1. Introduction
Food products are exposed to external conditions throughout the production chain, i.e., harvesting, post-processing, distribution, transportation, storage, and delivery to the final consumer [
1]. In the last decade, food packaging has been developing new biodegradable active and smart materials that extend shelf life, maintain quality, safety, and integrity, and avoid microorganisms’ proliferation and food oxidative reactions by incorporating active substances [
2,
3]. Along with protecting food products from environmental conditions and mechanical forces, active packaging plays an active role in quality and food preservation during the distribution process [
3,
4].
The use of biopolymers in packaging has increased considerably over the past few years due to their sustainable feedstock, biodegradability, and similar processing characteristics to existing thermoplastics [
5]. Starch is one of the most researched and widely used raw materials in the production of biodegradable films due to its high bioavailability, low cost, biodegradability, and renewability [
6]. Moreover, starch possesses good film-forming properties and chemical stability [
7,
8,
9,
10]. In addition, considering the scarcity of fossil fuel resources, starch is considered an alternative renewable agricultural resource with excellent film-forming ability, non-toxicity, and good gel stability. Furthermore, it can be made by standard techniques, such as solution casting and extrusion, and thus processed as a thermoplastic material using a plasticizer (urea, glycerol, sorbitol, glycerin) and water. However, starch-based films have poor resistance to water due to their hydrophilic nature. Therefore, blending starch with other polymers is a possible way of developing starch films [
11,
12].
On the other hand, wheat gluten has film-forming properties, and its high bioavailability and low cost makes it highly suitable for the preparation of biodegradable polymers [
13]. Wheat gluten has good oxygen and carbon dioxide barrier properties in dry conditions, controlling cellulosic materials’ inherent gas permeability [
14].
In order to confer active properties to biodegradable films, several substances can be incorporated into the biopolymers, such as essential oils (EOs) [
15,
16]. Many research studies have shown that the use of EOs inhibits the growth of a wide variety of Gram-negative bacteria, such as
Escherichia coli,
Pseudomonas aeruginosa, and
Salmonella typhimurium, as well as Gram-positive bacteria including
Staphylococcus aureus,
Listeria monocytogenes,
Streptococcus pyogenes, and
Alicyclobacillus acidoterrestris [
17,
18]. Additionally, other reports have demonstrated the high antioxidant activity of EOs, which ranges from 70% to 95% inhibition of free radicals of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(-3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), due to the presence of hydroxyl groups (–OH) in its chemical structure [
13]. Due to the aforementioned antimicrobial properties and antioxidant capacity, EOs have been widely used to develop active packaging for food [
19]. Among the most studied EOs are cinnamon and turmeric, which are composed of a wide range of volatile components such as terpenes, alcohols, acids, esters, epoxies, aldehydes, ketones, amines, and sulfides, among others, responsible for its strong biological activity [
20,
21,
22,
23]. The high antimicrobial and antioxidant properties of cinnamon EOs have been attributed mainly to cinnamaldehyde, cinnamate, cinnamic acid, trans-cinnamaldehyde, cinnamyl acetate, eugenol, L-borneol, camphor, caryophyllene oxide, b-caryophyllene, L-bornyl acetate, E-nerolidol, α-cubebene, α-terpineol, terpinolene, and α-thujene [
24]. In the case of turmeric EOs, the high bioactivity has been attributed to curcuminoids that consists of curcumin and two related compounds: demethoxy curcumin and bisdemethoxycurcumin [
25].
Since most EOs are volatile compounds, they require the use of manufacturing methods that are carried out at room temperature to preserve their bioactivity. In this sense, the most commonly used method for a laboratory biopolymer film formation is solvent casting that largely depends on polymer solubility [
26]. This method consists of three basic steps: first, biopolymer solubilization in a solvent, which is chosen according to biopolymer chemical structure; second, the solution is poured into molds, which are usually Teflon-coated glass plates; and third, the casted solution is heat dried, wherein the solvent is evaporated to obtain a polymer film adhered to the molds with a homogeneous and continuous microstructure [
27,
28,
29]. Lastly, the physical and chemical properties of the film are dependent on casting solution composition, wet casting thickness and drying conditions (e.g., temperature and relative humidity) [
30].
In this context, the objective of this research was to develop active films based on starch and wheat gluten containing cinnamon and turmeric EOs by the solvent casting method. Initially, different formulations were prepared varying the EOs and biopolymers concentrations. Then, the active films were characterized in terms of their moisture content (MC), water solubility (WS), water absorption capacity (WAC), contact angle, morphology, color, thermogravimetric analysis (TGA), antioxidant capacity (DPPH IC50 and ABTS IC50) and water vapor permeability (WVP). Thereafter, the active films were applied on baby carrots to increase their shelf life and maintain their quality.
2. Materials and Methods
2.1. Materials
Wheat flour was purchased from the local city market, glycerol was provided by Panreac, and EOs were purchased from NowFoods.
2.2. Wheat Starch and Gluten Extraction
Starch extraction was carried out forming a suspension of wheat flour and distilled water, which was washed several times with distilled water and filtered with muslin cloth, which allowed the starch to pass through. The starch suspension was kept at rest in a container at a temperature of 5 °C for 12 h for starch decantation. After the decanting time, the aqueous phase was removed from the vessel and the starch was washed with distilled water. Once the starch-washing stage was completed, the starch was recovered for subsequent drying in the sun. Once it was completely dry, it was milled and sieved to reduce its particle size and make it uniform. The calculation of the starch extraction yield was made between the starch obtained and the total wheat used.
For gluten extraction, a dough was first formed from wheat flour and 2% NaCl, and the dough was kneaded until it had a consistency sticky to the touch. The dough was left to rest for 5 min, washed with water for the necessary time to remove the starch, and then the clean gluten was weighed and taken as a percentage of the initial dough. To obtain the dry gluten, the gluten obtained from was dried in an oven at 100 °C for 5 h to constant weight, then cooled and weighed.
2.3. Active Film Development
Starch films were obtained based on the solution casting method, relying on previous studies with minor modifications, making several formulations and varying the bioactive compounds [
31]. The film-forming dispersion was prepared by placing the powdered starch in a 250 mL conical flask containing distilled water; the values used are described in
Table 1. The dispersion was heated in a water bath at 90 °C for 30 min while stirring at 500 rpm for the starches to gelatinize completely. Subsequently, the heating was stopped, and the dispersion was allowed to cool to 40 °C. 25% glycerol was added to the starch and stirred at 150 rpm for 20 min. After cooling, 3% of the EO (Cinnamon essential oil or Turmeric essential oil), depending on the formulations, was added to the dispersion in relation to the weight of the starch. The film-forming dispersion was then poured onto a Teflon to obtain the wheat starch-based films [
32].
Gluten films were obtained following the methodology of Olabarrieta et al. [
33], with minor modifications, making different formulations as shown in
Table 1, where the wheat gluten solution was prepared under stirring conditions by mixing wheat gluten powder and glycerol in ethanol. Deionized water was then added to the solution formed. The solution was heated at 75 °C for 20 min, then stirred for 10 min. After cooling, the EO (Cinnamon essential oil or Turmeric essential oil), depending on the formulations, was added. Finally, the solution was poured onto a Teflon for drying to obtain the wheat gluten-based films.
2.4. Film Characterization
2.4.1. Thickness
Before testing, the thickness of all films was measured using a digital micrometer (S00014, Mitutoyo, Corp., Kawasaki, Japan) with ±0.001 mm accuracy. Measurements were performed and averaged at five different points, two in each end and one in the middle.
2.4.2. Morphology
Cross-sections and surface sections of biodegradable films were studied using an optical microscope (Zeiss Primo Star HD cam, Jena, Germany) integrated into a high-definition camera with 40× magnification. The microphotographs were analyzed and processed through Image Pro-Plus version 5.1 computer program.
2.4.3. Color
The optical parameters (luminosity (
L∗),
a∗,
b∗, chrome (C∗ab), and hue angle (h∗ab)) of films were measured with a MINOLTA Spectro-colorimeter (Minolta Co., Tokyo, Japan). Total color differences (ΔΕ) with respect to the control film were also determined using Equation (1).
2.4.4. Moisture Content
The films were conditioned to a relative humidity of 53%. Moisture content was determined using a convection oven at 60 °C until a constant weight was obtained. Moisture was calculated as the ratio of the wet weight to the dry weight. The test was carried out in triplicate.
2.4.5. Water Solubility
This was determined by immersing the films in distilled water at a film:water ratio of 1:10 for 48 h. The samples were exposed to a natural convection oven for 24 h at 60 °C to remove free water and were placed in a desiccator with P2O5 at 25 °C for 2 weeks to remove the tightly bound water. The solubility of the films was calculated from the initial and final weights. The test was performed in triplicate.
2.4.6. Water Absorption Capacity
The test was carried out according to ASTM-D570 standard, using 25 mm by 60 mm specimens, which were placed in an oven at a temperature of 30 °C for 24 h to allow the samples to dry. After this time, they were immersed in distilled water for 2 h and weighed again to determine the amount of water absorbed.
2.4.7. Water Vapor Permeability (WVP)
This was determined according to the protocol reported by Ortega-Toro et al. [
31], following the ASTM E96-95 standard method (ASTM, 1995) with some modifications; a relative humidity gradient of 53% to 100% at a temperature of 25 °C was used. The films were selected for WVP testing based on the lack of physical defects. Distilled water was placed in Payne permeability cups to expose the film to 100% RH on one side. Once the films were secured, each cup was placed in a relative humidity-balanced cabinet at 25 °C. The RH of the cabinets (53%) was constant, using supersaturated solutions of magnesium nitrate-6-hydrate. The free film surface during film formation was exposed to the lowest relative humidity to simulate the actual application of the films on high water activity products when stored at intermediate relative humidity. The glasses were periodically weighed (0.0001 g) and the water vapor transmission (WVTR) was determined from the slope obtained from the regression analysis of the weight loss versus time data, once a steady state is reached, divided by the film area.
2.4.8. Contact Angle
For the measurement of the contact angle, a space with a background and lighting suitable for taking photographs with light contrast was provided. Fruit peel or rind slices of 1 cm2 were placed on a flat level surface. A 0.1 mL drop was dropped onto the surface and photographs were taken at 10 s, 30 s and 60 s. Image analysis was then carried out using Adobe Photoshop to determine the contact angle formed between the emulsion drop and the analyzed surface. The greater the contact angle, the greater the wettability of the emulsion on the surface.
2.4.9. Thermal Properties
The thermal properties were studied by thermogravimetric analysis (TGA) under nitrogen atmosphere in a Thermobalance TG-STDA Mettler Toledo model TGA/STDA851e/LF/1600 analyzer. TGA curves were obtained after conditioning the samples in the sensor for 5 min at 30 °C. The samples were then heated from 50 °C to 600 °C at a heating rate of 10 °C/min [
10,
34,
35]. The first derivatives of thermogravimetry (DTG) curves, expressing the weight loss rate as the function of time, were also obtained using TA analysis software. All tests were carried out in triplicate.
2.4.10. Antioxidant Activity
The antioxidant capacity of the films was determined using the 2,2-diphenyl-1-pikryl-hydroxyl (DPPH) reduction method [
14]. 30 µL of samples diluted in water (1:10 for powder films) were mixed with 1 mL of 0.1 mM DPPH in methanol. The mixture was vortexed and allowed to stand at room temperature in the dark (40 min) before measuring absorbance at 517 nm. In the same way, 30 μL of samples were diluted in water (1:10 for the powder films) and 1 mL of ammonium solution of 2.2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) from Sigma Aldrich
® (St. Louis, MI, USA); the solutions were diluted in mixed methanol. After six minutes of reaction in the dark, the absorbance at 734 nm was monitored using a spectrophotometer (UV Visible Thermo Scientific Genesys 10S, Dreieich, Germany). The results were expressed in the IC
50 parameter, which allows measuring the DPPH and ABTS+ radical scavenging capacity. The lower the IC50 value, the greater the antioxidant power it will have in the analyzed sample.
2.5. Application of Active Films on Baby Carrots
The application of active films based on starch and wheat gluten and containing essential oils of cinnamon and turmeric was carried out on baby carrots. Batches of 5 samples were evaluated for each coating in the study, and a negative blank without coating was also considered. Samples were stored at 25 °C and 75% RH for two weeks and monitored for weight loss, appearance, and fungal growth.
2.6. Statistical Analysis
Data for each test were analyzed statistically. Analysis of variance (ANOVA) was used to assess significance in the difference between factors and levels. Averages were evaluated using Fisher’s Least Significant Difference (LSD) test with 95% confidence. Data were analyzed using Statgraphics Plus for Windows 5.1 software (Manugistics Corp., Rockville, MD, USA).