2.2. Chemical Structure of dCs-ε-CL and dCs-ε-CL(MSA)
The first stage of our research was to obtain a chitosan derivative containing long aliphatic side chains. This structure of the copolymer, following previous observations, should not only reduce the solubility of chitosan in water, allow obtain blends with many synthetic polyesters, such as polycaprolactone but also increase the antibacterial activity of chitosan due to the possibility of a stronger destructive effect on the bacterial cell wall [
62]. These types of chains were introduced into the molecule through the grafting reaction of chitosan with ε-caprolactone. This process was carried out using two methods. In the first method, copolymerization was carried out in a DMSO solution, carrying out ROP of ε-caprolactone initiated by the zinc complex Zn[(acac)(LPhe)H
2O] containing ligands composed of acetylacetonate groups and Schiff bases (LPhe) obtained during the condensation of phenylalanine with pyridine carboxaldehyde [
63]. The choice of the initiator was determined by the practical lack of cytotoxicity and the confirmed high efficiency of this initiator during the polymerization of ROP lactide or lactide copolymerization, as well as its additionally confirmed high bactericidal activity [
64]. Polymerization was carried out at different molar ratios of glucopyranoside repeating units of chitosan/ε-caprolactone, obtaining chitosan grafted through an amine group with a caproyl chain (dCs-ε-CL). In the second method used, the process was carried out in a methanesulfonic acid environment, modified by the method described earlier, to obtain copolymers dCs-ε-CL(MSA) containing caproyl chains grafted onto the hydroxyl groups of chitosan [
65].
The chemical structures obtained copolymers of dCs-ε-CL and dCs-ε-CL(MSA) were verified by
1H-NMR and FTIR spectra. In the
1H-NMR spectrum, as illustrated in
Figure 2a for dCs-ε-CL, the peaks at 2.8–3.0, 3.4–3.8 ppm were assigned to f, g, h and i protons in Cs units according to the previous assignment of signals in the poor chitosan spectrum (
Figure 1). The e, a, b, c, d signals were shown at the peaks of protons -(CO)OC
H2-, (CO)C
H2, (CO)CH
2CH2-, (CO)CH
2CH
2CH2- and (CO)CH
2CH
2CH
2CH2- in caproyl units respectively (
Figure 2a). This spectrum also shows the second series of signals e', a', b', c', and d', which were assigned to analogous protons of the appropriate groups of caproyl units terminating the grafted PCL chains.
In the case of the dCs-ε-CL(MSA) spectrum, in addition to the signals e, a, b, d, and c mentioned above, there were also very strong analogous signals E, A, C, and B+D (
Figure 2b). These are signals characteristic of high-molecular-weight PCL [
66]. They correspond to the protons of the caproyl units of the grafted PCL.
The copolymers obtained in the ROP reaction initiated with the zinc (II) complex, despite the stoichiometric excess of caprolactone being used up to five times different, ultimately contained a similar, quite small amount of attached caprolactone units. Based on the ratio of signal intensities i to (A+a)/2 (
Figure 2a), the molar ratio of glucopyranoside chitosan units to caproyl units was calculated and amounted to approx 1:40. At the same time, the presence of intense signals related to the protons of the end group units of the created caproyl chains indicates the short length of the PCL sequences. The calculated average length of the created caproyl blocks (from the intensity of the a and a' signals) was only about 5 caproyl units. At the same time, among the signals related to the ring units, there are no visible signals f' related to the presence of protons of –C
H(NH
2)- glucopyranoside rings groups, but there are also visible f signals (2.9 ppm) assigned to the signals of protons –C
H(NH-CO(CH
2)
5OCO(CH
2)
5)
n–, formed as a result of the growth of the caproyl chain on the amino groups. The above data show that during the grafting reaction initiated with the zinc (II) complex, a chitosan copolymer was obtained with short caproyl sequences grafted on the amino groups. The hydroxyl groups of the chitosan ring obtained via ROP initiated with the zinc (II) complex did not undergo the grafting reaction (
Figure 2a).
The chitosan copolymer obtained in a reaction involving methanesulfonic acid has a completely different structure. In this case, we obtained copolymers with long caproyl blocks (signals: E, A, B, and C+D). In the spectrum of this compound is also a strong f' signal (3.05 ppm) associated with the presence of protons of the -CH(NH2)- groups, which indicates that the growth of caproyl blocks took place mainly on hydroxyl groups. The molar ratio of glucopyranoside chitosan units to caprolactone units ranged from 10 to 20, depending on the starting stoichiometric excess of ε-caprolactone used. The presence of weak signals e, a, b, c, and d, located practically identically as in the previous spectrum, show that some of the amino groups were subject to a grafting reaction, which resulted in the formation of a relatively small amount of caproyl sequences (up to about 10% of the total caproyl units) grafted onto the amino groups.
The rate of the chitosan grafting process was also observed by following changes in the FTIR spectrum, as illustrated in
Figure 3. For the Cs, the bands at 890 cm
-1 corresponded to the C-O stretching vibrations of glycoside in polysacharide structure and 1147 cm
-1 was related to asymmetric C-O stretching vibrations of C-O-C. The bands at 1614 cm
−1 and 1640 cm
−1 were attributed to deformation vibrations C-H in amine groups. In the case of the dCs-ε-CL spectrum, the signals related to the amino groups become less intensive, meanwhile signals related to C=O stretching vibrations of amide (I) bands appeared. Peaks at 2862 cm
−1 and 2945 cm
−1 were related to C-H stretching vibrations of Cs which have been intensified in copolymer spectrum accumulated by C-H stretching vibrations of CL units. At 3445 cm
−1, O-H and N-H stretching bands of chitosan overlapped These signals occur in all tested copolymers, which suggests that in the case of dCs-ε-CL(MSA), the growth of grafted PCL chains takes place only on part of the hydroxyl groups of chitosan units. The characteristic absorbance of the PCL ester bond appeared at 1725 cm
-1. The copolymers of dCs-ε-CL and dCs-ε-CL(MSA) presented absorbance bands at 1725, 1640, and 1614 cm
-1, which were assigned to the characteristic bands of ester in PCL, amide I band, and amino groups in Cs, respectively. The appearance of this signal in the spectra of dCs-ε-CL and dCs-ε-CL(MSA) confirms the grafting process of PCL onto Cs through its amino groups [
67,
68,
69]. Due to the much higher signal intensity at 1725 cm
-1 of the spectrum of the copolymer obtained during the grafting reaction with methanesulfonic acid, the higher content of caproyl units in this copolymer compared to the analogous one, obtained with the zinc complex was also confirmed.
2.3. Chemical Structure of Schiff Base dCsSB-PCA; dCsSB-SFD and Copolymers dCsSB-PCA-ε-CL; dCsSB-SFD-ε-CL
Fabrication of the antibacterial material was carried out by condensation reaction of deacetylated chitosan (dCs) with 2-pyridine carboxaldehyde (PCA) or sodium 4-formylbenzene-1,3-disulfonate aldehyde (SFD) via Schiff base formation. This reaction was carried out with a 20% stoichiometric excess of aldehyde (calculated concerning amino groups of chitosan). In this way, chitosan derivatives with significantly different properties were obtained. A Schiff base obtained from pyridine carboxaldehyde (CsSB-PCA), was slightly soluble in water and easily soluble in selected organic solvents. The chitosan derivative obtained by condensation reaction with the sodium salt of 4-formylbenzene-1,3-disulfonate (dCsSB-SFD) was practically insoluble in organic solvents.
The structure of the obtained chitosan-based Schiff bases was confirmed by
1H-NMR (
Figure 4 and
Figure 5). Comparing the obtained modified chitosan CsSB-PCA with Cs, the appearance of new signals was noticed. Signal 1 at 8.8 ppm corresponded to the imine protons (-N=C
H-ArN)-, while those at 8.4; 8.1; 7.9, and 7.7 ppm correspond to the 6, 5, 3, and 4 protons on the pyridine ring residues, respectively. The appearance of signal 7 (4.75 ppm) associated with the proton of chitosan units near the imide group (-C
H-(N=CH-ArN) was also noticed. However, the signal f (2.8 ppm) associated with the analogous proton near the amino group was still visible (-C
H-NH
2). The degree of substitution (DS) was calculated from the ratio between the integrated resonances of the hydrogen at carbon 1 in the imine groups and the sum of protons (f + 7) of the glucopyranoside ring. It was calculated that despite the large excess of aldehyde, only about 45% of the amino groups reacted.
The
1H NMR spectrum of dCsSB-SFD was obtained in D
2O (
Figure 5) because the product was insoluble in DMSO. Also, in this case, signals analogous to the spectrum discussed earlier appeared too. Signal 1 (9.3 ppm) was assigned to protons – N= C
H – Ar(SO
2)
2, signal 5 (3.8 - 4.0 ppm) to protons of the chitosan ring units -C
H(N=CH-Ar(SO
2)
2)-. A quite strong characteristic signal of the protons of the chitosan units connected to the amino group, the signal f -C
H(NH
2) was also still visible. In this spectrum, there were also signals of the protons of the aromatic ring 4 and 2,3. The degree of amino group substitution was estimated at approximately 55%. In the next step, it was decided to obtain PCL-grafted chitosan containing the discussed imide side groups. For this purpose, the Schiff bases dCsSB-PCA and dCsSB-SFD reacted with ε-CL using zinc complex Zn[(acac)(LPhe)]H
2O as an ROP initiator. The described method of chitosan copolymerization by ε-CL grafting with the presence of methanesulfonic acid could not be used in this case, because the acidic reaction environment caused rapid hydrolysis of the Schiff base. The reverse procedure was also difficult to carry out due to the insolubility of chitosan/caprolactone copolymers in solvents of the aldehydes.
The
1H NMR spectra of obtained copolymers are pictured in
Figure 6 and
Figure 7. In the spectrum of dCsSB-PCA-ε-CL (
Figure 6), all signals appeared in the previously presented spectrum of the output Schiff base (
Figure 4).
The protons described as the letters f, g, h, and j on the HNMR spectrum correspond to the chitosan moiety chain. The signals described in the numbers of this spectrum are associated with the Schiff base. The peak at 8.8 ppm corresponds to imine protons 7, and the signals at 8.1, 8.8, 7.9, and 8.3 ppm are associated with the aromatic protons on the pyridine ring residues. The protons described as the letters a, b, c, d, and e are assigned to caproyl units of PCL. However, unlike the previously presented dCs-εCL spectrum (
Figure 2a), the signals assigned to the protons of the end groups of the caproyl block (a', b' c', d' and e') were either very weak or invisible. This proves that the average length of the caproyl blocks in the dCsSB-PCA-εCL copolymer was much longer than in the dCs-εCL. There was also a clear f'' signal (3.2 ppm) assigned to the protons of the -C
H- groups of chitosan units located in the vicinity of the caproyl chains. However, a strong f signal associated with the protons of the -C
H(NH
2)- groups was still visible. The estimated degree of substitution of amino groups D in the obtained dCsSB-PCA-εCL, calculated according to equation; D = (f''+ 7)/(f+f''+ 7) x100% was approximately 70% in total (almost 60% substitution with Shiff bases and about 10% PCA chain). The composition of the obtained copolymer was estimated based on the intensity of signals f', f, 7, 1 and a is; 47 mol. % chitosan units, 37 mol.% Schiff base, 16 mol.% caproyl units.
The obtained
1H-NMR spectrum of the dCsSB-SFD-ε-CL with assigned signals is presented in
Figure 7.
And in this case, based on the intensity of the signals of the caproyl units a, b, c, d, and e, and the end groups of the caproyl blocks (signals b', c', d' and e'), it can be seen that the average length of the block is similar to the length of this obtained during the grafting reaction of chitosan itself, initiated by the zinc (II) complex. There is an f' signal in the spectrum, indicating the growth of caproyl blocks on amino groups. Signal 5 is also well visible coming from the protons of the -CH- group of chitosan linked to the imide group of the formed Schiff base. Moreover, signals characteristic of chitosan (g, h, j) and Schiff base (3, 2, 4, and 1) can be observed too. The composition of the obtained copolymer estimated based on the intensity of signals f', 5, 1, and a was; 40 mol% chitosan units, 20 mol% Schiff base, and 40 mol% caproyl units. The FTIR spectra of dCs, PCA, and the obtained Schiff base (dCsSB-PCA) and their copolymer with ε-CL (dCsSB-PCA-ε-CL) are shown in
Figure 8a. The main changes that can be highlighted between the spectra of dCs and dCsSB-PCA are the presence of bands at 1571 cm
−1 and 1432 cm
−1 related to the stretching vibrations of the pyridine ring. The appearance of these bands is due to the introduction of a pyridine moiety into the chitosan backbone. In addition, the presence of a band at 776 cm
−1 was also noticed, corresponding to the out-of-plane bending of C-H in the pyridine ring. This spectral data indicated that the chitosan modified by PCA had been successfully prepared. For the copolymer of dCsSB-PCA-ε-CL, the characteristic absorbance of the ester in PCL was demonstrated at 1731 cm
-1, which implied the success of grafting PCL onto dCsSB-PCA [
10,
56,
68].
Figure 8b shows the FTIR spectra of dCs, SFD, and the obtained Schiff base (dCsSB-SFD) and their copolymer with ε-CL (dCsSB-SFD-ε-CL). The characteristic bands at 1190 cm
-1 and 1030 cm
-1 are attributed to the S=O antisymmetric and S=O symmetric stretching bands. The small but significant characteristic peak at 960 cm
-1 for the sulfonated samples may be due to the S–O–C stretching vibrations. For the dCsSB-SFD-ε-CL copolymer, the characteristic C=O band was observed to be slightly shifted to lower values at 1704 cm
-1, which confirms the grafting of PCL onto dCsSB-SFD [
35,
69].
2.4. Hydrogel Blends with Carrageenan, Swelling Properties
Due to the possibility of using the developed copolymers in cosmetics and medicine, in many applications (moisturizing creams, dressings, drug-releasing systems), a particularly desirable property of such biodegradable and bactericidal materials is also the ability to create hydrogels with high water absorption above 1000%. For this purpose, the properties of carrageenan were used to create blends with this polysaccharide by dissolving it in water and mixing it with aqueous solutions of the produced chitosan derivatives. After the freeze-drying procedure, after removing virtually all the water, a porous solid form of unswelling hydrogel was obtained and used for water absorption tests. In addition to polymer composites containing chitosan derivatives, these tests also included chitosan and carrageenan themselves for comparison.
The swelling properties are considered an important feature for the transportation of drugs from hydrogels because it has a great influence on the pore sizes of hydrogels [
70]. The swelling ratio (SR) of prepared hydrogels as a function of time at room temperature and at 36 °C are presented in
Figure 9. All tested materials belong to the group of high-swelling hydrogels [
71]. Based on the obtained results it was found that at room temperature all the obtained blends had a higher swelling ratio than both chitosan and κ-carrageenan. For all tested hydrogels, an increase in temperature also resulted in a visible increase in the degree of swelling.
The observed temperature-responsive swelling behavior is mainly related to the phenomenon of the association/dissociation of hydrogen bonding between the cations, amino groups, carbonyl groups, sulfates, and hydroxyl groups [
72]. The highest SR is observed for dCs-ε-CL (MSA): CG 50:50 blends - containing the highest amount of caproyl and the longest average length of caproyl blocks, much higher compared to analogous blends with chitosan grafted with caproyl in the ROP reaction initiated by the zinc (II) complex) dCs-ε-CL: CG 50:50. For this reason, it seems that the presence of long, flexible chains of hydrophobic caproyl blocks that form hydrogen bonds between carboxyl groups and cations contained in both the carrageenan chain (sulfone groups) and the modified chitosan (amine, imine groups) have a decisive influence on the observed degree of swelling. and sulfonates). At a slightly higher temperature, human skin temperature (36 °C), this difference decreased significantly. The degree of specification of the dCs-ε-CL: CG 50:50 blend is very similar to the blend. However, at an elevated temperature, the highest SR dCs-ε-CL (MSA): CG 50:50 was observed.
In the case of blends containing Cs with chitosan Schiff base, the dCsCB-SFD-ε-CL: CG 50:50 shows a higher degree of swelling. The reason might be that dCsCB-SFD-ε-CL: CG 50:50 is more hydrophilic than the dCsCB-PCA-ε-CL: CG 50:50. The highest SR for dCsCB-SFD-ε-CL: CG 50:50 was recorded after 24 hours (SR = 1260 %). At a temperature of 36 °C, after 3 hours, the Cs hydrogel blends containing Schiff base, or the only Cs gradually degraded and dissolved. To summarize, the swelling properties of the hydrogels strongly depend on composition. The analyzed hydrogels create a three-dimensional macromolecular network capable of absorbing water beyond their volume, which makes it attractive to use as drug carriers, for wound healing and in tissue engineering [
73,
74].
2.5. Thermal Properties
The thermal properties of ĸ-carrageenan, chitosan, and materials obtained based on them were investigated by DSC method (
Figure 10).
Table 2 contains the glass transition and melting temperatures of the semi-crystalline phase. Because chitosan and ĸ- carrageenan can easily absorb water, the second run method is adopted to eliminate the influence of water. From the DSC curve of CG, it was possible to determine the glass transition temperature T
g=88.2 °C and it is similar to what has been reported for ĸ-carrageenan powder [
75]. The glass transition temperature for Cs was determined as T
g 173 °C. It is often difficult to determine the glass transition temperature of chitosan based on DSC curves, and there is a large dispersion between the values in the literature [
76]. Nevertheless, the T
g value obtained here (173.0°C) is comparable to the T
g reported by Martínez-Camacho et al. (170.9 °C) [
77] and Pourjavadi et al. (188°C) [
50]. Two transitions can be observed in the DSC curve of ε-caprolactone grafted chitosan dCs-ε-CL, which coincides with the glass transition temperature of the ε-caprolactone and chitosan block. A different effect is observed for chitosan grafted with ε-caprolactone in the presence of methanesulfonic acid dCs-ε-CL(MSA). Here, in the DSC studies, only the melting point for dCs-ε-CL(MSA) was observed at 50.6 °C (corresponding to the ε-CL chain fragment). This is because dCs-ε-CL(MSA) contains much longer ε-CL chains in its structure than dCs-ε-CL. The obtained Schiff bases only show the glass transition temperature, respectively 141.4 °C for dCsSB-PCA and 102.4°C for dCsSB-SFD, which indicated that the modification of chitosan with aldehyde effectively decreased the glass temperature of chitosan. After the Schiff base grafting reaction with ε-caprolactone two T
g is observed for dCsSB-PCA-ε-CL respectively -38 °C and 143 °C. The occurrence of two transitions is related to ε-caprolactone and chitosan block. In the case of dCsSB-SFD-ε-CL, two glass transition temperatures are also observed, but they are shifted to higher temperatures (20.4 °C and 155.4 °C). This is related to intermolecular interactions such as hydrogen bonding, which may limit the mobility of the polymer [
78].
All prepared blends were compatible as confirmed by DSC. The dCs-ε-CL: CG 50:50 blend shows a slight increase in the glass transition temperature compared to dCs-ε-CL. Such an increase may be caused by intermolecular interactions like hydrogen bonding between the sulfonate groups of ĸ-carrageenan and the hydroxyl groups of chitosan. For dCs-ε-CL(MSA): CG 50:50 blend the endothermic peak is shifted towards a lower temperature and the heat enthalpy has a lower value than for the dCs-ε-CL(MSA). The presence of the sulfonate group in the blends causes a lower melting point in DSC measurements. The same effect was described by T. Tanaka et al. for poly(vinyl alcohol)/ĸ-carrageenan blends [
79]. It was found that the dCsCB-PCA-ε-CL: CG 50:50 blend shows a broad glass transition (T
g=124.1 °C ΔT
g=82.6 °C). In the case of dCsCB-SFD-ε-CL: CG 50:50, glass transition temperatures (-62.2°C and 146.7 °C) are observed related to the presence of blocks derived from ε-CL and dCsCB-SFD. No glass transition temperature from CG was observed.
In conclusion, the lack of glass transition temperature of carrageenan and the occurrence of hydrogen bonding interactions between the functional groups of modified chitosan and ĸ-carrageenan ensure the preparation of compatible blends.
2.6. Antibacterial and Antifungal Evaluation
Following the assumptions of the conducted research, the presented group of synthesized copolymers based on chitosan and their blends with carrageenan, especially those containing Schiff bases, are characterized by bactericidal and fungicidal activity against a wide spectrum of strains. All results presented below were obtained at a polymer concentration of 0.1 mg/mL. The concentration of the compounds was chosen at which the antibacterial activity of chitosan itself was practically unnoticeable.
Figure 11 shows the antibacterial activity of the tested polymers against the E. coli strain. The greatest growth inhibition compared to the control was observed for samples containing Schiff bases. After 24 h, the growth of this strain decreased from 9.00 log
10 CFU/ml to 5.68 log
10 CFU/ml and 5.66 log
10 CFU/ml for the dCsSB-PCA and dCsSB-SFD, respectively. E. coli growth was inhibited also by copolymers of Schiff bases with ε-CL and reached 6.23 log
10 CFU/ml for dCsSB-PCA-ε-CL and 6.66 log
10 CFU/ml for CsSB-SFD-ε-CL. Importantly, blends of these copolymers with CG show greater growth inhibition than the copolymers themselves and are 5.81 log
10 CFU/ml dCsSB-PCA-ε-CL: CG 50:50 and 5.96 log
10 CFU/ml CsSB-SFD-ε-CL: CG 50:50. Despite many literature reports proving the antibacterial activity of chitosan and carrageenan [
59,
80,
81,
82,
83,
84] only a slight decrease in the growth of the E. coli strain was observed at concentration 0.1mg/mL. The antibacterial activity of this strain after 48 h is shown in
Figure S1 Supplementary Materials. The results were similar to those after 24 hours.
The results regarding antibacterial activity against P. aeruginosa are presented in
Figure 12. Similarly to the above, materials containing Schiff bases have the highest activity inhibiting the growth of this strain. Only these materials inhibit the growth of P. aeruginosa to a greater extent than the control. After 24 h, the growth of this strain decreased from 7.35 log
10 CFU/ml to 5.56 log
10 CFU/ml for dCsSB-PCA and 5.58 log
10 CFU/ml for dCsSB-SFD. For chitosan, carrageenan, dCs-ε-CL and dCs-ε-CL(MSA) copolymers and blends of these copolymers with carrageenan (dCsSB-PCA-ε-CL: CG 50:50 and CsSB-SFD-ε-CL: CG 50:50), a greater growth of this strain was observed compared to the control. In
Figure S2 Supplementary Materials the results after 48h are presented, they also show no significant changes compared to 24 h.
Figure 13 presents the antibacterial activity against the S. epidermidis strain. The strongest growth inhibition was observed in the case of 24 h contact with dCsSB-PCA and dCsSB-SFD. The activity of this strain for these samples decreased from 8.84 log
10 CFU/ml to 3.92 log
10 cfu/ml and 4.11 log
10 CFU/ml, respectively. A large decrease in activity was also observed for dCsSB-PCA-ε-CL to 4.21 log
10 CFU/ml and dCsSB-SFD-ε-CL to 4.31 log
10 CFU/ml. For this strain, both after 24 h and 48 h (
Figure S3 Supplementary Materials), all tested samples showed lower growth compared to the control.
In the case of antibacterial activity against the S. aureus strain shown in
Figure 14, in addition to the inhibitory effect of substances containing Schiff bases, the effect of the copolymer of dCs-ε-CL(MSA) can be observed. Activity decreased from 7.80 log
10 CFU/ml to 6.11 log
10 cfu/ml. The inhibitory effect of blends dCsSB-PCA-ε-CL: CG 50:50 and CsSB-SFD-ε-CL: CG 50:50 is also significant, with the activity of this strain decreasing to 5.51 log
10 CFU/ml and 5.45 log
10 CFU/ml, respectively. Similarly to the strains analyzed above, the bactericidal activity of S. aureus remains at a similar level after 48 h (
Figure S4 Supplementary Materials).
The antifungal activity against two selected strains C. albicans and A. brasiliensis is shown in
Figure 14 and
Figure 15. The growth inhibition of the C. albicans strain (
Figure 15) was strongest in the culture with the addition of Schiff bases; copolymers of Schiff bases with ε-CL and blends of these copolymers with CG. After 24 hours, an increase in the strain was observed for chitosan (6.56 log
10 CFU/ml), carrageenan (6.54 log
10 CFU/ml), dCs-ε-CL (6.57 log
10 CFU/ml), DCs-ε-CL(MSA) (6.54 log
10 CFU/ml) and dCs-ε-CL: CG 50:50 (6.27 log
10 CFU/ml) compared to the control (5.58 log
10 CFU/ml). After 48 h (
Figure S5 Supplementary Materials), a decrease in antifungal activity was observed for all tested polymers. Interestingly, one of the greatest inhibitions of C. albicans growth was recorded for the dCS-ε-CL (4.48 log
10 CFU/ml) with a control of 6.91 log
10 CFU/ml.
Figure 16 shows the results of activity tests against the A. brasiliensis strain after 24h. Only for the dCsSB-PCA and dCsSB-SFD, respectively, the same level (3.61 log
10 CFU/ml) or minimal decrease (3.52 log
10 CFU/ml) in activity was observed as for the control (3.61 log
10 CFU/ml). For the remaining samples, the obtained values exceeded the values of the control sample. After 48h (
Figure S6 S
Supplementary Materials), a further decrease in activity was observed for dCsSB-PCA (3.48 log
10 CFU/ml) and dCsSB-SFD (3.45 log
10 CFU/ml), as well as for dCsSB-PCA-ε-CL (3.45 log
10 CFU/ml); dCsSB-SFD-ε-CL (3.45 log
10 CFU/ml); dCsSB-PCA-ε-CL: CG 50:50 (3.36 log
10 CFU/ml) and dCsSB-SFD-ε-CL: CG 50:50 (3.79 log
10 CFU/ml) compared to the control (4.83 log
10 CFU/ml).
In conclusion, antibacterial and antifungal effects of the obtained polymers relative to pure Cs and CG, in all samples, a reduction in the growth of the tested microorganisms was observed. Both obtained Schiff bases (dCsSB-PCA; dCsSB-SFD) have a strong effect of inhibiting the growth of cells of the analyzed strains of bacteria and fungi. This effect is maintained in the case of copolymers of these Schiff bases (dCsSB-PCA-ε-CL; dCsSB-SFD-ε-CL as well as blends of these copolymers with CG (dCsSB-PCA-ε-CL: CG 50:50; dCsSB-SFD-ε-CL: CG 50:50).
The conducted research confirmed that the antimicrobial properties of chitosan can be improved by chemical modification of the Cs structure. The two reactive groups -NH
2 and -OH offer vast opportunities for chemical modification. These groups allow the formation of several functional derivatives via reactions such as sulfonation and amination [
85].