Effect of the application of an edible film with turmeric (Curcuma longa L.) on the oxidative stability of meat

Abstract The aim of this study was to develop an edible alginate‐based film produced with turmeric (EFT), as an active compound, and evaluate its antioxidant capacity for application in fresh pork loin, beef loin, and chicken breast. The EFT was characterized by barrier parameters, color, and mechanical, structural, and antioxidant properties. Meat samples with and without EFT were stored at 4°C and analyzed at 2‐day intervals. The meat samples with EFT showed significant differences (p < .05) in color (CIE L*a*b*) and exhibited lower TBARS values compared with those without EFT. The addition of turmeric in the film, besides affecting its physicochemical and structural properties, contributed an important antioxidant effect for the meat.

Nowadays, some research focused on the development and characterization of edible films that included curcumin as a bioactive compound, due to its antioxidant and antimicrobial properties (Liu, Cai, Jiang, Wu, & Le, 2016;Ma, Du, & Wang, 2017;Ma, Ren, & Wang, 2017;Roșu et al., 2017). However, until now, no edible films have been developed that included complete turmeric addition, nor has its use been documented for application in fresh meat. The objective of this research was to characterize and evaluate the antioxidant capacity of edible sodium alginate films with turmeric on fresh pork, beef, and poultry to prolong the shelf life of the meat during refrigeration storage.

| Materials
Reagents were purchased from the following vendors: sodium

| Preparation of the edible film with turmeric (EFT)
The EFT-forming solution was prepared by dispersing alginate and turmeric in distilled water, in a 1.0:0.13 ratio (%, w/w) at 70°C for 30 min under constant stirring. One percent glycerol was added and vigorously mixed for another 30 min. The mixture was poured into polyethylene plates (64 cm 2 ) and placed in a dehydrator (Excalibur 4900, Spain) at 41ºC for 12 hr. Finally, 15 ml 1% (w/v) calcium chloride (dry film formulation) was added. Then, they were stored at 53% relative humidity for analyzing physical and mechanical properties.

| Thickness
The EFT thickness was measured with a micrometer (Model MDC-1 "SB," AMES, USA) to the nearest 0.001 mm at five random positions.

| Moisture content (MC) and solubility in water (WS)
MC was determined by the EFT weight loss after oven drying at 105°C for 24 hr (ASTM, 2007). WS was determined as described by Gontard, Guilbert, and Cuq (1993), with slight modifications. The MC and WS determinations were both performed in triplicate.

| Antioxidant capacity
The EFT antioxidant activity was performed with the 2,2-diphenyl-1-picrylhydracil (DPPH) method, according to Brand-Williams, Cuvelier, and Berset (1995) and Dashipour et al. (2015). A homogenized sample of 25 mg EFT with 5 ml distilled water was prepared with constant agitation. The extract (0.1 ml) was mixed with 3.9 ml DPPH solution (0.1 mM methanol solution) and incubated for 60 min in the dark at room temperature. The absorbance was measured at 517 nm. The percentage of DPPH radical uptake activity was calculated using equation (1).
where A control is absorbance obtained from the blank, and A sample is the absorbance obtained from the sample.

| Color
The color was determined with a CR-400 Minolta Chroma Meter colorimeter (Konika Minolta, Valencia, Spain). The CIELab color scale was used for measurements: L* = 0 (black) to L* = 100 (white), −a* (green), +a* (redness), −b* (blue), and +b* (yellow). The EFT color was measured by placing the sample on the surface of the standard plate. (1) The meat samples were measured immediately after taking them out of their packages.

| Transparency
The relative transparency property was measured with a UV-Vis spectrophotometer (Thermo Fisher Scientific Inc., UK); the readings were performed at 600 nm. The EFT sample was cut into rectangular pieces (4 1 cm) and placed directly in a cell test of the spectrophotometer. The measurements were made using air as a reference. It was calculated with equation (2) (Han & Floros, 1997).
where A 600 is the absorbance at 600 nm, and δ is the thickness of the film (mm).
WVP was determined as described by Fabra et al. (2017).

| Oxygen permeability (OP)
Oxygen permeability obtained from the oxygen transmission rate (OTR) was calculated using an OXTRAN Model 2/21 ml Mocon system (Lippke, Neuwied, Germany). The EFT samples were previously purged with nitrogen at moisture equilibrium before exposure to 10 ml/min oxygen flow. The exposure zone during the test was 5 cm 2 for each EFT sample. To obtain the OP, the EFT thickness and the difference in the partial pressure of oxygen were considered. The experiments at 23°C and 53% RH were developed.

| Infrared spectroscopy
EFT structural characterization was examined with a Fouriertransform infrared spectrophotometer (FTIR; Bruker, Vertex, Wisconsin, USA) using the attenuated total reflectance (ATR) sampling method. Sixty-four scans were used with a resolution of 4 cm −1 in the spectral region of 4000-400 cm −1 . The spectra were obtained in triplicate.

| Preparation of meat samples
The raw meat was purchased from a local market. The pork and beef samples were obtained from the loin muscle (Longissimus dorsi) free of fat content; while the chicken samples were acquired from the breast (Pectoralis major) and obtained 24 and 8 hr postmortem, respectively. The meat was cut in pieces of 10 g (4 × 2 × 1 cm) and fully wrapped with the EFT, and the meat samples without film packaging were considered as the control. A total of 81 samples were prepared for each type of meat and treatment (with EFT and without EFT).
The analyses were performed in triplicate. Finally, all samples were stored at 4°C ± 0.5°C for analysis in two-day intervals, until an acceptable estimated shelf life of meat was reached. Before pH, color, and thiobarbituric acid reactive substances (TBARS) measurements, the film was removed off from meat.

| pH
pH of the meat samples was measured with a potentiometer (Thermo Fisher Scientific Inc., UK); a 10 g sample was homogenized with 100 ml distilled water. The extract was filtered using cotton gauze to remove the connective tissue.

| Lipid oxidation
The TBARS assay was used to determine lipid oxidation in the meat samples (Tarladgis, Watts, Younathan, & Dugan, 1960). Ten g meat with or without EFT was homogenized with 15 ml double-distilled water at 50°C and 5 ml of PG-EDTA (0.5% PG and 0.5% EDTA). Next, 77.5 ml double-distilled water and 2.5 ml 4 N HCl were added to the extract. The solution was transferred to a Kjeldahl flask and heated with steam distillation until 50 ml condensate was obtained. Five ml of this condensate was removed and mixed with 5 ml 0.02 M 2-TBA solution to be incubated in boiling water for 35 min. The solution was cooled at room temperature, and the absorbance was measured at 532 nm. The blank contained 5 ml double-distilled water with 5 ml (2) Transparency = A 600 ∕ , the 2-TBA solution. TBARS values were expressed as mg malonaldehyde/kg meat (mg MDA kg −1 meat).

| Statistical analysis
A completely randomized design was used, with a factorial arrangement in the treatments, the meat factor with three levels (pork, beef, and chicken) and the film factor with two levels (with and without EFT). The data were analyzed under a mixed-effects model and subjected to a one-way analysis of variance (ANOVA) using the statistical software SAS® (Statistical Analysis System, USA). The mean tests were performed using Tukey's test. The significance level was set at p < .05.

| Characterization of the EFT
Previous results in the development of the EFT (data not shown) indicated that the higher the amount of added turmeric, the greater the antioxidant activity. However, a 1.0:0.15 ratio (%, w/w) of turmeric did not show statistically significant differences (p < .05) in the antioxidant activity compared to a 1.0:0.13 ratio. Addition of higher turmeric concentrations to the edible film was not favorable for its application in meat, a factor closely related to consumers' purchasing habits and decisions. The optical properties were modified considerably, with a decrease in transparency, an increase in the variables a* and b*, and altered light scattering. Thus, the 1.0:0.13 (%, w/w) alginate/turmeric ratio was chosen for its appearance in the EFT development; it provided a slight yellow color ( Figure 1).
Additionally, the film exhibited homogeneity and flexibility, aspects that also contributed to its application in meat.

| Thickness
The developed EFT thickness was 0.096 mm (Table 1), a value greater than that reported by Musso, Salagado, and Mauri (2017), which was 0.051 mm for a gelatin-based film with curcumin. This film thickness difference is probably related to the constituents as well as the hydrophobic molecule content of the film-forming polymer.
The film exhibited low MC (23.83%; Table 1), similar to that reported by Musso et al. (2017), with 22.1% in gelatin films with curcumin. On the other hand, Senturk Parreidt, Müller, and Schmid (2018) consider alginate films as sacrificial moisture agents, because the moisture in the food is preserved and the moisture contained in the film evaporates. Nevertheless, Gontard et al. (1993) indicate that amino groups and proteins form hydrogen bonds with hydroxyl (OH) groups of water molecules, which causes greater susceptibility to hydration that influences the solubility, MC, and water permeability of the film. This factor represents a drawback to the film, since effective moisture transfer control is desirable, especially when in contact with fresh meat.
The EFT was completely soluble in water (Table 1). Murad. Karim, Bhat, Uthumporn, and Chew (2011) reported similar results in sodium alginate films; they dissolve in 100% water after 1 min of contact. The high alginate solubility is likely due to its hydrophilic nature, which is determined by alternating units of α-D-mannuronic acid and β-L-guluronic acid. Fabra et al. (2018) indicated that pure alginate films have certain limitations due to their high solubility under medium-high RH conditions. Although turmeric has very low aqueous solubility, its solubility improves significantly when it binds alginate, as reported by Dey and Sreenivasan (2014), who formed pharmaceutical conjugates of alginate-curcumin. Thus, since highly soluble alginate-based films with glycerol and turmeric were incorporated, the solubility of the latter compound improved and resulted in complete film solubility.

| Antioxidant capacity
DPPH radicals are widely used to test the antioxidant activity of certain active compounds, such as electron-donating agents. The function of the antioxidant agent is to form a stable radical and eliminate it by electron donation (Dawidowicz, Wianowska, & Olszowy, 2012). Table 1 shows the EFT antioxidant capacity, estimated from the percentage of DPPH elimination. In general, the EFT antioxidant activity was similar to that obtained by Ma, Ren, et al. (2017), who reported F I G U R E 1 The appearance of the edible film based on alginate with turmeric, in a 1.0:0.13 ratio (%,w/w); (a) front and (b) back an activity of 35.16% for films made with tara gum/active polyvinyl alcohol and curcumin, and by Kim, Baek, and Song (2018), with 32.96% in alginate films with black chokeberry extract. Yang, Lee, Won, and Song (2016) note that the antioxidant activity of plant extracts is associated with the number of phenolic compounds; that is, a higher extract concentration generally increases DPPH radical uptake. Additionally, phenolic compounds interact with the major component of the matrix, which plays an important role in the functional properties of these films. Further, the present interactions contribute to improve the WVP and, in some cases, reduce solubility (Fabra et al., 2018).
The antioxidant activity of turmeric is mediated by curcumin, mainly by the phenolic hydroxyl; a small fraction may come from the CH 2 site (Priyadarsini et al., 2003). Under alkaline conditions, the phenolic OH group can be converted to the phenolic oxygen anion, which is a likely reaction site of free radicals that generates a phenyl radical. However, since the film has desirable antioxidant properties, some curcumin phenolic groups were possibly not used to form radicals. Therefore, the EFT is a promising option to prolong the shelf life of food, especially meat. It represents an active container, and the release or absorption of the active component in the food or in the environment is not desirable. Additionally, it can be controlled because it is found in low quantities and not present directly in the meat. It would also help improve food quality, since it would provide protection against color deterioration and lipid oxidation (López-de-Dicastillo, Gómez-Estaca, Catalá, Gavara, & Hernández-Muñoz, 2012).

| Color and transparency
The addition of turmeric dispersed in the film-forming solution and gave it a yellow coloration. The color was from curcumin, a hydrophobic polyphenol derived from turmeric, which generates a yellow-orange hue (Almeida et al., 2018). The brightness value was 93.41 ± 0.74, similar to that obtained by Musso et al. (2017), which was 93.3 in gelatin-based films with 0.02 (%, w/v) curcumin. The a* value for the EFT was 6.53 ± 0.18, similar to that reported by Ma, Du, et al. (2017), with 6.05 in tara rubber films that incorporated curcumin (with a slight orange color). The b* value (degree of yellowness) obtained in the EFT was 43.94 ± 1.60, a value very close to that reported by Wang, Xue, and Zhang (2019), with 40.4 for films with nanoparticles composed of sodium caseinate loaded with curcumin and meina. Priyadarsini (2014) explains that the yellow coloration in film depends on curcumin solubility in water (due to its hydrophobic nature). Active films contain natural pigments that are expressed as colored compounds and induce changes in color parameters (Bitencourt, Fávaro-Trindade, Sobral, & Carvalho, 2014). This phenomenon indicates that the addition of 0.13% turmeric to the edible film significantly impacted light scattering, a fact that was confirmed in the appearance, transparency, and color that the film adopted.
On the other hand, the EFT transparency was 7.78%, a value higher than that obtained by Ramos et al. (2012). In that study, the values oscillated between 1.35% and 3.09% in films based on whey protein isolate. This discrepancy is probably due to the fact that curcumin, a diaroylmethane responsible for the characteristic yellow color in turmeric, exhibits lower opacity of light transmission (Gürses, 2019). Fabra et al. (2018) note extracts that contain polyphenols tend to selectively absorb light, especially at low wavelengths, a phenomenon that imparts color to the edible film and increases opacity. This change provides a protective barrier against UV light that could contribute to decreased meat deterioration and prevent the oxidation of myoglobin and the lipids present in the food.

| Water vapor permeability
The WVP in an edible film is a parameter that demonstrates the transfer of moisture in the material and in the food. WVP is dependent on the type of polymer, structural composition of alginate (mannuronic acid/guluronic acid), concentration of the cross-linked agent, and storage conditions such as RH and temperature. Therefore, in food packaging, it is desirable that the WVP value be as low as possible to avoid moisture loss in food (Gontard, Guilbert, & Cuq, 1992).
Curcumin has a long chain of carbon and hydrophobicity in the benzene rings that decreases the WVP value (Wang et al., 2019).
Nevertheless, Musso et al. (2017) found that the hydrophobic TA B L E 1 Physical and chemical characterization of the edible film based on alginate added with turmeric (1.0:0.13 ratio (%, w/w))

Variables
Edible film

| Oxygen permeability
OP in edible films has a direct effect on the quality and shelf life of the food, so a lower OP value represents better barrier capacity against oxygen in the packaging material (Ma, Ren, et al., 2017).
The OP value obtained was relatively low (Table 1) compared with those obtained by Ma, Du, et al. (2017), which ranged between 7 and 8.51 cm 3 mm m −2 atm −1 día −1 day -1 for films that contained curcumin, and from 42.7 to 187 cm 3 mm m −2 atm −1 día −1 day -1 for commercial high-density and low-density polyethylene films.
These results may be associated with bond interactions between curcumin and the carbohydrate matrix, which can prolong or limit oxygen access . Generally, phenolic compounds that are incorporated into carbohydrate-based films have the capacity to form hydrogen bonds between the carboxyl groups, a phenomenon that induces a cross-linking effect that decreases OP (Fabra, Hambleton, Talens, Debeaufort, & Chiralt, 2011). Overall, the EFT presented a good barrier against oxygen, even better than commercial plastic films.

| Mechanical properties
The values for TS, EAB, and Young's modulus are shown in Table 1.
The TS value for the EFT was 8.26 MPa, slightly higher than that obtained by Wang & Zhang (2019), which was 7.4 MPa in NaCas/zein films loaded with curcumin. These data indicate that the EFT was stronger in order to support extensibility by external tensile stress before breaking. Kalaycıoğlu, Torlak, Akın-Evingür, Özen, and Erim (2017) observed that the addition of turmeric extracts in chitosan films increases the TS value 1.5 times compared to film without the extract. This resistance depends on the polymer, the internal union, and the possible interactions between the matrix and the active compound (Gilbert, Cheng, & Jones, 2018).
For EAB, the EFT elongation was 35.94%, higher than that reported by Ma, Ren, et al. (2017) and Wang & Zhang (2019), which are 29.88 and 30.8% in tara gum films added with curcumin and caseinate/zein films with curcumin, respectively. Bitencourt et al. (2014) observed that at 50 g of turmeric ethanol extract/100g of gelatin (a higher concentration than used in this study), the EAB value significantly increases. Noronha, de Carvahlo, Lino, and Barreto (2014) indicate that the presence of phenolic compounds in the film could increase EAB, potentially due to interactions between curcumin and alginate, a phenomenon that would generate a more flexible and cohesive matrix.
For Young's modulus value, although there are no determined values for each food, a high value indicates that a material is strong. In this study, the EFT value was 42.33 ± 1.79 MPa, which was lower compared to that reported by Kalaycıoğlu et al. (2017) in chitosan films with turmeric extract (2064 ± 155 MPa) and with turmeric flour films from the dye extraction residue (values obtained between 246.8 ± 46.8 and 875 ± 15.7 MPa; Maniglia, de Paula, Domingos, & Tapia-Blácido, 2015). This result could be due to the hydrophobicity of the matrix caused by the incorporation of turmeric, which causes changes in the cross-linking and decreases Young's modulus.
Overall, the goal is to develop flexible films that adhere perfectly to the surface of the food, as a "second layer," to preserve the shape, texture, and integrity of the product (Rosa, 2019). The EFT has the potential to be applied to meats as a packaging system that maintains properties such as appearance, color, water retention capacity, lipid stability, and microbial spoilage.

| Infrared spectroscopy
The infrared spectroscopy technique was used to analyze molecular changes mediated by the addition of turmeric. Figure 2 shows the spectra of the alginate films with and without turmeric. The line shapes were very similar, and there were only slight band shifts.
Therefore, the lack of observable drastic changes suggests that the chemical stability of the film is not compromised by the presence of turmeric. In general, both spectra showed bands associated with the functional groups C = O, C-O-C, and COOH (all present in the alginate spectrum). Additionally, the bands associated with the presence of the turmeric had low absorption values (Figure 2b), because turmeric is present at a lower concentration.

| Determination of pH value
Meat pH is related to quality and freshness characteristics, such as color, tenderness (Jayasena et al., 2013), microbial growth, and the addition of antioxidants (Battisti et al., 2017). EFT significantly affected the pH of pork and beef (Table 2; p < .05). The pH value in the meat, with and without EFT, during refrigeration storage increased gradually, but in those with EFT, this increase was lower. In chicken breast meat, there were no significant differences in pH with or without EFT (Table 2; p < .05).
The increase in pH during storage can be attributed to protein denaturation and the accumulation of alkaline byproducts such as ammonia, amines, and trimethylamine, all of which are produced during amino acid degradation by autolytic or microbial reactions (Lorenzo, Batlle, & Gómez, 2014). Likewise, the pH tendency in meat with EFT is probably due to the phenolic compounds present in the edible film and its inhibitory effect on microbial growth during storage (Ehsani, Jasour, Hashemi, Mehryar, & Khodayari, 2014). Indeed, curcumin has antibacterial functions in addition to its antioxidant effects (Chan, Ng, Tan, & Low, 2011).
The pH results in pork with EFT were similar to minced pork wrapped in gelatin film with catechin-lysozyme (Kaewprachu, Osako, Benjakul, & Rawdken, 2015); the wrapped pork exhibited a pH increase from 5.7 to 6.4 during 4 days of storage. In beef with edible film, Amiri, Aminzare, Azar, and Mehrasbi (2019)  after 15 days of storage for samples of beef wrapped in corn starch films with clove and cinnamon essential oils. Pirsa and Shamusi (2019) observed similar behavior in pH for chicken meat, with a value of 7.38 after 10-day storage with cellulose-polypyrrole-ZnO films.

| Color
Changes in meat color during storage are important to consumers' acceptance of and decision to buy the product. The mean values of CIE L*, a*, and b* of the meat with and without EFT during storage are shown in Table 2. Notably, all beef and chicken samples were significantly affected (p < .05) in the L* value. This effect may be due to the oxygen barrier property of the EFT, which may have delayed oxygen diffusion and its reaction with myoglobin. In beef, the luminosity value increased, while the opposite effect was observed in chicken in the presence of the EFT. This discrepancy could be explained by factors such as the composition of glycolytic fibers in the muscle that is generally associated with greater luminosity (Kim, Cho, & Han, 2013) and the degree of association of myoglobin with oxygen that is related to the muscle pH. Where there is the highest degree of association, the pH value is lower, and the meat will appear "bright" (Hoving-Bolink et al., 2000). The pork values were not different with or without the EFT.
Low a* values indicate a loss in the redness of the meat, which is associated with the oxidation of myoglobin to metmyoglobin (Warner, 2014). Specifically, an increase in oxymyoglobin causes negative color changes and is generally related to a reduction in freshness (Serrano-León et al., 2018). This discoloration is strongly linked to the myoglobin content in the muscles; pork has a type of oxidic-glycolytic muscle IIX, and beef contains an oxidative muscle type I with a high myoglobin content (Astruc, 2014). However, prolonged storage times cause the iron in the heme ring to oxidize to the reduced ferric state (MbFe 3+ ), a phenomenon that induces browning of the meat (Baron & Andersen, 2002). In this study, there were no significant differences with or without EFT in pork and beef during refrigerated storage, data that suggest the effectiveness of using EFT to maintain color integrity in this type of meat. In chicken with EFT, the a* value increased, since the chicken breast, being mostly type B fibers, contains relatively little myoglobin (Nishida & Nishida, 1985).
The mean b* values during storage in pork and beef samples with and without EFT were not significantly different. However, for chicken with EFT (Table 2), b* significantly increased (p < .05).
This change may be caused by the presence of curcuminoids (yellow color) in the edible film. Zhang and Guo (2016) found that the incorporation of spice extracts increases yellowing in fresh chicken meat.

| Lipid oxidation
The mean TBARS values of the meat with and without EFT are presented in Table 2. Overall, the EFT produced a significant effect in the different meat. The change in TB.ARS values during refrigeration storage in pork (a), beef (b), and chicken (c) is presented in Figure 3.  indicate that the inclusion of natural extracts rich in phenolic compounds helps to delay lipid oxidation in meats.

| CON CLUS ION
Edible films have attributes beyond being biodegradable and biocompatible, advantages they hold over traditional plastic containers.
Incorporating turmeric into alginate-based films produced interactions between turmeric compounds and the polymer matrix. These interactions influenced the mechanical and barrier properties. The addition of turmeric increased the antioxidant capacity, results that indicate great potential to be applied as a packaging material and extend the shelf life of pork (12 days), beef (16 days), and chicken (12 days) in refrigeration storage, with a particular benefit to reducing lipid oxidation. Consequently, the development of the EFT is proposed as a promising technology to increase shelf life and preserve its quality in different meat.

ACK N OWLED G M ENTS
We gratefully acknowledge the Consejo Nacional de Ciencia y Tecnología (CONACYT) and the Colegio de Postgraduados, Campus Córdoba, Veracruz for the support provided for the development and financing of the present investigation.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest for this article.