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Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films
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Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films L. Karam a, M. Casetta a, N.E. Chihib a, F. Bentiss a,b, U. Maschke a, C. Jama a,∗ a b
UMET-PSI, CNRS UMR 8207, ENSCL, BP 90108, F-59652 Villeneuve d’Ascq Cedex, France Laboratoire de Catalyse et de Corrosion des Matériaux (LCCM), Faculté des Sciences, Université Chouaib Doukkali, B.P. 20, M-24000 El Jadida, Morocco
a r t i c l e
i n f o
Article history: Received 17 September 2015 Revised 8 April 2016 Accepted 15 April 2016 Available online xxx Keywords: Surface modification Nitrogen plasma Nisin Experimental design Functional polymers Antimicrobial packaging
a b s t r a c t In order to prevent microbial contamination and food-borne illnesses, antimicrobial active packaging represents an innovative option. The aim of this paper was to optimize the experimental parameters of the plasma treatment. Such pre-treatment can be used to develop an active packaging film by adsorbing a natural antimicrobial peptide “Nisin” on the surface of a commonly used polymer in the agro-food sector “low density polyethylene (LDPE)”. Cold nitrogen (N2 ) plasma treatment was used to functionalize LDPE and generate a hydrophilic polymer suitable for the adsorption of the peptide. The experimental design technique allowed determining the optimal conditions of the plasma treatment. The lowest contact angle and highest hydrophilic character were obtained for a gas flow rate of 500 cm3 /min, a generator power of 300 W and an exposure time of 300 s. The surface characterization techniques X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), contact angle and surface free energy measurements were used to confirm the surface modification after cold plasma treatment. The antimicrobial tests permitted to validate the efficiency of the active packaging films. The plasma treated films showed higher antimicrobial activity after nisin adsorption as compared to the native ones. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Recent and continuous foodborne microbial outbreaks are driving a search for innovative ways to inhibit microbial growth in the foods while maintaining quality, freshness, and safety. One option is to adsorb antimicrobial peptides on the packaging polymers to impart functional active properties and an increased margin of safety and quality. Polymer materials are inexpensive, easy to process, and exhibit excellent bulk and mechanical properties. However, their chemical inertness and their low surface energy represent generally a great barrier for their food packaging applications [1]. Plasma treatments have been used to expand the applications and transform these materials into highly valuable finished products. They can increase the wettability, polar groups, surface energy and improve the printability, adhesion, mechanical and barrier properties of polymer films [1–5]. From one side, these particular advantages of plasma treated films are highly desirable in food packaging applications to minimize leakage, reduce the risk of microbial contamination, and improve package integrity. From another side, the increase in hydrophilicity can improve the adsorption of peptides on surfaces as reported in previous studies
∗
Corresponding author. Tel.: +33 320 434 482; fax: +33 320 436 584. E-mail address: [email protected] (C. Jama).
[6–14]. Popelka et al. and Bílek et al. investigated antibacterial treatments on cold plasma pretreated low density polyethylene (LDPE) foils using benzalkonium chloride [15] and allylamine [16], respectively. Nisin is a well-known antimicrobial peptide, produced by Lactococcus lactis subsp. Lactis and widely used in the food industry as a safe and natural preservative [17]. It has an antimicrobial activity against a broad spectrum of Gram-positive bacteria and food pathogens such as Listeria innocua, Listeria monocytogenes, Staphylococcus aureus and Clostridium botulinum [18,19]. Therefore, the aim of this study was to assess the possibility of elaborating an antimicrobial polymer by adsorbing nisin on nitrogen (N2 ) plasma treated low density polyethylene (LDPE) film. This entailed the determination of the optimum conditions for using nitrogen plasma treatment, the use of physico-chemical methods to confirm the surface modification after the plasma treatment, the adsorption of nisin on both treated and native films, and finally the determination of the antimicrobial activity and/or the ability of nisin to retain its activity on the N2 plasma treated films. 2. Materials and methods 2.1. Film preparation Low density polyethylene (LDPE) was obtained from Polimeri Europa, France SAS (Density of 0.922 g/cm3 and thickness of
http://dx.doi.org/10.1016/j.jtice.2016.04.018 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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- a 2k fractional factorial design, k being the number of studied variables, - 6 axial points at a distance of α = 1.68 from the design center, - 3 replicates of the center point. Experiments were carried out randomly to provide protection against the extraneous factors, which could affect the measured response. For statistical calculations, the variables Ui were coded as Xi according to the following transformation (Eq. (1)):
Xi =
(Ui − U0 ) U
(1)
where Xi is the dimensionless coded value of the variable Ui , U0 represents the value of Ui at the center point and U is the step change. The experimental values associated to the coded levels of the different variables are given in Table 1. The quadratic model for predicting the optimal conditions was expressed according to Eq. (2):
Ypred = β0 +
n
βi Xi +
i=1
n
βi j Xi X j +
n
βii Xi2
(2)
i=1
i< j
where Y is the predicted response, β 0 is the value of the fitted response at the center point, β i , β ii and β ij correspond to the linear, quadratic and interaction terms respectively.
Fig. 1. Schematic representation of the experimental set-up.
70 μm). LDPE films were cut into square shape (2 cm × 2 cm) and washed with ethanol in an ultrasonic bath to remove possible dusts or any oily compounds adsorbed on the film surface. They were then dried in an oven at 55 °C for 3 h. Those films were either used directly or treated for nisin adsorption.
2.2.2. Surface characterization Contact angle and surface free energy measurements: Static contact angle measurements of the samples were carried out at room temperature on a Digidrop goniometer (GBX, France) using pure water for the optimization step. A 5 μL drop of water was applied onto the sample surface and the contact angle formed with the surface was instantaneously measured. Triplicate tests were performed for the films, immediately after the plasma treatment, and at least six different measurements were made on each sample surface. The average values for contact angles and the standard deviation were then calculated. The contact angles were also determined using two other test liquids (formamide and diiodomethane) in order to calculate the surface free energy values with the Owens–Wendt model [20–22]. This method takes into account dispersive and polar components of the surface energy and using different test liquids, it is possible to determine the solid surface free energy (γ ) as the sum of polar (γ p ) and dispersive (γ d ) contributions [21,23–25]. X-ray photoelectron spectroscopy (XPS): X-ray photoelectron spectroscopy (XPS) experiments were carried out using a Kratos Analytical AXIS UltraDLD spectrometer. A monochromatized aluminium source (Al Kα = 1486.6 eV) was used for excitation. The X-ray beam diameter was around 1 mm. The analyser was operated at constant pass energy of 160 eV for survey spectra using an analysis area of approximately 700 μm × 300 μm. The electron take off angle was 90°. Charge compensation was applied to compensate for the charging effect occurring during the analysis. The C 1s hydrocarbon (285.0 eV) binding energy (BE) was used as internal reference. The spectrometer BE scale was initially calibrated
2.2. Plasma treated films 2.2.1. Design of experiments Plasma treatments were performed in LDPE samples were plasma treated in a EUROPLASMA CD 1200 set-up reactor using cold radiofrequency plasma (13.56 MHz) fitted with a capacitively coupled, parallel-electrode system with an automatic matching device. A schematic representation of the experimental set-up is shown in Fig. 1. A pure nitrogen glow discharge was generated in an aluminium reactor chamber with a continuous out power ranging from 0 to 600 W. The chamber was pumped down to 10.7 Pa using a pump Edwards (80 m3 /h), and the N2 gas was introduced into the chamber. When the pressure became constant, the generator was switched on and adjusted to a certain power value, which gave rise to a continuous glow discharge. The sample holder was placed in the center between the electrodes without any bias. The distance between the electrodes is equal to 10 cm. Optimization of the plasma treatment was carried out using response surface methodology (RSM). The influence of three main process parameters was studied namely the nitrogen flow, the time of treatment and the power during the plasma treatment. A central composite design (CCD), consisting of 17 experimental runs, was used including:
Table 1 Coded and real values of experimental parameters used for the CCD. Coded variables
X1 X2 X3
Parameter
Levels
3
U1 , nitrogen flow (cm /min) U2 , time of treatment (s) U3 , power (W)
−α
−1
0
+1
+α
100 30 100
180 85 180
300 165 300
420 245 420
500 300 500
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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against the Ag 3d5/2 (368.2 eV) level. Pressure was in the 10−10 Torr range during the experiments. Quantification and simulation of the experimental photopeaks was carried out using CasaXPS software. Quantification took into account a non-linear Shirley background subtraction [26]. Time-of-flight secondary ion mass spectrometry (ToF-SIMS): ToFSIMS spectra measurements were carried out using a ToF-SIMS V instrument (ION-TOF GmbH, Germany). This instrument is equipped with a Bi liquid metal ion gun (LMIG). Pulsed Bi3 + primary ions have been used for analysis (25 keV, 0.4 pA). Mass spectra were taken for each sample, from an area of 500 μm × 500 μm (30 scans) using 256 × 256 pixel random rasters. These experimental conditions allowed staying within the static conditions since primary ion dose did not exceed 1012 ions/cm2 . Pulsed low energy flood gun (20 eV) were used for charge neutralization. Average mass resolution was about 50 0 0 at m/z = 55.05 (C4 H7 + ) which allowed the separation of ion fragments under investigation. 2.3. Nisin-functionalized films 2.3.1. Nisin adsorption on films Nisin adsorption was carried out on the native and the plasma treated films. A pure grade of nisin was donated by Dupont Health and Nutrition (United Kingdom). Activity was indicated as 5.2 × 107 IU/g nisin solutions were prepared by dissolving 1.0 mg/ml of nisin in HCl (0.01 M). Solutions were freshly prepared and filtered (0.22 μm) before each experiment. Each film was immersed in 20 ml of nisin solution (1.0 mg/ml) and it was agitated at 8 °C for 16 h. After that, the samples were removed from solution and briefly rinsed in sterile distilled water to remove non adsorbed nisin. All the tests were done after drying the films in sterile Petri dishes at 25 °C for 24 h. 2.3.2. Assessment of the antimicrobial activity of nisin-functionalized films The antimicrobial tests were carried out against Listeria innocua. Pre-cultures were performed by inoculating a single colony in 10 ml of Brain Heart broth. The cultures were made by inoculating 10 ml of Brain Heart broth with 100 μl of the pre-culture. Pre-cultures and cultures were incubated at 37 °C for 24 h. Qualitative antibacterial tests were done using a modified agar diffusion assay [27]. Mueller Hinton agar medium was seeded with the indicator micro-organism, L. innocua. The face up of the film to be tested was placed on the agar surface. Bioassay plates containing experimental samples were kept at 4 °C for 4 h to initiate nisin diffusion and were then incubated at 37 °C for 24 h. Nisin activity was assessed as an inhibition of the indicator bacterium growth under and around the film. The quantitative inhibitory effect of the two nisinfunctionalized films was carried out, at room temperature, by putting each film in 5 ml of L. innocua cell suspension of ca.106 CFU/ml. After 30 min of contact time, the samples were enumerated by plating onto Luria Bertani agar and incubating for 24 h at 37 °C. In each experiment, a control test of L. innocua (without the test-film) was realized under the same conditions. The viable and culturable counts of L. innocua, were determined and used to assess the antimicrobial activity of the films. The results were expressed in terms of logarithm of the difference in population (DP) according to the following equation (Eq. (3)) [28]:
log DP = log (N0 /N ) = (log N0 ) − (log N )
(3)
where N0 and N are, respectively, the bacterial population (colonyforming units per milliliter) before and after exposure of bacteria culture to nisin-functionalized films for 30 min. Those experiments were made in triplicates and all the values were expressed as a mean value ± the standard deviation.
3
Table 2 CCD in coded and real values with the experimental responses obtained for the different runs. Experiments
X1
X2
X3
U1
U2
U3
Yexp (°)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
−1 +1 −1 +1 −1 +1 −1 +1 −α +α 0 0 0 0 0 0 0
−1 −1 +1 +1 −1 −1 +1 +1 0 0 −α +α 0 0 0 0 0
−1 −1 −1 −1 +1 +1 +1 +1 0 0 0 0 −α +α 0 0 0
180 420 180 420 180 420 180 420 100 500 300 300 300 300 300 300 300
85 85 245 245 85 85 245 245 165 165 30 300 165 165 165 165 165
180 180 180 180 420 420 420 420 300 300 300 300 100 500 300 300 300
52.7 50.8 50.9 51.6 49.8 50.6 49 49.5 46.1 47.9 45.8 48.9 56.1 47.1 50.4 52.7 50.3
Table 3 Coefficients of the model estimated with Modde7.0. Coefficients
β0 β1 β2 β3 β 11 β 22 β 33 β 12 β 13 β 23
51.14 0.458 0.900 0.346 −1.475 −1.351 1.935 −0.961 −0.936 −0.504
3. Results and discussion 3.1. Optimization of nitrogen plasma treatment The experimental design technique was used to determine the optimal conditions of the plasma treatment to obtain the best hydrophilicity. This property was evaluated through water contact angle measurements. The experimental conditions (Ui) applied for the 17 experiments and the experimental results obtained for the water contact angle (Y) are shown in Table 2. Modde7.0 software developed by Umetrics was used for regression and graphical analysis of the obtained experimental data. The statistical significance of the main, quadratic and interaction effects of the variables was determined by analysis of variance (ANOVA) and a multiple regression analysis was performed to fit the experimental data (Yexp ) to the second-order polynomial equation (Eq. (2)). The determination coefficient R2 describes the fraction of variation of the response explained by the model. The coefficient of prediction Q2 describes the fraction of variation of the response that can be predicted by the model. The initial determination coefficient was too low because R² was around 65%. To improve the statistical significance of the model, three experiments were removed from the experimental design namely experiments 1, 7 and 14. R² is now equal to 95.7%, indicating that less than 5% of the total variation is not explained by the model. The contact angle is also well predicted by the model because the coefficient of prediction Q² is equal to 88%. After checking the reliability of the models, the significance of the different model terms can be discussed. Their values are listed in Table 3. First, the power of the plasma treatment seems to be the less significant factor. So it was decided to define the power (X3) as a
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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Fig. 2. Contour plot showing the evolution of the contact angle as a function of the nitrogen flow and the time of treatment for a constant power of 300 W.
Fig. 3. Contour plot showing the evolution of the contact angle as a function of the nitrogen flow and the power of the plasma treatment for a constant time of treatment of 300 s.
constant to plot the different response surfaces. By studying the different figures obtained by varying progressively the power, it was observed that the minimum contact angle was obtained for a power ranging between 150 and 300 W (variation lower than 1° in that range). The power was thus fixed at the center of the experimental domain, namely 300 W, to plot the evolution of the contact angle in function of the nitrogen flow and the time of treatment (Fig. 2). A minimum contact angle is obtained for the lowest nitrogen flow (100 cm3 /min) and the lowest time of treatment (30 s) and the predicted value for the contact angle is around 38.2°. Once the optimum value of each parameter has been determined, an experiment has to be done applying these conditions to compare the experimental result with the predicted one in order to validate the mathematical model associated to the experimental design. However, with a 100 cm3 /min nitrogen flow, a 30 s treatment time and a 300 W power, the experimental values obtained for the contact angle were much higher than the predicted value and the results were not reproducible as well. To explain this phenomenon, it can be assumed that a low gas flow combined with a short time of treatment does not allow reaching an equilibrium state inside the reactor (large volume of 350 l), which leads to a non-uniform treatment at the films surface. Thus, the response surfaces were considered again and Fig. 2 shows that a contact angle value relatively close to the minimum value previously predicted could be obtained by applying the opposite experimental conditions which are the highest values for the nitrogen flow and the time of treatment. That is why a new response surface has been plotted by fixing the time of treatment at 300 s (the highest value) and varying the nitrogen flow and the power (Fig. 3). In that case, the lowest contact angle is obtained for the highest nitrogen flow (which confirms the results given by Fig. 2) and for a power ranging from 300 to 450 W. So the new selected conditions were: a 500 cm3 /min nitrogen flow, a 300 s treatment time and a 300 W power, giving a predicted value of 43° for the contact angle.
Table 4 The total solid surface free energy (γ ), the polar (γ p ) and dispersive (γ d ) components of native and N2 plasma treated LDPE films.
3.2. Surface characterization after nitrogen plasma treatment 3.2.1. Surface wettability An experiment was done applying these conditions and a contact angle of 45.1 ± 2.2° was obtained allowing the validation of the experimental design. Surface wettability was evaluated by contact angle and surface free energy measurements. The water contact angle decreased clearly from 101.8 ± 1.4° for the untreated polymer
Surface energy (mJ/m2 )
Native N2 plasma treated
γ
γp
γd
34.7 ± 0.5 57.5 ± 1.7
0.0 ± 0.0 24.3 ± 0.9
34.7 ± 0.5 33.1 ± 0.7
to 45.1 ± 2.2° after optimization of the nitrogen plasma treatment. Moreover, the surface free energy increased from to 34.7 ± 0.5 to 57.5 ± 1.7 mJ/m2 and was mainly related to the increase of its polar component from zero to 24.3 ± 0.9 mJ/m2 (Table 4). The decrease in water contact angle and the increase of the surface free energy confirmed the hydrophilic character of the modified film [23]. This can be essentially attributed to the introduction of functional polar groups by cold plasma treatment as confirmed by the increase of the polar component. Similar results were obtained in previous studies [20,25,29,30]. 3.2.2. Surface composition XPS analysis was used to determine the chemical changes occurred on the surface (Fig. 4) and the elemental composition of the films, expressed as atomic concentrations. Compared with the untreated film (Fig. 4a), it can be clearly seen that after the N2 plasma treatment (Fig. 4b), two obvious peaks, namely the peak at 400.1 eV corresponding to N 1s and the peak at 532.2 eV corresponding to O 1s appear. The native film contained mainly carbon (99.4%) and traces of oxygen impurities (0.6%). After the plasma treatment, a decrease in the relative atomic concentration of carbon to 83.7% was associated with the increase of oxygen content (9.8%) and the incorporation of nitrogen (6.5%). The N 1s peak can be related to the plasma functionalization that occurs by the interaction of active gas species (nitrogen containing species) with the outermost region of the LDPE films [31,32]. Such interaction promotes chain scission and subsequent formation of free radicals that act as insertion points of the active species. However, the presence of oxygen on the surface can be explained by some functionalization that can be achieved during and/or after nitrogen plasma treatment. The free radicals generated during the treatment can react with residual oxygen in the plasma reactor and the remaining ones after the treatment can react with oxygen when the surface is exposed to the ambient atmosphere [3].
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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C3 H8 O+ (m/z = 60.06), C3 H2 NO+ (m/z = 68.01), were detected. Some representative secondary ion fragments are presented in Fig. 5. The obtained molecular ion species allow the identification of specific nitrogen plasma fragmentation and were in agreement with the XPS results. Both techniques permitted to confirm the nitrogen plasma surface modification highlighted by the presence of O and N and the introduction of polar hydrophilic groups to the surface.
3.3. Assessment of the antimicrobial activity of nisin treated films
Fig. 4. XPS survey spectra of (a) the native and (b) the nitrogen plasma treated LDPE films.
For more specific characterization of the surface chemical structure, ToF-SIMS analysis was performed. The characteristic ionized fragments of native LDPE films were hydrocarbonated ions (Cx Hy + ) such as C2 H5 + (m/z = 29.04), C3 H8 + (m/z = 44.06), C4 H7 + (m/z = 55.05), C5 H7 + (m/z = 67.05), C7 H11 + (m/z = 95.09). After the nitrogen plasma treatment, additional nitrogen- and/or oxygenbased fragments (Cx Hy N+ , Cx Hy O+ , Cx Hy NO+ ) such as CH2 NO+ (m/z = 44.01), CH4 NO+ (m/z = 46.03), C2 H2 NO+ (m/z = 56.01),
The antimicrobial activity of nisin-functionalized films was assessed qualitatively by the agar diffusion assay (Fig. 6) and quantitatively by comparing the log reduction of the viable and culturable L. innocua cells after 30 min of contact with both films (Fig. 7). Before nisin adsorption, the control films had no antibacterial activity since no inhibition was observed for native and plasma treated films. Similarly, the control culture with no addition of nisin-films showed a stable viable count along the experiment (data not shown). After nisin adsorption, the native LDPE film displayed a spot-like irregular antibacterial activity in some points of contact between the film and agar plate (Fig. 6a). In contrast, the N2 plasma film presented an almost complete regular inhibition under the film (discontinued by few colonies growth) in addition to a slight activity spread beyond the film perimeter (Fig. 6b). This activity can only be related to nisin adsorption on both films since the control films showed no activity. The quantitative tests permitted to further confirm the higher activity on the modified surfaces
Fig. 5. Representative positive ion fragments detected on the films: nitrogen- and oxygen-based fragments (CH2 NO+ and CH4 NO+ ) were detected on the plasma treated films (- - -) while only hydrocarbonated fragments (C3 H8 + ) were detected on the native films (—).
Fig. 6. Antimicrobial activity assay against L. innocua of native film + nisin (a) and N2 plasma treated film + nisin (b).
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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la Recherche were acknowledged for funding XPS/ToF-SIMS spectrometers within the Pôle Régional d’Analyses de Surface. We are also grateful for Dupont Health and Nutrition (United Kingdom) for donating the pure nisin for our research work. References
Fig. 7. Effect of nisin-functionalized films on the culturability loss of L. innocua. N0 and N are, respectively, the colony-forming units per milliliter before and after exposure of bacteria culture to nisin-functionalized films for 30 min. Error bars represent the standard deviation of the mean of three experiments.
(Fig. 7). The untreated and nitrogen plasma films induced, respectively, 0.5 and 0.9 log reduction of the viable count of L. innocua. Those results can be explained by the hydrophilic character of the plasma treated surface. In our previous work, nisin interactions, conformation, adsorption amount and antimicrobial activity on hydrophilic and hydrophobic surfaces were studied [13]. The higher nisin activity and adsorbed amount recorded on the hydrophilic polymers as compared to the hydrophobic ones were highly correlated to the type of interactions and the peptide conformation on those surfaces [14]. A larger change in peptide conformation induced by the interactions with the hydrophobic polymer may account for the lower activity of nisin observed on those surfaces [8,9]. In the present paper, nitrogen plasma treatment permitted to retain and improve nisin activity after its adsorption on the surfaces. It can offer thus a potential option for setting up hydrophilic bioactive polymers. 4. Conclusions This study, addressing the challenges of surface treatments, materials’ chemistry and those characteristic of microbiology and biochemistry (antimicrobial activity, adsorption of peptides…), allowed to determine the optimum conditions for using cold nitrogen plasma on LDPE polymer and then to functionalize it with nisin to impart its antimicrobial properties. The physico-chemical characterization techniques (XPS, ToF-SIMS, contact angle, surface free energy) confirmed the surface modification by plasma treatment. The measured antimicrobial activity makes it possible to consider the use of this film for active food packaging applications. In addition the N-functionalities generated by nitrogen plasma are highly desirable for immobilisation of different types of bioactive molecules for both agro-food and biomedical applications [33–36]. Also, the achieved methodology in this paper can be used to optimize various gases plasma or different plasma polymerization processes to further investigate surface characterization and interfacial interactions and then improve the antimicrobial performance of the films. Finally, other studies are needed to test their efficiency in real food applications. Acknowledgments This work was supported financially by the ‘‘Action de Recherche Concertée d’Initiative Régionale (ARCIR) No. 33517, de la région Nord Pas de Calais’’ (France) and by a PhD fellowship within the Erasmus Mundus Program. The Fonds Européen de Développement Régional (FEDER), CNRS, Région Nord Pas-de-Calais and Ministère de l’Education Nationale de l’Enseignement Supérieur et de
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[34] Duday D, Vreuls C, Moreno M, Frache G, Boscher ND, Zocchi G, Archambeau C, Van De Weerdt C, Martial Jo, Choquet P. Atmospheric pressure plasma modified surfaces for immobilization of antimicrobial nisin peptides. Surf Coat Technol 2013;218:152–61. [35] Goddard M, Hotchkiss JH. Polymer surface modification for the attachment of bioactive compounds. Prog Polym Sci 2007;32:698–725. [36] Chu P, Chen J, Wang L, Huang N. Plasma-surface modification of biomaterials. Mater Sci Eng R Rep 2002;36:143–206.
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Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films L. Karam a, M. Casetta a, N.E. Chihib a, F. Bentiss a,b, U. Maschke a, C. Jama a,∗ a b
UMET-PSI, CNRS UMR 8207, ENSCL, BP 90108, F-59652 Villeneuve d’Ascq Cedex, France Laboratoire de Catalyse et de Corrosion des Matériaux (LCCM), Faculté des Sciences, Université Chouaib Doukkali, B.P. 20, M-24000 El Jadida, Morocco
a r t i c l e
i n f o
Article history: Received 17 September 2015 Revised 8 April 2016 Accepted 15 April 2016 Available online xxx Keywords: Surface modification Nitrogen plasma Nisin Experimental design Functional polymers Antimicrobial packaging
a b s t r a c t In order to prevent microbial contamination and food-borne illnesses, antimicrobial active packaging represents an innovative option. The aim of this paper was to optimize the experimental parameters of the plasma treatment. Such pre-treatment can be used to develop an active packaging film by adsorbing a natural antimicrobial peptide “Nisin” on the surface of a commonly used polymer in the agro-food sector “low density polyethylene (LDPE)”. Cold nitrogen (N2 ) plasma treatment was used to functionalize LDPE and generate a hydrophilic polymer suitable for the adsorption of the peptide. The experimental design technique allowed determining the optimal conditions of the plasma treatment. The lowest contact angle and highest hydrophilic character were obtained for a gas flow rate of 500 cm3 /min, a generator power of 300 W and an exposure time of 300 s. The surface characterization techniques X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), contact angle and surface free energy measurements were used to confirm the surface modification after cold plasma treatment. The antimicrobial tests permitted to validate the efficiency of the active packaging films. The plasma treated films showed higher antimicrobial activity after nisin adsorption as compared to the native ones. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Recent and continuous foodborne microbial outbreaks are driving a search for innovative ways to inhibit microbial growth in the foods while maintaining quality, freshness, and safety. One option is to adsorb antimicrobial peptides on the packaging polymers to impart functional active properties and an increased margin of safety and quality. Polymer materials are inexpensive, easy to process, and exhibit excellent bulk and mechanical properties. However, their chemical inertness and their low surface energy represent generally a great barrier for their food packaging applications [1]. Plasma treatments have been used to expand the applications and transform these materials into highly valuable finished products. They can increase the wettability, polar groups, surface energy and improve the printability, adhesion, mechanical and barrier properties of polymer films [1–5]. From one side, these particular advantages of plasma treated films are highly desirable in food packaging applications to minimize leakage, reduce the risk of microbial contamination, and improve package integrity. From another side, the increase in hydrophilicity can improve the adsorption of peptides on surfaces as reported in previous studies
∗
Corresponding author. Tel.: +33 320 434 482; fax: +33 320 436 584. E-mail address: [email protected] (C. Jama).
[6–14]. Popelka et al. and Bílek et al. investigated antibacterial treatments on cold plasma pretreated low density polyethylene (LDPE) foils using benzalkonium chloride [15] and allylamine [16], respectively. Nisin is a well-known antimicrobial peptide, produced by Lactococcus lactis subsp. Lactis and widely used in the food industry as a safe and natural preservative [17]. It has an antimicrobial activity against a broad spectrum of Gram-positive bacteria and food pathogens such as Listeria innocua, Listeria monocytogenes, Staphylococcus aureus and Clostridium botulinum [18,19]. Therefore, the aim of this study was to assess the possibility of elaborating an antimicrobial polymer by adsorbing nisin on nitrogen (N2 ) plasma treated low density polyethylene (LDPE) film. This entailed the determination of the optimum conditions for using nitrogen plasma treatment, the use of physico-chemical methods to confirm the surface modification after the plasma treatment, the adsorption of nisin on both treated and native films, and finally the determination of the antimicrobial activity and/or the ability of nisin to retain its activity on the N2 plasma treated films. 2. Materials and methods 2.1. Film preparation Low density polyethylene (LDPE) was obtained from Polimeri Europa, France SAS (Density of 0.922 g/cm3 and thickness of
http://dx.doi.org/10.1016/j.jtice.2016.04.018 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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- a 2k fractional factorial design, k being the number of studied variables, - 6 axial points at a distance of α = 1.68 from the design center, - 3 replicates of the center point. Experiments were carried out randomly to provide protection against the extraneous factors, which could affect the measured response. For statistical calculations, the variables Ui were coded as Xi according to the following transformation (Eq. (1)):
Xi =
(Ui − U0 ) U
(1)
where Xi is the dimensionless coded value of the variable Ui , U0 represents the value of Ui at the center point and U is the step change. The experimental values associated to the coded levels of the different variables are given in Table 1. The quadratic model for predicting the optimal conditions was expressed according to Eq. (2):
Ypred = β0 +
n
βi Xi +
i=1
n
βi j Xi X j +
n
βii Xi2
(2)
i=1
i< j
where Y is the predicted response, β 0 is the value of the fitted response at the center point, β i , β ii and β ij correspond to the linear, quadratic and interaction terms respectively.
Fig. 1. Schematic representation of the experimental set-up.
70 μm). LDPE films were cut into square shape (2 cm × 2 cm) and washed with ethanol in an ultrasonic bath to remove possible dusts or any oily compounds adsorbed on the film surface. They were then dried in an oven at 55 °C for 3 h. Those films were either used directly or treated for nisin adsorption.
2.2.2. Surface characterization Contact angle and surface free energy measurements: Static contact angle measurements of the samples were carried out at room temperature on a Digidrop goniometer (GBX, France) using pure water for the optimization step. A 5 μL drop of water was applied onto the sample surface and the contact angle formed with the surface was instantaneously measured. Triplicate tests were performed for the films, immediately after the plasma treatment, and at least six different measurements were made on each sample surface. The average values for contact angles and the standard deviation were then calculated. The contact angles were also determined using two other test liquids (formamide and diiodomethane) in order to calculate the surface free energy values with the Owens–Wendt model [20–22]. This method takes into account dispersive and polar components of the surface energy and using different test liquids, it is possible to determine the solid surface free energy (γ ) as the sum of polar (γ p ) and dispersive (γ d ) contributions [21,23–25]. X-ray photoelectron spectroscopy (XPS): X-ray photoelectron spectroscopy (XPS) experiments were carried out using a Kratos Analytical AXIS UltraDLD spectrometer. A monochromatized aluminium source (Al Kα = 1486.6 eV) was used for excitation. The X-ray beam diameter was around 1 mm. The analyser was operated at constant pass energy of 160 eV for survey spectra using an analysis area of approximately 700 μm × 300 μm. The electron take off angle was 90°. Charge compensation was applied to compensate for the charging effect occurring during the analysis. The C 1s hydrocarbon (285.0 eV) binding energy (BE) was used as internal reference. The spectrometer BE scale was initially calibrated
2.2. Plasma treated films 2.2.1. Design of experiments Plasma treatments were performed in LDPE samples were plasma treated in a EUROPLASMA CD 1200 set-up reactor using cold radiofrequency plasma (13.56 MHz) fitted with a capacitively coupled, parallel-electrode system with an automatic matching device. A schematic representation of the experimental set-up is shown in Fig. 1. A pure nitrogen glow discharge was generated in an aluminium reactor chamber with a continuous out power ranging from 0 to 600 W. The chamber was pumped down to 10.7 Pa using a pump Edwards (80 m3 /h), and the N2 gas was introduced into the chamber. When the pressure became constant, the generator was switched on and adjusted to a certain power value, which gave rise to a continuous glow discharge. The sample holder was placed in the center between the electrodes without any bias. The distance between the electrodes is equal to 10 cm. Optimization of the plasma treatment was carried out using response surface methodology (RSM). The influence of three main process parameters was studied namely the nitrogen flow, the time of treatment and the power during the plasma treatment. A central composite design (CCD), consisting of 17 experimental runs, was used including:
Table 1 Coded and real values of experimental parameters used for the CCD. Coded variables
X1 X2 X3
Parameter
Levels
3
U1 , nitrogen flow (cm /min) U2 , time of treatment (s) U3 , power (W)
−α
−1
0
+1
+α
100 30 100
180 85 180
300 165 300
420 245 420
500 300 500
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against the Ag 3d5/2 (368.2 eV) level. Pressure was in the 10−10 Torr range during the experiments. Quantification and simulation of the experimental photopeaks was carried out using CasaXPS software. Quantification took into account a non-linear Shirley background subtraction [26]. Time-of-flight secondary ion mass spectrometry (ToF-SIMS): ToFSIMS spectra measurements were carried out using a ToF-SIMS V instrument (ION-TOF GmbH, Germany). This instrument is equipped with a Bi liquid metal ion gun (LMIG). Pulsed Bi3 + primary ions have been used for analysis (25 keV, 0.4 pA). Mass spectra were taken for each sample, from an area of 500 μm × 500 μm (30 scans) using 256 × 256 pixel random rasters. These experimental conditions allowed staying within the static conditions since primary ion dose did not exceed 1012 ions/cm2 . Pulsed low energy flood gun (20 eV) were used for charge neutralization. Average mass resolution was about 50 0 0 at m/z = 55.05 (C4 H7 + ) which allowed the separation of ion fragments under investigation. 2.3. Nisin-functionalized films 2.3.1. Nisin adsorption on films Nisin adsorption was carried out on the native and the plasma treated films. A pure grade of nisin was donated by Dupont Health and Nutrition (United Kingdom). Activity was indicated as 5.2 × 107 IU/g nisin solutions were prepared by dissolving 1.0 mg/ml of nisin in HCl (0.01 M). Solutions were freshly prepared and filtered (0.22 μm) before each experiment. Each film was immersed in 20 ml of nisin solution (1.0 mg/ml) and it was agitated at 8 °C for 16 h. After that, the samples were removed from solution and briefly rinsed in sterile distilled water to remove non adsorbed nisin. All the tests were done after drying the films in sterile Petri dishes at 25 °C for 24 h. 2.3.2. Assessment of the antimicrobial activity of nisin-functionalized films The antimicrobial tests were carried out against Listeria innocua. Pre-cultures were performed by inoculating a single colony in 10 ml of Brain Heart broth. The cultures were made by inoculating 10 ml of Brain Heart broth with 100 μl of the pre-culture. Pre-cultures and cultures were incubated at 37 °C for 24 h. Qualitative antibacterial tests were done using a modified agar diffusion assay [27]. Mueller Hinton agar medium was seeded with the indicator micro-organism, L. innocua. The face up of the film to be tested was placed on the agar surface. Bioassay plates containing experimental samples were kept at 4 °C for 4 h to initiate nisin diffusion and were then incubated at 37 °C for 24 h. Nisin activity was assessed as an inhibition of the indicator bacterium growth under and around the film. The quantitative inhibitory effect of the two nisinfunctionalized films was carried out, at room temperature, by putting each film in 5 ml of L. innocua cell suspension of ca.106 CFU/ml. After 30 min of contact time, the samples were enumerated by plating onto Luria Bertani agar and incubating for 24 h at 37 °C. In each experiment, a control test of L. innocua (without the test-film) was realized under the same conditions. The viable and culturable counts of L. innocua, were determined and used to assess the antimicrobial activity of the films. The results were expressed in terms of logarithm of the difference in population (DP) according to the following equation (Eq. (3)) [28]:
log DP = log (N0 /N ) = (log N0 ) − (log N )
(3)
where N0 and N are, respectively, the bacterial population (colonyforming units per milliliter) before and after exposure of bacteria culture to nisin-functionalized films for 30 min. Those experiments were made in triplicates and all the values were expressed as a mean value ± the standard deviation.
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Table 2 CCD in coded and real values with the experimental responses obtained for the different runs. Experiments
X1
X2
X3
U1
U2
U3
Yexp (°)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
−1 +1 −1 +1 −1 +1 −1 +1 −α +α 0 0 0 0 0 0 0
−1 −1 +1 +1 −1 −1 +1 +1 0 0 −α +α 0 0 0 0 0
−1 −1 −1 −1 +1 +1 +1 +1 0 0 0 0 −α +α 0 0 0
180 420 180 420 180 420 180 420 100 500 300 300 300 300 300 300 300
85 85 245 245 85 85 245 245 165 165 30 300 165 165 165 165 165
180 180 180 180 420 420 420 420 300 300 300 300 100 500 300 300 300
52.7 50.8 50.9 51.6 49.8 50.6 49 49.5 46.1 47.9 45.8 48.9 56.1 47.1 50.4 52.7 50.3
Table 3 Coefficients of the model estimated with Modde7.0. Coefficients
β0 β1 β2 β3 β 11 β 22 β 33 β 12 β 13 β 23
51.14 0.458 0.900 0.346 −1.475 −1.351 1.935 −0.961 −0.936 −0.504
3. Results and discussion 3.1. Optimization of nitrogen plasma treatment The experimental design technique was used to determine the optimal conditions of the plasma treatment to obtain the best hydrophilicity. This property was evaluated through water contact angle measurements. The experimental conditions (Ui) applied for the 17 experiments and the experimental results obtained for the water contact angle (Y) are shown in Table 2. Modde7.0 software developed by Umetrics was used for regression and graphical analysis of the obtained experimental data. The statistical significance of the main, quadratic and interaction effects of the variables was determined by analysis of variance (ANOVA) and a multiple regression analysis was performed to fit the experimental data (Yexp ) to the second-order polynomial equation (Eq. (2)). The determination coefficient R2 describes the fraction of variation of the response explained by the model. The coefficient of prediction Q2 describes the fraction of variation of the response that can be predicted by the model. The initial determination coefficient was too low because R² was around 65%. To improve the statistical significance of the model, three experiments were removed from the experimental design namely experiments 1, 7 and 14. R² is now equal to 95.7%, indicating that less than 5% of the total variation is not explained by the model. The contact angle is also well predicted by the model because the coefficient of prediction Q² is equal to 88%. After checking the reliability of the models, the significance of the different model terms can be discussed. Their values are listed in Table 3. First, the power of the plasma treatment seems to be the less significant factor. So it was decided to define the power (X3) as a
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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Fig. 2. Contour plot showing the evolution of the contact angle as a function of the nitrogen flow and the time of treatment for a constant power of 300 W.
Fig. 3. Contour plot showing the evolution of the contact angle as a function of the nitrogen flow and the power of the plasma treatment for a constant time of treatment of 300 s.
constant to plot the different response surfaces. By studying the different figures obtained by varying progressively the power, it was observed that the minimum contact angle was obtained for a power ranging between 150 and 300 W (variation lower than 1° in that range). The power was thus fixed at the center of the experimental domain, namely 300 W, to plot the evolution of the contact angle in function of the nitrogen flow and the time of treatment (Fig. 2). A minimum contact angle is obtained for the lowest nitrogen flow (100 cm3 /min) and the lowest time of treatment (30 s) and the predicted value for the contact angle is around 38.2°. Once the optimum value of each parameter has been determined, an experiment has to be done applying these conditions to compare the experimental result with the predicted one in order to validate the mathematical model associated to the experimental design. However, with a 100 cm3 /min nitrogen flow, a 30 s treatment time and a 300 W power, the experimental values obtained for the contact angle were much higher than the predicted value and the results were not reproducible as well. To explain this phenomenon, it can be assumed that a low gas flow combined with a short time of treatment does not allow reaching an equilibrium state inside the reactor (large volume of 350 l), which leads to a non-uniform treatment at the films surface. Thus, the response surfaces were considered again and Fig. 2 shows that a contact angle value relatively close to the minimum value previously predicted could be obtained by applying the opposite experimental conditions which are the highest values for the nitrogen flow and the time of treatment. That is why a new response surface has been plotted by fixing the time of treatment at 300 s (the highest value) and varying the nitrogen flow and the power (Fig. 3). In that case, the lowest contact angle is obtained for the highest nitrogen flow (which confirms the results given by Fig. 2) and for a power ranging from 300 to 450 W. So the new selected conditions were: a 500 cm3 /min nitrogen flow, a 300 s treatment time and a 300 W power, giving a predicted value of 43° for the contact angle.
Table 4 The total solid surface free energy (γ ), the polar (γ p ) and dispersive (γ d ) components of native and N2 plasma treated LDPE films.
3.2. Surface characterization after nitrogen plasma treatment 3.2.1. Surface wettability An experiment was done applying these conditions and a contact angle of 45.1 ± 2.2° was obtained allowing the validation of the experimental design. Surface wettability was evaluated by contact angle and surface free energy measurements. The water contact angle decreased clearly from 101.8 ± 1.4° for the untreated polymer
Surface energy (mJ/m2 )
Native N2 plasma treated
γ
γp
γd
34.7 ± 0.5 57.5 ± 1.7
0.0 ± 0.0 24.3 ± 0.9
34.7 ± 0.5 33.1 ± 0.7
to 45.1 ± 2.2° after optimization of the nitrogen plasma treatment. Moreover, the surface free energy increased from to 34.7 ± 0.5 to 57.5 ± 1.7 mJ/m2 and was mainly related to the increase of its polar component from zero to 24.3 ± 0.9 mJ/m2 (Table 4). The decrease in water contact angle and the increase of the surface free energy confirmed the hydrophilic character of the modified film [23]. This can be essentially attributed to the introduction of functional polar groups by cold plasma treatment as confirmed by the increase of the polar component. Similar results were obtained in previous studies [20,25,29,30]. 3.2.2. Surface composition XPS analysis was used to determine the chemical changes occurred on the surface (Fig. 4) and the elemental composition of the films, expressed as atomic concentrations. Compared with the untreated film (Fig. 4a), it can be clearly seen that after the N2 plasma treatment (Fig. 4b), two obvious peaks, namely the peak at 400.1 eV corresponding to N 1s and the peak at 532.2 eV corresponding to O 1s appear. The native film contained mainly carbon (99.4%) and traces of oxygen impurities (0.6%). After the plasma treatment, a decrease in the relative atomic concentration of carbon to 83.7% was associated with the increase of oxygen content (9.8%) and the incorporation of nitrogen (6.5%). The N 1s peak can be related to the plasma functionalization that occurs by the interaction of active gas species (nitrogen containing species) with the outermost region of the LDPE films [31,32]. Such interaction promotes chain scission and subsequent formation of free radicals that act as insertion points of the active species. However, the presence of oxygen on the surface can be explained by some functionalization that can be achieved during and/or after nitrogen plasma treatment. The free radicals generated during the treatment can react with residual oxygen in the plasma reactor and the remaining ones after the treatment can react with oxygen when the surface is exposed to the ambient atmosphere [3].
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C3 H8 O+ (m/z = 60.06), C3 H2 NO+ (m/z = 68.01), were detected. Some representative secondary ion fragments are presented in Fig. 5. The obtained molecular ion species allow the identification of specific nitrogen plasma fragmentation and were in agreement with the XPS results. Both techniques permitted to confirm the nitrogen plasma surface modification highlighted by the presence of O and N and the introduction of polar hydrophilic groups to the surface.
3.3. Assessment of the antimicrobial activity of nisin treated films
Fig. 4. XPS survey spectra of (a) the native and (b) the nitrogen plasma treated LDPE films.
For more specific characterization of the surface chemical structure, ToF-SIMS analysis was performed. The characteristic ionized fragments of native LDPE films were hydrocarbonated ions (Cx Hy + ) such as C2 H5 + (m/z = 29.04), C3 H8 + (m/z = 44.06), C4 H7 + (m/z = 55.05), C5 H7 + (m/z = 67.05), C7 H11 + (m/z = 95.09). After the nitrogen plasma treatment, additional nitrogen- and/or oxygenbased fragments (Cx Hy N+ , Cx Hy O+ , Cx Hy NO+ ) such as CH2 NO+ (m/z = 44.01), CH4 NO+ (m/z = 46.03), C2 H2 NO+ (m/z = 56.01),
The antimicrobial activity of nisin-functionalized films was assessed qualitatively by the agar diffusion assay (Fig. 6) and quantitatively by comparing the log reduction of the viable and culturable L. innocua cells after 30 min of contact with both films (Fig. 7). Before nisin adsorption, the control films had no antibacterial activity since no inhibition was observed for native and plasma treated films. Similarly, the control culture with no addition of nisin-films showed a stable viable count along the experiment (data not shown). After nisin adsorption, the native LDPE film displayed a spot-like irregular antibacterial activity in some points of contact between the film and agar plate (Fig. 6a). In contrast, the N2 plasma film presented an almost complete regular inhibition under the film (discontinued by few colonies growth) in addition to a slight activity spread beyond the film perimeter (Fig. 6b). This activity can only be related to nisin adsorption on both films since the control films showed no activity. The quantitative tests permitted to further confirm the higher activity on the modified surfaces
Fig. 5. Representative positive ion fragments detected on the films: nitrogen- and oxygen-based fragments (CH2 NO+ and CH4 NO+ ) were detected on the plasma treated films (- - -) while only hydrocarbonated fragments (C3 H8 + ) were detected on the native films (—).
Fig. 6. Antimicrobial activity assay against L. innocua of native film + nisin (a) and N2 plasma treated film + nisin (b).
Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018
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la Recherche were acknowledged for funding XPS/ToF-SIMS spectrometers within the Pôle Régional d’Analyses de Surface. We are also grateful for Dupont Health and Nutrition (United Kingdom) for donating the pure nisin for our research work. References
Fig. 7. Effect of nisin-functionalized films on the culturability loss of L. innocua. N0 and N are, respectively, the colony-forming units per milliliter before and after exposure of bacteria culture to nisin-functionalized films for 30 min. Error bars represent the standard deviation of the mean of three experiments.
(Fig. 7). The untreated and nitrogen plasma films induced, respectively, 0.5 and 0.9 log reduction of the viable count of L. innocua. Those results can be explained by the hydrophilic character of the plasma treated surface. In our previous work, nisin interactions, conformation, adsorption amount and antimicrobial activity on hydrophilic and hydrophobic surfaces were studied [13]. The higher nisin activity and adsorbed amount recorded on the hydrophilic polymers as compared to the hydrophobic ones were highly correlated to the type of interactions and the peptide conformation on those surfaces [14]. A larger change in peptide conformation induced by the interactions with the hydrophobic polymer may account for the lower activity of nisin observed on those surfaces [8,9]. In the present paper, nitrogen plasma treatment permitted to retain and improve nisin activity after its adsorption on the surfaces. It can offer thus a potential option for setting up hydrophilic bioactive polymers. 4. Conclusions This study, addressing the challenges of surface treatments, materials’ chemistry and those characteristic of microbiology and biochemistry (antimicrobial activity, adsorption of peptides…), allowed to determine the optimum conditions for using cold nitrogen plasma on LDPE polymer and then to functionalize it with nisin to impart its antimicrobial properties. The physico-chemical characterization techniques (XPS, ToF-SIMS, contact angle, surface free energy) confirmed the surface modification by plasma treatment. The measured antimicrobial activity makes it possible to consider the use of this film for active food packaging applications. In addition the N-functionalities generated by nitrogen plasma are highly desirable for immobilisation of different types of bioactive molecules for both agro-food and biomedical applications [33–36]. Also, the achieved methodology in this paper can be used to optimize various gases plasma or different plasma polymerization processes to further investigate surface characterization and interfacial interactions and then improve the antimicrobial performance of the films. Finally, other studies are needed to test their efficiency in real food applications. Acknowledgments This work was supported financially by the ‘‘Action de Recherche Concertée d’Initiative Régionale (ARCIR) No. 33517, de la région Nord Pas de Calais’’ (France) and by a PhD fellowship within the Erasmus Mundus Program. The Fonds Européen de Développement Régional (FEDER), CNRS, Région Nord Pas-de-Calais and Ministère de l’Education Nationale de l’Enseignement Supérieur et de
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Please cite this article as: L. Karam et al., Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.04.018