Zinc migration and its effect on the functionality of a low density polyethylene-ZnO nanocomposite film

Food Packaging and Shelf Life 20 (2019) 100301

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Zinc migration and its effect on the functionality of a low density polyethylene-ZnO nanocomposite film ⁎

Nattinee Bumbudsanpharokea,1, Jeongin Choib,1, Hyun Jin Parkb, , Seonghyuk Koa, a b

T

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Department of Packaging, College of Science and Technology, Yonsei University, Gangwon-do, 26493, Republic of Korea Department of Biotechnology, College of Life Science and Biotechnology, Korea University, Seoul, 02841, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: ZnO nanoparticles Migration Food simulants Antimicrobial activity Food contact material

The interest in incorporating ZnO nanoparticles (ZnO-NPs) into food contact materials is increasing due to its attractive functions such as ultraviolet (UV) blocking and antimicrobial activity. Despite their benefits, the stability and functionality of ZnO-NPs could be altered when they come into contact with foodstuff through migration. Hence, we investigated Zn migration and its effect on the functional properties of low density polyethylene (LDPE)-ZnO nanocomposite films. The migration of Zn from nanocomposite films into food simulants (distilled water, 4% acetic acid (w/v), 50% ethanol (v/v) and n-heptane) was conducted at 70 °C for 30 min according to the Korea standard and specifications for food utensils, containers and packages conditions. The presence of Zn in the food simulants was verified by inductively coupled plasma-optical emission spectrometry. Different concentrations of dissolved Zn were observed ranging from 0.006 to 3.416 mg L−1 (except for heptane) and the level of migrated Zn was found as a function of the ZnO-NPs content in the nanocomposite film. However, the highest amount of migrated Zn measured in this study was lower than the specific migration limit regulated by the European Commission. In addition, the UV light absorption and the antimicrobial activity of the LDPE-ZnO nanocomposite film were significantly affected by the dissolution of ZnO, particularly in acetic acid. Therefore, particular attention is requested to use when LDPE-ZnO nanocomposite films are used for food packaging, especially acidic food, because Zn can likely migrate, and thereby their UV blocking and antimicrobial functions could be no longer effective.

1. Introduction Hybrid nano-enabled packaging using inorganic nanomaterials, such as silver, nanoclay, copper, zinc oxide and titanium dioxide, has attracted considerable attention as it provides unique features such as antibacterial function, barrier enhancement and ultraviolet (UV) absorption. From a chemical standpoint, the integration of inorganic nanoscale fillers into organic polymer matrices can be processed through several pathways depending on the interactions between the moieties. Nanomaterials are stably inlaid in polymer matrices without chemical interactions or anchor to the surface by strong/weak chemical bonds such as covalent, ionic or hydrogen bonds (Kickelbick, 2003). However, as a food contact material, these two dissimilar phases could be separated and leached via chemical interactions especially with food components, causing a loss in stability and functionality. Many reports revealed the potential migration of nanomaterial from polymer nanocomposites into food simulants and real foods (Bott, Störmer,

Wolz, & Franz, 2012, November; Cushen, Kerry, Morris, Cruz-Romero, & Cummins, 2013; Lin et al., 2014; Song, Li, Lin, Wu, & Chen, 2011), which can adversely affect the quality of packaging materials and the human safety (Bradley, Castle, & Chaudhry, 2011). Among the various metal oxide nanomaterials, ZnO nanoparticles (ZnO-NPs) show the greatest antimicrobial performance against numerous microorganisms (Jones, Ray, Ranjit, & Manna, 2008) and are more affordable and less toxic to human health compared to silver nanoparticles (AgNPs) (Chaudhry et al., 2008; Espitia et al., 2012). In addition, when composited with polymeric materials, ZnO-NPs further improve packaging properties, including stability, mechanical strength and barrier properties (Emamifar, Kadivar, Shahedi, & SoleimanianZad, 2011; Jones et al., 2008; Kompany, Mirza, Hosseini, PingguanMurphy, & Djordjevic, 2014; Tankhiwale & Bajpai, 2012). Many studies have evaluated the potential migration of ZnO-NPs from packaging into foodstuff related to safety issues (Huang, Tang, Luo, Zhang, & Qin, 2017; Panea, Ripoll, Gonzalez, Fernandez-Cuello, & Alberti, 2014;

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Corresponding authors. E-mail addresses: [email protected] (H.J. Park), [email protected] (S. Ko). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.fpsl.2019.100301 Received 11 October 2018; Received in revised form 9 January 2019; Accepted 15 January 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. a) FTIR spectra and b) FE-SEM surface micrographs of LDPE and LDPE-ZnO nanocomposites (1 and 5 wt.%) with EDS spectra. Table 1 Precision of the ICP-OES analysis represented by the mean recovery (%) and RSD (%) of the samples measured (six replicates) with different food simulants. Spiked concentration of Zn 0.05 mg L−1

0.5 mg L−1

5 mg L−1

Food simulant

Mean Recovery (%)

RSD (%)

Mean Recovery (%)

RSD (%)

Mean Recovery (%)

RSD (%)

Distilled water 4% acetic acid (w/v) 50% ethanol (v/v)

102 101 103

2.5 1.7 2.3

102 93 87

0.8 1.0 1.9

98 94 100

0.9 2.1 2.4

on AgNPs as they can be oxidised and dissolved into silver ions (Ag+) when they come in contact foods. Although the presence of these migrated Ag species in food provides significant antimicrobial activity, they are toxic to human cells due to the potential bioaccumulation that is the same as for microorganisms (AshaRani, Kah, Mun, Hande, &

Polat, Fenercioglu, Turhan, & Guclu, 2018); however, there has been no report about the effect of migration on packaging functionality. Migration studies on nanomaterials have been actively conducted because of the negative influence on the quality and safety of the packaging products (Bradley et al., 2011), but they were mostly focused 2

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water (Millipore, Billerica, Massachusetts, USA); glacial acetic acid (Daejung Chemicals & Metals Co., Ltd, Gyeonggi-Do, Korea); ethanol (Duksan Pure Chemicals, Gyeonggi-Do, Korea); n-heptane (95%, Samchun Chemical Co., Ltd., Seoul, Korea). A multi-element tuning solution at a concentration of 100 μg/mL was used (AccuStandard Inc., New Haven, Connecticut, USA). Nitric acid (Duksan Pure Chemicals, Gyeonggi-Do, Korea) was employed in the acid digestion. 2.2. Preparation of LDPE-ZnO nanocomposite films The LDPE-ZnO nanocomposite films with different ZnO-NPs content (0, 1 and 5 wt.%) were prepared by multilayer extrusion with a T-die extruder (30 mm diameter, L/D = 30, Hankook E.M Ltd., Incheon, Korea). The extrusion temperature ranged between 190 and 230 °C and the screw speed was 40 rpm. In order to yield various concentrations of ZnO in the nanocomposite films, pure LDPE and ZnO masterbatch (consisting of LDPE loaded with 10 wt.% of ZnO-NPs) were blended for final concentration at 1 and 5 wt.% ZnO-NPs content. Neat LDPE film was prepared in the same way for use as reference sample. Prior to processing, LDPE and ZnO masterbatch pellets were dried in a convection oven at 80 °C for 24 h and then mixed in a sealed plastic bag by manual tumbling before being fed into the extruder. Three different compositions of LDPE-ZnO nanocomposite films with a controlled thickness of 60–70 μm were obtained.

Fig. 2. Concentrations of migrated Zn from LDPE-ZnO nanocomposite film into food simulants (distilled water, 4% acetic acid and 50% ethanol).

Table 2 Solubility of ZnO-NPs in different food simulants under same condition as for the migration test. Food simulant

Solubility (%)

Distilled water 4% acetic acid (w/v) 50% ethanol (v/v) n-heptane

9.45 ± 0.65 86.87 ± 2.41 1.64 ± 0.65 0.01 ± 0.00

2.3. Characterisation of LDPE-ZnO nanocomposite films FTIR spectra were obtained with a Perkin Elmer Spectrum 65 spectrophotometer in attenuated total reflection mode with a C/ZnSe crystal. Each spectrum was obtained in transmittance mode with 32 scans and a resolution of 2 cm−1 in the 400–4000 cm−1 range. The surface morphology of each prepared film was observed with a field-emission scanning electron microscope (FE-SEM Quanta FEG 250, FEI, Hillsboro, Oregon, USA). Prior to the analysis, the specimens were sputtered with platinum/palladium using a Cressington Sputter Coater 108 auto (Cressington Scientific Instruments, Watford, UK). Elemental analysis was performed using an energy-dispersive X-ray spectrometer (EDS, Ametek, Berwyn, Pennsylvania, USA) integrated in the FE-SEM. Transparency of the films was determined using a V-600 UV–vis spectrophotometer (Jasco, Tokyo, Japan). The spectra of the films were obtained in the 200–800 nm range.

Valiyaveettil, 2008; Hannon et al., 2016). Several researches have demonstrated the migration of Ag species from commercial polymeric food packaging materials and interestingly, the maximum values of migrated Ag were detected in acidic solutions (Echegoyen & Nerin, 2013; Huang et al., 2011; Ntim, Thomas, Begley, & Noonan, 2015; Song et al., 2011). Similar to AgNPs, Ti is leachable from polyethylene-based nanocomposite films into food simulants depending on the immersion time and the content of additives in the film (Lin et al., 2014). Even though public concern about human exposure to ZnO is increasing together with the use of ZnO-NPs in food packaging, the data about Zn migration into food simulants are insufficient and evaluation studies on the functionality (i.e. antimicrobial activity) of packaging after migration are still lacking. The aim of this study is to investigate the mechanism of Zn migration from low density polyethylene (LDPE)-ZnO nanocomposite films into food simulants (distilled water, 4% acetic acid (w/v), 50% ethanol (v/v) and n-heptane) and its effect on the UV blocking and antibacterial functions of the films. The migration test was conducted in accordance with the Korea standards and specifications for food utensils, containers and packages (MFDS, 2015). In addition, the effect of Zn migration on the material properties was assessed through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transparency tests and antimicrobial activity tests against Staphylococcus aureus and Escherichia coli.

2.4. Method validation To verify that there is no loss of Zn during the migration experiment and the inductively coupled plasma-optical emission spectrometry (ICPOES) analysis, the validation was performed by determining limit of detection (LOD), limit of quantification (LOQ), recovery and repeatability of the method. LOD and LOQ were obtained by substituting the ICP-OES signal and standard deviation (SD) values of six different blank samples into the respective formulas 3.3 SD/s and 10 SD/s, where s is the slope of the calibration curve. Recovery and repeatability were determined by spiking each one of the Zn standard solutions (0.05, 0.5 and 5 mg L−1) into four different food simulants. Before ICP-OES analysis, all the samples were digested with acid and all experiments were performed with six replicates (n = 6).

2. Materials and methods 2.5. Solubility test 2.1. Materials The solubility of ZnO-NPs in the four food simulants was evaluated under the same condition as for the migration test. About 17 mg of ZnONPs powder, which equals its amount in the 5 wt.% loading nanocomposite film, was added to 200 mL of each food simulants; the mixtures were stirred at room temperature overnight and centrifuged (15,000×g, 30 min) for three times. Then, the supernatant was taken and analysed with a ICP-OES spectrometer (Perkin Elmer Optima 8300,

LDPE (Lutene LB7500, LG Chemical Co., Yeosu, Korea) with a melt flow index of 7.5 g/10 min (ASTM D1238) and a density of 0.918 g cm−3 was employed in this study. A white and opaque spherical powder of ZnO-NPs with 10–20 nm in diameter (Nano, Future and Life, Inc., Daejon, Korea) was used for nanocomposite film preparation and solubility tests. Four types of food simulants were purchased: Milli-Q 3

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Fig. 3. a) FE-SEM surface and b) cross-section micrographs of LDPE-ZnO nanocomposite films before and after migration test with 4% acetic acid.

4

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based on 2 mL per 1 cm2; hence, 200 mL of food simulants was added to the vials, which were sealed with closures. The samples were placed in a dry oven at 70 °C for 30 min, except for the case of n-heptane, which was kept at room temperature (25 °C) for 1 h. Next, the vials were taken out of the dry oven and the film samples were removed. To determine the amount of Zn, food simulants were digested with the hot-plate method. Each food simulant solution acquired after migration was placed on a heating stirrer and acidized with ultra-high pure nitric acid until 3–4 drops of solution remained. Finally, remained solution was massed up to 10 mL with 1% HNO3 and analysed by ICP-OES. The conditions of ICP-OES were as follows: radio-frequency (RF), 40 MHz; RF generator power, 1500 W; plasma gas flow, 12 L min−1; auxiliary gas flow, 0.2 L min−1; nebuliser gas flow, 0.55 L min−1.

2.7. Antimicrobial activity test The antibacterial activity of LDPE-ZnO composite films was evaluated by adapting the ASTM method (ASTM E2149-0) and JIS method (JIS Z 2801:2000) against both Gram-negative and Gram-positive bacteria, Escherichia coli (E. coli, ATCC 8739) and Staphylococcus aureus (S. aureus, ATCC 6538 P), respectively. Briefly, each film sample (50 × 50 mm) was sterilised with UV light, and then 400 μL bacterial inoculum in nutrient broth (2.5–10 × 105 CFU mL−1, where CFU indicates the colony-forming units) was dropped onto both control and test samples. Each sample was covered with a 40 × 40 mm sterilised Stomacher® 400 Poly-bag film, and then incubated at 35 ± 1 °C and 90% ± 5% RH for 24 h. Next, the test samples and the covered films were carefully washed with 10 mL of nutrient broth in a sterilised Stomacher® pouch. For viable cell enumeration, 1 mL from the washing was pipetted into a test tube containing 9 mL of nutrient broth and mixed thoroughly. Then, 1 mL from this test tube was taken with a new pipette and placed into another test tube containing 9 mL of nutrient broth. This procedure was repeated to achieve the 10-fold serial dilutions. Subsequently, 400 μL of each microbial suspension was dispensed onto an agar plate and incubated at 35 ± 1 °C and 90% ± 5% RH. After 24 h of incubation, the CFUs in a serially diluted petri dish with 30–300 colonies were counted. Antibacterial activity was calculated as a percentage of colonies reduction according the ASTM method (ASTM E2149-0), as the following equation:

Fig. 4. Transparency of the LDPE and LDPE-ZnO nanocomposite films before and after migration test.

Shelton, CT). The solubility was obtained by substituting the ICP-OES values into the following formula:

%Solubility =

V × 100 W

(1) −1

where V is the amount of Zn (mg L ) given by ICP-OES analysis and W is the weight of the ZnO-NP powder used in the solubility test. 2.6. Migration test Distilled water, 4% acetic acid (w/v), 50% ethanol (v/v) and nheptane were used to simulate aqueous, acidic, alcoholic and fatty foods, respectively. Two samples of each film (2.5 × 10 cm) were used for each test condition. Before the immersion into the food simulants, the film samples were washed with distilled water and dried by fixing them to a glass holder inserted in a 250 mL glass vial. The amount of food simulant for the migration test was determined by surface area

Fig. 5. Antimicrobial activity of LDPE and LDPE-ZnO nanocomposite films before and after migration test with the acidic food simulant. 5

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(A − B ) ⎤ Antibacterial activity (%) = ⎡ × 100 A ⎦ ⎣

0.006–0.013 mg L−1 in 50% ethanol, while no Zn was detected in nheptane. Furthermore, the concentration of migrated Zn from total ZnO in the nanocomposite films was 0.017–0.058 μg cm−2 in distilled water, 0.035–6.831 μg cm−2 in 4% acetic acid, and 0.011–0.026 μg cm−2 in 50% ethanol. These results can be explained by the solubility measurement (Table 2), which shows the same tendency. The solubility of ZnO-NPs was significantly higher (86.87%) in 4% acetic acid than in the other simulants; 9.45%, 1.64% and 0.01% in distilled water, 50% ethanol and n-heptane, respectively. ZnO-NPs exhibited maximum values of both migration and solubility in 4% acetic acid. Previous studies reported that the dissolution of engineered ZnO-NPs is highly dependent on the pH of contact media (Miao et al., 2010). Moreover, the dissolution of ZnO-NPs occurs rapidly in artificial lysosomal fluids (pH 5.5) but halts in interstitial fluids (pH 7.4) (Cho et al., 2012). Furthermore, Ozaki, Kishi, Ooshima, Hase, and Kawamura (2016) examined the migration of Zn from food contact plastics into food simulants (distilled water, 4% acetic acid and 20% ethanol) and found that Zn migration was highest in 4% acetic acid due to the higher tendency of ionisation. To confirm the dissolving behaviour of ZnO-NPs in food simulants, especially 4% acetic acid, FE-SEM analysis of the surfaces and crosssections of the nanocomposite films after migration testing was conducted. As shown in Fig. 3a, in untreated samples, the ZnO-NPs were well dispersed and spread over the surface. However, these particles disappeared from the surface of treated samples implying the detaching of ZnO-NPs during migration, which coordinates with the solubility result from Table 2. On the other hand, the ZnO-NPs inside LDPE matrix were preserved, as visible from the cross-section micrographs in Fig. 3b. Based on these results, we suggest that the migration of Zn from nanocomposite film is definitely influenced by food simulants. More exactly, solubility of ZnO-NPs in each food simulant affects the stability of ZnO-NPs embedded in the polymer matrix. Higher solubility allows a greater amount of Zn to be released from the composite film. This finding is supported by the fact that ZnO-NPs were not detected in nheptane because of the low solubility of metal and metal salts in nonpolar solvents (Hongjun, Zang, & Tang, 2014; Störmer, Bott, Kemmer, & Franz, 2017). Considering these references, the state of nanoparticles migrated from polymer matrices into food simulants is mainly in the ionic form due to the dissolution caused by the solvent entering into polymer through diffusion (Damm & Munstedt, 2008; Jokar & Rahman, 2014). Zn was detected in all food simulants at levels between 0.006 and 3.416 mg L−1. However, considering the concentration of ZnO-NPs contained in the nanocomposite films, Zn was released only at a rate of 0.00001%–0.007%. Furthermore, Zn was detected in the neat LDPE film (Fig. 2). Therefore, these very low migration levels of Zn are supposed to be safe according to the specific migration limit (SML) for soluble ionic zinc (5 mg kg−1 food) set out by the European Plastics Regulation (EU) 2016/1416 amending and correcting Regulation (EU) 10/2011 (European Commission, 2016; European Commission, 2011). European Food Safety Authority (EFSA) has adopted scientific opinions that coated and uncoated ZnO-NPs are not migrated as nanoform from polyolefin, and thus its safety evaluation should be focused on the migration of soluble ionic zinc (EFSA CEF Panel, 2015, 2016). Based on these opinions, EFSA has approved uncoated ZnO-NPs as additives for plastic materials and articles intended to food contact according to the European Plastics Regulation (EU) No 10/2011with the restriction that these nanoparticles may only be used in unplasticized polymers in compliance with the current applicable SML for Zn. In addition, a previous in vivo study including mice reported that ZnO-NPs could be toxic when they accumulate in organs at a concentration of 300 mg kg−1 (Sharma, Singh, Pandey, & Dhawan, 2012).

(2)

where A and B are the average numbers of viable cells in the control and test samples, respectively, after 24 h. 2.8. Statistical analysis The statistical significance was evaluated using Duncan test and one-way analysis of variance (ANOVA). The level of significance was set at p < 0.05 level for all analyses. The data are presented as mean ± standard deviation (SD). All analyses were performed using Statistical Package for the Social Science (SPSS, Version 20.0 SPSS Inc., Chicago, Illinois, USA) software. 3. Results and discussion 3.1. Characterisation of LDPE-ZnO nanocomposite films Prior to the investigation of Zn migration and its effect on the functionality of LDPE-ZnO nanocomposite films, they were characterised by FTIR and FE-SEM to confirm the presence of ZnO. Fig. 1a displays the infrared spectra of neat LDPE and LDPE-ZnO nanocomposites prepared with different ZnO-NPs contents (0, 1 and 5 wt.%). FTIR spectra show the typical bands of polyolefin (719, 1463, 2848 and 2916 cm−1), corresponding to carbon–hydrogen and carbon–carbon bonds (Gulmine, Janissek, Heise, & Akcelrud, 2002). The characteristic ZnO vibrational peak (around 434 cm−1) (Li & Cao, 2011) is clearly visible after the introduction of ZnO into the LDPE film and its intensity increases with the increase of ZnO content. The surface morphology of LDPE-ZnO nanocomposite films was observed by FE-SEM and the micrographs are presented in Fig. 1b. The ZnO powder used in this study is agglomerate form with dimensions > 100 nm, but it consists of aggregated ZnO-NPs with diameter < 100 nm. Under the shear force from the extrusion process, the ZnO-NPs were well dispersed with nanoscale dimension and distributed all over the surface and inside of polymer films. As seen in the 1 wt.% ZnO sample, the smaller ZnO-NPs were spotted in the form of partially embedded and protrude out of the surface at lower concentration. The higher the loading amount of ZnO, the larger the agglomerated particles attributed to the cluster of ZnO-NPs appeared, shown as whiteclouded clots with non-uniform distribution. The presence of ZnO-NPs on the surface was confirmed by the EDS spectra. 3.2. Validation The calculated LOD and LOQ were 0.1 and 0.3 ng mL−1, respectively. As shown in Table 1, the ranges of spike recoveries for accuracy and relative standard deviations (RSDs) for repeatability were 87%–103% and 0.8%–2.5%, respectively. The recovery and repeatability data for the n-heptane case are not shown because the migration test results were lower than the LOQ. The recovery and RSD results of this experiment showed similar to the other migration studies conducted with ICP (Lin et al., 2014; Ntim et al., 2015; Song et al., 2011). 3.3. Migration of Zn from LDPE-ZnO nanocomposite films To understand the influence of food simulants on the properties of nanocomposite films, the solubility and stability of ZnO in the LDPE matrices were evaluated. Fig. 2 shows the amount of migrated Zn from nanocomposite films into food simulants; the migration increased with the content of ZnO-NPs embedded into LDPE films. A similar relationship has been observed between silver ions released from polyethylene films containing AgNPs (1, 3 and 5 wt.%) (Zapata et al., 2011). Additionally, the migration levels of Zn were 0.009–0.029 mg L−1 in distilled water, 0.017–3.416 mg L−1 in 4% acetic acid and 6

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to dissolution on the surface profoundly diminished the functional performances of LDPE-ZnO nanocomposite films. The UV blocking capability decreased from 30 to 40% of initial value. Whereas the antimicrobial activity against both S.aureus and E.coli significantly dropped from about 99% to 20% reduction.

3.4. Effect of migration on the functionality of LDPE-ZnO nanocomposite films Addition of nanoparticles to polymer matrices provides new specific properties to the neat polymer. ZnO-NP exhibits an excellent antibacterial activity over a wide spectrum of bacterial species and significant UV light blocking (Nair, Nirmala, Rekha, & Anukaliani, 2011; Sirelkhatim et al., 2015). However, the migration phenomena could negatively affect these properties due to the loss of the active substance. To determine the effect of Zn migration on the functionality of LDPEZnO nanocomposite films, light transmittance and antimicrobial activity were investigated. Different sets of samples, untreated and treated neat LDPE films and nanocomposite films (1 and 5 wt.% ZnO-NP loading) were studied. The treated films were prepared by exposure to the 4% acetic acid according to the migration test condition. Fig. 4 shows the transparency measured with the UV–vis spectrophotometer. The opaque white ZnO-NP inclusion in LDPE gradually decreases the film transparency, particularly in the UV range (200–400 nm), similar to that reported previously (Becheri, Durr, Lo Nostro, & Baglioni, 2008), because the band-gap energy of ZnO lies in the UV range (Lu, Fei, Xin, Wang, & Li, 2006). This offers an application of ZnO nanocomposite films as an alternative UV blocking material. With 1 wt.% ZnO-NPs incorporation, the transparency of neat LDPE decreased by about 30% in the UV range, whereas a maximum UV blocking efficacy of about 80% was achieved with a 5 wt.% loading content. Moreover, the loss of active ZnO-NPs due to dissolution from the migration test negatively affected the initial UV blocking performance of the nanocomposite. However, the primitive functionality of the nanocomposite film for UV blocking was conserved because the migration took place mainly at the surface, whereas the active ZnO-NPs inside the film were preserved as displayed in the FE-SEM cross-section micrograph (Fig. 3b). Basically, the exact antibacterial mechanism of ZnO-NPs is still unclear. Many mechanisms of ZnO-NPs have been proposed such as the direct damaging of bacterial cell wall from the interaction with nanoparticles, the formation of reactive oxygen species (ROS), and the release of Zn2+ ions (Shi et al., 2014). Fig. 5 shows the antimicrobial activity of LDPE-ZnO composite films before and after the migration test. The nanocomposite films with 1 and 5 wt.% ZnO-NPs exhibited a significant reduction in the growth of S. aureus and E. coli after 24 h of incubation compared to the neat LDPE film. The result indicates that Gram-positive bacteria had lower resistance on antimicrobial activity than Gram-negative bacteria. One of the main reasons for this different sensitivity is the difference of cell membrane structure (Espitia et al., 2012; Shi et al., 2014). However, the performance of both the samples was poor when the bacterial growth was suppressed after Zn migration. Therefore, as observed in Fig. 3a, the loss of surface ZnO-NPs by Zn migration directly affects the bactericidal property of the nanocomposite films.

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4. Conclusion In this study, the migration behaviour of ZnO-NPs from LDPE-ZnO nanocomposite films into four types of food simulants and its effect on the functionality of nanocomposite films for food packaging were investigated. The results revealed that the migration was directly influenced by the solubility of ZnO in food simulants and the loading content of ZnO-NPs in the nanocomposite films. The migrated Zn was observed at a mg L−1 level in all the food simulants, excluding n-heptane, with the maximal value in 4% acetic acid with 5 wt.% ZnO-NP loading. However, the amount of migrated Zn ion was lower than the specific migration level suggested by European Plastics Regulation (EU) 10/ 2011 and related amendments, which is considered as non-toxic level for human health. FE-SEM micrographs showed that the migration of Zn occurred mainly from the dissolution of ZnO-NPs on the surface of the nanocomposite films. Nevertheless, the loss of active particles due 7

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