3.1 Physical properties of the films
Table 1 shows the water-related properties of the films. The moisture contents varied from 7.03–7.89%, with no significant difference arising from the incorporation of the CNCs, MEO, and CEO. As EOs are hydrophobic in nature, it was expected that the moisture content of the films would be reduced. However, this was not observed in the present work, probably as a result of the low concentrations of EOs tested.
Table 1
Water-related properties of arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils
Film | Moisture content (%) | Solubility (%) | WVP (10− 7 g H2O∙m− 1∙h− 1∙Pa− 1) |
AA/CWN | 7.89 ± 1.88 | 26.41 ± 2.93a | 3.98 ± 0.25a |
AA/CWN/CNC | 7.42 ± 1.86 | 18.74 ± 2.33b | 3.21 ± 0.12b |
AA/CWN/CNC/MEO1* | 7.03 ± 0.91 | 16.62 ± 2.61b | 3.28 ± 0.15b |
AA/CWN/CNC/MEO2 | 7.26 ± 1.16 | 14.23 ± 3.41bc | 3.15 ± 0.18b |
AA/CWN/CNC/MEO3 | 7.45 ± 1.45 | 12.10 ± 3.44c | 2.92 ± 0.60b |
AA/CWN/CNC/CEO1 | 7.09 ± 1.71 | 17.45 ± 2.43b | 3.11 ± 0.26b |
AA/CWN/CNC/CEO2 | 7.87 ± 2.57 | 14.92 ± 1.14bc | 3.06 ± 0.38b |
AA/CWN/CNC/CEO3 | 7.54 ± 0.70 | 12.60 ± 1.23c | 2.97 ± 0.20b |
AA: arrowroot starch; CWN: carnauba wax nanoemulsion; CNC, cellulose nanocrystals; MEO: Mentha spica essential oil; CEO: Cymbopogon martini essential oil; WVP: water vapor permeability. |
*Essential oils were added at the concentrations of 0.1% (MEO1 or CEO1), 0.2% (MEO2 or CEO2), or 0.3% (MEO3 or CEO3). |
Values in the same column followed by at least one common letter (or not followed by any letters) are not significantly different according to the Tukey test (p < 0.05). |
The solubility of the films in water varied from 12.1–26.4%, decreasing significantly with the addition of CNCs and increasing concentrations of either of the EOs. The reduction in water solubility of the AA-based films following the incorporation of CNCs was a result of the formation of a three-dimensional (3D) cellulose network through hydrogen bonding between the starch and CNC molecules. Three-dimensional networks reduce the solubility of biopolymers, reinforcing the structure and restricting the interactions between the polymer and water molecules (Noshirvani et al. 2018).
The reduction in water solubility of the films by the addition of the EOs was probably due to the hydrophobic nature of these molecules and their low affinity to water molecules (Ma and Wang 2016). The same observation has been reported for starch films incorporated with Syzygium aromaticum EO (Sousa et al. 2019) and chitosan films incorporated with Citrus limonia EO (Oliveira Filho et al. 2020b).
The addition of CNCs to the AA/CWN film reduced its WVP (from 3.98 to 3.21 10− 7 g H2O∙m− 1∙h− 1∙Pa− 1), similar to results reported in the literature (Abdollahi et al. 2013; Pereda et al. 2014; Sogut 2020). According to El Miri et al. (2015), CNCs limit the mobility of water molecules through the film matrix, resulting in a reduction in the WVP of the nanocomposite film.
The incorporation of the various concentrations of CEO or MEO did not alter the WVP of the films. This was similar to the results reported for whey protein films incorporated with oregano EO (Zinoviadou et al. 2009) and AA-based films incorporated with Piper aduncum EO (Valadares et al. 2020).
The addition of EOs was expected to reduce the WVP of the films owing to the hydrophobic nature of the oils, as previously observed for the water solubility property (Table 1). However, because WVP is a function of both solubility and diffusivity (Santos et al. 2014), the lack of a significant variation in the WVP may be due to a concomitant increase in water molecule diffusivity resulting from discontinuities in the matrix caused by the incorporated EO molecules (as shown by the scanning electron micrographs discussed below).
3.2 Tensile properties of the films
The film thickness increased significantly with the addition of CNCs, CEO, and MEO, similar to the results reported for films composed of whey protein isolate, CNCs, and bergamot EO (Sogut 2020) and those made up of chitosan, CNCs, and palm oil (Pereda et al. 2014). The increase in thickness of the films may be related to the increase in the amount of solids present in the nanocomposites (de Souza Coelho et al. 2020), with differences in homogeneity within the biopolymer matrices, and could also be due to interactions between the components used in the formulation of the nanocomposites (Sogut 2020).
The stress properties of the films are listed in Table 2. The incorporation of CNCs increased the tensile strength of the AA/CWN film from 3.0 to 5.3 MPa. This increase can be due to interactions between the CNCs and starch molecules and the reinforcement effect from voltage transference at the CNC–starch interface (de Mesquita et al. 2010; Khan et al. 2012). These interactions strengthen the 3D network of the nanocomposite film by creating nanofillers, which improve the mechanical properties of the film and limit the movement of the biopolymer chains (Jouyandeh et al. 2019).
Table 2
Tensile properties and thermal properties of arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils
Film | Thickness (mm) | Tensile strength (MPa) | Elongation at break (%) | Tonset (°C) | Tmax (°C) |
AA/CWN | 0.134 ± 0.022c | 3.00 ± 0.60c | 247.5 ± 32.8a | 272.5 | 314.3 |
AA/CWN/CNC | 0.147 ± 0.310b | 5.30 ± 0.68a | 125.7 ± 42.1b | 271.8 | 313.4 |
AA/CWN/CNC/MEO1 | 0.166 ± 0.040a | 4.25 ± 0.40b | 123.6 ± 39.9b | 276.9 | 316.4 |
AA/CWN/CNC/MEO2 | 0.182 ± 0.140a | 4.17 ± 0.53b | 122.5 ± 23.1b | 280.9 | 317.5 |
AA/CWN/CNC/MEO3 | 0.188 ± 0.007a | 4.06 ± 0.35b | 117.3 ± 47.7b | 293.1 | 317.8 |
AA/CWN/CNC/CEO1 | 0.168 ± 0.017a | 4.28 ± 0.49b | 133.2 ± 61.9b | 275.8 | 315.8 |
AA/CWN/CNC/CEO2 | 0.162 ± 0.034a | 4.07 ± 0.29b | 118.1 ± 39.3b | 278.9 | 316.4 |
AA/CWN/CNC/CEO3 | 0.173 ± 0.006a | 4.23 ± 0.69b | 106.7 ± 7.73b | 286.8 | 317.3 |
AA: arrowroot starch; CWN: carnauba wax nanoemulsion; CNC, cellulose nanocrystals; MEO: Mentha spica essential oil; CEO: Cymbopogon martini essential oil; Tonset: starting decomposition temperature; Tmax: maximum decomposition temperature. |
*Essential oils were added at the concentrations of 0.1% (MEO1 or CEO1), 0.2% (MEO2 or CEO2), or 0.3% (MEO3 or CEO3). |
Values of thickness, tensile strength, and elongation at break in the same column followed by at least one common letter are not significantly different according to the Tukey test (p < 0.05). |
The tensile strength of the films with EOs incorporated was lower than that of the AA/CWN/CNC film. However, all films were superior in tensile strength to the control film (AA/CWN without CNCs and EOs). The changes in the mechanical properties were likely due to the presence of discontinuities in the polymer matrix caused by the EO molecules (Atarés and Chiralt 2016), corroborating the film structures seen in the scanning electron micrographs and the theory of an increase in diffusivity as being responsible for the non-reduction in WVP (Table 1). Thus, EOs increase the extensibility, flexibility, and mobility of films and decrease their cohesive strength (Mahcene et al. 2020). Whereas the elongation at break was significantly reduced from 247–125.7% with the addition of CNCs, it was not affected by the addition of EOs. The CNC-mediated increase in tensile strength and reduction in elongation at break of the films have also been reported by other authors (Dai et al. 2020; de Souza Coelho et al. 2020; Yadav et al. 2016).
3.3 Thermogravimetric analysis
Figure 1 shows the TGA curves and their first derivatives. The starting and maximum decomposition temperatures (Tonset and Tmax) are shown in Table 2. Weight loss of the samples occurred in three major stages. The first stage occurred during the temperature range of 25–250°C and was related to the evaporation of water molecules (52–92°C), glycerol (200–250°C), and other volatile low-molecular-weight components. The second stage occurred between 270 and 350°C and was related to the thermal degradation of starch and CNCs, which occur at similar temperatures (Rico et al. 2016). The last stage occurred from 350 to 490°C (Freitas et al. 2016; Milanovic 2010) and was caused by the degradation of CWN and thermally stable compounds present in the EOs (Alizadeh et al. 2017; Sousa et al. 2019).
The films with MEO and CEO incorporated showed higher Tmax values than the other films, indicating that the addition of these oils had improved the thermal stability of the nanocomposites (Table 2). These results were corroborated by other studies that showed that the improvement in thermal properties led to the higher homogeneity observed in the biopolymer matrix (Noshirvani et al. 2017; Sousa et al. 2019).
3.4 Optical properties
The optical properties of the films are listed in Table 3. The incorporation of EOs and CNCs did not change the L* parameter (luminosity) of the films. Moreover, the hue values ranged from 89.34° to 90.30° (between red and yellow), indicating that the films were yellow in color (Table 3).
Table 3
Color attributes and opacity of arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils
Film | L* | h° | C* | Opacity |
AA/CWN | 80.49 ± 0.99 | 89.88 ± 0.67 | 16.96 ± 1.85a | 1.22 ± 0.01h |
AA/CWN/CNC | 81.89 ± 0.27 | 89.53 ± 0.59 | 13.75 ± 0.22b | 2.01 ± 0.03g |
AA/CWN/CNC/MEO1 | 81.03 ± 0.91 | 89.52 ± 0.31 | 14.32 ± 2.09b | 2.54 ± 0.02d |
AA/CWN/CNC/MEO2 | 81.53 ± 0.83 | 90.11 ± 0.46 | 15.15 ± 0.76a | 2.63 ± 0.02c |
AA/CWN/CNC/MEO3 | 81.31 ± 0.22 | 89.34 ± 0.19 | 16.46 ± 0.20a | 2.70 ± 0.01b |
AA/CWN/CNC/CEO1 | 81.92 ± 0.60 | 90.23 ± 0.41 | 13.74 ± 1.63b | 2.27 ± 0.02f |
AA/CWN/CNC/CEO2 | 81.58 ± 0.34 | 90.21 ± 0.46 | 15.91 ± 1.31a | 2.45 ± 0.03e |
AA/CWN/CNC/CEO3 | 81.58 ± 0.28 | 90.30 ± 0.25 | 16.07 ± 0.65a | 2.99 ± 0.01a |
AA: arrowroot starch; CWN: carnauba wax nanoemulsion; CNC, cellulose nanocrystals; MEO: Mentha spica essential oil; CEO: Cymbopogon martini essential oil; L*: luminosity; h°: hue angle; C*: chroma. |
*Essential oils were added at the concentrations of 0.1% (MEO1 or CEO1), 0.2% (MEO2 or CEO2), or 0.3% (MEO3 or CEO3). |
Values in the same column followed by at least one common letter (or not followed by any letters) are not significantly different according to the Tukey test (p < 0.05). |
The C* value decreased with the addition of CNCs, indicating that the nanocrystals lowered the color intensity of the films. By contrast, the value increased with the addition of 0.2% and 0.3% EOs, indicating that the oils made the film coloring more intense. Similar phenomena have been reported for agar films (Shankar et al. 2015) and starch films (de Souza Coelho et al. 2020). In another study, the yellowish coloration of corn and wheat starch films was attributed to the lemon EO added (Song et al. 2018).
The addition of CWN, CNCs, and EOs decreased the transparency of the AA-based films. The opacity values increased from the AA/CWN film to the AA/CWN/CNC/MEO3 and AA/CWN/CNC/CEO3 films (1.22 to 2.70 and 2.99, respectively). This increase may be due to the strong interaction between the CNCs and the starch matrix as well as light scattering by the nanocrystals (Li et al. 2018).
The increased opacity could also be due to the hinderance of light passage through the film as a result of CNC accumulation within the matrix (Abdollahi et al. 2013), as evidenced by the CNC aggregates observed in the scanning electron micrographs (Fig. 2). Similar results have been reported by other authors (de Souza Coelho et al. 2020; Thomas et al. 2020). Meanwhile, the decrease in film transparency caused by the addition of EOs was probably due to the dispersion of light by the oil droplets in the film matrix, as previously described for other films (Oliveira Filho et al. 2019; Sousa et al. 2019).
Figure 3 shows the light transmission rate of the films. All of the fabricated films were found to be strong barriers against UV light (200–350 nm) not exceeding 0.1%; that is, they provided a 100% barrier to UV light. In the visible light region (380–780 nm), the light transmission rate of the control film (AA/CWN) was 24.0–65.4%, but this decreased to a range of 26.0–55.9% in the AA/CWN/CNC film. The addition of EOs also caused a slight reduction in light transmittance rates compared with that of the AA/CWN/CNC film (Fig. 3). The best light barrier performance was observed for films incorporated with CNCs and EOs at the highest concentration (0.3%), which was due to the increased opacity (reported in Table 3). Reductions in the UV–Vis light transmission rate have also been observed for chitosan films incorporated with Citrus limonia EO (Oliveira Filho et al. 2020b) and potato starch films incorporated with CNCs (Oliveira et al. 2017). Therefore, AA/CWN/CNC films with MEO or CEO can be used as food packaging materials, as they have excellent light barrier function.
3.5 Characterization of the film microstructures
Figure 2 shows the surface and cross-sectional microstructures of the AA-based films containing CWN, reinforced with CNCs, and supplemented with MEO or CEO. The AA/CWN film had a dense and regular surface with some lipid clusters present. In the film containing CNCs, the surface was rougher and more opaque, which was attributed to the nanocrystal aggregates, as observed in other studies on starch films containing CNCs (Johar and Ahmad 2012; Silva et al. 2019). None of the films had obvious cracks or discontinuities in their microstructures, and the addition of CNCs positively impacted the traction and barrier properties of the films (Tables 1–3).
As shown in Fig. 2, compared with the AA/CWN/CNC film, the films with EOs had lower amounts of CNC aggregates and regular and compact structures, probably as a result of the uniform distribution of the droplets within the emulsion and good compatibility between the matrices. These characteristics indicated that the emulsion was stable and there was no phase separation or droplet aggregation during the preparation and drying of the films. This may have been due to interactions between the CNCs and EOs that electrostatically stabilized the oil droplets, giving rise to Pickering emulsions (Zhang et al. 2017; Zhou et al. 2018).
With higher concentrations of EOs in the films, the microstructure of the cross-sections was slightly heterogeneous and less compact, with some holes in the films (especially with 0.3% EOs, as a result of the oil droplets (Pastor et al. 2013; Zhou et al. 2018)). The characteristics of oil droplets in a Pickering emulsion can affect the immobilization of the emulsion (Ribeiro-Santos et al. 2017). Overall, the findings of the film microstructure corroborated the results previously described for the properties that were improved with the addition of CNCs and EOs.
3.6 Antifungal activity
Table 4 shows the antifungal activities of the films against R. stolonifer and B. cinerea. As expected, the AA/CWN and AA/CWN/CNC films did not show antifungal activity against the two fungi studied, corroborating previous results obtained with films based on starch, waxes, and nanocellulose (Ochoa et al. 2017; Raigond et al. 2019; Salmieri et al. 2014).
Table 4
Antifungal activity of arrowroot-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils
Film | Diameter of inhibition zone (mm) |
Rhizopus stolonifer | Botrytis cinerea |
AA/CWN | 0.0 ± 0.0e | 0.0 ± 0.0f |
AA/CWN/CNC | 0.0 ± 0.0e | 0.0 ± 0.0f |
AA/CWN/CNC/MEO1 | 16.0 ± 1.0d | 19.0 ± 0.7e |
AA/CWN/CNC/MEO2 | 20.8 ± 1.1c | 25.9 ± 2.0d |
AA/CWN/CNC/MEO3 | 25.2 ± 2.0b | 33.7 ± 1.6b |
AA/CWN/CNC/CEO1 | 18.7 ± 0.7c | 24.3 ± 0.8d |
AA/CWN/CNC/CEO2 | 24.1 ± 1.6b | 30.4 ± 0.6c |
AA/CWN/CNC/CEO3 | 29.8 ± 0.8a | 36.3 ± 0.7a |
AA: arrowroot starch; CWN: carnauba wax nanoemulsion; CNC, cellulose nanocrystals; MEO: Mentha spica essential oil; CEO: Cymbopogon martini essential oil. |
*Essential oils were added at the concentrations of 0.1% (MEO1 or CEO1), 0.2% (MEO2 or CEO2), or 0.3% (MEO3 or CEO3). |
Values in the same column followed by at least one common letter are not significantly different according to the Tukey test (p < 0.05). |
By contast, the films with EOs incorporated showed obvious antifungal activity that was directly proportional to the EO concentration used. The diameters of the inhibition zones against R. stolonifer increased from 16.0 to 25.2 mm for films with MEO and from 18.7 to 29.8 mm for films with CEO. Those against B. cinerea increased from 19.0 to 33.7 mm for films with MEO and from 24.3 to 29.8 mm for films with CEO. Thus, B. cinerea was more sensitive than R. stolonifer to the EOs studied. The antifungal effect of MEO is related to its chemical composition, mainly of carvone, which has high antimicrobial activity (Soković et al. 2009). By contrast, the antifungal activity of CEO is related to the synergistic effects of its major compounds: geraniol, linalool, neral, and mirceno (da Rocha Neto et al. 2019). Taken together, these results confirmed that MEO and CEO act as antifungal agents. Therefore, these EOs in combination with a film composed of AA, CWN, and CNCs provide a functional film material.