3.2. FTIR spectroscopy of CS/PVOH/xAC, CS/PVOH/xTO@AC films
FTIR spectra of all obtained CS/PVOH/xAC and CS/PVOH/xTO@AC films as well as pure CS/PVOH film are plotted in
Figure 3.
In the FTIR plot of pure CS/PVOH film (see line (1) in
Figure 3) there are obtained reflections of both CS and PVOH polymers. The large band at 3443 cm
-1 is assigned to stretching vibration of hydroxyl groups of both CS and PVOH [
29]. At 3400 cm
-1 is assigned the primary stretching vibration of amino groups of CS [
29]. The reflections at 1637 cm
−1, 1550 cm
−1, 1260 cm
−1, 1080 cm
−1, 900 cm
−1 are red shift of C-H stretching, C-O stretching, N-H bending and O-H stretching of CS due to the strong relaxation with PVOH chains [
32]. In the case of both CS/PVOH/xAC (see lines (2), (3) and (4) in
Figure 3) and CS/PVOH/xTO@AC (see lines (5), (6) and (7) in
Figure 3) FTIR plots the characteristic reflections of AC are observed in advance to CS/PVOH matrix reflections. The band at 1260 cm
-1 is assigned to the stretching vibration of C-O groups. The bad with a maximum at about 3420–3440 cm
−1 is assigned to the O–H stretching mode of hydroxyl groups of the adsorbed water molecules [
28]. With a careful glance in CS/PVOH/xAC and CS/PVOH/xTO@AC FTIR plot it is resulted that in the case of CS/PVOH/xTO@AC the reflection at 1260 cm
-1 is increased implying a bigger relaxation and interplay between CS/PVOH N-H groups and TO@AC C-O groups. In advance in the case of CS/PVOH/xTO@AC FTIR plots the reflection of the primary stretching vibration of amino groups of CS at 3400 cm
-1 are more intense than in the case of CS/PVOH/xAC FTIR plots where the same reflection almost disappeared. This is also implying the higher relaxation of N-H groups of CS/PVOH matrix with the O-H groups of TO@AC nanohybrid.
Thus, by both XRD analysis and FTIR spectroscopy it is resulted the higher dispersion and relaxation of TO@AC nanohybrid with CS/PVOH matrix in comparison to pure AC with CS/PVOH matrix.
3.6. Antibacterial properties of CS/PVOH/xAC and CS/PVOH/xTO@AC films- Agar diffusion test
The antibacterial activity of various film materials against four bacterial strains (E. coli, S. aureus, S. enterica, and L. monocytogenes) was investigated in this study. The films tested included CS, CS/PVOH, CS/PVOH/5AC, CS/PVOH/10AC, CS/PVOH/15AC, CS/PVOH/5TO@ AC, CS/PVOH/10TO@ AC, and CS/PVOH/15TO@ AC. The inhibitory activity of the film materials against the bacteria was determined by measuring the clear zone diameters. The results showed varying levels of antibacterial activity among the film materials, indicating the potential for improved efficacy through the addition of specific components.
Table 3 and
Figure 6 provide an assessment of the antibacterial effectiveness of the studied nanoreinforced CS/PVOH based packaging films.
Table 3.
Antimicrobial activity of active films against food pathogenic bacteria E. coli, S. aureus, S. enterica and L. monocytogenes.
Table 3.
Antimicrobial activity of active films against food pathogenic bacteria E. coli, S. aureus, S. enterica and L. monocytogenes.
Film material |
E. coli |
S. aureus |
S. enterica |
L. monocytogenes |
Inhibition1 (diameter of clear zone) |
Inhibition1 (diameter of clear zone) |
Inhibition1 (diameter of clear zone) |
Inhibition1 (diameter of clear zone) |
CS |
3.07 ± 0.22a
|
3.56 ± 0.43a
|
3.40 ± 0.32a
|
2.03 ± 0.26a
|
CS/PVOH |
3.63 ± 0.35ab
|
3.72 ± 0.25ab
|
3.49 ± 0.11ab
|
3.78 ± 0.52ab
|
CS/PVOH/5AC |
0 ± 0c
|
0 ± 0c
|
0 ± 0c
|
0 ± 0ac
|
CS/PVOH/10AC |
0 ± 0c
|
0 ± 0c
|
0 ± 0c
|
0 ± 0ac
|
CS/PVOH/15AC |
0 ± 0c
|
0 ± 0c
|
0 ± 0c
|
0 ± 0ac
|
CS/PVOH/5TO@AC |
3.08 ± 0.26abd
|
5.43 ± 0.49abd
|
4.13 ± 0.68abd
|
2.66 ± 0.36a
|
CS/PVOH/10TO@AC |
3.10 ± 0.29abd
|
3.63 ± 0.46abd
|
3.00 ± 0.16abd
|
3.44 ± 0.30ad
|
CS/PVOH/15TO@AC |
3.84 ± 0.60abd
|
4.53 ± 0.36abd
|
6.07 ± 0.18e
|
3.86 ± 0.91ad
|
The results indicate that the film material composed of CS) exhibits some antimicrobial activity against the tested bacteria, with varying levels of effectiveness. Notably, it demonstrates moderate inhibition against
S. aureus and
S. enterica, while displaying lower activity against
L. monocytogenes. However, its efficacy against
E. coli is relatively limited. CS, is known for its antibacterial effects against a wide range of bacteria. This property is attributed to its cationic characteristics, specifically the positively charged ammonium (NH
4+) groups that interact with the negatively charged elements of bacterial cell walls. Furthermore, the importance of chitosan’s cationic nature in contributing to its antibacterial activity has been emphasized by Giannakas et al. (2022) and Unuabonah et al. (2018) [
15,
35].
The CS/PVOH film material displayed diverse antibacterial activity depending on the bacterial strain, with the greatest inhibitory activity against L. monocytogenes. Moreover, CS/PVOH films showed comparable inhibitory activity to CS against S. aureus and S. enterica. This suggests that the addition of PVOH to CS enhances the film’s ability to inhibit bacterial growth.
On the other hand, the film materials containing AC in various concentrations (CS/PVOH/5AC, CS/PVOH/10AC, and CS/PVOH/15AC) show no antimicrobial activity against any of the tested bacteria. This indicates that the inclusion of activated carbon in the film formulation does not contribute to its antimicrobial properties.
However, when TO complexed with AC (TO@AC) is incorporated into the CS/PVOH film material, the antimicrobial activity is notably improved.
The clear zone diameters ranged from 2.66 to 6.07, with the highest inhibition observed against S. enterica. Specifically, the presence of 5%TO@AC enhances the film’s effectiveness against S. aureus and S. enterica. Increasing the concentration of TO@AC to 10% and 15% in CS/PVOH films displayed varying degrees of inhibitory activity against the bacteria, with CS/PVOH/15%TO@AC demonstrating the highest inhibitory activity against S. enterica, followed by S. aureus. The results clearly demonstrate an increased inhibitory effect on bacterial growth with higher concentrations of TO@AC nanohybrids. The results of the statistical analysis revealed significant differences in the antibacterial activity of the film materials against the tested bacteria (p < 0.05) and Tukey HSD post-hoc comparisons were conducted to identify the specific differences.
For the tested bacteria, pairwise comparisons showed that all CS/PVOH films containing TO@AC in different concentrations, exhibited significantly higher inhibitory activity compared to CS/PVOH films incorporated only with AC (p < 0.05). In the case of S. enterica, the CS/PVOH/15% TO@AC film displayed the highest inhibitory activity, with significant differences observed when compared to all the tested materials (p < 0.05). These statistical results confirm that the addition of thyme oil complexed with activated carbon (TO@AC) significantly enhances the antibacterial activity of the CS/PVOH films, particularly against S. aureus and S. enterica.
AC, also known as activated charcoal, is a highly porous form of carbon that has various applications due to its adsorption properties. It is extensively utilized in the field of air pollution control and wastewater treatment for the removal of various pollutants. This is primarily attributed to its remarkable characteristics, such as large surface area and high adsorption capacity.
While AC is commonly used for its ability to remove impurities, toxins and odors, its antimicrobial activity against bacteria is limited. A notable concern associated with AC is the formation of biofilm on carbon particles by bacteria, leading to increased resistance to disinfection processes and the potential for the carbon itself to act as a source of bacterial contamination [
36,
37].
AC primarily works by adsorbing (binding) substances to its porous surface. As mentioned, before it can effectively remove organic compounds, toxins, and certain gases through this adsorption process. However, its ability to kill or inhibit the growth of bacteria is not significant. The porous structure of AC provides a large surface area for bacteria to attach to, but it does not possess inherent antimicrobial properties. The adsorption process can physically trap bacteria within the carbon’s pores, preventing them from spreading. This property is utilized in certain applications, such as water and air filters, to remove bacteria and improve cleanliness. However, it’s important to note that activated carbon alone does not actively kill bacteria [
38].
Based on the above, AC seems that may have the ability to limit the migration properties of an antimicrobial agent through adsorption. When an antimicrobial agent is incorporated or impregnated into activated carbon, the activated carbon’s porous structure physically traps and binds the antimicrobial agent, preventing or limiting its release into the surrounding environment. In the present work, the antimicrobial activity of chitosan, is completely lost when it is incorporated into activated carbon. This suggests that the specific characteristics of chitosan and its interaction with activated carbon may lead to the loss of its antimicrobial properties.
There could be several reasons why chitosan loses its antimicrobial activity when incorporated into activated carbon. One possibility is that the adsorption process of chitosan onto activated carbon might alter its chemical structure or inhibit its interaction with microorganisms, thereby diminishing its antimicrobial effectiveness. Another reason could be related to the pore size or surface chemistry of the activated carbon. If the pore size of the activated carbon is not suitable for retaining chitosan or if the surface chemistry of the activated carbon hinders the interaction between chitosan and microorganisms, it could lead to a loss of antimicrobial activity.
However, our results demonstrated an increase in antimicrobial activity when thyme oil was incorporated into the CS/AC matrix. The presence of thyme oil led to a notable enhancement in the inhibitory effect against S. aureus and S. enterica. The antimicrobial efficacy observed suggests a synergistic effect between the constituents of the composite matrix, particularly chitosan, activated carbon, and thyme oil.
It’s worth noting that the effectiveness of activated carbon in limiting the migration properties of an antimicrobial agent can vary depending on the specific characteristics of the antimicrobial agent and the properties of the activated carbon. In the case of chitosan, further investigation and experimentation would be required to understand why its antimicrobial activity is completely lost when incorporated into activated carbon.
In this study, CS served as the base material for the film, and its inherent antibacterial activity likely played a crucial role in controlling microbial growth, which was further amplified by the addition of thymol-modified activated carbon (TO@AC). The incorporation of specific components, such as thyme oil, can enhance the antibacterial efficacy of CS/PVOH films. However, the addition of activated carbon did not exhibit any inhibitory effects on the tested bacteria. These findings emphasize the need to optimize formulations and concentrations to maximize the antibacterial activity of film materials for various applications, including food packaging and biomedical devices. Azmi et al. (2022) provide insights into the various factors influencing the antibacterial properties of clay, which may be a significant avenue for future exploration if considering clay-based nanohybrids for antimicrobial applications [
39].
3.7. Visual Evaluation of the obtained Active Coatings against enzymatic browning of fresh bananas
In the
Figure 6 representative photos during the experimental period of 8 days of the uncoated bananas and bananas coated with CS/PVOH, CS/PVOH15AC and CS/PVOH/15TO@AC coatings are shown. It must be mentioned that the CS/PVOH15AC and CS/PVOH/15TO@AC samples are chosen to apply as bananas coatings as it was shown to have the highest water/oxygen barrier properties and the highest antioxidant activity. With a first glance it is obvious that all the CS/PVOH, CS/PVOH15AC and CS/PVOH/15TO@AC coatings succeed to protect bananas form the enzymatic browning in comparison to uncoated bananas [
24]. This result is probably due to the CS/PVOH and
CS/PVOH/15AC hydrogel coatings, which probably succeeded in protecting the fresh bananas from enzymatic browning growth. With a more careful glance it is obtained that CS/PVOH/15AC and CS/PVOH/15TO@AC coatings are succeeded to protect more effectively the bananas form enzymatic browning. The extension of life duration was confirmed only by visible observations, and it was around 2 to3 days longer for bananas coated with CS/PVOH/15AC and CS/PVOH/15TO@AC coatings as compared to the respective uncoated bananas and bananas coated with pure CS/PVOH coating.
In
Figure 7 they are plotted the average values of % weigh loss of uncoated and uncoated bananas. It is obvious that the group of bananas coated with CS/PVOH/15TO@AC coating exhibited the lower % weight loss during the experimental period of 8 days. The group of bananas coated with CS/PVOH/15AC coating exhibited higher % weight loss values than bananas coated with CS/PVOH/15TO@AC coating but much lower % weight loss values than bananas coated with CS/PVOH coating. So, it is concluded that the % weight loss values sequence following the trend:
uncoated bananas>CS/PVOH coated bananas>CS/PVOH/15AC coated bananas> CS/PVOH/15TO@AC coated bananas. The results of % weight loss of fresh bananas during the 8 days period of storage agrees with the results of the visual evaluation of active coating in the protection of fresh bananas against the enzymatic browning deterioration process.
Figure 6.
photos during the experimental period of 8 days of the uncoated bananas and bananas coated with CS/PVOH, CS/PVOH15AC and CS/PVOH/15TO@AC coatings.
Figure 6.
photos during the experimental period of 8 days of the uncoated bananas and bananas coated with CS/PVOH, CS/PVOH15AC and CS/PVOH/15TO@AC coatings.
Figure 7.
average % weight loss values of uncoated and bananas coated with CS/PVOH, CS/PVOH/15AC and CS/PVOH/15TO@AC coatings.
Figure 7.
average % weight loss values of uncoated and bananas coated with CS/PVOH, CS/PVOH/15AC and CS/PVOH/15TO@AC coatings.