of NPs with bacteria [14, 38-40] and the release of Zn²⁺ ions [41, 42]. For ROS whose amount generated should increase with the concentration of ZnO-NPs [12, 42], they are expected to oxidize the lipid membrane of the cell wall of *E. coli*. The direct interaction between ZnO-NPs and the cell surface affects the permeability of the membrane [14, 40], allows the internalization of NPs [38] and induce oxidative stress in bacteria cell [43]. The solubility of Zn²⁺ ions in the micro-organisms medium could be the cause in the toxicity of ZnO-NPs [13, 19]. Research has shown that ZnO-NPs possess better activity toward *E. coli* as Gram-negative bacteria than toward *S. aureus* as Gram-positive bacteria. The high resistance of *S. aureus* to ZnO-NPs can be interpreted to the structural differences in the bacterial membranes and the intracellular antioxidant content of *S. aureus* [44].

This study is aimed at exploring the feasibility of producing AB-LDPE films by coating with ZnO-NPs in the last step of the extrusion process. The use of LDPE-g-AM to enhance the distribution of ZnO-NPs on the surface of the polymeric film and decrease in their agglomeration will be evaluated. To the best of our knowledge, this coating method has never been used to develop AB coated films with ZnO-NPs. The main objectives are to evaluate the AB efficiency of the prepared films and its ability to impede the growth of food-borne bacteria such as *E. coli* and *S. aureus* at different times after films preparation and after being treated in an ultrasonic bath. The distribution and the concentration of the ZnO-NPs on the surface of the coated-films before and after treatment were investigated by EDS-SEM (Energy-dispersive X-ray spectroscopy). Finally, the ABA of these films was compared to that of the films after thin pure LDPE layer lamination and to those of ZnO-NPs blended with LDPE via the melt compounding.

## 6.3 Methodology

### 6.3.1 Materials

In this study different polymeric matrices were used. Surpass FPs 317-A Octene sLLDPE film resin obtained from Nova chemicals (Calgary, Alberta, Canada) with a melt flow index of 4 g/10 min. Bynel 42E703 adhesive resin was purchased from DuPont (Mississauga, Ontario, Canada) which is an LDPE-g-AM resin with a melt flow index and maleic anhydride (MAH) content of 5.7g/10 min and 0.08 (wt %). Commercially produced ZnO nanofillers were purchased from SkySpring Nanomaterials Inc (Houston, TX, USA) and were used without any additional
purification where the average diameter is 20 nm, and the specific surface area is 30 to 50 m²/g. Cultures of non-pathogen *E. coli* (DH5α) and pathogen *S. aureus* (54-73), Luria Bertani (LB) broth, which is a nutritional-rich medium, LB agar and phosphate-buffered saline (PBS) solutions, used during the AB tests were obtained from the Department of Microbiology, Infectiology, and Immunology at Université de Montréal.

## 6.3.2 Preparation of ZnO-coated LDPE films

LDPE films were prepared by melt blending in a tightly intermeshing co-rotating twin screw extruder (TSE) (Leistritz ZSE-18HP-40D, Germany) having a screw diameter of 18 mm and L/D = 40. All the LDPE films were produced by using constant speed screw of 100 rpm, temperature profiles as shown in Table 6.1 and a cast film die connected to the TSE. The ZnO-NPs were spread, at the exit of the die, manually with the help of a strainer. To laminate the coated-LDPE films, the dipping method was used. A xylene solution was prepared at 120 °C, with 2 % wt of FPSs 317A resin. Once the resin is completely dissolved, the solution is cooled to 70 °C, and the films were dipped for 1-2 second then dried under the hood. In fact FPS-T1, FPS-T2 and FPS-T3 films were processed from FPSs 317A resin at 190, 200 and 220 °C, respectively. Whereas, Bynel-T3 films were produced from Bynel 42E703 resin at 220 °C.

<table><caption>Table 6.1: Different temperature profiles used to produce the LDPE films by the TSE, where T1= 190, T2= 200 and T3= 220 °C.</caption><thead><tr><th>Zones</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th></tr></thead><tbody><tr><td>FPS-T1</td><td>190 °C</td><td>190 °C</td><td>190 °C</td><td>190 °C</td><td>190 °C</td><td>190 °C</td><td>190 °C</td><td>190 °C</td><td>190 °C</td></tr><tr><td>FPS-T2</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td></tr><tr><td>FPS-T3</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>220 °C</td><td>220 °C</td><td>220 °C</td><td>220 °C</td><td>220 °C</td></tr><tr><td>Bynel-T3</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>200 °C</td><td>220 °C</td><td>220 °C</td><td>220 °C</td><td>220 °C</td><td>220 °C</td></tr></tbody></table>
**6.4 Characterization**

**6.4.1 Antibacterial tests**

The AB efficiency of the nanocomposites films were quantified using the strains *E. coli* (DH5α) and *S. aureus* (54-73). These bacteria were grown in LB broth under constant agitation for 24 h at 37 °C, to reach a density of 10⁹ Colony Forming Units (CFU)/mL. The bacteria culture was diluted in the same medium after 24 h, in order to reach a density of 10⁵ CFU/mL. AB tests were conducted on the films produced by ZnO coating and melt mixing according to the standard ISO 22196:2011 and as described in Abdali and Ajji's work [3]. Both active and blank samples were cut out into discs of 4 to 5 cm in diameter. ISO 22196:2011 standards require that the surface of contact between the two polymer layers should be between 400 and 1600 mm². Blank discs should be sterilized by washing with 70 % ethanol before the test, whereas active films were sterilized under UV light (λ = 254 nm) for 20 minutes. The two discs (one blank and the other active) are sandwiched together after 200 µL of 10⁵ CFU/mL culture is deposited between them. Both films are rubbed against each other, so that the surface is contaminated. These are placed in a Petri dish and incubated at 37 °C for a period of 24 h. The next day, both discs are quenched in a test tube with 2 mL of PBS solution. Then, for each tube, six serial dilutions of PBS solution (10⁻¹ to 10⁻⁶) were conducted. Three samples were taken from each of the six dilutions and 10 µL droplets were applied onto the LB agar plates which were incubated overnight at 37 °C for the counting of the surviving bacteria (CFU/mL). Each sample is made in triplicate, along with the negative control containing two blank discs. In order to study how well the ZnO-NPs adhere to the film, the ABA of the ZnO-coated LDPE films before and after shaking were investigated. The samples were immersed in a beaker with distilled water and were shaken at a constant temperature in an ultrasonic bath (Thermo Fischer, 40 KHz) for 20 minutes. All the AB tests were conducted in the Department of Microbiology, Infectology, and Immunology at Université de Montréal.
### 6.4.2 Morphology

The ZnO-NPs distribution on the surface of LDPE films was assessed using a Field Emission Gun-Scanning Electron Microscopy JSM-7600TFE (FEG-SEM) (Calgary, Alberta, Canada) operating at 5-10 KV. The analysis of the surface composition was performed in the same SEM equipment under energy-dispersive X-ray spectroscopy (EDS) mode operated at 10 KV.

## 6.5 Results and discussion

<figure><img src="image_23.png" /><figcaption>Figure 6.1: Inhibitory effects of different FPS and Bynel films toward *E. coli* and *S. aureus* at 1 month post-preparation.</figcaption></figure>
The ABA of LDPE films was tested against two bacterial strain, and was measured by viable cell counts after incubation for 24 h at 37 °C, and the results are shown in Figure 6.1. The results showed a complete eradication of the *E. coli* population when compared with the control samples and a reduction rate higher than 99.9 %. Whereas for *S. aureus*, for both FPS and Bynel films, a reduction higher than 2 logs (CFU/mL) was observed for all the samples. These results also proved that the increase in the extrusion temperature from 190 (FPS-T1) to 220 °C (FPS-T3) allowed to improve the AB activity of these films. In fact the increase in the processing temperature has probably allowed to impregnate higher concentration of ZnO-NPs on the surface of the coated LDPE films. The increase of the concentration of ZnO-NPs on the surface decreased the *S. aureus* population from 1.5 × 10⁵ CFU/mL for FPS-T1 films, to 4.3 × 10³ CFU/mL for FPS-T3 films processed at 190 and 220 °C, respectively. This study showed that the ZnO- coated LDPE films exhibited increased ABA on inhibiting the growth of Gram-negative (*E. coli*) than the Gram-positive (*S. aureus*) bacteria. Several studies indicate that Gram-positive (*S. aureus*) bacteria have better resistance to ZnO-NPs due to the intracellular content of *S. aureus* and the structural difference in the bacterial membranes compared to *E. coli* [44].

To observe and compare the ZnO-NPs distribution across the surface of the LDPE films, FPS-T3 and Bynel-T3 were analyzed by SEM, since these two samples were processed at the same conditions (200-220 °C). The SEM micrographs, presented in Figure 6.2a and b, allowed the observation of the distribution of the ZnO-NPs on the surface of the coated films. The results demonstrated obviously that the distribution of the ZnO-NPs on the surface of the Bynel-T3 film was more uniform, whereas agglomerations were observed on the surface of FPS-T3, possibly because of the strong interactions between ZnO-NPs. In fact, Bynel-T3 was processed from LDPE-g-AM. The presence of MAH acts as a disruptor of agglomerations on the surface of the Bynel-T3 samples and as a compatibilizer [45]. Thus, dispersion of ZnO-NPs was improved for Bynel-T3 samples, showing smaller aggregates. Moreover, the use of PE-g-AM has probably reinforced the bonding of ZnO-NPs to the surface of Bynel-T3 films. EDS analysis results, presented in Figure 6.2c and d allowed to estimate the concentration of ZnO-NPs on the surface of these films by using the mapping mode in the SEM-EDS equipment. The concentration of ZnO-NPs on the surface of Bynel-T3 films was around 17 wt % a little bit higher than the concentration of FPS-T3 films which was around 14 wt %. These results are congruent with the
hypothesis proposed above saying that the presence of MAH attached more ZnO-NPs to the film surface and caused a good interaction between LDPE surface and ZnO-NPs [6].

<figure><img src="image_24.png" /><figcaption>Figure 6.2: SEM and EDS analysis (by SEM) of the surface of (a), (c): FPS-T3 and (b), (d): Bynel-T3 films. For EDS samples were analyzed with a mapping mode operating at 15 KV.</figcaption></figure>
To the best of our knowledge, it was the first time that this method was used to produce coated polymeric films with ZnO-NPs or with others AB metallic NPs. Otherwise, several methods have been used to produce ZnO coatings on different polymeric substrate. Thin films of ZnO and ZnO-Ag were deposited on polyethylene terephthalate (PET) and silicon (Si) substrates by reactive DC magnetron sputtering [30]. In another study, ZnO thin films were applied to biaxially oriented poly(lactic) acid (BOPLA) and polypropylene (BOPP) by atomic layer deposition at low temperatures [23]. In an earlier study, ZnO suspensions were coated on the surface of PVC film. After drying, the film was pressed at 100 °C for 10 min and then, it was peeled carefully after cooling at room temperature [18]. In the three studies listed above, the ABA of the coated polymeric films was evaluated by different methods. One of the interesting results is the one related to the PET substrate. In that study, it was found that the samples having thinner coatings (50 and 100 nm) exhibited less ABA than the thicker coatings (200-600 nm), which were not only able to decrease the growth rate but also reduce the amount of bacteria cells observed at the end of the experiments by 45 %. This result is extremely useful for food packaging applications because it may reduce bacterial presence [30].
<figure><img src="image_25.png" /><figcaption>Figure 6.3: Inhibitory effects of FPS-T3 and Bynel-T3 films towards *E. coli* and *S. aureus* at 1, 5 and 8 months after their preparation.</figcaption></figure>

After their preparation, the FPS and Bynel films were stored, and their ABA was tested at 5 and 8 months following the same steps to verify if their ABA will change with time.

The results presented in Figure 6.3 proved that after 5 and 8 months, FPS-T3 and Bynel-T3 samples retained their AB properties, and the ZnO-NPS were able to decrease the *E. coli* population by more than 99.9 %. For the Gram-positive strain (*S. aureus*), at 1 month after its preparation, the FPS-T3 films decreased the bacterial growth by approximately 4 logs (CFU/mL) to reach a population of 4.3 10³ CFU/mL. 4 months later, the *S. aureus* population was around 1.5 10⁴ CFU/mL, but the inhibition was considerable since the control population was reduced by 3 logs (CFU/mL). At 8 months after the films preparation, the FPS-T3 films reduced the bacterial
population to 9.8 10⁵ CFU/mL reducing thus the population by 1 log (CFU/mL). The slight loss of the AB activity of the FPS-T3 films between 1 and 8 months, can be attributed to a partial loss of the ZnO-NPs during storage. For the Bynel-T3 sample, the first test performed at 1 month post-preparation, has revealed a reduction rate of 3 logs (CFU/mL), and then for the subsequent tests, the reduction rate exceeded 99.9 %. This finding consolidated what was discussed above; ZnO-NPs were well impregnated on the surface of the PE-g-MA. Considering the quantity of ZnO-NPs that can be lost by handling the samples, sufficient concentration of ZnO-NPs was still well attached to the surface of the film 8 months after its preparation reducing thus more than 99.9 % of the bacterial population.
<figure><img src="image_26.png" /><figcaption>Figure 6.4: Inhibitory effects of treated and untreated FPS-T3 and Bynel-T3 films toward *E. coli* and *S. aureus*. The films were shacked in an ultrasonic bath during 20 minutes and then dried in air.</figcaption></figure>

To confirm if the ZnO-NPs are well attached to LDPE films, the films were shaken in an ultrasonic bath for 20 minutes and then dried in air. During their preparation for the AB test, and before their inoculation, the films were sterilized by UV light during 20 minutes.

Figure 6.4 reports the AB properties of the treated and untreated films. According to the results, for the Gram-negative bacteria, the washed FPS-T3 films have reduced the *E. coli* growth by just 1 log (CFU/mL), whereas before washing the same film was able to kill all the bacterial population (more than 99.9 %). By contrast, Bynel-T3 films still displayed a high ABA and a decrease of more than 99.9 % of the bacterial density was achieved before and after washing. For
the *S. aureus* strain, the washed FPS-T3 films did not exhibit antibacterial properties when compared to the same film before washing that provided 2 logs (CFU/mL) reduction, while the washed Bynel-T3 films reduced the bacterial density by about 3 logs (CFU/mL). These films had lost much of their effectiveness since, before washing, they reduced the *S. aureus* density by approximately 7 logs (CFU/mL).

<figure><img src="image_27.png" /><figcaption>Figure 6.5: SEM and EDS analysis of the surface of (a), (c): FPS-T3 and (b), (d): Bynel-T3 films surface after washing 20 minutes in a ultrasonic bath. EDS samples were analyzed with a mapping mode operating at 15 KV.</figcaption></figure>
Taking into account the previous results, it is imperative to determine the amount of ZnO-NPs that remained on the surface of the films after the washing. EDS analysis, presented in Figure 6.5c and d allowed to estimate ZnO-NPs concentrations on the surface of these films by using the SEM-EDS mapping mode. The concentration of ZnO-NPs on the surface of the washed FPS-T3 and Bynel-T3 films was around 7.7 and 4.5 wt %, respectively. The observation of the SEM images consolidated the results discussed previously saying that the distribution of ZnO-NPs on the surface of the films is more uniform in the case of the Bynel-T3 films which was well illustrated in Figure 6.5a and b. It's obvious that after washing, a large amount of ZnO-NPs has been removed. According to EDS results, the surface of the washed Bynel-T3 films was characterized by a ZnO-NPs concentration of 4.5 wt %, which is far below the concentration on the surface of washed FPS-T3 films. Even though, the ABA of the washed Bynel-T3 films was higher than that of the washed FPS-T3 films for both bacterial strain. In fact, the washed Bynel-T3 films decreased more than 99.9 % of the *E. coli* population, whereas the washed FPS-T3 ones reduced the same bacterial population by only 1 log (CFU/mL). For *S. aureus*, the washed Bynel-T3 films reduced the bacterial density by about 3 logs (CFU/mL), while no ABA was recorded for the washed FPS-T3 films.

From the discussion above, it can be concluded that the distribution of the ZnO-NPs has a large effect on their ABA. A lower concentration with better distribution was more efficient toward both bacterial strains. This effect was due to the use of PE-g-AM that increased the compatibility between the polymeric matrix and ZnO-NPs, allowing a better adhesion to the surface with the formation of smaller ZnO-NPs agglomerates. Thereby, more surface area was available to cope with the bacteria. In fact, one of the parameters involved in ZnO-NPs antibacterial properties is the specific surface area. It increases for reduced particle size, thus enhancing particle surface reactivity [14, 15, 42, 46]. Moreover, generation of $H_2O_2$ is mainly dependent on the surface area of ZnO-NPs [42, 47]. Padmavathy and Vijayaraghavan [42] studied the AMA of ZnO suspensions against *E. coli* with various particle size (12 nm, 45 nm and 2 µm), using a standard microbial method. They concluded that using small nanoparticles, thus having a larger surface area, will generate a higher concentration of ROS on the surface and will result in greater ABA. More precisely, for smaller ZnO-NPs, more particles are needed to cover the bacterial colony (2 µm), resulting in a larger number of ROS, killing bacteria more effectively [42]. By analogy, it
can be assumed that smaller agglomeration of the ZnO-NPs, could provide more surface area and
more ROS to ensure better ABA.

But if the ZnO-NPs will be blended with LDPE via the melt compounding, are they going to retain
the same AB properties? To answer this question, a master batch with a 20 wt % of ZnO- NPs was
prepared. Then, they were diluted to produce cast films with 5 wt % ZnO-NPs, the first one was
processed from the Bynel resin and the second was a blend of FPS and Bynel resin. The results of
the AB evaluation did not show any interesting performance neither for Gram-positive nor the
Gram-negative bacteria.

These results were predictable since the extrusion process as well as solvent casting are more
suitable for migratory AB systems where the release of a volatile AB agent from the packaging
material to the packaging headspace or the surface of the food product is required.

It is obvious that only when the ZnO-NPs are coated on the surface of the LDPE films and remain
in direct contact with bacteria that they ensure efficient ABA against both Gram-positive and
Gram-negative bacteria. It remains to be seen if the sprayed ZnO-NPs will retain their activity if
they were laminated with another polymer layer or their effectiveness will be lost.
<figure><img src="image_28.png" /><figcaption>Figure 6. 6: (a) Inhibitory effects of different FPS and Bynel films towards *E. coli* and *S. aureus*. The films were coated by dipping in FPS 317A /xylene solution. (b) Distribution of the thickness of the coating layer in µm.</figcaption></figure>
The results of AB tests of the laminated Bynel and FPS films presented in

Figure 6. 6a show that the dipped FPS-T3 films reduced the *E. coli* population by just 1 log (CFU/mL), while the dipped Bynel-T3 was still efficient toward the bacteria and destroyed more than 99.9 % of the bacterial density. For *S. aureus*, the reduction has not even reached 1 log (CFU/mL) for both films. FPS-T3 films registered only 1 log (CFU/mL) reduction, while before lamination they were able to reduce more than 99.9 % from the bacterial population. The distribution of the thickness of the coating layer is presented in

Figure 6. 6b and it shows that the average thickness was around 8 µm for each side of the dipped films. According to these results, one can assume that, for coating thicknesses that does not exceed 8 µm, the coated Bynel-T3 could retain their activity toward *E. coli*. These films were characterized by a good adhesion of the ZnO-NPs to the LDPE surface. So to estimate the limit of this hypothesis, FPS-T3 were laminated with a hot press with PE films having different thickness (18, 21 and 38 µm). Then the ABA of these films were evaluated. The results (not shown here) showed that the films did not register any ABA toward *E. coli* and *S. aureus*, compared to the control samples. It is obvious that when the thickness of the lamination layer increased beyond 8 µm, the ZnO-NPs will be far away from the bacterial cells and lose their AB performance. To be efficient toward bacteria, ZnO-NPs should be in direct contact or close to the bacteria cells to ensure a good ABA.

## 6.6 Conclusions

This study presented a new approach to coat LDPE films with metallic AB agents. Both LDPE and LDPE-g-AM coated films showed considerable inhibition against *E. coli* and *S. aureus*. For the FPS films, the efficiency of the ZnO-NPs toward *S. aureus* was improved by increasing the processing temperature. SEM images of ZnO-coated LDPE films clearly showed that, in the case of the use of LDPE-g-AM, the distribution of the ZnO-NPs was more uniform, showing smaller aggregates. In this case, the compatibility between the polymeric matrix and the metal oxide NPs increased where the NPs were well attached to the surface of the film. The AB tests showed that the FPS film retained the same AB efficiency toward *E. coli* between 1 and 8 months post-
preparation. For *S. aureus*, the AB properties of the FPS-T3 films were slightly reduced, which may be due to a partial loss of the ZnO-NPs during storage or during samples preparation for the AB tests, since ZnO-NPs are not well attached to LDPE film surface. For the Bynel-T3 films, the ABA was improved between 1 and 8 months. When treated by shaking in water, the FPS-T3 films lost their AB properties toward *E. coli* and *S. aureus* by contrast to Bynel-T3 films that were able to keep a reduction rate higher than 3 logs (CFU/mL). To maintain desired ABA, it is recommended that the Bynel-T3 should be laminated with a film layer whose thickness does not exceed 8 µm. This coating approach guaranteed a good ABA of the ZnO-NPs that were absent in the case where NPs were compounded with the polymer in the melt state.

## Acknowledgment

Financial support of 3S Pack NSERC/Saputo/Prolamina Industrial Research Chair from the natural Sciences and Engineering research council Canada and Industrial partners is gratefully acknowledged.

## Compliance with Ethical Standards:

**Funding:** This study was funded by 3S Pack NSERC/Saputo/Prolamina Industrial Research Chair from the natural Sciences and Engineering research council Canada and Industrial partners.

**Conflict of Interest:** The authors declare that they have no conflict of interest.

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# CHAPITRE 7 DISCUSSION GÉNÉRALE

La validation de l'activité antibactérienne des oxydes métalliques en suspensions est une étape indispensable avant leur incorporation au sein de matrices polymériques pour former de nouveaux nanocomposites antibactériens. Cette validation s'est montrée indispensable à cause de la non-reproductibilité des résultats des tests antibactériens sur des mats de nanofibres de PLA à base de ces nanoparticules. De plus, cette étape est primordiale puisqu'une fois incorporés dans des nanocomposites, l'efficacité des oxydes métalliques a été comparée, une évaluation de leur activité quand ils ne sont pas influencés par la présence d'autres éléments (polymère) était cruciale. S'il est vrai que plusieurs études ont rapporté l'efficacité de ces oxydes, leurs propriétés intrinsèques (taille, propriétés de surface, présence de groupements fonctionnels), les méthodes de quantification de l'activité antibactérienne, ainsi que les souches bactériennes exploitées étaient assez variables et donc il était difficile de faire des extrapolations. L'influence des propriétés intrinsèques du ZnO (taille, concentration, propriétés de surface) ainsi que celles des souches bactériennes (Gram-négatif, Gram-positif) a été largement étudiée, mais rares sont les études qui se sont focalisées sur les prétraitements que peuvent recevoir les suspensions d'oxydes métalliques avant leur contamination. Un intérêt spécial a été porté aux vibrations ultrasonores comme prétraitement pour les solutions des nanoparticules. En effet, il est connu que l'un des problèmes majeurs survenus lors de l'utilisation des nanoparticules de ZnO est leur tendance à l'agglomération en solutions. Celle-ci découle essentiellement de leur énergie de surface ainsi que de leur hydrophobicité assez élevée. Durant l'une des rares études portant sur l'effet des vibrations ultrasonores sur les suspensions de ZnO, une contamination avait eu lieu avant le traitement des suspensions. En outre, cette étude s'est intéressée principalement à l'effet que peuvent avoir ces vibrations sur les interactions physiques entre les nanoparticules de ZnO et la membrane bactérienne. Les tests *in vitro* menés au cours de ce projet ont montré que les vibrations ultrasonores combinées avec la stérilisation des suspensions ont engendré un effet synergique sur l'amélioration de l'efficacité antibactérienne des nanoparticules de ZnO. Cette amélioration s'est manifestée par une diminution considérable des CMBs des suspensions d'oxydes métalliques. Les vibrations ultrasonores ont probablement aidé à dissocier les agglomérats des nanoparticles de
ZnO et à mieux les solubiliser en ions $Zn^{2+}$. Ainsi, une meilleure internalisation des nanoparticules et une concentration plus élevée en $Zn^{2+}$ peuvent être la cause de l'augmentation du taux d'inhibition de la bactérie *E. coli* par les suspensions de ZnO.

Les résultats de l'étude comparative menée sur les nanoparticules de ZnO et de ZnO-UVM-7 ont également souligné l'amélioration de l'activité antibactérienne engendrée par le dopage de la silice par des nanoparticules de ZnO. Ces résultats sont en cohérence avec la littérature et peuvent être expliqués principalement par la diminution de l'agglomération et du diamètre de ces nanoparticules, ce qui augmente la surface active disponible des oxydes métalliques.

Le procédé d'électrofilage a été sélectionné d'une part en raison de sa simplicité, la facilité de son utilisation et son moindre coût et d'autre part en vue des avantages que peuvent apporter les structures en nanofibres tels qu'une surface spécifique et un rapport d'aspect assez élevés. Parmi les divers polyesters dégradables aliphatiques, le PLA a été considéré comme l'un des matériaux biodégradables les plus intéressants et les plus prometteurs pour le développement de matériaux d'emballage antibactériens. Comme il a reçu le statut GRAS (Generally Recognized As Safe) par la FDA (Food and Drug Administration), le PLA a été approuvé pour les utilisations dans les emballages alimentaires, y compris les applications de contact direct. En outre, il convient également à la production de nanofibres électrofilées minces et uniformes et peut être dissout dans de nombreux solvants courants.

Malgré la simplicité du procédé d'électrofilage, l'obtention des fibres à base de nanoparticules de ZnO était une tâche complexe en raison de la mauvaise solubilisation et de l'agglomération des nanoparticules de ZnO en suspensions. Vu que la concentration finale des nanoparticules de ZnO quantifiée dans les nanofibres de PLA était variable d'un échantillon à un autre, il fallait au moins utiliser le même mat de fibres pour les échantillons d'un même triplicata pour une teneur donnée en ZnO. Ce détail a rendu la tâche plus fastidieuse, puisque l'allongement du temps de l'électrofilage favorisait davantage la sédimentation des nanoparticules au fond de la seringue pendant le procédé, diminuant ainsi la concentration finale en nanoparticules de ZnO dans les nanofibres obtenues. Ainsi, l'instabilité de ces suspensions a exigé une étude de leurs propriétés rhéologiques. Les résultats ont prouvé que les suspensions de ZnO et de TFE représentent un milieu favorable pour la dégradation du PLA en solution, ce qui s'est traduit par une diminution importante de la viscosité des solutions. Cette chute de viscosité était encore plus prononcée au
dessus d'une certaine concentration seuil de ZnO où l'agglomération est favorisée davantage. Cela pourrait avoir des répercussions négatives sur la fonction antibactérienne des nanofibres de PLA, puisque des billes de PLA ont été formées en même temps que les nanofibres avec formation de gros agglomérats. Ceci pourrait probablement modifier la surface spécifique disponible des nanoparticules. En effet, la validation de cette baisse de la viscosité par une mesure directe du poids moléculaire des solutions avant et après l'exposition aux nanoparticules de ZnO n'était pas possible à cause de la présence des nanoparticules en solutions.

Par conséquent, pour développer des nanofibres à base de nanoparticules de ZnO, il faut choisir judicieusement le solvant, ainsi que les teneurs adéquates en ZnO permettant à la fois de réduire la prolifération des souches bactériennes tout en évitant leur agglomération. Pour être efficaces au sein d'un emballage alimentaire antibactérien, ces nanofibres à base de ZnO doivent être intégrées à la couche interne de l'emballage qui est généralement constituée de PE. Cette condition représente un frein quant à l'utilisation des ces nanofibres comme partie intégrante d'un emballage alimentaire actif et ce en raison de la faible adhésion des nanofibres due à la différence de polarité et au manque d'affinité entre le PLA et le PE. Pour surmonter ce frein, l'électrofilage peut se faire directement sur des films de PE ayant déjà subis un traitement plasma ou corona. Un autre élément qui peut faire obstacle à l'exploitation des nanofibres dans un emballage alimentaire actif est le fait que leur efficacité exige strictement un contact direct avec l'aliment emballé (tel que discuté dans la littérature). Ceci pourrait limiter l'utilisation de ces nanofibres à certains types d'emballage ayant un contact direct avec l'aliment dont les emballages sous vide pour la viande fraîche, le poulet ou le poisson. Néanmoins, malgré ces obstacles, les demandes pour des emballages alimentaires à la fois biodégradables et antibactériens pouvant allonger la durée de conservation des aliments ne cesse de croître et aujourd'hui elle est plus forte que jamais. Dans la dernière partie de ce travail, on s'est focalisé sur le développement de films antibactériens en combinant le procédé d'extrusion qui demeure le procédé le plus couramment utilisé pour développer les films d'emballages et la technique de revêtement. Les films actifs à base de PE ont été sélectionnés grâce à leurs bonnes propriétés de scellage et leur transparence qui sont deux caractéristiques absentes pour les mats de nanofibres à base de PLA.

Une attention particulière a été accordée à l'utilisation des revêtements à base de nanoparticules dans les emballages alimentaires. La nouvelle méthode de revêtement des films de PE proposée dans ce projet, est une méthode simple et ne nécessite l'utilisation ni de solvants ni d'équipements
spécifiques. Un tel revêtement maximise l'efficacité antibactérienne des nanoparticules de ZnO qui sont disponibles à la surface du film de PE en contact direct avec la surface contaminée. Ce nouveau procédé convient parfaitement aux agents antibactériens non-migrateurs comme le ZnO. Les résultats *in vitro* ont prouvé que l'activité antibactérienne de ces films est Gram-dépendante avec une plus grande sensibilité de la bactérie *E. coli* comparativement à *S aureus*. L'augmentation de la température au cours du procédé d'extrusion ainsi que l'utilisation d'un grade de PE greffé avec l'anhydride maléique ont largement favorisé l'attachement ainsi que la distribution uniforme des nanoparticules de ZnO à la surface des films de PE. Ces films ont retenu leur efficacité antibactérienne vis-à-vis de la bactérie *E. coli* après 8 mois de leur préparation avec un taux de réduction qui dépassait 99.9 %.

Quand ces films ont été laminés avec une fine couche de PE pur, ils ont conservé un taux d'inhibition considérable pour *E. coli* seulement dans les cas où l'épaisseur des films de laminage ne dépassait pas 8 µm. En effet, cette épaisseur constitue probablement la distance maximale à travers laquelle les espèces réactives générées par les nanoparticules de ZnO pourraient migrer pour être en contact avec les parois cellulaires. L'idée derrière ce procédé de laminage est de limiter le contact direct entre les nanoparticules et la surface de l'aliment emballé pour le rendre plus sécuritaire. Des recherches approfondies devraient être menées pour consolider cette idée, développer ce procédé de revêtement pour l'amener à l'échelle industrielle et élaborer ainsi un emballage actif qui soit plus sécuritaire. Une autre alternative serait d'exploiter un troisième élément autre que la matrice polymérique et l'oxyde antibactérien pour stimuler la migration des nanoparticules de ZnO à la surface des films pendant leur formation.

L'évaluation de l'activité antibactérienne des nanoparticules d'oxydes métalliques à travers les trois parties de ce travail de recherche a permis de comparer leurs performances antibactériennes en solution et en nanocomposites en exploitant des matrices polymériques et des procédés différents.

Les huiles essentielles ont été aussi exploitées durant ce travail comme agent antibactériens. Le thymol a été incorporé à la matrice polymérique à l'état fondu. Les mélanges de PE et de thymol ont été réalisés à l'aide du procédé d'extrusion et du mélangeur interne. Les films de PE incorporés avec le thymol n'ont pas prouvé une activité antibactérienne importante et durable à cause de la grande volatilité du thymol.
Il est vrai que le secteur des emballages alimentaires pourrait largement bénéficier des nanocomposites antibactériens pour réduire ou inhiber la prolifération de la flore d'altération ou de la flore pathogène à la surface des aliments emballés. Néanmoins des préoccupations concernant la sécurité de ces nanoparticules doivent être considérées.

En effet, l'exposition aux nanoparticules présentes dans les emballages alimentaires peut se produire à travers le contact avec la peau, l'inhalation mais surtout l'ingestion des nanoparticules qui ont migré vers l'aliment. Par exemple, selon une étude menée par Sahrma et al. [86], il s'est avéré que les nanoparticules de ZnO pourraient avoir une toxicité sur les cellules épidermiques humaines, même si le ZnO ("bulk") n'est pas toxique. Malgré cela, on peut constater que les données scientifiques disponibles sur la migration des nanoparticules des emballages vers les aliments sont bien limitées. Ces études sont généralement menées à l'aide de simulant de denrées alimentaires, et non pas sur des vrais aliments. Aussi, la réglementation canadienne concernant l'utilisation des nanoparticules de ZnO n'est pas encore claire. Citons que les seules données disponibles sur le taux de migration des nanoparticules de ZnO proviennent d'une réglementation européenne et spécifient la concentration maximale autorisée de ZnO à 25 mg/Kg d'aliment [87]. Un niveau indéfini de la toxicité des nanoparticules et le manque de connaissance sur leur effet sur la santé humaine et sur l'évaluation des risques pourraient expliquer le manque actuel d'emballages à base de nanoparticules de ZnO sur le marché Nord-Américain. Bien que les États-Unis soient un des principaux pays leaders dans le développement de ressources de sécurité en nanomatériaux pour les aliments et les emballages alimentaires, une approche réglementaire définitive sur l'utilisation des nanomatériaux au contact des aliments est encore en cours de développement par la FDA. Par ailleurs, chaque gouvernement doit prendre des mesures de coopération internationale pour la mise en place d'un système d'alerte, car les nanoparticules peuvent être difficiles à détecter dans les produits emballés importés et transférés d'un pays à l'autre [88].
# CHAPITRE 8 CONCLUSIONS ET RECOMMANDATIONS

## 8.1 Conclusions

Ce travail de recherche s'est concentré sur la production de nanocomposites à base de nanoparticules de ZnO en vue d'une potentielle utilisation comme matériaux d'emballage alimentaire à caractère antibactérien. Pour ce faire, l'évaluation des propriétés antibactériennes des poudres à base de ZnO a d'abord été effectuée en solutions. Par la suite, deux méthodes d'incorporation différentes ont été testées et l'efficacité antibactérienne des nanocomposites obtenus a été examinée.

La première partie de cette étude a évalué l'effet des prétraitements tels que les vibrations ultrasonores et la stérilisation sur l'activité antibactérienne de suspensions de poudres de ZnO. Les résultats ont démontré que ces propriétés sont améliorées grâce à un effet synergique des vibrations ultrasonores et de la stérilisation. Cette amélioration s'est traduite par une diminution considérable des CMBs, où l'effet bactériostatique/bactéricide a été constaté pour des concentrations beaucoup plus faibles pour deux poudres différentes d'oxydes de zinc. Le dopage de la silice avec les nanoparticules de ZnO avait lui aussi contribué à l'amélioration de cette activité. L'incorporation des nanoparticules de ZnO au sein des nanofibres de PLA produites par le procédé d'électrofilage a indiqué que l'agglomération des nanoparticules peut constituer un obstacle pour l'optimisation des propriétés antibactériennes des nanofibres.

La deuxième partie s'est focalisée sur les propriétés rhéologiques des solutions d'électrofilage et l'effet qu'elles peuvent avoir sur la morphologie des nanofibres de PLA/ZnO. Les résultats ont montré que le PLA se dégrade en solution en présence du TFE et des nanoparticules de ZnO. Cette dégradation, qui se traduit par une chute considérable de la viscosité en solution, est favorisé par l'augmentation de la concentration en nanoparticules de ZnO en solutions et par l'allongement de la durée de conservation des solutions. Les nanofibres produites à partir de solutions de très faible viscosité, ont engendré la formation simultanée de nanofibres et de nano-billes avec la présence de gros agglomérats de ZnO. Les résultats suggèrent que le choix du chloroforme comme solvant, parmi ceux testées, permet d'obtenir des suspensions d'électrofilage stables avec une viscosité constante au cours du temps.
Finalement la troisième partie de ce travail a permis le développement d'une nouvelle méthode de revêtement à base de ZnO, assez facile à reproduire. Les résultats ont prouvé que pour une optimisation maximale de leur activité antibactérienne, les nanoparticules de ZnO doivent être à la surface des films plastiques en contact direct avec la surface contaminée. Cette activité a été conservée même 8 mois après la production des films. Par ailleurs, si la surface du revêtement est séparée de la surface contaminée par une fine couche de polymère pure, les nanoparticules de ZnO peuvent conserver une action antibactérienne acceptable à condition que l'épaisseur de cette couche ne dépasse pas 8 µm. Les résultats ont aussi souligné que l'activité des nanoparticules de ZnO est Gram-dépendante.

## 8.2 Recommandations

Les points suivants sont recommandés pour les travaux futurs :

1) Évaluer les propriétés antibactériennes des suspensions de ZnO contre *S. aureus* et l'effet des prétraitements (vibrations ultrasonores, stérilisation) sur l'amélioration de ces propriétés.

2) Tester l'efficacité des nanoparticules de ZnO dont la surface a été modifiée quand elles sont incorporées dans des nanofibres de PLA pour atténuer leur tendance à l'agglomération et favoriser leur action antibactérienne.

3) Élaborer des fibres de PLA/ZnO à l'aide d'un procédé simultané d'électrofilage de PLA/électro-pulvérisation de ZnO pour maximiser la présence des nanoparticules de ZnO à la surface des nanofibres de PLA.

4) Tester l'efficacité antibactérienne des nanofibres développées en présence de nanoparticules de ZnO modifiées ainsi que celle produites par le procédé simultané d'électrofilage/électro-pulvérisation vis-à-vis des bactéries d'origine pathogène dont *E. coli* O157:H7, *S. Thyphimurium*, ou *L. monocytogenes*.

5) Évaluer les propriétés antibactériennes des nanofibres de PLA/ZnO *in situ* sur des aliments tels que la viande, le poulet ou le poisson frais.

6) Déposer, à l'aide du procédé de co-extrusion, la couche de laminage qui protégera le film de PE revêtu avec du ZnO pour uniformiser et minimiser l'épaisseur sur toute la surface.
7) Tester l'activité des films de LDPE/ZnO sur d'autres bactéries d'origine pathogène dont E. coli O157:H7, S. Thyphimurium, ou L. monocytogenes et notamment des souches provenant de la banque American Type Culture Collection (ATCC).

8) Tester l'activité antibactérienne des films produits par revêtement de ZnO *in situ* sur des aliments tels que la viande, le poulet ou le poisson frais et évaluer l'allongement de la DLC engendré.

9) Étudier le taux de migration ou de libération suite à l'utilisation des films avec un revêtement de ZnO.

10) Évaluer l'effet des films produits par revêtement de ZnO sur les propriétés organoleptiques des aliments testés en termes d'odeur, saveur, aspect visuel et texture.

11) Incorporer les nanoparticules de ZnO au PE à l'état fondu en utilisant un troisième composé qui agira de sorte de favoriser la migration des nanoparticules de ZnO à la surface de ZnO.
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