2.1. Chemical–Physical Characterization of the Zn-Modified Clays
In
Figure 1a, the FT-IR/ATR spectra of ZnMMT and its precursors is reported. It is worth noting that the ATR peaks of ZnMMT were almost similar to those of MMT, i.e., a band at ~3620 cm
−1 attributed to Al-OH group stretching and a band at 1028 cm
−1, attributed to stretching vibration of Si-O [
28]. No trace of zinc nitrate was detected in the ZnMMT sample. Thermogravimetric analysis, TGA and DTGA, reported in
Figure 1c and 1e, respectively, also evidenced a strong similarity between MMT and ZnMMT clays. The residue of ZnMMT at 800 °C was 89.6% and the first thermal event, centered at 76 °C and corresponding to 4% weight loss, was due the loss of interlayer water, while the second event at 491 °C could be linked to the loss of coordination water, with a weight loss equal to 6.3%. ATR spectra of ZnZEO and its precursors were reported in
Figure 1b. ATR of zinc acetate showed the band of OH stretching at 3075 cm
−1 and the asymmetric and symmetric stretching of COO
−, falling at 1547 and 1435 cm
−1. Moreover, ATR analysis of ZnZEO evidenced that the calcination allowed for the almost total acetate removal. The band at 976 cm
−1 present in ZEO spectrum was related to the Si–O–Si or Si–O–Al vibrations in tetrahedra or alumino- and silicon–oxygen bridges [
12]. In ZnZEO system, in addition to the typical absorptions of clay, the analysis evidenced the presence of bands at about 3350 cm
−1, which could be relate to the stretching vibration of OH present in Zn(OH)
2 species. Other modes originating from this compound were observed at lower wavenumbers (in the range 1100–900 cm
−1) [
29]. TGA and DTGA of ZnZEO and its precursors are reported in
Figure 1d and
Figure 1f, respectively. Zinc acetate thermogravimetric analysis showed a first event linked to the presence of physically adsorbed water at about 115 °C (weight loss of 14%), a second event probably linked to coordination water at 175 °C (weight loss of 14%), and a third event at 278 °C (weight loss of 57%), ascribable to the transformation of anhydrous zinc acetate to ZnO, a process occurring at about 300 °C [
30]. As far as the ZEO sample is concerned, the first event at 182 °C was relevant to desorption of physically adsorbed water or other volatiles within the zeolite cages, as well as the water located in zeolite cavities and bound to the non-framework cations (weight loss of 10.7%). The second event, at about 348 °C (weight loss 3.4%), could be associated to the water loss from hydration complexes formed with the exchangeable cations [
31]. TGA (and DTGA) traces of the Zn-modified clay evidenced an anticipation of the first event, falling at about 141 °C, probably linked to the presence of Zn(OH)
2, in agreement with what was observed by XRD analysis. The second event disappeared, while a third event, not present in the ZEO thermogram, fell at about 594 °C. In the literature, a third weight loss in the range (450–500 °C) for zeolites was reported to be due to the structural water and the destruction of the zeolite structure [
32].
As far as XRD analyses are concerned, MMT and ZnMMT showed the same XRD pattern (
Figure 2a), suggesting a simple cation exchange process without structural modifications of the clay mineral. In
Figure 2b, the XRD pattern of the zeolite 4A used in the experiments is reported together with the indexes of the main diffraction peaks, according to the reference 00-043-0142 PDF file (International Centre for Diffraction Data,
https://www.icdd.com/, accessed on 30 June 2024). Simple calcination at 500 °C did not modify the zeolite structure while the Zn-modified zeolite (ZnZEO) presented several differences compared to the initial zeolite 4A (
Figure 2c). Specifically, changes in the relative intensity of almost all the zeolite 4A diffraction peaks were observed together with a shift of all the peaks to higher 2Θ° values. The shift was confirmed adding a corundum internal standard. Alswatha et al. [
33] observed the same modification of the diffraction pattern, suggesting a small compression of the zeolite crystalline structure after the treatment used to modify the zeolite with Zn (ZnZEO), without changing the cubic structure. New peaks were observed after the treatment of the zeolite with Zn at 11.98, 15.78, 31.39, 32.00, 35.83, and 54.64 2Θ° (
Figure 2d) and were attributed to Zn(OH)
2 (PDF n°00-020-1437) and ZnO (PDF n°01-079-0205), respectively.
XPS analysis proved to be particularly useful for understanding the changes in the clays surface composition. Unfortunately, composites containing thymol or carvacrol cannot be examined by XPS due to the volatility of these organic molecules. The atomic percentages of the elements detected on the surfaces of the investigated samples are reported in
Table 1. Comparing XPS data relevant to MMT and ZnMMT samples, it can be observed the disappearance of the calcium signal in ZnMMT obtained with the Zn exchange procedure suggesting that calcium ions were replaced with zinc ones. The atomic percentages relevant to C1s, O1s, Al2p, Si2p, and Mg1s signals remained substantially unchanged in MMT and ZnMMT samples. No nitrogen signal relevant to Zn(NO
3)
2 reagent was detected on ZnMMT. As far as ZEO samples are concerned, XPS analysis suggests that the introduction of zinc can be associated both to the exchange with sodium ions and to an increase of the O1s/Si2p ratio, due to the ZnO and Zn(OH)
2 species, revealed also by XRD analysis. Moreover, the ZEO-Zn(Ac)
2 calcination at 500 °C induced a decrease of the C/Zn ratio from 1.6 to 1.2, indicating the removal of acetate molecules in agreement with what observed by ATR-FTIR analysis.
FT-IR/ATR spectra of thymol and carvacrol, reported in
Figure 3a,b, showed a band at about 3200 and 3360 cm
−1, corresponding to phenolic O–H stretching involved in H bonds. The stretching of C–H fell in the 3000–2850 cm
−1 range. The C=C stretching (1622 cm
−1), –OH bending (1360 and 1362 cm
−1), and C–O stretching (1242 and 1250 cm
−1) were typical of phenolic groups of thymol [
34] and carvacrol [
35]. Moreover, a distinction between the two regioisomer spectra can be made considering the fingerprint region: the meta-substitution (thymol) brings the aromatic C–H bending absorption at 1058 cm
−1, while the orthosubstitution (carvacrol) brings it at 994 cm
−1, as reported in the literature [
36]. As far as the hybrid composites ATR analyses are concerned, no significant variations in the thymol and/or carvacrol and ZnMMT and/or ZnZEO IR spectra were detected. However, in ZnMMT-active molecule spectra, a broadening of the Al–OH stretching band characteristic of MMT-based clay was observed. This can be related to the H-bonds instauration between the clay and the active molecules, as hypothesized in the discussion of TGA results (see below) and as previously reported by Essifi et al. [
13].
As far as TGA analyses are concerned (
Figure 3c,d), thymol and carvacrol compounds showed a decomposition peak centered at 183 °C [
12] and 230 °C [
37], respectively. Concerning the hybrid composites prepared (i.e., ZnMMT–thymol, ZnMMT–carvacrol, ZnZEO–thymol, and ZnZEO–carvacrol), two main mass losses were detected, i.e., a first weight loss below 220 °C (probably due to active molecule and/or water/volatiles evaporation) and a second weight loss between 300 and 600 °C. Similar results were also reported by Essifi et al. [
13], which ascribed this second stage to the desorption of the active molecules interacting with the clay surface through hydrogen bonds between OH groups of thymol or carvacrol and OH groups of the hosting clay.
2.2. Antioxidant Activity Evaluation of Thymol and Carvacrol
The antioxidant activity of thymol and carvacrol is well documented in the literature by different studies that describe the concentration-dependent scavenging effect [
38,
39,
40]. The ABTS and DPPH antioxidant assays were performed on both thymol and carvacrol free molecules. As far as the ABTS test (
Figure 4a), both molecules showed excellent antioxidant activity at maximum of the tested concentrations (300 μg/mL) with values of 97.2 ± 0.3% and 99.2 ± 0.7% for thymol and carvacrol, respectively. In this test, carvacrol provided a major antioxidant effect in the range 300–10 μg/mL in comparison to thymol, which shows a similar but less intense concentration-dependent trend. On the other hand, in the DPPH radical scavenging test (
Figure 4b), samples provided a low antioxidant activity even at the higher concentration (100 μg/mL), with the highest RSA value (%) equal to 20.3 ± 0.1% and 21.1 ± 0.1% for thymol and carvacrol, respectively. For both molecules, a relationship between the percentages of the RSA values and the concentrations was observed, in agreement with previously reported data [
38,
39].
ABTS and DPPH tests have been also carried out on thymol and carvacrol loaded Zn-modified clays to verify if the molecules’ antioxidant activity can be modified by the presence of the clay matrix. In
Figure 4, concentrations on X-axes are relevant to carvacrol or thymol loaded in the clays and evaluated by GC-MS.
ABTS test showed a decrease in antioxidant activity compared to free thymol and carvacrol, demonstrating the presence of a matrix effect.
The DPPH assay’s results are reported in
Figure 4b. RSA% values for concentrations between 100 and 25 μg/mL remained substantially unchanged with respect to what observed for free molecules. On the other hand, at concentrations lower than 18.9 μg/mL, ZnMMT systems showed an almost stable antioxidant activity always better than ZnZEO systems.
2.5. Antimicrobial Activity
The inhibition zones produced by thymol, carvacrol, and their composite materials, evaluated at a concentration of 50 mg mL
−1 of organic molecule, are shown in
Table 3 and
Table 4. One-way ANOVA analysis showed a significant effect (
p ≤ 0.05) of the type of organic molecules or their composite materials on the inhibition zone values. Carvacrol and thymol produced inhibition zones against all spoiler and pathogenic strains, but with higher values against pathogenic bacteria than spoilage bacteria. The most sensitive strains were
Erwinia persicina ITEM 17997 and
Staphylococcus aureus DSM 799, whereas the most resistant strains were
Pseudomonas chicorii ITEM 17296 and
P. aeruginosa DSM 939, for spoiler and pathogenic strains, respectively. Clays (MMT or ZEO) did not show antibacterial activity against all target strains. MMT and ZEO–clay hybrids loaded with carvacrol and thymol showed, in most cases, inhibition zones comparable to pure compounds. MMT–carvacrol showed inhibition zones significantly higher than carvacrol for
P. chicorii ITEM 17296 and
Escherichia coli ATCC 35401, whereas MMT–thymol showed inhibition zones significantly higher than thymol for all spoiler strains, and also for pathogenic strains of
E. coli ATCC 35401,
Listeria monocytogenes DSM 20600, and
Sta. aureus DSM 799 (
Table 3). ZEO–carvacrol showed inhibition zones significantly higher than carvacrol for
P. putida ITEM 17297,
P. chicorii ITEM 17296,
P. aeruginosa DSM 939,
Salmonella enterica ATCC 13311, and
Sta. aureus DSM 799, whereas ZEO–thymol showed inhibition zones significantly higher than thymol for
P. chicorii ITEM 17296,
P. aeruginosa DSM 939, and
Sta. aureus DSM 799 (
Table 4).
Clay hybrids loaded with essential oil compounds such as thymol and carvacrol showed improved antibacterial activity as compared to pure essential oil compounds. However, few studies evaluated the antibacterial activity of clay hybrids similar to those characterized in our work. In particular, Cometa et al. [
12] found that ZEO4A/thymol hybrid showed better antibacterial activity than thymol, at the same concentration of the essential oil compound (75 mM), against
P. aeruginosa DSM 939. Bernardos et al. [
41] found lower MIC values of MMT loaded with thymol, eugenol, or carvacrol than pure compounds against
Sta. aureus. MMT-Thy was in most cases more active than MMT-Car against bacterial strains. This result was also confirmed by Zhong et al. [
42] with palygorskite loaded with thymol or carvacrol against
E. coli and
Sta. aureus.
Regarding Zn-modified clays, ZnZEO showed no inhibition zones against target strains, whereas ZnMMT showed inhibition zones against all strains except for
P. aeruginosa DSM 939. ZnMMT–carvacrol produced inhibition zones significantly higher than MMT–carvacrol for all strains, except
Sal. enterica ATCC 13311. ZnMMT–thymol produced inhibition zones significantly higher than MMT–thymol for many of strains, except for
Erw. persicina ITEM 17997,
E. coli ATCC 35401,
P. aeruginosa DSM 939, and
Sta. aureus DSM 799 (
Table 3). Zn-modified ZEO hybrids showed in most cases a reduction in antibacterial activity compared to ZEO hybrids without zinc. An improvement in antibacterial activity was detected for ZnZEO–carvacrol against
E. coli ATCC 35401 and
Sta. aureus DSM 799, and for ZnZEO–thymol against
Erw. persicina ITEM 17997,
P. putida ITEM 17297,
E. coli ATCC 35401, and
L. monocytogenes DSM 20600. The inhibition zones produced by clay hybrids were generally improved in Zn-modified clay hybrids. In this respect, carvacrol incorporated onto ZnO/palygorskite nanoparticles showed lower MIC values against
E. coli than carvacrol/palygorskite composite material [
43]. The most interesting result we found was the different antibacterial activity of Zn-modified ZEO and MMT hybrids. ZnMMT hybrids always showed better antimicrobial performances than ZnZEO hybrids. This result could be explained by the higher zinc release from the MMT hybrids than ZEO clay materials. Regarding the antibacterial mechanism of action, Zn and monoterpenes could act synergically targeting the structural integrity of the cell, the electron transport system, various enzymatic activities, the nucleic acid, protein and cell wall synthesis, and membrane functionality [
2,
44].
Our findings are in accordance with those of Li et al., which demonstrated the improving in antibacterial activity of
Eucalyptus citriodora essential oil, lacking in both carvacrol and thymol, in the presence of zinc ions against
E. coli O157:H7 [
45]. Conversely, Windiasti et al. demonstrated a synergistic effect of carvacrol and ZnO nanoparticles against
Campylobacter jejuni, where carvacrol damaged the cell membrane followed by the cell leakage induced by ZnO [
46].
To compare ZEO and MMT hybrids, the ratio among inhibition zones of hybrids against pure organic molecule was calculated (
Table 5). Zn-modified MMT hybrids generally showed average antibacterial activity higher than that displayed by Zn-modified ZEO hybrids.
For this reason, a deep evaluation of the antibacterial activity of Zn-modified MMT clays was performed through the determination of MIC values in comparison to pure carvacrol and thymol, loading blank disks with of 20 μL of different samples in the range of 50–3.15 mg mL−1 of organic molecule.
Carvacrol and thymol showed a MIC value lower than 3.15 mg mL
−1 for
Erw. persicina ITEM 17997 and
Pectobacterium carotovorum subsp.
carotovorum LMG 2404, and of 25 mg mL
−1 against
P. putida ITEM 17297 and
P. chicorii ITEM 17296. For bacterial pathogens, carvacrol showed a MIC value lower than 3.15 mg mL
−1 for both
E. coli strains and
Sta. aureus DSM 799, of 6.3 mg mL
−1 against
L. monocytogenes DSM 20600 and
Sal. enterica ATCC 13311, and of 25 mg mL
−1 against
P. aeruginosa DSM 939; thymol showed a MIC value of 6.3 mg mL
−1 against
P. aeruginosa DSM 939, and lower than 3.15 mg mL
−1 for the remaining pathogens. In the case of ZnMMT loaded with carvacrol and thymol, MIC values were lower than 3.15 mg mL
−1 for all strains. Only
P. aeruginosa DSM 939 showed higher MIC values, specifically 25 mg mL
−1 for ZnMMT–carvacrol and 12.5 mg mL
−1 for ZnMMT–thymol (
Table S1).
The comparison of the antimicrobial activity between carvacrol and thymol showed that the average activity displayed by carvacrol for all concentrations and for all target strains resulted to be the 62.70 ± 17.76% for pathogens (range of 31.88–86.29%) and 84.85 ± 32.40% for spoilers (range of 59.62–132.42%).
To understand the effect of the hybrid material type (ZnMMT–carvacrol or ZnMMT–thymol) on the inhibition zones values, measured at 25 mg mL
−1 of carvacrol or thymol equivalent concentration, the Shapiro–Wilk test followed by a bilateral T Student test was applied. For both spoiler and pathogenic strains, replicate values of inhibition zones, produced by ZnMMT–carvacrol and ZnMMT–thymol, were significantly different for
p < 0.05. On these premises, the average inhibition zones of ZnMMT–carvacrol and ZnMMT–thymol were compared independently by the carvacrol- or thymol-equivalent concentration showing higher antibacterial activity of ZnMMT–thymol than ZnMMT–carvacrol against eight out of ten bacterial strains (
Figure 8).
Based on these results, antimicrobial activity assays moved from the measurement of inhibition zones to the calculation of minimal inhibitory concentration (MIC) of pure thymol and ZnMMT–thymol in broth culture medium. The MIC values were obtained by means of the difference in absorbance values (OD
600nm) between the beginning and the end of incubation, attributing the presence of growth when this difference was ≥0.1 (
Figure 9).
Thymol produced MIC values of 20 mM (corresponding to 3 mg thymol mL
−1) against
E. coli ATCC 35401,
L. monocytogenes DSM 20600,
Erw. persicina ITEM 17997,
P. chicorii ITEM 17296,
P. fluorescens ITEM 19245, and
P. putida ITEM 17297, and of 10 mM (corresponding to 1.5 mg thymol mL
−1) against
E. coli ATCC 8739,
Sal. enterica ATCC 13311,
Sta. aureus DSM 799, and
Pec. carotovorum sub.
carotovorum LMG 2404. The MIC value for
P. aeruginosa DSM 939 was higher than 20 mM thymol, as highlighted by the red arrow in
Figure 9. The viability assay carried out at the end of 24 h incubation informed us that thymol produced MBC values of 20 mM against all strains, except for
Erw. persicina ITEM 17997 and
P. aeruginosa DSM 939, which were more resistant (MBC values higher than 20 mM).
Based on these values, a new test was set up aiming at the evaluation of Zn–MMT–thymol antibacterial activity. To compare the thymol antibacterial performance with that of ZnMMT–thymol, the average loading of thymol in Zn–MMT was considered 28.5%. The tests were therefore carried out at the same thymol concentration in hybrid material as those of pure thymol. Due to the turbidity produced by ZnMMT–thymol, we proceeded to measure the viable cells directly in the control broth medium and in that supplemented with ZnMMT–thymol at 0.5xMIC values. The results are reported in
Table 6.
The ZnMMT–thymol showed stronger antimicrobial activity than thymol in solution, probably due to the presence of the Zn ions loaded in MMT that were largely released during 24 h at 37 °C (
Figure 6 and
Figure 7). At the thymol concentration of 20 mM (3 mg mL
−1),
P. aeruginosa were still alive at a load of approximately 8 log cfu mL
−1, while at the same concentration of thymol included in the ZnMMT–thymol, no viable cells were found. We can guess that this improvement is the result of synergistic or additive antimicrobial activity between thymol and zinc ions.