Materials
Plantago major seeds and fenugreek seeds were bought from the local market in Urmia, Iran. 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), CeO2 NPs, CMC powder, and glycerol were purchased from Sigma Aldrich, USA. Distilled water and other chemical and reagents were analytical grades purchased from representatives of reputable companies in Tehran, Iran.
Preparation Of Nanocomposite Films
The casting method was used to prepare PMSG-CMC nanocomposite films containing FSEO and CeO2 NPs. First, 0.5 grams of Plantago major powder was dissolved in 50 mL of distilled water at a temperature of 80°C. Then, 1 gram of carboxymethyl cellulose was dissolved separately in 50 mL of distilled water at room temperature. The resulting solutions were combined and stirred for 1 hour. Then cerium oxide nanoparticles were added and mixing was done again for 30 minutes. After the temperature of the solution dropped to 35 degrees, fenugreek plant essential oil and finally glycerol were added to the solution. Finally, 50 mL of film solutions were poured into 8 cm plates and dried at room temperature. The control film was made using only carboxymethyl cellulose and Plantago major in the same conditions. Subsequent to drying, the film was taken out from the plate surface and assessed.
Characterization And Properties Of The Film
Moisture content
The moisture content of the nanocomposite films was evaluated by the oven-drying method. The weight of the nanocomposite films was calculated before and after drying in a conventional oven. The samples remained at 110 ºC for 24 h. They were determined in triplicate, and the results were reported in percentage [24].
Moisture Absorption
Moisture absorption of the nanocomposite films was evaluated based on the Sadeghnezhad et al. method [25]. Initially, pieces of the dried nanocomposite films (20\(\times 20\)mm) were kept in an oven at 50°C for 24h and weighed. Then, they were put in the desiccator comprising K2SO4 solution with RH 97% at 20–25°C. Then, the weight of the samples was measured at proper time intervals until it reached a constant weight. Ultimately, the moisture absorption was measured by the following equation:
$$Moisture absorption=\frac{{W}_{f}-{W}_{0}}{{W}_{0}}\times 100$$
1
Where Wf is the weight of the final weight of the sample, and W0 is the initial weight of the sample.
Water Vapor Permeability (WVP)
To calculate the WVP of the samples, the water vapor transmission rate (WVTR) should be determined first. WVTR was measured by the standard cup method ASTM (1995) with some modifications. At first, 8 g silica gel was filled into the special aluminum cups, then slightly larger pieces (than the diameter of the cups) of films were sealed in the cup. Then 3 g water-free CaSO4 was added to each cup. Using water-free CaSO4 led to zero relative humidity in the cups. The cups were put in the desiccator with K2SO4 saturated solution. K2SO4 produces a constant relative humidity of 97%. The desiccator was kept inside the oven at 25°C for the whole research period. The weight of the samples was measured every 1 hour for 5 days. The WVTR was calculated by the following equation where the slope was the slope of the resulting line of the weight gain curve over time determining the linear regression, A (cm2) was the area exposed in the cup:
$$WVTR \left(g {day}^{-1}{mm}^{-2}\right)=\frac{\text{s}\text{l}\text{o}\text{p}\text{e}}{ \text{A}}$$
2
Afterward, the WVP of the films was calculated by the following equation:
$$WVP=(WVTR \times L)/\varDelta p$$
3
Where L\(=\) the thickness of the film (m), ∆p= water vapor pressure difference through the film [26, 27].
Opacity
The nanocomposite films were cut into rectangular pieces (2\(\times 2\)mm) and placed on the top of the cuvette. The absorbance of the empty cuvette and the samples were recorded at 600 nm by applying a UV-visible spectrophotometer (Model 80-2088-64, Pharmacia LKB Biochrom, Cambridge, UK). The opacity content of the films was calculated by the following equation [28, 29]:
$$Opacity=\frac{Abc600}{X}$$
4
where Abc600 is the absorbance value at 600 nm, and X represents the film thickness (mm).
Antioxidant Activity
According to Amiri et al. [30] 0.25 g of each film sample was added to the 4 mL pure water and vibrated for 5 min, later the mixture was added to 1 mL DPPH solution and incubated at room temperature for 30 min and calculated the absorbance at 517 nm. Free radicals scavenging activity (FRSA) was calculated as follows:
$$FRSA=\frac{{Abs}_{control}-{Abs}_{sample}}{{Abs}_{control}}\times 100$$
5
Antimicrobial Test
Amiri et al. [31] method was used to assess the antibacterial activity of the films. Disk diffusion method was utilized, and Escherichia coli PTCC1763 (E. coli) and Staphylococcus aureus PTCC 1431 (S. aureus) were recognized. 20 mL tryptic soy broth was used to inoculate these bacterial strains. They were incubated for 37 h at 37 °C. Next, cultured microorganisms were put in the centrifuge for 10 min. Then, a ten-fold dilution of uncultivated tryptic soy broth was performed. 2\(\times 2\)cm2 pieces of film samples were prepared and put on a particular plate of Muller Hinton Agar which was previously inoculated with 0.1 mL of 105-106 CFU/mL pathogens. Afterward, the plates were incubated for 24 h at 37 °C, and the inhibition zone areas around the films were measured by a digital caliper.
Mechanical Properties
The thickness of the biofilms was determined by applying a digital micrometer. Measurements were performed in five different places for each film and reported their average value [32]. Tensile Strength (TS) and elongation at break (EB) were determined using an Instron Universal Testing Machine (TA. XT plus texture analyzer, Stable Microsystems, UK) according to the standard (ASTM19 D882). The procedure was performed with 100 mm/min crosshead speed and 20 mm initial grip separation. For measurement of the mechanical properties, the samples were cut into rectangular strips (\(0.5\times 0.5 cm\)). The TS and EB were calculated by the following equations:
$$TS\left(MPa\right)=\frac{{F}_{max}}{A}$$
6
$$EB\left(\%\right)=\frac{\varDelta L}{{l}_{0}}\times 100$$
7
Fourier-transform Infrared (FTIR) Spectroscopy
FTIR is a molecular oscillation spectroscopy to evaluate molecular structure and composite composition. The prepared films were utilized directly for FTIR analysis. They were recorded with (Spectrum two, Perkin Elmer, USA) at the 400–4000 cm− 1 wavenumber with the resolution of 4 cm− 1 [33].
Thermogravimetric Analysis (TGA)
The thermal stability of the biofilms (10–15 mg) was estimated by the thermo-gravimetric analyzer (Linseis model Sta Pt-1000 Germany) by heating from 25 °C to 700 °C under the nitrogen flow condition (10 °C/min). The heating rate was set at 60 mL/min [26].
X-ray Diffraction (XRD) Analysis
The structure of extended crystalline biofilms was determined by an X-ray diffractometer instrument (Siemens, model D 5005, Baden Wurttemberg, Germany). The setting of the generator of the X-rays was 40 kV and 40 mA. XRD patterns were achieved at 2\(\theta =10-70 ℃\) with a scan rate of 1 degree per minute [26].
Field Emission Scanning Electron Microscopy (FESEM)
A field emission scanning electron microscope (XL30 ESEM-FEG, Philip, Netherland) was used to observe film surface, broken surface, cross-section, and nanocomposites performing at different magnifications. Initially, the samples were coated with gold nanoparticles before observations [25].
Statistical analysis
Statistical analyses were performed based on a completely randomized design using a one-way analysis of variance (ANOVA) procedure followed by Tukey’s test with Minitab software version 20 (Minitab Inc., State College, PA, USA) at first error α\(\le\)0.05 to determine significant differences. The experimental design is shown in Table 1.
Table 1
Samples with different concentrations of CeO2 NPs and FSEO and abbreviations
Samples
|
Abbreviation
|
CeO2 NPs (%W/W)
|
FSEO (%W/W)
|
1
|
NPs 0/EO 0
|
0
|
0
|
2
|
NPs 0/EO 4
|
0
|
4
|
3
|
NPs 0/EO 8
|
0
|
8
|
4
|
NPs 2.5/EO 4
|
2.5
|
4
|
5
|
NPs 2.5/EO 8
|
2.5
|
8
|
6
|
NPs 5/EO 4
|
5
|
4
|
7
|
NPs 5/EO 8
|
5
|
8
|
8
|
NPs 2.5/EO 0
|
2.5
|
0
|
9
|
NPs 5/EO 0
|
5
|
0
|
CeO2 NPs: Cerium oxide nanoparticles and FSEO: Fenugreek seeds essential oil |