1. Introduction
Edible films are thin layers of semi-solid materials that are assembled to improve the properties of fresh foods. They are generally recognized as safe (GRAS) [
1]. The materials utilized to produce these films include polymers such as proteins and polysaccharides, whose function is to approach the barrier to O
2 and CO
2. However, they are poor barriers to water vapor. The lipids incorporated into these films form a hydrophobic barrier that reduces water vapor permeability, protects fruits and other foods from damage due to friction, and gives these edible coatings greater flexibility and cohesion [
2]. The main function of these films when applied to fresh products is to delay maturation and the eventual senescence that occurs after harvesting. This is achieved by regulating the gas exchange, water vapor, moisture, and light, as well as preventing the volatilization of flavor compounds [
3,
4].
One of the polysaccharides most often used in the manufacturing of film-forming dispersions and thus edible coatings is XG, which is produced from the fermentation process of
Xanthomonas campestris. XG is GRAS according to the FDA. Because it remains stable under changes in temperature and pH, it aids in obtaining emulsions that can reduce surface tension and ease the integration or addition of bioactive compounds as a controlled release system for edible coatings [
5].
Recently, the demand for improving the functionality of edible films to meet consumer requirements has led to the development and application of novel materials that, due to their functional properties, extend food preservation for longer periods. This has given rise to the elaboration of nanocomposite films, which consist of a natural polymer matrix with added organic/inorganic fillers that have at least one magnitude on the nanometric scale [
6]. Nanotechnology is commonly used to improve the properties of edible coatings by generating renewed materials that present enhanced mechanical, thermal, and water vapor barrier properties [
7]. In this regard, García-Betanzos et al. [
8] demonstrated that incorporating SLN made with Candeuba
® wax (mixture of
Euphorbia Cerifera and
Copernicia Cerifera) improved the mechanical and gas barrier properties of films when added to an XG matrix. In the same way, an increase in the shelf life was observed when guava fruit was coated with these systems [
9]. The incorporation of SLN and protein into the mechanical properties of edible coatings has been studied. The observations showed that adding SLN decreased the WVT [
10], whereas adding Tween 20 as an emulsifier agent in the elaboration of nanoparticles resulted in a reduction in tensile strength. Edible coatings prepared with lipids only do not have good mechanical properties and are weaker, but adding a lipid to films made with polysaccharides improves the water vapor barrier and increases the film’s strength, adhesion, and elastic properties [
11].
SLN are aqueous colloidal dispersions, ranging in size from 40 to 1000 nm [
12], which consist of lipids that are solid at room temperature. Hot homogenization is the most commonly used technique for producing SLN since this process does not require solvents other than water and is based on the formation of a nanoemulsion at high temperatures. Solid particles are expected to be formed by the cooling of the sample to room temperature or below. The surface area behavior of SLN is different from that of particles with a micrometric size, showing higher diffusion rates and better transport properties due to its low viscosity [
13]. As mentioned above, SLN have been shown to enhance the properties of edible films and coatings. For this reason, this study was designed to analyze the impact of using candelilla wax as the lipid phase in SLN, as well as the effects of various polysaccharide types and plasticizer concentrations on the aforementioned material properties.
Candelilla wax (
E. antisyphilitica Zucc.) is an endemic species found in the semiarid regions of northern Mexico and Texas. It is composed of 49–50%
n-alkanes with 29–33% carbons, 20–29% high-molecular-weight esters, 12–14% alcohols and sterols, and 7–9% free acids [
14,
15]. It is considered one of the most effective waxes for blocking moisture migration [
16]. The addition of candelilla wax to edible coatings was reported in the conservation of blackberry fruits by employing guar gum. This coating helped to increase the fruit´s shelf life. Candelilla wax and Arabic gum nanocoatings have also been developed as a vehicle for phytomolecules for use with tarbush. Products containing this substance were found to increase the shelf life of Golden Delicious apples during 8 weeks of refrigeration and 4 weeks of commercial storage at the industrial level [
16]. However, some reports suggest that coatings employing candelilla wax in the dispersal phase are breakable due to the high melting point of this material (68.5–72.5 °C) while its mechanical and thermal properties and moisture barrier have not yet been studied in combination with polysaccharides such as XG and CMC.
The main aim of this work was to study a nanocomposite edible film by adding candelilla wax SLN to an XG and CMC continuous matrix in order to analyze its impact on the mechanical, optical, thermal, and barrier properties of films. Candelilla wax nanoparticles have never been used to strengthen edible films. The literature suggests that reducing particle size may have significant effects on the brittleness, color, and water vapor permeability due to the larger contact surface and hydrophobic nature of this material.
2. Materials and Methods
2.1. Materials
PVA-205 polyvinyl alcohol (m: 4.6–5.4 mPa·s) was used as the stabilizer (Sigma-Aldrich®, St. Louis, MO, USA). Xanthan gum derived from Xanthomonas campestris (Mw ≈ 2 × 10−6 g/mol and ηint = 7627 mL/g) and sodium carboxymethyl cellulose Cekol® 30,000 (Mw: 240.20 g/mol) were used to form the film (CP Kelco, Mexico City, Mexico). Glycerol (98%) was used as the plasticizer agent (Droguería Cosmopolita, Mexico City, Mexico), whereas Candelilla REAL® wax (melting point: 68–72.5 °C) purchased from Multiceras® S.A. de C.V. (Monterrey, Mexico) was used as the solid lipid. Distilled water was acquired from Milli-Q equipment (Millipore® Corporation, Bedford, MA, USA). All other reagents were analytical grade.
2.2. Solid Lipid Nanoparticle Preparation
The SLN were prepared using both a lipid and an aqueous phase. The first phase consisted of melting 100 g/L of candelilla wax at 90 °C while a PVA-205 solution (50 g/L) was heated up to the same temperature. Both phases were homogenized using the high-shear stirring technique (Ultra-Turrax
® T5; KikalborTechnik, Germany, with an S25N-25G, IKA
® disperser element) during 3 to 5 min cycles at 2094.4 s
−1. The solidification of the SLN occurred during the cooling of the suspension at 25 °C [
16,
17].
2.3. Dynamic Light Scattering (DLS)
A Z-sizer 4 (Zetasizer Nano Series, Malvern Ltd., Enigma Business Park, Grovewood Road, Malvern, UK) was employed to determine the particle size (PS), polydispersion index (PDI), and zeta potential (ζ). The evaluation was carried out for pure candelilla wax SLN (100 g/L) for 5 weeks. The PS and PDI were measured using a laser light-scattering technique at a 90° fixed angle and a temperature of 25 °C. The zeta potential was evaluated by electrophoretic movement with a Z-sizer 4 at 90 ° (Zetasizer Nano Series). The dilutions were performed with Milli-Q® water and the results obtained were normalized via polystyrene standard dispersion (ζ = −55 mV). All measurements were taken in triplicate at room temperature.
2.4. Film-Forming Dispersions
The edible film-forming dispersions were prepared by dispersing 3 g/L of each polysaccharide in distilled water using a stirrer (Eurostar Power Control Visc, IKA® Werke, Staufen, Germany) at room temperature for 10 min and a speed of 104.72 s−1. Subsequently, two concentrations of SLN (20 and 60 g/L) were added to study their impact on the mechanical, optical, and barrier properties, as well as the thermal behavior and film microstructure. Two concentrations of plasticizer (10 or 30 g/L) were added to the film dispersions.
2.5. Film Formation
Once the dispersions were obtained, 22.5 mL of each was cast in a Teflon plate (192.5 cm
2) with circular geometry. The dispersions were stored for 24 h in an acrylic chamber at a temperature of 35 °C and 60% of RH to allow them to dry. The thickness was evaluated with a digital micrometer (Mitutoyo Model 293-348, Kanagawa, Japan) with an error of 0.001 mm. To calculate the thickness, 9 measurements taken at different positions of the film were used. All films were manufactured following the same method to ensure the variations were a function of the storage conditions and formulations only. The formulations tested are shown in
Table 1.
2.6. Water Vapor Permeability (WVP)
The WVP was evaluated at 10, 25, and 35 ± 2 °C using the ASTM E96/E96M-05 method with minor variations. Briefly, 3 g of silica gel was added to glass vials to ensure 0% of RH within each container. Subsequently, each vial was covered with films of a 1.5 cm diameter. The samples were conditioned in acrylic chambers using saturated salt solutions (NaBr–60% RH; NaCl–70% RH; and KCl–85% RH). The weight variations of the vials were documented over a period of 205 h. Each determination was evaluated in triplicate. The film previously determined thickness was utilized for all WVP estimations.
2.7. Mechanical Properties
A Brookfield texture analyzer (TA-CT3, Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) was utilized to evaluate the mechanical properties of the films using the ASTM D882-02 method. Before performing the tests, the films were cut into rectangular shapes (10 mm wide × 40 mm long) and conditioned at 60, 70, and 85% of RH and 10, 25, or 35 °C for 48 h. After this time, the mechanical properties were evaluated using the TA-DGF accessory (Dual-Grip Fixture for tensile testing of thin films or integrity of seals for packaging). The crosshead speed was set at 50 mm/min. The tensile strength (TS), elongation at breaking (E), and Young’s modulus (YM) were obtained. All assays were performed in triplicate at room temperature.
2.8. Whiteness Index of Films
The optical properties of the films were determined with an Agrocolor
® colorimeter (Apollinaire Ltd., Agrotechnology, Serqueux, France), and the R (red), B (blue), and G (green) parameters were obtained, which were then converted to L*, a*, and b* coordinates (L*: lightness; a*: red-green; b*: yellow-blue). The films’ color determinations were contrasted against a blank and both sides of the film were evaluated in duplicate to calculate the total color difference (Δ
E). Determining the whiteness index (
WI) did not require any background and was calculated as follows:
where Δ
L*, Δ
a*, and Δ
b* represent the variations in the color parameters for the SLN–polysaccharide films vs. the white tile (
L = 97.75,
a = −0.49,
b = 1.96) [
9,
18,
19].
2.9. Differential Scanning Calorimetry (DSC)
The thermal properties of the bulk components, pure SLN, and non-conditioned edible films were evaluated using a Diamond Differential Scanning Calorimeter (Perkin Elmer Instruments, Waltham, MA, USA). For this technique, 10 mg of each specimen was precisely weighed in a 40 μL aluminum pan and then hermetically sealed. The heating tests were staged from −20 to 150 °C (10 °C/min). An empty aluminum pan was utilized as a blank and each thermograph was baseline-corrected.
2.10. Scanning Electron Microscopy (SEM)
Morphological analyses of the prepared films with different concentrations of SLN, glycerol, and polysaccharides were conducted using a high-resolution, cold-field scanning electron microscope (SEM) (Hitachi, SU-8230, Tokyo, Japan) with a BSE + BSE (U) detector, an acceleration voltage of 2.5 kV, and an average deceleration mode of 15 kV. The emission current was 5 Å with a working distance of 3.7 mm.
2.11. Statistical Analysis
An analysis of variance (ANOVA) was carried out to evaluate the effect of the formulation, temperature, and RH. All statistical differences between batches were analyzed with a Tukey test. The responses evaluated were the film thickness, tensile strength (TS), elongation at breaking (E), Young’s modulus (YM), total color difference (ΔE), and whiteness index (WI). Minitab® (Minitab® Statistical Software 17 Inc., Centre, PA, USA) software was used to carry out the analysis of variance.
4. Conclusions
The SLN formulated in this work remained sterically stable for 5 weeks due to the use of PVA as a surfactant compound. The incorporation of candelilla wax SLN into the XG and CMC networks improved the mechanical properties of the films since the geometry and rigidity of the particles caused changes in stiffness, elasticity, and flexibility. Likewise, the natural crystallinity of the SLN modified the hygroscopicity of the films and the structural arrangement of the continuous matrices, thus enhancing the water vapor barrier properties. However, the addition of submicron-sized candelilla wax particles resulted in a yellowish coloration of the films, which was more pronounced when 60 g/L of SLN was added. This directly impacted the ΔE and WI parameters. The best results were observed in the formulations with 20 g/L of SLN added, especially those containing 30 g/L of glycerol and 3 g/L of XG, as the microstructural analysis showed a better distribution of nanoparticles along the matrix that resulted in a low WVP. Adding 30 g/L of plasticizer decreased the intermolecular forces and improved the mobility of the polymeric chain, resulting in low ΔH values and, therefore, films that were more flexible and elastic that can maintain their physical structure by avoiding deformations and ruptures when applied to fresh foods. In conclusion, candelilla wax is a good option for preparing nanocomposite films since its interaction with the polysaccharide used as the continuous matrix improved the properties of the films. Most nanocomposites are eco-friendly, novel materials, as is the case with the nanocoating developed and studied by our group. These characteristics, together with the improvements in the mechanical, water vapor barrier, and structural properties, allow nanocoatings based on SLN to preserve the shelf life of whole fruits. Climacteric fruits, which are characterized by a high respiration rate, can be a good option to experiment “in vivo” with the conservative effect of these nanocoatings since their excellent barrier properties can have a sluggish effect on the respiration rate, thus delaying ripening and senescence. We suggest the application of the developed nanocoatings to fruits such as pears, apples, guavas, tomatoes, plums, and figs.