1. Introduction
In the last few years, advances in the production of edible coatings and films using different compounds from renewable sources has been provided. In fact, it is well known that barrier, mechanical and optical properties of films and coatings depend on both the chemical-physical properties of utilized compounds and their specific interactions [
1,
2]. Chitosan, derived from chitin deacetylation is one of the most widely studied and used biopolymers for the preparation of edible films and coatings due to its excellent film forming ability, biodegradability and antimicrobial properties [
3]. Sodium caseinate is soluble casein obtained through the acidic precipitation of milk with subsequent solubilization in sodium hydroxide [
4]. Sodium caseinate shows good film-forming and thermoplastic properties due to its random coil structure, and its ability to form weak intermolecular interactions [
5,
6]. However, it does not confer antimicrobial properties.
Panela cheese is a type of fresh cheese made from pasteurized cow’s milk very popular in Mexico, where its production accounted for 418,560 tons in 2018 [
7]. In addition, it is the most popular Hispanic-style cheese consumed in the USA [
8]. It is white, with a porous, soft and fluffy texture with low fat content. The shelf life of fresh cheeses is about 15–18 days, due to its high moisture content and nutrients availability [
9].
Cheese packaging is practiced to minimize weight loss due to moisture loss, and to prevent microbial contamination and spoilage without affecting the cheese composition [
10,
11]. Edible coatings are applied directly to the surface of the food, where a thin layer forms after drying, while the films dry separately to form a material that is then used to cover the food.
Essential oils (EOs) are concentrated oily liquids obtained from plant materials such as flowers, buds, leaves and bark, which contain a mixture of volatile aromatic compounds including terpenes, terpenoids (oxygenated terpenes) and phenols [
12,
13]. EOs have shown antimicrobial properties against foodborne pathogens, i.e., bacteria, molds and their associated toxins [
14]. The antimicrobial activity of essential oils may be regulated by more than one mechanism of action, including changes in the fatty acid profile of the cell membrane, damage of the cytoplasmic membrane and disruption of the proton-motive force [
13,
15]. However, due to the high volatility and diffusivity of essential oils in foods, the development of strategies able to increase their retention and release control, are the main challenges that need to be solved. EOs encapsulation is a promising technique that offers numerous advantages, such as ease of handling, stability, protection against oxidation, improved distribution, solubility, controlled release, with little or no effect on the organoleptic properties of foods to which they are applied with improved bioavailability [
1,
16].
Mesoporous silica nanoparticles (MSNs) are a good system for encapsulating essential oils due to their porous structure, chemical stability, biocompatibility, adjustable pore size and porosity, simple and low-cost synthesis, and possible expansion for industrial use. Furthermore, silica is biologically inert, and can break down into relatively harmless silicic acid by-products [
17,
18].
The aim of this study was to evaluate the physicochemical, mechanical and barrier properties of sodium caseinate and chitosan films, added with mesoporous silica particles filled with oregano essential oil (OEO). In addition, this formulation was tested as coating of fresh Panela cheese and the microbiological and physicochemical changes were evaluated throughout 15-days storage period.
2. Materials and Methods
2.1. Materials
Sodium caseinate was purchased from Fonterra Group (Auckland, the Netherland), Chitosan Powder (90% deacetylation; viscosity 50–800 mPa·s) was obtained from Chemsavers (Bluefield, VA, USA). Anhydrous glycerol was purchased from J.T. Baker (Radnor, PA, USA); lactic acid (85%), cetyltrimethylammonium bromide (CTAB) and tetraethyl orthosilicate (98%) (TeOS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fresh oregano leaves (Lippia graveolens Kunth) were collected in Cerrito Parado, Tolimán (Querétaro, México). A voucher specimen was authenticated and deposited in the Ethno-botanical Collection of the Herbarium of Querétaro “Dr. Jerzy Rzedowski” (QMEX), located at the Faculty of Natural Sciences, University of Querétaro, Mexico (voucher specimen: E. Hernández-Hernández No. 1). Potato dextrose agar, plate count agar and casein peptone were acquired from BD Bioxon (Estado de Mexico, México).
All bacteria tested were obtained from the microbial collection of the Food Biotechnology Laboratory, DIPA, Universidad Autónoma de Querétaro (Mexico). Selected bacteria were those relevant in microbial contamination of fresh cheese, Listeria innocua and Salmonella Saintpaul. The strains were stored at −60 °C in sterile glycerol. The bacteria were activated in nutrient broth (Bioxon) at 37 °C for 24 h.
2.2. Extraction of Oregano Essential Oil (OEO)
The oregano leaves were air-dried at room temperature for 3 days in a dark chamber. The dry material was stored in black polyethylene bags until use [
19]. Dry oregano leaves (700 g) were suspended in 5 L of distilled water and subjected to hydrodistillation for 2 h, using a Clevenger-type apparatus (Cristalab, CDMX, México). The obtained OEO was dried with anhydrous sodium sulfate, sterilized by filtration using a Swinnex unit of 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA), and then stored in sealed vials protected from light at 4 °C for later use [
20].
2.3. MSN-OEO Preparation
The MSN were synthesized, as previously reported [
21], with modifications. In a solution of 240 mL of distilled water and 1.75 mL of 2 N NaOH, 0.5 g of CTAB was dispersed by using an ultrasonic bath at room temperature. The sample was heated up to 80 °C under constant stirring and then 2.5 mL of TeOS was added drop by drop during 5 min, and vigorously stirred for 2 h at 80 °C. The solid product was obtained by filtration, then washed with ethanol, and finally calcined at 500 °C for 5 h to remove the surfactant. The obtained MSNs (0.1 g) were dispersed into 2 mL of ethanol containing 0.1 g of the OEO. The final mixture was stirred overnight in a fume cupboard at room temperature to remove the ethanol.
The amount of OEO in the MSN-OEO particles was determined by suspending 5.0 mg of the samples in 10 mL of ethyl acetate, followed by centrifugation (IKA, Wilmington, NC, USA) at 10,000×
g for 10 min. The supernatant containing the released OEO was quantified from a calibration curve constructed with different OEO concentrations in ethyl acetate, and measuring absorbance at 270 nm using a spectrophotometer (UV-1800 Shimadzu, Kioto, Japan) [
22]. The released OEO was subtracted from the total OEO to obtain the amount of OEO in the MSN.
2.4. Preparation of SC:CH Film Forming Solution (FFS)
The SC solution was prepared at 0.90% (w/v) in distilled water and stirred at 40 °C for 30 min, then the pH was adjusted to 5.3. The solution of chitosan 1% (w/v) in lactic acid (1% v/v) was stirred for 60 min at 80 °C, once dissolved, the pH was adjusted to 5.8; each solution was kept under stirring until complete dissolution. To produce the FFS, SC (360 mg) was added to the CH at 4:1 and 8:1 (SC:CH w/w) ratios, under stirring at 700 rpm. The MSN-OEO was suspended in distilled water (20 mg/mL) at pH = 10 and added to the FFS (3% relative to the total mass of SC plus CH, w/w). Glycerol was added as plasticizer at 30% of SC (w/w), and the mixture was adjusted to pH 5.3, followed by gently stirring for 20 min. Finally, the FFS was homogenized using an Ultra-Turrax (IKA T25, Staufen, Germany) at 9500 rpm for 2 min.
2.5. Characterization of the FFSs and Films
2.5.1. ζ-Potential and Particle Size Measurement
One ml of each FFS was analyzed for ζ-potential and particle size by using a Zetasizer Nano-ZSP (Malvern Instrument, Worcestershire, UK). ζ-potential was calculated through the electrophoretic mobility by applying a voltage of 200 mV and using the Helmholtz-Smoluchowski equation. Hydrodynamic size was obtained from dynamic ligh scattering by using a He–Ne laser (wavelength of 633 nm) and a detector angle of 173°.
2.5.2. Film Preparation
Films were prepared by the casting method. Each FFS (45 mL) was poured into 8 cm diameter polyester Petri dishes and dried at 38 °C and 50% relative humidity (RH) in an environmental chamber (Binder, KBF 115, Tuttlingen, Germany), for 18 h. Film thickness was measured with a digital micrometer (Mitutoyo, 293–185, Kawasaki, Japan) at five random points over the film, and the mean ± standard deviation (SD) of five replicates was reported.
2.5.3. Mechanical Properties
Film tensile strength (TS), elongation at break (EB) and Young’s modulus (YM) were measured by using an Universal testing instrument (Instron Engineering, model No. 5543A, Norwood, MA, USA) as previously described [
23]. Film samples were cut, using a sharp razor blade, into 10 mm wide and 40 mm length strips equilibrated overnight at 50% ± 5% RH and 23 ± 2 °C in an environmental chamber. Five samples of each film type were tested.
2.5.4. Barrier Properties
Film permeabilities to gas (O2 and CO2) and water vapor (WV) were determined by using a MultiPerm apparatus (Extrasolution s.r.l., Pisa, Italy). Duplicate samples of each film were conditioned for 2 days at 50% RH before measurement. Aluminum masks were used to reduce the film test area to 5 cm2, whereas the testing was performed at 25 °C under 50% RH.
2.5.5. Solubility
The solubility of films was determined according to Vahedikia et al. [
24]. The initial dry matter of samples was determined by placing the films in a desiccator containing calcium sulfate at 25 °C and 0% relative humidity (RH) for 24 h. The films were then immersed in 50 mL distilled water and then placed in a shaker incubator at 25 °C and stirred for 24 h at 250 rpm. The film solubility (%) was calculated using Equation (1):
2.5.6. Scanning Electron Microscopy (SEM)
Microstructural analysis of cross-sections of the films was carried out by using SEM, in a JEOL JSM-5410 (Tokyo, Japan) electron microscope. Pieces of 5 mm × 5 mm were cut from films and mounted on copper stubs. Films were fixed on the support using double side adhesive tape. Samples were gold coated and observed using an accelerating voltage of 10 kV.
2.5.7. Color and Transparency
The color of the films was evaluated according to the CIELAB method, using a Minolta CR400 colorimeter (Minolta, Osaka, Japan) with the parameters L* = lightness, a* = green to red color component and b* = blue to yellow color component. The color was standardized using a white reference plate.
The transparency of films was determined according to Lin et al. [
6], with modifications. The edible film was cut into strips (1 cm × 4 cm), and the top and bottom edges of the specimen were fixed on the surface of a quartz cuvette with adhesive tape, and the transmittance at 600 nm was measured using a spectrophotometer. The transparency was calculated using Equation (2):
where: T = transparency; A = absorbance; δ = thickness.
2.5.8. Atomic Force Microscopy (AFM)
The surface roughness of the coating used on Panela cheese (SC:CH, 1:8 with and without SMN.OEO) was estimated by atomic force microscopy (AFM), according to Escamilla-García et al. [
25], by means of an atomic force microscope (di Multimode V, Veeco, Plainview, NY, USA) in contact mode. Film pieces of 0.5 cm × 0.5 cm were used, and the resonance frequency of scanning was 286–362 kHz with a spring constant of 20–80 N/m, scanning speed of 1 Hz and resolution of 256 × 256 pixels. The results were analyzed using the Gwyddion Version 2.53 software (Okružní, Czech Republic). The roughness was obtained by evaluating the square root of the deviation from an average plane of the peaks and surface valleys (Rq) (Equation (3). The mean absolute value of surface height deviations from the middle plane (Ra), was estimated from Equation (4).
where Ra and Rq indicate the roughness (nm), Zi and Zj are the height difference of i and j relative to the heights average, and N is the number of points on the image.
2.5.9. Antimicrobial Activity
Antimicrobial activity was evaluated following Hernández-Hernández et al. [
20]. Briefly, 10 mL of trypticase soy agar (0.8%
w/
v (Bioxon) was inoculated with 200 µL of
L. innocua solution (10
7 CFU/mL) or 100 µL of
S. Saintpaul (10
7 CFU/mL), subsequently poured onto plates containing solidified agar (1.5%
w/
v).
Disks were prepared from films of SC:CH at 8:1 ratio (w/w) with and without MSN-OEO of 25 mm in diameter. Additionally, PVDF membrane disks of 25 mm in diameter (Darmstadt, Germany) were impregnated with 8 mg of MSN-OEO or 75 µL of OEO diluted at 25% (w/v) with Tween 80 at 10% (v/v). One disk of each was gently placed on top of the soft agar layer and OEO were allowed to diffuse for 2 h, at 4 °C and then incubated at 37 °C for 48 h. The growth inhibition zone, which included the disk diameter, was measured using Vernier callipers.
2.6. Application as a Coating on Panela Cheese
The coating was applied on samples of Panela cheese using a spray gun (Husky, Lincoln, NE, USA) the samples were sprayed 2 times at 2 min interval. After the coating process, all samples were drained on stainless steel screens and air-dried in a laminar flow cabinet for 20 min, and then were put into plastic “clam-shell” containers (Industry standard, 9756Z, Pactiv Corp., Zapopan, México) and stored at 4 °C. The cheese was subjected to physicochemical and microbiological analyses at 0 day, 5 days, 10 days and 15 days of storage. Uncoated cheeses were used as control, and were stored and analyzed at the same times.
2.6.1. Moisture Content, pH and Titratable Acidity
The moisture content (MC) in Panela cheese was analyzed by moisture analyzer XM50 (Precisa Gravimetrics AG, Dietikon, Switzerland) by using 1 g of homogenized cheese, and moisture was expressed as weight %. Titratable acidity and pH were evaluated following the Mexican standard NOM-243-SSA1 [
26]. The pH was detected by using 1 g of cheese homogenized in 10 mL of distilled water, and from each sample, 3 pH measurements were taken using a calibrated potentiometer Orion (Star A211, Thermo Scientific, Waltham, MA, EUA). Titratable acidity was evaluated using 18 g of homogenized cheese in 36 mL of distilled water, followed by titration with 0.1 N NaOH until the appearance of a pink color for at least 30 s. The results are expressed as% lactic acid (
w/
w).
2.6.2. Microbiological Analysis
Molds and yeast, and mesophilic aerobic bacteria were quantified according to the Mexican standard [
26]. Panela cheese with and without coating at 0 day, 5 days, 10 days and 15 days of storage were analyzed in triplicate.
Molds and Yeasts
For the population of molds and yeasts the cheese was homogenized followed by serial decimal dilutions; then 1 mL of each dilution was poured into potato dextrose agar (Bioxon), previously adjusted to pH = 3.5 ± 0.1 by using 10% tartaric acid (J.T. Baker), and incubated at 25 ± 1 °C. Colonies (CFU/g) were evaluated after 3 days of incubation, and results were reported as Log10 CFU/g.
Mesophilic Aerobic Bacteria
The population of mesophilic aerobic bacteria of the cheese samples was performed using plate count agar, pouring 1 mL of decimal dilutions and incubating for 48 h at 35 °C. Results were reported as Log10 CFU/g.
2.7. Statistical Analysis
Experiments were made in triplicate, and analysis of variance (ANOVA) was performed using the JMP software 13.0 (SAS Institute, Cary, NC, USA). Tukey’s multiple range test was used, differences at p < 0.05 were considered significant and are indicated with different letters.