Essential Oils of Garlic and Oregano Incorporated in Cellulose Acetate Films : Antimicrobial Activity and Physical Properties

Polymers of natural origin and their derivatives are currently used as biomaterials because they are easily available and their properties can be tailored to meet specific requirements. The essential oils are widely used as antimicrobials.  The objective of this study was to evaluate the in-vitro antimicrobial efficiency of cellulose acetate (CA) films incorporated with the essential oils of garlic (GR) and oregano (OR) on the microorganisms Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Salmonella choleraesuis and Pseudomonas aeruginosa and characterize the films as to their mechanical, optical and structural properties. Four treatments were evaluated, Control, Film 1 (50 ml OR.100 g-1 CA) Film 2 (50 ml OR + 30 ml GR.100 g-1 CA) and Film 3 (50 ml OR + 50 ml GR.100 g-1 CA). The concentration of oils influenced the mechanical parameters of maximum load, relative deformation at maximum load and elastic modulus, resulting in weaker, less rigid and more flexible films. There was an increase in L* and b* in films incorporated with garlic and oregano essential oil. The films incorporated with a mixture of oregano and garlic essential oils exhibited inhibition against all organisms tested.


Film conditioning and thickness
All the films were stored at a controlled temperature of 23 ± 2 °C and 50 ± 5 % relative humidity for 48 h before analysis according to the (ASTM, 2000). The average film thickness was measured by reading at ten distinct points, randomly selected in each test body, using a Mitutoyo digital micrometer (0.01 mm precision, Mitutoyo Sul Americana, Brazil).

Scanning electron microscopy (SEM)
The Morphological characterization of films was performed by Scanning Electronic Microscopy (SEM) on equipment HITACHI TM 3000 (Japan). Three 1 cm diameter samples of each treatment were assembled in aluminum stubs with double carbon tape and metalized in a gold evaporator. Pictures were obtained with an acceleration voltage of 20 kV.

Mechanical properties
The films were subjected to mechanical tests according to the ASTM (2002) method using an Instron Universal Testing machine model 3367 (Instron Corporation, USA). For tensile tests, maximum load (N), relative deformation at maximum load (%) and elastic modulus (MPa) were evaluated. The test bodies were cut into strips (150 x 25mm) with an initial separation between grips of 100mm, 1kN load cell and a velocity of 50 mm/min.

Optical Properties
The opacity of the films was determined using a Cary 50 UV -Visible spectrophotometer (Varian, Australia) (Giménez et al., 2009). The films were cut (3x3 cm) Research, Society and Development, v. 9, n. 10, e329108304, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8304 7 and a control film was used as the reference in the measurements. The absorbance spectra of the films were recorded at 600 nm and the opacity calculated by Equation 1:

Opacity
(1) where, Abs600 is absorbance value at 600 nm, and X is film thickness (mm).

In-vitro antimicrobial activity of the films
Mueller-Hinton agar was prepared and sterilized according to the instructions of the manufacturer. In a laminar flow hood, Mueller-Hinton agar was poured into 9 cm internal diameter Petri dishes with a volume from 25 to 30 ml. The lids of the plates were ajar to prevent moisture formation on their inner surface. After solidification, all plates were covered and placed in an incubator 35 °C for 24 hours.

Statistical Analyses
The results were analyzed in the SISVAR software (Ferreira, 2014) employing the Univariate Statistical Analysis (ANOVA) and Tukey test considering 5% significance level.

Scanning electron microscopy (SEM)
In general, cellulose acetate films incorporated with the essential oils of oregano and garlic presented insoluble points of dispersion ( Figure 1). In Figure 1A and 1B cellulose, acetate agglomerates can be observed. Figure 1B shows points that possibly refer to globules of oregano essential oil that were not fully dispersed in the film matrix. Figures 1C and 1D show an alteration in the film matrix, Research, Society and Development, v. 9, n. 10, e329108304, 2020 (CC BY 4

Mechanical Properties
Film thickness did not differ significantly (p > 0.05), with a mean value of 40 µm ± 3. The variation of the oil concentration factor significantly (p ≤ 0.05) influenced the parameters maximum load, relative deformation upon maximum load, and elastic modulus ( Table 2). The control film showed a higher maximum load than the active films, i.e. with the addition of essential oils, the films became less resistant. In contrast, there was an increase in the relative deformation upon maximum load. This indicates that the addition of oils provided the film with a higher stretch capacity compared to the control. It was observed that the treatment with the mixture of oils resulted in decreased stiffness (p = 0.05) and control treatments and Film 1 have significantly the same stiffness. Research, Society and Development, v. 9, n. 10, e329108304, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8304 Table 2. Maximum load (ML), relative deformation at maximum load (D) and elastic modulus (EM) of the films.
The change in the film matrix with lipid agglomerates and porosity was observed in the micrographs (Figure 1), which may have affected the mechanical properties of the films. As

Optical Properties
The visual aspect is related to the color and transparency of the films, attributes that impact the appearance of the packaging, and product acceptability by the consumer. The active films showed a sharp increase in opacity and the control film presented the highest transparency (Table 3). In addition, it was observed that opacity increased with increased essential oil concentrations. Yang and Paulson (2000), Han Lyn and Nur Hanani (2020), and Shojaee-Aliabadi et al. (2013) report that increased film opacity arises from the dispersion of light produced by the lipid droplets dispersed in the emulsion, causing a milky/whitish film appearance. Thus, the different opacities observed in Table 3 can be justified by the presence of non-miscible material, forming heterogeneous films, since the hydrophobicity of essential Research, Society and Development, v. 9, n. 10, e329108304, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8304 oils can hinder their interaction with the CA matrix, as also evidenced in the previous analyzes. Table 3. Opacity, color parameters (L*, a*, b*) and the total color difference (ΔE) of the films.