Development of PLA-Based Active Packaging Films Plasticized with a Deep Eutectic Solvent and Enriched with Sumac (Rhus coriaria L.) Extract

Abstract

In this study, the plasticizing effect of a deep eutectic solvent (DES) on polylactic acid (PLA) films mixed with sumac extract (SE) was investigated. DES (ChCl-Lev), consisting of choline chloride (ChCl) and levunic acid (Lev), was used. Chemical analysis confirmed the synthesis of ChCl–Lev and demonstrated the effective integration of the ChCl–Lev and SE system into PLA films. Incorporation of ChCl–Lev into the film led to an approximately 76% increase in elongation at break. This increase continued at approximately 49% in the film with 1% SE (SE1_ChCl-Lev10) additive. Antioxidant activity increased with SE content, reaching ABTS and DPPH scavenging activities of 96% and 83% at 1% SE (SE1_ChCl-Lev10) and 98% and 91% at 10% SE (SE10_ChCl-Lev10). Furthermore, antibacterial activity increased significantly as SE concentration increased; the film containing 10% SE showed strong inhibition against Staphylococcus aureus at a rate of 96.95 ± 0.32, while the inhibition rate against Escherichia coli also increased to 26.68 ± 2.89, whereas no antibacterial activity was observed in pure PLA. Considering these findings, it is anticipated that films produced using an innovative strategy could be a potential candidate for packaging, active food contact materials, and biomedical applications.

1. Introduction

Active packaging materials fortified with polyphenol-dense extracts are receiving growing attention due to their ability to suppress microbial proliferation and slow oxidative deterioration in food products [1]. Phenolic compounds are structurally diverse secondary metabolites synthesized by plants. Due to their significant antioxidant capacity, they are considered important in promoting and maintaining human health. Within the food industry, the predominant synthetic antioxidant compounds encompass butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG), tert-butylhydroquinone (TBHQ), which are routinely incorporated to mitigate oxidative deterioration [2]. Nevertheless, contemporary research on this category of packaging, which seeks to address consumers’ substantial concerns regarding health and environmental sustainability, predominantly emphasizes the incorporation of natural constituents. Representative examples of such natural constituents encompass aromatic herbs, as well as fruits and vegetables, which possess notable antioxidants and antimicrobial properties [3].

Recent studies have elucidated the pronounced antioxidant and antimicrobial properties exhibited by various Rhus coriaria L. (sumac) extracts [2]. Sumac, a widely employed culinary spice, constitutes a characteristic component of traditional Mediterranean diets [4]. Sumac is further characterized by its substantial anthocyanin content, and its associated health benefits include antipyretic properties as well as cardioprotective effects. Anthocyanins, recognized for their pronounced antioxidant capacity, have increasingly been regarded as pivotal bioactive constituents in the formulation of advanced food-packaging films [5]. A previous study demonstrated that the incorporation of sumac extract into active-packaging film matrices resulted in improved antioxidant and antimicrobial functionalities [6]. Active polymer packaging development involves key foundational processes, notably the integration of bioactive agents and the careful selection of a suitable polymer matrix [7].

PLA, due to its wide applicability, inherent biodegradability, and favorable environmental profile, is considered superior to many other polymer matrices [8]. Despite its numerous benefits, the brittleness and inadequate mechanical performance of PLA constrain its wider utilization. To address these limitations, plasticizers are integrated into polymer matrices, thereby enhancing the flexibility and overall mechanical properties of PLA. Although widely used, di(2-ethylhexyl) phthalate (DEHP) volatility, migration, and health risks have prompted the search for safer and more efficient plasticizer alternatives [9].

DES have attracted increasing attention as a new class of natural and environmentally benign solvents or plasticizers [10]. DES’s comprise hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) that form extensive self-associated hydrogen-bond networks. DESs also offer several advantages, including low cost, reduced or negligible toxicity, non-volatility, environmental benignity, and biodegradability [11]. Recent studies have widely examined the use of DESs to modify various properties of chitosan-based films, as well as the effects of DES-based plasticizers on the mechanical behavior of starch films [12,13,14,15]. However, the application of DESs as plasticizers in PLA-based films has been addressed in only a limited number of studies. Uzun et al. reported that Lev-based DESs functioned effectively as plasticizers for PLA films, enhancing both their mechanical and barrier properties [9].

This study aims to develop PLA-based biodegradable packaging films incorporating SE as a bioactive filler. Sumac was selected due to its widespread natural distribution in the Eastern Black Sea region of Türkiye (Artvin) and its pronounced antioxidant activity [16]. It also investigates the plasticizing effect of a DES composed of ChCl and Lev. A review of the literature indicates that the plasticizing behavior of DESs in PLA films incorporating natural bioactive fillers has received limited attention, with existing studies mainly focusing on starch- and chitosan-based film systems. Accordingly, the present study aims to offer new insights into this area. Furthermore, to the best of our knowledge, no prior studies have reported on the plasticizing effect of DESs in PLA-based active films containing SE. The combined influences of the bioactive component and the DES were evaluated through water contact angle, mechanical, chemical, thermal, antioxidant, and antibacterial analyses.

2. Materials and Methods

2.1. Materials

PLA provided by Total Corbion Luminy^® LX175 was used in granule form in this work. Chloroform (Fisher Chemical, MA, USA), Choline chloride (99% purity, Acros, MA, USA), Levulinic acid (99% purity, Macklin, Shanghai, China), Ethanol, Methanol, Potassium persulfate, 2.2-diphenyl-1-picrylhydrazyl(DPPH),2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma (Darmstadt, Germany). The plant materials used in the present study fruits of sumac were collected from Artvin, Türkiye. The fruits were subsequently powdered and stored at 25 °C until analysis.

2.2. Preparation of Ethanol Extract of Sumac

For the preparation of the ethanol extract, 25 g of sumac was combined with 250 mL of ethanol and stirred on a magnetic stirrer for 10 h. The mixture was then filtered, and the filtrate was evaporated. In this way, the ethanolic sumac product was obtained and stored at −18 °C until use [17].

2.3. Preparation of ChCl-Lev Used as a Plasticizer

DES plasticizer was prepared by mixing ChCl and Lev at a molar ratio of 1:2. The mixture was heated at 60 °C under continuous magnetic stirring until a homogeneous and transparent liquid was obtained. For crystallization assessment, the transparent ChCl-Lev was stored at room temperature for 48 h, during which no crystallization or phase separation was observed, indicating good physical stability of the system.

2.4. Characterization of ChCl-Lev

The density of ChCl–Lev was determined at 25 °C using a pycnometer with a volume of 25 mL. The pycnometer was filled with ChCl–Lev to the volume mark and its mass was recorded using an analytical balance. The density of ChCl-Lev was determined at 25 °C using a pycnometer with a volume of 25 mL. The pycnometer was filled with ChCl-Lev up to the volume mark and weighed using an analytical balance. The chemical structures of HBA (ChCl), HBD (Lev), and ChCl–Lev were characterized by ATR–FTIR spectroscopy (Bruker Scientific LLC., Billerica, MA, USA) at a spectral resolution of 4 cm⁻¹ in the range of 400–4000 cm⁻¹.

2.5. Preparation of Film

The films were prepared using the solvent casting method, and a summary is given in Figure 1. Before film preparation, PLA pellets were dried in an oven at 50 °C for 24 h to remove residual moisture. For film production, 2.4 g of PLA was dissolved in 60 mL of chloroform under continuous stirring for 1 h. Subsequently, the plasticizer (ChCl–Lev) was added at a concentration of 10 wt% relative to the PLA content, and this concentration was kept constant across all film formulations. This concentration was chosen based on a previous study that determined it to be the optimum plasticizer content [9]. Afterwards, SE was added to the films in different proportions by weight relative to PLA (1%, 3%, 5%, 7%, and 10%) and mixed for 1 h. The prepared film-forming solutions were cast into glass Petri dishes and allowed to dry at 25 °C for 48 h. Following solvent evaporation, the films were carefully removed from the glass Petri dishes and conditioned in a climate chamber at 25 °C and 53% relative humidity until further characterization. The films were named as follows: control film (PLA), film containing 10% ChCl-Lev (ChCl-Lev10), and films containing 1%, 3%, 5%, 7%, and 10% SE and 10% ChCl-Lev (SE1_ChCl-Lev10, SE3_ChCl-Lev10, SE5_ChCl-Lev10, SE7_ChCl-Lev10, and SE10_ChCl-Lev10).

2.6. Film Characterization

2.6.1. Thickness

The thickness of PLA films containing varying proportions of SE was measured using a digital micrometer (Mitutoyo, IP65, Takatsu-ku, Kawasaki-shi, Kanagawa, Japan) with an accuracy of ±0.0001 mm, by taking measurements at five different points on each film.

2.6.2. FTIR

FTIR spectra of the raw materials and films were recorded using a Bruker Tensor 37 spectrophotometer equipped with an ATR accessory, (Bruker Scientific LLC., Billerica, MA, USA) over the wavenumber range of 400–4000 cm⁻¹ with a spectral resolution of 4 cm⁻¹.

2.6.3. Mechanical Tests

The tensile properties of the specimens were evaluated using the SHIMADZU AG-IC (Shimadzu Co., Tokyo, Japan) instrument in accordance with ASTM D882 standards. The films were cut into specimens with dimensions of 10 mm × 50 mm, and five specimens were tested for each film.

2.6.4. Differential Scanning Calorimetry (DSC)

The film samples were dried in a desiccator containing silica gel for three weeks to eliminate residual moisture. Subsequently, the samples were heated from 5 to 200 °C at a rate of 10 °C/min under a nitrogen atmosphere with a flow rate of 20 mL/min [9].

The degree of crystallinity (Xc) of PLA was calculated using the following:

$$\mathrm{Xc} = {\displaystyle \frac{\mathsf{\Delta }HM}{\left(1-\Phi \right)\mathsf{\Delta }\mathrm{H}0}} \times 100$$

where ϕ is the PLA weight fraction of the dispersed phase, ∆H_m is the melting enthalpy (J/g) from the DSC curve, and ∆H₀ is the heat of fusion for completely crystallized PLA (93.0 J/g) [18].

2.6.5. Water Contact Angle (WCA) Analysis

Contact angle measurements were carried out over a range of 0–180° using an Ossila, (Sheffield, United Kingdom) contact angle goniometer. For each film, measurements were taken at three different locations.

2.6.6. Microstructure Analysis

The microstructures of the PLA-films were examined at ×500 magnification using a (Carl Zeiss Ind. Metrology GmbH, Oberkochen, Germany) scanning electron microscope (SEM).

2.6.7. Antioxidant Capacity

DPPH Radical Scavenging Assay

The DPPH radical scavenging activity of the films was measured at 517 nm using a UV-Vis spectrophotometer (Scinco-NEOSYS200, Gangnam-gu, Seoul). Approximately 0.1 g of each film sample was taken and dissolved in 3 mL of ethanol for a period of 3 h. A volume of 50 μL of the diluted sample was mixed with 1.45 mL of a DPPH solution at a concentration of 0.2 mmol. The mixture was then incubated in the dark at room temperature for a period of 30 min.

The absorbances of the film solutions were then measured in comparison with the control. and the DPPH radical scavenging activity was calculated using the following equation:

$$\mathrm{DPPH} \mathrm{scavenging} \mathrm{activity} (\%) ={\displaystyle \frac{{\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{film}}}{{\mathrm{A}}_{\mathrm{control}}}}\times 100$$

where ${\mathrm{A}}_{\mathrm{control}}$ and ${\mathrm{A}}_{\mathrm{film}}$ represent the absorbance values of the DPPH solution in the absence and presence of film samples, respectively [2].

ABTS Radical Scavenging Assay

The ABTS radical scavenging activity of the films was determined using a modified version of the method described by [19]. The ABTS solution (7 mmol/L) was prepared by reacting it with potassium persulfate (2.45 mmol/L). and the mixture was incubated in the dark at 25 °C for 16 h to generate the ABTS^•+ radical cation. The ABTS^•+ solution was diluted with ethanol to an absorbance of 0.70 (±0.02) at 734 nm. Approximately 0.1 g of each film sample was taken and dissolved in 3 mL of ethanol for 3 h. A volume of 50 μL of the diluted sample was mixed with 1.45 μL of ABTS^•+ solution and incubated in the dark for 30 min.

The ABTS radical capture activity of the films was calculated using the following equation:

$$\mathrm{ABTS} \mathrm{scavenging} \mathrm{activity} (\%) ={\displaystyle \frac{{\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{film}}}{{\mathrm{A}}_{\mathrm{control}}}}\times 100$$

where ${\mathrm{A}}_{\mathrm{control}}$ and ${\mathrm{A}}_{\mathrm{film}}$ denote the absorbance values of the ABTS solution in the absence and presence of the film extract, respectively.

2.6.8. Antibacterial Activity

Preparation of Test Microorganisms

Antibacterial activity testing was conducted following the protocol reported by Özcan et al., with slight modifications [20]. The antibacterial activity of the films was determined by utilizing the Escherichia coli ATCC 25922 (Gram-negative bacteria) and Staphylococcus aureus ATCC 29213 (Gram-positive bacteria) strains. For this purpose, a loopful of culture was taken from Mueller–Hinton Agar (MHA) medium and transferred to Mueller–Hinton Broth (MHB), then activated by incubating at 37 ± 2 °C for 18–24 h. The turbidity of the resulting suspensions was then adjusted to the 0.5 McFarland (DEN-1B, BIOSAN) standard using MHB.

Determination of Antibacterial Activity

A quantity of 200 mg film samples was cut and sterilized for a duration of one hour on both sides using UV light. The samples were then transferred to a sterile Falcon tube containing 20 mL Mueller–Hinton Broth. The S. aureus ATCC 29213 and E. coli ATCC 25922 strains were cultivated for a period of 18–24 h, following which they were diluted to a final inoculum concentration of 1.0 × 10⁵ colony-forming unit (CFU)/mL. Thereafter, the inoculum was introduced into the Falcon tubes containing the films. The tubes were then subjected to an incubation process at a temperature of 37 °C for a duration of 24 h, conducted within a shaking water bath that operated at a frequency of 120 rpm. Thereafter, the colonies that had formed on Mueller–Hinton Agar were enumerated in accordance with the pour plate technique. The results were expressed in terms of CFU/mL and as a percentage decrease relative to the PLA (blank sample).

The following formula was utilized to calculate the number of colonies.

$$\mathrm{N}=\mathrm{C}/[\mathrm{V}\times \left(n1+0.1\times n2\right)\times \mathrm{d}]$$

where:

N = Number of microorganisms in 1 mL of the sample

C = Total number of colonies counted on all Petri dishes used for enumeration (plates containing between 15 and 300 colonies are considered)

V = Volume plated on all Petri dishes used for counting (mL)

n1 = Number of Petri dishes counted at the first selected dilution

n2 = Number of Petri dishes counted at the second selected dilution

d = Dilution factor corresponding to the first of the two consecutive dilutions used for counting

The following formula was utilized to calculate inhibition.

$$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{R}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{A}-\mathrm{B}\right)\times 100/\mathrm{A}$$

where:

A = Is the number of viable microorganisms before treatment

B = Is the number of viable microorganisms after treatment

3. Results and Discussion

3.1. ChCl-Lev Characterization Results

In this study, the density of ChCl-Lev, used as a plasticizer, was found to be 1.082 g/cm³. This value is consistent with similar studies in the literature. Low-density DESs are desirable as plasticizers, as high-density DESs are difficult to mix with polymers and complicate processing during solution mixing [9,21]. The FTIR spectrum of ChCl–Lev exhibited a characteristic absorption band at approximately 1700 cm⁻¹, corresponding to the C=O stretching vibration of the carboxyl group, along with a broad band around 3060 cm⁻¹ attributed to O–H stretching vibrations (Figure 2a). These spectral features indicate the formation of a new compound through intermolecular hydrogen-bond interactions between hydroxyl groups and carboxyl functionalities [9].

3.2. FTIR Results of the Films

The FTIR spectra presented in Figure 2b show the characteristic absorption bands of ChCl_Lev10 and SE1_Chcl-Lev10–SE10_ChCl-Lev10 films. The FTIR spectrum of the neat PLA film exhibited characteristic absorption peaks at approximately 2946–2997 cm⁻¹, attributed to –CH– stretching vibrations and representing the strongest absorption bands, along with a C=O carbonyl stretching band at 1747 cm⁻¹. The absorption bands at 1456 cm⁻¹ were associated with symmetric and asymmetric CH₃ bending vibrations, while the band at 1385 cm⁻¹ was related to CH₃ stretching vibrations [22].

The characteristic absorption bands of neat PLA were preserved in films containing ChCl–Lev and SE; nevertheless, noticeable changes and shifts in the intensity of specific bands were observed. The broad absorption band observed in the ChCl–Lev10 film in the 3200–3500 cm⁻¹ region is attributed to the –OH groups of Lev. The increased intensity of this band compared to that of neat PLA indicates the successful incorporation and homogeneous dispersion of Lev within the PLA matrix. The increased intensity of bands around 3200 cm⁻¹ observed in SE-containing films is attributed to the O–H stretching vibrations of phenolic compounds. The pronounced and intense absorption band observed at 1730 cm⁻¹ is attributed to the stretching vibrations of the carbonyl (C=O) group [5,8].

3.3. Mechanical Properties of the Films

As shown in

Table 1. Mechanical properties of PLA-based active packaging films.

Samples	TS (MPa) ± SD	EaB(%) ± SD	Thickness (mm) ± SD
neat PLA	23.28 ± 0.87	8.91 ± 2.06	0.122 ± 0.001
ChCl-Lev10	22.05 ± 1.04	15.75 ± 1.14	0.126 ± 0.002
SE1_ChCl-Lev10	21.20 ± 0.59	13.35 ± 1.16	0.127 ± 0.001
SE3_ChCl-Lev10	19.50 ± 0.93	6.82 ± 1.42	0.130 ± 0.001
SE5_ChCl-Lev10	17.55 ± 2.55	5.13 ± 1.90	0.171 ± 0.004
SE7_ChCl-Lev10	16.83 ± 0.79	4.34 ± 0.81	0.172 ± 0.003
SE10_ChCl-Lev10	13.98 ± 1.07	3.79 ± 0.79	0.177 ± 0.002

Abbreviations: EaB, elongation at break; TS, tensile strength; SD, standard deviations.

and Figure 3, the tensile test results demonstrate that neat PLA exhibits the characteristic behavior of a brittle polymer, characterized by extremely low elongation at break (EaB). While the incorporation of Lev as a plasticizer did not significantly affect the tensile strength, an approximately twofold increase in elongation at break was observed compared to neat PLA. A slight decrease in tensile strength was observed due to increased polymer chain mobility resulting from the addition of plasticizers [23]. In films containing low levels of SE (1%), a slight decrease in TS values was observed, while a significant increase in EaB values occurred (e.g., 8.91% → 13.35%). This suggests that SE exhibits a plasticizer-like effect between PLA chains, increasing chain mobility and improving film ductility. It has been reported in previous studies that the addition of different extracts (e.g., green tea and rice straw extracts) to PLA leads to a reduction in tensile strength as well as a decrease in elongation at break [3,24,25]. The increase in elongation at break in the SE1_ChCl-Lev10 film can be attributed to the synergistic effect of SE and ChCl-Lev. At high extract concentrations (5%,7% and 10%), significant decreases were observed in TS and EaB values (17.55 → 13.98 MPa and 5.13 → 3.79%). This suggests that excessive extract addition may be related to phase separation, poor interfacial interaction, or disruption of matrix integrity. This observation is further supported by the SEM micrographs. The present finding aligns well with previously published studies [26].

3.4. DSC Results of the Films

DSC analysis was conducted to assess the influence of SE and ChCl–Lev on the thermal behavior of the PLA matrix (Figure 4). The thermal transition parameters obtained from the second heating cycle of the DSC measurements—namely, the glass transition temperature (Tg), cold crystallization temperature (Tc), melting temperature (Tm), melting enthalpy (ΔHm), and degree of crystallinity (Xc)—are summarized in

Table 2. DSC results of PLA-based active packaging films.

Samples	Tg (°C)	Tc (°C)	ΔHc (J/g)	Tm (°C)	ΔHm (J/g)	Xc (%)
Neat PLA	52.93	126.93	40.39	153.90/158.08	31.25	33.60
ChCl-Lev10	40.72	101.77	38.90	138.24/152.04	37.67	45.04
SE1_ChCl-Lev10	45.76	110.08	38.25	143.88/153.54	32.90	39.74
SE3_ChCl-Lev10	44.48	109.74	38.35	144.01/153.32	33.08	40.88
SE5_ChCl-Lev10	43.94	113.05	31.86	144.44/153.18	30.13	38.11
SE7_ChCl-Lev10	44.69	118.19	28.89	146.51/153.87	28.57	37.01
SE10_ChCl-Lev10	43.59	114.14	29.13	144.83/153.65	27.83	37.40

. Neat PLA is known to display a distinctive double melting behavior in DSC thermograms. This phenomenon, typically appearing as a minor endothermic event followed by a dominant melting peak, is generally associated with a melt—recrystallization—remelting mechanism. In this process, less ordered or imperfect α′ crystallites melt at lower temperatures and subsequently reorganize into more stable crystalline structures. These newly formed, well-ordered α crystals then undergo melting at higher temperatures, giving rise to the second endothermic peak, which is therefore ascribed to the melting of the thermodynamically more stable α phase [27]. Neat PLA film showed the highest Tg value (50.02 °C), and the addition of ChCl-Lev as a plasticizer to PLA lowered the Tg value. Subsequently, a slight increase in the Tg value was observed with the addition of SE. The lowest Tg value was found in the SE10_ChCl-Lev10 film at 43.59. Plasticizers disrupt polymer chains by reducing intermolecular forces and increasing free volume, which leads to a decrease in the glass transition temperature (Tg), consistent with previous studies [9,28,29]. The increase in Tg with the addition of SE (SE1_ChCl-Lev10) can be interpreted as the interactions between the filler and the polymer matrix limiting molecular mobility and chain flexibility at the interface regions [30].

The degree of crystallization (Xc) of pure PLA increased significantly from 33% to approximately 45% in the formulation containing SE and ChCl–Lev10. The highest Xc value was observed at 45% in the ChCl-Lev10 film. This finding suggests that ChCl–Lev may function as a nucleating agent, promoting crystal formation, which is consistent with previous studies on the plasticizing effects of DESs [9]. Going into more detail, this behavior can be attributed to the good compatibility between the matrix and the filler, where strong interfacial interactions allow the filler to act as nucleation sites for polymer chain ordering, thereby increasing the crystallinity of the composite [30,31]. The decrease in Xc% with increasing SE content (SE7_ChCl–Lev10 and SE10_ChCl–Lev10), together with the comparatively lower mechanical performance of these films, supports this interpretation.

3.5. WCA Analysis

WCA results are shown in Figure 5. A lower water contact angle reflects enhanced surface wettability, indicating that the films possess a more hydrophilic character and an increased affinity for water, which is associated with pronounced hygroscopic behavior [32]. The surface wettability of neat PLA and films incorporating ChCl–Lev with varying SE contents was assessed using water contact angle measurement. Neat PLA exhibited a water contact angle (WCA) value of 73.4, close to the values reported in the literature [22,33]. The incorporation of ChCl–Lev10 and SE reduced the WCA to values between 67.64 and 42.51, reflecting increased surface hydrophilicity relative to neat PLA. Although ChCl–Lev, used as a plasticizer, contains the relatively hydrophobic Lev moiety, its high content of hydrophilic carboxyl groups promotes water retention and increases surface hydrophilicity [9]. In addition, ChCl exhibits inherently hydrophilic behavior and enhances the water-holding capacity of films [34]. The reduced WCA observed for the films can therefore be attributed to these combined effects. On the other hand, the WCA value is strongly influenced by the surface characteristics of the film, including surface heterogeneity and roughness [35]. As seen in the SEM images, particularly in films containing 5% and higher SE, increased surface roughness and heterogeneity become more pronounced, and these morphological changes support the observed decrease in WCA values.

3.6. SEM Analysis

The microstructure of neat PLA and films containing ChCl–Lev10 with 1%, 3%, 5%, 7%, and 10% SE was examined at ×500 magnification, and the corresponding micrographs are presented in Figure 6 and Figure 7. As shown in Figure 6a, the neat PLA film exhibits a smooth and homogeneous surface.

In the ChCl–Lev10 and SE1_ChCl–Lev10 films, the overall morphology was largely retained; however, increasing the SE content—particularly at levels of 5% and above-resulted in pronounced phase separation and void formation. This behavior indicates limited compatibility of SE with the PLA matrix at higher loadings, leading to deterioration of surface integrity. The corresponding reduction in crystallinity observed for SE7_ChCl–Lev10 and SE10_ChCl–Lev10 films, in agreement with thermal analysis results, can be attributed to saturation-induced phase separation within the polymer matrix. Previous studies have reported that saturation effects arising from increased filler content in the matrix may lead to phase separation in film structures, which is also consistent with the observed increase in pore size, as excessive filler loading can disrupt matrix continuity and promote the formation and coalescence of voids [22,36].

3.7. Antioxidant Capacity

Antioxidant properties of PLA-based samples were evaluated using ABTS and DPPH radical scavenging methods (

Table 3. ABTS radical scavenging activity (%) and DPPH radical scavenging activity (%) of PLA-based activated packaging films.

Samples	ABTS Radical Scavenging Activity (%)	DPPH Radical Scavenging Activity (%)
Neat PLA	17.48 ± 0.75	14.23 ± 1.11
ChCl-Lev10	20.47 ± 1.05	18.85 ± 0.93
SE1_ChCl-Lev10	96.57 ± 0.77	83.19 ± 0.63
SE3_ChCl-Lev10	97.33 ± 1.05	90.49 ± 1.13
SE5_ChCl-Lev10	97.43 ± 1.19	90.51 ± 1.10
SE7_ChCl-Lev10	97.58 ± 1.13	91.30 ± 0.72
SE10_ChCl-Lev10	98.34 ± 0.94	91.55 ± 0.59

).

The neat PLA sample exhibited lower antioxidant activity compared to the doped films, with scavenging activity of 17.48 ± 0.75% for ABTS and 14.23 ± 1.11% for DPPH. In the ChCl-Lev10 sample prepared with the addition of Lev, an increase in both radical scavenging activities was observed; ABTS and DPPH activities were determined as 20.47 ± 1.05% and 18.85 ± 0.93%, respectively. With the addition of SE to the films, a significant increase in radical scavenging activity was observed in both tests.

A more significant enhancement in antioxidant activity was observed with the incorporation of SE into the films. For the ABTS radical scavenging assay, the SE1_ChCl-Lev10 film showed a substantial improvement, achieving 96.57 ± 0.69%. As the SE concentration increased, the ABTS scavenging activity further improved, reaching 97.33 ± 1.05% in SE3_ChCl-Lev10, 97.43 ± 1.19% in SE5_ChCl-Lev10, and 97.58 ± 1.13% in SE7_ChCl-Lev10. The highest value was observed in the SE10_ChCL-Lev10 film, which demonstrated an impressive ABTS scavenging activity of 98.34 ± 0.94%. Similarly, for the DPPH radical scavenging activity, the SE1_ChCl-Lev10 film exhibited a scavenging ability of 83.19 ± 0.86%. This activity increased progressively with higher SE concentrations, reaching 90.49 ± 1.13% in SE3_ChCl-Lev10, 90.51 ± 1.10% in SE5_ChCl-Lev10, and 91.30 ± 0.72% in SE7_ChCl-Lev10. The highest DPPH scavenging activity of 91.55 ± 0.59% was recorded in the SE10_ChCl-Lev10 film.

The antioxidant activity of PVA-based nanofibers containing sumac extract was tested by scavenging DPPH free radicals, and the addition of sumac extract at concentrations of 10%, 20%, and 30% resulted in increases in antioxidant activity of 41.04%, 42.98%, and 46.32%, respectively [5]. In another study, guar gum and sumac solutions were mixed in ratios of 1:1, 1:2, and 1:4 (w/w), and the antioxidant activities of the films were determined using DPPH and ABTS methods. Increasing the sumac weight in the films to ratios of 1, 2, and 4 resulted in DPPH activities of 51.55%, 88.24%, and 93.83%, respectively, and ABTS activities of 75.56%, 99.56%, and 99.12%, respectively. Furthermore, this study reported that the DPPH radical scavenging activities of the films were lower than those of ABTS [2].

The results of the antioxidant analyses clearly demonstrate that the incorporation of SE into the PLA matrix significantly enhances the free radical scavenging ability of the resulting composites. While neat PLA possesses inherently low antioxidant activity, the substantial increase observed in SE-loaded samples indicates that SE is the dominant contributor to the radical-neutralizing capability of these materials. This finding is consistent with previous studies reporting that plant-derived extracts rich in polyphenols or flavonoids can effectively improve the oxidative stability of biodegradable polymers [2,5,37].

The slight increase observed in the ChCl-Lev10 sample indicates that Lev alone exhibits limited radical scavenging efficiency. Nevertheless, its presence may indirectly modulate the overall antioxidant performance by enhancing the dispersion, compatibility, or mobility of SE within the polymer matrix. Improved miscibility can promote more effective interactions between active components and free radicals, which may account for the markedly higher scavenging activities observed in the SE–Lev formulations. This possible synergistic contribution should be further clarified through comprehensive structural, morphological, and molecular-level investigations.

The consistently high ABTS and DPPH scavenging activities (exceeding 96% and 90%, respectively) in PLA_SE composites indicate that SE maintains its bioactive functionality after processing. This is an important finding as thermal processing during biopolymer fabrication can often degrade natural antioxidant compounds. The preserved activity suggests that the processing conditions used in this study were suitable for protecting SE’s active components. or that the components themselves possess sufficient thermal stability.

Another notable observation is the concentration–dependence of antioxidant activity. Although even low levels of SE incorporation yielded substantial improvement. Increasing SE content up to 10% resulted in further enhancement, particularly in ABTS scavenging activity. This trend aligns with the expectation that higher concentrations of phenolic compounds provide a greater number of hydrogen-donating functional groups. which can neutralize free radicals more effectively. However, beyond a certain threshold. additional SE may no longer proportionally increase activity due to saturation effects or limited diffusivity within the PLA matrix. Therefore, identifying the optimal SE content is critical for balancing performance. cost. and material processability.

Overall, the findings highlight the strong potential of SE-loaded PLA composites as functional materials with enhanced oxidative stability. Such improvements are particularly relevant for applications in packaging, biomedical devices, and active food-contact materials. Where resistance to oxidation plays a crucial role in preserving product integrity and prolonging shelf life. Future work should focus on evaluating the long-term stability of the antioxidant effect, the controlled release behavior of bioactive compounds, and the impact of SE incorporation on mechanical, thermal, and barrier properties to establish a comprehensive understanding of the material’s applicability.

3.8. Antibacterial Activity

The antibacterial effects of neat PLA, ChCl-Lev10, SE1_ChCl-Lev10 and SE10_ChCl–Lev10 samples on E. coli ATCC 25922 (Gram-negative bacteria) and S. aureus ATCC 29213 (Gram-positive bacteria) are presented in

Table 4. Antibacterial activity of neat PLA, ChCl-Lev10, SE1_ChCl-Lev10 and SE10_ChCl-Lev10 against S. aureus and E. coli.

Samples	S. aureus (% Inhibition)	E. coli (% Inhibition)
Neat PLA	-	-
ChCl-Lev10	12.26 ± 6.12	nd
SE1_ChCl-Lev10	17.99 ± 0.52	7.01 ± 1.63
SE10_ChCl-Lev10	96.95 ± 0.32	26.68 ± 2.89

nd: not detected

.

Neat PLA exhibited no detectable antibacterial activity against S. aureus and E. coli. This finding is consistent with previous studies reporting that PLA, despite being a biodegradable polymer, does not possess antibacterial properties. Previous research has shown that PLA needs to be modified with functional or active components to limit microbial growth [38,39,40]. The incorporation of ChCl-Lev into the PLA matrix did not exhibit any inhibitory effect against E. coli, whereas an inhibition of approximately 12% was observed against S. aureus. In contrast, the SE1_ChCl–Lev10 formulation, containing 1% SE, demonstrated a limited antibacterial activity against both bacterial species. In the SE10_ChCl-Lev10 film, which contains the highest additive loading, the inhibition values increased for both microorganisms, with a pronounced antibacterial effect observed against S. aureus, reaching an inhibition rate of 96%. These results clearly demonstrate that the SE10_ChCl-Lev10 composition has a strong and broad-spectrum antibacterial effect. It has been widely reported in the literature that the addition of various components (organic acids, essential oils, natural extracts, nanoparticles, or ionic/eutectic systems) to PLA-based films significantly increases antibacterial efficacy compared to neat PLA [41,42].

4. Conclusions

In this study, biodegradable and active PLA-based films plasticized with a choline chloride–based DES/ChCl–Lev and enriched with SE were successfully developed and systematically characterized. The results demonstrated that ChCl–Lev functioned as an effective green plasticizer for PLA, significantly enhancing film flexibility, while maintaining acceptable mechanical integrity. The addition of SE imparted pronounced functional properties to the PLA films. Antioxidant analyses revealed a substantial enhancement in radical scavenging activity with increasing SE content, with ABTS and DPPH inhibition values exceeding 98% and 91%, respectively, at the highest SE loading. These findings confirm that the bioactive compounds present in SE retained their functionality after film processing and acted as the dominant contributors to the antioxidant performance of the films. Antibacterial activity results further highlighted the synergistic effect of ChCl-Lev and SE. While neat PLA exhibited no antibacterial activity and ChCl-Lev alone showed only limited inhibition, SE-enriched films demonstrated concentration-dependent antibacterial behavior. Notably, the SE10_ChCl-Lev10 film exhibited strong inhibition against S. aureus (≈96%) and measurable activity against E. coli, indicating broad-spectrum antibacterial potential.

Overall, this study presents a novel and sustainable strategy for producing multifunctional PLA-based films by combining a green DES with a natural plant extract. The resulting materials exhibit improved mechanical flexibility alongside strong antioxidants and antibacterial activities, making them promising candidates for active food packaging, biomedical applications, and other food-contact materials. Future studies should focus on long-term stability, controlled release behavior of bioactive compounds, and real-food application tests to further validate their practical applicability.