3.1. Homopolymer Synthesis and Modification
PIA was synthesized in water at 60 °C, as described previously. The yield was 60% and the number average molecular weight (MW) and its polydispersity index were 59700 Da and 1.9, respectively. It is important to mention that the relationship between hydrodynamic volume and molecular weight in the polymer is different from the standards. Therefore, these data should be taken as an estimation of MW.
Figure 1 shows the proton spectrum of PIA, where methylene protons at 2.0 ppm corresponding to the main chain and methylene protons at 3.5 ppm assigned to –CH
2–COOH are easily identified.
Modified polymers MeFPIAx were prepared at two different temperatures (90 and 120 °C). The characteristic peaks are identified in the figure; the CH2 of the main chain at 2.3 ppm and the methylene protons of –CH2–COO– at 3.5 ppm are slightly shifted to higher displacement as a result of the higher rigidity. The methylene protons near to ester (O–CH2–) appear at 4.0 ppm, while the CH2 protons bonded to the thiazole group appear at 3.0 ppm. The CH3 protons of thiazole group emerge at 2.3 ppm along with the methylene protons of the main chain. The degree of modification was determined by 1H-NMR spectroscopy by comparison of triazole protons at 8.8 ppm and the methylene protons of –CH2–COO–. The results were 28% and 60% for FPIA1 and FPIA2, respectively. The increment in the temperature produces a higher degree of modification. After that, the total quaternization of the pendant heterocycles groups was achieved in both cases. There are no signals at ca. 8.8 ppm corresponding to thiazol protons and new peaks appear at ca. 10 ppm from the protons of both thiazolium groups.
Figure 2 displays, as an example, the FTIR spectra of PIA, FPIA1, and MeFPIA1. PIA homopolymer spectrum shows one characteristic band at 1695 cm
−1 (C=O), which should be assigned to the carboxyl group. The spectrum of FPIA1, polymer with lower degree of modification, shows other characteristics bands, such as the following: 3082 cm
−1 (=C–H thiazole), 2928 cm
−1 (C–H), 1720 cm
−1 (C=O), 1547 cm
−1 (C=N thiazole), and 1117 cm
−1 (C–O) [
24]. Considering these signals, it could be concluded that the functionalization of PIA was achieved. Additionally, it could be seen in the MeFPIA1 spectrum that the band corresponding to the C=N bond was displaced at 1572 cm
−1 (C=N
+ thiazolium) owing to the quaternization.
Additionally, ζ-potential values for charged polymers were determined. Measurements were made from solutions of 1 mg/mL of polymer in MilliQ water (18 mΩ). The values were −60 mV for PIA, −52 mV for MeFPIA1, and −9 mV for MeFPIA2. The negative ζ-potential observed in PIA due to the presence of carboxylate groups was shifted to less negative as the degree of modification increases. Therefore, the results confirm that a greater degree of modification was obtained with the increasing reaction temperature, as MeFPIA2 presented higher ζ-potential than MeFPIA1. These negative values were changed with the addition of a few drops of hydrochloric acid solution 0.1 M, where the ζ-potential of the polymers presented less negative values, as the free carboxylic groups were protonated. Therefore, both MeFPIA homopolymers have a dual-nature, and the presence of positive and negative charges along the side chain can produce some variations in conformation, giving the possibility to be considered as zwitterionic polymers [
50]. These zwitterions, including polyampholytes and polybetaines, are macromolecules with oppositely charged groups (cationic and anionic) along the chain or side chain that show different behaviors depending on their environment. Considering this definition, the charges could be distributed throughout the chain in different ways [
51] and, in this case, is a random distribution. Typically, cation groups are quaternized ammonium, and anionic groups are sulfonates, carboxylates, and phosphonates [
52]. The chain’s ionization depends on the pH of the environment, which has a marked effect on the polymer’s conformational behavior. The use of a carboxylate as the anion at acidic environments resulted in the protonation and neutralization of the monomer units [
51]. Zwitterionic polymers have great potential as anti-fouling coating, between other functionalities, because they can resist nonspecific protein adsorption, bacterial adhesion, and biofilm formation [
53].
After the polymers were characterized, they were incorporated into gelatin- and starch-based films with glycerol as plasticizer. Half of the formulations made incorporated dopamine as a crosslinking agent to improve their properties.
Table 1 shows the names and the description of each film formulation; those without polymer and dopamine were considered as control films.
3.2. Mechanical Properties
The tensile test results are presented in
Table 2, and the increase in TS and EB was observed with the addition of MeFPIAx (GS-MeFPIA1 and GS-MeFPIA2), showing a statistically significant variation (
p < 0.05). Gelatin is a very valuable biopolymer; however, its poor mechanical properties limit its application as a packaging material [
54]. The considerable improvement in TS and EB may be due to the presence of increasing electrostatic forces and hydrogen bonding interactions between the gelatin macromolecules and carboxyl groups of MeFPIAx, constituting a stable network with enhanced mechanical properties [
55].
The difference between the films with both functionalized polymers is that MeFPIA1 had a greater number of free carboxyl groups capable of interacting with the protein chains. Gelatin has an amphoteric character because of the functional groups of the amino acids and the terminal amino and carboxyl groups created during collagen hydrolysis [
56], so they are able to form links with polymer chains. No differences were observed on YM values.
Similar results were obtained with the incorporation of dopamine into the mixture. Significant differences (
p < 0.05) were found in films GS-MeFPIA1D and GS-MeFPIA2D compared with the control sample with only dopamine without modified polymers (Gel-D). GS-MeFPIA1D and GS-MeFPIA2D films showed higher values of EB, probably owing to the increase of interactions generated by both polymers (as mentioned above) and dopamine added to form network points [
57]. Dopamine belongs to the catechol family and its incorporation in almost any polymer matrix induces the reaction with the functional groups on the polymer backbone in a weak alkaline environment [
55]. Although there was a slight increase in TS, as expected, this difference was not significant. Moreover, a decrease in YM was observed. This result could be explained as dopamine may interfere between strong interactions between gelatin and polymer chains.
3.3. Color Properties
Color properties are shown in
Table 3 and a clear change in color values was observed with the addition of MeFPIAx. The presence of MeFPIA2 in the formulation caused a decrease in the luminosity (decrease in L* values) of the samples. Moreover, films with MeFPIA1 and MeFPIA2 turned greenish (decrease in a* values) and yellowish (increase in b* values), the most pronounced increase in the yellow color of the films was observed with the addition of MeFPIA2. Therefore, the color change ΔE* is significant for both polymers and perceptible to the human eye, as can be seen in
Figure 3; this change was more abrupt for the polymer with less negative potential.
Moreover, the addition of dopamine produced a change even greater in all the parameters studied. The L* value of samples was significantly reduced. Samples with dopamine tend to be reddish (increase in a* values), even more in the sample with MeFPIA2. Moreover, an increase in b* values towards yellow was observed in all the samples.
Generally, colorless films are desirable for food packaging, but sometimes, transparency could be a drawback owing to the light exposure, causing food deterioration. Therefore, films with such a color could protect food from light degradation [
58]. In this way, the incorporation of MeFPIAx could be advantageous.
3.4. Experimental Water Vapor Permeability (Pwexp)
The quality of most food products deteriorates via moisture absorption, and this can occur between food and the atmospheric environment, so it is important to characterize the barrier properties of films against water vapor [
59]. The flow of water through polymeric films does not occur through pores, but can be understood considering that the process occurs in three stages: (i) sorption of water vapor at the surface layer of the film in contact with the highest internal r.h.; (ii) diffusion of the permeant molecules through the film; and (ii) desorption of water vapor from the other surface of the film, where the lowest external r.h. occurs [
60]. Hence, the chemical structure, polarity, degree of crystallinity, density, crosslinking degree, molecular weight, and polymerization, as well as the presence of plasticizers, are factors to take into account because they will affect water vapor permeability [
45].
As can be seen in
Table 4, the incorporation of MeFPIAx caused an increase in
Pwexp. Considering the mechanical properties, it was observed that the incorporation of polymer increased the elongation of the materials; therefore, as the polymeric chains have more mobility, the passage of water vapor through the films is facilitated [
61]. Moreover, when samples with MeFPIAx are compared to films with MeFPIAx and dopamine, it could be seen that dopamine incorporation slightly reduced the
Pwexp values. This could be explained with the fact that dopamine might increase polymer chains’ interactions, making the passage of water vapor more difficult. This effect was not so significant in those films that do not contain MeFPIAx, as the number of interactions was lower.
3.5. Swelling Properties
The knowledge of the water or a solution transport towards the interior of some materials during swelling presents significant importance in different fields, such as medicine (drug delivery systems), environment (removal of contaminants), and agriculture (fertilizer delivery).
Figure 4 displays the swelling behavior for the different films in PBS at 25 °C and 37 °C.
Control films showed the highest degree of swelling, as can be seen in
Figure 4A, but they were the weakest matrices. Swelling of native gelatin-based films cannot be easily determined because of slow dissolution in water [
62]. Over time, those films lost their ability to swell as well as their integrity, being dissolved in the media, as occurred at 37 °C, where the swelling process could not be concluded. Hence, cross-linking agents are necessary to introduce covalent bonds into the network, preventing dissolution of the gels [
62]. Swelling capacity depends on many factors, such as the properties of the swelling liquid (pH, temperature, nature, and concentration of salts and properties, among others) and properties of the matrix (gelatin/starch ratio) itself (degree of degradation, presence of impurities, and conditions under which gelatin films were dried, among others) [
63].
Films with MeFPIA1 and MeFPIA2 shown in
Figure 4B,C, respectively, presented a lower degree of swelling compared with the control; in all the tested conditions, they were more stable, owing to the appearance of new interactions, mainly because of the presence of free carboxyl groups. It should be noted that, during the swelling test, the solubility of some of the components might have occurred, and this could be the case of dopamine. At the beginning, its release was not too fast, because the solution absorption rate was higher than the release of the additive; however, as time progressed, dopamine was slowly released to the media. It is possible that, as dopamine was released to the medium, free sites on the polymer chains appeared, able to interact with solution molecules, and retained it. In this study, samples containing dopamine better preserved their integrity during the test time. Although dopamine did not fully crosslink with polymer chains, as expected, because it was released into the swelling medium, it increased the number of bonds, especially with MeFPIAx, which contains carboxyl and ammonium groups to interact, providing greater stability [
56].
The conversion temperature for gelatin is determined as melting point (gel to sol process), and the gel is converted into a solution as the temperature rises from 30 °C to 40 °C [
32]. Among all the variables examined, the temperature increase from 25 °C to 37 °C produced an increase in the swelling process. This effect was caused by a rapid approach to the gel melting point as the temperature was raised. Thus, films swelled at 37 °C were softer and weaker than films swelled at 25 °C, indicating a lesser resistance to swelling [
56].
Moreover, the results showed that the crosslinking was favored with the addition of modified MeFPIAx polymers and dopamine as the number of interactions increased. Then, it could be seen that the addition of MeFPIAx not only improved the mechanical properties of gelatin, but also decreased the absorption capacity of the solution molecules during the swelling process.
It is well-established that, when the behavior of water or solution absorption in polymers is considered a truly Fickian diffusion, the plot of (
Wt −
W0)/
W0 versus
t1/2 should be linear up to almost a 60% increase in hydrogel mass, irrespective of any dependence of the diffusion coefficient on the moisture concentration [
64]. In this way, and according to Fick’s second law and considering one-dimensional diffusion, the solution content as a function of time can be fitted with the power law equation when n is equal to 0.5, and the solution transport follows Fickian diffusion.
Consequently, and by plotting (
Wt −
W0)/
W0 versus
t1/2 at initial stages of swelling, the Fickian behavior can be analyzed. The corresponding plots are displayed in
Figure 5. As can be observed, the solution absorption curves of films for all the formulations were not linear up to a 60% increase in hydrogel mass. Therefore, as can be seen in the figure, most of the different formulations did not follow one-dimensional Fickian diffusion, so the values that n takes should be different to 0.5, and data should be adjusted by a non-Fickian or anomalous diffusion process.
It is described that dynamic factors, including rearrangements in the polymer structure, as a response to the sorption and diffusion process of solution, can be responsible for deviations from ideal Fick’s behavior. Indeed, these structural changes have their corresponding relaxation time constants. Consequently, the experimental data were fitted using a biexponential function in Equation (4). This equation expresses that the swelling process is produced through two different processes, one faster than the other. Data fitted quite well to the behavior of films (see lines of
Figure 4).
The results of the different relaxation process that contributes to the solution uptake are collected in
Table 5, where φ
1 and φ
2 are the fractions of solutions contributing to processes 1 and 2; this is Δh
1/h
∞ and Δh
2/h
∞ [
65]. In all the curves, the square correlation was higher than 0.999, which indicates the adjustment accuracy.
Table 5 shows that parameters for GS and GS-D at 37 °C were not determined (n.d.), because the films were disintegrated in the solution and could not be fitted.
The two processes, suggested by the biexponential model, can be attributed to a fast solution absorption mechanism more associated with the interaction of solution with the polymeric matrix, and a slow process related to the solution, less associated with the matrix, and that could be filling empty cavities and forming large multilayers of solution.
3.6. Antioxidant Activity
Figure 6 exhibits the results of antioxidant activity in different solutions. Based on the results, it could be seen that there was a decrease in the absorbance of the samples against control and, therefore, an increase in the RSA% value, caused by the antioxidant action of the developed films.
These values were even higher in films with dopamine, and an increase was seen with the increasing water content of solution B. It is well known that gelatin is soluble in water and in aqueous solutions of poly-hydric alcohols; however, it is practically insoluble in less polar organic solvents such as acetone, carbon tetrachloride, ethanol, ether, benzene, dimethylformamide, and most other nonpolar organic solvents [
54]. Considering this, as the water content in solution B increased, so does the affinity with water, and samples swelled more rapidly, releasing dopamine more easily to the medium, increasing the antioxidant power of the dopamine. The increase in antioxidant activity in samples with dopamine added was because of the fact that many studies have indicated that dopamine was a strong free-radical inhibitor, which reacts with free radicals [
42]. The dopamine molecule has a phenolic hydroxyl group in the structure (like most synthetic and natural antioxidants), and phenolic antioxidants have been recognized to function as hydrogen donors, acting like a free radical scavenger [
66]. Surprisingly, when the assay was performed in solution B (water solution), the formulations with MeFPIA2 showed significant antioxidant activity even without the presence of dopamine. This fact could be because of a higher hydrogen donating potential of the network formed.