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Incorporation of zinc oxide nanoparticles improved the mechanical, water vapor barrier, UV-light barrier, and antibacterial properties of PLA-based nanocomposite films
Materials Science & Engineering C 93 (2018) 289–298
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Incorporation of zinc oxide nanoparticles improved the mechanical, water vapor barrier, UV-light barrier, and antibacterial properties of PLA-based nanocomposite films Shiv Shankara, Long-Feng Wangb, Jong-Whan Rhima,
T
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a Center for Humanities and Sciences, Bionanocomposite Research Center, Department of Food and Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea b Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China
A R T I C LE I N FO
A B S T R A C T
Keywords: ZnO nanoparticles PLA Nanocomposite films Antibacterial activity
Zinc oxide nanoparticles (ZnO NPs) were synthesized using zinc chloride and NaOH and they were incorporated to prepare PLA/ZnO NPs composite films. The SEM images showed that the ZnO NPs were cubical in shape with size ranged from 50 to 100 nm, and the PLA/ZnO NPs composite films were smooth and compact. The composite films exhibited strong UV-light barrier property with a slight decrease in the transparency. The thickness, tensile strength, and water vapor barrier property of the films increased significantly after incorporation of ZnO NPs. The TS of PLA films increased by 37.5%, but the WVP decreased by 30.5% from 3.11 × 10−11 to 2.16 × 10−11 g m/m2·Pa·s when 0.5 wt% of ZnO NPs was incorporated. The composite films exhibited potent antibacterial activity against food-borne pathogenic bacteria, Escherichia coli and Listeria monocytogenes. The developed films were applied to the packaging of a minced fish paste and showed strong antibacterial function. The prepared composite films could be used as antibacterial and UV-light barrier films for food packaging and biomedical applications.
1. Introduction
toughness and ductility [8]. To overcome such shortcomings, PLA has been blended with nanofillers such as nano-clay, nano-cellulose, and nano-metals to improve film properties with additional functional properties like antimicrobial and UV-light screening properties [9]. Among the metallic or metal oxide nanofillers, zinc oxide nanoparticles (ZnO NPs) are interesting since they possess a large surface area and several unusual properties such as non-toxicity, availability, low cost, stability, high ultraviolet absorption capacity, and strong antimicrobial activity [10]. Accordingly, ZnO NPs have been considered as a potential candidate for reinforcing materials of the polymer matrix in the food packaging applications [10–13]. ZnO NPs can be synthesized using various methods such as laser ablation, micro-emulsion, solid state, sonochemical, and thermal decomposition methods [14–17]. Production of ZnO NPs with uniform size, shape, and specific functional properties is needed for their various applications. There are various reports on the preparation of PLA/ZnO NPs, but in most cases, the composite films have been produced using the melt extrusion method [18–20]. To the best of our knowledge, a few reports are available in the literature on the preparation of PLA/ZnO NPs composite film using solution casting method without the use of surface
The demand for biodegradable packaging materials obtained from renewable resources has increased in recent years due to their environmentally-friendliness and sustainability [1,2]. Among the renewable source-based biodegradable plastics, poly(lactide) or poly(lactic acid) (PLA) is one of the most promising materials since it is versatile, thermoplastic, biodegradable, and biocompatible with superior mechanical properties, good processability, optical clarity, and low cost [3,4]. PLA is a linear aliphatic polymer that was produced either by the ring opening polymerization of lactide or by the lactic acid condensation. Since PLA is a highly transparent and rigid plastics with high mechanical properties, it has been used for the fabrication of various products such as trays, cups, tubs, and films [5,6]. Though PLA is considered biodegradable by many definitions and can degrade into water, carbon dioxide, and other small molecules under compost conditions, the biodegradation rate of PLA is relatively slow in the environmental or landfill conditions [7]. However, PLA has some limitations for food packaging applications due to its low gas and water vapor barrier properties, weak thermal stability, high rigidity, and low
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Corresponding author. E-mail address: [email protected] (J.-W. Rhim).
https://doi.org/10.1016/j.msec.2018.08.002 Received 24 October 2017; Received in revised form 3 July 2018; Accepted 1 August 2018 Available online 03 August 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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films were peeled off from the glass plate and preconditioned at 25 °C and 50% RH for 48 h in a constant temperature humidity chamber (model FX 1077, Jeio Tech Co., Ltd., Ansan, Gyeonggi-do, Korea) to normalize the moisture content of the films before further analysis. The control PLA films were prepared by the same method without addition of ZnO NPs. The films prepared with 0.5, 1.0, and 1.5 wt% of ZnO NPs were designated as PLA/ZnO NPs0.5, PLA/ZnO NPs1.0, and PLA/ZnO NPs1.5, respectively.
modifiers [21]. Jayaramudu et al. (2014) prepared PLA/ZnO NP composite film using various content of commercial ZnO NPs (2, 4, and 6 wt %) as a filler using solution casting method and found that the tensile strength (TS) increased by 10% when 2 wt% of the filler was incorporated. However, when the concentration of the filler increased > 2 wt%, the TS decreased rather than pure PLA film. Therefore, in this study, we investigated the effect of ZnO NPs prepared by the laboratory on the properties of PLA/ZnO NPs composite films at concentrations of < 2 wt% (0.5, 1.0 and 1.5 wt%). The ZnO NPs were prepared in a gram-scale using a simple homogeneous precipitation method at low temperature (50–60 °C) without the use of surfactants or chelating agents without high-temperature annealing, and PLA/ZnO NPs composite films were prepared using a solution casting method. Therefore, the main objectives of the present work were to develop PLA/ZnONPs composite films using a solution casting method and to characterize their properties using various analytical techniques. The effect of ZnO NPs concentration on structural, mechanical, thermal, water vapor barrier, and antibacterial properties was also evaluated.
2.4. Characterization of composite films 2.4.1. Morphology and optical properties The microstructure of the film surface was observed using a field emission scanning electron microscope (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan) with an accelerating voltage of 5.0 kV. The light transmittance of the film samples was determined using a UV–visible spectrophotometer (Mecasys Optizen POP Series UV/Vis spectrophotometer, Seoul, Korea) in the wavelength of 200–700 nm. The percent transmittance at 660 nm (T660) and 280 nm (T280) was used to evaluate the transparency and UV barrier property of the films, respectively [10].
2. Material and methods 2.1. Materials
2.4.2. FTIR and XRD Fourier transform infrared (FTIR) spectrum of the film samples was measured using an attenuated total reflectance-Fourier transform infrared (AT-FTIR) spectrophotometer (TENSOR 37 spectrophotometer, Billerica, MA, USA) operated at a resolution of 4 cm−1. For the X-ray diffraction analysis, the film samples were cut into rectangular shapes (2 × 2 cm) and directly placed on the ray exposing stage and XRD analysis was performed using an X-ray diffractometer (PANalytical X'pert pro MRD diffractometer, Amsterdam, Netherlands) at diffraction angles between 2θ = 30–80° with a scanning speed of 0.4°/min at room temperature.
PLA (poly(L-lactide), Biomer® L9000; average molecular weight = 200 kDa, weight-average molecular weight/number-average molecular weight = 1.98) was procured from Biomer Inc. (Krailling, Germany). PLA resins were dried under vacuum at 60 °C for 24 h before use. Chloroform was procured from Daejung Chemicals & Metals Co., Ltd. (Siheung, Gyeonggi-do, Korea). Zinc chloride was purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan). Tryptic soy broth (TSB), brain heart infusion broth (BHI), and agar powder were purchased from Duksan Pure Chemicals Co., Ltd. (Ansan, Gyeonggi-do, Korea). For the test of antimicrobial activity of films, two types of food-borne pathogenic bacteria, Listeria monocytogenes ATCC 15313 and Escherichia coli O157: H7 ATCC 43895, were obtained from Korean Collection for Type Culture (KCTC, Seoul, Korea). Frozen hair-tail minced fish was donated from OurHome Co., Ltd. (Yongin, Gyeonggi-do, Korea).
2.4.3. Differential scanning calorimetry The melt-crystallization of PLA/ZnONPs composite films was investigated using a differential scanning calorimeter (DSC Q100, TA Instruments, New Castle, DE, USA) under dynamic nitrogen atmosphere (60 mL/min). Film samples (~5 mg) were taken in aluminum standard pans and first heated at a rate of 5 °C/min from 30 °C to 200 °C, held for 2 min and cooled to −50 °C with a scanning rate of 10 °C/min. The second heating curves of DSC were recorded with a heating rate of 5 °C/ min in the temperature range of −50 to 200 °C. The glass-transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm) of film samples were determined from the second heating curves. The Tm and Tc were taken as the peak values of the respective endotherms, and Tg was taken as the mid-point of the heat capacity changes. The transition temperatures, the enthalpy of crystallization (ΔHc) and enthalpy of fusion (ΔHm), were calibrated with indium as the standard and an empty aluminum pan as a reference. The degree of crystallinity (Xc) of the PLA and its nanocomposite films was estimated using the following equation:
2.2. Preparation of ZnO NPs Zinc oxide nanoparticles (ZnO NPs) were prepared using zinc chloride and sodium hydroxide following the method of Shankar & Rhim with slight modification [22]. For the preparation of ZnO NPs, 13.6 g of zinc chloride was dissolved in 960 mL of distilled water with stirring and heating using a magnetic stirrer for 10 min at 50–60 °C. Then, ~40 mL of 5 M sodium hydroxide solution was added drop-wise and continued heating for 2 h at 60 °C. The ZnO NPs in the form of white precipitate was collected by centrifugation and washed three times with distilled water. The precipitate was washed with ethanol for two times and dried in an oven at 70 °C for 6 h. The surface morphology of ZnO NPs was analyzed using a field emission scanning electron microscopy (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan).
X c (%) = 100 × (∆Hm − ∆Hc)/ ∆Hm0
2.3. Preparation of PLA/ZnO NPs composite films
where the ΔHm0 was the theoretical enthalpy of 93 J/g for fully crystalline PLA [24].
PLA-based films were prepared using a solution casting method [23]. First, different concentration of ZnO NPs (0.5, 1.0 and 1.5 wt% of PLA) were dispersed in 100 mL of chloroform by ultrasonication using a water bath sonicator (FS140 Ultra Cleaner, Fisher Scientific, Pittsburg, PA, USA) and homogenized for 2 min at 8000 rpm using a high-speed homogenizer (T25 basic, Ika Labotechnik, Janke & Kunkel GmbH & Co., KG Staufen, Germany). Then, 4 g of PLA was dissolved in the ZnO NPs solution with stirring for 24 h at room temperature using a magnetic stirrer. The solutions were cast evenly onto a leveled Teflon coated glass plate (24 cm × 30 cm) and allowed to dry in a fume hood at room temperature (25 °C) for 2 days. The completely dried nanocomposite
2.4.4. Thermal stability The thermal stability of film samples was evaluated using a thermogravimetric analyzer (Hi-Res TGA 2950, TA Instrument, New Castle, DE, USA). The film samples were heated from 30 to 600 °C at the rate of 10 °C/min under a nitrogen atmosphere (60 mL/min). A derivative form of TGA (DTG) was obtained using differentials of TGA values, which was calculated using a central finite difference method [25]. The maximum decomposition temperature (Tmax) of films was determined from the DTG curve, and the char content and the weight loss 290
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packaging test was performed using a minced fish paste as a model food. The minced fish paste was prepared by mixing 4 parts of minced fish meat of the hair-tail fish with 1 part of wheat flour using a mixer (KitchenAid Ultra Power Series 4.5-Quart Stand Mixer SM90, KitchenAid, St Joseph, MI, USA). The paste was sheeted using a home noodle sheeter to get fish paste sheet with the thickness of 4 mm. The fish paste sheets were wrapped in an aluminum foil and cooked using an autoclaving at 120 °C for 15 min, then cooled to room temperature and cut into rectangular pieces of 2 cm × 2 cm. The fish paste pieces were immersed into the bacterial suspension containing ~104 CFU/mL of L. monocytogene and E. coli for 1 min and dried in the air for 10 min. The samples were then packaged individually in the test film (PLA/ ZnONPs1.0) pouches (size of 5 cm × 5 cm), which were sealed using a heat sealer (HJ-300-2, PACKTOWN. Co., Seoul, Korea). The packaged fish paste samples were stored at 5 ± 1 °C for 10 days, and microbial growth in the samples was evaluated at 0, 3, 6, and 10 days of storage. The microbial growth was estimated by a total viable colony count method. For this, the samples were aseptically transferred to a sterilized centrifuge tube containing 10 mL of sterile normal saline and vortexed for 2 min. The suspension was serially diluted, and 100 μL of diluted samples were spread on TSB and BHI agar plates and incubated at 37 °C for 24 h. The growth of the bacteria was determined by counting the number of bacterial colonies.
(%) was determined from the TGA curve [26]. 2.4.5. Tensile properties of films The thickness of the films was determined using a hand-held micrometer (Dial Thickness Gauge 7301, Mitutoyo, Tokyo, Japan) with an accuracy of 0.001 mm. The mechanical properties of the films were determined using an Instron Universal Testing Machine (Model 5565, Instron Engineering Corporation, Canton, MA, USA) at room temperature (22 ± 2 °C) according to the standard test method of ASTM D 88288. The tensile strength (TS), elongation at break (E), and elastic modulus (EM) were evaluated following the method of Shankar et al. and the results were presented in MPa, %, and GPa, respectively [27]. For measuring the TS, each film was cut into rectangular strips (2.54 cm × 15 cm) using a precision double blade cutter (Model LB.02/ A, Metrotech, SA, San Sebastain, Spain). The machine was operated in the tensile mode with an initial grip separation of 50 mm and a crosshead speed of 50 mm/min. The TS was determined by dividing the maximum load (N) by the initial cross-sectional area (m2) of the films. The E was determined by dividing the extension at the rupture of the film by the initial length of the film (50 mm) multiplied by 100. The EM was determined from the slope of the linear portion of the stress-strain curve, which corresponds to the stress divided by the strain of the film sample. Ten measurements were carried out for each film, and the average values were presented.
2.6. Statistical analysis
2.4.6. Water vapor permeability Water vapor permeability (WVP) of the film samples was determined gravimetrically according to the method of ASTM E96-95 with slight modification [28]. Test film (7.5 cm × 7.5 cm) was mounted on a WVP cup (2.5 cm of depth and 6.8 cm in diameter) which was filled with 18 mL of distilled water and sealed to prevent the leakage of water vapor. The assembled cup was kept in a humidity chamber (model FX 1077, Jeio Tech Co. Ltd., Ansan, Korea) controlled at 25 °C and 50% RH with an air movement of 198 m/min. Change in weight of the cup was determined by measuring the weight at 6 h interval for 48 h. Water vapor transmission rate (WVTR) in g/m2·s was determined from the slope of the plot of weight loss vs. time, then the WVP of the film was calculated in g·m/m2·Pa·s as follows:
Film properties were measured with individually prepared films in triplicate, as the replicated experimental units. Statistical analysis was done by one-way analysis of variance (ANOVA), and the significance of each mean value was determined (p < 0.05) with Duncan's multiple range tests using the SPSS software (SPSS Inc., Chicago, IL, USA). The values were presented as mean ± SD (standard deviation). 3. Results and discussion 3.1. Morphology The ZnO NPs obtained were white colored powder with the yield of 32.4% (4.4 g ZnO NPs from 13.6 g ZnCl2 with the conversion rate of 54.2%). The SEM image of ZnO NPs (Fig. 1) showed that the particles were in a cubic form with the size of 50–100 nm. The average diameter of the ZnO NPs calculated using ImageJ software was 56.1 ± 18.6 nm. All the films were transparent, smooth-surfaced, homogeneous, and flexible. The surface structure of PLA and PLA/ZnO NPs composite films was observed using FE-SEM as shown in Fig. 2. The neat PLA film exhibited a smooth and compact surface, while PLA/ZnO NPs films showed a slightly rough surface with nanoparticles distributed in the polymer matrix. The roughness of the film increased with increase in
WVP = (WVTR×L)/Δp where L was the thickness of the film (m) and Δp was the partial water vapor pressure difference (Pa) across the film. For the determination of Δp, the actual water vapor pressure underneath the film sample was calculated following the method of Gennadios et al. [28]. 2.4.7. Antimicrobial activity The antibacterial activity of composite films was determined against food-borne pathogenic bacteria, E. coli (Gram-negative) and L. monocytogenes (Gram-positive). The antimicrobial tests were performed using a viable colony count method [25]. For the surface sterilization, the film samples used for the antimicrobial test were irradiated with UV light for 20 min. A colony of E. coli and L. monocytogenes were inoculated in BHI and TSB broth, respectively, and subsequently incubated at 37 °C for 16 h. Each culture broth was centrifuged at 2000 rpm for 10 min, and the bacterial cell pellets were suspended in 100 mL of sterile BHI and TSB broth, respectively, and diluted 10 times with sterile distilled water. 50 mL of diluted broth (106–107 CFU/mL) was taken into 100 mL conical flask containing 200 mg of the film sample and subsequently incubated at 37 °C for 12 h with mild shaking. The same diluted broth without film sample was used as the control. The cell viability of test microorganisms was calculated by counting bacterial colonies on the plates at 0, 3, 6, 9, and 12 h of incubation. 2.5. Packaging test
Fig. 1. SEM image of ZnO NPs.
To test the antibacterial efficiency of the composite films, a 291
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PLA
PLA/ZnO NPs0.5
PLA/ZnO NPs1.0
PLA/ZnO NPs1.5
Fig. 2. SEM images of PLA and PLA/ZnO NPs composite films.
highly transparent against both UV and visible light as observed high transmittance of light between 250 and 700 nm. However, the transmittance of light decreased significantly after incorporation of ZnO NPs. The decrease in transmittance of the PLA films was dependent on the content of ZnO NPs. The decline in the light transmittance was mainly due to the prevention of light passage by the light impenetrable particles dispersed in the polymer matrix. It is interesting to note that the light transmittance of the PLA/ZnO NPs composite films decreased more profoundly in the UV light range compared with the visible light
the concentration of ZnO NPs. Since the ZnO NPs were dispersed in the PLA dissolved chloroform solution without any surface treatment, the ZnO NPs appeared on the surface of the film, which was clearly shown at higher concentration of ZnO NPs. 3.2. Light transmittance The light transmittance of the composite film was evaluated in the range of 200–700 nm of wavelength (Fig. 3). The neat PLA film was
Fig. 3. Light transmittance spectra of PLA and PLA/ZnO NPs composite films. 292
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Fig. 4. (a) FTIR and (b) XRD spectra of PLA and PLA/ZnO NPs composite films.
range. This indicated that the PLA/ZnO NPs composite films prevented UV penetration with a slight sacrificing the transparency of the film. The decrease in transmittance in UV-light range was due to the strong UV-light absorption property of ZnO NPs [10,29,30]. Similar results of UV light screening effect by the ZnO NPs have also been found with various types of biopolymer-based films such as gelatin, agar, carrageenan, and carboxymethyl cellulose [10,30]. The PLA/ZnO NPs composite films with UV screening capacity, especially for UV-B (280–320 nm) and UV-A (320–400 nm), can be properly used for the application of UV-light prevention food packaging to prevent a photocatalytic reaction in the packaged foods [31].
3.3. FTIR and XRD analysis FTIR analysis of the composite films was performed to test the interactions between PLA and ZnONPs, and the results were shown in Fig. 4a. The peak at the wavenumber of 3658 cm−1 in the PLA film corresponded to OeH stretching vibration. The peak at 2931 cm−1 was associated with the stretching vibrations of a CH3 group of saturated hydrocarbons [32]. The intense peak at 1750 cm−1 was attributed to the C]O stretching vibration of the ester group in the PLA molecules [33]. The peaks between 1456 and 1365 cm−1 represented the asymmetric and the symmetric CH3 deformation vibration. The peaks at 1182 and 1084 cm−1 assigned to the symmetric and asymmetric stretching of the complex CeOeC group, respectively [34]. The peak at 871 cm−1 can be assigned to the amorphous phase and the peak at 293
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Table 1 DSC parameters of the neat PLA and PLA/ZnO NPs composite films. Films
Tg (°C)
Tc (°C)
Tm (°C)
χc (%)
PLA PLA/ZnO NPs0.5 PLA/ZnO NPs1.0 PLA/ZnO NPs1.5
54.3 54.3 54.3 54.3
99.7 99.7 99.7 99.7
167.7 167.7 167.7 167.7
12.7 15.9 18.5 20.4
± ± ± ±
0.4 0.2 0.3 0.7
observed in the range of 30–80°. On the other hand, PLA/ZnONPs composite films exhibited clear diffraction peaks in the range of 30–40°, which were due to the characteristic diffraction peaks of ZnONPs (100), (002), and (101). Virovska et al. also found the characteristic diffraction peaks for ZnO at 31.8°, 34.5°, and 36.4°, which were corresponding to (100), (002), and (101) crystal planes when nano-sized ZnO was incorporated into PLA film [36]. 3.4. DSC analysis Fig. 5a shows the DSC thermograms of the neat PLA and PLA/ ZnONPs composite films. All the film samples exhibited characteristic exothermic crystallization of a semi-crystalline polymer. The calorimetric parameters determined from the DSC thermograms were summarized in Table 1. There was no change in the glass transition temperature (Tg), cold crystallization temperature (Tc), and melting temperature (Tm) of PLA film after incorporation of ZnONPs up to 1.5 wt%. These results indicated that the ZnONPs were compatible with the polymer matrix to form well miscible composite films. However, there was a possibility that such compatibility might be attributed to the small amount of NPs inclusion. The DSC test results indicated that the PLA film matrix was mainly composed of an amorphous structure. However, the crystallinity (χc) of PLA film increased slightly from 12.7% up to 20.4% after blending with ZnONPs. Murariu et al. also found that the addition of ZnO nanofillers, surface-treated or not, increased the crystallinity of the PLA matrix [24]. This might be due to heterogeneous nucleation of PLA or nanofillers in PLA acting as a nucleating agent to enhance a cold crystallization of PLA chains [37,38]. 3.5. Thermal stability Fig. 5b and c show the TGA and DTG thermograms of the composite films, respectively. All films exhibited a single-step of thermal decomposition at 230–385 °C. The decomposition of the neat PLA film started at about 275 °C and ended at about 385 °C and showed maximum decomposition at 362.5 °C. However, the decomposition temperature of PLA/ZnO NPs shifted to the low temperature and shifted further as the concentration of ZnO NPs increased. The maximum decomposition temperatures of PLA/ZnO NPs0.5, PLA/ZnO NPs1.0, and PLA/ZnO NPs1.5 were 327.5, 310, and 298 °C, respectively. The thermal stability of ZnO NPs-incorporated nanocomposite films decreased significantly compared with the neat PLA film since the ZnO had an important degrading effect on PLA at high temperature [24,39]. This result was consistent with the TGA profile obtained when Jayaramudu et al. added 2, 4, and 6 wt% ZnO NPs to the PLA matrix [21]. Fig. 5. (a) DSC, (b) TGA, and (c) DTG analysis of PLA and PLA/ZnO NPs composite films.
3.6. Mechanical properties
756 cm−1 to the crystalline phase of PLA [35]. The addition of ZnONPs did not change in any peaks from the PLA except the increase in the intensity of the peak at 3654 cm−1. The result indicated that only secondary forces like weak hydrogen bonds were formed between the ZnONPs and PLA polymer matrix. The X-ray diffraction patterns of the PLA-based films are presented in Fig. 4b. No specific diffraction peak in the neat PLA film was
The mechanical properties of neat PLA and PLA/ZnO NPs composite films were presented in Table 2. The average thickness of the neat PLA film was 48.2 ± 8.7 μm, and it was increased significantly (p < 0.05) after blending with ZnO NPs. The thickness of PLA/ZnO NPs composite films increased with the concentration of ZnO NPs that might be due to the increase in solid content associated with ZnO NPs or might be attributed to the presence of ZnO NPs on the surface of the films leading to rough surfaced films [40]. 294
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Table 2 Tensile properties and water vapor permeability of the neat PLA and PLA/ZnO NPs composite films.1 Films
Thickness (μm)
PLA PLA/ZnO NPs0.5 PLA/ZnO NPs1.0 PLA/ZnO NPs1.5
48 53 56 60
± ± ± ±
9a 9ab 10bc 7c
TS (MPa) 42.5 58.4 54.8 52.9
± ± ± ±
E (%)
11.0a 5.2c 4.3bc 7.6b
4.9 4.5 4.4 4.4
± ± ± ±
EM (GPa) 1.5a 0.3a 0.4a 0.4a
2.49 2.57 2.46 2.44
± ± ± ±
0.22ab 0.16b 0.09ab 0.23a
WVP (×10−11 g·m/m2·Pa·s) 3.11 2.16 2.66 2.68
± ± ± ±
0.09c 0.39a 0.13b 0.25b
1 The values are presented as mean ± standard deviation. Any two means in the same column followed by the same superscript (a, b, c) are not significantly (p > 0.05) different by Duncan's multiple range tests.
Fig. 6. Antibacterial activity of PLA and PLA/ZnO NPs composite films.
film when ZnO NPs were added up to 0.5 wt%, however, when this concentration was exceeded, TS decreased, which might be due to the aggregation of ZnO NPs [41]. Jayaramudu et al., reported a 10% increase in the TS of PLA composites when 2 wt% of ZnO NPs was incorporated into PLA, and the TS was decreased when added by > 2 wt% of ZnONPs [21]. The effectiveness of the reinforcement and dispersion
The TS of the neat PLA film was 42.5 MPa, and it increased significantly to 58.4, 54.8, and 52.9 MPa, respectively, when 0.5, 1.0, and 1.5 wt% of ZnO NPs was incorporated. The increase in the TS of the films was highest when 0.5 wt% of ZnO NPs was incorporated, which showed 37.5% of increase compared with the neat PLA film. Li & Li, also found an increase in the TS of high-density polyethylene (HDPE)
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Fig. 7. Growth of E. coli and L. monocytogenes in the packaged minced fish cake.
composite films, a composite film with the lowest content of ZnO NPs (PLA/ZnO NPs0.5) showed the lowest WVP of 2.16 × 10−11 g·m/ m2·Pa·s. The WVP of composite films increased with an increase in the concentration of ZnO NPs incorporation, however, the WVP of composite films was still lower than that of neat PLA film. Some controversial results on the effect of ZnO NPs on the WVP of ZnO NPsincorporated nanocomposite films have been reported. Petchwattana et al., reported the WVP of the poly(butylene succinate) film had decreased when ZnO NPs was used as reinforcing nanofiller [43]. They explained that the decrease in the WVP of the composite films was due to the increased tortuous path of water vapor created by the impermeable nanofillers with low concentration. On the contrary, Marra et al., reported the WVP of PLA film had increased when ZnO NPs were incorporated, and they explained that the ZnO NPs modify the free volume at the interfaces of nanoparticles and polymer matrix resulting in an increase in the permeability for the polar water molecules [44]. Such different effect on the WVP of ZnO NPs may be attributed to various factors such as the type of polymers, compatibility between the polymer matrix and the nanofillers, and filler concentration. Especially, the concentration of nanofillers has been known to have great influence on the WVP of the composite films. Usually, the WVP of the nanocomposite films decreased significantly when a low concentration of
of nanofillers in the polymer matrix are the main controlling parameters for effective stress transfer at the interface of the matrix and the filler and ultimately increase the TS of the polymeric biocomposite materials [12,40]. The elongation at break (E) of PLA/ZnO NPs composite films was slightly decreased with the increase in the concentration of ZnO NPs, however, the decrease was not significantly different (p > 0.05). The E of PLA film has been found to be decreased significantly when it was composited with nano clay due to the restriction of polymer chain movement by the nanofillers [42]. The nanofiller incorporated into the PLA matrix limited the movement of the PLA chain segments and generated an interactive force against the PLA chains. This was evidenced by increased TS but decreased E. Similar to the TS, the EM of PLA composite film increased slightly when 0.5 wt% of ZnO NPs was incorporated, but it decreased when a higher concentration of ZnO NPs was incorporated. 3.7. Water vapor permeability The WVP of composite films are presented in Table 2. The WVP of the neat PLA film was 3.11 × 10−11 g·m/m2·Pa·s, which decreased significantly (p < 0.05) after blending with ZnO NPs. Among the 296
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food-borne pathogenic bacteria, L. monocytogenes and E. coli. They decreased the bacterial count on minced fished paste and increased the shelf life. The PLA/ZnO NPs composite films have a high potential to be used as a functional active food packaging and biomedical applications using their antimicrobial and UV-light barrier properties.
nanofillers was added, however, it increased when a high concentration of nanofillers was added. This is likely that the nanofillers at high concentration formed agglomerated structure without homogeneous dispersion in the polymer matrix [2,45]. 3.8. Antimicrobial activity
Acknowledgment The antimicrobial activity of the composite films was tested against food-borne pathogenic bacteria, E. coli and L. monocytogenes, and the results are shown in Fig. 6. As expected, the neat PLA film did not show any antimicrobial activity against both bacteria. However, PLA/ZnO NPs composite films exhibited distinctive antimicrobial activity against both E. coli and L. monocytogenes, depending on the type of bacteria and concentration of ZnO NPs. Similar to the control groups (without any film or neat PLA film), the number of L. monocytogenes count increased up to 6 h of incubation in the PLA/ZnO NPs composite film groups, and afterward, their growth started to decrease slowly. In the case of E. coli, the growth of bacteria was constant up to 3 h, and started to decline after that. The intensity of antibacterial activity was dependent on the concentration of ZnO NPs in the composite films. The initial increase of the microbial count with the PLA/ZnONPs composite films was presumably due to the slow release of ZnO NPs from the PLA matrix [46]. Fig. 6 also showed that the ZnO NPs incorporated PLA films exhibited stronger antimicrobial activity against Gram-negative bacterium (E. coli) than Gram-positive one (L. monocytogenes). Though the exact mechanism of antibacterial action by the ZnONPs has not been completely explained yet, some probable mechanisms of antimicrobial activity by ZnO NPs have been proposed. The first one was explained by the direct contact of ZnONPs with cell walls of microorganisms to result in the destruction of bacterial cell integrity [47]. The second one was regarding the release of Zn2+ ions to interfere with DNA replication and protein synthesis, and the third one was on the formation of reactive oxygen species to destroy bacterial cells [48]. In addition, the toxicity of ZnO NPs against microorganisms was known to depend on various factors such as the size of ZnONPs, the composition of growth media, and other physiochemical parameters [49].
This research was supported by the Agriculture Research Center (ARC 710003) program of the Ministry of Agriculture, Food and Rural Affairs, Korea, and Korea Research Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016H1D3A1903910). References [1] T.V. Duncan, Applications of nanotechnology in food packaging and food safety: barrier materials, antimicrobials and sensors, J. Colloid Interface Sci. 363 (2011) 1–24. [2] J.W. Rhim, H.M. Park, C.S. Ha, Bio-nanocomposites for food packaging applications, Prog. Polym. Sci. 38 (2013) 1629–1652. [3] R.E. Drumright, P.R. Gruber, D.E. Henton, Polylactic acid technology, Adv. Mater. 12 (2000) 1841–1846. [4] D. Garlotta, A literature review of poly(lactic acid), J. Polym. Environ. 9 (2001) 63–84. [5] H. Eslami, M.R. Kamal, Elongational rheology of biodegradable poly(lactic acid)/ poly[(butylene succinate)-co-adipate] binary blends and poly(lactic acid)/poly [(butylene succinate)-co-adipate]/clay ternary nanocomposites, J. Appl. Polym. Sci. 127 (2013) 2290–2306. [6] I.S.M.A. Tawakkal, M.J. Cran, J. Miltz, S.W. Bigger, A review of poly (lactic acid)based materials for antimicrobial packaging, J. Food Sci. 79 (2014) R1447–R1490. [7] Y. Rudeekit, J. Numnoi, M. Tajan, P. Chaiwutthinan, T. Leejarkpai, Determining biodegradability of polylactic acid under different environments, J. Met. Mater. Miner. 18 (2008) 83–87. [8] M. Harada, T. Ohya, K. Iida, H. Hayashi, K. Hirano, H. Fukuda, Increased impact strength of biodegradable poly(lactic acid)/poly(butylene succinate) blend composites by using isocyanate as a reactive processing agent, J. Appl. Polym. Sci. 106 (2007) 1813–1820. [9] S. Therias, J.F. Larche, P.O. Bussiere, J.L. Gardette, M. Murariu, P. Dubois, Photochemical behavior of polylactide/ZnO nanocomposite films, Biomacromolecules 13 (2012) 3283–3291. [10] S. Shankar, X. Teng, G. Li, J.W. Rhim, Preparation, characterization, and antimicrobial activity of gelatin/ZnO nanocomposite films, Food Hydrocoll. 45 (2015) 264–271. [11] E. Darezereshki, M. Alizadeh, F. Bakhtiari, M. Schaffie, M. Ranjbar, A novel thermal decomposition method for the synthesis of ZnO nanoparticles from low concentration ZnSO4 solutions, Appl. Clay Sci. 54 (2011) 107–111. [12] A.M. Díez-Pascual, A.L. Díez-Vicente, ZnO-reinforced poly(3-hydroxybutyrate-co-3hydroxyvalerate) bionanocomposites with antimicrobial function for food packaging, ACS Appl. Mater. Interfaces 6 (2014) 9822–9834. [13] O. Tari, A. Aronne, M.L. Addonizio, S. Daliento, E. Fanelli, P. Pernice, Sol–gel synthesis of ZnO transparent and conductive films: a critical approach, Sol. Energy Mater. Sol. Cells 105 (2012) 179–186. [14] P. Banerjee, S. Chakrabarti, S. Maitra, B.K. Dutta, Zinc oxide nanoparticles – sonochemical synthesis, characterization and application for photo-remediation of heavy metal, Ultrason. Sonochem. 19 (2012) 85–93. [15] M. Pudukudy, Z. Yaakob, Facile solid state synthesis of ZnO hexagonal nanogranules with excellent photocatalytic activity, Appl. Surf. Sci. 292 (2014) 520–530. [16] R.K. Thareja, S. Shukla, Synthesis and characterization of zinc oxide nanoparticles by laser ablation of zinc in liquid, Appl. Surf. Sci. 253 (2007) 8889–8895. [17] O.A. Yildirım, C. Durucan, Synthesis of zinc oxide nanoparticles elaborated by microemulsion method, J. Alloys Compd. 506 (2010) 944–949. [18] A. Marra, C. Silvestre, D. Duraccio, S. Cimmion, Polylactic acid/zinc oxide biocomposite films for food packaging application, Int. J. Biol. Macromol. 88 (2016) 254–262. [19] M. Murariu, A. Doumbia, L. Bonnaud, A.L. Dechief, Y. Paint, M. Ferreira, C. Campagne, E. Devaux, P. Dubois, High-performance polylactide/ZnO nanocomposites designed for films and fibers with special end-use properties, Biomacromolecules 12 (2011) 1762–1771. [20] R. Pantani, G. Gorrasi, G. Vigliotta, M. Murariu, P. Dubois, PLA-ZnO nanocomposite films: Water vapor barrier properties and specific end-use characteristics, Eur. Polym. J. 49 (2013) 3471–3482. [21] J. Jayaramudu, K. Das, M. Sonakshi, G. Siva Mohan Reddy, B. Aderibigbe, R. Sadiku, S.S. Ray, Structure and properties of highly toughened biodegradable polylactide/ZnO biocomposite films, Int. J. Biol. Macromol. 64 (2014) 428–434. [22] S. Shankar, J.W. Rhim, Facile approach for large-scale production of metal and metal oxide nanoparticles and preparation of antibacterial cotton pads, Carbohydr. Polym. 163 (2017) 137–145. [23] S. Shankar, J.W. Rhim, Tocopherol-mediated synthesis of silver nanoparticles and preparation of antimicrobial PBAT/silver nanoparticles composite films, LWT Food
3.9. Packaging test The antimicrobial function of the PLA and PLA/ZnO NPs composite films tested by packaging cooked minced fish paste with those films and monitoring the growth of E.coli and L. monocytogenes, and the results are shown in Fig. 7. As expected, both types of food-borne pathogenic bacteria packaged with the neat PLA film were grown continuously during the storage. On the contrary, the test groups packaged with PLA/ ZnO NPs1.0 composite film showed distinctive antibacterial activity. The number of viable colonies of both types of bacteria was reduced to zero in 10 days of storage. This indicated that the PLA/ZnO NPs composite film possessed a strong bactericidal activity to prevent the growth of the bacteria in the packaged fish paste. The antimicrobial activity of the PLA/ZnO NPs composite films was presumably due to the direct contact of the microorganisms with the ZnO NPs in the film or Zn2+ ions diffused out from the film. Wang & Rhim, also reported a similar antimicrobial activity against L. monocytogenes for the minced fish paste packaged with GSE (grapefruit seed extract) incorporated PLA film [47]. 4. Conclusion The PLA/ZnO NPs composite films prepared using a solvent casting method were flexible and smooth, and had high UV-light screening properties without much sacrificing the transparency. The nanocomposite films also showed increased water vapor barrier and mechanical properties. However, the thermostability of the composite films was decreased compared with the neat PLA film. The PLA/ZnO NPs composite films exhibited potent antibacterial activity against 297
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[37] Z. Chu, T. Zhao, L. Li, J. Fan, Y. Qin, Characterization of antimicrobial poly(Lactic acid)/nano-composite films with silver and zinc oxide nanoparticles, Dent. Mater. 10 (2017) 659. [38] Y. Du, T. Wu, N. Yan, M.T. Kortschot, R. Farnood, Fabrication and characterization of fully biodegradable natural fiber-reinforced poly(lactic acid) composites, Compos. Part B 56 (2014) 717–723. [39] H. Abe, N. Takahashi, K.J. Kim, M. Mochzuki, Y. Doi, Thermal degradation processes of end-capped poly(L-lactide)s in the presence and absence of residual zinc catalyst, Biomacromolecules 5 (2004) 1606–1614. [40] J. Ahmed, Y.A. Arfat, E. Castro-Aguirre, R. Auras, Mechanical, structural and thermal properties of Ag-Cu and ZnO reinforced polylactide nanocomposite films, Int. J. Biol. Macromol. 86 (2016) 885–892. [41] S.C. Li, Y.N. Li, Mechanical and antibacterial properties of modified nano-ZnO/ high-density polyethylene composite films with a low doped content of nano-ZnO, J. Appl. Polym. Sci. 116 (2010) 2965–2969. [42] J.W. Rhim, S.I. Hong, S.K. Ha, Tensile, water vapor barrier and antimicrobial properties of PLA/nanoclay composite films, LWT Food Sci. Technol. 42 (2009) 612–617. [43] N. Petchwattana, S. Covavisaruch, S. Wibooranawong, P. Naknaen, Antimicrobial food packaging prepared from poly(butylene succinate) and zinc oxide, Measurement 93 (2016) 442–448. [44] A. Marra, C. Silvestre, D. Duraccio, S. Cimmino, Polylactic acid/zinc oxide biocomposite films for food packaging application, Int. J. Biol. Macromol. 88 (2016) 254–262. [45] I.Y. Jeon, J.B. Baek, Nanocomposites derived from polymers and inorganic nanoparticles, Dent. Mater. 3 (2010) 3654–3674. [46] L.F. Wang, J.W. Rhim, Grapefruit seed extract incorporated antimicrobial LDPE and PLA films: effect of type of polymer matrix, LWT Food Sci. Technol. 74 (2016) 338–345. [47] L. Zhang, Y. Ding, M. Povey, D. York, ZnO nanofluids - a potential antibacterial agent, Prog. Nat. Sci. 18 (2008) 939–944. [48] M. Li, L. Zhu, D. Lin, Toxicity of ZnO nanoparticles to Escherichia coli: mechanism and the influence of medium components, Environ. Sci. Technol. 45 (2011) 1977–1983. [49] L. Zhang, Y. Jiang, Y. Ding, M. Povey, D. York, Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids), J. Nanopart. Res. 9 (2007) 479–489.
Sci. Technol. 72 (2016) 149–156. [24] M. Murariu, A. Doumbia, L. Bonnaud, A.L. Dechief, Y. Paint, M. Ferreira, C. Campagne, E. Devaux, P. Dubois, High-performance polylactide/ZnO nanocomposites designed for films and fibers with special end-use properties, Biomacromolecules 12 (2011) 1762–1771. [25] S. Shankar, J.W. Rhim, Amino acid mediated synthesis of silver nanoparticles and preparation of antimicrobial agar/silver nanoparticles composite films, Carbohydr. Polym. 130 (2015) 353–363. [26] J.P. Reddy, J.W. Rhim, Characterization of bionanocomposite films prepared with agar and paper-mulberry pulp nanocellulose, Carbohydr. Polym. 110 (2014) 480–488. [27] S. Shankar, L.F. Wang, J.W. Rhim, Preparations and characterization of alginate/ silver composite films: effect of types of silver particles, Carbohydr. Polym. 146 (2016) 208–216. [28] A. Gennadios, C.L. Weller, C.H. Gooding, Measurement errors in water vapor permeability of high permeable, hydrophilic edible films, J. Food Eng. 21 (1994) 395–409. [29] M.E. Calvo, J.R. Smirnov, H. Miguez, Novel approaches to flexible visible transparent hybrid films for ultraviolet protection, J. Polym. Sci. B Polym. Phys. 50 (2012) 945–956. [30] P. Kanmani, J.W. Rhim, Properties and characterization of bionanocomposite films prepared with various biopolymers and ZnO nanoparticles, Carbohydr. Polym. 106 (2014) 190–199. [31] T. Bintsis, E. Litopoulou-Tzanetaki, R.K. Robinson, Existing and potential applications of ultraviolet light in the food industry - a critical review, J. Sci. Food Agric. 80 (2000) 637–645. [32] R. Al-Itry, K. Lamnawar, A. Maazouz, Rheological, morphological, and interfacial properties of compatibilized PLA/PBAT blends, Rheol. Acta 53 (2014) 501–517. [33] P. Qu, Y. Goa, G.F. Wu, L.P. Zhang, Nanocomposites of poly (lactic acid) reinforced with cellulose nanofibrils, Bioresources 5 (2010) 1811–1823. [34] X. Gong, L. Pan, C.Y. Tang, L. Chen, Z. Hao, W.C. Law, X. Wang, C.P. Tsui, C. Wu, Preparation, optical and thermal properties of CdSe–ZnS/poly(lactic acid) (PLA) nanocomposites, Compos. Part B 66 (2014) 494–499. [35] R. Auras, B. Harte, S. Selke, An overview of polylactides as packaging materials, Macromol. Biosci. 4 (2004) 835–864. [36] D. Virovska, D. Paneva, N. Manolova, I. Rashkov, D. Karashanova, Photocatalytic self-cleaning poly(L-lactide) materials based on a hybrid between nanosized zinc oxide and expanded graphite or fullerene, Mater. Sci. Eng. 60 (2016) 184–194.
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Incorporation of zinc oxide nanoparticles improved the mechanical, water vapor barrier, UV-light barrier, and antibacterial properties of PLA-based nanocomposite films Shiv Shankara, Long-Feng Wangb, Jong-Whan Rhima,
T
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a Center for Humanities and Sciences, Bionanocomposite Research Center, Department of Food and Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea b Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China
A R T I C LE I N FO
A B S T R A C T
Keywords: ZnO nanoparticles PLA Nanocomposite films Antibacterial activity
Zinc oxide nanoparticles (ZnO NPs) were synthesized using zinc chloride and NaOH and they were incorporated to prepare PLA/ZnO NPs composite films. The SEM images showed that the ZnO NPs were cubical in shape with size ranged from 50 to 100 nm, and the PLA/ZnO NPs composite films were smooth and compact. The composite films exhibited strong UV-light barrier property with a slight decrease in the transparency. The thickness, tensile strength, and water vapor barrier property of the films increased significantly after incorporation of ZnO NPs. The TS of PLA films increased by 37.5%, but the WVP decreased by 30.5% from 3.11 × 10−11 to 2.16 × 10−11 g m/m2·Pa·s when 0.5 wt% of ZnO NPs was incorporated. The composite films exhibited potent antibacterial activity against food-borne pathogenic bacteria, Escherichia coli and Listeria monocytogenes. The developed films were applied to the packaging of a minced fish paste and showed strong antibacterial function. The prepared composite films could be used as antibacterial and UV-light barrier films for food packaging and biomedical applications.
1. Introduction
toughness and ductility [8]. To overcome such shortcomings, PLA has been blended with nanofillers such as nano-clay, nano-cellulose, and nano-metals to improve film properties with additional functional properties like antimicrobial and UV-light screening properties [9]. Among the metallic or metal oxide nanofillers, zinc oxide nanoparticles (ZnO NPs) are interesting since they possess a large surface area and several unusual properties such as non-toxicity, availability, low cost, stability, high ultraviolet absorption capacity, and strong antimicrobial activity [10]. Accordingly, ZnO NPs have been considered as a potential candidate for reinforcing materials of the polymer matrix in the food packaging applications [10–13]. ZnO NPs can be synthesized using various methods such as laser ablation, micro-emulsion, solid state, sonochemical, and thermal decomposition methods [14–17]. Production of ZnO NPs with uniform size, shape, and specific functional properties is needed for their various applications. There are various reports on the preparation of PLA/ZnO NPs, but in most cases, the composite films have been produced using the melt extrusion method [18–20]. To the best of our knowledge, a few reports are available in the literature on the preparation of PLA/ZnO NPs composite film using solution casting method without the use of surface
The demand for biodegradable packaging materials obtained from renewable resources has increased in recent years due to their environmentally-friendliness and sustainability [1,2]. Among the renewable source-based biodegradable plastics, poly(lactide) or poly(lactic acid) (PLA) is one of the most promising materials since it is versatile, thermoplastic, biodegradable, and biocompatible with superior mechanical properties, good processability, optical clarity, and low cost [3,4]. PLA is a linear aliphatic polymer that was produced either by the ring opening polymerization of lactide or by the lactic acid condensation. Since PLA is a highly transparent and rigid plastics with high mechanical properties, it has been used for the fabrication of various products such as trays, cups, tubs, and films [5,6]. Though PLA is considered biodegradable by many definitions and can degrade into water, carbon dioxide, and other small molecules under compost conditions, the biodegradation rate of PLA is relatively slow in the environmental or landfill conditions [7]. However, PLA has some limitations for food packaging applications due to its low gas and water vapor barrier properties, weak thermal stability, high rigidity, and low
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Corresponding author. E-mail address: [email protected] (J.-W. Rhim).
https://doi.org/10.1016/j.msec.2018.08.002 Received 24 October 2017; Received in revised form 3 July 2018; Accepted 1 August 2018 Available online 03 August 2018 0928-4931/ © 2018 Elsevier B.V. All rights reserved.
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films were peeled off from the glass plate and preconditioned at 25 °C and 50% RH for 48 h in a constant temperature humidity chamber (model FX 1077, Jeio Tech Co., Ltd., Ansan, Gyeonggi-do, Korea) to normalize the moisture content of the films before further analysis. The control PLA films were prepared by the same method without addition of ZnO NPs. The films prepared with 0.5, 1.0, and 1.5 wt% of ZnO NPs were designated as PLA/ZnO NPs0.5, PLA/ZnO NPs1.0, and PLA/ZnO NPs1.5, respectively.
modifiers [21]. Jayaramudu et al. (2014) prepared PLA/ZnO NP composite film using various content of commercial ZnO NPs (2, 4, and 6 wt %) as a filler using solution casting method and found that the tensile strength (TS) increased by 10% when 2 wt% of the filler was incorporated. However, when the concentration of the filler increased > 2 wt%, the TS decreased rather than pure PLA film. Therefore, in this study, we investigated the effect of ZnO NPs prepared by the laboratory on the properties of PLA/ZnO NPs composite films at concentrations of < 2 wt% (0.5, 1.0 and 1.5 wt%). The ZnO NPs were prepared in a gram-scale using a simple homogeneous precipitation method at low temperature (50–60 °C) without the use of surfactants or chelating agents without high-temperature annealing, and PLA/ZnO NPs composite films were prepared using a solution casting method. Therefore, the main objectives of the present work were to develop PLA/ZnONPs composite films using a solution casting method and to characterize their properties using various analytical techniques. The effect of ZnO NPs concentration on structural, mechanical, thermal, water vapor barrier, and antibacterial properties was also evaluated.
2.4. Characterization of composite films 2.4.1. Morphology and optical properties The microstructure of the film surface was observed using a field emission scanning electron microscope (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan) with an accelerating voltage of 5.0 kV. The light transmittance of the film samples was determined using a UV–visible spectrophotometer (Mecasys Optizen POP Series UV/Vis spectrophotometer, Seoul, Korea) in the wavelength of 200–700 nm. The percent transmittance at 660 nm (T660) and 280 nm (T280) was used to evaluate the transparency and UV barrier property of the films, respectively [10].
2. Material and methods 2.1. Materials
2.4.2. FTIR and XRD Fourier transform infrared (FTIR) spectrum of the film samples was measured using an attenuated total reflectance-Fourier transform infrared (AT-FTIR) spectrophotometer (TENSOR 37 spectrophotometer, Billerica, MA, USA) operated at a resolution of 4 cm−1. For the X-ray diffraction analysis, the film samples were cut into rectangular shapes (2 × 2 cm) and directly placed on the ray exposing stage and XRD analysis was performed using an X-ray diffractometer (PANalytical X'pert pro MRD diffractometer, Amsterdam, Netherlands) at diffraction angles between 2θ = 30–80° with a scanning speed of 0.4°/min at room temperature.
PLA (poly(L-lactide), Biomer® L9000; average molecular weight = 200 kDa, weight-average molecular weight/number-average molecular weight = 1.98) was procured from Biomer Inc. (Krailling, Germany). PLA resins were dried under vacuum at 60 °C for 24 h before use. Chloroform was procured from Daejung Chemicals & Metals Co., Ltd. (Siheung, Gyeonggi-do, Korea). Zinc chloride was purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan). Tryptic soy broth (TSB), brain heart infusion broth (BHI), and agar powder were purchased from Duksan Pure Chemicals Co., Ltd. (Ansan, Gyeonggi-do, Korea). For the test of antimicrobial activity of films, two types of food-borne pathogenic bacteria, Listeria monocytogenes ATCC 15313 and Escherichia coli O157: H7 ATCC 43895, were obtained from Korean Collection for Type Culture (KCTC, Seoul, Korea). Frozen hair-tail minced fish was donated from OurHome Co., Ltd. (Yongin, Gyeonggi-do, Korea).
2.4.3. Differential scanning calorimetry The melt-crystallization of PLA/ZnONPs composite films was investigated using a differential scanning calorimeter (DSC Q100, TA Instruments, New Castle, DE, USA) under dynamic nitrogen atmosphere (60 mL/min). Film samples (~5 mg) were taken in aluminum standard pans and first heated at a rate of 5 °C/min from 30 °C to 200 °C, held for 2 min and cooled to −50 °C with a scanning rate of 10 °C/min. The second heating curves of DSC were recorded with a heating rate of 5 °C/ min in the temperature range of −50 to 200 °C. The glass-transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm) of film samples were determined from the second heating curves. The Tm and Tc were taken as the peak values of the respective endotherms, and Tg was taken as the mid-point of the heat capacity changes. The transition temperatures, the enthalpy of crystallization (ΔHc) and enthalpy of fusion (ΔHm), were calibrated with indium as the standard and an empty aluminum pan as a reference. The degree of crystallinity (Xc) of the PLA and its nanocomposite films was estimated using the following equation:
2.2. Preparation of ZnO NPs Zinc oxide nanoparticles (ZnO NPs) were prepared using zinc chloride and sodium hydroxide following the method of Shankar & Rhim with slight modification [22]. For the preparation of ZnO NPs, 13.6 g of zinc chloride was dissolved in 960 mL of distilled water with stirring and heating using a magnetic stirrer for 10 min at 50–60 °C. Then, ~40 mL of 5 M sodium hydroxide solution was added drop-wise and continued heating for 2 h at 60 °C. The ZnO NPs in the form of white precipitate was collected by centrifugation and washed three times with distilled water. The precipitate was washed with ethanol for two times and dried in an oven at 70 °C for 6 h. The surface morphology of ZnO NPs was analyzed using a field emission scanning electron microscopy (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan).
X c (%) = 100 × (∆Hm − ∆Hc)/ ∆Hm0
2.3. Preparation of PLA/ZnO NPs composite films
where the ΔHm0 was the theoretical enthalpy of 93 J/g for fully crystalline PLA [24].
PLA-based films were prepared using a solution casting method [23]. First, different concentration of ZnO NPs (0.5, 1.0 and 1.5 wt% of PLA) were dispersed in 100 mL of chloroform by ultrasonication using a water bath sonicator (FS140 Ultra Cleaner, Fisher Scientific, Pittsburg, PA, USA) and homogenized for 2 min at 8000 rpm using a high-speed homogenizer (T25 basic, Ika Labotechnik, Janke & Kunkel GmbH & Co., KG Staufen, Germany). Then, 4 g of PLA was dissolved in the ZnO NPs solution with stirring for 24 h at room temperature using a magnetic stirrer. The solutions were cast evenly onto a leveled Teflon coated glass plate (24 cm × 30 cm) and allowed to dry in a fume hood at room temperature (25 °C) for 2 days. The completely dried nanocomposite
2.4.4. Thermal stability The thermal stability of film samples was evaluated using a thermogravimetric analyzer (Hi-Res TGA 2950, TA Instrument, New Castle, DE, USA). The film samples were heated from 30 to 600 °C at the rate of 10 °C/min under a nitrogen atmosphere (60 mL/min). A derivative form of TGA (DTG) was obtained using differentials of TGA values, which was calculated using a central finite difference method [25]. The maximum decomposition temperature (Tmax) of films was determined from the DTG curve, and the char content and the weight loss 290
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packaging test was performed using a minced fish paste as a model food. The minced fish paste was prepared by mixing 4 parts of minced fish meat of the hair-tail fish with 1 part of wheat flour using a mixer (KitchenAid Ultra Power Series 4.5-Quart Stand Mixer SM90, KitchenAid, St Joseph, MI, USA). The paste was sheeted using a home noodle sheeter to get fish paste sheet with the thickness of 4 mm. The fish paste sheets were wrapped in an aluminum foil and cooked using an autoclaving at 120 °C for 15 min, then cooled to room temperature and cut into rectangular pieces of 2 cm × 2 cm. The fish paste pieces were immersed into the bacterial suspension containing ~104 CFU/mL of L. monocytogene and E. coli for 1 min and dried in the air for 10 min. The samples were then packaged individually in the test film (PLA/ ZnONPs1.0) pouches (size of 5 cm × 5 cm), which were sealed using a heat sealer (HJ-300-2, PACKTOWN. Co., Seoul, Korea). The packaged fish paste samples were stored at 5 ± 1 °C for 10 days, and microbial growth in the samples was evaluated at 0, 3, 6, and 10 days of storage. The microbial growth was estimated by a total viable colony count method. For this, the samples were aseptically transferred to a sterilized centrifuge tube containing 10 mL of sterile normal saline and vortexed for 2 min. The suspension was serially diluted, and 100 μL of diluted samples were spread on TSB and BHI agar plates and incubated at 37 °C for 24 h. The growth of the bacteria was determined by counting the number of bacterial colonies.
(%) was determined from the TGA curve [26]. 2.4.5. Tensile properties of films The thickness of the films was determined using a hand-held micrometer (Dial Thickness Gauge 7301, Mitutoyo, Tokyo, Japan) with an accuracy of 0.001 mm. The mechanical properties of the films were determined using an Instron Universal Testing Machine (Model 5565, Instron Engineering Corporation, Canton, MA, USA) at room temperature (22 ± 2 °C) according to the standard test method of ASTM D 88288. The tensile strength (TS), elongation at break (E), and elastic modulus (EM) were evaluated following the method of Shankar et al. and the results were presented in MPa, %, and GPa, respectively [27]. For measuring the TS, each film was cut into rectangular strips (2.54 cm × 15 cm) using a precision double blade cutter (Model LB.02/ A, Metrotech, SA, San Sebastain, Spain). The machine was operated in the tensile mode with an initial grip separation of 50 mm and a crosshead speed of 50 mm/min. The TS was determined by dividing the maximum load (N) by the initial cross-sectional area (m2) of the films. The E was determined by dividing the extension at the rupture of the film by the initial length of the film (50 mm) multiplied by 100. The EM was determined from the slope of the linear portion of the stress-strain curve, which corresponds to the stress divided by the strain of the film sample. Ten measurements were carried out for each film, and the average values were presented.
2.6. Statistical analysis
2.4.6. Water vapor permeability Water vapor permeability (WVP) of the film samples was determined gravimetrically according to the method of ASTM E96-95 with slight modification [28]. Test film (7.5 cm × 7.5 cm) was mounted on a WVP cup (2.5 cm of depth and 6.8 cm in diameter) which was filled with 18 mL of distilled water and sealed to prevent the leakage of water vapor. The assembled cup was kept in a humidity chamber (model FX 1077, Jeio Tech Co. Ltd., Ansan, Korea) controlled at 25 °C and 50% RH with an air movement of 198 m/min. Change in weight of the cup was determined by measuring the weight at 6 h interval for 48 h. Water vapor transmission rate (WVTR) in g/m2·s was determined from the slope of the plot of weight loss vs. time, then the WVP of the film was calculated in g·m/m2·Pa·s as follows:
Film properties were measured with individually prepared films in triplicate, as the replicated experimental units. Statistical analysis was done by one-way analysis of variance (ANOVA), and the significance of each mean value was determined (p < 0.05) with Duncan's multiple range tests using the SPSS software (SPSS Inc., Chicago, IL, USA). The values were presented as mean ± SD (standard deviation). 3. Results and discussion 3.1. Morphology The ZnO NPs obtained were white colored powder with the yield of 32.4% (4.4 g ZnO NPs from 13.6 g ZnCl2 with the conversion rate of 54.2%). The SEM image of ZnO NPs (Fig. 1) showed that the particles were in a cubic form with the size of 50–100 nm. The average diameter of the ZnO NPs calculated using ImageJ software was 56.1 ± 18.6 nm. All the films were transparent, smooth-surfaced, homogeneous, and flexible. The surface structure of PLA and PLA/ZnO NPs composite films was observed using FE-SEM as shown in Fig. 2. The neat PLA film exhibited a smooth and compact surface, while PLA/ZnO NPs films showed a slightly rough surface with nanoparticles distributed in the polymer matrix. The roughness of the film increased with increase in
WVP = (WVTR×L)/Δp where L was the thickness of the film (m) and Δp was the partial water vapor pressure difference (Pa) across the film. For the determination of Δp, the actual water vapor pressure underneath the film sample was calculated following the method of Gennadios et al. [28]. 2.4.7. Antimicrobial activity The antibacterial activity of composite films was determined against food-borne pathogenic bacteria, E. coli (Gram-negative) and L. monocytogenes (Gram-positive). The antimicrobial tests were performed using a viable colony count method [25]. For the surface sterilization, the film samples used for the antimicrobial test were irradiated with UV light for 20 min. A colony of E. coli and L. monocytogenes were inoculated in BHI and TSB broth, respectively, and subsequently incubated at 37 °C for 16 h. Each culture broth was centrifuged at 2000 rpm for 10 min, and the bacterial cell pellets were suspended in 100 mL of sterile BHI and TSB broth, respectively, and diluted 10 times with sterile distilled water. 50 mL of diluted broth (106–107 CFU/mL) was taken into 100 mL conical flask containing 200 mg of the film sample and subsequently incubated at 37 °C for 12 h with mild shaking. The same diluted broth without film sample was used as the control. The cell viability of test microorganisms was calculated by counting bacterial colonies on the plates at 0, 3, 6, 9, and 12 h of incubation. 2.5. Packaging test
Fig. 1. SEM image of ZnO NPs.
To test the antibacterial efficiency of the composite films, a 291
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PLA
PLA/ZnO NPs0.5
PLA/ZnO NPs1.0
PLA/ZnO NPs1.5
Fig. 2. SEM images of PLA and PLA/ZnO NPs composite films.
highly transparent against both UV and visible light as observed high transmittance of light between 250 and 700 nm. However, the transmittance of light decreased significantly after incorporation of ZnO NPs. The decrease in transmittance of the PLA films was dependent on the content of ZnO NPs. The decline in the light transmittance was mainly due to the prevention of light passage by the light impenetrable particles dispersed in the polymer matrix. It is interesting to note that the light transmittance of the PLA/ZnO NPs composite films decreased more profoundly in the UV light range compared with the visible light
the concentration of ZnO NPs. Since the ZnO NPs were dispersed in the PLA dissolved chloroform solution without any surface treatment, the ZnO NPs appeared on the surface of the film, which was clearly shown at higher concentration of ZnO NPs. 3.2. Light transmittance The light transmittance of the composite film was evaluated in the range of 200–700 nm of wavelength (Fig. 3). The neat PLA film was
Fig. 3. Light transmittance spectra of PLA and PLA/ZnO NPs composite films. 292
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Fig. 4. (a) FTIR and (b) XRD spectra of PLA and PLA/ZnO NPs composite films.
range. This indicated that the PLA/ZnO NPs composite films prevented UV penetration with a slight sacrificing the transparency of the film. The decrease in transmittance in UV-light range was due to the strong UV-light absorption property of ZnO NPs [10,29,30]. Similar results of UV light screening effect by the ZnO NPs have also been found with various types of biopolymer-based films such as gelatin, agar, carrageenan, and carboxymethyl cellulose [10,30]. The PLA/ZnO NPs composite films with UV screening capacity, especially for UV-B (280–320 nm) and UV-A (320–400 nm), can be properly used for the application of UV-light prevention food packaging to prevent a photocatalytic reaction in the packaged foods [31].
3.3. FTIR and XRD analysis FTIR analysis of the composite films was performed to test the interactions between PLA and ZnONPs, and the results were shown in Fig. 4a. The peak at the wavenumber of 3658 cm−1 in the PLA film corresponded to OeH stretching vibration. The peak at 2931 cm−1 was associated with the stretching vibrations of a CH3 group of saturated hydrocarbons [32]. The intense peak at 1750 cm−1 was attributed to the C]O stretching vibration of the ester group in the PLA molecules [33]. The peaks between 1456 and 1365 cm−1 represented the asymmetric and the symmetric CH3 deformation vibration. The peaks at 1182 and 1084 cm−1 assigned to the symmetric and asymmetric stretching of the complex CeOeC group, respectively [34]. The peak at 871 cm−1 can be assigned to the amorphous phase and the peak at 293
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Table 1 DSC parameters of the neat PLA and PLA/ZnO NPs composite films. Films
Tg (°C)
Tc (°C)
Tm (°C)
χc (%)
PLA PLA/ZnO NPs0.5 PLA/ZnO NPs1.0 PLA/ZnO NPs1.5
54.3 54.3 54.3 54.3
99.7 99.7 99.7 99.7
167.7 167.7 167.7 167.7
12.7 15.9 18.5 20.4
± ± ± ±
0.4 0.2 0.3 0.7
observed in the range of 30–80°. On the other hand, PLA/ZnONPs composite films exhibited clear diffraction peaks in the range of 30–40°, which were due to the characteristic diffraction peaks of ZnONPs (100), (002), and (101). Virovska et al. also found the characteristic diffraction peaks for ZnO at 31.8°, 34.5°, and 36.4°, which were corresponding to (100), (002), and (101) crystal planes when nano-sized ZnO was incorporated into PLA film [36]. 3.4. DSC analysis Fig. 5a shows the DSC thermograms of the neat PLA and PLA/ ZnONPs composite films. All the film samples exhibited characteristic exothermic crystallization of a semi-crystalline polymer. The calorimetric parameters determined from the DSC thermograms were summarized in Table 1. There was no change in the glass transition temperature (Tg), cold crystallization temperature (Tc), and melting temperature (Tm) of PLA film after incorporation of ZnONPs up to 1.5 wt%. These results indicated that the ZnONPs were compatible with the polymer matrix to form well miscible composite films. However, there was a possibility that such compatibility might be attributed to the small amount of NPs inclusion. The DSC test results indicated that the PLA film matrix was mainly composed of an amorphous structure. However, the crystallinity (χc) of PLA film increased slightly from 12.7% up to 20.4% after blending with ZnONPs. Murariu et al. also found that the addition of ZnO nanofillers, surface-treated or not, increased the crystallinity of the PLA matrix [24]. This might be due to heterogeneous nucleation of PLA or nanofillers in PLA acting as a nucleating agent to enhance a cold crystallization of PLA chains [37,38]. 3.5. Thermal stability Fig. 5b and c show the TGA and DTG thermograms of the composite films, respectively. All films exhibited a single-step of thermal decomposition at 230–385 °C. The decomposition of the neat PLA film started at about 275 °C and ended at about 385 °C and showed maximum decomposition at 362.5 °C. However, the decomposition temperature of PLA/ZnO NPs shifted to the low temperature and shifted further as the concentration of ZnO NPs increased. The maximum decomposition temperatures of PLA/ZnO NPs0.5, PLA/ZnO NPs1.0, and PLA/ZnO NPs1.5 were 327.5, 310, and 298 °C, respectively. The thermal stability of ZnO NPs-incorporated nanocomposite films decreased significantly compared with the neat PLA film since the ZnO had an important degrading effect on PLA at high temperature [24,39]. This result was consistent with the TGA profile obtained when Jayaramudu et al. added 2, 4, and 6 wt% ZnO NPs to the PLA matrix [21]. Fig. 5. (a) DSC, (b) TGA, and (c) DTG analysis of PLA and PLA/ZnO NPs composite films.
3.6. Mechanical properties
756 cm−1 to the crystalline phase of PLA [35]. The addition of ZnONPs did not change in any peaks from the PLA except the increase in the intensity of the peak at 3654 cm−1. The result indicated that only secondary forces like weak hydrogen bonds were formed between the ZnONPs and PLA polymer matrix. The X-ray diffraction patterns of the PLA-based films are presented in Fig. 4b. No specific diffraction peak in the neat PLA film was
The mechanical properties of neat PLA and PLA/ZnO NPs composite films were presented in Table 2. The average thickness of the neat PLA film was 48.2 ± 8.7 μm, and it was increased significantly (p < 0.05) after blending with ZnO NPs. The thickness of PLA/ZnO NPs composite films increased with the concentration of ZnO NPs that might be due to the increase in solid content associated with ZnO NPs or might be attributed to the presence of ZnO NPs on the surface of the films leading to rough surfaced films [40]. 294
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Table 2 Tensile properties and water vapor permeability of the neat PLA and PLA/ZnO NPs composite films.1 Films
Thickness (μm)
PLA PLA/ZnO NPs0.5 PLA/ZnO NPs1.0 PLA/ZnO NPs1.5
48 53 56 60
± ± ± ±
9a 9ab 10bc 7c
TS (MPa) 42.5 58.4 54.8 52.9
± ± ± ±
E (%)
11.0a 5.2c 4.3bc 7.6b
4.9 4.5 4.4 4.4
± ± ± ±
EM (GPa) 1.5a 0.3a 0.4a 0.4a
2.49 2.57 2.46 2.44
± ± ± ±
0.22ab 0.16b 0.09ab 0.23a
WVP (×10−11 g·m/m2·Pa·s) 3.11 2.16 2.66 2.68
± ± ± ±
0.09c 0.39a 0.13b 0.25b
1 The values are presented as mean ± standard deviation. Any two means in the same column followed by the same superscript (a, b, c) are not significantly (p > 0.05) different by Duncan's multiple range tests.
Fig. 6. Antibacterial activity of PLA and PLA/ZnO NPs composite films.
film when ZnO NPs were added up to 0.5 wt%, however, when this concentration was exceeded, TS decreased, which might be due to the aggregation of ZnO NPs [41]. Jayaramudu et al., reported a 10% increase in the TS of PLA composites when 2 wt% of ZnO NPs was incorporated into PLA, and the TS was decreased when added by > 2 wt% of ZnONPs [21]. The effectiveness of the reinforcement and dispersion
The TS of the neat PLA film was 42.5 MPa, and it increased significantly to 58.4, 54.8, and 52.9 MPa, respectively, when 0.5, 1.0, and 1.5 wt% of ZnO NPs was incorporated. The increase in the TS of the films was highest when 0.5 wt% of ZnO NPs was incorporated, which showed 37.5% of increase compared with the neat PLA film. Li & Li, also found an increase in the TS of high-density polyethylene (HDPE)
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Fig. 7. Growth of E. coli and L. monocytogenes in the packaged minced fish cake.
composite films, a composite film with the lowest content of ZnO NPs (PLA/ZnO NPs0.5) showed the lowest WVP of 2.16 × 10−11 g·m/ m2·Pa·s. The WVP of composite films increased with an increase in the concentration of ZnO NPs incorporation, however, the WVP of composite films was still lower than that of neat PLA film. Some controversial results on the effect of ZnO NPs on the WVP of ZnO NPsincorporated nanocomposite films have been reported. Petchwattana et al., reported the WVP of the poly(butylene succinate) film had decreased when ZnO NPs was used as reinforcing nanofiller [43]. They explained that the decrease in the WVP of the composite films was due to the increased tortuous path of water vapor created by the impermeable nanofillers with low concentration. On the contrary, Marra et al., reported the WVP of PLA film had increased when ZnO NPs were incorporated, and they explained that the ZnO NPs modify the free volume at the interfaces of nanoparticles and polymer matrix resulting in an increase in the permeability for the polar water molecules [44]. Such different effect on the WVP of ZnO NPs may be attributed to various factors such as the type of polymers, compatibility between the polymer matrix and the nanofillers, and filler concentration. Especially, the concentration of nanofillers has been known to have great influence on the WVP of the composite films. Usually, the WVP of the nanocomposite films decreased significantly when a low concentration of
of nanofillers in the polymer matrix are the main controlling parameters for effective stress transfer at the interface of the matrix and the filler and ultimately increase the TS of the polymeric biocomposite materials [12,40]. The elongation at break (E) of PLA/ZnO NPs composite films was slightly decreased with the increase in the concentration of ZnO NPs, however, the decrease was not significantly different (p > 0.05). The E of PLA film has been found to be decreased significantly when it was composited with nano clay due to the restriction of polymer chain movement by the nanofillers [42]. The nanofiller incorporated into the PLA matrix limited the movement of the PLA chain segments and generated an interactive force against the PLA chains. This was evidenced by increased TS but decreased E. Similar to the TS, the EM of PLA composite film increased slightly when 0.5 wt% of ZnO NPs was incorporated, but it decreased when a higher concentration of ZnO NPs was incorporated. 3.7. Water vapor permeability The WVP of composite films are presented in Table 2. The WVP of the neat PLA film was 3.11 × 10−11 g·m/m2·Pa·s, which decreased significantly (p < 0.05) after blending with ZnO NPs. Among the 296
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food-borne pathogenic bacteria, L. monocytogenes and E. coli. They decreased the bacterial count on minced fished paste and increased the shelf life. The PLA/ZnO NPs composite films have a high potential to be used as a functional active food packaging and biomedical applications using their antimicrobial and UV-light barrier properties.
nanofillers was added, however, it increased when a high concentration of nanofillers was added. This is likely that the nanofillers at high concentration formed agglomerated structure without homogeneous dispersion in the polymer matrix [2,45]. 3.8. Antimicrobial activity
Acknowledgment The antimicrobial activity of the composite films was tested against food-borne pathogenic bacteria, E. coli and L. monocytogenes, and the results are shown in Fig. 6. As expected, the neat PLA film did not show any antimicrobial activity against both bacteria. However, PLA/ZnO NPs composite films exhibited distinctive antimicrobial activity against both E. coli and L. monocytogenes, depending on the type of bacteria and concentration of ZnO NPs. Similar to the control groups (without any film or neat PLA film), the number of L. monocytogenes count increased up to 6 h of incubation in the PLA/ZnO NPs composite film groups, and afterward, their growth started to decrease slowly. In the case of E. coli, the growth of bacteria was constant up to 3 h, and started to decline after that. The intensity of antibacterial activity was dependent on the concentration of ZnO NPs in the composite films. The initial increase of the microbial count with the PLA/ZnONPs composite films was presumably due to the slow release of ZnO NPs from the PLA matrix [46]. Fig. 6 also showed that the ZnO NPs incorporated PLA films exhibited stronger antimicrobial activity against Gram-negative bacterium (E. coli) than Gram-positive one (L. monocytogenes). Though the exact mechanism of antibacterial action by the ZnONPs has not been completely explained yet, some probable mechanisms of antimicrobial activity by ZnO NPs have been proposed. The first one was explained by the direct contact of ZnONPs with cell walls of microorganisms to result in the destruction of bacterial cell integrity [47]. The second one was regarding the release of Zn2+ ions to interfere with DNA replication and protein synthesis, and the third one was on the formation of reactive oxygen species to destroy bacterial cells [48]. In addition, the toxicity of ZnO NPs against microorganisms was known to depend on various factors such as the size of ZnONPs, the composition of growth media, and other physiochemical parameters [49].
This research was supported by the Agriculture Research Center (ARC 710003) program of the Ministry of Agriculture, Food and Rural Affairs, Korea, and Korea Research Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016H1D3A1903910). References [1] T.V. Duncan, Applications of nanotechnology in food packaging and food safety: barrier materials, antimicrobials and sensors, J. Colloid Interface Sci. 363 (2011) 1–24. [2] J.W. Rhim, H.M. Park, C.S. Ha, Bio-nanocomposites for food packaging applications, Prog. Polym. Sci. 38 (2013) 1629–1652. [3] R.E. Drumright, P.R. Gruber, D.E. Henton, Polylactic acid technology, Adv. Mater. 12 (2000) 1841–1846. [4] D. Garlotta, A literature review of poly(lactic acid), J. Polym. Environ. 9 (2001) 63–84. [5] H. Eslami, M.R. Kamal, Elongational rheology of biodegradable poly(lactic acid)/ poly[(butylene succinate)-co-adipate] binary blends and poly(lactic acid)/poly [(butylene succinate)-co-adipate]/clay ternary nanocomposites, J. Appl. Polym. Sci. 127 (2013) 2290–2306. [6] I.S.M.A. Tawakkal, M.J. Cran, J. Miltz, S.W. Bigger, A review of poly (lactic acid)based materials for antimicrobial packaging, J. Food Sci. 79 (2014) R1447–R1490. [7] Y. Rudeekit, J. Numnoi, M. Tajan, P. Chaiwutthinan, T. Leejarkpai, Determining biodegradability of polylactic acid under different environments, J. Met. Mater. Miner. 18 (2008) 83–87. [8] M. Harada, T. Ohya, K. Iida, H. Hayashi, K. Hirano, H. Fukuda, Increased impact strength of biodegradable poly(lactic acid)/poly(butylene succinate) blend composites by using isocyanate as a reactive processing agent, J. Appl. Polym. Sci. 106 (2007) 1813–1820. [9] S. Therias, J.F. Larche, P.O. Bussiere, J.L. Gardette, M. Murariu, P. Dubois, Photochemical behavior of polylactide/ZnO nanocomposite films, Biomacromolecules 13 (2012) 3283–3291. [10] S. Shankar, X. Teng, G. Li, J.W. Rhim, Preparation, characterization, and antimicrobial activity of gelatin/ZnO nanocomposite films, Food Hydrocoll. 45 (2015) 264–271. [11] E. Darezereshki, M. Alizadeh, F. Bakhtiari, M. Schaffie, M. Ranjbar, A novel thermal decomposition method for the synthesis of ZnO nanoparticles from low concentration ZnSO4 solutions, Appl. Clay Sci. 54 (2011) 107–111. [12] A.M. Díez-Pascual, A.L. Díez-Vicente, ZnO-reinforced poly(3-hydroxybutyrate-co-3hydroxyvalerate) bionanocomposites with antimicrobial function for food packaging, ACS Appl. Mater. Interfaces 6 (2014) 9822–9834. [13] O. Tari, A. Aronne, M.L. Addonizio, S. Daliento, E. Fanelli, P. Pernice, Sol–gel synthesis of ZnO transparent and conductive films: a critical approach, Sol. Energy Mater. Sol. Cells 105 (2012) 179–186. [14] P. Banerjee, S. Chakrabarti, S. Maitra, B.K. Dutta, Zinc oxide nanoparticles – sonochemical synthesis, characterization and application for photo-remediation of heavy metal, Ultrason. Sonochem. 19 (2012) 85–93. [15] M. Pudukudy, Z. Yaakob, Facile solid state synthesis of ZnO hexagonal nanogranules with excellent photocatalytic activity, Appl. Surf. Sci. 292 (2014) 520–530. [16] R.K. Thareja, S. Shukla, Synthesis and characterization of zinc oxide nanoparticles by laser ablation of zinc in liquid, Appl. Surf. Sci. 253 (2007) 8889–8895. [17] O.A. Yildirım, C. Durucan, Synthesis of zinc oxide nanoparticles elaborated by microemulsion method, J. Alloys Compd. 506 (2010) 944–949. [18] A. Marra, C. Silvestre, D. Duraccio, S. Cimmion, Polylactic acid/zinc oxide biocomposite films for food packaging application, Int. J. Biol. Macromol. 88 (2016) 254–262. [19] M. Murariu, A. Doumbia, L. Bonnaud, A.L. Dechief, Y. Paint, M. Ferreira, C. Campagne, E. Devaux, P. Dubois, High-performance polylactide/ZnO nanocomposites designed for films and fibers with special end-use properties, Biomacromolecules 12 (2011) 1762–1771. [20] R. Pantani, G. Gorrasi, G. Vigliotta, M. Murariu, P. Dubois, PLA-ZnO nanocomposite films: Water vapor barrier properties and specific end-use characteristics, Eur. Polym. J. 49 (2013) 3471–3482. [21] J. Jayaramudu, K. Das, M. Sonakshi, G. Siva Mohan Reddy, B. Aderibigbe, R. Sadiku, S.S. Ray, Structure and properties of highly toughened biodegradable polylactide/ZnO biocomposite films, Int. J. Biol. Macromol. 64 (2014) 428–434. [22] S. Shankar, J.W. Rhim, Facile approach for large-scale production of metal and metal oxide nanoparticles and preparation of antibacterial cotton pads, Carbohydr. Polym. 163 (2017) 137–145. [23] S. Shankar, J.W. Rhim, Tocopherol-mediated synthesis of silver nanoparticles and preparation of antimicrobial PBAT/silver nanoparticles composite films, LWT Food
3.9. Packaging test The antimicrobial function of the PLA and PLA/ZnO NPs composite films tested by packaging cooked minced fish paste with those films and monitoring the growth of E.coli and L. monocytogenes, and the results are shown in Fig. 7. As expected, both types of food-borne pathogenic bacteria packaged with the neat PLA film were grown continuously during the storage. On the contrary, the test groups packaged with PLA/ ZnO NPs1.0 composite film showed distinctive antibacterial activity. The number of viable colonies of both types of bacteria was reduced to zero in 10 days of storage. This indicated that the PLA/ZnO NPs composite film possessed a strong bactericidal activity to prevent the growth of the bacteria in the packaged fish paste. The antimicrobial activity of the PLA/ZnO NPs composite films was presumably due to the direct contact of the microorganisms with the ZnO NPs in the film or Zn2+ ions diffused out from the film. Wang & Rhim, also reported a similar antimicrobial activity against L. monocytogenes for the minced fish paste packaged with GSE (grapefruit seed extract) incorporated PLA film [47]. 4. Conclusion The PLA/ZnO NPs composite films prepared using a solvent casting method were flexible and smooth, and had high UV-light screening properties without much sacrificing the transparency. The nanocomposite films also showed increased water vapor barrier and mechanical properties. However, the thermostability of the composite films was decreased compared with the neat PLA film. The PLA/ZnO NPs composite films exhibited potent antibacterial activity against 297
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