Physicochemical and functional characterization of gelatin edible film incorporated with fucoidan isolated from Sargassum tenerrimum

Abstract Biodegradable films were created with fish gelatin and fucoidan extracted from Sargassum tenerrimum using 30% glycerol as a plasticizer. The gelatin films were incorporated with fucoidan (2.5%, 5%, 7.5%, and 10%), respectively. Results presented that the average thickness of films ranged from 0.12 to 0.147 mm. Tensile strength (TS) was decreased from 29.27 to 3.46 MPa by adding the fucoidan except for the gelatin/fucoidan 10% (5.35 MPa) sample. The results showed that the physical characteristics (the contact angle (Ɵ), water solubility, opacity, and moisture content) of the films significantly changed depending on different fucoidan concentrations. FTIR and SEM analysis confirmed the interaction of fucoidan with gelatin in the composite film. Furthermore, adding 10% fucoidan showed high DPPH radical scavenging activity (65%) than other treatments. Therefore, incorporation of fucoidan extracted from brown algae (Sargassum tenerrimum) with fish gelatin films improved thermal stability, anti‐oxidative, and antibacterial characteristics in addition to enhanced mechanical and protective properties, to be used as a bioactive edible film in the food packaging industry.


| INTRODUC TI ON
In the last decade, synthetic plastics are widely used in the food industry as packaging polymers, but the use of these polymers due to their very slow biodegradation has caused many environmental problems (Jouki et al., 2013). Since microbial contamination is one of the main reasons for spoilage and deterioration of food quality, film production has attracted a lot of attention in the food packaging industry (Clarke et al., 2017). The use of edible and biodegradable films has been considered one of the active alternative packaging methods for nondestructible plastic films (Rui et al., 2017). Oral films maintain the quality and shelf life of food by controlling the transfer of moisture, carbon dioxide, oxygen, and food additives (Arfat et al., 2014).
Gelatin is a protein obtained from the hydrolysis of collagen in the skin, bones, and connective tissue of animals, including livestock, poultry, and aquatic animals. Fish gelatin is also a good source of biopolymers for film packaging. It is biodegradable and has good film properties (Etxabide et al., 2017). In recent years, due to the prevalence of bovine disease and the rejection of pig gelatin by religious groups, research on other raw sources of gelatin has increased (Nagarajan et al., 2017). Gelatin can be extracted from fishery waste such as bone and fish skin. The physical and chemical properties of fish gelatin, including gel resistance, melting and boiling temperatures, and rheological properties are weaker than those of mammalian gelatin (Sow et al., 2018). Studies on film production from fish gelatin have shown that gelatin produced from fish also has excellent film-forming properties (Qiao et al., 2017).
Gelatin, with its unique properties, has many applications in the food, pharmaceutical, medical and photographic industries, including the ability to produce a gel, its adhesion, clarity, and thickening properties (Das et al., 2017).
Lipid oxidation is a major contributor to reducing the quality of foods by negatively affecting their sensory properties, shelf-life, and nutritional value. To prevent such oxidative damage, synthetic antioxidants are most commonly used as food additives. The toxicity of synthetic antioxidants butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) have paved the way for the search for unique natural antioxidants (Wang et al., 2022).
Gelatin-based packaging films can be used as an antimicrobial and antioxidant agent to improve the quality and shelf life of packaged foods (Benbettaïeb et al., 2019). On the other hand, seaweed is a rich source of bioactive compounds that can produce many types of metabolites with a wide range of biological activities. Compounds with antioxidant, antiviral, and antimicrobial activity have been identified in brown, red, and green algae (Cox et al., 2010). Seaweeds are limited due to their abundance as a suitable alternative to terrestrial resources (Khalil et al., 2017). One of their bioactive compounds is fucoidan, which is obtained from brown algae and is a polysaccharide . Applicable properties of fucoidan include film formation, antimicrobial, antioxidant, biodegradable, anticoagulant, anti-inflammatory, and wound healing properties and are used in the medical industry (Gomaa et al., 2018;Hifney et al., 2017;Perumal et al., 2018). Sulfated fucoidan derived from brown seaweeds such as Porphyra, Laminaria japonica, Undaria pinnatifida, Sargassum polycystum, Kjellmaniella crassifoliacan can be as a potential source of natural antioxidants, as it has excellent free radical scavenging, hydroxyl radical scavenging activity, superoxide anion radical scavenging, and ferric reducing power activities (Xu & Wu, 2021). Packaging with antioxidant releasing system is a kind of food preservation method, in which an antioxidant incorporated into the package will increase the shelf-life of foods (Govindaswamy et al., 2018). Fish gelatin being a carrier of functional components, it has been proposed to combine fucoidan derived from brown seaweed in gelatin to produce an edible film. This research has been considered because no research has been done on the gelatin/fucoidan film extracted from Persian Gulf algae.
Other chemicals such as ethanol and acetone were obtained from Merck company.

| Extraction of fucoidan from Sargassum tenerrimum
Brown algae (Sargassum tenerrimum) were collected from the Persian Gulf (Iran) and used for the extraction of fucoidan. Algae were first washed with seawater and twice in sterile distilled water to remove sediment and foreign substances; then, air dried under shade and powdered using an electric mill. Dried algae powder (20 g) was treated with 80% ethanol (200 mL) at room temperature in a magnetic stirrer to remove proteins and pigments; then washed with acetone and centrifuged at 5000× g for 10 min. Dried algae were extracted by dis-

| Preparation of gelatin-fucoidan films
Lyophilized gelatin 4% (w/v) was dissolved in distilled water under continuous stirring in a magnetic stirrer at room temperature for 15 min.
Glycerol (30% w/w) was added as a plasticizer and stirred for 10 min.
The film-forming solutions were made by adding 0 (control gelatin), 2.5%, 5%, 7.5%, and 10% of fucoidan. Solutions were homogenized for 30 min using a high-speed magnetic stirrer, and then 15 mL of it was poured into circular plastic plates at 25°C and dried for 24 h. The dried films were then manually separated and used for further analysis.

| Film thickness
A digital micrometer (with an accuracy of 0.001 mm, Mitutoyo Corporation) was used to measure the thickness of the films.
Measurements were repeated randomly at five points in each film.
The average thickness of these points was used to determine the physical and mechanical properties of the films (Gibis et al., 2012).

| Mechanical properties
Tensile strength (TS) and elongation at break (E%) of films were measured using Instron Universal Testing Machine (Model A1 700, Gotech) using method number d882-01 approved by ASTM (ASTM, 2002). The films were cut into rectangles measuring 2.5 × 10 cm 2 and stored in a desiccator containing magnesium nitrate (to create a relative humidity of 53%) at 25°C for 48 h. The samples were then placed between the two jaws of the device. The initial distance between the two jaws and the movement speed of the upper jaw were determined to be 50 mm and 50 mm/min, respectively, and the data were recorded by a computer (Ghanbarzadeh & Almasi, 2011 where Wo: initial weight of the sample, Wf; weight of the undissolved desiccated residue.

| The contact angle (Ɵ)
The contact angle of the films was measured by the sessile drop method, which is a common method for determining the hydrophobicity of solid surfaces. For this purpose, 5 μL of deionized water was placed on the samples and the contact angle of the drop with the film was reported at the initial time. In this test, 5 replications were considered for each sample.

| Opacity
The light transmission spectrum of the samples was measured at 200-800 nm by a spectrophotometer (Lambda 25, Perkine Elmer) in 5 replications. The device was calibrated with an empty tube. Then the samples (1 × 4 cm 2 ) were pasted into the tube and placed in a spectrophotometer. The following formula was also used to calculate the opacity of the films (Abdollahi et al., 2013): where Abs 600 = value of absorbance at 600 nm, 'x' = the film thickness (mm).

| Scanning electron microscopy (SEM)
Morphology of films was displayed using a scanning electron microscope (Philips XL30, Netherlands). Films were cut into 3 × 3 cm and glued to an aluminum base with silver glue. The bases were coated with gold in a coating/spraying apparatus. The surface of the samples was imaged at different magnifications.

| Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) spectra of films were determined using FTIR Spectrometer (Perkin-Elmer, Spectrum Rxi) in the range of 400 to 4000 cm −1 and in the resolution of 4 cm −1 .

| Thermal gravimetric analysis (TGA)
The dried films were analyzed. from 25 to 600°C with a heating rate of 10°C/min in a nitrogen gas at a flow rate of 20 mL/min using TGA-7 (Perkin Elmer).

| Antioxidative activities of gelatinfucoidan films
DPPH radical scavenging activity of films was determined according to the method presented by Govindaswamy et al. (2018). Film solution was prepared by solubilizing the treatments in distilled water to obtain a 5 mg/mL protein concentration. Then, methanolic DPPH solution (1 mL, 0.1 mM) was added to each treatment, mixed well, and placed for 2 h in the dark at room temperature. The absorbance was read at 517 nm using a UV-Vis spectrophotometer (Shimadzu UVmini-1240) distilled water was used as control. DPPH radical scavenging activity was measured based on the following formula: DPPH radical scavenging activity ( % ) = 1 − Abs sample Abs control × 100

| Microbial analysis
Three circular pieces were cut from each film (1 cm in diameter).

Staphylococcus aureus (ATCC 29737) and
Escherichia coli (ATCC 10536) strains were obtained from the microbial collection of the University of Tehran (Iran). Some of each bacterium was removed using a sterile loop from sterile ampoules and added to 10 mL of BHI Broth culture medium. The culture medium was incubated for 24 h at 37°C. The tubes were linearly cultured on a Nutrient Agar medium using a sterile loop. The plates were autoclaved and incubated for 24 h at 37°C. 3-5 well-isolated colonies were transferred to tubes containing 5 mL of physiological saline. The turbidity of the suspensions was examined by a spectrophotometer at 625 nm. Each tube was suspended on its culture medium. Then, the films were cut into circular disks (10 mm in diameter). the films were placed in a culture medium inoculated with the suspension at appropriate intervals, and the plates were incubated at 37°C for 24 h. Then inhibition zone diameter around the film was reported in millimeters.

| Statistical analysis
All experiments were done in triplicates and the results are indicated as an average mean ± SD. A significant difference between the means was defined by Duncan's multiple comparison test using the statistical software Statistical Package for the Social Sciences Version 26 (SPSS Inc.).

| Physical properties (solubility, moisture, contact angle, film thickness)
The results of the physical properties of the prepared films are presented in Table 1. Results showed that the moisture content of films significantly increased with the addition of fucoidan from 12.02% in the gelatin treatment to 20.80% in the treatment containing 10% fucoidan. The moisture content represents the total volume occupied by the film network microstructure by water molecules, while the solubility index is related to the hydrophilicity of the films (Jiang et al., 2010). The lower amounts of moisture in gelatin films compared to gelatin-fucoidan films are probably due to the reduction of capacity and voids in the polymer substrate. This result also indicates that gelatin showed more hydrophobic properties than protein and fucoidan films. Solubility is one of the important properties of biodegradable films that can determine the degree of resistance of the film to water, especially in humid environments such as meat foods (Gimenez et al., 2009 The water solubility of gelatin-fucoidan films significantly increased (P < 0.05) with the incorporation of fucoidan (2.5%, 5%, 7.5%, and 10%) which could be due to the formation of more interactions between biopolymers, leading to lower availability of hydroxyl groups to absorb the water molecules. Earlier, Hosseini et al. (2013) reported similar results for the edible film produced by gelatin and chitosan.
In another research, using seaweed (Turbinaria ornata) extract incorporated with fish gelatin films reported reduced water solubility (Rattaya et al., 2009). Cross-linking of gelatin composite film with other biopolymers decreases low molecular fractions, which reduce the water solubility (Wang et al., 2022).
The fucoidan polysaccharide cross-links by electrostatic interactions with low molecular fractions of gelatin, which stabilizes the gelatin-fucoidan film matrix, and thus limits the water solubility.
The results of the contact angle of the smooth surface of produced films showed that the composite films containing fucoidan did not display a significant difference compared to the control film, but by increasing in fucoidan level, the contact angle of the composite films decreased somewhat. This is probably due to hydrophilic groups at the biopolymer level. Thickness is one of the most important parameters used to evaluate the mechanical properties and opacity of films. The mean thickness of edible films ranged from 0.12 to 0.147 mm (Table 2) and it was increased with the addition of fucoidan significantly (p ≤ 0.05). A similar trend in the thickness of fish gelatin films in combination with polysaccharides and carrageenan (Pranoto et al., 2007) chitosan, and essential oil (Wu et al., 2015) was declared by some researchers.
The addition of other macromolecules such as polysaccharides, film preparation method, and drying process are factors influencing the thickness of gelatin-based films. Our results are in agreement with other studies (Hoque et al., 2010;Govindaswamy et al., 2018;Lupina et al., 2022;Ratna Aprilia et al., 2022). They found that a low increase in the thickness is usually due to the protruding construction

| Film color and opacity
Film color is an important factor because of its direct effect on the appearance of packaged products and consumer acceptance (Bortom et al., 2008). Transparency of films is an important feature of food packaging in terms of its direct relationship with the appearance of product (Abdullah et al., 2022). The greater the transparency of biodegradable films and similar to synthetic polymers, the greater the use of this type of packaging material.
The transparency of the films can affect the oxidation rate of fats and the quality of the packaged product (Abdullah et al., 2022).
Results showed that the lightness (L*) value of films was higher in gelatin films, (Table 2) Similar results were also considered by Peng and Li (2014) in the chitosan-based film using thyme, lemon, and cinnamon essential oil.
Furthermore, the incorporation of 2% gellan and k-carrageenan to fish gelatin film significantly decreased lightness (L*) as the films, which were darker than the control treatment, whereas a* and b* values did not reduce significantly (Said et al., 2021).
Materials used in the packaging industry must be able to protect food from the effects of light, especially UV radiation. Protein-based films have a great UV barrier property due to the presence of high amounts of aromatic amino acids that absorb UV light (Ningruma et al., 2021). However, the plasticizers used in the film formulation compete with the polymers for hydrogen bonds, disrupting the homogeneity of the network and forming transparent films (Begines et al., 2022).
The opacity of gelatin-fucoidan films varied with adding the fucoidan (

| Mechanical Properties
Mechanical properties of prepared films are studied to evaluate the film stiffness, stretching ability, and film strength. The results of mechanical properties are presented in Table 3. Adding the fucoidan to the gelatin film resulted in significant changes in its mechanical properties so that the tensile strength (TS) of films was initially decreased by adding the fucoidan and the highest amount was observed in the gelatin film treatment (29.27 MPa).
The TS of gelatin-fucoidan films decreases by adding fucoidan at low levels and then increased by increasing fucoidan (7.5 and 10%) from 3.93 to 5.35 MPa (p ≤ .05) ( Table 3).
In agreement with this study, an increase in TS with incorporating polysaccharides to fish gelatin film (Pranoto et al., 2007); Hydrogen bonds in the layers mainly improve TS protein films.
Moreover, the properties of gelatin-based film layers strongly depend on the softeners and film thickness (Wang et al., 2022).
The results showed that the strength and flexibility of layers will change with changes in the amounts and ratio of protein to polysaccharides. Pranoto et al. (2007) found that the optimal level of gelatin and polysaccharide for better interaction between them was 2%. In our study, the composition of gelatin-fucoidan 10% was prepared as a compact film with better tensile properties. The average EAB values of gelatin-fucoidan films increased from 47.31% to 62.47% with the increase in fucoidan concentration (Table 3).
It is important to study the different mechanical properties of films because of the need for edible polymers to have the desired strength and elasticity and to be free from defects such as cavities and small fractures.  Pranoto et al. (2007) found that the incorporation of gellan also improves the internal structure to give the compact and dense appearance, however, this initiated some linkages and resided between fibrillar zones of fish gelatin through polyelectrolyte association.

| FTIR spectroscopy analysis
FTIR analysis of fucoidan showed the primary absorptive peaks were associated with glycosidic structures which were attributed to C-O and C-C stretching vibrations of the pyrenoid ring and the C-H group. The FTIR spectrum showed a peak at 3423 cm −1 which was related to a hydroxyl stretching vibration. Another peak at 2925 was consistent with the stretching vibration of the C-H bond of the pyrenoid ring. Previous studies showed that the wide signal at 1250 cm −1 represents the total sulfate esters in the polysaccharides (Barros et al., 2013;Synytsya et al., 2010).
To examine the functional groups, structure, and interactions of gelatin with other molecules, FTIR spectra of gelatin-fucoidan films are analyzed (Said et al., 2021). In this study, amide-A-related peaks at 3280 and 3282 cm −1 were discovered in gelatin and gelatinfucoidan films (Figure 2).
These peaks correlate to NH stretching and hydrogen bonding.
Moreover, in FTIR analysis of gelatin-based films, amide B peak at 29332-2934 cm −1 approves C-H stretching and -NH3+, which is specific to gelatin. Similarity, amide B peaks were considered at 2928 cm − 1 in skin fish gelatin film incorporated with herbal extract (Nagarajan et al., 2013;Tongnuanchan et al., 2013). Using fucoidan in film formulation had shifted this peak to a lower wave number (2932 cm −1 ) because of asymmetrical stretching vibration of C-H in the CH2 group. Amide I peak was observed at 1633 cm −1 , which links to C=O stretching and hydrogen bonding coupled with COO− (Aewsiri et al., 2009). A peak was like the present study related to amide I was reported in the tilapia skin gelatin layer made with kcarrageenan and gellan at 1650 cm −1 . Amide III peak at 1238 cm −1 was created due to vibrations in the C-N and N-H groups of bound amide or vibration of CH 2 groups of glycine. Similarly, the peaks that occurred at 921 cm −1 were characteristic of gelatin molecules.

| Thermal gravimetric analysis (TGA) of prepared films
Thermal gravimetric analysis curves shown in Figure 3 represented four main stages of decomposition and weight loss in all films. The initial weight loss observed in all films at lower temperatures is probably due to the reduction or evaporation of free water, bound water, and also volatile compounds absorbed in the films. According to Figure 3, in a fucoidan film, the first step involves the removal of physically absorbed water, while the second stage deals with the removal of chemisorbed water concurrently with the destruction of the fucoidan molecule. The destruction of the polysaccharide macromolecule is followed by stages three and four (Matusiak et al., 2022).
Fucoidan film began to degrade thermally around 520°C as a result of the random breakage of glycosidic linkages. The remaining mass at the end of this process (600°C) was changed into ash, which contains minerals recognized in fucoidan like phosphates, carbonates, and sulfates (Saravana et al., 2018). In gelatin film ( Figure 3b) the most important temperature for the beginning of

| Antioxidative activities of gelatinfucoidan films
DPPH free radical scavenging test was used to estimate the antioxidant capacity of the fucoidan, gelatin, and gelatinfucoidan films. Previous findings reported that the antioxidant F I G U R E 1 SEM image of the morphology of fucoidan (a), gelatin (b), and gelatin-fucoidan 5% (c) films.
In agreement with our study, incorporating essential oils in gelatin films also enhanced the DPPH radical scavenging activity from 25% to 100%.

| Antimicrobial properties of prepared films
The antimicrobial activity of gelatin film as well as gelatin-fucoidan films with different concentrations against Staphylococcus aureus and Escherichia coli were evaluated and compared with co-amoxiclav, gentamicin, and tetracycline antibiotics ( been substantiated by identifying the nucleic acids and proteins (Wang et al., 2022) released after the treatment of microorganisms with sulfated polysaccharides. Another hypothesis is the antibacterial effect of fucoidans by trapping nutrients. Cationic minerals, for example, are explained in the nutrient medium by negatively charged molecules of sulfated polysaccharides, which leads to reduced bioavailability of nutrients to microorganisms. Therefore, according to the results, it can be seen that the bacteriostatic effect on the studied bacteria is due to fucoidan, which prevents the bacteria from receiving adequate nutrition, which leads to inhibition of bacteria (Ayrapetyan et al., 2021).

| CON CLUS ION
In this work, the effect of adding fucoidan on improving the prop-

ACK N OWLED G M ENTS
The authors thank the Gorgan University of Agricultural Sciences and Natural Resources.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors report no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.

E TH I C S S TATEM ENT
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.