2.1. Color, Transparency, and Optical Properties
The effect of the added essential oils on the transparency, chromaticity, and refractive index of the edible films prepared from a chitosan–zein 50:50% blend are presented in Table 1
. Adding the EOs into the chitosan films significantly affected (p
< 0.05) the a* (redness/greenness) and b* (yellowness/blueness) values of the films’ surface. When EFs were incorporated with OR and CN, the a* values showed 27% and 6% more negative values, indicating a redness tendency, whereas the EF with AN added showed the highest a* value among the treatments (greenness tendency). All EFs incorporating EOs increased their b* values, indicating a yellowness tendency. Polysaccharide films are usually colorless, essential oils show a slightly yellow appearance, and the incorporation of EOs into polysaccharide-based EFs have been reported to affect color and transparency [4
AS showed the highest transparency value and the lowest refractive index of all of the EOs incorporated into an EF. Nevertheless, all edible films presented a refractive index between 1.35 and 1.55, and because they fall in the range 1.35–1.70 they are classified as transparent [15
]. The refractive index of EFs incorporating CN or OR showed similar values compared to the EFs without an EO. However, the refractive index of the EF incorporating AS was significantly different (p
< 0.05) from the values of the EF without an EO, and the EF with CN. Nevertheless, even small changes in the refractive index indicate that EO addition led to structural changes [16
]. The visual properties of a film are an important factor for consumer acceptability.
2.2. Physical Properties
EF thickness (e) and filmogenic suspension density (ρ) changed with EO addition, achieving higher thickness values as compared to the control sample (Table 2
). This effect can be partly related to the filmogenic suspension density, because a higher density was determined within these samples. In this regard, Bonilla et al. [17
] working with basil reported that the higher density of a filmogenic suspension containing an EO was due to larger molecular contact between the chitosan’s CH groups and the oil compounds, weakening the polymer chain aggregation forces, and producing a more open matrix leading to a higher film thickness.
The film prepared without an EO tended to show higher surface roughness than the others; this could be due to the formation of Z agglomerates, resulting from hydrogen or disulfide bonding and hydrophobic interactions promoted by the pH at which the films’ suspensions were prepared. This effect was noted by Escamilla et al. [18
] when working with EFs without EO addition, and was confirmed by Guo et al. [19
The addition of EOs could reduce such interactions, decreasing agglomerates’ formation and promoting a smoother surface. The roughness (Ra, Rq) of the EF decreased with the addition of an EO, in agreement with Atarés et al. [20
]. These authors worked with films made from soy protein isolate with added ginger and cinnamon EOs and reported that heating enables the integration of the EOs into the protein matrix, resulting in smoother surfaces.
The addition of EOs improved the water vapor barrier property of the chitosan-zein (CT-Z) EF, achieving permeability of 1.2, 1.5, and 1.6 g mm h−1
for AS, OR, and CN, respectively (Table 2
). Aguirre et al. [21
], working with whey protein isolate and the oregano EO, suggested a protein–EO interaction that immobilized the protein chain, producing a more ordered and tightly crosslinked structure, and consequently a lower permeability. On the other hand, the interaction of EO components such as ethers, ketones, and aldehydes with the OH groups of polymers increased the EFs’ hydrophobicity, thereby improving the water vapor barrier property [22
Another study produced sodium caseinate films incorporating the cinnamon or ginger EOs, and the cinnamon EO was homogenously distributed in the protein matrix, whereas the ginger oil droplets showed agglomeration [20
]. These authors concluded that structural differences linked to the oil type were the result of complex interactions taking place among lipids, proteins, and solvents. Another study reported that water vapor permeability (WVP) depends on different structural factors, such as the kind of matrix, the composition and amount of oil added, interactions with the polymers, and the hydrophilic–hydrophobic balance in the matrix [23
Nevertheless, one report concluded that a decrease in the WVP of chitosan (CT)-based films with a lemongrass, thyme, or cinnamon EO added was due to an increase in the tortuosity factor of the water transfer within the film matrix [24
]. Bonilla et al. [17
] reported a decrease in the WVP of CT films with an increasing concentration of the basil EO, suggesting that the water molecules’ diffusivity decreased because of the hydrophobic nature of the EOs that predominated over the cohesion forces of the CT matrix.
The addition of EOs decreased the elastic modulus and increased the hardness of the EF without an EO. All EFs incorporating an EO did not show a significant difference in the elastic modulus, whereas the AS EF exhibited the highest hardness value (Table 3
). The addition of lemongrass, rosemary pepper, and basil EOs decreased the elastic modulus of a cellulose-based EF, suggesting that the interactions of EF polymers with an EO were similar to those exerted by plasticizers [25
]. An EF made from fish skin gelatin with added CN, basil, plai, and lemon EOs showed a decreased elastic modulus, but a larger elongation at break due to the replacement of protein–protein interactions by EO addition in the film network [26
]. This report agrees with that of Zinoviadou [26
], who worked with whey protein with the oregano EO added. In this regard, the three-dimensional structure of proteins stabilized by hydrogen and disulfide bonds should be disrupted to obtain separate, entangled macromolecules to achieve plastic-like properties. The EO and other plasticizers are able to reduce the inter- and intra-molecular interactions and increase films’ flexibility depending on the oil and protein compatibility [20
2.3. X-ray Diffraction
To confirm structural changes in an EF matrix, X-ray diffraction and Raman spectroscopy analyses were conducted. EFs prepared without EO addition showed two well-defined peaks, one at 7.5° and the other at 20° (2θ); however, regarding EO-containing films, two peaks, one at 2.7° and another at about 10° (2θ), can be noted.
Based on Equation (14), AS addition promoted higher crystallinity than the other EOs added to the EFs, which was probably associated with higher components miscibility in the films’ matrix [27
]. On the other hand, OR added to an EF barely decreased the crystallinity (% C
: 21.0 ± 0.4 vs. 22.4 ± 0.5) (Figure 1
). According to Sánchez-Gonzalez et al. [28
], an EO added to an EF based on CT only increased the crystallinity up to 50%, whereas glycerol increases resulted in a lower EF crystallinity due to the higher mobility of the polymer chains [27
Thus, it is suggested that the presence of OR leads to a decreased crystallinity of the prepared films. This phenomenon corresponds to newly formed interactions between CT and OR that slightly destroy the original crystalline structure, whereas AS and CN incorporation increased the crystallinity, suggesting that AS and CN reinforced CT films, leading to more dense crystalline domains in comparison to pure CT and CT–OR; similar results have been reported by Jahed et al. [29
2.4. Raman Spectroscopy
Raman spectroscopy showed that, for all EFs with an EO added, the signal at 1745 cm−1
disappeared (Figure 2
a), which, according to Gizem-Gezer et al. [30
], is a characteristic Z signal that corresponds to the functional group O=S=O. This was attributed to interactions between the protein and EO components.
The signal at 1666 cm−1
was associated with the partial acetylation of the NH2
group of CT [31
]; it also disappeared in the presence of an EO.
In relation to CN edible films, cinnamaldehyde, which is comprised of a mono-substituted benzene ring, an aldehyde functional group, and a conjugated double bond, reacted with CT, promoting a nucleophilic addition reaction typical of aldehydes (Figure 2
b). When analyzing the EF’s structural components, the carbonyl group (C=O) provides a reactive site for nucleophilic addition, with a Schiff base (N=C) formation.
The presence of carbonyl and amino primary groups with a free electron pair allows for the formation of a Schiff base due to the electron-deficient carbonyl carbon. This was confirmed by the disappearance of the characteristic carbonyl (C=O) signal at 1745.9 cm−1
when the CN EO was added, which involved a signal decrease at 998–953 cm−1
and at 1081–1083.7 cm−1
, both characteristic of C–O–C bonds. This finding agrees with those reported by Gao et al. [32
], who studied reactions between aldehydes and CT, nucleophilic addition being the most typical reaction.
The spectrogram of the edible film containing the OR EO (Figure 3
a) did not show characteristic signals at 1081, 1440, and 1745.9 cm−1
associated with the C–O–C, N=N, and O=S=O bonds, respectively. Limonene (an olefin) is the main active component of the OR EO, which in the presence of CT experiences olefin metathesis allowing for the synthesis of small and polymeric molecules by scission and the regeneration of C=C molecules [33
], which were detected at 1597 cm−1
, whereas the signals listed above disappeared.
The EF with the AS EO added showed signal disappearance at 729.74 (C–S), 1110–1150 (C–O–C), 1440 (N=N), and 1745 (C=O). These signal losses indicate the interaction of EF components (CT-Z) with the AS EO, causing bond disruption. The characteristic tyrosine signal disappeared upon the addition of the AS EO (821 cm−1
), as well as the signal at 1745.9 cm−1
that is related to the Z functional group O=S=O reacting with CT [30
] (Figure 3
b). This implies the disruption of the protein’s secondary structure (α
] so that protein structural changes due to EO addition allow for a tyrosine reaction with other film components. The reduced signal at 1619.4–1655.4 cm−1
indicates chemical interactions due to C=N bonds losses. The addition of each EO resulted in a signal disappearance at 729.74 cm−1
, characteristic of the C–S aliphatic group of cysteine, suggesting the reaction of each one of the three EOs with the CT-Z EF.
From the Raman spectra, the addition of any EO confirms the X-ray analysis, since an EF’s structure was modified through the formation of a Schiff base, metathesis of CT, or disappearance of CS bonds.
2.5. Antifungal Activity
All EFs with an EO added showed inhibitory effects against Rhizopus
sp. and Penicillium
sp., whereas AS and CN showed an inhibitory effect on Penicillium
sp., though a lower antifungal effect was observed for Rhizopus
sp. (Table 4
). The inhibitory effect of the AS EO was attributed to anethole, its main bioactive compound [8
], which is effective against mycotoxigenic fungi, such as Rhizopus
sp. and Penicillium
]. Among the EOs, OR showed the smallest inhibitory effect on each of the two tested fungi. The amount of each EO used to conduct the antifungal tests was 15.6 ppm, which is below the daily ingestion limit of 250 ppm [35
]. Thus, higher doses may be more effective against these fungi without exceeding the permitted level. Concentrations of 100 ppm of anethole inhibited Aspergillus flavus
and Aspergillus parasiticus
, whereas a complete inhibition was achieved using concentrations of 100 ppm and 200 ppm, respectively [36
Anethole targets a fungi’s mitochondrial defense system against oxidative stress [37
]. The antifungal activity of the EO has been observed at different stages of a fungi’s life cycle, including at spore germination, the formation of penetrating structures, and mycelium and sporulation development [38
]. Limonene is the active ingredient of OR, and it causes changes in a microbial cell membrane’s properties, increasing its fluidity, and leading to altered permeability and homeostasis loss. In addition, the EO components of OR denature enzymes responsible for germination and sporulation. A concentration of 500 ppm of OR produced a fungistatic effect on A. flavus [28
Cinnamaldehyde is the active compound of CN, and its mechanism of action involves cellular ultrastructural changes, including organelles disappearance and solidification and the degeneration of the cell wall and cytoplasm. An inhibitory concentration of 0.5% (v
) was found against A. niger [38
Although EOs’ mechanism of action is not well-defined yet, their inhibitory effect has been generally associated with their hydrophobicity, which causes changes in the permeability, ion transport, and solubilization of lipid components of the cell membrane. More studies are needed to evaluate the combined effect of the tested EOs to ascertain whether there are synergistic effects against a variety of fungi.
Microbial food spoilage is one of the main problems of the food industry, and more than 50% of fruits and fruit products, are spoilt by fungi; in the baking industry, these losses represent between 1% and 3%. In addition, antifungal packaging has been proposed to extend the safety and shelf life of ready-to-eat foods [39
]. Thus, active edible films may represent a good alternative for food preservation in the baking and fruit industry, and are suitable for direct food products consumption.