Seasonal Changes in Chemical Profile and Antioxidant Activity of Padina pavonica Extracts and Their Application in the Development of Bioactive Chitosan/PLA Bilayer Film

Citation: ˇ Cagalj, M.; Fras Zemljiˇc, L.; Kraševac Glaser, T.; Mežnar, E.; Sterniša, M.; Smole Možina, S.; Razola-Díaz, M.d.C.; Šimat, V. Seasonal Changes in Chemical Profile and Antioxidant Activity of Padina pavonica Extracts and Their Application in the Development of Bioactive Chitosan/PLA Bilayer Film. Foods 2022,11, 3847. https:// doi.org/10.3390/foods11233847 Academic Editor: Fernanda Vitória Leimann Received: 18 October 2022 Accepted: 23 November 2022 Published: 28 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article Seasonal Changes in Chemical Profile and Antioxidant Activity of Padina pavonica Extracts and Their Application in the Development of Bioactive Chitosan/PLA Bilayer Film Martina ˇ Cagalj 1, Lidija Fras Zemljiˇc 2, Tjaša Kraševac Glaser 2, Eva Mežnar 3, Meta Sterniša 3, Sonja Smole Možina 3, María del Carmen Razola-Díaz 4and Vida Šimat 1,* 1University Department of Marine Studies, University of Split, Ru ¯ dera Boškovi´ca 37, 21000 Split, Croatia 2Laboratory for Characterization and Processing of Polymers, Faculty of Mechanical Engineering, University of Maribor, Smetanova 17, 2000 Maribor, Slovenia 3 Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia 4 Department of Nutrition and Food Science, Campus of Cartuja, University of Granada, 18071 Granada, Spain *Correspondence: [email protected] Abstract: Seaweeds are a potentially sustainable source of natural antioxidants that can be used in the food industry and possibly for the development of new sustainable packaging materials with the ability to extend the shelf-life of foods and reduce oxidation. With this in mind, the seasonal variations in the chemical composition and antioxidant activity of brown seaweed (Padina pavonica) extracts were investigated. The highest total phenolic content (TPC) and antioxidant activity (measured by ferric reducing/antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and oxygen radical absorbance capacity (ORAC)) were found for P. pavonica June extract. The TPC of 26.69 ± 1.86 mg gallic acid equivalent/g, FRAP of 352.82 ± 15.41 µ mole Trolox equivalent (TE)/L, DPPH of 52.51 ± 2.81% inhibition, and ORAC of 76.45 ± 1.47 µ mole TE/L were detected. Therefore, this extract was chosen for the development of bioactive PLA bilayer film, along with chitosan. Primary or quaternary chitosan was used as the first layer on polylactic acid (PLA) films. A suspension of chitosan particles with entrapped P. pavonica extract was used as the second layer. X-ray photoelectron spectroscopy confirmed the presence of layers on the material surface. The highest recorded antioxidant activity of the newly developed films was 63.82% inhibition. The developed functional films exhibited antifogging and antioxidant properties, showing the potential for application in the food industry. Keywords: functional PLA films; seaweed and chitosan bilayer; sustainable natural antioxidants; microwave-assisted extraction 1. Introduction Seaweed Padina pavonica belongs to the genus Padina, family Dictyotaceae, order Dictyotales, and class Phaeophyceae. Currently, there are 60 taxonomically recognized species names in the genus Padina. The species of this genus are widely distributed from tropical to temperate seas [ 1 ]. Due to its availability and proven biological potential [ 2 – 4 ], P. pavonica is a good choice for the production of biologically active extracts that can be used in many food industry processes, including packaging. Moreover, the chemical composition and biological activity of seaweeds vary depending on the season, growth phase, and other environmental factors [ 5 , 6 ]. In this regard, P. pavonica extracts were found to have the strongest antibacterial activity against human and fish pathogens during the period from April to September [7]. The highest antitumor activity of fucoidans was found in samples collected in June [ 8 ]. Since there are no reports of seasonal changes in the chemical profiles or antioxidant activity of this species, it is important to determine the best time to harvest Foods 2022,11, 3847. https://doi.org/10.3390/foods11233847 https://www.mdpi.com/journal/foods Foods 2022,11, 3847 2 of 18 seaweeds and prepare extracts for further exploitation. It is also important to choose the right extraction method to extract higher amounts of bioactive compounds and obtain stronger bioactivity [3]. In the food industry, new sustainable packaging materials should be developed and integrated with new packaging solutions to reduce the environmental footprint (both in terms of biodegradability and the origin of raw materials for packaging production). Apart from environmental aspects, new packaging solutions should also be functional and provide direct benefits to consumers by extending food shelf-life, ensuring food safety, and monitoring food quality [ 9 ]. New bio-based coatings are the trend for implementing active packaging concepts [ 10 ]. Active coated films with antioxidant properties interact with food, change its conditions, and control its quality [11]. On the other hand, biopolymers used for the development of new packaging solutions should be economical, abundant, and come from renewable sources. Polylactic acid (PLA), a thermoplastic polyester that is biodegradable, is obtained from renewable sources. PLA is one of the most widely used bioplastics due to its mechanical and physical properties. To achieve the active functionality and biodegradability of food packaging, the most commonly used biopolymers are polysaccharides, such as chitosan [ 12 – 15 ]. Chitosan, which is derived from the waste of marine crustaceans (shrimp, shellfish, crabs, and lobsters), is abundant, natural, and biodegradable [ 16 ]. However, chitosan itself has some limitations, such as low antioxidant capacity. Therefore, it has been shown that colloidal complexes based on the combination of chitosan and other substances, such as surfactants, polyphenols, etc., can overcome these drawbacks [17,18]. The variations in the chemical profile and biological activity of P. pavonica during seasonal growth, as well as the use of seaweed extracts for the development of functional PLA films alone or in combination with chitosan have not yet been investigated. In our previous research, the general concept of packaging materials with layer-by-layer colloidal chitosanextract-polyphenol coatings for polypropylene and polyethylene was developed [ 18 – 20 ]. Therefore, the aim of this study was: (i) to determine the changes in total phenolic content (TPC) and chemical profile of P. pavonica during seasonal growth (from May to September); (ii) to determine the antioxidant activity of P. pavonica extracts; (iii) to develop and physicochemically characterize PLA films with bilayers of primary or quaternary chitosan in combination with P. pavonica extract—for the first time in this formulation; and (iv) to determine the antioxidant activity of the developed active packaging. 2. Materials and Methods 2.1. Collection, Extraction, and Compound Analyses Padina pavonica was sampled in the Adriatic Sea off the southern coast of the island ˇ Ciovo (43.493389 ◦ N, 16.272505 ◦ E) from May to September 2020. The depth range was from 20 to 80 cm. A YSI Pro2030 probe (Yellow Springs, OH, USA) was used for the sea temperature and salinity measurements during the sampling (Figure 1). Seaweed samples were washed with tap water and freeze-dried (FreeZone 2.5, Labconco, Kansas City, MO, USA). Freeze-dried seaweed samples were pulverized and extracted based on our previous research [ 3 ]. The seaweed powder was mixed with 50% ethanol (Gram-Mol, Zagreb, Croatia) in a 1:10 (w/v) seaweed-to-solvent ratio. Microwave-assisted extraction (MAE) was performed in an advanced microwave extraction system (ETHOS X, Milestone Srl, Sorisole, Italy) at 200 W and 60 ◦ C for 15 min. After the extraction, samples were centrifuged for 8 min at 5000 rpm and room temperature and filtered. The ethanol was evaporated at 50 ◦C in a rotary evaporator and the remaining water extract was freeze-dried. Foods 2022,11, 3847 3 of 18 Foods 2022, 11, 3847 3 of 18 Figure 1. Sea temperature and salinity measured during harvesting of P. pavonica. Freeze-dried seaweed samples were pulverized and extracted based on our previous research [3]. The seaweed powder was mixed with 50% ethanol (Gram-Mol, Zagreb, Croatia) in a 1:10 (w/v) seaweed-to-solvent ratio. Microwave-assisted extraction (MAE) was performed in an advanced microwave extraction system (ETHOS X, Milestone Srl, Sorisole, Italy) at 200 W and 60 °C for 15 min. After the extraction, samples were centrifuged for 8 min at 5000 rpm and room temperature and filtered. The ethanol was evaporated at 50 °C in a rotary evaporator and the remaining water extract was freeze-dried. Total phenolic content (TPC) was determined by the Folin–Ciocalteu method [21], previously described by Čagalj et al. [5]. In brief, 125 µL of Folin–Ciocalteu reagent and 1.5 mL of distilled water were combined with 25 µL of the sample. After stirring the mixture for a minute, 375 µL of a 20% sodium carbonate solution and 475 µL of distilled water were added. Samples were kept at room temperature in the dark for two hours. Absorbance was measured at 765 nm. The results were expressed as mg gallic acid equivalents (GAE)/g of freeze-dried extract. The analysis of compounds from P. pavonica was performed by dissolving 3 mg of freeze-dried extract in 1 mL of methanol/water (v/v) (Merck KGaA, Darmstadt, Germany) using the ACQUITY Ultra Performance LC system equipped with a photodiode array detector with binary solvent manager (Waters Corporation, Milford, MA, USA) series with a mass detector Q/TOF micromass spectrometer (Waters) with electrospray ionization (ESI) source operating in negative mode (UPLC-PDA-ESI-QTOF). The conditions were as follows: capillary voltage, 2300 kV; source temperature, 100 °C; cone gas flow, 40 L/h; desolvation temperature, 500 °C; desolvation gas flow, 11,000 L/h; and scan range, m/z 50– 1500. Individual compounds’ separation was performed using an ACQUITY UPLC BEH Shield RP18 column (1.7 µm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA) at 40 ◦C. Water containing 1% acetic acid (A) and acetonitrile (B) (Merck KGaA, Darmstadt, Germany) was used for the elution gradient test and applied as follows: 0 min, 1% B; 2.3 min, 1% B; 4.4 min, 7% B; 8.1 min, 14% B; 12.2 min, 24% B; 16 min, 40% B; 18.3 min, 100% B, 21 min, 100% B; 22.4 min, 1% B; 25 min, 1% B. The injected sample volume was 2 µL and the used flow rate was 0.6 mL/min. The wavelength of 280 nm was used to monitor the compounds. MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for the integration and data elaboration [5,22]. 2.2. Collection, Extraction, and Compound Analyses The antioxidant activity of P. pavonica extracts was measured by using three different methods with different mechanisms of action. Ferric-reducing/antioxidant power (FRAP) method is based on electron transfer, while 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and oxygen radical absorbance capacity (ORAC) methods are based on Figure 1. Sea temperature and salinity measured during harvesting of P. pavonica. Total phenolic content (TPC) was determined by the Folin–Ciocalteu method [ 21 ], previously described by ˇ Cagalj et al. [ 5 ]. In brief, 125 µ L of Folin–Ciocalteu reagent and 1.5 mL of distilled water were combined with 25 µ L of the sample. After stirring the mixture for a minute, 375 µ L of a 20% sodium carbonate solution and 475 µ L of distilled water were added. Samples were kept at room temperature in the dark for two hours. Absorbance was measured at 765 nm. The results were expressed as mg gallic acid equivalents (GAE)/g of freeze-dried extract. The analysis of compounds from P. pavonica was performed by dissolving 3 mg of freeze-dried extract in 1 mL of methanol/water (v/v) (Merck KGaA, Darmstadt, Germany) using the ACQUITY Ultra Performance LC system equipped with a photodiode array detector with binary solvent manager (Waters Corporation, Milford, MA, USA) series with a mass detector Q/TOF micromass spectrometer (Waters) with electrospray ionization (ESI) source operating in negative mode (UPLC-PDA-ESI-QTOF). The conditions were as follows: capillary voltage, 2300 kV; source temperature, 100 ◦ C; cone gas flow, 40 L/h; desolvation temperature, 500 ◦ C; desolvation gas flow, 11,000 L/h; and scan range, m/z 50–1500. Individual compounds’ separation was performed using an ACQUITY UPLC BEH Shield RP18 column (1.7 µ m, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA) at 40 ◦ C. Water containing 1% acetic acid (A) and acetonitrile (B) (Merck KGaA, Darmstadt, Germany) was used for the elution gradient test and applied as follows: 0 min, 1% B; 2.3 min, 1% B; 4.4 min, 7% B; 8.1 min, 14% B; 12.2 min, 24% B; 16 min, 40% B; 18.3 min, 100% B, 21 min, 100% B; 22.4 min, 1% B; 25 min, 1% B. The injected sample volume was 2 µ L and the used flow rate was 0.6 mL/min. The wavelength of 280 nm was used to monitor the compounds. MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for the integration and data elaboration [5,22]. 2.2. Collection, Extraction, and Compound Analyses The antioxidant activity of P. pavonica extracts was measured by using three different methods with different mechanisms of action. Ferric-reducing/antioxidant power (FRAP) method is based on electron transfer, while 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and oxygen radical absorbance capacity (ORAC) methods are based on hydrogen atom transfer [ 23 ]. The freeze-dried extracts were dissolved in 50% ethanol prior to the antioxidant activity assays. The used FRAP method was previously described by Benzie and Strain [ 24 ] with modifications described in ˇ Cagalj et al. [ 5 ]. In brief, the microplate wells were filled with 300 µ L of FRAP reagent solution and the absorbance at 592 nm was measured. Four minutes after adding 10 µ L of the sample to the microplate wells, the absorbance Foods 2022,11, 3847 4 of 18 change was measured. Measured absorbances were compared with the readings obtained for the standard Trolox solutions. The FRAP results were expressed as micromoles of Trolox equivalents/liter of extract (µmole TE/L). The ability to scavenge DPPH radicals was measured using the method previously described by Milat et al. [ 25 ]. DPPH radical solution (290 µ L) was pipetted to microplate wells and absorbance was measured at 517 nm. One hour after addition of 10 µ L of the sample, the decrease in the absorbance was measured. The results were expressed as the percentage of DPPH radical inhibition (% inhibition). The extracts were diluted 200-fold before performing the ORAC method. The method used was previously described by Burˇcul et al. [ 26 ]. In brief, the mixture of 150 µ L of fluorescein and 25 µ L of the sample (or Trolox for standard or puffer for blank) was pipetted to microplate wells and thermostated at 37 ◦ C for 30 min. Following the addition of 25 µ L of 2,2 0 -Azobis(2-amidinopropane) dihydrochloride (AAPH), measurements were made every minute for 80 min at excitation and emission wavelengths of 485 and 520 nm. ORAC results were expressed in µmole TE/L. The TPC and antioxidant assays were performed in triplicate. The absorbance of the extracts’ color was subtracted before the calculations. The extract with the highest antioxidant activity was selected for chitosan/PLA bilayer film development. 2.3. Development of Chitosan/Polylactic Acid Bilayer Films 2.3.1. Preparation of Bilayer Solutions For this study, the solutions listed in Table 1were prepared. Primary (low-molecularweight chitosan (50 to 190 kDa), poly (D-glucosamine); Sigma-Aldrich, St. Louis, MO, USA) and quaternary (Chitosan Quaternary Ammonium Salt; CD Bioparticles, Shirley, NY, USA) chitosan solutions of 1% and 2% (w/v) were prepared by adding deionized water to chitosan powder with a few drops of absolute acetic acid ( ≥ 99.8%; Sigma-Aldrich, St. Louis, MO, USA) to dissolve the powder. The solutions were stirred overnight, and the pH was adjusted to 4.0 with acetic acid. Sodium tripolyphosphate (TPP; Sigma-Aldrich, St. Louis, MO, USA) was suspended in deionized water and stirred overnight to prepare a 0.2% (w/v) solution. The main purpose of setting pH = 4 was to ensure the same conditions for both chitosans. It should be taken into account that there are still 20% (the degree of substitution is 80%) of the primary groups in quaternary chitosan, which could be protonated in an acidic environment. Table 1. Sample description of prepared solutions. Solutions Abbreviation 1% primary chitosan CH 1% quaternary chitosan QCH 2% primary chitosan 2%CH 2% quaternary chitosan 2%QCH Primary Chitosan particles CHP’s Quaternary Chitosan particles QCHP’s P. pavonica June extract PPAV Primary chitosan particles with captured extract CHP’sPPAV Quaternary Chitosan particles with captured extract QCHP’sPPAV Sodium tripolyphosphate TPP Before preparation of the chitosan particles, the freeze-dried P. pavonica June extract was dissolved in 50% ethanol (99.8%, GC; Sigma-Aldrich, St. Louis, MO, USA) to prepare a solution with a concentration of 10 mg/mL. The chitosan particles with the captured Foods 2022,11, 3847 5 of 18 extract were then prepared using the ionic gelation technique. The prepared TPP and P. pavonica extract solutions were simultaneously added to a fixed volume of 1% (w/v) chitosan solution (primary and quaternary) to obtain a 5:1 weight ratio between chitosan and TPP, which was chosen according to our previous research [ 27 ]. The particles formed spontaneously under continuous stirring for 1 h at room temperature. The final pH of the chitosan particle dispersions with PPAV was adjusted to 4.0 with acetic acid under slow mixing. 2.3.2. Application of Chitosan Solutions and Particles to PLA Films The prepared chitosan solutions and chitosan particle dispersions were applied to the PLA films (Optimont ® PLA-Folie, Bleher Folientehnik GmbH, Ditzingen, Germany), which were cleaned and air-dried before application. The layers were applied directly to the surfaces of PLA in a roll-to-roll printing process using a printing table and a magnetic roller (Johannes Zimmer, Kufstein, Austria) at rolling speed level 3 and magnetic strength level 1. All previously prepared solutions were stirred before application and applied to the film surfaces in a fixed volume (primary and quaternary chitosan: 4 mL, chitosan particles with embedded extracts: 4 mL). Both layers were, thus, prepared under the same conditions and air-dried after each individual layer. The first layer consisted of 2% primary or 2% quaternary chitosan and the second layer consisted of chitosan particle dispersions (CHPs or QCHPs) with captured P. pavonica June extract. The description of the samples and their names are listed in Table 2. Table 2. PLA film samples description. Description of the Samples Sample Name References PLA with no layers PLA PLA applicated with 2% CH PLA + 2%CH PLA applicated with 2% QCH PLA + 2%QCH Samples with primary chitosan as first layer PLA applicated with 2% CH and CHP’sPPAV P3 PLA applicated with 2% CH and QCHP’sPPAV P4 Samples with quaternary chitosan as first layer PLA applicated with 2% QCH and CHP’sPPAV P9 PLA applicated with 2% QCH and QCHP’sPPAV P10 2.3.3. Physical and Chemical Properties of Chitosan/PLA Bilayer Films The zeta potential (ZP) and hydrodynamic diameter (HD) of the prepared particle dispersions were determined using the particle size analyzer (Litesizer 500, Anton Paar, Graz, Austria) at 25 ◦ C. ZP was measured by electrophoretic light scattering (ELS), which measures the velocity of particles in the presence of an electric field. HD was measured by dynamic light scattering (DLS). The speed of this motion depends on the size of the particles; smaller particles move faster than larger ones. Before analysis, the dispersion was stirred and, if necessary, adjusted to pH 4 with acetic acid. To perform the measurements, the diluted sample was placed in an Omega cuvette for ZP and size. Data were collected using Kalliope software (Anton Paar, Graz, Austria). Contact angles were measured using a goniometer (Data Physics, Fidelstadt, Germany) to estimate the surface wettability of layered molding compounds. Milli-Q water (5 µ L) was carefully placed on the test film surface. A goniometer with static contact angle (SCA) 20 software was used to determine SCA at room temperature. Analyses were performed in triplicates. The chemical composition of the chitosan/PLA bilayer films was analyzed using the X-ray photoelectron spectroscopy (XPS) instrument model TFA-XPS (Physical Electronics, Munich, Germany). The spectrometer was equipped with a hemispherical electron analyzer Foods 2022,11, 3847 6 of 18 and a monochromatic X-ray source with Al K α 1.2 radiation with a photon energy of 1486.6 eV. The excitation area of the sample was 400 µ m 2 . The emitted photoelectrons were measured at a departure angle of 45 ◦ . During the XPS measurements, an electron gun was used to neutralize the surface charge. The survey spectra were measured at a transit energy of 187 eV and an energy step of 0.4 eV. MultiPak v8.1c software (Physical Electronics, Munich, Germany) was used to analyze the measured spectra. Chitosan/PLA bilayer films were examined with a scanning electron microscope (SEM) using the JSM-IT800 instrument (Jeol, Tokyo, Japan). The PLA films were cut and glued to a double-sided conductive carbon tape, placed on a holder, and sprayed with gold to ensure conductivity and prevent charging effects. The samples were examined with an accelerating voltage of 5 kV and a variable working distance at comparable magnification. Images were acquired using a secondary electron detector. 2.4. Antioxidant Potential of Chitosan/PLA Bilayer Films The antioxidant activity of the chitosan/PLA bilayer films was tested using 2,2 0 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) reagent. The assay is based on the spectrophotometric determination (UV-VIS) of the decolorization of the reagent in the presence of an antioxidant. The ABTS reagent (7 mM; Sigma-Aldrich, St. Louis, MO, USA) was prepared in 2.45 mM potassium persulfate (Sigma-Aldrich, St. Louis, MO, USA) and diluted with phosphate-buffered saline (PBS; Gibco, Life Technologies, Grand Island, NY, USA). Absorbance was measured at 734 nm and 25 ◦ C at time points of 0 min, 15 min, and 60 min after addition of 0.1 g of film sample to 3.9 mL of ABTS solution. The solution was shaken during the extraction. The results are given as percentage of inhibition [18]. 2.5. Statistical Analysis Results are expressed as mean ± standard deviation. The statistically significant difference between P. pavonica extracts’ TPC and antioxidant activity over the months was determined by analysis of variance (one-way ANOVA), followed by a least significant difference test at the 95% confidence level [ 28 ]. Analyses were performed using Statgraphics Centurion-Ver.16.1.11 (StatPoint Technologies, Inc., Warrenton, VA, USA). 3. Results and Discussion Padina pavonica was harvested from May to September during the period when its thallus grows in the Adriatic Sea. In particular, P. pavonica’s thallus detaches every winter and regrows in spring. After September and during the winter, this seaweed is in the form of rhizoids, filamentous thalli, or sporelings until spring comes and the conditions are suitable for its full regrowth [ 29 ]. Thus, its seasonal growth is from May till September. For this reason, we aimed to investigate the difference during thallus growth to see if there are significant changes in chemical profile and antioxidant activity during this algae’s seasonal growth. Knowing the perfect harvesting time can contribute to the knowledge needed for the possible cultivation or farming of this species and its potential exploitation. 3.1. Compound Analyses of P. pavonica Extracts The results of the TPC for P. pavonica harvested from May to September are shown in Figure 2. The highest TPC value was determined for the extract of P. pavonica harvested in June. Overall, the results varied between 11.88 ± 0.51 and 26.69 ± 1.86 mg GAE/g. The lowest TPC was found for the May sample. There are many factors that can affect the TPC in seaweeds. TPC can vary due to seasonal variations in salinity, sea temperature and light intensity, different geographic locations, and biological factors, such as algal life cycle, size, age, and the presence of predators [ 6 ]. To avoid the effects of geographic location, the seaweed samples in this study were collected from the same location and depth each month. The sea temperature and TPC results showed no correlation. The results of this study are in accordance with Bernardini et al. [ 30 ], who reported a TPC of 27.0 mg GAE/g Foods 2022,11, 3847 7 of 18 in P. pavonica extract. In addition, Sofiana et al. [ 31 ] reported a TPC value of 20.34 mg GAE/g in the ethanolic extract of P. pavonica. Foods 2022, 11, 3847 7 of 18 intensity, different geographic locations, and biological factors, such as algal life cycle, size, age, and the presence of predators [6]. To avoid the effects of geographic location, the seaweed samples in this study were collected from the same location and depth each month. The sea temperature and TPC results showed no correlation. The results of this study are in accordance with Bernardini et al. [30], who reported a TPC of 27.0 mg GAE/g in P. pavonica extract. In addition, Sofiana et al. [31] reported a TPC value of 20.34 mg GAE/g in the ethanolic extract of P. pavonica. Figure 2. Total phenolic content of P. pavonica extracts. a–d different letters denote statistically significant difference. Padina pavonica extracts were subjected to quali-quantitative analysis of polar compounds using LC-ESI-QTOF-MS in negative ion mode. The chromatograms of the basic peaks are shown in Figure 3. The results are listed in Table 3, along with their retention time, score (%), molecular formulae, observed and theoretical m/z, and error (ppm). Fortyseven compounds were tentatively identified. For all compounds, the error was lower than 5 ppm and the score was higher than 90%. All compounds were identified considering previous research [5] and the PubChem database. The amount of each compound was calculated based on the peak areas and expressed as a percentage. Figure 3. Chromatograms of the UPLC-PDA-ESI-QTOF analyses of P. pavonica. Figure 2. Total phenolic content of P. pavonica extracts. a–d different letters denote statistically significant difference. Padina pavonica extracts were subjected to quali-quantitative analysis of polar compounds using LC-ESI-QTOF-MS in negative ion mode. The chromatograms of the basic peaks are shown in Figure 3. The results are listed in Table 3, along with their retention time, score (%), molecular formulae, observed and theoretical m/z, and error (ppm). Forty-seven compounds were tentatively identified. For all compounds, the error was lower than 5 ppm and the score was higher than 90%. All compounds were identified considering previous research [ 5 ] and the PubChem database. The amount of each compound was calculated based on the peak areas and expressed as a percentage. Foods 2022, 11, 3847 7 of 18 intensity, different geographic locations, and biological factors, such as algal life cycle, size, age, and the presence of predators [6]. To avoid the effects of geographic location, the seaweed samples in this study were collected from the same location and depth each month. The sea temperature and TPC results showed no correlation. The results of this study are in accordance with Bernardini et al. [30], who reported a TPC of 27.0 mg GAE/g in P. pavonica extract. In addition, Sofiana et al. [31] reported a TPC value of 20.34 mg GAE/g in the ethanolic extract of P. pavonica. Figure 2. Total phenolic content of P. pavonica extracts. a–d different letters denote statistically significant difference. Padina pavonica extracts were subjected to quali-quantitative analysis of polar compounds using LC-ESI-QTOF-MS in negative ion mode. The chromatograms of the basic peaks are shown in Figure 3. The results are listed in Table 3, along with their retention time, score (%), molecular formulae, observed and theoretical m/z, and error (ppm). Fortyseven compounds were tentatively identified. For all compounds, the error was lower than 5 ppm and the score was higher than 90%. All compounds were identified considering previous research [5] and the PubChem database. The amount of each compound was calculated based on the peak areas and expressed as a percentage. Figure 3. Chromatograms of the UPLC-PDA-ESI-QTOF analyses of P. pavonica. Figure 3. Chromatograms of the UPLC-PDA-ESI-QTOF analyses of P. pavonica. Foods 2022,11, 3847 8 of 18 Table 3. The list of compounds detected in P. pavonica extracts analyzed by UPLC-PDA-ESI-QTOF. No. RT (min) Observed m/z Theorical m/z Error (ppm) Score (%) Molecular Formulae Tentative Compound May (%) June (%) July (%) August (%) September (%) 1 0.27 343.0367 343.0368 −0.3 94.07 C20 H4N6O1a,9b-Dihydrophenanthro [9,10-b]oxirene-2,3,4,7,8,9-hexacarbonitrile 7.61 6.45 4.78 6.42 6.63 2 0.29 201.0239 201.0247 −4.0 98.89 C4H10 O92-(1,2,2,2-Tetrahydroxyethoxy)ethane-1,1,1,2-tetrol 7.62 7.13 5.76 6.72 7.05 3 0.34 141.0157 141.0161 −2.8 91.01 C2H2N6O2Diazidoacetic acid 0.82 1.27 1.30 0.80 1.05 4 0.35 181.0709 181.0712 −1.7 100 C6H14 O6D-Sorbitol 1.56 1.14 1.35 1.12 2.44 5 0.39 317.0516 317.0509 2.2 90.44 C12 H14 O10 D-glucaric acid derivate 0.50 0.45 0.55 0.52 1.62 6 16.60 343.2122 343.2121 0.3 95.77 C18 H32 O610,11-Dihydroxy-9,12-dioxooctadecanoic acid 0.15 0.14 0.12 0.13 0.13 7 16.84 487.3426 487.3423 0.6 96.96 C30 H48 O5Esculentic acid 0.36 0.00 0.00 0.00 0.00 8 16.96 275.2012 275.2011 0.4 100 C18 H28 O2Stearidonic acid (C18:4n-3) isomer a 0.08 0.08 0.04 0.16 0.41 9 17.10 309.2056 309.2066 −3.2 96.09 C18 H30 O46,9-Octadecadienedioic acid 0.20 0.33 0.17 0.26 0.32 10 17.16 285.2066 285.2066 0.0 90.36 C16 H30 O4Hexadecanedioic acid 0.32 0.14 0.07 0.14 0.21 11 17.17 277.2168 277.2168 0.0 90.14 C18 H30 O2gamma-Linolenic acid isomer a (C18:3n-6) 0.12 0.11 0.08 0.30 0.53 12 17.19 295.2276 295.2273 1.0 100 C18 H32 O39,10-Epoxyoctadecenoic acid isomer a (vernolic acid) 0.45 0.20 0.47 0.29 0.14 13 17.22 429.30090 429.3005 0.9 91.64 C27 H42 O424-Keto-1,25-dihydroxyvitamin D3 isomer a n.d.* 0.02 n.d. 0.01 0.03 14 17.27 247.1712 247.1698 5.7 100 C16 H24 O22,4,6-Triisopropyl benzoic acid 0.17 0.60 1.09 0.77 0.48 15 17.30 297.2426 297.2430 −1.3 98.84 C18 H34 O310-Oxooctadecanoic acid isomer a 1.14 0.43 n.d. 0.34 0.29 16 17.34 287.2212 287.2222 −3.5 90.62 C16 H32 O410,16-Dihydroxyhexadecanoic acid isomer a 0.07 0.09 0.10 0.09 0.16 17 17.35 287.2211 287.2222 −3.8 90.73 C16 H32 O410,16-Dihydroxyhexadecanoic acid isomer b 0.03 0.16 0.67 0.58 1.40 18 17.37 199.16890 199.1698 −4.5 92.54 C12 H24 O2Lauric acid 0.60 0.63 0.57 0.57 0.43 19 17.38 243.1952 243.1960 −3.3 90.78 C14 H28 O33-hydroxymyristic acid 0.68 0.32 0.37 0.34 0.40 20 17.42 293.2117 293.2117 0.0 80.96 C18 H30 O313-ketooctadecadienoic acid isomer a 0.33 0.22 0.57 0.29 0.12 21 17.43 293.2117 293.2117 0.0 87.56 C18 H30 O313-ketooctadecadienoic acid isomer b 0.46 1.93 2.47 1.95 3.15 22 17.44 295.2276 295.2273 1.0 100 C18 H32 O39,10-Epoxyoctadecenoic acid isomer b (vernolic acid) 1.00 0.34 n.d. 0.29 0.24 23 17.51 269.2110 269.2117 −2.6 98.63 C16 H30 O33-Oxohexadecanoic acid 1.29 1.05 1.11 1.12 2.11 24 17.51 225.1847 225.1855 −3.6 95.99 C14 H26 O2Myristoleic acid 1.53 1.44 1.27 1.33 1.03 25 17.56 275.2007 275.2011 −1.5 36.37 C18 H28 O2Stearidonic acid (C18:4n-3) isomer b 0.72 0.66 0.40 1.09 2.19 26 17.58 275.2010 275.2011 −0.4 93.59 C18 H28 O2Stearidonic acid (C18:4n-3) isomer c 1.13 4.46 4.38 3.85 2.88 27 17.59 277.2159 277.2168 −3.2 91.36 C18 H30 O2gamma-Linolenic acid isomer b (C18:3n-6) 0.01 0.08 0.03 0.07 0.04 28 17.60 213.18450 213.1855 −4.7 92.41 C13 H26 O2Tridecanoic acid 0.82 0.75 0.62 0.72 0.54 29 17.61 257.2108 257.2117 −3.5 95.16 C15 H30 O311-Hydroxypentadecanoic acid 0.43 0.16 n.d. 0.17 0.22 30 17.63 251.2010 251.2011 −0.4 100 C16 H28 O27-cis,10-cis-hexadecadienoic acid 0.72 0.94 1.07 1.12 0.61 31 17.64 297.2429 297.2430 −0.3 97.33 C18 H34 O310-Oxooctadecanoic acid isomer b 0.88 0.42 n.d. 0.55 0.39 Foods 2022,11, 3847 9 of 18 Table 3. Cont. No. RT (min) Observed m/z Theorical m/z Error (ppm) Score (%) Molecular Formulae Tentative Compound May (%) June (%) July (%) August (%) September (%) 32 17.66 239.2004 239.2011 −2.9 98.8 C15 H28 O2Myristoleic acid methyl ester 3.69 3.23 2.67 3.06 2.39 33 17.70 301.2156 301.2168 −4 98.12 C20 H30 O2Eicosapentanoic acid isomer a (C20:5n-3) 0.96 3.26 3.49 3.39 2.36 34 17.75 277.2171 277.2168 1.1 50.48 C18 H30 O2gamma-Linolenic acid isomer c (C18:3n-6) 2.75 5.28 6.90 5.60 5.16 35 17.77 227.2005 227.2011 −2.6 94.05 C14 H28 O2Tetradecanoic acid (C14:0) 5.16 4.13 4.47 4.01 3.13 36 17.80 271.2266 271.2273 −2.6 97.75 C16H32O3Hydroxy-palmitic acid 3.43 1.40 1.76 1.46 1.91 37 17.85 253.2159 253.2168 −3.6 99.61 C16 H30 O2Palmitoleic acid (C16:1n-7) 10.10 9.25 9.71 9.28 8.84 38 17.91 279.2319 279.2324 −1.8 98.14 C18 H32 O2Octadeca-10,12-dienoic acid (C18:2n-6) isomer a 1.49 1.68 2.55 2.08 2.32 39 17.93 241.2168 241.2168 0.0 100 C15 H30 O2Pentadecanoic acid (C15:0) 2.89 2.65 2.39 2.57 2.49 40 17.97 279.2324 279.2324 0.0 91.12 C18 H32 O2Octadeca-10,12-dienoic acid (C18:2n-6) isomer b 1.93 1.84 1.91 1.73 1.58 41 18.00 267.2329 267.2324 1.9 100 C17 H32 O29-Heptadecenoic acid (C17:1n-8) 2.81 2.88 2.47 2.70 2.86 42 18.07 255.2318 255.2324 −2.4 99.95 C16 H32 O2Hexadecanoic acid (palmitic acid) isomer a (C16:0) 0.24 0.31 0.39 0.35 0.45 43 18.08 255.2318 255.2324 −2.4 99.44 C16 H32 O2Hexadecanoic acid (palmitic acid) isomer b (C16:0) 9.40 8.43 9.19 8.67 8.11 44 18.10 281.2472 281.2481 −3.2 99.96 C18 H34 O2Oleic acid (C18:1n-9) 13.85 12.59 11.68 11.81 11.48 45 18.21 269.2474 269.2481 −2.6 97.24 C17 H34 O2Heptadecanoic acid (C17:0) 3.78 3.64 3.27 3.59 3.33 46 18.33 283.2629 283.2637 −2.8 99.97 C18 H36 O2Octadecanoic acid (stearic acid) C18:0 4.33 3.76 3.74 3.65 3.40 47 18.54 311.2944 311.295 −1.9 92.83 C20 H40 O2Arachidic acid 0.55 0.46 0.53 0.49 0.49 * n.d.—not detected. Foods 2022,11, 3847 16 of 18 antioxidant activity. This extract was selected for the development of chitosan/PLA bilayer films. This is the first report of testing such a formulation. A macromolecular solution of chitosan (primary and quaternary chitosan) was applied as the first layer on PLA films, and a second layer consisted of a suspension of chitosan particles with entrapped PPAV extract. XPS spectra confirmed that both layers, first as chitosan macromolecular solutions and second as PPAV extract embedded into chitosan particles, were successfully deposited on the film surface, with some desorption. In addition, the hydrophilicity of the films was reduced, which is very important for ensuring food safety and quality due to the anti-fog effect. An increase in antioxidant activity in the range of 48–65% was observed for all functionalized films. The developed films exhibited anti-fogging and antioxidant properties, confirming the development of the active concept and the potential use of these films for food packaging solutions. Author Contributions: Conceptualization, V.Š., M. ˇ C. and L.F.Z.; methodology, M. ˇ C., L.F.Z., S.S.M. and M.d.C.R.-D.; formal analysis, M. ˇ C., T.K.G., E.M. and M.d.C.R.-D.; resources, V.Š. and L.F.Z., data curation, M. ˇ C., T.K.G. and M.d.C.R.-D.; writing—original draft preparation, M. ˇ C., V.Š., L.F.Z. and M.S.; writing—review and editing, M. ˇ C., V.Š. and S.S.M.; supervision, V.Š. and L.F.Z.; funding acquisition, V.Š. and L.F.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research is supported by the PRIMA program under project BioProMedFood (Project ID 1467). The PRIMA program is supported by the European Union. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data are available from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Guiry, M.D. AlgaeBase. Available online: https://www.algaebase.org (accessed on 31 August 2022). 2. Pérez, M.; Falqué, E.; Domínguez, H. Antimicrobial Action of Compounds from Marine Seaweed. Mar. Drugs 2016 ,14, 52. [CrossRef] [PubMed] 3. ˇ Cagalj, M.; Skroza, D.; Tabanelli, G.; Özogul, F.; Šimat, V. Maximizing the Antioxidant Capacity of Padina Pavonica by Choosing the Right Drying and Extraction Methods. Processes 2021,9, 587. [CrossRef] 4. Al-Enazi, N.M.; Awaad, A.S.; Zain, M.E.; Alqasoumi, S.I. Antimicrobial, Antioxidant and Anticancer Activities of Laurencia catarinensis,Laurencia majuscula and Padina pavonica Extracts. Saudi Pharm. J. 2018,26, 44–52. [CrossRef] 5. ˇ Cagalj, M.; Skroza, D.; Razola-Díaz, M.D.C.; Verardo, V.; Bassi, D.; Frleta, R.; Generali´c Mekini´c, I.; Tabanelli, G.; Šimat, V. Variations in the Composition, Antioxidant and Antimicrobial Activities of Cystoseira Compressa during Seasonal Growth. Mar. Drugs 2022,20, 64. [CrossRef] [PubMed] 6. Mekini´c, I.G.; Skroza, D.; Šimat, V.; Hamed, I.; ˇ Cagalj, M.; Perkovi´c, Z.P. Phenolic Content of Brown Algae (Pheophyceae) Species: Extraction, Identification, and Quantification. Biomolecules 2019,9, 244. [CrossRef] [PubMed] 7. Ben Ali, A.I.; Ktari, L.; Boudabbous, A.; El Bour, M. Seasonal Variation Of Antibacterial Activity Of The Brown Alga Padina Pavonica (L) Thivy Collected From Northern Coast Of Tunisia. Bull. L’institut Natl. Des Sci. Technol. 2010,37, 111–116. 8. Men’shova, R.V.; Ermakova, S.P.; Rachidi, S.M.; Al-Hajje, A.H.; Zvyagintseva, T.N.; Kanaan, H.M. Seasonal Variations of the Composition, Structural Features, and Antitumor Properties of Polysaccharides from Padina Pavonica (Lebanon) as a Function of Composition. Chem. Nat. Compd. 2012,47, 870–875. [CrossRef] 9. Soltani Firouz, M.; Mohi-Alden, K.; Omid, M. A Critical Review on Intelligent and Active Packaging in the Food Industry: Research and Development. Food Res. Int. 2021,141, 110113. [CrossRef] 10. Bastarrachea, L.; Wong, D.; Roman, M.; Lin, Z.; Goddard, J. Active Packaging Coatings. Coatings 2015,5, 771–791. [CrossRef] 11. Wyrwa, J.; Barska, A. Innovations in the Food Packaging Market: Active Packaging. Eur. Food Res. Technol. 2017 ,243, 1681–1692. [CrossRef] 12. Kehinde, B.A.; Joy, O.S.; Ishrat, M.; Kehinde, O.; Ashaolu, T.J. Use of Biopolymers for Packaging of Functional Foods. In Functional Foods; Wiley: New York, NY, USA, 2022; pp. 477–509. [CrossRef] 13. Garcia-Garcia, D.; Carbonell-Verdu, A.; Arrieta, M.P.; López-Martínez, J.; Samper, M.D. Improvement of PLA Film Ductility by Plasticization with Epoxidized Karanja Oil. Polym. Degrad. Stab. 2020,179, 109259. [CrossRef] Foods 2022,11, 3847 17 of 18 14. Deshmukh, K.; Basheer Ahamed, M.; Deshmukh, R.R.; Khadheer Pasha, S.K.; Bhagat, P.R.; Chidambaram, K. Biopolymer Composites With High Dielectric Performance: Interface Engineering. In Biopolymer Composites in Electronics; Elsevier: Amsterdam, The Netherlands, 2017; pp. 27–128. [CrossRef] 15. García Ibarra, V.; Sendón, R.; Rodríguez-Bernaldo de Quirós, A. Antimicrobial Food Packaging Based on Biodegradable Materials. In Antimicrobial Food Packaging; Elsevier: Amsterdam, The Netherlands, 2016; pp. 363–384. [CrossRef] 16. Rathod, N.B.; Bangar, S.P.; Šimat, V.; Ozogul, F. Chitosan and Gelatine Biopolymer-based Active/Biodegradable Packaging for the Preservation of Fish and Fishery Products. Int. J. Food Sci. Technol. 2022. [CrossRef] 17. Ajdnik, U.; Zemljiˇc, L.F.; Plohl, O.; Pérez, L.; Trˇcek, J.; Braˇciˇc, M.; Mohan, T. Bioactive Functional Nanolayers of Chitosan–Lysine Surfactant with Singleand Mixed-Protein-Repellent and Antibiofilm Properties for Medical Implants. ACS Appl. Mater. Interfaces 2021,13, 23352–23368. [CrossRef] [PubMed] 18. Potrˇc, S.; Sterniša, M.; Smole Možina, S.; Knez Hrnˇciˇc, M.; Fras Zemljiˇc, L. Bioactive Characterization of Packaging Foils Coated by Chitosan and Polyphenol Colloidal Formulations. Int. J. Mol. Sci. 2020,21, 2610. [CrossRef] [PubMed] 19. Zemljiˇc, L.F.; Plohl, O.; Vesel, A.; Luxbacher, T.; Potrˇc, S. Physicochemical Characterization of Packaging Foils Coated by Chitosan and Polyphenols Colloidal Formulations. Int. J. Mol. Sci. 2020,21, 495. [CrossRef] 20. Potrˇc, S.; Fras Zemljiˇc, L.; Sterniša, M.; Smole Možina, S.; Plohl, O. Development of Biodegradable Whey-Based Laminate Functionalised by Chitosan–Natural Extract Formulations. Int. J. Mol. Sci. 2020,21, 3668. [CrossRef] [PubMed] 21. Amerine, M.A.; Ough, C.S. Methods for Analysis of Musts and Wines; Wiley: New York, NY, USA, 1980. 22. Verni, M.; Pontonio, E.; Krona, A.; Jacob, S.; Pinto, D.; Rinaldi, F.; Verardo, V.; Díaz-de-Cerio, E.; Coda, R.; Rizzello, C.G. Bioprocessing of Brewers’ Spent Grain Enhances Its Antioxidant Activity: Characterization of Phenolic Compounds and Bioactive Peptides. Front. Microbiol. 2020,11, 1831. [CrossRef] [PubMed] 23. Granato, D.; Shahidi, F.; Wrolstad, R.; Kilmartin, P.; Melton, L.D.; Hidalgo, F.J.; Miyashita, K.; van Camp, J.; Alasalvar, C.; Ismail, A.B.; et al. Antioxidant Activity, Total Phenolics and Flavonoids Contents: Should We Ban in Vitro Screening Methods? Food Chem. 2018,264, 471–475. [CrossRef] 24. Benzie, I.F.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 1996,239, 70–76. [CrossRef] 25. Milat, A.M.; Boban, M.; Teissedre, P.L.; Šešelja-Perišin, A.; Juri´c, D.; Skroza, D.; Generali´c-Mekini´c, I.; Ljubenkov, I.; Volarevi´c, J.; Rasines-Perea, Z.; et al. Effects of Oxidation and Browning of Macerated White Wine on Its Antioxidant and Direct Vasodilatory Activity. J. Funct. Foods 2019,59, 138–147. [CrossRef] 26. Burˇcul, F.; Generali´c Mekini´c, I.; Radan, M.; Rollin, P.; Blaževi´c, I. Isothiocyanates: Cholinesterase Inhibiting, Antioxidant, and Anti-Inflammatory Activity. J. Enzym. Inhib. Med. Chem. 2018,33, 577–582. [CrossRef] 27. Glaser, T.K.; Plohl, O.; Vesel, A.; Ajdnik, U.; Ulrih, N.P.; Hrnˇciˇc, M.K.; Bren, U.; Fras Zemljiˇc, L. Functionalization of Polyethylene (PE) and Polypropylene (PP) Material Using Chitosan Nanoparticles with Incorporated Resveratrol as Potential Active Packaging. Materials 2019,12, 2118. [CrossRef] [PubMed] 28. Šimat, V.; Vlahovi´c, J.; Soldo, B.; Generali´c Mekini´c, I.; ˇ Cagalj, M.; Hamed, I.; Skroza, D. Production and Characterization of Crude Oils from Seafood Processing By-Products. Food Biosci. 2020,33, 100484. [CrossRef] 29. Benita, M.; Dubinsky, Z.; Iluz, D. Padina pavonica: Morphology and Calcification Functions and Mechanism. Am. J. Plant Sci. 2018 , 9, 1156–1168. [CrossRef] 30. Bernardini, G.; Minetti, M.; Polizzotto, G.; Biazzo, M.; Santucci, A. Pro-Apoptotic Activity of French Polynesian Padina Pavonica Extract on Human Osteosarcoma Cells. Mar. Drugs 2018,16, 504. [CrossRef] [PubMed] 31. Sofiana, M.S.J.; Safitri, I.; Helena, S.; Warsidah, W. Phytochemical Screening, Total Phenolic Content and Antioxidant Activity of Tropical Brown Macroalgae Padina Pavonica Hauck from Kabung Island, West Kalimantan. Saintek Perikan. Indones. J. Fish. Sci. Technol. 2021,17, 32–36. [CrossRef] 32. Generali´c Mekini´c, I.; Šimat, V.; Boti´c, V.; Crnjac, A.; Smoljo, M.; Soldo, B.; Ljubenkov, I.; ˇ Cagalj, M.; Skroza, D. Bioactive Phenolic Metabolites from Adriatic Brown Algae Dictyota dichotoma and Padina pavonica (Dictyotaceae). Foods 2021 ,10, 1187. [CrossRef] [PubMed] 33. Sudha, G.; Balasundaram, A. Determination of Bioactive Components in the Methanolic Extract of Padina pavonica Using GC-MS. World J. Pharm. Res. 2018,7, 991–997. [CrossRef] 34. Sudha, G.; Balasundaram, A. Analysis of Bioactive Compounds in Padina pavonica Using HPLC, UV-VIS and FTIR Techniques. J. Pharmacogn. Phytochem. 2018,7, 3192–3195. 35. Uslu, L.; Sayin, S.; Naz, M.; Taskin, E.; Soyler, O.; Saygili, I.; Cetin, Z.; Dinler, Z.M.; Isik, O. Proximate Analysis and Fatty Acid Profile of Some Brown Macroalgae Collected from the Northeastern Mediterranean Coast. Fresenius Environ. Bull. 2021 ,30, 9433–9437. 36. El Shoubaky, G.A.; El Rahman Salem, E.A. Active Ingredients Fatty Acids as Antibacterial Agent from the Brown Algae Padina pavonica and Hormophysa triquetra.J. Coast. Life Med. 2014,2, 431–438. [CrossRef] 37. Kosani´c, M.; Rankovi´c, B.; Stanojkovi´c, T. Brown Macroalgae from the Adriatic Sea as a Promising Source of Bioactive Nutrients. J. Food Meas. Charact. 2019,13, 330–338. [CrossRef] 38. Abdul Khalil, H.P.S.; Tye, Y.Y.; Saurabh, C.K.; Leh, C.P.; Lai, T.K.; Chong, E.W.N.; Nurul Fazita, M.R.; Mohd Hafiidz, J.; Banerjee, A.; Syakir, M.I. Biodegradable Polymer Films from Seaweed Polysaccharides: A Review on Cellulose as a Reinforcement Material. Express Polym. Lett. 2017,11, 244–265. [CrossRef] Foods 2022,11, 3847 18 of 18 39. Galus, S.; Arik Kibar, E.A.; Gniewosz, M.; Kra´sniewska, K. Novel Materials in the Preparation of Edible Films and Coatings—A Review. Coatings 2020,10, 674. [CrossRef] 40. Albertos, I.; Martin-Diana, A.B.; Burón, M.; Rico, D. Development of Functional Bio-Based Seaweed (Himanthalia Elongata and Palmaria Palmata) Edible Films for Extending the Shelflife of Fresh Fish Burgers. Food Packag. Shelf Life 2019 ,22, 100382. [CrossRef] 41. Andrade, M.A.; Barbosa, C.H.; Souza, V.G.L.; Coelhoso, I.M.; Reboleira, J.; Bernardino, S.; Ganhão, R.; Mendes, S.; Fernando, A.L.; Vilarinho, F.; et al. Novel Active Food Packaging Films Based on Whey Protein Incorporated with Seaweed Extract: Development, Characterization, and Application in Fresh Poultry Meat. Coatings 2021,11, 229. [CrossRef]