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Effect of nanocomposite packaging containing ZnO on growth of Bacillus subtilis and Enterobacter aerogenes
Materials Science and Engineering C 58 (2016) 1058–1063
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Effect of nanocomposite packaging containing ZnO on growth of Bacillus subtilis and Enterobacter aerogenes Hakimeh Esmailzadeh a, Parvaneh Sangpour b,⁎, Farzaneh Shahraz a, Jalal Hejazi c, Ramin Khaksar a a b c
National Nutrition and Food Technology Research Institute, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran Nanotechnology and Advanced Materials Department, Materials and Energy Research Center, Karaj, Iran Department of Biochemistry and Nutrition, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 25 August 2015 Accepted 22 September 2015 Available online 25 September 2015 Keywords: Nanocomposite ZnO Antimicrobial activity Bacillus subtilis Enterobacter aerogenes
a b s t r a c t Recent advances in nanotechnology have opened new windows in active food packaging. Nano-sized ZnO is an inexpensive material with potential antimicrobial properties. The aim of the present study is to evaluate the antibacterial effect of low density Polyethylene (LDPE) containing ZnO nanoparticles on Bacillus subtilis and Enterobacter aerogenes. ZnO nanoparticles have been synthesized by facil molten salt method and have been characterized by X-ray diffraction (XRD), and scanning electron microscopy (SEM). Nanocomposite films containing 2 and 4 wt.% ZnO nanoparticles were prepared by melt mixing in a twin-screw extruder. The growth of both microorganisms has decreased in the presence of ZnO containing nanocomposites compared with controls. Nanocomposites with 4 wt.% ZnO nanoparticles had stronger antibacterial effect against both bacteria in comparison with the 2 wt.% ZnO containing nanocomposites. B. subtilis as Gram-positive bacteria were more sensitive to ZnO containing nanocomposite films compared with E. aerogenes as Gram-negative bacteria. There were no significant differences between the migration of Zn ions from 2 and 4 wt.% ZnO containing nanocomposites and the released Zn ions were not significantly increased in both groups after 14 days compared with the first. Regarding the considerable antibacterial effects of ZnO nanoparticles, their application in active food packaging can be a suitable solution for extending the shelf life of food. © 2015 Published by Elsevier B.V.
1. Introduction Long shelf life and least processing are of the most desirable approaches in food technology. Microbial contamination is one of the major limiting factors in extending the shelf life of food products. An appropriate solution to overcome this problem is application of antimicrobial packaging. Antimicrobial packaging is the most common type of active food packaging system which can inhibit or retard the growth of spoilage microorganisms and can extend the shelf life of food [1]. Recent advances in the field of nanotechnology have attracted many attentions of researchers to design more efficient active packaging systems. When particle size is reduced to nanoscale, the resulting materials exhibit different physical and chemical properties compared with their macroscale counterparts. Materials in nano-size have larger surface to volume ratio; therefore they are able to inactive more microbial cells, which confers higher efficiency [2]. Antimicrobial materials can be classified as organic or inorganic. Inorganic antimicrobials such as metal and metal oxides are stable at high pressures and temperatures; as a result they can tolerate the harsh process conditions [3]. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (P. Sangpour).
http://dx.doi.org/10.1016/j.msec.2015.09.078 0928-4931/© 2015 Published by Elsevier B.V.
Most of the recent studies in the field of antimicrobial nanocomposites, have concentrated on silver nanoparticles and many of them have reported promising impacts. In a review by Marambio-Jones et al. an extended investigation of studies on silver nanoparticles has showed that silver can be a potent antimicrobial agent against different microorganisms. However silver is an expensive material with a well-defined toxicity to human cells which makes the use of it difficult in the food industry [4]. Thus investigators are looking for less expensive and safer nanomaterials such as zinc. However the studies about the antimicrobial effects of these metal nanoparticles are scarce and sometimes controversial. For example in a study by Jin et al. ZnO nanoparticles has showed an antimicrobial effect in growth media, but not in polystyrene film [5]. Zinc is an abundant element with a large distribution, which is essential for the proper function of many metalloenzymes. Zinc oxide is one of the five compounds of zinc that are currently listed as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). Some recent investigations have also proposed antimicrobial properties of ZnO nanoparticles and these nanoparticles have several advantages compared with silver nanoparticles such as lower price, white appearance and UV blocking properties [5–6]. Low density poly ethylene (LDPE) is widely used in food packaging because of its flexibility, transparency, thermo stability and low cost.
H. Esmailzadeh et al. / Materials Science and Engineering C 58 (2016) 1058–1063
Both ZnO nanoparticles and LDPE are stable at high temperatures; furthermore melt mixing is an appropriate method for production of ZnO nanocomposite [7]. The theory behind this investigation is that ZnO containing nanocomposite, as an inexpensive and safe material, can confer antibacterial properties which may be used as an efficient method to extend the shelf life of foods. The objective of present study is to investigate the anti-microbial effect of zinc oxide containing nanocomposite on Bacillus subtilis, one of the most important spoiling bacteria in the food industry which can cause spoilage of bread and Enterobacter aerogenes, pathogenic bacteria that found in water, vegetable and meat. 2. Materials and methods 2.1. Preparation of ZnO nanoparticles ZnO nanoparticles have been synthesized using molten salt method [8]. Sodium hydroxide, potassium hydroxide and Zinc chloride were obtained from Merck, Germany. Stochiometric amount of these materials were mixed and put into the Teflon cup and then sealed. Mixed powders were heated up to the 200 ± 5 °C (above eutectic of NaOHKOH (175 °C)) for 30 min in an electric oven and then it was cooled down to room temperature. The resultant product washed with hot deionized water and centrifuged at 4000 rpm at 25 °C for 15 min to remove the alkali metal salts. Then, the sediment dried at 100 °C for 3 h to obtain ZnO nanoparticles. 2.2. Characterization of ZnO nanoparticles The phase composition and the microstructure of ZnO nanoparticles were analyzed by X-ray diffractometer (XRD, Philips, X'Pert). The particle size and morphology of the samples were determined by scanning electron microscopy (SEM, Tescan). The Brunauer–Emmett–Teller (BET) surface area of the powders was analyzed by nitrogen adsorption in a Micromeritics ASAP 2020 nitrogen adsorption apparatus (BEL Japan Inc.). 2.3. Preparation of nanocomposite films Insertion of nanoparticles into low density poly ethylene matrix has been accomplished using a twin-screw extruder with a screw diameter of 42 mm. Film grade LDPE resin pellets (300 g) were directly mixed with ZnO nanoparticles (6 g and 12 g respectively for 2 and 4 wt.% ZnO containing nanocomposites) and the mixture was fed into a twin-screw extruder machine. The heating profile was set to six heating zones of the twin-screw extruder including 90 °C, 160 °C, 175 °C, 155 °C, 155 °C, and 150 °C. Then the nanoparticle containing granules, converted into the nanocomposite films with average thickness of 0.1 mm using a hot press with pressure of 120 Kg/cm2 at 200 °C.
2.5. Metal ion releasing measurement Zinc ions releasing in to culture media determined using inductively coupled plasma- optical emission spectrometry (ICP-OES) (varian vistapro Australia). For this purpose, 10 cm2 of nanocomposite films were immersed in 10 mL of TSB broth for 1, 7 and 14 days. Then 100 μL of 70% nitric acid was added to TSB broth and centrifuged at 5000 rpm for 5 min at room temperature. The upper transparent phase was isolated then deionized distilled water was added up to 10 mL. Finally the samples introduced to ICP-OES to determine the quantity of Zn ions migrating into TSB broth.
2.6. Statistical analysis Statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL). All P-values were two-tailed and the level of statistical significance was set at p b 0.05. Between-group differences were analyzed using analysis of variance (ANOVA).
3. Results 3.1. X-ray diffraction Fig. 1 shows the X-ray diffraction of ZnO nanopowders synthesize by molten salt method. As it is obvious, the ZnO phase with wurtzite structure was completely synthesized only after 30 min in molten hydroxide media. The formation reaction of zinc oxide could be sketched as: ZnCl2 þ NaOH þ KOH→ZnO þ NaCl þ KCl þ H2 O:
ð1Þ
According to the JCPDS card No. 36-1451 characteristic peaks can be observed at 2θ values of 31.7°, 34.4°, 36.2°, 56.6°, 62.9°, 67.9°, 69.0° corresponding to (1 0 0), (0 0 2), (1 0 1), (1 1 0), (1 0 3), (1 1 2), (2 0 1) of zinc oxide, wurtzite phase. The absence of remaining peaks indicates the purity of the samples. Sharp diffraction peaks shown in Fig. 1 indicate good crystallinity of ZnO nanoparticles. The broadening of the peaks indicates that the particles are in nanometer scale. According to Scherrer formula Eq. (1), where D stands for the average grain size, λ stands for the X-ray wavelength (0.15405 nm), and θ and β are the diffraction angle and full-width at half maximum of an observed peak, respectively. D ¼ 0:891λ=β cosθ
ð1Þ
The average crystalline size of the particles is 39.7 nm.
2.4. Antimicrobial tests B. subtilis (American type culture collection 6051) and E. aerogenes (American type culture collection 13,048) were obtained from Iranian the Research Organization for Science and Technology. For antimicrobial tests, the bottom of 8 cm plates was coated by the nanocomposite films, then 100 μl of 106 cfu/mL microorganism containing suspension which was inoculated into 10 mL of tryptic soy broth (TSB Merck, Germany), were poured. The plates were incubated in 37 °C (the optimum temperature for the bacterial growth) and bacterial growth was monitored turbidometrically each 2 h (up to 24 h) at the wavelength of 600 nm with a Bioscreen C. Then the growth curves of microorganisms were plotted.
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Fig. 1. XRD pattern of ZnO nanoparticles.
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Fig. 2. SEM micrograph of ZnO nanoparticles.
3.2. Scanning electron microscopy
3.4. Growth curves of bacteria
Morphology, structure and size of the ZnO nanoparticles are investigated by SEM. The SEM images (Fig. 2) show that these nanoparticles have star-like shapes. The mean length of the stars and their diameter of particles are below 400 and 50 nm, respectively. During reaction time (30 min), agglomerated nanoparticles, which are almost flower and star like in shape, can be observed in Fig. 2. It is known that zinc oxide structure, as a polar crystal, consists of hexagonally close-packed oxygen and zinc atoms. There are three main crystal planes in these structures: a top tetrahedron corner exposed polar zinc (0001) plane, six symmetric no polar {1010 planes parallel to the [0001] direction, and a basal polar oxygen (000 1) plane. We speculated that the hydroxide salt provides(OH-) and helps to form zinc complexes acting as growth units. Due to high enough amount of alkali anions(OH-), the ZnO nuclei are enclosed by negatively charged species. Therefore, the flower-like structures can be promoted without any surfactant or capping agent via molten salt method.
The growth curves of B. subtilis and E. aerogenes in presence of ZnO containing and control nanocomposites are shown in Figs. 3 and 4. The curves show the relationship between microbial population and optical density (OD) versus time of incubation. 3.5. Metal ion releasing measurement The quantities of Zn ions released from nanocomposite into the broth during the time intervals are presented in Table 1. As it is seen release of Zn2+ ions from matrix of the films into the broth was not significantly increased during the time and the greatest amount of migration was obtained in the first day. The Zn ions released for both concentrations after 14 days was about 1.4 ppm. 4. Discussion 4.1. Morphology and size of nanoparticles
3.3. Brunauer–Emmett–Teller surface area The Brunauer–Emmett–Teller (BET) surface area of the nano powders was analyzed by nitrogen adsorption. The specific surface area of the as-synthesized ZnO nano architecture is evaluated to be approximately 15.1 m2 g−1 by BET equation.
According to our SEM results the synthesized ZnO nanoparticles have star-like shapes and based on our XRD results their average crystalline size is 39.7 nm. Effect of morphology, size and concentration on antibacterial properties of nanoparticles is presented in Table 2. It is generally accepted that there is an inverse relationship between the
Fig. 3. Growth curve of B. subtilis in the presence of 2 and 4 wt.% ZnO containing nanocomposites.
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Fig. 4. Growth curve of E. aerogenes in the presence of 2 and 4 wt.% ZnO containing nanocomposites. As it is shown, growth of both microorganisms decreased in the presence of 2 and 4 wt.% ZnO containing nanocomposites compared with controls. Nanocomposites with 4 wt.% ZnO nanoparticles had stronger antibacterial effect against both bacteria as compared with the 2 wt.% ZnO containing nanocomposites. B. subtilis as a Gram-positive bacteria was more sensitive to ZnO containing nanocomposite films in comparison with E. aerogenes as a Gram-negative bacteria.
size of nanoparticles and their antimicrobial activity. A possible mechanism for this observation is that smaller particles have higher surface area to volume ratio and thus they have greater bioactivity [9]. The specific surface area of the synthesized ZnO nano architecture in our study is approximately 15.1 m2 g− 1 (estimated by BET equation), which is greater in comparison with that of ZnO nano-flowers in a similar study by Becker et al. [10]. In a more recent study by Stankovic et al., the antibacterial activity of four different sizes of ZnO nanoparticles against Gram positive Staphylococcus aureus and Gram-negative Escherichia coli has been investigated. The authors concluded that ZnO powder with an average diameter around 30 nm has the greatest antibacterial activity compared with other larger nanoparticles [11]. Although investigating the effect of size on antibacterial effect of ZnO nanoparticle was not of the objectives of the present study, the observed antibacterial effects with an average crystalline size of 39.7 nm, can confirm the findings of previous studies about effectiveness of small sized nanoparticles in inhibition of bacterial growth. As it can be seen in Table 2 only a few studies have investigated the effect of morphology on antibacterial properties of ZnO nanoparticles and there is no general agreement about the effect of shape on bioactivity of ZnO nanoparticles. As an example in the study by Stankovic et al. nanospherical particles showed higher antibacterial activity in comparison with hexagonal prisms and ellipsoid forms whereas in a research by Talebian et al. flower-like ZnO nanoparticles showed the highest photocatalytic inactivation [11]. 4.2. Growth curves of bacteria The results showed that B. subtilis as a Gram-positive bacteria is more sensitive to ZnO nanoparticles in comparison with E. aerogenes as a Gram-negative bacteria. Our findings are in line with several previous studies which have investigated the antibacterial effect of metal and metal oxide nanoparticles on various Gram-positive and Gram-negative bacteria. In an investigation by Li et al., it has been shown that ZnO coated poly vinyl chloride (PVC) film can inhibit the growth of both E. coli and S. aureus. However the antibacterial effect was more pronounced against the Gram-positive S. aureus [14]. In another study, it has been reported that ZnO nanoparticles exhibit more toxicity against Gram-positive S. aureus than Gramnegative E. coli and Pseudomonas aeruginosa [15]. Several mechanisms have been proposed for the antibacterial effects of metal based nanoparticles such as ZnO. ZnO nanoparticles or powders in aqueous solution can produce various reactive oxygen species (ROS) such as hydroxyl radicals (•OH), singlet oxygen or superoxide anion (O2•−), and hydrogen peroxide (H2O2) [16]. The main proposed factors for NP-induced oxidative stress include: (a) the oxidative properties of
the NP themselves which are attributable to their physicochemical properties including surface reactivity, particle size, surface charge and chemical composition, and (b) oxidant generation upon interaction of NP with cellular materials including mitochondrial respiration, and NADPH oxidase system [17]. More over these nanoparticles are able to interact with thiol groups of proteins in the cell wall and to increase the membrane permeability which consequently can result in rupture and leakage of cell content [18]. The differences between Gram-positive and Gram-negative bacteria sensitivity to ZnO nanoparticles are mostly attributable to the differences in cell wall structure. Gram-positive bacteria such as B. subtilis have a thick peptidoglycan layer that contains teichoic acids. Presence of phosphodiester bonds between teichoic acid monomers gives an overall negative charge to the Gram-positive bacteria, while Gram-negative bacteria such as E. aerogenes have a thin peptidoglycan layer surrounded by phospholipids and lipopolysaccharides; [19–20] therefore positively charged ions have more tendency to attach to Gram-positive bacteria as compared with Gram-negative bacteria [21]. As it can seen in Table 2 it seems there is a direct relationship between concentration of nanoparticles in the composite and its antibacterial effect. In the present study nanocomposites with 4 wt.% ZnO had stronger antibacterial effect against both bacteria as compared with the 2 wt.% ZnO containing nanocomposites. This finding is in line with several previous studies. In a study by Gharoy-Ahangar et al. antibacterial effect of three concentrations of ZnO nanoparticles (1, 3 and 5 wt.%) in polyvinyl alcohol (PVA) biopolymer were compared and the results showed that the antibacterial properties of the films improved by increasing nanoparticle content up to 5 wt% [22]. In another study by Jalal et al. it has been shown that increasing the concentration of ZnO nanofluid resulted in a stronger antibacterial activity. The proposed mechanism for this observation by the authors is increased generation of H2O2 by higher concentrations of ZnO nanoparticles [23]. Although higher concentrations of nanoparticles may increase antibacterial activity of nanocomposites, safety issues and inappropriate mechanical properties with higher concentrations are limiting factors [24–25]. Further studies are needed to investigate the most appropriate concentrations of ZnO Table 1 The quantity of Zn ions (mean ± SD) released from nanocomposite LDPE films in to the broth. Concentration
Storage time
Film type
Ion (ppm)
(Days)
LDPE + 2% nano-ZnO
LDPE + 4% nano-ZnO
Zn2+
1 7 14
1.301 ± 0.034 1.376 ± 0.016 1.454 ± 0.009
1.424 ± 0.012 1.428 ± 0.011 1.456 ± 0.009
12 nm particles had greater antibacterial effect against Escherichia coli ZnO suspension with concentration of 5 to 100 mM effectively inhibited bacterial growth Escherichia coli 0.01 to 100 mM [9]
Spherical shape
Various Gram-positive and Gram-negative strains 4–7 mM
ZnO flower-like showed the highest photocatalytic inactivation The antibacterial activity of ZnO increased with decreasing crystallite size The antibacterial activity of the ZnO nanoparticles was inversely proportional to the size of the nanoparticles in S. aureus Escherichia coli Staphylococcus aureus 20 ppm
Spherical particles Commercial ZnO powder with prismatic structures [12] Rod-like Spherical shape Flower-like [13] Nanostructures
Escherichia coli Staphylococcus aureus 1–5 mM Ellipsoid forms
Concentration Studied bacteria Size
Length: 1 μm Diameter: 100 nm Length: 500–600 nm Diameter: 100 nm Diameter: 30 nm Wide particle size distribution 76 nm 65 nm 45 nm 12 nm 25 nm 88 nm 142 nm 212 nm 12 nm 45 nm 2 μm
Morphology
[11] Hexagonal prisms
Ref.
nanoparticles which can be used in active food packagings regarding the above mentioned concerns. 4.3. Metal ion releasing measurement There is no significant difference between the migration of ZnO nanoparticles in 2 and 4 wt.% concentrations. In agreement with our results, Emamifar et al. found that there is no relationship between concentration of ZnO nanoparticle in the polymer and its entrance into the orange juice. Unexpectedly, in their study the quantities of metal ions released from nanocomposite containing 0.25% ZnO nanoparticle was a bit higher than the nanocomposite with 1% ZnO nanoparticle [2]. Based on our knowledge which is in agreement by others [26] the mechanism of silver ions releasing is consisted based on 3 basic processes: 1 — diffusion of water into the composite, 2 — reaction between silver and water molecules and formation of silver ions and 3 — migration of silver ions from composite which can result in the releasing of ions from composite into the aqueous environment. The releasing of silver ions requires the diffusion of the ions through the interconnected amorphous regions of polymers. The amorphous layers near the surface are able to confine more water, resulting in greater release of silver ions. It is quite probable that the migration of zinc ions which has been shown in the present study is following the same pattern. However the quantities of Zn ions migrating into TSB broth are very low. According to the findings of Zapata et al. the releasing of ions from polymer matrix is related to the polarity of the polymer. In polar polymers such as polyamide easier diffusion of water through the polymer matrix result in greater migration of ions compared with nonpolar polymers such as polyethylene, due to oxidation of nanoparticles [27]. Zinc has been classified as a GRAS substance by FDA and it doesn't seem that Zn ions in the concentrations as few as 1.4 ppm can be considered as a health concern; therefore the application of LDPE nanocomposite containing ZnO nanoparticles in food packaging is quite conceivable; however our knowledge about safety of metal based nanomaterials in foods is scarce and further studies in this area are needed. 5. Conclusion Based on our results, 2 and 4 wt.% ZnO containing nanocomposites can reduce the growth of both B. subtilis and E. aerogenes. However, 4 wt.% ZnO containing nanocomposite had stronger inhibitory effect. Moreover B. subtilis as Gram-positive bacteria was more sensitive to ZnO containing nanocomposite films compared with E. aerogenes as Gram-negative bacteria. One of the most promising applications of these nanocomposites can be in the packaging of foods to control the microbial load during storage and to extend their shelf life. The migration of Zn ions from nanocomposites was negligible in the present study, as a result the usage of such nanocomposites can be considered as safe for active food packaging applications. Acknowledgments The authors would like to thank the Research and Technology Council of National Nutrition (721391010), Food Technology Research Institute and Materials and Energy Research Center (MERC) for the financial support. Assistance of Dr. Khanlarkhani is greatly acknowledged.
Padmavathy and Vijayarghavan
Raghupathi et al.
Talebian et al.
Stankovic et al.
References
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Table 2 Antibacterial activity of ZnO nanoparticles with different shapes, sizes and concentrations.
Nanospherical particles showed the highest antibacterial activity With increasing with increasing the concentration of ZnO nanospheres – up to 5 mM, cell viability of bacteria reduced
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Effect of nanocomposite packaging containing ZnO on growth of Bacillus subtilis and Enterobacter aerogenes Hakimeh Esmailzadeh a, Parvaneh Sangpour b,⁎, Farzaneh Shahraz a, Jalal Hejazi c, Ramin Khaksar a a b c
National Nutrition and Food Technology Research Institute, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran Nanotechnology and Advanced Materials Department, Materials and Energy Research Center, Karaj, Iran Department of Biochemistry and Nutrition, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 25 August 2015 Accepted 22 September 2015 Available online 25 September 2015 Keywords: Nanocomposite ZnO Antimicrobial activity Bacillus subtilis Enterobacter aerogenes
a b s t r a c t Recent advances in nanotechnology have opened new windows in active food packaging. Nano-sized ZnO is an inexpensive material with potential antimicrobial properties. The aim of the present study is to evaluate the antibacterial effect of low density Polyethylene (LDPE) containing ZnO nanoparticles on Bacillus subtilis and Enterobacter aerogenes. ZnO nanoparticles have been synthesized by facil molten salt method and have been characterized by X-ray diffraction (XRD), and scanning electron microscopy (SEM). Nanocomposite films containing 2 and 4 wt.% ZnO nanoparticles were prepared by melt mixing in a twin-screw extruder. The growth of both microorganisms has decreased in the presence of ZnO containing nanocomposites compared with controls. Nanocomposites with 4 wt.% ZnO nanoparticles had stronger antibacterial effect against both bacteria in comparison with the 2 wt.% ZnO containing nanocomposites. B. subtilis as Gram-positive bacteria were more sensitive to ZnO containing nanocomposite films compared with E. aerogenes as Gram-negative bacteria. There were no significant differences between the migration of Zn ions from 2 and 4 wt.% ZnO containing nanocomposites and the released Zn ions were not significantly increased in both groups after 14 days compared with the first. Regarding the considerable antibacterial effects of ZnO nanoparticles, their application in active food packaging can be a suitable solution for extending the shelf life of food. © 2015 Published by Elsevier B.V.
1. Introduction Long shelf life and least processing are of the most desirable approaches in food technology. Microbial contamination is one of the major limiting factors in extending the shelf life of food products. An appropriate solution to overcome this problem is application of antimicrobial packaging. Antimicrobial packaging is the most common type of active food packaging system which can inhibit or retard the growth of spoilage microorganisms and can extend the shelf life of food [1]. Recent advances in the field of nanotechnology have attracted many attentions of researchers to design more efficient active packaging systems. When particle size is reduced to nanoscale, the resulting materials exhibit different physical and chemical properties compared with their macroscale counterparts. Materials in nano-size have larger surface to volume ratio; therefore they are able to inactive more microbial cells, which confers higher efficiency [2]. Antimicrobial materials can be classified as organic or inorganic. Inorganic antimicrobials such as metal and metal oxides are stable at high pressures and temperatures; as a result they can tolerate the harsh process conditions [3]. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (P. Sangpour).
http://dx.doi.org/10.1016/j.msec.2015.09.078 0928-4931/© 2015 Published by Elsevier B.V.
Most of the recent studies in the field of antimicrobial nanocomposites, have concentrated on silver nanoparticles and many of them have reported promising impacts. In a review by Marambio-Jones et al. an extended investigation of studies on silver nanoparticles has showed that silver can be a potent antimicrobial agent against different microorganisms. However silver is an expensive material with a well-defined toxicity to human cells which makes the use of it difficult in the food industry [4]. Thus investigators are looking for less expensive and safer nanomaterials such as zinc. However the studies about the antimicrobial effects of these metal nanoparticles are scarce and sometimes controversial. For example in a study by Jin et al. ZnO nanoparticles has showed an antimicrobial effect in growth media, but not in polystyrene film [5]. Zinc is an abundant element with a large distribution, which is essential for the proper function of many metalloenzymes. Zinc oxide is one of the five compounds of zinc that are currently listed as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). Some recent investigations have also proposed antimicrobial properties of ZnO nanoparticles and these nanoparticles have several advantages compared with silver nanoparticles such as lower price, white appearance and UV blocking properties [5–6]. Low density poly ethylene (LDPE) is widely used in food packaging because of its flexibility, transparency, thermo stability and low cost.
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Both ZnO nanoparticles and LDPE are stable at high temperatures; furthermore melt mixing is an appropriate method for production of ZnO nanocomposite [7]. The theory behind this investigation is that ZnO containing nanocomposite, as an inexpensive and safe material, can confer antibacterial properties which may be used as an efficient method to extend the shelf life of foods. The objective of present study is to investigate the anti-microbial effect of zinc oxide containing nanocomposite on Bacillus subtilis, one of the most important spoiling bacteria in the food industry which can cause spoilage of bread and Enterobacter aerogenes, pathogenic bacteria that found in water, vegetable and meat. 2. Materials and methods 2.1. Preparation of ZnO nanoparticles ZnO nanoparticles have been synthesized using molten salt method [8]. Sodium hydroxide, potassium hydroxide and Zinc chloride were obtained from Merck, Germany. Stochiometric amount of these materials were mixed and put into the Teflon cup and then sealed. Mixed powders were heated up to the 200 ± 5 °C (above eutectic of NaOHKOH (175 °C)) for 30 min in an electric oven and then it was cooled down to room temperature. The resultant product washed with hot deionized water and centrifuged at 4000 rpm at 25 °C for 15 min to remove the alkali metal salts. Then, the sediment dried at 100 °C for 3 h to obtain ZnO nanoparticles. 2.2. Characterization of ZnO nanoparticles The phase composition and the microstructure of ZnO nanoparticles were analyzed by X-ray diffractometer (XRD, Philips, X'Pert). The particle size and morphology of the samples were determined by scanning electron microscopy (SEM, Tescan). The Brunauer–Emmett–Teller (BET) surface area of the powders was analyzed by nitrogen adsorption in a Micromeritics ASAP 2020 nitrogen adsorption apparatus (BEL Japan Inc.). 2.3. Preparation of nanocomposite films Insertion of nanoparticles into low density poly ethylene matrix has been accomplished using a twin-screw extruder with a screw diameter of 42 mm. Film grade LDPE resin pellets (300 g) were directly mixed with ZnO nanoparticles (6 g and 12 g respectively for 2 and 4 wt.% ZnO containing nanocomposites) and the mixture was fed into a twin-screw extruder machine. The heating profile was set to six heating zones of the twin-screw extruder including 90 °C, 160 °C, 175 °C, 155 °C, 155 °C, and 150 °C. Then the nanoparticle containing granules, converted into the nanocomposite films with average thickness of 0.1 mm using a hot press with pressure of 120 Kg/cm2 at 200 °C.
2.5. Metal ion releasing measurement Zinc ions releasing in to culture media determined using inductively coupled plasma- optical emission spectrometry (ICP-OES) (varian vistapro Australia). For this purpose, 10 cm2 of nanocomposite films were immersed in 10 mL of TSB broth for 1, 7 and 14 days. Then 100 μL of 70% nitric acid was added to TSB broth and centrifuged at 5000 rpm for 5 min at room temperature. The upper transparent phase was isolated then deionized distilled water was added up to 10 mL. Finally the samples introduced to ICP-OES to determine the quantity of Zn ions migrating into TSB broth.
2.6. Statistical analysis Statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL). All P-values were two-tailed and the level of statistical significance was set at p b 0.05. Between-group differences were analyzed using analysis of variance (ANOVA).
3. Results 3.1. X-ray diffraction Fig. 1 shows the X-ray diffraction of ZnO nanopowders synthesize by molten salt method. As it is obvious, the ZnO phase with wurtzite structure was completely synthesized only after 30 min in molten hydroxide media. The formation reaction of zinc oxide could be sketched as: ZnCl2 þ NaOH þ KOH→ZnO þ NaCl þ KCl þ H2 O:
ð1Þ
According to the JCPDS card No. 36-1451 characteristic peaks can be observed at 2θ values of 31.7°, 34.4°, 36.2°, 56.6°, 62.9°, 67.9°, 69.0° corresponding to (1 0 0), (0 0 2), (1 0 1), (1 1 0), (1 0 3), (1 1 2), (2 0 1) of zinc oxide, wurtzite phase. The absence of remaining peaks indicates the purity of the samples. Sharp diffraction peaks shown in Fig. 1 indicate good crystallinity of ZnO nanoparticles. The broadening of the peaks indicates that the particles are in nanometer scale. According to Scherrer formula Eq. (1), where D stands for the average grain size, λ stands for the X-ray wavelength (0.15405 nm), and θ and β are the diffraction angle and full-width at half maximum of an observed peak, respectively. D ¼ 0:891λ=β cosθ
ð1Þ
The average crystalline size of the particles is 39.7 nm.
2.4. Antimicrobial tests B. subtilis (American type culture collection 6051) and E. aerogenes (American type culture collection 13,048) were obtained from Iranian the Research Organization for Science and Technology. For antimicrobial tests, the bottom of 8 cm plates was coated by the nanocomposite films, then 100 μl of 106 cfu/mL microorganism containing suspension which was inoculated into 10 mL of tryptic soy broth (TSB Merck, Germany), were poured. The plates were incubated in 37 °C (the optimum temperature for the bacterial growth) and bacterial growth was monitored turbidometrically each 2 h (up to 24 h) at the wavelength of 600 nm with a Bioscreen C. Then the growth curves of microorganisms were plotted.
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Fig. 1. XRD pattern of ZnO nanoparticles.
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Fig. 2. SEM micrograph of ZnO nanoparticles.
3.2. Scanning electron microscopy
3.4. Growth curves of bacteria
Morphology, structure and size of the ZnO nanoparticles are investigated by SEM. The SEM images (Fig. 2) show that these nanoparticles have star-like shapes. The mean length of the stars and their diameter of particles are below 400 and 50 nm, respectively. During reaction time (30 min), agglomerated nanoparticles, which are almost flower and star like in shape, can be observed in Fig. 2. It is known that zinc oxide structure, as a polar crystal, consists of hexagonally close-packed oxygen and zinc atoms. There are three main crystal planes in these structures: a top tetrahedron corner exposed polar zinc (0001) plane, six symmetric no polar {1010 planes parallel to the [0001] direction, and a basal polar oxygen (000 1) plane. We speculated that the hydroxide salt provides(OH-) and helps to form zinc complexes acting as growth units. Due to high enough amount of alkali anions(OH-), the ZnO nuclei are enclosed by negatively charged species. Therefore, the flower-like structures can be promoted without any surfactant or capping agent via molten salt method.
The growth curves of B. subtilis and E. aerogenes in presence of ZnO containing and control nanocomposites are shown in Figs. 3 and 4. The curves show the relationship between microbial population and optical density (OD) versus time of incubation. 3.5. Metal ion releasing measurement The quantities of Zn ions released from nanocomposite into the broth during the time intervals are presented in Table 1. As it is seen release of Zn2+ ions from matrix of the films into the broth was not significantly increased during the time and the greatest amount of migration was obtained in the first day. The Zn ions released for both concentrations after 14 days was about 1.4 ppm. 4. Discussion 4.1. Morphology and size of nanoparticles
3.3. Brunauer–Emmett–Teller surface area The Brunauer–Emmett–Teller (BET) surface area of the nano powders was analyzed by nitrogen adsorption. The specific surface area of the as-synthesized ZnO nano architecture is evaluated to be approximately 15.1 m2 g−1 by BET equation.
According to our SEM results the synthesized ZnO nanoparticles have star-like shapes and based on our XRD results their average crystalline size is 39.7 nm. Effect of morphology, size and concentration on antibacterial properties of nanoparticles is presented in Table 2. It is generally accepted that there is an inverse relationship between the
Fig. 3. Growth curve of B. subtilis in the presence of 2 and 4 wt.% ZnO containing nanocomposites.
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Fig. 4. Growth curve of E. aerogenes in the presence of 2 and 4 wt.% ZnO containing nanocomposites. As it is shown, growth of both microorganisms decreased in the presence of 2 and 4 wt.% ZnO containing nanocomposites compared with controls. Nanocomposites with 4 wt.% ZnO nanoparticles had stronger antibacterial effect against both bacteria as compared with the 2 wt.% ZnO containing nanocomposites. B. subtilis as a Gram-positive bacteria was more sensitive to ZnO containing nanocomposite films in comparison with E. aerogenes as a Gram-negative bacteria.
size of nanoparticles and their antimicrobial activity. A possible mechanism for this observation is that smaller particles have higher surface area to volume ratio and thus they have greater bioactivity [9]. The specific surface area of the synthesized ZnO nano architecture in our study is approximately 15.1 m2 g− 1 (estimated by BET equation), which is greater in comparison with that of ZnO nano-flowers in a similar study by Becker et al. [10]. In a more recent study by Stankovic et al., the antibacterial activity of four different sizes of ZnO nanoparticles against Gram positive Staphylococcus aureus and Gram-negative Escherichia coli has been investigated. The authors concluded that ZnO powder with an average diameter around 30 nm has the greatest antibacterial activity compared with other larger nanoparticles [11]. Although investigating the effect of size on antibacterial effect of ZnO nanoparticle was not of the objectives of the present study, the observed antibacterial effects with an average crystalline size of 39.7 nm, can confirm the findings of previous studies about effectiveness of small sized nanoparticles in inhibition of bacterial growth. As it can be seen in Table 2 only a few studies have investigated the effect of morphology on antibacterial properties of ZnO nanoparticles and there is no general agreement about the effect of shape on bioactivity of ZnO nanoparticles. As an example in the study by Stankovic et al. nanospherical particles showed higher antibacterial activity in comparison with hexagonal prisms and ellipsoid forms whereas in a research by Talebian et al. flower-like ZnO nanoparticles showed the highest photocatalytic inactivation [11]. 4.2. Growth curves of bacteria The results showed that B. subtilis as a Gram-positive bacteria is more sensitive to ZnO nanoparticles in comparison with E. aerogenes as a Gram-negative bacteria. Our findings are in line with several previous studies which have investigated the antibacterial effect of metal and metal oxide nanoparticles on various Gram-positive and Gram-negative bacteria. In an investigation by Li et al., it has been shown that ZnO coated poly vinyl chloride (PVC) film can inhibit the growth of both E. coli and S. aureus. However the antibacterial effect was more pronounced against the Gram-positive S. aureus [14]. In another study, it has been reported that ZnO nanoparticles exhibit more toxicity against Gram-positive S. aureus than Gramnegative E. coli and Pseudomonas aeruginosa [15]. Several mechanisms have been proposed for the antibacterial effects of metal based nanoparticles such as ZnO. ZnO nanoparticles or powders in aqueous solution can produce various reactive oxygen species (ROS) such as hydroxyl radicals (•OH), singlet oxygen or superoxide anion (O2•−), and hydrogen peroxide (H2O2) [16]. The main proposed factors for NP-induced oxidative stress include: (a) the oxidative properties of
the NP themselves which are attributable to their physicochemical properties including surface reactivity, particle size, surface charge and chemical composition, and (b) oxidant generation upon interaction of NP with cellular materials including mitochondrial respiration, and NADPH oxidase system [17]. More over these nanoparticles are able to interact with thiol groups of proteins in the cell wall and to increase the membrane permeability which consequently can result in rupture and leakage of cell content [18]. The differences between Gram-positive and Gram-negative bacteria sensitivity to ZnO nanoparticles are mostly attributable to the differences in cell wall structure. Gram-positive bacteria such as B. subtilis have a thick peptidoglycan layer that contains teichoic acids. Presence of phosphodiester bonds between teichoic acid monomers gives an overall negative charge to the Gram-positive bacteria, while Gram-negative bacteria such as E. aerogenes have a thin peptidoglycan layer surrounded by phospholipids and lipopolysaccharides; [19–20] therefore positively charged ions have more tendency to attach to Gram-positive bacteria as compared with Gram-negative bacteria [21]. As it can seen in Table 2 it seems there is a direct relationship between concentration of nanoparticles in the composite and its antibacterial effect. In the present study nanocomposites with 4 wt.% ZnO had stronger antibacterial effect against both bacteria as compared with the 2 wt.% ZnO containing nanocomposites. This finding is in line with several previous studies. In a study by Gharoy-Ahangar et al. antibacterial effect of three concentrations of ZnO nanoparticles (1, 3 and 5 wt.%) in polyvinyl alcohol (PVA) biopolymer were compared and the results showed that the antibacterial properties of the films improved by increasing nanoparticle content up to 5 wt% [22]. In another study by Jalal et al. it has been shown that increasing the concentration of ZnO nanofluid resulted in a stronger antibacterial activity. The proposed mechanism for this observation by the authors is increased generation of H2O2 by higher concentrations of ZnO nanoparticles [23]. Although higher concentrations of nanoparticles may increase antibacterial activity of nanocomposites, safety issues and inappropriate mechanical properties with higher concentrations are limiting factors [24–25]. Further studies are needed to investigate the most appropriate concentrations of ZnO Table 1 The quantity of Zn ions (mean ± SD) released from nanocomposite LDPE films in to the broth. Concentration
Storage time
Film type
Ion (ppm)
(Days)
LDPE + 2% nano-ZnO
LDPE + 4% nano-ZnO
Zn2+
1 7 14
1.301 ± 0.034 1.376 ± 0.016 1.454 ± 0.009
1.424 ± 0.012 1.428 ± 0.011 1.456 ± 0.009
12 nm particles had greater antibacterial effect against Escherichia coli ZnO suspension with concentration of 5 to 100 mM effectively inhibited bacterial growth Escherichia coli 0.01 to 100 mM [9]
Spherical shape
Various Gram-positive and Gram-negative strains 4–7 mM
ZnO flower-like showed the highest photocatalytic inactivation The antibacterial activity of ZnO increased with decreasing crystallite size The antibacterial activity of the ZnO nanoparticles was inversely proportional to the size of the nanoparticles in S. aureus Escherichia coli Staphylococcus aureus 20 ppm
Spherical particles Commercial ZnO powder with prismatic structures [12] Rod-like Spherical shape Flower-like [13] Nanostructures
Escherichia coli Staphylococcus aureus 1–5 mM Ellipsoid forms
Concentration Studied bacteria Size
Length: 1 μm Diameter: 100 nm Length: 500–600 nm Diameter: 100 nm Diameter: 30 nm Wide particle size distribution 76 nm 65 nm 45 nm 12 nm 25 nm 88 nm 142 nm 212 nm 12 nm 45 nm 2 μm
Morphology
[11] Hexagonal prisms
Ref.
nanoparticles which can be used in active food packagings regarding the above mentioned concerns. 4.3. Metal ion releasing measurement There is no significant difference between the migration of ZnO nanoparticles in 2 and 4 wt.% concentrations. In agreement with our results, Emamifar et al. found that there is no relationship between concentration of ZnO nanoparticle in the polymer and its entrance into the orange juice. Unexpectedly, in their study the quantities of metal ions released from nanocomposite containing 0.25% ZnO nanoparticle was a bit higher than the nanocomposite with 1% ZnO nanoparticle [2]. Based on our knowledge which is in agreement by others [26] the mechanism of silver ions releasing is consisted based on 3 basic processes: 1 — diffusion of water into the composite, 2 — reaction between silver and water molecules and formation of silver ions and 3 — migration of silver ions from composite which can result in the releasing of ions from composite into the aqueous environment. The releasing of silver ions requires the diffusion of the ions through the interconnected amorphous regions of polymers. The amorphous layers near the surface are able to confine more water, resulting in greater release of silver ions. It is quite probable that the migration of zinc ions which has been shown in the present study is following the same pattern. However the quantities of Zn ions migrating into TSB broth are very low. According to the findings of Zapata et al. the releasing of ions from polymer matrix is related to the polarity of the polymer. In polar polymers such as polyamide easier diffusion of water through the polymer matrix result in greater migration of ions compared with nonpolar polymers such as polyethylene, due to oxidation of nanoparticles [27]. Zinc has been classified as a GRAS substance by FDA and it doesn't seem that Zn ions in the concentrations as few as 1.4 ppm can be considered as a health concern; therefore the application of LDPE nanocomposite containing ZnO nanoparticles in food packaging is quite conceivable; however our knowledge about safety of metal based nanomaterials in foods is scarce and further studies in this area are needed. 5. Conclusion Based on our results, 2 and 4 wt.% ZnO containing nanocomposites can reduce the growth of both B. subtilis and E. aerogenes. However, 4 wt.% ZnO containing nanocomposite had stronger inhibitory effect. Moreover B. subtilis as Gram-positive bacteria was more sensitive to ZnO containing nanocomposite films compared with E. aerogenes as Gram-negative bacteria. One of the most promising applications of these nanocomposites can be in the packaging of foods to control the microbial load during storage and to extend their shelf life. The migration of Zn ions from nanocomposites was negligible in the present study, as a result the usage of such nanocomposites can be considered as safe for active food packaging applications. Acknowledgments The authors would like to thank the Research and Technology Council of National Nutrition (721391010), Food Technology Research Institute and Materials and Energy Research Center (MERC) for the financial support. Assistance of Dr. Khanlarkhani is greatly acknowledged.
Padmavathy and Vijayarghavan
Raghupathi et al.
Talebian et al.
Stankovic et al.
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Table 2 Antibacterial activity of ZnO nanoparticles with different shapes, sizes and concentrations.
Nanospherical particles showed the highest antibacterial activity With increasing with increasing the concentration of ZnO nanospheres – up to 5 mM, cell viability of bacteria reduced
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