Physicochemical and Antimicrobial Properties of Whey Protein-Based Films Functionalized with Palestinian Satureja capitata Essential Oil

: The present study aimed to produce bio-active packaging materials made of whey proteins (WPs) and essential oil (EO) extracted from Thymbra ( Satureja capitata , L.), one of the most popular Palestinian wild plants. In this study, two different Thymbra leaves from Nablus and Qabatiya in Palestine were collected and analyzed for EOs by gas chromatography and mass spectrometry. Based on the analysis, two EOs, namely, TEO1 and TEO2, were extracted, and it was found that both samples primarily contain γ -terpinene and carvacrol, whereas p -cymene was detected only in TEO1. The antimicrobial activity of TEO1 and TEO2 was evaluated by microbroth microdilution assays against pathogenic bacteria and yeast. Based on the results, TEO1 exhibited potent antimicrobial activity against the test strains. Besides, TEO1 was chosen to functionalize WP-based ﬁlms at different concentrations (0.1%, 0.4%, and 0.8% v / v of Film Forming Solutions). Film mechanical property investigation showed a marked reduction in the tensile strength and Young’s modulus at 0.8% TEO1. In contrast, its elongation at break value was signiﬁcantly ( p < 0.05) increased due to the plasticizing effect of the EO. Moreover, the ﬁlm transparency was found to be signiﬁcantly ( p < 0.05) reduced by increasing TEO1 concentrations. Finally, microbiological investigations indicated that ﬁlm antimicrobial activity against both gram-positive and gram-negative bacteria increased dose-dependently. The overall results open interesting perspectives for employing these ﬁlms as preservative materials in food packaging.


Introduction
Food packaging has a crucial role in protecting food products from the risk of pathogen growth, leading to an extension of food product shelf life [1,2]. So far, polyethylene terephthalate, polyethylene, and polyvinylchloride are the most common petrol-derived plastics, commonly used due to their availability at a relatively low cost [2], and because they possess excellent technical properties such as hardness, formability, rigidity, elasticity, heat, and chemical resistance [3]. Meanwhile, these conventional plastics are not biodegradable, and are thus considered today as being the most polluting materials on our planet [4]. Hence, scientists have started developing new strategies and production of alternative materials, such as bio-based polymers obtained from agricultural or food wastes. In this context, whey from the cheese industry is considered one of the most interesting and valuable food wastes to be recycled. Whey is the liquid that is formed following casein coagulation in the dairy industries, possessing a high organic content and a high percentage of proteins. As whey is rich in several nutrients important for the obtainment of further bioproducts, it would be advisable to recycle whey-containing liquid waste through an eco-friendly method. However, as a huge amount of whey goes unused, or is even disposed of illegally, researchers should develop innovative methods to get the maximum benefits from its use. In fact, whey proteins (WPs) derived from the cheese industry waste stream possess a high nutritional value and properties such as solubility in water and the ability to act as emulsifiers, which have been exploited for the manufacturing of transparent, flexible, colorless, and odorless bioplastics with poor moisture and O 2 barrier properties. [5]. In the year 2017, the production of milk in the EU totaled 170 billion kg, 93% of which resulted in dairy products, which includes cheese (37%), butter (30%), cream (13%), fresh milk (11%), acidified milk (4%), milk powder (2%), and other minor products. Each liter of processed milk results, on an average, in 2.5 L of whey-containing wastewater, while about 9-10 L of whey is produced per kg of cheese [6]. Whey contains two main proteins, ß-lactoglobulin, and α-lactalbumin, endowed with important properties such as solubility, foaming, and gelling [7], making them of great interest for different biotechnological applications. In particular, WPs can give rise to edible bioplastic materials [8][9][10][11][12].
Aiming to improve food packaging features, many studies have exploited natural additives with antimicrobial and antioxidant activity to produce bio-active materials [13][14][15]. Interestingly, essential oils (EOs) extracted from different plants and edible herbs are considered potential additives for food packaging applications because of their biological features [1,13,16,17].
agricultural or food wastes. In this context, whey from the cheese industry is co one of the most interesting and valuable food wastes to be recycled. Whey is t that is formed following casein coagulation in the dairy industries, possessin organic content and a high percentage of proteins. As whey is rich in several important for the obtainment of further bioproducts, it would be advisable t whey-containing liquid waste through an eco-friendly method. However, as amount of whey goes unused, or is even disposed of illegally, researchers should innovative methods to get the maximum benefits from its use. In fact, whey (WPs) derived from the cheese industry waste stream possess a high nutritional v properties such as solubility in water and the ability to act as emulsifiers, which h exploited for the manufacturing of transparent, flexible, colorless, and odorless bi with poor moisture and O2 barrier properties. [5]. In the year 2017, the productio in the EU totaled 170 billion kg, 93% of which resulted in dairy products, which cheese (37%), butter (30%), cream (13%), fresh milk (11%), acidified milk (4 powder (2%), and other minor products. Each liter of processed milk result average, in 2.5 L of whey-containing wastewater, while about 9-10 L of whey is p per kg of cheese [6]. Whey contains two main proteins, ß-lactoglobulin, lactalbumin, endowed with important properties such as solubility, foaming, an [7], making them of great interest for different biotechnological applications. In p WPs can give rise to edible bioplastic materials [8][9][10][11][12].
Aiming to improve food packaging features, many studies have exploited additives with antimicrobial and antioxidant activity to produce bio-active mate 15]. Interestingly, essential oils (EOs) extracted from different plants and edible h considered potential additives for food packaging applications because of their b features [1,13,16,17].
Satureja capitata, an aromatic plant of the Lamiaceae family, commonly "Thymbra" (Figure 1) and also known in Arabic as "za'atar rumi", "za'atar fra "za'atar farsi" [18] (meaning "Roman hyssop", "European hyssop", and hyssop", respectively), attracted our attention since it possesses some interesting Thymbra has been used for more than 100 years by the Palestinians as a spice food, as well as an alternative medicine to treat tonsillitis. In fact, many scientifi proved that this plant has powerful antibacterial, antifungal, and antiviral activiti further investigations have shown that the extract from this plant can protec coughing, respiratory infections, diarrhea, and digestive problems [19,20]. There study has been addressed to extract Thymbra EOs (TEOs), to determine their com and to produce bioactive materials by incorporating the extracted TEOs into W films. Thymbra has been used for more than 100 years by the Palestinians as a spice in some food, as well as an alternative medicine to treat tonsillitis. In fact, many scientific studies proved that this plant has powerful antibacterial, antifungal, and antiviral activities, while further investigations have shown that the extract from this plant can protect against coughing, respiratory infections, diarrhea, and digestive problems [19,20]. Therefore, this study has been addressed to extract Thymbra EOs (TEOs), to determine their composition, and to produce bioactive materials by incorporating the extracted TEOs into WP-based films.

Thymbra Essential Oil Extraction
Thymbra leaves were obtained both from a local shop in Nablus and directly collected from Qabatiya mountains in Palestine. The first ones were already dry, whereas the second ones were previously dried in the oven (Huanghua Faithful Instrument Co., Ltd., Tianjin, China) at 30 • C for 24 h before EO extraction. Then, TEOs were extracted from both dried leaf samples by steam-distillation for 3 h using a Clevenger type apparatus, according to a previously described method [21], and respectively named TEO1 and TEO2. CaCl 2 was finally added to remove the remaining water, and both TEO samples were kept in a dark glass bottle at 4 • C before their characterization and use.

Determination of Thymbra Essential Oil Composition
Yields (%) of extracted TEOs were calculated by dividing the TEO weight by the weight of Thymbra leaves. TEOs were then analyzed with a gas chromatography-mass spectrometry (Shimadzu QP-5000 GC-MS, Kyoto, Japan). The GC apparatus was equipped with a Rtx-5 MS column (30 m long, 0.25 µm thickness, and 0.250 mm inner diameter), whereas helium was utilized as a carrier gas at a flow rate of 1 mL/min, and the injector temperature was 220 • C. The oven temperature started from 50 • C (1 min hold) to 130 • C (5 • C/min), then to 250 • C (10 • C/min) and, finally, it was kept isothermally for 15 min (the transfer line temperature was 290 • C). For GC-MS detection, an electron ionization system, with detector volts of 1.7 KV, was used. A scan rate of 0.5 s, and a scan speed of 1000 amu/s was applied, covering a mass range from 38 to 450 M/Z. The chemical ingredients of the volatile oil were identified by correlating their mass spectra with the reference ones present of the mass spectrometry data center of the National Institute of Standards and Technology (NIST) [22].

Antimicrobial Activity of Essential Oils Extracted from Thymbra Leaves
Minimal inhibitory concentration (MIC) values of TEO were evaluated by using the method previously described [23] against different microorganisms such as S. aureus, E. coli, K. pneumoniae, P. vulgaris, E. faecium, P. aeruginosa, and C. albicans. In addition, the clinical pathogen MRSA was used to evaluate TEO antimicrobial activity. Briefly, TEOs were two-fold serially diluted in the freshly prepared sterile MHB medium and dispensed into a 96-well micro-titration plate. After that, each well was inoculated with bacterial inoculums, which were prepared from adjusted 0.5 McFarland microbial suspensions. Regarding the C. albicans, the same procedure was used by using RPMI media instead of Mueller-Hinton. After mixing, the 96-well micro-titration plates were incubated at 37 • C for 24 h for those plates inoculated with the test bacterial strains, and for about 48 h for those plates inoculated with C. albicans. MIC values were determined as the lowest antimicrobial agent concentration that prevents the visible microorganism growth. The experiment was carried out in triplicates and against each tested microbe, three controls were included. Two of them were negative controls, one consisting of the used MHB medium alone, and the other one consisting of MHB medium with the diluted TEOs to check the sterility of these materials. The third control used was a positive control consisting of antibiotics with known antimicrobial activity: ampicillin, ciprofloxacin, or fluconazole.

Zeta Potential and Particle Size Measurements of Whey Protein Film-Forming Solutions (FFSs)
To determine the FFS particle size and stability, 1.0 mL of each FFS was analyzed by a Zetasizer Nano-ZSP (Malvern ® , Worcestershire, UK) as reported in Abdalrazeq et al. [8]. Three measurements of each replicate were carried out.

Antimicrobial Activity of Whey Protein Film-Forming Solutions
S. enteriditis 706 RIVM, S. enterica ATCC ® 14028, S. aureus ATCC 29213, and E. faecalis ATCC 29212 were grown in MHB or on TSA plates. All the strains were grown overnight at 37 • C. Afterward, the bacteria were diluted in fresh media as described below. To test the antimicrobial activity of the FFS, microbroth microdilution assay [24,25] followed by a colony count assay were carried out. Briefly, 50 µL of bacterial cells at the final concentration of 2 × 10 6 CFU/mL were plated into 96-well plates. After that, 50 µL of 2-fold serial dilution of the FFSs were added and, upon 24 h of incubation, 100 µL of each sample were plated on TSA Petri dishes. The plates were incubated at 37 • C for 24 h and, at the end of the incubation, colony counting was performed. FFSs in the absence of TEO, as well as the bacterial cells alone, were used as control samples. Experiments were carried out in triplicate.

Whey Protein-Based Film Preparation
For film preparation, FFSs were prepared as reported in Abdalrazeq et al. [8] by dissolving WP isolate (5%, w/v) in distilled water and by adding 50% GLY (w/w of WPs) to the FFS. Hence, different concentrations of TEO1 (0.1%, 0.4% or 0.8% v/v) were added or not to FFS aliquots. The films were prepared by casting the FFS samples (25 mL) onto 8 cm diameter polyethylene Petri dishes and by incubating the solutions in a climatic chamber set up at 25 • C and 45% relative humidity for 24 h.

Film Opacity Determination
Film opacity was performed following the method of Shevkani et al. [26]. More in detail, 1 cm × 4 cm film strips were let to adhere perfectly to a cuvette wall. Absorbance was detected at 600 nm and the opacity was obtained as, where A 600nm and X were the absorbance recorded at 600 nm and the film thickness (mm), respectively.

Film Thickness and Mechanical Properties
WP-based film strips with a dimension of 1 cm × 8 cm were obtained with a sharp scissor and then equilibrated for 48 h at 25 • C and 50% relative humidity into a glass chamber containing a saturated solution of Mg(NO 3 ) 2 before the characterization. Film thickness was measured by means of a micrometer (Electronic digital micrometer, DC-516, sensitivity 0.001 mm, Metrology Co., Pontoglio, Italy) at six different points of each film, whereas the determination of film tensile strength (TS), elongation at break (EB), and Young's modulus (YM) were performed on five specimens of each sample (5 cm gage length, 1 kN load and 5 mm/min speed) with an Instron universal testing instrument model no. 5543A (Instron Engineering Corp., Norwood, MA, USA).

Film Moisture Content and Uptake
Samples of each film (2 cm × 2 cm) were weighed and dried at 105 • C in an oven for 24 h. Analyses in triplicate were made and film moisture content was obtained as: where W 1 is the initial weight of the film and W 2 is the film weight after drying at 105 • C. Moisture uptake was measured gravimetrically in triplicate following the methodology described by Sartori and Menegalli [27]. Briefly, 20 mm-sided square film samples were dried at 105 • C for 24 h, conditioned at 23 ± 2 • C into a desiccator containing a saturated Mg(NO 3 ) 2 solution, and weighed. The moisture uptake was obtained as: where Ws and Wd are the weight of swollen and dried films, respectively.

Antimicrobial Properties of Whey Protein Films
Bacterial cells were grown in Muller Hinton Broth (MHB, Becton Dickinson Difco, Franklin Lakes, NJ, USA)and on TSA. In all the experiments, bacteria were inoculated and grown overnight in MHB at 37 • C. The next day, bacteria were transferred to a fresh medium and grown to the mid-logarithmic phase. The antimicrobial activity of the WP-based films derived from FFSs, containing or not containing TEO1, was tested by the previously described method [25]. Briefly, bacterial cells were diluted into tryptic soy broth till 2 × 10 7 CFU/mL and spread onto TSA plates. A 1.5 cm 2 square of the edible film was placed into an inoculated plate to allow full contact with the agar surface. Following incubation at 37 • C for 24 h, the microorganism growth underneath the film was investigated.

Statistical Analysis
Statistical analyses were carried out utilizing JMP software 5.0 (SAS Institute, Cary, NC, USA, version 10.0). The analysis of variance was performed, and the means were compared with the Tukey-Kramer HSD test. Differences were considered significant at p < 0.05. Significant differences highlighted by Student's t-Test were indicated as * p < 0.05 or ** p < 0.01.

Chemical Composition of Thymbra Essential Oils
It is well known that several parameters affect the percentage yield of plant EO extraction, including soil, genetics, geographic origin, maturity degree, different organs, temperature, and relative humidity conditions, extract distillation time, and, finally, pressure and temperature of distillation [21,23,28]. In the present study, the TEO1 percentage yield was found to be 2.36%, whereas the TEO2 yield was 3.07%. In addition, notable quantitative and qualitative differences were observed in the components identified by analyzing with GC-MS the two different extracted TEOs. In fact, as shown in Table 1, the major identified volatile compounds detected in TEO1 were γ-terpinene, carvacrol, and p-cymene with the following percentages, 38.95%, 22.96%, and 19.53%, respectively, whereas in TEO2 samples the γ-terpinene content was higher, with a percentage of 57.81%, and no p-cymene was detected.

Antimicrobial Activity of Thymbra Essential Oil Samples
As reported in Table 2, TEO antimicrobial activity was investigated against gramnegative bacteria (E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. vulgaris ATCC 8427, and P. aeruginosa ATCC 9027), and gram-positive bacteria (E. faecium ATCC 700221, S. aureus ATCC 25923, and the clinical pathogen S. aureus MRSA). Besides, TEO antimicrobial activity was also tested against C. albicans ATCC 90028. MIC 100 values of both TEO samples revealed a powerful antimicrobial property towards both gram-positive and gram-negative bacteria, as well as against the fungal strain, even though TEO1 showed a stronger inhibitory effect than TEO2 against all the studied microorganisms. it is also possible to see that the investigated Palestinian TEOs exhibited stronger antimicrobial activity in comparison with that exerted by TEOs extracted from Turkish Thymbra previously analyzed by Baydar et al. [19]. In fact, the Turkish Thymbra EOs inhibited the microorganism growth at concentrations <1/100 (v/v), whereas the Palestinian TEOs showed antimicrobial effects at concentrations <1/800 (TEO1) or <1/400 (TEO2) (v/v).

Characterization of the Film-Forming Solution Containing Thymbra Essential Oil
The particle size and zeta potential values of WP-based FFSs containing 0.8% TEO1 indicate a notable reduction in the particle size, most likely due to the formation of emulsions in the presence of EO, and that FFS stability slightly decreased by increasing TEO1 concentration (Figure 2).

Characterization of the Film-Forming Solution Containing Thymbra Essential Oil
The particle size and zeta potential values of WP-based FFSs containing 0.8% TEO1 indicate a notable reduction in the particle size, most likely due to the formation of emulsions in the presence of EO, and that FFS stability slightly decreased by increasing TEO1 concentration (Figure 2). To determine the ability of WP-containing FFSs to inhibit microbial growth, MIC100 values were calculated by carrying out dose-response experiments. WP FFSs prepared in the absence of TEO1 showed a slight antimicrobial activity towards all the strains tested. Conversely, the FFSs containing the lowest concentration tested (0.1%) of TEO1 were able to totally suppress the bacterial growth in the case of the gram-negative bacteria S. enteriditis 706 RIVM, S. enterica subsp. enterica serovar Typhimurium (ATCC ® 14028) and the gram-positive S. aureus ATCC 29213 ( Figure 3A-C, respectively). Moreover, in the case of E. faecalis ATCC 29212, FFS containing 0.1% TEO was found to inhibit the bacterial growth by about 50%, whereas complete inhibition was observed in the presence of 0.4% TEO1 ( Figure 3D). To determine the ability of WP-containing FFSs to inhibit microbial growth, MIC 100 values were calculated by carrying out dose-response experiments. WP FFSs prepared in the absence of TEO1 showed a slight antimicrobial activity towards all the strains tested. Conversely, the FFSs containing the lowest concentration tested (0.1%) of TEO1 were able to totally suppress the bacterial growth in the case of the gram-negative bacteria S. enteriditis 706 RIVM, S. enterica subsp. enterica serovar Typhimurium (ATCC ® 14028) and the grampositive S. aureus ATCC 29213 ( Figure 3A-C, respectively). Moreover, in the case of E. faecalis ATCC 29212, FFS containing 0.1% TEO was found to inhibit the bacterial growth by about 50%, whereas complete inhibition was observed in the presence of 0.4% TEO1 ( Figure 3D).

Opacity, Thickness, and Mechanical Properties of Films Functionalized with Thymbra Essential Oil
The transparency of EO-containing films is affected by the oil volume fraction and droplet size distribution in the FFS, as well as by the droplet rearrangement during FFS drying [29,30]. Moreover, the solvent evaporation of the solvent during the FFS drying may cause changes in the emulsion structure (i.e., creaming, aggregation, and/or coalescence), influencing the film optical properties [31]. The results illustrated in Figure 4 show a considerable decrease in the WP-based film transparency by increasing TEO1 concentration. lected in three independent experiments. (A) is S. enteriditis RIVM 706, (B) is S. enterica s terica serovar Typhimurium ATCC ® 14028, (C) is S. aureus ATCC ® 29213, (D) is E. faecal 29212.

Opacity, Thickness, and Mechanical Properties of Films Functionalized with Thymb Essential Oil
The transparency of EO-containing films is affected by the oil volume frac droplet size distribution in the FFS, as well as by the droplet rearrangement du drying [29,30]. Moreover, the solvent evaporation of the solvent during the FF may cause changes in the emulsion structure (i.e., creaming, aggregation, and/o cence), influencing the film optical properties [31]. The results illustrated in Figur a considerable decrease in the WP-based film transparency by increasing TEO1 tration.  These findings are very similar with those previously reported by Galus et al. [30], who prepared and characterized WP-based films containing rapeseed oil. In addition, Figure 5 indicates that no differences were observed among the thickness of the WP-films prepared in the absence or presence of different TEO1 concentrations, whereas the mechanical feature investigation of TEO1-containing films demonstrated a progressive decrease in film TS by increasing TEO1 concentrations. Conversely, a marked decrease in the film YM value and an appreciable increase in the EB were observed only at the highest TEO1 concentration tested. All these data clearly demonstrate an increased plasticizing effect triggered by TEO1 addition to the WP-based FFSs. It is worthy to note that the results obtained in this study are close with those obtained on whey protein isolate-based films into which different concentrations of tarragon oil were added [32]. In fact, an improvement of the technological attitude of the films, e.g., enhanced mechanical properties and higher transparency were found following the tarragon oil incorporation [32].
Coatings 2021, 11, 1364 9 of 12 TEO1 concentration tested. All these data clearly demonstrate an increased plasticizing effect triggered by TEO1 addition to the WP-based FFSs. It is worthy to note that the results obtained in this study are close with those obtained on whey protein isolate-based films into which different concentrations of tarragon oil were added [32]. In fact, an improvement of the technological attitude of the films, e.g., enhanced mechanical properties and higher transparency were found following the tarragon oil incorporation [32].

Moisture Content and Uptake of Whey Protein Films Containing Thymbra Essential Oil
Moisture content of WP-based films progressively decreased following the addition of increasing concentrations of TEO1 into the originating FFSs (Table 3). It is worthy to note that even at very low concentrations (0.1%), TEO1 was effective to reduce the film moisture content. These results reflected those from Galus et al. [30], who investigated WP-based materials containing rapeseed oil. On the other hand, slight, even though not statistically significant, increases of moisture uptake were also observed. Similar data were obtained by Salarbashi et al. [33], who analyzed moisture uptake of soluble soybean/polysaccharide films incorporated with Zataria multiflora Boiss (Shirazi thyme).

Moisture Content and Uptake of Whey Protein Films Containing Thymbra Essential Oil
Moisture content of WP-based films progressively decreased following the addition of increasing concentrations of TEO1 into the originating FFSs (Table 3). It is worthy to note that even at very low concentrations (0.1%), TEO1 was effective to reduce the film moisture content. These results reflected those from Galus et al. [30], who investigated WP-based materials containing rapeseed oil. On the other hand, slight, even though not statistically significant, increases of moisture uptake were also observed. Similar data were obtained by Salarbashi et al. [33], who analyzed moisture uptake of soluble soybean/polysaccharide films incorporated with Zataria multiflora Boiss (Shirazi thyme). 3.6. Determination of the Antimicrobial Activity of Whey Protein Films Containing Thymbra Essential Oil Figure 6 shows the antimicrobial activity of the WP-based films, prepared in the absence or presence of TEO1, towards S. enteriditis 706 RIVM, S. enterica ATCC ® 14028, S. aureus ATCC 29213, and E. faecalis ATCC 29212 strains, observed by placing small squares (1.5 cm × 1.5 cm) of each film on a confluent bacterial plate in full contact with the agar surface [34,35].
reported in the same column (Tukey-Kramer test, p < 0.05) 3.6. Determination of the Antimicrobial Activity Of Whey Protein Films Containing Thymbra Essential Oil Figure 6 shows the antimicrobial activity of the WP-based films, prepared in the ab sence or presence of TEO1, towards S. enteriditis 706 RIVM, S. enterica ATCC ® 14028, aureus ATCC 29213, and E. faecalis ATCC 29212 strains, observed by placing small square (1.5 cm × 1.5 cm) of each film on a confluent bacterial plate in full contact with the aga surface [34,35]. After 24 h at 37 °C, it was observed that the WP-based film itself was unable to inhib bacterial growth (Figure 6). On the contrary, the films containing TEO1 at the highest con centration tested (0.8%) showed a strong antimicrobial activity towards S. enteriditis 70 RIVM, S. enterica ATCC ® 14028 as well as against S. aureus ATCC 29213. Interestingly, was also possible to observe a zone of inhibition surrounding the films activated wit TEO1, thus, suggesting the ability of TEO1 to diffuse from the film into the agar matri Furthermore, no antimicrobial activity of the films was observed against E. faecalis ATC 29212 even at the highest TEO1 concentration tested. The data presented in this paper ar comparable to those obtained by Bleoancă et al. [36] who produced films from high pre sure thermally treated whey protein concentrate into which the extracts were added. Th authors have demonstrated the ability of the film matrix to vehicular the bioactive mol cules, as they were endowed with good antimicrobial properties as demonstrated by th inhibition zones towards three fresh food spoilage microorganisms such Torulopsis stellat Geotrichum candidum, and Bacillus subtilis [36]. After 24 h at 37 • C, it was observed that the WP-based film itself was unable to inhibit bacterial growth (Figure 6). On the contrary, the films containing TEO1 at the highest concentration tested (0.8%) showed a strong antimicrobial activity towards S. enteriditis 706 RIVM, S. enterica ATCC ® 14028 as well as against S. aureus ATCC 29213. Interestingly, it was also possible to observe a zone of inhibition surrounding the films activated with TEO1, thus, suggesting the ability of TEO1 to diffuse from the film into the agar matrix. Furthermore, no antimicrobial activity of the films was observed against E. faecalis ATCC 29212 even at the highest TEO1 concentration tested. The data presented in this paper are comparable to those obtained by Bleoancă et al. [36] who produced films from high pressure thermally treated whey protein concentrate into which the extracts were added. The authors have demonstrated the ability of the film matrix to vehicular the bioactive molecules, as they were endowed with good antimicrobial properties as demonstrated by the inhibition zones towards three fresh food spoilage microorganisms such Torulopsis stellata, Geotrichum candidum, and Bacillus subtilis [36].

Conclusions
EOs extracted from two different samples of Palestinian Thymbra (Satureja capitata, L.) leaves were analyzed for their composition and antimicrobial activity. Among these, the most promising antimicrobial agent was incorporated into WP-based edible films and evaluated for its ability to control the microbial growth. TEO1, showing higher antimicrobial properties most likely due to the presence of p-cymene, was included in the FFSs and the derived films were shown to possess improved mechanical properties, decreased moisture content, and exhibited a marked antimicrobial activity towards S. enteriditis, S. enterica and S. aureus. Regarding the E. faecalis bacterial strain, while FFSs were found to exert pronounced antimicrobial properties, no noteworthy effects of the films were detected. Furthermore, the observed zone of inhibition surrounding the films activated with TEO1 suggested the ability of TEO1 to diffuse into the agar matrix. Therefore, TEO1 may represent an effective bioactive additive of protein-based materials to be used in food packaging against specific spoilage bacterial strains. To this purpose, more investigations are still required to effectively examine the ability of WP-based bio-active packaging material functionalized with TEO1 in extending the shelf-life of some food.