3.2. Basic Morphological and Textural Properties of the Films
The thickness of films is given in
Table 3. No net decrease or increase in thickness was found in the samples with increasing addition of the extract, these values fluctuated in the samples. The results also do not show statistically significant differences (
p > 0.05), so it can be said that the increasing percentage of extract did not affect the thickness of the resulting package. In the case of comparison with previous studies, there was both a significant increase in thickness values after the addition of plant extracts [
15,
34] but there were also findings that resulted in a decrease [
35,
36].
The results of textural properties are shown in
Table 4. The interaction of hydrocolloids and other additives such as plasticizers, water and antimicrobial substances has the greatest influence on the textural properties of edible coatings [
37]. The 5CH
LBO, 5CH
LPE, 5CH
LHR and 10CH
LHR samples are statistically significantly different (
p < 0.05) from the 20CH
LPE sample that had the highest strength (0.10 ± 0.02 MPa). No statistically significant difference (
p > 0.05) was found between the other samples. The comparison of results with the control sample CH
L did not resulted in statistically significant differences (
p > 0.05) between the samples with the addition of extracts. The increase in strength can be caused by interactions between plant extracts that contain phenolic acids and their esters. These compounds can react with the hydrophilic groups present in the chitosan matrix, and this interaction can lead to stronger adhesion between the plant extracts and the chitosan molecules. These interactions can cause an increase in strength [
38]. In previously published articles, it has been found that some additives incorporated into the chitosan matrix both increase and decrease the strength of the prepared packages. The explanation is similar to that described above: the increase in strength is due to stronger interactions between additives and chitosan, and the decrease in strength is due to weak interactions [
39].
The prepared chitosan packaging was characterized by high flexibility, which was observed by the handling of the packaging itself and subsequently confirmed by measuring the flexibility, where the results are presented in
Table 2. Flexibility decreased with the addition of the extract. Compared to the CH
L sample, the lowest value of elasticity was found in the 20CH
LBO sample, but no statistically significant difference was found (
p > 0.05). The results correspond to the results of the force; due to the interaction between phenolic acids and chitosan, the flexibility is not so high in most samples [
38]. It has also been found, when compared with the results of measuring the strength and elasticity of packages made of κ-carrageenan and ι-carrageenan with the addition of lapacho tea extract, that chitosan packages are more flexible and also have a lower strength value [
40].
The results of gas barrier properties are summarized in
Table 5. Water vapor transmission rate of all tested samples was higher than 1000 g/m
2 day, which is a relatively high value that limits the use of this material for packaging products that need to be protected from moisture. However, this value is similar for chitosan-based materials [
41,
42,
43]. Similar values of WVTR were measured for bleached Kraft paper [
44]. The results also showed that the addition of plant extracts increased the WVTR value of the chitosan-based material by an average of: 3.84 ± 1.14% for 5% addition of blueberry extract, 9.63 ± 0.23% for 5% addition of parsley extract, 8.05 ± 1.64% for 5% addition of red grapes extract. The lowest average value of WVTR for the material with the addition of plant extract 1270.0 ± 25.3 g/m
2 day was achieved for film with 5% addition of blueberry extract, the highest average value of 1724.6 ± 30.8 g/m
2 day was achieved for material with 20% addition of red grapes extract.
Changes of oxygen permeability caused by the addition of plant extract are opposite to the changes of WVTR according to the plant extract addition. The reduction in oxygen permeability compared to chitosan films without the addition of plant extract was on average 21.30 ± 3.15% for 5% addition of blueberry extract, 16.11 ± 1.70% for 5% addition of parsley extract and 13.85 ± 3.97% for 5% addition of red grapes extract. The lowest average value of oxygen permeability of the material with the addition of plant extract 4.1 mL/m
2 day 0.1 MPa was measured for the material with 20% addition of blueberry extract, the highest average value of 13.7 mL/m
2 day 0.1 MPa was achieved for a film with 5% addition of red grapes extract. The resulting values are relatively low and correspond to permeability values for commonly used packaging materials based on, e.g., polyamide, polyethylene terephthalate [
45].
Similar and completely different effects of the addition of plant extracts on the barrier properties of final materials have been published [
46,
47]. The increase in permeability may be caused by the destabilization of the original chitosan matrix by extract components that may act as plasticizers [
48]. The reduction in permeability is then explained mainly by the possibility of crosslinking between components of the extracts and the polymer matrix [
49].
For the purposes of wider use of this material in food packaging, it would be good to combine the material with barrier materials preventing the penetration of moisture into the packaged product. An example may be the incorporation of waxes, oils, etc., directly into the material [
47,
50]. A disadvantage of this method may be a significant reduction in oxygen transmission rate [
48].
3.3. Basic Compositional and Structural Analysis
Data for water content, solubility and swelling degree are summarized in
Table 6. In the case of water content, a decrease in water content was observed in the samples after the addition of the extracts, but significant (
p < 0.05) differences in comparison with the control sample (CH
L) were noticed among the following samples: 10CH
LBO, 5CH
LPE and 20CH
LPE. The reduction of the water content in the packaging with the addition of extracts is due to the formation of hydrogen bonds, which in turn reduce the availability of hydroxyl groups and amino groups and thus limit the interaction of chitosan with water [
51,
52].
The solubility results did not differ significantly with the obtained values, and it can therefore be solved that the addition of extracts from red grape, blueberry and parsley pomace does not affect the solubility. Similar results were found in research by Bourbon et al. [
53].
In the analysis of swelling degree, the highest values were reached in the case of the CH
L sample (the control sample without the addition of extract), in the samples where extracts were added, the values of the swelling degree decreased, but in comparison with CH
L the differences were not statistically significant (
p > 0.05). In all other samples there was a gradual decrease in the value of the swelling degree, but only between the samples 5CH
LHR and 20CH
LHR was a statistically significant (
p < 0.05) difference found. For the swelling degree, the reduction is caused similarly to the water content. When the presence of polyphenolic substances blocks the active groups of chitosan available for water adsorption [
24], these results are confirmed because for samples with the lowest swelling degree (20CH
LBO and 20CH
LHR), the highest value of the content of polyphenolic substances were found (see section
Content of the Antioxidant Compounds in the Films).
Furthermore, the Fourier transform infrared spectroscopy was used to provide a closer look on how the addition of plant extracts alters the chemical structure of the prepared chitosan film. The FTIR spectra of all analyzed films are shown in
Figure 1. It can be seen that the presence of a plant extract does not induce any distinct spectral features (such as the occurrence of new absorption bands) that could be directly assigned to the molecular structure of the extract components. In other words, the structure of the films as observed by FTIR is primarily governed by the matrix composition of the film. In this structure, characteristic FTIR vibrations that correspond to the three matrix components can be found in all spectra. Firstly, chitosan as the main film-forming components is reflected by: (i) absorptions originating from amide linkage in acetylated amine groups, in particular C=O stretch in amides at 1640 cm
−1 (referred to as Amide I), N-H in-plane bend at 1535 cm
−1 (Amide II), C-N stretch at 1310 cm
−1 (Amide III), (ii) vibrations attributed to deacetylated amine groups (NH
2 bend at 1570 cm
−1, less pronounced N-H stretches at 3350 and 3270 cm
−1 overlapped by intensive -OH stretch of all matrix components and moisture), and (iii) vibrations of the oxygen containing groups, namely asymmetric C-O-C stretching in glycosidic bond (1150 cm
−1) and C-O stretching at 1070 and 1030 cm
−1. Secondly, presence of lactic acid is manifested by: (i) characteristic bands of carboxylic groups (C = O stretch in carboxylic groups at 1725 cm
−1, less intensive shoulder of H-bonded dimers at 2600 cm
−1, C-O stretch at 1220 cm
−1) and (ii) methyl vibrations (clearly visible asymmetric C-H stretch in CH
3 at 2983 cm
−1, symmetric “umbrella” bend at 1377 cm
−1). Last but not least, glycerol presence is reflected by the (i) characteristic absorption of -CH
2- groups (asymmetric and symmetric C-H stretches at 2935 and 2875 cm
−1, respectively, CH
2 scissoring bends at 1454 and 1415 cm
−1) and (ii) contribution to −OH related vibrations in 3000–3500 cm
−1 (O-H stretches) and in 1150–800 cm
−1 (C-O stretches), where the presence of glycerol is usually accompanied by characteristic asymmetric stretches at 924 and 852 cm
−1.
At first sight, it may seem that the addition of plant extracts has negligible effect and may not be observed in the FTIR spectra. However, the effect of the extracts on the FTIR spectra may be revealed when an advanced processing is applied on the spectra. We have used the whole-spectra Principal Component Analyses for this purpose. In this technique the set of original variables (absorbances measured at individual wavenumbers) is replaced by the new set of variables (principal components, calculated as specific linear combinations of the original variables) with the same total variance (i.e., the same overall information on the observed system) but with different spread of the information among these variables. In order to reveal the effect of individual extracts, three separate Principal Component Analyses were performed for the three sets of films with a particular extract added. The results of these analyses are shown in
Figure 2 in form of the respective two-dimensional factor planes of the two principal components that covers the highest relative variance (PC1 and PC2) together with their spectral loadings. The loading shows how the component is composed from the original data, in other words, it illustrates what are the main spectral variations among the analyzed spectra from the view of this component.
Apparently, PCA analysis revealed the spectral features that clearly distinguish FTIR spectra of different films. In
Figure 2a, it can be seen that the clusters that represent films with different content of the blueberry extract are separated in the plot mainly via component PC2, whereby the increasing content of the extract results in a more negative value of this component. From the loading of this component, it can be seen that an increase of the content of blueberry extract is reflected in the spectrum mainly by a decrease in the signal of lactic acid (note the positive loading of PCA at 1720, 1217, 1120 and 1180 cm
−1) or its salt (positive loading of an asymmetric stretch of -COO- at 1530 cm
−1). On the other hand, in the negative loading of PC2 that correlate with the content of the blueberry extract, spectral features can be found that were previously ascribed to anthocyanin, such as absorption at 1435 cm
−1 (C-N in anthocyanin) or the flavonoid C-O-C stretch at 1070 cm
−1 [
54]. Similarly, also the clusters that represent PCA coordinates of the films prepared with different contents of parsley extract are well separated in the PC1-PC2 coordinate plot (see
Figure 2b), mainly distinguished by the value of PC1 component. Once again, from the loading of this component, a negative correlation between the contents of the extract and of the lactic acid is evident. The most prominent spectral features in negative loading of this component (marked in
Figure 2b) well correspond with the vibrations that were already found in parsley essential oil [
55]. Finally,
Figure 2c shows results of PCA for the films prepared with different content of red grapes extract. Clusters representing these films are once again well separated from the CH
L matrix film. Again, increase in the extract is linked with decrease of the signal of lactic acid and lactate in FTIR spectra of the films (see the negative loading of PC1), while its increase enhances the signal (represented by the positive loading of PC1), which is in a good agreement with a spectrum published for the biostimulant prepared from red grapes recently [
56].
3.4. Content of the Antioxidant Compounds in the Films
Phenols and phenolic acids are metabolites of plants with the highest antioxidant activity [
57]. The content of phenolic acids in the experimentally produced packaging is summarized in
Table 7. The CH
L sample was found to contain total polyphenols 0.12 ± 0.01 mg of gallic acid/g. Small amounts of polyphenols have also been found in previous research [
58,
59,
60]. The measurement of content of a certain polyphenol in the package without the addition of extracts may have been due to the formation of chromogens that are formed by the reaction of Folin–Ciocalteu reagent with non-phenolic reducing agents and that can subsequently be detected by spectrophotometer measurements [
59]. The addition of blueberry, parsley and grape by-products extracts showed an increase in content of phenolic acids in samples with extracts. Total polyphenols contents in the extracts used to make the packaging was as follows: blueberry extract 0.26 ± 0.00 mg gallic acid/mL; parsley extract 0.04 ± 0.00 mg gallic acid/mL; grape extract 0.12 ± 0.00 mg gallic acid/mL.
5CHLBO, 10CHLBO, and 20CHLBO were statistically significantly different (p < 0.05) from CHL, meaning that the addition of blueberry extract has a high effect on the total polyphenol content. The addition of parsley by-product extract increased TPC, but a statistically significant difference (p < 0.05) was found only between the 20CHLPE and CHL samples, where the content of polyphenols in the 20CHLPE sample was about half that of the 20CHLBO and 20CHLHR samples. Red grape by-product extract affected the total content of polyphenols the most, as the 20CHLHR sample had the highest content of polyphenols (0.96 ± 0.05 mg gallic acid/g), but no statistically significant difference (p > 0.05) was found between the 20CHLHR and 20CHLBO samples.
In previous research, a higher TPC content was found in packages with the addition of blueberry ash fruit extract, macadamia peel extract and banana peel extract [
61], which may be affected by the procedure extract preparation—ratio of solid material and solvent, type of solvent and amount of addition to the matrix of film-forming solution [
62].
Aside from the determination of total polyphenol content, the selected common representatives of the polyphenolic substances were directly assayed in the prepared films. The results of the determination of individual polyphenolic substances in the samples are given in
Table 8 and
Table 9. All samples were analyzed for the presence of rosemary acid, chlorogenic acid, as well as epigallocatechin, epicatechin gallate and epicatechin.
In the case of packages with the addition of plant extracts, it was found that rosemary acid is found in the highest concentration in samples with the addition of parsley extract, the sample 20CHLPE reached a concentration of 0.0206 ± 0.0009 mg rosemarinic acid/g.
Chlorogenic acid was found at the highest concentrations in samples with the addition of blueberry extract (20CHLBO: 0.0781 ± 0.0061 mg/g), the result was statistically significantly different (
p < 0.05) from the values in all other samples. Thus, it was confirmed that the extracts of moldings blueberries occurred from crossing of chlorogenic acid, compared with cranberries, black currants, strawberries, red currants, raspberries and blackberries, had the second highest value [
63]. Thus, it was confirmed that the extracts of blueberries crossed with chlorgenic acid had the second highest content, as compared with cranberries, black currants, strawberries, red currants, raspberries, and blackberries [
63].
Epigallocatechin was also found in most of samples with the addition of blueberry extract and similar results were obtained in the determination of epicatechin. Epicatechin gallate was found in the highest concentration in samples with the addition of red grape extract. Epigallocatechin and epicatechin belong to the group of flavan-3-ols that occur in red grapes [
19].
3.5. Evaluation of the Antioxidant Properties of the Films
Aside from the determination and identification of the components with antioxidant activity, direct monitoring of antioxidant properties of the films was performed as well. These properties have been characterized by performing three different assays, each with different reaction conditions that can affect obtained results. Combination of three methods certainly provides more accurate perception about the experimentally produced edible packaging antioxidant properties. The obtained results confirmed that the addition of extracts increased the antioxidant properties of the chitosan films. The antioxidant activity of the extracts used to prepare the packaging samples was as follows: blueberry extract 0.176 ± 0.001 μmol Trolox/mL; 73.18 ± 0.44% ABTS; grape extract 0.021 ± 0.001 μmol Trolox/mL; 23.35 ± 0.19% ABTS; parsley extract 0.0 ± 0.0 μmol Trolox/mL; 6.32 ± 0.22% ABTS.
The results of the antioxidant properties are summarized in
Table 10. The FRAP method showed the highest results for the 20CH
LBO sample (2.45 ± 0.06 μmol Trolox/g), where it must be emphasized that the addition of by-product extracts had a statistically significant impact (
p < 0.05) on the FRAP method results, regardless of the addition concentration. The best antioxidant properties measured by the ABTS method were found in the sample 20CH
LHR (the sample with the highest antioxidant property, though, antioxidant properties of other samples can be defined as poor, accordingly) and similarly to the FRAP method, a statistically significant difference (
p < 0.05) was found between all samples compared to CH
L, except for the sample 5CH
LBO. DPPH showed the same trend as FRAP and ABTS. The highest result was determined in the sample 20CH
LBO (5.39 ± 0.06%) and this result is statistically significantly different (
p < 0.05) from all other DPPH results.
As expected, to some extent the antioxidant activity was also found in a control sample without the addition of extracts, where the main component is chitosan. Chitosan belongs to the compounds with the properties of inhibiting reactive oxygen species (ROS) and can prevent lipid oxidation in food, but is also in biological systems [
64,
65]. The antioxidant activity of edible packaging with the addition of extracts was higher because plant extracts are a good source of antioxidant compounds. In general, red fruits are very good sources of antioxidant compounds, as they contain anthocyanins that have these properties
16. Higher antioxidant activity was also observed in packaging with the addition of red grape by-product extract. It is mentioned in the literature that if the extract from the marc is used in higher concentrations, then its antioxidant activity is comparable to synthetic BHT [
66]. Parsley is also one of the plants with antioxidant properties [
67], but in comparison with other samples, the antioxidant activity was sometimes up to half lower in packages with the addition of parsley extract.
3.7. Release of the Active Components from the Films
According to the legislation, simulant A was chosen to determine the migration values of active substances, it is a simulation of the transition of active substances into foods with a hydrophilic character which, according to EU Regulation No. 10/2011, includes the following food commodities with the hydrophilic character: nuts in paste or cream, fresh vegetables, fish, fresh meat and processed meat products, fried potatoes, donuts, preparations for making soups, etc. The results of migration tests for manufactured packaging are shown in
Table 11 (polyphenol content) and
Table 12 (antioxidant properties). The values of the results of polyphenols showed that there is a migration of polyphenolic substances, which was confirmed by the analysis of antioxidant activity, where higher values were measured with the addition of extracts from by-products of blueberries, parsley and red grapes.
For polyphenols, the highest migration value was recorded for the 20CHLHR sample (0.016 ± 0.000 mg gallic acid/mL), which is statistically significantly (p < 0.05) different from all other measurements of polyphenol migration.
In determining the antioxidant activity, the samples 20CHLHR, 20CHLBO and 20CHLPE achieved the best results, as described above, due to the higher concentration of extracts used for the production, so these packages can be characterized as most suitable for subsequent application to packaged foods.
In general, when migrating substances from packaging to food, it is more likely to avoid migration in the case of film-forming components—e.g., migration of monomers of PET materials, etc., that are undesirable and must meet limits [
71]. In contrast, the migration of active components such as polyphenols and antioxidants is desirable because they can contribute to improving the properties of packaged foods, prolong their shelf life, prevent oxidation, and thus function as a package with active properties [
12].
Non-correlating results were found for antioxidant activity, i.e., in different methods (ABTS, FRAP and DPPH) the same sample did not always show the highest value of antioxidant activity, for these samples the effect of solvent is excluded, as the same solvent was used everywhere to detect migration of active substances, but the results are influenced by the conditions and the course of reactions of individual methods. For the FRAP method, the analysis is performed at low pH values (3.6) compared to the DPPH and ABTS methods, where the pH value was not adjusted. In the ABTS method, color loss is determined spectrophotometrically after the addition of an antioxidant to the blue-green chromophore ABTS
·+, so the antioxidant reduces ABTS
·+ to ABTS and decolorizes it [
72]. The DPPH method uses a stable free radical, and in the presence of an antioxidant compound it can donate a hydrogen atom; lead to a reduction and decolorization of the dark purple solution; in the case of DPPH this radical does not always react with the same compounds as in the case of ABTS; and the stability of the ABTS solution is also much lower than that of DPPH [
72,
73].