concentration of 1.1 µg/mL. The release of carvacrol was faster from coated active films, with a plateau reached after 30 and 40 days for nanoparticles and β-cyclodextrin, respectively. This faster release of carvacrol from coated films than impregnated ones could be attributed (i) to the lower thickness of the layer containing the same quantity of carvacrol (21 or 23 µm against 398 µm) decreasing the diffusion pathway between carvacrol place and headspace, and (ii) to the porosity of the coated layer generated by the solvent evaporation (see Section 3.2.1) (Ulloa et al., 2017; Yang et al., 2020). However, as already observed from encapsulant results (Section 3.1.2), the release was higher for nanoparticles (96 % after 80 days) than β-cyclodextrin (58 % after 80 days). This maximum corresponded to carvacrol headspace concentrations of 1.3 and 0.8 µg/L for nanoparticles and β-cyclodextrin-coated films, respectively, which were sufficient to reach the minimal inhibitory concentration (between 4.62 × 10⁻² µg/mL and 5 µg/mL) (Arfa et al., 2007; Kurek et al., 2013). The slower release of coated carvacrol encapsulated in β-cyclodextrin compared to nanoparticles was due to

<figure><img src="image_8.png" /><figcaption>Fig. 5. Scanning electron microscopy images of PHBV film, PHBV film with 0.05 % w/w of impregnated carvacrol, PHBV film coated with 0.05 % w/w of carvacrol encapsulated in PHBV nanoparticles and PHBV film coated with 0.05 % w/w of carvacrol encapsulated in β-cyclodextrin, with a surface view ((A)–(D) respectively) and cross-sectional view ((E)–(H) respectively). The scale bar represented 100 µm.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Carvacrol release from PHBV film impregnated with carvacrol (A), PHBV film coated with carvacrol encapsulated in PHBV nanoparticles (B) and PHBV film coated with carvacrol encapsulated in β-cyclodextrin (C) into headspace at 30 °C and 100 % RH in a closed system. Dots represented experimental data, while lines represented simulated data.</figcaption></figure>

the necessity of high relative humidity to open β-cyclodextrin and liberate the entrapped carvacrol (Kurek et al., 2017). Therefore, contrary to nanoparticles, the carvacrol release from β-cyclodextrin necessitated two steps: the diffusion of water from headspace through the polymer, which turned out to be low ($D = 3.3 \times 10^{-13} \text{ m}^2/\text{s}$) (Crétois et al., 2014), then diffusion of carvacrol from PHBV into headspace, as already shown on AITC encapsulated in β-cyclodextrin (Kurek et al., 2017). These release results were similar to those observed by Wicochea-Rodríguez et al. (2019), which showed a release of 75 % of carvacrol encapsulated in β-cyclodextrin and coated with soy protein isolate on paper after 50 days at 30 °C and 80 % relative humidity.

The partition coefficient values between headspace and film (Fig. 4) also confirmed a trend to lower affinity of carvacrol (16 times more) for nanoparticles coated films ($2.4 \times 10^{-3}$, or $log K = -2.6$) (and consequently a higher release) than β-cyclodextrin coated films ($1.5 \times 10^{-4}$, or $log K = -3.8$). These values were approximately 3 times higher than those obtained from encapsulants (Section 3.1.2), likely due to the higher concentration of carvacrol in encapsulants compared to films. Moreover, the partition coefficient of carvacrol between headspace and impregnated films was evaluated to be $2.7 \times 10^{-2}$ (or $log K = -1.6$), so it was around 10 times higher than the one for nanoparticle-coated films. This difference was principally due to the fact that only the coating layer was taken into account in the partition coefficient calculation for coated films, which only represented 3.2 % of the total film mass. It should be

noticed that the partition coefficient was probably higher for impregnated film, and the equilibrium is not totally reached. These partition coefficient values were higher than the ones obtained by Kurek et al. (2013) on carvacrol (between $1 \times 10^{-4}$ and $6.8 \times 10^{-8}$) between headspace and different chitosan-based films, carvacrol probably has a higher affinity for chitosan than PHBV.

The model adjustment to release carvacrol in headspace conducted to apparent diffusivities of $1.6 \times 10^{-14} \text{ m}^2/\text{s}$ for impregnated films; $1.1 \times 10^{-16} \text{ m}^2/\text{s}$ for nanoparticles coated films; and $1.0 \times 10^{-16} \text{ m}^2/\text{s}$ for β-cyclodextrin coated films (so more than 100 time lower than for impregnated films). This higher carvacrol diffusivity from impregnated than coated films was probably due to a difference of structure and crystallinity generated by the process production (thermo-mechanical process for impregnated film and solvent process for coated films), as already pointed out by Bruni et al., (2024). These values remained in the same order of magnitude as those reported by Kurek et al. (2014), for which the carvacrol diffusivities from chitosan film were between $6.7 \times 10^{-16} \text{ m}^2/\text{s}$ and $5.5 \times 10^{-13} \text{ m}^2/\text{s}$ in environmental conditions between 20 and 30 °C and 75–96 % relative humidity.

### 3.2.3. Carvacrol release from PHBV films into food simulants

Fig. 7 shows the carvacrol release from PHBV film (i) impregnated with carvacrol, (ii) coated with carvacrol encapsulated in nanoparticles, and (iii) coated with carvacrol encapsulated in β-cyclodextrin into the food simulant A and D1 at 30 °C.

The total carvacrol release increased with time in both food simulants until reaching a plateau after 2 h (95 % of the initial carvacrol) and 8 h (58 % of the initial carvacrol) using food simulant D1 and A, respectively for nanoparticles coated on PHBV; after 8 h (93 % of the initial carvacrol) and 24 h (84 % of the initial carvacrol) using food simulant D1 and A respectively for carvacrol encapsulated with β-cyclodextrin and coated on PHBV, while the plateau was not reached after 240 h for the impregnated film in both food simulants (66 and 49 % of the initial carvacrol for food simulant D1 and A respectively). Therefore, the release of carvacrol was faster and higher in food simulant D1 than A for the three types of films due to the higher solubility of carvacrol in ethanol compared to water (Chen et al., 2014). Moreover, as observed in Section 3.2.2, the total release was faster for carvacrol encapsulated in nanoparticles and coated on PHBV than for carvacrol encapsulated with β-cyclodextrin and coated on PHBV (due to the necessity of water diffusion for β-cyclodextrin), this latest showing itself a faster release than impregnated carvacrol into PHBV film (due to higher thickness of layer containing carvacrol). These results are similar to those reported by other authors (Bruni et al., 2024; Buonocore et al., 2005; Jipa et al., 2012; Vasile & Baican, 2021). For example, Requena et al. (2017) showed 96 % and 22 % carvacrol release from coated PHBV film into food simulant D1 and A, respectively, after 10–20 h at 20 °C.

Due to the volatility of the carvacrol, the released molecules were distributed between the food simulant (100 mL) and the headspace of the cell (50 mL). Fig. 7 shows that generally, half of the released carvacrol was recovered in the food simulant, while the other half was present in the headspace, resulting in a partition coefficient between headspace and food simulant of approximately 2. Therefore, the concentration of carvacrol in the headspace was at equilibrium (or after 240 h for impregnated films) between 2.7 and 8.1 µg/mL (data not shown), which was enough to reach the minimal inhibitory concentration in air (between $4.62 \times 10^{-2} \text{ µg/mL}$ and 5 µg/mL) (Arfa et al., 2007; Kurek et al., 2013). However, the concentration of carvacrol in food simulant was at equilibrium between 0.3 and 3.1 µg/mL (Fig. 7), while the minimal inhibitory concentration of carvacrol in liquid media was known to be between 16 µg/mL and 1000 µg/mL (Requena et al., 2017) and was consequently never reached. Therefore, this active packaging strategy could be chosen to limit the microorganism's growth on food surface (through headspace), otherwise, the quantity of carvacrol in the film should be increased at least 10 times more (film production around 0.3 % instead of 0.03 %) to reach the minimal inhibitory concentration
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Carvacrol release from PHBV film impregnated with carvacrol, PHBV film coated with carvacrol encapsulated in nanoparticles and PHBV film coated with carvacrol encapsulated in β-cyclodextrin into food simulant A (10 % (v/v) ethanol) and D1 (50 % (v/v) ethanol), and the corresponding headspace at 30 °C in a closed system.</figcaption></figure>

in food (around 20 µg/mL). However, the admissible daily intake of the molecules should be taken into consideration, that is, 1800 µg/person/day (FAO/WHO, 2001) for structural class I substances, such as carvacrol. It means that, for 100 mL of food consumed per day, the concentration of carvacrol should not exceed 18 µg/mL to assure consumer safety, this concentration being close to the lower limit of minimal inhibitory concentration. Moreover, the rapid release of carvacrol from coated films (carvacrol encapsulated in nanoparticle or β-cyclodextrin) makes these formulations better for short shelf life (8 h at 30 °C corresponding to one week at 4 °C), while the slower release of carvacrol from impregnated films make this formulation better for longer shelf life foods (10 days at 30 °C corresponding to more than six months at 4 °C), according to regulation (European Standard No 10/, 2011). It should be noticed that the antimicrobial efficiency of active packaging needs to be validated on contaminated real food matrix, the food complexity being

not fully reproduced by food simulant, and the microbial inhibition being based on literature data in this present study.

The partition coefficient between carvacrol present in food simulant and films was presented in Fig. 4 and showed a higher value with simulant D1 (1.4 × 10⁻²; 4.0 × 10⁻³; 4.1 × 10⁻³, corresponding to a log K of - 1.9; - 2.4 and - 2.4 respectively) than A (2.9 × 10⁻³; 4.0 × 10⁻⁴; 1.2 × 10⁻³, corresponding to a log K of - 2.5; - 3.4 and - 2.9 respectively) for carvacrol impregnated in PHBV films, carvacrol encapsulated in PHBV nanoparticles and coated on PHBV films and carvacrol encapsulated in β-cyclodextrin and coated on PHBV films respectively, proving the higher affinity of the molecule for ethanol than water again. As in Section 3.2.2, the partition coefficient was higher for impregnated films than coated films (due to the higher volume of film). From the literature, the same order of magnitude was obtained for the partition coefficient. For example, Torres et al. (2014) showed a partition coefficient for
thymol between ethanol 10% (simulant A) and LLDPE of $1.2 \times 10^{-2}$ during impregnation under pressure.

## 4. Conclusion

In this study, the release profile of carvacrol encapsulated in different carriers (PHBV nanoparticles and beta-cyclodextrin) and coated on PHBV films was successfully evaluated in the headspace and different food simulants (A and D1) and compared with the release profile of carvacrol impregnated in PHBV films.

The results showed an encapsulation efficiency of 66%, 31% and 2% using beta-cyclodextrin, PHBV nanoparticles and halloysite, respectively, while the carvacrol release at 30 °C in headspace reached 45% of the initial carvacrol amount in 30 days from beta-cyclodextrin encapsulant and more than 90% of the initial carvacrol in 10 days from PHBV nanoparticles. The film's production generated a 30% loss of carvacrol for both coated film and the impregnated one, but the remaining carvacrol was homogeneous and distributed through the coating layer or film. The active coated films showed a faster release in the headspace, with a plateau reached after 30 and 40 days, in comparison to impregnated films, where the plateau was not yet reached after 80 days. However, since the affinity of carvacrol is lower with PHBV than beta-cyclodextrin, the equilibrium was reached at a higher carvacrol concentration in the headspace from PHBV nanoparticles or non-encapsulated films. The release of carvacrol in food simulants also demonstrated a faster and higher release in food simulant D1 than in A due to the higher affinity of carvacrol for ethanol.

Moreover, all these release experiments (headspace, food simulants) demonstrated that the minimal inhibitory concentration could be reached in the headspace. Consequently, carvacrol encapsulated (PHBV nanoparticles or beta-cyclodextrin) and coated on film could be an optimal strategy for products with a short shelf life (one week), while impregnated films could be a strategy for longer food shelf life (a few months). A shelf-life test on foods under real conditions (temperature, etc.) could be interesting in order to validate the use of this active film in prolonging food shelf life and reducing food loss and waste. Moreover, the industrial scalability of encapsulation process, as well as film production still need to be explored.

## CRediT authorship contribution statement

**Nathalie Gontard:** Validation, Conceptualization, Writing – review & editing, Funding acquisition. **Valérie Guillard:** Validation, Investigation, Conceptualization, Writing – review & editing, Supervision, Funding acquisition. **Aynura Rzayeva:** Investigation, Data curation, Writing – original draft, Formal analysis. **Mina Hariri:** Investigation, Data curation, Writing – review & editing, Formal analysis. **Fanny Coffigniez:** Validation, Writing – original draft, Supervision, Funding acquisition, Conceptualization, Investigation, 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 project was conducted thanks to the financial support provided by the Ministry of Science and Education of the Republic of Azerbaijan.

## Data availability

Data will be made available on request.

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