Production and Characterization of k-Carrageenan Films Incorporating Cymbopogon winterianus Essential Oil as New Food Packaging Materials

The global production of synthetic plastics from petroleum-based raw ingredients exceeds 150 million metric tons. The environment is threatened by tons of plastic waste, thus endangering wildlife and the public’s health. These consequences increased the interest in biodegradable polymers as potential substitutes for traditional packaging materials. This study aimed to produce and characterize k-carrageenan films incorporating Cymbopogon winterianus essential oil, in which citronellal was determined to be the major compound (41.12%). This essential oil presented remarkable antioxidant activity, as measured through DPPH (IC50 = 0.06 ± 0.01%, v/v; AAI = 85.60 ± 13.42) and β-carotene bleaching (IC50 = 3.16 ± 0.48%, v/v) methods. The essential oil also showed antibacterial properties against Listeria monocytogenes LMG 16779 (diameter of inhibition zone = 31.67 ± 5.16 mm and MIC = 8 µL/mL), which were also observed when incorporated in the k-carrageenan films. Moreover, scanning electron microscopy showed the reduction of the biofilms of this bacterium, and even its inactivation, due to visible destruction and loss of integrity when the biofilms were created directly on the developed k-carrageenan films. This study also revealed the quorum sensing inhibition potential of Cymbopogon winterianus essential oil (diameter of violacein production inhibition = 10.93 ± 0.81 mm), where it could impede intercellular communication and, hence, lower violacein synthesis. The produced k-carrageenan films were transparent (>90%) and slightly hydrophobic (water contact angle > 90°). This work demonstrated the viability of using Cymbopogon winterianus essential oil to produce k-carrageenan bioactive films as new food packaging materials. Future work should focus on the scale-up production of these films.


Introduction
Plastic-based pollutants have had a significant negative impact on Earth, severely exposing all biotic and abiotic components. Polyurethane (PUR), polystyrene (PS), polyamines (PA), polyvinyl chloride (PVC), poly(-caprolactone) (PCL), polyethylene terephthalate (PET), and many other types of plastics are used in daily life. Due to their advantageous physicochemical properties, they have been widely employed for several applications, namely, packaging, commodities, and hygiene goods [1,2].
Each year, 19 to 23 million tons of plastic waste are dumped into the environment. Recycling plastic presents challenges, especially for single-use plastics. A third of the world's pollution deposits come from it, which harms the environment, costs a lot to clean, The C. winterianus essential oil chemical composition was studied through Gas Chromatography coupled to Mass Spectrometry (GC-MS) (ISO 7609:1985). The program ran at a temperature of 190 • C for 6 min, before increasing to 190 • C by 2 • C/min, and then to 220 • C by 4 • C/min. After remaining for 10 min at 220 • C, the program switched to its final two modes, which were 4 • C/min up to 240 • C, then staying at that temperature for 10 min. The carrier gas was helium, with injection volumes of 0.1 µL to the Flame Ionization Detector (FID) and 0.1 µL to the Mass Selective Detector (MSD) at head pressures of 33 Psi (FID) and 25.5 Psi (MSD) [20,21].
For the DPPH method, 100 µL of several concentrations of the essential oil (5, 3.75, 2.5, 1.25, 0.5, and 0.25%, v/v) were added to 3.9 mL of three DPPH methanolic solutions (0.2, 0.1242 and 0.08 mM). A mixture of 3.9 mL of each DPPH solution and 100 µL of methanol was used as a negative control. Then, these mixtures remained in the dark at room temperature for 90 min, and their absorbances were measured at 517 nm. The essential oil antioxidant activity was determined as the percentage of inhibition (% Inhibition). Additionally, the IC 50 was calculated by plotting the essential oil concentrations versus their % Inhibition. The Antioxidant Activity Index (AAI) was calculated, thus allowing the classification of essential oil antioxidant activity (AAI ≤ 0.5-poor; 0.5 < AAI ≤ 1.0moderate; 1.0 < AAI < 2.0-strong; AAI ≥ 2.0-very strong). The assays were performed in duplicate [22].
For the β-carotene bleaching method, 500 µL of a β-carotene solution (20 mg/mL in chloroform), 1 mL of chloroform, 40 µL of linoleic acid, and 400 µL of Tween 40 were mixed. Then, the chloroform was evaporated with a rotavapor. Finally, 100 mL of distilled water saturated with oxygen was added, thus creating an emulsion. Subsequently, 5 mL of this emulsion was mixed with 300 µL of several essential oil dilutions, as mentioned above, for the DPPH method. The negative control consisted of a mixture of 300 µL of methanol with 5 mL of the emulsion. These mixtures were incubated at 50 • C for 1 h, and, finally, their absorbances were measured at 470 nm. An emulsion without β-carotene was used as a blank. The essential oil antioxidant activity was determined as % Inhibition of β-carotene oxidation, and its IC 50 was also determined [23].

Antibacterial and Anti-Quorum Sensing Activities Evaluation
The antibacterial activity of the C. winterianus essential oil was evaluated against several bacterial species: Gram-negative (Salmonella Typhimurium ATCC 13311, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922), and Gram-positive (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Listeria monocytogenes LMG 16779). Stock cultures of bacterial species were stored at −80 • C using 20% (v/v) glycerol. Brain-Heart Infusion agar (BHI) was used to culture the bacteria 24 h prior to the antibacterial tests [23].
For the solid diffusion assay, several bacterial colonies were removed from the agar plates and resuspended in a sterile saline solution (NaCl, 0.85% w/v). Its turbidity was adjusted to 0.5 McFarland (1-2 × 10 8 colony-forming units/mL (CFU/mL)). Müeller-Hinton agar (MHA) plates were seeded with these inoculums. An amount of 15 µL of the essential oil was impregnated in sterile cellulose discs (6 mm) and then placed in inoculated Petri dishes. After incubating for 24 h at 37 • C, the inhibition zones were checked and measured using a digital pachymeter [23].
The resazurin microtiter assay was employed to determine the essential oil Minimum Inhibitory Concentrations (MIC). Using Müeller-Hinton broth (MHB), serial two-fold essential oil dilutions (32,16,8,4, 2, 1, 0.5, and 0.25 µL/mL) were made in a 96-well plate (50 µL/well). The resazurin indicator solution (10 µL, 0.1% w/v) was added to each well, and, finally, 30 µL of MHB was also added. After that, 10 µL of the bacterial suspensions (0.5 McFarland) were added to the wells. The plates were made in triplicate and incubated at 37 • C for 24 h. A visual review allowed us to understand that the color change from purple to pink or colorless was positive. The lowest concentration at which the color shift was observed was found to be the MIC value [23].
Using the biomonitor bacterium, Chromobacterium violaceum ATCC 12472, the antiquorum sensing activity of C. winterianus essential oil was evaluated. Luria-Bertani (LB) agar plates were inoculated using a bacterial suspension adjusted to an Optical Density (OD 620nm ) of 1 after the bacterial aerobic growth at 30 • C using LB broth. Then, 15 µL of essential oil was impregnated in sterile cellulose discs (6 mm), which were then placed on the inoculated Petri dishes. After incubating for 24 h at 30 • C, the inhibition of the violacein pigment production around the disc (a ring of colorless, but living, cells) was checked and measured using a digital pachymeter [23,24].

Production of Bioactive Films
The solvent casting technique was used to produce the bioactive films. Initially, 1 g of k-carrageenan was added to 50 mL of distilled water, and this mixture was maintained at 50 • C for 10 min under magnetic stirring to complete the dissolution of the k-carrageenan. To further investigate the effect of incorporating the C. winterianus essential oil, 3 different volumes (62.5 µL, 125 µL, and 250 µL) were added to the previous mixture, which was stirred again at 50 • C for 7 min. Finally, 250 µL of glycerol (plasticizer) was added, and the mixture was stirred again under the same conditions. Control films were also prepared without adding the essential oil. Approximately 15 g of the filmogenic solution was placed in polystyrene Petri dishes, and the solvent was left to evaporate for 6 h at 40 • C. Finally, the films were peeled off from the plates and stored at standard room conditions (temperature = 23 ± 2 • C and relative humidity (RH) = 50 ± 5%) [23].
Tensile strength (N/m), tensile index (N.m/g), peak elongation (%), and elastic modulus (MPa) were determined using a tensile tester, with the initial gap set at 50 mm and with a constant rate of elongation of 10 mm/min (ISO 1924/2) [26].
The optical properties (transparency and color) were evaluated using a Color Touch 2 model ISO spectrophotometer with the following condition: illuminant D65 and observer angle of 10 • . The CIE L*a*b* color space enabled the accurate measurements of the films' color through the evaluation of the lightness (L*), redness/greenness (a*), and yellowness/blueness (b*) coordinates. These measurements were made on several randomly selected points of the films [26].

Contact Angles and Surface Free Energy
The contact angles were measured using three reference liquids (deionized water, ethyleneglycol, and diiodomethane) by applying the sessile drop method. The surface free energy of the films (total, dispersive, and polar components) was calculated with the equipment software that provides the surface tension components of the used liquids. At least six measurements were considered, and the surface free energies of the films were determined through the Owens, Wendt, Rabel, and Kaelble (OWRK) approach [23].

Barrier Properties
Water Vapor Permeability (WVP) (g/Pa.day.m) and Water Vapor Transmission Rate (WVTR) (g/m 2 .day) were analyzed by placing the films on the top of cups containing 15 g of anhydrous CaCl 2 (desiccant). During 48 h, the weight increase was checked every 2 h, and the test cups were kept at standard room conditions, as mentioned above (ASTM E96-00) [23].
The samples were cut and fixed on the upper part of test tubes, in which 5 mL of edible sunflower seeds oil were added. Then, they were positioned upside-down above a cellulosic filter paper, previously dried (24 h, 105 • C) and weighed to evaluate the Oil Permeability (OP) (g.mm/m 2 day). The thickness of the films, the filter paper weight increase, the storage time (24 h), and the effective contact area were used to calculate the OP [27].

Antioxidant Activity
The methods described above for C. winterianus essential oil were used to quantify the antioxidant activity of the films, with minor changes. Adding 2.9 mL of a 0.1 mM DPPH solution to 3 discs of the films (6 mm) allowed the measurement of the absorbances at 517 nm every 30 min over 5 h against methanol as a blank. A mixture of the DPPH solution (2.9 mL) and 100 µL methanol was used as a control [27].
By using the β-carotene bleaching method described above for the essential oil, the lipid peroxidation inhibition capacity of the films was evaluated, and the sample was replaced with 3 discs of the films (6 mm) [27].
2.6.6. Antibacterial and Anti-Quorum Sensing Activities The films' antibacterial and anti-quorum sensing activities were assessed using the solid diffusion method, outlined above for C. winterianus essential oil. Shortly, discs of the films (6 mm) were cut in an aseptic environment and placed on the surface of inoculated Petri dishes [23]. They were also monitored using an optical microscope to verify the bacterial growth inhibition after the incubation period [27]. The results were achieved through three independent assays.

Anti-Biofilm Properties
The anti-biofilm capacity of the films against Listeria monocytogenes LMG 16,779 was assessed. For that purpose, the bacterium was grown overnight in Tryptic Soy Broth (TSB) at 250 rpm and 37 • C. Then, the suspension's turbidity was adjusted to an OD 610nm of 0.7, and 300 µL of this suspension was placed on discs of the films (1 cm 2 ) that were put on 12-well plates, followed by the addition of 700 µL of TSB. After incubating at 37 • C for 24 h, the biofilms were washed twice with a sterile saline solution and fixed with 2.5% (v/v) glutaraldehyde for 4 h at 4 • C. Afterward, samples were rewashed with a Phosphate Buffered Saline (PBS) solution and then dehydrated, using a sequence of increasing ethanol concentration solutions. Finally, the samples were dried overnight in a desiccator. The samples were spray-coated with gold, using a metal evaporator, and then were observed through Scanning Electron Microscopy (SEM) (voltage of 20.0 kV and 120.0 A emission) [22,23].

Statistical Analysis
The results were shown as mean ± standard deviation (SD). The raw data were analyzed using the Student's t-test (Microsoft Excel ® , One Microsoft Way, Redmond, WA, USA), assuming that the continuous variables had a normal distribution. Significant differences were taken if the p-value was 0.05 or lower (95% confidence level).

Chemical Composition of C. winterianus Essential Oil
The GC-MS analysis of the essential oil allowed to identify 73 compounds, which correspond to more than 98% of its chemical composition (Table 1). Citronellal, a monoterpenic aldehyde, was identified as the compound present in the highest amount, representing 41.12%, followed by geraniol (19.97%), citronellol (11.94%), along with limonene, geranyl acetate, citronellyl acetate, and elemol, with relative percentages of 3.50%, 2.63%, 2.0%, 1.98%, respectively (Table 1). These results are in accordance with what was previously reported about essential oil-bearing grasses of the genus Cymbopogon, focused on Cymbopogon nardus [19,22,[28][29][30][31]. Citronellal is present in several plants, including those of the Cymbopogon genus. It has considerable value in the perfume, cosmetic, and aromatherapy sectors and is the component that gives the essential oil a lemony scent [19]. Citronellal and citronellol are employed as intermediates in synthesizing 1-menthol, α-tocopherol, and irones, which are important products in the fragrance industry [32]. Citronellal has many therapeutic benefits, including antifungal, anthelmintic, anticancer, and antioxidant effects [33,34].
Monoterpenes and sesquiterpenes predominate among the identified compounds ( Table 1). The most prevalent types of terpenes are called monoterpenes [35]. They are a vast and diversified class of naturally occurring compounds, many of which are present in essential oils because of their low molecular weight. Several monoterpenes have medicinal benefits that include antimicrobial, anti-inflammatory, antioxidant, antipruritic, and analgesic effects [36]. Sesquiterpenes have a richer aroma, are more stereochemically diverse, are less volatile than terpenes, and have antibacterial and anti-inflammatory properties [37,38].

Antioxidant Activity of the Essential Oil
All foods decay while in storage, since they are perishable, even at varying rates [9]. The reactions that occur during lipid oxidation can produce off-odors, off-flavors, as well as texture and color changes, which affect the consumer's acceptance and choice [39]. The DPPH method, based on the reduction of reactions and the β-carotene bleaching test, linked to lipid peroxidation, stand out among the known in vitro techniques for assessing the antioxidant capacities of natural products. Thus, these two methods were applied in the evaluation of the antioxidant activity of the essential oil (Table 2) [40].
Results are presented as mean ± SD.
C. winterianus essential oil exhibits important properties, such as antiseptic, antiinflammatory, and antioxidant [29]. Regarding the results obtained through the DPPH assay, the essential oil displayed a solid antioxidant activity, which is consistent with the previous reports [30]. Moreover, citronellal, the major compound of the essential oil, is a monoterpenic aldehyde, known to possess antioxidant activity. Monoterpenoids, the most abundant compounds in the essential oil, are also recognized, due to their antioxidant properties [36].
One of the determining elements for use in food packaging is the capacity to decrease lipid oxidation, and it is clear from the results of the IC 50 that very small amounts of C. winterianus essential oil are required to inhibit lipid oxidation by 50%. Therefore, it can be concluded that this essential oil can inhibit lipid peroxidation, which can also be related to the high amount of citronellal found in the essential oil (41.12%) and the other monoterpenoids present [36].
To sustainably contribute to the food chain, research has expanded in combining essential oils into packaging produced from materials derived from renewable and biodegradable sources. By having this promising result, the C. winterianus essential oil has the potential to be used in active food packaging, whose organoleptic properties are highly dependent on the preservation of the lipid content. [41].

Antibacterial and Anti-Quorum Sensing Properties of the Essential Oil
Concerning the antibacterial activity of the C. winterianus essential oil, Gram-positive bacteria, particularly Listeria monocytogenes LMG 16779, were more susceptible than Gramnegative ones (Table 3). By determining their MIC values (Table 3), the antibacterial activity of the essential oil was further assessed. The lowest MIC values were found for Grampositive bacteria, specifically for Listeria monocytogenes LMG 16,779 and Enterococcus faecalis ATCC 29212, thus supporting the findings from the solid diffusion assay. Gram-positive bacteria are known to be more susceptible than Gram-negative ones, since they do not have an external membrane [42]. Table 3. Antibacterial and anti-quorum sensing properties of the C. winterianus essential oil.

Strains
Diameter of Inhibition Zones (mm) 1  The opportunistic pathogen Listeria monocytogenes leads to life-threatening infections in both humans and animals. This facultative intracellular bacterium is common in nature, and it contaminates a wide variety of surfaces through cross-contamination during the development of biofilms in industrial machinery, plumbing, and other surfaces, thus reaching the final product for consumption [43]. Eating foods that have been contaminated can result in illnesses [44]. Therefore, in the context of food packaging, the activity that In a sort of microbial communication, called quorum sensing, bacteria communicate and work together through various chemical signals, called auto-inducers, to keep a certain process in balance and impact populations. Additionally, it affects how genes are expressed in response to cell density, and triggers the activation of several transcription factors in bacteria [45]. This essential oil was able to inhibit the violacein production by Chromobacterium violaceum ATCC 12,472 (Table 3); thus, it was possible to confirm its anti-quorum sensing capacity.

FTIR Spectra of the Films
The results of the FTIR analysis of the films are shown in Figure 1. The FTIR spectrum of the k-carrageenan film ( Figure 1A) showed several representative bands: at 3000-3500 cm −1 , corresponding to O-H stretching, and at 1220-845 cm −1 , known as the fingerprint region of carbohydrates [46]. The peak at 1220 cm −1 corresponds to the asymmetric stretching of ester sulfate groups (O=S=O), 1030 cm −1 is assigned to C-O and C-OH stretching, and 930 cm −1 corresponds to C-O-C stretching in 3,6-anhydro-D-galactose. The band at 845 cm −1 is related to C-O-SO 3 stretching in (1-3)-D-galactose. The intense bands at 1592 cm −1 and 1382 cm −1 are probably related to the structural water deformation band [46].
The fingerprint area in FTIR spectra is in the range between 400 cm −1 and 1500 cm −1 . It typically has many peaks, thus making it challenging to distinguish between them and pick out individual bonds. However, since each compound's fingerprint region is distinct, it is possible to distinguish one compound from another [47].
The FTIR spectra of the k-carrageenan films incorporating the essential oil ( Figure 1B-D) showed that the existing peaks were higher, even though no new peaks have appeared, thus indicating that the essential oil compounds were present as their concentration increased. The observed peaks corresponded to the O-H bond at 3400 cm −1 , the C-H bond at 2900 cm −1 , and the C=C bond at 1650 cm −1 . Generally, a difference in the peaks' intensity was noticed. As the volume of the essential oil incorporated in the films increased, their bands also increased in the FTIR spectra, thus showing that the essential oil was present in the films. Since the concentration of the C. winterianus essential oil is much lower than that of k-carrageenan, the peaks corresponding to citronellal (the major compound) are superimposed with those of k-carrageenan, as shown in Figure 1.
do not have an external membrane [42]. Table 3. Antibacterial and anti-quorum sensing properties of the C. winterianus essential oil.

Strains
Diameter of Inhibition Zones (mm) The opportunistic pathogen Listeria monocytogenes leads to life-threatening infections in both humans and animals. This facultative intracellular bacterium is common in nature, and it contaminates a wide variety of surfaces through cross-contamination during the development of biofilms in industrial machinery, plumbing, and other surfaces, thus reaching the final product for consumption [43]. Eating foods that have been contaminated can result in illnesses [44]. Therefore, in the context of food packaging, the activity that the C. winterianus essential oil demonstrated against Listeria monocytogenes LMG 16,779 is interesting because it would inhibit its growth.
In a sort of microbial communication, called quorum sensing, bacteria communicate and work together through various chemical signals, called auto-inducers, to keep a certain process in balance and impact populations. Additionally, it affects how genes are expressed in response to cell density, and triggers the activation of several transcription factors in bacteria [45]. This essential oil was able to inhibit the violacein production by Chromobacterium violaceum ATCC 12,472 (Table 3); thus, it was possible to confirm its antiquorum sensing capacity.

FTIR Spectra of the Films
The results of the FTIR analysis of the films are shown in Figure 1. The FTIR spectrum of the k-carrageenan film ( Figure 1A) showed several representative bands: at 3000-3500 cm −1 , corresponding to O-H stretching, and at 1220-845 cm −1 , known as the fingerprint region of carbohydrates [46]. The peak at 1220 cm −1 corresponds to the asymmetric stretching of ester sulfate groups (O=S=O), 1030 cm −1 is assigned to C-O and C-OH stretching, and 930 cm −1 corresponds to C-O-C stretching in 3,6-anhydro-Dgalactose. The band at 845 cm −1 is related to C-O-SO3 stretching in (1-3)-D-galactose. The intense bands at 1592 cm −1 and 1382 cm −1 are probably related to the structural water deformation band [46].
The fingerprint area in FTIR spectra is in the range between 400 cm −1 and 1500 cm −1 . It typically has many peaks, thus making it challenging to distinguish between them and  Table 4 shows the effects of incorporating the essential oil on the films' structural, mechanical, and optical properties. The k-carrageenan films' thickness varied from 38.51 to 64.93 mm, with the incorporation of the C. winterianus essential oil significantly affecting (p-value < 0.05) the thickness of the resulting films, as well as the grammage, due to the difference in the total solid contents of the films [26]. Visually, it was feasible to confirm that the roughness increases as the amount of essential oil in the films increases. The surface roughness may increase because of the essential oils' incorporation into the polymer matrix, thus affecting their thickness [48]. Tensile strength, tensile index, peak elongation, and elastic modulus were evaluated as the films' main mechanical properties that depend on the type of the polymer matrix, the sort of additives, and the interactions between them [49]. The ability to endure a tensile force is measured by assessing the breakpoint of a specimen under tensile stress [50].

Physical Properties of the Films
Increasing the content of the essential oil in the films causes a reduction in tensile strength and tensile index, which significantly changed (p-value < 0.05) when the volume was 250 mL, but did not change significantly (p-value > 0.05) for the other essential oil volumes. In the film network, weaker polymer-essential oil connections have partially replaced stronger polymer-polymer interactions, which is a possible reason for the reduction [51,52]. Peak elongation is the increase in the specimen's length, from its starting to its breakpoint length, which is related to the elasticity of a polymeric material. In this study, peak elongation significantly increased (p-value < 0.05) as the volume of essential oil also increased to 62.5 µL and 125 µL. The peak elongation obtained for the film with 250 µL of C. winterianus essential oil was lower than that of the control, thus suggesting the existence of fewer interconnections within the film structure, thus limiting its flexibility. As previously observed, the elastic modulus decreased with the increase of the essential oil content incorporated in the films, hence resulting in less rigid films [53]. Adding the essential oil to the polymer may not result in a homogenous combination, giving the films a roughness that creates minuscule weak areas and makes them more prone to breaking [48,49]. When combining polysaccharides and essential oils, the mechanical properties are frequently reduced [50,51,54].
The mechanical properties of the k-carrageenan films now developed are weaker when compared with plastic films used in the food industry, such as low-density polyethylene (LDPE) [55].
Since color and transparency directly affect the consumers' acceptance, the optical properties of the films were evaluated (Table 4). Adding C. winterianus essential oil did not significantly modify (p-value > 0.05) the L*, a*, and b* coordinates. The incorporation of essential oil decreased the transparency values; nonetheless, the films showed high transparency values, between 92% and 96%, which might be related to the amorphous arrangement in k-carrageenan films and connected to the light scattering brought on by the distribution of the essential oil droplets inside the biopolymer matrix [56]. This conclusion is consistent with findings from other studies, since adding more essential oil often makes film less transparent [53,54].

Contact Angle and Surface Free Energies
The most popular technique for determining the hydrophobicity or hydrophilicity of a surface is to measure the water contact angle. A hydrophobic surface has a water contact angle superior to 90 • , whereas a hydrophilic one has a water contact angle inferior to 90 • [57]. The values of the contact angles were obtained on both sides of the films and are listed in Table 5. It was crucial to carefully examine both sides of the films, because they displayed extremely different behaviors. This can be important when deciding which side to pack food on. If it is desirable to pack food with more water content, then the hydrophobic side must be turned inward to prevent water loss. Generally, there were no changes in the hydrophobicity of the films regarding the upper surface. Moreover, the upper surface is hydrophobic, contrary to the lower one, which has a contact angle inferior to 90 • , and this tends to decrease with the increase of essential oil volumes. It was also noticed that the water contact angles obtained in the control films were similar for both sides, whereas this was not the case with the addition of the essential oil. Looking at the films with essential oil, a decrease in hydrophobicity on the bottom surfaces was observed, while, on the upper ones, the hydrophobicity tends to increase. Essential oils are hydrophobic, due to their non-polar chemical structure, which is the reason why incorporating them into hydrophilic polymer matrices increases the barrier properties [52]. In addition to the essential oil's natural hydrophobicity, another explanation for the rise in water contact angle may be related to the roughness of the films, which may also increase their hydrophobicity, since the surface of the structures is crucial in determining a surface's hydrophobicity [58].
There are numerous applications for surface free energy, which establishes how solids will perform in the presence of liquids. High surface free energy materials are often easily wettable by any liquid [59]. Measurements of contact angles were frequently used to calculate the material's surface free energy for some liquids [60]. The total surface free energy of the films varied from 13.95 to 38.60 mN/m, and, analyzing its evolution, along with the increase of C. winterianus essential oil, it increased relatively to the control film. The polar component decreased with the addition of the essential oil, with no statistical significance (p-value > 0.05). The dispersive element also tended to increase with the addition of the essential oil. The total surface free energy values obtained from the films were comparable to some petroleum-derived plastics, such as polypropylene [61].

Barrier Properties
Food products should be transported, handled, and commercialized in a protective environment, created by food packaging materials. By serving as an effective barrier to moisture and gases, they should help extend the shelf life of perishable foods [62].
The barrier properties of the films developed in this work were evaluated in terms of barrier-to-water vapor and oil (Table 6). Regarding the water vapor permeability of the films, it was observed that adding the essential oil did not significantly change (p-value > 0.05) the WVTR and WVP of the k-carrageenan films. These results are aligned with the values obtained for the water contact angles of the films, which indicated that the essential oil did not affect the hydrophobicity of the films, and suggests that the barrier properties to water vapor will also not be affected. Considering the oil permeability results, they lacked a clear pattern and were somewhat dispersed; however, they did not show statistically significant results (p-value > 0.05). A previous study with k-carrageenan films reported values of oil permeability of 0.37 and 0.97 g.mm/m 2 .day, which are lower than those obtained in the present research [63].
It has been previously established that water retention in polysaccharide-based films is connected to potential interactions between polysaccharide molecules and glycerol hydroxyl groups, thus resulting in a more compact polymeric matrix [64].

Antioxidant Activity of the Films
The results of the films' antioxidant activity determined by the DPPH assay are shown in Figure 2. The control film did not show antioxidant activity, which does not happen with the films incorporating essential oil. An antioxidant activity pattern was observed for films containing the essential oil. After the initial phase, they remained steady, but after 2 h, an increase in the antioxidant activity was verified. After 2.5 h of reaction, the films with 250 µL of essential oil showed 6%, and, at 4 h, they showed 5% of DPPH free radicals inhibition. The films containing 62.5 and 125 µL of essential oil showed only one major peak: almost 1.5% and 2.8% inhibition of DPPH free radicals, respectively. [63].
It has been previously established that water retention in polysaccharide-based films is connected to potential interactions between polysaccharide molecules and glycerol hydroxyl groups, thus resulting in a more compact polymeric matrix [64].

Antioxidant Activity of the Films
The results of the films' antioxidant activity determined by the DPPH assay are shown in Figure 2. The control film did not show antioxidant activity, which does not happen with the films incorporating essential oil. An antioxidant activity pattern was observed for films containing the essential oil. After the initial phase, they remained steady, but after 2 h, an increase in the antioxidant activity was verified. After 2.5 h of reaction, the films with 250 µL of essential oil showed 6%, and, at 4 h, they showed 5% of DPPH free radicals inhibition. The films containing 62.5 and 125 µL of essential oil showed only one major peak: almost 1.5% and 2.8% inhibition of DPPH free radicals, respectively. The results of the β-carotene bleaching test, in which the ability of the films to inhibit lipid peroxidation was evaluated, are presented in Table 7. The findings demonstrated that films containing the essential oil exhibit a high level of lipid peroxidation inhibition, and, for that reason, they have the potential to be used as substitutes for traditional food packaging materials, particularly for foods with high-fat content.
Regarding the anti-quorum properties of the films, it was noticed that the essential oil capacity to inhibit bacterial intercellular communication was not maintained when it was incorporated in k-carrageenan films.

Anti-Biofilm Activity of Film against Listeria monocytogenes
Since the C. winterianus essential oil primarily affected the growth of L. monocytogenes LMG 16779, and because Listeria monocytogenes is a well-known foodborne pathogen, the antibiofilm activity of the films incorporating 250 µL of essential oil were evaluated through SEM (Figure 4). These films were chosen because the increase in the volume of essential oil incorporated in the films would present a better antibiofilm effect, since the essential oil has an antibacterial activity for this bacterium [65,66]. Figure 4A presents the Listeria monocytogenes biofilm formed in the control film, showing several layers of bacteria. As shown in Figure 4B, biofilms formed on the surface of k-carrageenan films containing 250 µL of essential oil are sparse and consist of just one layer of cells. In addition, it was also visible that the cell integrity was lost (examples marked in red). Figure 4C shows that the number of bacteria decreased when the biofilms were formed on the bioactive k-carrageenan films. Some bacteria have compromised cell membrane integrity, but there were also some bacteria with good integrity for comparison (examples marked in blue). The results of the β-carotene bleaching test, in which the ability of the films to inhibit lipid peroxidation was evaluated, are presented in Table 7. The findings demonstrated that films containing the essential oil exhibit a high level of lipid peroxidation inhibition, and, for that reason, they have the potential to be used as substitutes for traditional food packaging materials, particularly for foods with high-fat content.

Anti-Biofilm Activity of Film against Listeria Monocytogenes
Since the C. winterianus essential oil primarily affected the growth of L. monocytogenes LMG 16779, and because Listeria monocytogenes is a well-known foodborne pathogen, the antibiofilm activity of the films incorporating 250 µL of essential oil were evaluated through SEM (Figure 4). These films were chosen because the increase in the volume of essential oil incorporated in the films would present a better antibiofilm effect, since the essential oil has an antibacterial activity for this bacterium [65,66].  Figure 4A presents the Listeria monocytogenes biofilm formed in the control film, showing several layers of bacteria. As shown in Figure 4B, biofilms formed on the surface of k-carrageenan films containing 250 µL of essential oil are sparse and consist of just one layer of cells. In addition, it was also visible that the cell integrity was lost (examples marked in red). Figure 4C shows that the number of bacteria decreased when the biofilms were formed on the bioactive k-carrageenan films. Some bacteria have compromised cell membrane integrity, but there were also some bacteria with good integrity for comparison (examples marked in blue).

Conclusions
This work demonstrated the viability of using C. winterianus essential oil to produce k-carrageenan bioactive and eco-friendly films that can be used as innovative food packaging materials, thus avoiding using conventional plastics. The produced films were transparent (>90%) and hydrophobic (water contact angle > 90°), and presented antioxidant activity related to free radical scavenging and lipid peroxidation inhibition. Moreover, the films inhibited the growth of planktonic and biofilm cells of Listeria monocytogenes, an important foodborne pathogen. Future work should focus on industrial production at a large scale of these films and study their biodegradability under different conditions. Funding: This work was developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology (FCT). FibEnTech-UBI is also financed by FCT under the scope of the project UIDB/00195/2020.

Conclusions
This work demonstrated the viability of using C. winterianus essential oil to produce kcarrageenan bioactive and eco-friendly films that can be used as innovative food packaging materials, thus avoiding using conventional plastics. The produced films were transparent (>90%) and hydrophobic (water contact angle > 90 • ), and presented antioxidant activity related to free radical scavenging and lipid peroxidation inhibition. Moreover, the films inhibited the growth of planktonic and biofilm cells of Listeria monocytogenes, an important foodborne pathogen. Future work should focus on industrial production at a large scale of these films and study their biodegradability under different conditions. Funding: This work was developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology (FCT). FibEnTech-UBI is also financed by FCT under the scope of the project UIDB/00195/2020. Data Availability Statement: Data will be available by request to the corresponding author.