Can Satureja montana Essential Oil Promote Edible Films Based on Plum Oil Cake into Antimicrobial and Antioxidant Food Packaging?

Abstract

This study adheres to the principles of the circular economy by valorising fruit processing waste—specifically, the oil cake remaining after the cold pressing of plum seeds—for the production of new biopolymer packaging material. This study investigates the effects of incorporating Satureja montana essential oil (SMeo) on the properties of plum oil cake (POC)-based biofilms for potential food packaging applications. The mechanical, physico-chemical, barrier, structural, thermal, and biological properties of the POC-based film were investigated. The results showed that the addition of SMeo had the greatest impact on improving the water vapor barrier permeability (up to 48%). Antimicrobial analyses showed outstanding results against bacteria, yeasts, and molds, with the most pronounced inhibition observed for A. ochraceus and S. aureus. On the other hand, structural analysis confirmed that the 3% SMeo sample underwent the greatest changes, as indicated by the appearance of new bonds originating from oil–biopolymer interactions. This observation was further supported by thermal analysis, which showed that films containing SMeo degraded more rapidly than the control in a dose-dependent manner. The reduction in tensile strength values (up to 35%) suggests that SMeo-loaded POC films are more suitable for use as coatings rather than standalone packaging materials.

1. Introduction

Modern society is fundamentally dependent on plastics, with projections from the World Economic Forum indicating that global plastic production is expected to triple by 2050 [1], although it significantly contributes to environmental pollution and landfill accumulation [2]. Studies have primarily emphasized the development of eco-friendly and sustainable biopolymer films utilizing natural and biodegradable resources in response to widespread concerns about the environmental impact of petroleum-based plastic packaging materials, including the natural resources depletion, energy crisis, global warming, and disposal issues [3]. The use of bio-based films from proteins, lipids and polysaccharides as an alternative to non-biodegradable polymeric films often exhibits a diverse range of applications in the food sector because of their superior biodegradability, biocompatibility, edibility, organoleptic features, and effective barrier properties [4] along with reasonable raw material costs, consumer-attractive optical characteristics, and convenient technology [5].

Adopting a circular economy framework redefines waste as a valuable resource. Food and agricultural by-products constitute a vast, underexploited biomass reservoir. Valorizing food and agricultural waste for the synthesis of biopolymer packaging coatings/films is strongly advocated from environmental, economic, and technological perspectives [6,7]. More than one-third of all food produced globally is lost or wasted, resulting in unnecessary resource consumption and missed opportunities for value recovery [8]. Approximately 14% of food produced worldwide is lost between harvest and sale, and 17% of global food production is thought to be wasted [9]. During the industrial processing of fruits, a large amount of waste and by-products, such as peels, stones, and seeds, are generated [10]. The deposition of these materials in landfill sites has significant negative food-security, economic and environmental impacts [11]. The valorization of such fruit waste is a growing focus, with by-products now being targeted for biorefinery, nutraceuticals, cosmetics, and sustainable packaging solutions [8,12].

The Prunus genus (Rosaceae family) contains more than 400 species, including apricot, peach, sour and sweet cherry, nectarine, European plum, etc. [8]. The main by-products of Prunus fruits are pomace and kernel. Pomace is a by-product from the processing, composed of the fruit’s cell wall components, stems, and seeds [13], commonly used in animal feeding or as fertilizer [9]. On the other hand, plum kernels are a cost-effective source of high-quality oils and proteins [14,15] which are irreversibly wasted during processing, but could offer a novel source for food, cosmetics, and pharmaceutical industries [16]. The stone of the Prunus species is separated into two parts: the hard outer shell and the softer, internal kernel or seed. Depending on the fruit, the stone’s seed makes up 15–30 weight percent of the total weight, with the shell making up the remaining 70%–85% [17]. One of the primary ingredients wasted in the fruit processing is the Prunus seeds. Given that seeds are a good source of proteins, dietary fibers, carbs, polyphenols, vitamins, unsaturated fatty acids, and other bioactive elements, this waste represents a significant loss [18,19].

Recent research outlines the potential for valorizing a wide variety of Prunus fruit by-products for biodegradable film and coating production. For example, in the work of [20], apricot kernel oil was incorporated into a chitosan film, which consequently improved the antioxidant and antimicrobial properties of the films. A similar experiment indicated that apricot kernel essential oil caused a bacteriostatic effect against L. monocytogenes in vitro [21]. In another work, kernel shells were used to reinforce the mechanical properties of films: Young’s modulus and tensile strength in starch films were improved with kernel shells addition along with film solubility and water vapor transmission rate reduction [22]. Another similar experiment revealed that the cherry shell powder managed to reinforce low-density polyethylene films [23].

As for waste being used to be part of smart biopolymer films, films that can monitor the evolution of quality decays and also act to increase shelf life, could serve as an example where Prunus maackii pomace was used to develop a biodegradable intelligent film with κ-carrageenan and hydroxypropyl methylcellulose [24]. In another work, plum peel extract was used in the development of smart chitosan films [25]. These researchers often discuss the suitability of wastes for extracting valuable compounds that are then turned into packaging films [26]. It is important to note that using bioactive compounds from Prunus fruit by-products to create active and smart films also has additional positive effects, including enhancing elongation at break, water resistance, and water vapor barrier [9]. This highlights a recognized Prunus species potential, with plum waste as a plausible but less documented example.

Plum by-products have also been investigated as a structural component for filmmaking, with encouraging results in the work of Li et al. [27], who used plum kernel protein isolate in conjunction with gum acacia to form a film, and Sheikh et al. [16], who synthesized composite films from plum kernel protein isolates, arrowroot starch, and plum kernel oil. The special type of biopolymer films—composite films composed of hydrocolloids and lipid mixtures—can be obtained by bilayering or emulsion technology, or simply by natural compositions of biomass used for film synthesis. In the work of Pantić et al. [28], for the first time, the whole plum oil cake was used to produce a composite biopolymer film. The process was optimized by choosing optimal processing parameters: pH, temperature and plasticizer addition. This work presents continuation of research in terms of plum oil cake (POC)-based biopolymer film activation with Satureja montana essential oil. Satureja montana (winter savoury or mountain savory), an aromatic plant widely known for its beneficial properties due to its essential oil composition, which is rich in monoterpenes such as thymol, carvacrol, p-cymene, geraniol, γ-terpinene, myrcene, and linalool [29]. Carvacrol is known for its strong antimicrobial activity, while the non-volatile fraction of the plant (phenolic acids, phenylpropanoids, tannins, fatty acids, and tocopherols) contributes to its antioxidant potential [30]. The integration of active components, in this case essential oils, into the packaging matrix guarantees improved agent stability and slow and steady release over time, which allows for longer-term control over microbial growth and antioxidant protection [31].

This paper will try to give an answer to the question of how effectively Satureja montana essential oil will promote edible films based on POC into antimicrobial and antioxidant food packaging. By analyzing mechanical, physico-chemical, barrier, structural, thermal, and biological properties, we will arrive at an answer as to whether the new material is more applicable as a film or coating in the food packaging sector.

2. Materials and Methods

2.1. Materials

Plum oil cake was provided by “All Nut” (Belgrade, Serbia) and stored under refrigeration until use. Satureja montana essential oil was obtained from Siempre Viva Oils (Niš, Serbia). All chemicals used for the preparation and characterization of POC films were of analytical grade and were procured from commercial suppliers, including Sigma–Aldrich (Darmstadt, Germany), Alfapanon (Bački Petrovac, Serbia), and Lach-Ner (Neratovice, Czech Republic).

2.2. Plum Oil Cake-Based Films Synthesis

The films were prepared as per our previous instructions [28] and according to the optimal parameters. In brief, film-forming suspension was obtained when POC powder was diluted in distilled water (10% w/v) with the addition of 20% glycerol (w/w of POC). The suspension pH value was set to 12 using 0.2 M NaOH, after which it was heated at 60 °C for 25 min. The suspension was filtered and cooled to room temperature. After cooling, Satureja montana essential oil was added in different concentrations (v/v) to the film-forming suspension, which was cast and left to air dry for 2 days at room conditions (23 ± 2 °C, 50 ± 5% RH) (Figure 1). The samples were labeled as indicated:

• C—control sample, plum oil cake-based film without essential oil addition.
• 1% SMeo—plum oil cake-based film with 1% (v/v) Satureja montana essential oil addition.
• 2% SMeo—plum oil cake-based film with 2% (v/v) Satureja montana essential oil addition.
• 3% SMeo—plum oil cake-based film with 3% (v/v) Satureja montana essential oil addition.

Figure 1. Synthesis route for active POC-based films with SMeo.

Figure 1. Synthesis route for active POC-based films with SMeo.

2.3. Plum Oil Cake-Based Films Characterization

2.3.1. Mechanical Properties

Film thickness was measured with an accuracy of 3 points by using a Digico 1 micrometer (Tesa, Renens, Switzerland).

Tensile strength and elongation at break were determined in accordance with standard ASTM D882-18:2018 [32] by using Instron 4301 (Instron Engineering, Canton, MA, USA). Test tubes were rectangular (15 × 75 mm), clamp distance was set to 50 mm, while cross head speed was 50 mm/min. The test was performed with each sample in ten replicates.

2.3.2. Physico-Chemical Properties

Physico-chemical properties were determined according to [28] in three replicates. In brief, water content was determined after drying film samples until constant weight and calculated as follows:

$$\mathrm{W}\mathrm{C}\left[\%\right]=\frac{({\mathrm{w}}_1-{\mathrm{w}}_2)\times 100}{{\mathrm{w}}_1}$$

(1)

where w₁ is the initial mas of film samples and w₂ is the mass of film samples when reached constant weight (dried films).

After determining WC, dried film samples (w₂) were placed in 50 mL of distilled water and kept at room temperature for 24 h. After 24 h soaking, the film samples were removed from water and again dried at 105 ± 2 °C until a constant weight was reached (w₃). Total soluble matter (% TSM) was calculated as follows:

$$\mathrm{T}\mathrm{S}\mathrm{M} \left[\%\right]=\frac{({\mathrm{w}}_2-{\mathrm{w}}_3)\times 100}{{\mathrm{w}}_2}$$

(2)

The swelling degree was determined when initial film samples (w₁) were immersed in deionized water for 20 s at room temperature. The wet samples were wiped with filter paper to remove excess liquid and weighed (w₄). The swelling degree (%) reflected as the amount of adsorbed water was calculated as follows:

$$\mathrm{S}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{e} \left[\%\right]=\frac{({\mathrm{w}}_4-{\mathrm{w}}_1)\times 100}{{\mathrm{w}}_1}$$

(3)

2.3.3. Water Barrier Properties

The water vapor transmission rate (WVTR) was determined according to standard ISO 2528:2017 [33] (23 ± 1 °C and 50 ± 2% RH) in a Binder KMF 240 air conditioning chamber (Tuttlingen, Germany). Three replicates were performed simultaneously.

Equation (4) was used to calculate the WVTR, while Equation (5) was used to express the water vapor permeability (WVP) of film samples:

$$\mathrm{WVTR} [{\mathrm{g}/\mathrm{m}}^{-2} {\mathrm{h}}^{-1}]={\displaystyle \frac{∆\mathrm{w}/∆\mathrm{t}}{\mathrm{Area}}}$$

(4)

$$\mathrm{WVP} [{\mathrm{g}/\mathrm{m}}^{-1} {\mathrm{s}}^{-1} {\mathrm{Pa}}^{-1}]={\displaystyle \frac{\mathrm{WVTR}\times \mathrm{Thickness}}{\mathrm{P} \mathrm{vap}.\mathrm{sat}.}}$$

(5)

where Δw/Δt is a change in weight over time, and P vap. sat. = saturated vapor pressure of pure water at 23 °C and 50% RH.

2.3.4. Structural Properties—Fourier-Transform Infrared Spectroscopy (FTIR)

Using a Nicolet IS10 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an attenuation total reflection (ATR) extension, structural properties were ascertained in accordance with method ASTM D5576:00 [34]. At a resolution of 4 cm⁻¹, all spectra were captured within the 4000–400 cm⁻¹ spectral region. Before each sample was analyzed, a background was recorded, and each sample was scanned 32 times. Omnic 8.1 software (Thermo Fisher Scientific, Waltham, MA, USA) was used for data processing.

2.3.5. Antioxidative Activity—DPPH Assay

Antioxidative activity was determined according to Šuput et al. [35]. In brief, a vial with 2 mL of a freshly made DPPH^• solution (0.0185 g DPPH^• in 50 mL ethanol) diluted in ethanol (1:5) was used to put a 1 cm² film sample and stirred for 24 h at room temperature in a dark chamber. A T80/T80+ UV-VIS spectrophotometer (PG Instruments Ltd., Lutterworth, UK) was used to measure the absorbance at 520 nm and quantify the residual DPPH^• concentrations in each sample following the removal of the solid film (DPPH^•_s). The sample without any film was used as a control sample (blank) (DPPH^•₀). The following formula was used to express the films’ antioxidant activity.

AO (%) = ((DPPH^•₀ − DPPH^•_s))/(DPPH^•₀) × 100

(6)

The test was performed in three replicates, and the result was presented as mean ± SD.

2.3.6. Thermal Analysis—Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) was employed to investigate the thermal properties and stability of plum oil cake-based films. The primary objective was to evaluate the effect of incorporating Satureja montana essential oil (SMEO) on the films’ thermal transitions, including melting/denaturation and decomposition behavior by using DSC Q20 (TA Instruments, New Castle, DE, USA) in a nitrogen (50 mL/min) atmosphere. Samples were hermetically sealed in aluminum pans and heated from −20 °C to 250 °C at a constant rate of 10 °C/min.

2.3.7. Antimicrobial Properties—Disc Diffusion Assay

The antimicrobial activity of the samples was determined using the disc diffusion method. The bacterial strains selected for testing were Escherichia coli ATCC 25922 as a representative of Gram-negative bacteria, Staphylococcus aureus ATCC 6598 as a representative of Gram-positive bacteria, and Bacillus cereus ATCC 11778 as a representative of sporogenic bacteria. In addition to antibacterial activity, antifungal activity was tested against Candida albicans as a representative of yeasts, and Aspergillus flavus, Aspergillus parasiticus, and Aspergillus ochraceus as representatives of molds. Aspergillus spp. were isolated from various food products on the Serbian market (A. flavus and A. ochraceus from corn flour, and A. parasiticus from walnuts).

Bacterial, yeast, and mould suspensions were prepared in 0.85% sterile saline. Bacterial strains were cultured on Mueller-Hinton Agar (MHA, Sigma Aldrich, Darmstadt, Germany) for 24 h at 37 °C (B. cereus at 30 °C for 48 h), while yeast and mould cultures were grown on Sabouraud Maltose Agar (SMA, Sigma Aldrich, USA) at 25 °C for 48 h or 7 days, respectively [36]. After incubation, a microbiological suspension of 10⁸ CFU/mL was prepared, as determined by the McFarland Standard (bioMérieux SA, Marcy l’Etoile, France) and a McFarland densitometer (Biosan SIA, Riga, Latvia). The final concentration used for the experiment was 10⁶ CFU/mL, obtained by preparing a series of decimal dilutions. For testing antibacterial activity, 15 mL of MHA was poured onto Petri plates, and for antifungal activity, 15 mL of SMA was used. Microbiological suspensions were inoculated onto the surfaces of the solidified and cooled media using a sterile swab.

Samples of biopolymer films were prepared using a sterile circular knife with a diameter of 6 mm and placed in the centre of the inoculated media. The Petri plates were then incubated at 37 °C for 24 h for bacteria (B. cereus at 30 °C for 48 h), and at 25 °C for 48 h and 7 days for yeasts and molds, respectively. The diameters of the inhibition zones were measured in millimetres. Control film without SMeo was used as a control. All tests were performed in duplicate.

2.3.8. Statistical Analysis

The mean value ± standard deviation was used to express all of the acquired data. Analysis of variance (ANOVA) and the post hoc Tukey HSD test were used to determine the significance of the effect and interaction of individual components for each response. Standard score analysis was used for the study of the experimental data. Comparing the original data with the highest and lowest values recorded for each output variable was the basis of the ranking system. The lowest values of MC, S, SD, and WVP, and the highest values of TS, EB, AO, and AM, were designated as the ideal output variable values. Software called StatSoft Statistica 10.0^® was used for all analyses.

3. Results and Discussion

3.1. Mechanical Properties

The visual appearance of a product and its packaging directly influences consumer purchasing decisions. The visual properties of obtained POC-based biofilms are shown in Figure 2. The films exhibited a brownish colour and varying degrees of transparency, which may be suitable for packaging natural ore eco-friendly products or as an inner layer in multilayer materials. At higher concentrations of SMeo, the formation of a network structure becomes evident, resulting from the aggregation of essential oil molecules along the polymer chains.

The film thickness is an important parameter that can influence other film properties (mechanical, barrier, physico-chemical) and its application area. The control film (0.145 ± 0.003 mm) was the thinnest, while the incorporation of SMeo resulted in a progressive increase in film thickness with rising oil concentration, reaching a maximum of 0.221 ± 0.023 mm for the 3% SMeo film. Although this increase was not statistically significant, the trend is consistent with earlier reports for essential oil-incorporated biofilms [37,38], where higher thickness values were attributed to changes in surface stresses and the presence of oil droplets dispersed within the polymer matrix.

The results in Figure 3 show that adding SMeo clearly affected the mechanical properties of POC-based films. The control film had the lowest elongation at break (39.95%), but the highest tensile strength (about 4.05 MPa), indicating a stable hydrogen-bonding network in the polymer. When 1% and 2% of the SMeo were added, elongation at break increased significantly (reaching about 115% at 2%SMeo), while tensile strength decreased, suggesting that the oil acted as a plasticizer and increased chain mobility [39]. At the highest SMeo concentration (3%), both EB and TS decreased, probably because an excessive amount of oil disrupted the structure and weakened cohesion in the matrix [40]. This is consistent with the FTIR results, where changes in the O–H/N–H stretching region indicated weaker hydrogen bonding.

In line with our findings, other studies have shown that oil-loaded biofilms obtained from oil–cake matrices change both structure and functionality. For example, Bulut et al. [41] reported a decrease in tensile strength (from 0.638 to 0.381 MPa) when Satureja montana and Ocimum basilicum essential oils were incorporated into pumpkin oil cake films. Similarly, Šuput et al. [42] demonstrated that enriching Camelina sativa oil cake films with rosemary and eucalyptus essential oils reduced tensile strength (from 2.28 MPa in the control to 1.29 MPa in the sample with 2% rosemary EO), while elongation at break increased (from 19.83% to 26.13%), confirming the plasticizing effect of essential oils.

A similar pattern was observed in our present findings of SMeo-incorporated POC films, although those films exhibited higher tensile strength and elongation at break, suggesting a stronger and more cohesive polymer network compared to other oil–cake-based matrices. This difference may be related to the specific composition of the POC matrix, which provides enhanced structural integrity while still allowing SMeo components to act as plasticizers [43].

Further improvement should focus on strengthening the mechanical properties of POC-based films, as other biofilms reported in the literature exhibit significantly higher tensile strength. For example, starch–chitosan composites reinforced with raw seaweed and sodium tripolyphosphate have achieved values up to 29.9 MPa, while ultrathin PLA films exhibited tensile strength in the range of 53–105 MPa [44,45]. These benchmarks highlight the need to improve the mechanical strength of POC films in terms of competitiveness and further application as films. Otherwise, in their current form, SMeo-loaded POC films are recommended as coatings for food preservation rather than as standalone packaging materials.

3.2. Physico-Chemical Properties of Biofilms

The evaluation of moisture content, total soluble matter (solubility in water), and swelling degree (swelling in water) provided valuable insight into how the incorporation of Satureja montana essential oil influenced the functional properties of POC films (

Table 1. Physico-chemical properties of POC films: control (C) and POC films with 1%, 2%, and 3% Satureja montana EO.

Sample	Thickness (mm)	Moisture Content (%)	Solubility (%)	Swelling Degree (%)	WVTR (g m−2 h−1)	WVP (g m−1 s−1 Pa−1) × 10−13
C	0.145 ± 0.003 a	9.47 ± 0.69 c	43.46 ± 0.61 b	86.82 ± 3.01 b	4.88 ± 0.14 a	6.76 ± 0.21 a
1% SMeo	0.183 ± 0.002 a	15.46 ± 1.55 bc	14.12 ± 1.09 ab	73.89 ± 0.47 a	4.24 ± 0.50 a	6.52 ± 0.26 a
2% SMeo	0.202 ± 0.033 a	14.23 ± 1.25 ab	11.66 ± 0.51 a	60.51 ± 0.76 c	3.95 ± 0.19 a	6.25 ± 0.15 a
3% SMeo	0.221 ± 0.023 a	10.57 ± 0.77 ab	10.10 ± 0.59 c	57.20 ± 1.14 c	2.85 ± 0.24 b	3.56 ± 0.20 b

Data are presented as mean values ± standard deviation. Different letters indicate significant differences among samples (p < 0.05).

). Since low resistance to water remains one of the main limitations for practical applications of biofilms, assessing these properties is essential for predicting their stability and potential application. In this context, and according to previous findings, the addition of a hydrophobic phase is expected to improve these properties by reducing solubility and swelling, thereby enhancing the water resistance of the films [16,37,41,46]. In line with those studies, the incorporation of SMeo into POC films decreased solubility (up to 77%) and swelling (up to 35%) compared to the control POC film, confirming the beneficial effect of the essential oil in reducing water affinity properties. On the other hand, films with antioxidant potential, such as SMeo-loaded POC films, require a certain degree of swelling to enable more efficient release of antioxidants.

On the other hand, moisture content did not follow a linear trend, with values ranging from 9.47% in the control to 15.46% in SMeo-loaded films, suggesting that oil incorporation modifies the film matrix in a more complex manner, possibly due to microstructural rearrangements and heterogeneous oil distribution. Regarding moisture content, the values observed were in line with lipid-loaded composite films [16,47], supporting their suitability for food packaging applications where reduced water affinity properties are desirable.

3.3. Water Barrier Properties

Biopolymer films are inherently water-sensitive due to their hydrophilic nature, and various strategies have been explored to address this limitation. One of the most common and effective approaches is the incorporation of essential oils, which not only increase the proportion of the hydrophobic phase but also enrich the edible film with biologically active and valuable compounds that can enhance its nutritional and functional profile [48]. For this reason, the use of essential oils is considered more favourable than other strategies, such as the addition of inert lipids or synthetic hydrophobic agents, which may improve barrier properties but do not provide additional bioactivity [49].

POC-based films already contain plum oil originating from the oil cake, which already provides a certain level of hydrophobicity and explains their relatively low WVTR (4.88 ± 0.14 g m⁻² h⁻¹). Incorporation of SMeo further strengthened this effect, with a clear dose-dependent trend (Table 1). The most pronounced improvement was recorded at 3% SMeo, where WVTR decreased by about 42% compared to the control. To obtain a clearer assessment of the material’s barrier properties, WVP was also calculated. Unlike WVTR, it accounts for film thickness and the water vapor pressure difference, making it easier to compare different film samples.

Observing the reduction in WVP among control and SMeo-loaded POC films (up to 48%), it can be concluded that this percentage of decrease is consistent with findings for other edible films enriched with hydrophobic agents, such as composite films obtained from plum protein and arrowroot starch enriched with plum kernel oil [16]. However, when considering the WVP values, it can be concluded that POC films showed lower permeability compared to many other reported biopolymer films, highlighting their strong potential as effective moisture barriers. In the study by Sheikh et al. [16], composite films made from plum protein and arrowroot starch enriched with plum kernel oil showed WVP values ranging from 20.07 to 10.67 × 10⁻¹³ g m⁻¹ s⁻¹ Pa⁻¹. Similarly, Zhang et al. [47,50] reported improved water vapour barrier properties in agar/maltodextrin-based films: the addition of beeswax and glycerol monostearate reduced WVP to 7.64 × 10⁻¹³ g m⁻¹ s⁻¹ Pa⁻¹, while formulations with 10% beeswax or 10% liquid paraffin achieved the lowest values, reaching 6.86 × 10⁻¹³ g m⁻¹ s⁻¹ Pa⁻¹. These comparisons show that SMeo-incorporated POC films exhibited lower WVP values than other hydrophobic-enriched edible films, thus proving strong potential for use in active food packaging.

3.4. Structural Properties—Fourier-Transform Infrared Spectroscopy (FTIR)

FTIR was used to investigate the structural interactions between the POC matrix and Satureja montana essential oil, providing valuable insights into the preservation of the polymer backbone during biofilm formation and the specific modifications in functional groups induced by oil incorporation. The FTIR spectra of the control film, POC films containing 1%, 2%, and 3% SMeo, and pure essential oil are presented in Figure 4a,b. The spectra revealed similar major absorption bands in all samples, including a broad O–H/N–H stretching vibration around 3300 cm⁻¹ associated with hydroxyl groups and adsorbed water, and amide-related absorptions at 1650 and 1540 cm⁻¹. However, distinct differences in peak intensities and shapes were observed depending on the concentration of SMeo, indicating changes in the hydrogen-bonding environment within the polymer matrix. The incorporation of essential oil had the most pronounced effect in the 3% SMeo sample, which was separately presented in Figure 4c.

In the fingerprint region ranging from 650 to 1800 cm⁻¹ (Figure 4a), all films exhibited characteristic bands of the POC matrix, including a strong C=O stretching at Amide I region (1700–1600 cm⁻¹), N–H bending and C–N stretching in Amide II region (1470–1570 cm⁻¹), and N–H deformation, -CH₂ wagging and C–N stretching at Amide III region (1200–1350 cm⁻¹) [51,52]. However, with increasing SMeo concentration, the absorbance intensities of these characteristic amide bands progressively decreased, indicating a gradual loss of ordered protein secondary structures (α-helix and β-sheet conformations) and weakening of intermolecular hydrogen bonding within the protein network. Moreover, the incorporation of SMeo introduced additional spectral features characteristic for the aromatic and phenolic constituents of Satureja montana essential oil, mainly carvacrol, thymol, and p-cymene. For the 3% SMeo sample, increased absorbance was observed at 1600–1585 cm⁻¹, corresponding to aromatic C=C stretching vibrations, while new bands appeared at 1381 and 1362 cm⁻¹, attributed to symmetric and asymmetric bending of the isopropyl–methyl group. These bands corresponded to carvacrol present in Satureja montana essential oil [53], as also shown in its spectra (SMeo in Figure 4a). Additional peaks were identified in the spectral region characteristic of carvacrol (862, 994, and 1173 cm⁻¹) and thymol (877, 945, 1044, and 1153 cm⁻¹) [53]. A distinct band near 811 cm⁻¹, associated with out-of-plane C–H bending of the aromatic ring, and absorptions in the 1055–1019 cm⁻¹ range corresponded to para-substituted phenyl vibrations from p-cymene, which were also present in Satureja montana EO [54].

The incorporation of SMeo in the 3% SMeo sample caused noticeable alterations (Figure 4b), specifically in the spectral region 3000–3500 cm⁻¹, which is characteristic of O–H and C–H stretching vibrations. The appearance of peaks at 2958, 2927, and 2869 cm⁻¹ corresponded to the asymmetric and symmetric stretching of aliphatic C–H groups from terpene components (e.g., carvacrol and thymol) [55]. Furthermore, the broad O–H stretching band became more intense and slightly shifted towards lower wavenumbers in the presence of essential oil, suggesting the establishment of intermolecular hydrogen bonding between the phenolic –OH groups of SMeo and the hydroxyl groups of the POC matrix [56]. These results are consistent with the findings of Asghar et al. [57], as similar outcomes were observed for thyme-, cinnamon-, ginger-, and basil-incorporated biofilms. This weakening is due to interference from the essential oil with the polymer’s hydrogen-bonding network, which disrupts the association between polymer segments and reduces overall cohesion. Therefore, this structural change was reflected in the mechanical properties, as the lowest tensile strength values were observed at the highest SMeo concentration.

FTIR analysis demonstrated concentration-dependent changes with Satureja montana essential oil incorporation, reflecting the presence of terpene and phenolic constituents. The results indicate that the essential oil interacts with the POC matrix mainly through hydrogen bonding and physical integration, while the structural integrity of the POC backbone was preserved. With increasing SMeo concentration, the intensity of characteristic aromatic and aliphatic absorption bands became more pronounced, indicating weaker interactions between polymer chains when more essential oil is present, which corresponds to the decrease in tensile strength at higher oil concentrations.

3.5. Thermal Analysis—Differential Scanning Calorimetry (DSC)

The DSC thermograms for the control POC film and the POC films containing SMeo are shown in Figure 5. The main thermal transitions and enthalpy values are summarized in

Table 2. Summary of thermal transitions from DSC Analysis.

Sample	Low-T Endotherm (Melting/Denaturation) (°C)	Enthalpy ΔH (J/g)	High-T Endotherm (Melting/Denaturation) (°C)	Enthalpy ΔH (J/g)	Decomposition Temperature (°C)
C	133.07	148.1	191.21	0.93	229.3
1% SMeo	134.02	1.5	171.58	166.1	220.24
2% SMeo	131.93	0.9	160.24	17.12	212.3
3% SMeo	128.52	118.0	194.9	2.1	213.9

. The DSC curve for the control film displayed a primary endothermic peak at 133.07 °C, with a high enthalpy change (ΔH) of 148.1 J/g. This transition was attributed to the evaporation of moisture trapped within the film, as well as the unfolding and initial denaturation of the native protein structure within the film matrix [58,59]. A minor, higher-temperature endothermic peak was observed at 191.21 °C, which may correspond to the melting of highly stable protein aggregates or ordered regions.

The incorporation of SMeo significantly influenced the thermal properties of the films. The most notable effect was the pronounced reduction in the enthalpy of the primary endotherm (133 °C) in all SMeo-containing films. Specifically, the ΔH values for samples 1% SMeo and 2% SMeo dropped to 1.5 J/g and 0.9 J/g, respectively, compared to 148.1 J/g for the control. The magnitude of this enthalpy reduction was remarkable, more than 99% for 1% SMeo and 99.4% for 2% SMeo compared to the control. This reduction indicated that the addition of SMeo caused pre-denaturation of the protein structure during film formation. This interpretation was supported by FTIR analysis—the characteristic Amide I (~1650 cm⁻¹) and Amide II (~1540 cm⁻¹) bands, although still present, displayed reduced absorbance intensities in the SMeo-loaded films. This reduction in absorbance suggested the partial disruption of α-helical and β-sheet conformations and weakening of hydrogen bonding within the protein matrix. This effect was most pronounced in the 3% SMeo sample, indicating that SMeo components acted as potent denaturants during the film-forming process, by disrupting the hydrogen bonding and electrostatic interactions that stabilize the native protein conformation [60].

In contrast, an unexpected effect was observed in the high-temperature region. The appearance of a new, sharper endothermic peak at 171.58 °C and 160.24 °C for the 1% and 2% SMeo films, with significantly higher enthalpy values (166.1 J/g and 17.12 J/g, respectively, compared to 0.93 J/g for the control), indicated the formation of new, ordered structures. We hypothesize that SMeo components, acting as cross-linking agents or facilitators of protein–protein interactions, promoted the formation of heat-stable protein aggregates and/or protein-lipid complexes. The lower temperature of this new transition compared to the control’s high-T peak (191.21 °C) suggests that these new structures, while more enthalpically stable (higher ΔH) were morphologically distinct and melted/decomposed at a different, lower temperature range.

The behaviour of the 3% SMeo film showed an intermediate denaturation enthalpy (118.0 J/g), along with a slight shift in the high-temperature transition to 194.9 °C, suggesting a different balance of interactions. At this higher concentration, the essential oil may create a more hydrophobic environment that slightly stabilises the protein structure against thermal denaturation, or the saturation of binding sites could prevent the extensive disruption observed at lower concentrations. This non-linear effect highlights the concentration-dependent complexity of SMeo’s interaction with the POC matrix.

Despite the formation of these energy-intensive structures, the overall thermal stability of the POC-based films was reduced. The onset of decomposition, indicated by rapid and irreversible weight loss, shifted from 229.3 °C in the control film to 212.3–220.24 °C in the SMeo-loaded samples. Similar effects have been reported for carvacrol- and thymol-enriched films, where the plasticizing action of essential oil constituents decreased thermal stability [61,62]. These findings indicated that the incorporation of SMeo had a dual effect on the thermal properties of POC films—it disrupted the native protein structure while simultaneously promoting the formation of different types of ordered structures. However, although SMeo facilitated the formation of enthalpy-stable intermediates, it lowered the threshold for final thermal degradation, consistent with observations in other essential-oil-loaded systems [63]. The reduced thermal resistance can be attributed to the lubricating effect of hydrophobic molecules [50], whose low molecular weight allows them to penetrate intermolecular spaces within the polymer network. This increases free volume and molecular mobility while weakening hydrogen bonding, thereby reducing the energy required for molecular motion and thermal breakdown.

3.6. Microbial Activity—Disc Diffusion Assay

The antimicrobial potential of POC-based biofilms with Satureja montana essential oil was evaluated against Gram-negative bacteria (E. coli), Gram-positive bacteria (S. aureus), spore-forming bacteria (B. cereus), yeast (C. albicans), and filamentous fungi (A. flavus, A. parasiticus, A. ochraceus) using the disc diffusion method (

Table 3. Antimicrobial activity of POC films: control (C) and POC films with 1%, 2%, and 3% Satureja montana EO.

Sample	Antimicrobial Activity of Biofilms (mm)
	E. coli	S. aureus	B. cereus	C. albicans	A. flavus	A. parasiticus	A. ochraceus
C	ND	ND	ND	ND	ND	ND	ND
1% SMeo	11.0 ± 1.4 a	17.5 ± 3.5 a	13.5 ± 2.1 a	16.5 ± 2.1 a	8.0 ± 0.0 a	9.5 ± 0.7 a	10.0 ± 0.0 a
2% SMeo	12.5 ± 1.4 a	31.75 ± 2.5 a	15.5 ± 4.9 a	20.5 ± 4.9 a	9.0 ± 0.0 a	10.5 ± 0.7 a	21.0 ± 0.0 a
3% SMeo	23.5 ± 0.7 b	45.0 ± 1.4 b	30.5 ± 2.5 b	38.0 ± 1.4 b	13.5 ± 1.4 b	19.0 ± 4.2 a	42.5 ± 3.5 a

Data are presented as mean values ± standard deviation. Different letters indicate significant differences among samples (p < 0.05); ND—not detected.

). The disc diffusion method is used to evaluate how POC-based biofilms in direct contact with a product can release active compounds to reduce, delay, or prevent microbial growth. According to Ponce et al. (2003) [64], bacterial sensitivity is classified based on the diameter of the inhibition zone: bacteria are considered not sensitive if the zone is smaller than 8 mm, sensitive if it is 9–14 mm, very sensitive if it is 15–19 mm, and extremely sensitive if it exceeds 20 mm. According to the results shown in Table 2, the control sample did not show any inhibition, confirming that the observed effects were completely dependent on SMeo concentration.

A clear dose-dependent response was observed across all tested microorganisms. S. aureus was highly sensitive, with an inhibition zone reaching 45.0 mm at 3% concentration, as well as B. cereus, with the inhibition zone up to 30.5 mm. E. coli exhibited lower susceptibility, consistent with the protective effect of its outer membrane against hydrophobic compounds [65]. Winter savoury essential oil demonstrated antimicrobial activity against various bacteria, particularly against Gram-positive and spore-forming bacteria, suggesting its potential as a natural antimicrobial agent in food preservation and therapeutic applications [66,67,68]. Satureja montana essential oil contains several monoterpenes—such as thymol, carvacrol, p-cymene, geraniol, γ-terpinene, myrcene, and linalool—but its antibacterial activity is mainly attributed to carvacrol, a phenolic monoterpene [29]. Carvacrol is known to disrupt bacterial cell membranes, increasing permeability and causing leakage of cellular contents, which leads to cell death. The phenolic structure of carvacrol, particularly its hydroxyl group, plays a central role in the antibacterial mechanism, both individually and in combination with other components, while the presence of other monoterpenes is thought to enhance the effect of carvacrol, for example, by modifying membrane lipophilicity and helping carvacrol reach its target more efficiently [69].

Figure 6 shows the inhibition zones of the POC-based active film containing 3% Satureja montana essential oil against S. aureus, C. albicans, and A. ochraceus, respectively. In addition to its antibacterial effects, the 3% SMeo sample demonstrated extremely sensitive inhibitory activity against yeasts (C. albicans) and filamentous fungi (A. flavus, A. parasiticus, and A. ochraceus). C. albicans also exhibited extremely sensitive susceptibility, with inhibition zones increasing from 16.5 mm at 1% to 38.0 mm at 3% of SMeo. Among filamentous fungi, A. ochraceus was the most sensitive (42.5 mm at 3%), followed by A. parasiticus and A. flavus, which showed sensitive to very sensitive inhibition. The antifungal efficacy of SMeo appears to be the result of the synergistic action of its various bioactive compounds, effectively inhibiting both yeast and filamentous fungi, and highlighting its potential as a natural preservative in food and pharmaceutical applications [70,71].

3.7. Antioxidative Activity—DPPH Assay

The DPPH free radical scavenging assay is one of the most widely used methods for evaluating the antioxidant capacity of natural compounds in vitro. Results from this assay are shown in Figure 7, which shows the antioxidant activity of POC films enriched with various concentrations of Satureja montana essential oil. A clear concentration-dependent increase in antioxidant capacity was observed. The control POC film, without essential oil, showed the lowest activity (~9.97%), while the addition of 1% and 2% SM significantly enhanced antioxidant properties (24.26% and 36.51%, respectively). The highest activity (41.95%) was achieved with 3% SMeo, which was statistically different from both the control and lower concentrations. These results highlight the strong radical scavenging potential of S. montana oil, attributed to its high content of bioactive compounds such as carvacrol, thymol, and other phenolics with well-documented antioxidant activity [29,71,72,73].

In the study by Bulut et al. [74], films based on pumpkin oil cake (PuOC) were prepared with the addition of different SMeo concentrations, but no significant increase in antioxidant activity was observed with higher SMeo concentrations. This was attributed to the reduced effectiveness of SMeo after incorporation into the PuOC biofilm, probably due to the loss of volatile compounds during film preparation, drying, and storage, as these compounds are highly sensitive and prone to evaporation. In contrast, the present findings suggest that POC is a more suitable matrix for SMeo incorporation, allowing the active antioxidant carriers to be effectively retained within the biopolymer film structure. Such a composition of POC-based films is promising for the development of active packaging materials with potential applications in food preservation.

3.8. Standard Score Analysis

Z-score analysis was used to evaluate all measured parameters—thickness, tensile strength, elongation at break, moisture content, solubility, swelling degree, water vapour permeance, and antioxidative and antimicrobial activities—to identify the most balanced formulation for biofilm synthesis. As shown in Figure 8, the 3% SMeo film achieved the highest composite Z-score (0.853), designating it as the optimal sample. This result indicates that despite shifts between individual properties the 3% SMeo formulation offers the most favorable overall balance of mechanical, structural, barrier, and bioactive characteristics.

4. Conclusions

The incorporation of Satureja montana essential oil into POC-based films was shown to influence their structural, thermal, mechanical, physico-chemical, water barrier, and biological properties. FTIR analysis confirmed that while the POC-polymer backbone remained intact, new interactions between oil constituents and the matrix introduced subtle but meaningful changes in the chemical composition. This was further confirmed by thermal analysis, which revealed that SMeo incorporation disrupted native protein structures and promoted the formation of new EO–biopolymer interactions, with the key outcome being a decrease in overall thermal stability with SMeo addition. These findings are firmly related to the mechanical properties of SMeo-loaded POC films. Addition of SMeo affected tensile strength in a dose-dependent manner, where an overplasticizing effect was achieved with the sample 3%SMeo. An excessive amount of oil weakened cohesion and decreased elongation at break values.

Water barrier properties were significantly improved, as demonstrated by the remarkable reduction in WVP and WVTR values, highlighting the role of hydrophobic SMeo components in preventing water permeation. Moreover, water affinity properties were shown to be reduced in terms of solubility and swelling, while controlled swelling, on the other hand, is suitable for the release of antioxidant compounds.

The key highlights were related to biological properties reflected as an effective inhibition of all tested microbiota—bacteria, yeast, and mold—with the most pronounced activity observed against S. aureus and A. ochraceus. Combined with moderate, dose-dependent antioxidant activity, the incorporation of SMeo clearly enhanced the bioactive potential of POC films, positioning them as sustainable, biologically enriched materials. Furthermore, Z-score analysis identified the 3% SMeo formulation as the optimal, most appropriately balancing all the abovementioned analyses.

The obtained results provide a clear answer to the question posed in the study’s title—SMeo can transform POC-based films into active food packaging materials intended for food quality and shelf-life preservation.