CuO nanocomposite

CuO nanocomposite

International Journal of Biological Macromolecules 109 (2018) 1219–1231 Contents lists available at ScienceDirect International Journal of Biologica...

3MB Sizes 0 Downloads 8 Views

International Journal of Biological Macromolecules 109 (2018) 1219–1231

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Enhanced Antibacterial effect of chitosan film using Montmorillonite/CuO nanocomposite Afsaneh Nouri a , Mohammad Tavakkoli Yaraki b,c,1 , Mohammad Ghorbanpour a,∗ , Shilpi Agarwal d , Vinod Kumar Gupta d,∗ a

Department of Chemical Engineering, University of Mohaghegh Ardabili, Ardabil, Iran Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, 15875-4413, Iran c Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore d Department of Applied Chemistry, University of Johannesburg, Johannesburg, South Africa b

a r t i c l e

i n f o

Article history: Received 27 September 2017 Received in revised form 10 November 2017 Accepted 18 November 2017 Available online 21 November 2017 Keywords: Bio-nanocomposite Chitosan Montmorillonite Copper oxide Antibacterial film Food packaging

a b s t r a c t Montmorillonite −copper oxide (MMT-CuO) nanocomposites were prepared by a facile and eco-friendly method and introduced into chitosan (Cs) matrix to enhance its optical, mechanical and antibacterial properties. The synthesized composites were characterized using diffuse reflectance spectroscopy (DRS), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and energy dispersive Xray (EDX) spectroscopy. The antimicrobial activity of MMT-CuO nanocomposites showed more than 99% mortality against two Gram-negative bacterium (E.coli (PTCC 1270), P.aeruginosa (PTCC 1430)) and two Gram-positive bacterium (S.aureus (PTCC1112) B.cereus (PTCC- 1015)). The effect of weight fraction of MMT-CuO nanocomposites (1, 3 and 5% w/w) as antibacterial nanofiller on physical, optical, mechanical, microstructural, and antibacterial properties of chitosan films were evaluated. The obtained data showed that introducing small amount MMT-CuO to chitosan films could enhance the mechanical, antibacterial properties, and decreased both water solubility and UV transition with the lowest effect on the transparency of the films. The incorporation of 3% w/w MMT-CuO-90 nanocomposite into the films increased the tensile strong (TS), and elongation at break (E%) values 58.5% and 52.4%, respectively while reduced the water vapor permeability and oxygen permeability about 55% and 32%, respectively. CSG3MMT-CuO90 films showed intense antibacterial activity against food borne pathogenic and more effective against S. aureus and B.cereus. than E.coli and P.aeruginosa. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Nowadays, food packaging industry is based on consumption of petroleum-based plastics (more than 300 million tons per year) [1] due to their high accessibility and low-cost, as well as excellent mechanical properties, good barrier to gasses (oxygen, carbon dioxide, anhydride and aroma compound) and thermal stability [2]. However, there are some environmental drawbacks of plastic packagings, such as limited disposal methods, accumulation of plastic waste in landfill and also exhausting fossil fuel resource [3–5]. Moreover, in recent years, the growing consumer’s trend to fresh

∗ Corresponding author. E-mail addresses: [email protected] (M. Ghorbanpour), [email protected], [email protected] (V.K. Gupta). 1 Current Affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. https://doi.org/10.1016/j.ijbiomac.2017.11.119 0141-8130/© 2017 Elsevier B.V. All rights reserved.

and safe food products with minimally processed and minimized food losses and also without preservative agents led to developing intelligent packaging which has more functionality than simple plastic-based packaging materials [6]. For these reasons, substituting of petrochemical-based plastic packaging material with biodegradable polymers has been of interest over the past few years [7]. Among different kinds of biopolymers, polysaccharides, especially chitosan, have been widely considered good candidates to be used as edible films and coating in food packaging sector due to its properties. These properties include non-toxicity, biodegradability, biocompatibility, excellent film forming ability, strong antimicrobial, antifungal activities (against a wide range of fungi, Gram-positive, and Gram-negative bacteria) and excellent carrier properties for various additives including nutrients, minerals, colors, vitamins, antioxidants, antifungal, antimicrobials agents [8–12]. Despite all of these desirable properties, the application of the chitosan is limited due to its low mechanical strength and

1220

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

thermal stability, and also high gas and water vapor permeability [13]. Therefore, improving the physical, mechanical and barrier properties of the chitosan film is essential for food packaging application. Blending with other polymers [14,15], using cross-linkers [16] and incorporation of micro or nanofiller into the chitosan matrix [17–20] are some of the different methods that have been utilized to enhance the chitosan properties. Among these methods, developing nanocomposites using nanomaterials have been studied extensively [21]. Montmorillonite (MMT) is one of the most popular layered silicate materials that have been used in chitosan-based nanocomposites due to its low cost, availability, high aspect ratios (100–1500) and high surface-to-volume ratio (700–800 m2 /g), as well as good miscibility with cationic polymers [22–24]. Significant improvements of mechanical, barrier and optical properties of chitosan film by addition of small amount of MMT (1–5 wt%) have been reported [25–27]. while MMT does not have antibacterial activity, it needs chemical modification to act as an antibacterial agent in biopolymer based food packing; however, this chemical modification might suppress the enhancement in tensile strength of final chitosan nanocomposite film [28,29]. Hence, antibacterial agents such as silver, copper or copper oxide nanoparticles could be added to the nanocomposite [30,31]. Nevertheless, the direct inclusion of copper ions in polymer film should be avoided due to the uncontrolled leaching and toxicity of copper ions which may accelerate the biochemical deterioration in foods. Fortunately, substitution of copper ions with exchangeable positively charged ions, such as Na+, K+,Mg2+ and Ca2+ in high cation exchange clay minerals (montmorillonite or Vermiculite) and stabilization onto these inorganic carriers prevents the burst release of copper ion [32,33]. Hence, This method allows copper and copper complexes to be used as an antibacterial agent in smart packaging for the food industry [32]. In our previous report [34], montmorillonite-copper oxide (MMT-CuO) nanocomposite was synthesized via a straight forward and facile method without any chemical solvents at different alkaline ion exchange times. The present study focuses on the preparation and characterization chitosan-based films by incorporation of MMT-CuO nanocomposites as a reinforcing and antibacterial agent. Based on this approach, different amount of MMT-CuO nanocomposite was added to the chitosan matrix via simple solution casting method. Then, the effect of incorporation of MMT-CuO nanofillers on the chitosan film morphology, crystallinity, thermal stability, mechanical, optical, barrier and antibacterial properties were investigated.

2. Materials and methods

2.2. Sample preparations Firstly MMT-CuO nanocomposite was prepared by a simple, cost-effective and eco-friendly method based on basic ionexchange that has been described in our previous work [34]. Briefly, MMT and CuSO4 ·5H2 O at the ratio of 1:1 (w/w) were placed in a muffle furnace for 20, 90 min at 550 ± 10 ◦ C. Then, the mixture was cooled to room temperature and washed with appropriate 0.1 M saline solution and deionized water repeatedly. Finally, the solution was filtered using Whatman No. 1 filter paper and allowed to dry at ambient temperature for 24 h. In order to prepare the nanocomposite film, a known amount of MMT powder and as-synthesized MMT-CuO nanocomposite were dispersed in 100 ml of 1% (v/v) acetic acid solution and stirred at room temperature overnight. Then, the solution was treated by ultrasonic probes (100W, on/off: 7/3 s) for 10 min. Afterward, 25% (w/w of dry chitosan base) glycerol as a plasticizer and 2 gr chitosan were added into the solution for 4 h with a mechanical stirring at room temperature until a homogeneous viscous solution was obtained. An ultrasonic treatment (400 W power, 10 min) was used to remove the formed air bubbles during the stirring. Chitosan film forming solutions (25 ml) were poured into the plastic Petri dishes and allowed to be dried in the incubator at 25 ± 0.7 ◦ C for 24 h. Finally, the dried films were peeled off and kept in polyethylene bags Scheme S1 shows the procedure of nanocomposite film preparation). All sample films were preconditioned at 25 ◦ C and 55% RH before further analysis. Table S1 presents the prepared sample and their formulations in this study. 2.3. Characterization techniques 2.3.1. Optical characteristics The UV–vis absorption spectra of Copper sulfate, MMT and MMT-CuO nanocomposites was measured by a UV–visible diffusive reflectance spectrophotometer (Sinco S4100, Korea), in the spectral range 200–1000 nm. Also, optical properties of the chitosan-based films were determined by UV–vis spectroscopy. A piece of each film sample was directly mounted between the two spectrophotometer magnetic cell holders. The absorbance spectra of the films were measured at a wavelength range of 190–890 nm using a UV–vis spectrophotometer (Nsnospec 2UV-A, German). Also, transparency of the samples was determined by measuring light transmittance at UV (T280nm ) and visible (T660nm ) regions. 2.3.2. X-Ray diffraction X-ray diffraction technique was carried out using a Philips powder diffractometer (type PW 1730, goniometer) equipped Cu-K␣ ´˚ in the 2␪ range from 1◦ to 15◦ with a step radiations (␭=1.5405 A) ◦ size 0.02 . Bragg’s law was used to calculate d001 of MMT and MMTCuO nanocomposite.

2.1. Materials Ca-montmorillonite (MMT) was obtained from Kansas Jam Company (Rasht, Iran). Medium molecular weight (Mw) Chitosan (310–375 kDa, with a degree of deacetylation greater than 75%) was purchased from Sigma–Aldrich Chemical Co., USA. Glacial acetic acid (HAc), Copper (II) sulfate pentahydrate (CuSO4 ·5H2 O), sodium chloride (NaCl) and glycerol as a plasticizer, MuellerHinton broth (MHA), nutrient agar and ethanol were obtained from Merck Co., Germany. The Gram-negative E.coli PTCC 1270 (PTCC, Persian Type Culture Collection), P.aeruginosa PTCC 1430 and Grampositive S.aureus PTCC1112 B.cereus PTCC 1015 bacterial strains were prepared by the Iranian Research Organization for Science and Technology. All the chemicals used were of analytical grade, and samples were prepared using deionized water.

2.3.3. Scanning electron microscopy (SEM) and elemental analysis Microstructures of the prepared MMT and MMT-CuO nanocomposite, as well as the surface of morphology the chitosan- based films, were characterized by FE-SEM analysis. Powder samples and small pieces of film were coated with a thin layer of gold and then mounted on a cylindrical aluminum specimen holder and were scrutinized using a Scanning Electron Microscopy (MIRA3TESCANXMU, Acc Volt. 15 kV, Czech Republic). Also, the EDX analysis was performed to assess elemental maps of the composite and determine the presence of copper oxide in MMT microstructure using energy dispersive X-ray spectrometer (MIRA3TESCAN-XMU, Czech Republic) which operated at an electron accelerating voltage of 15 kV.

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

2.3.4. Film thickness Film thickness was measured using a hand-held digital Electronic micrometer (Mitotuyo No. 7327, Tokyo, Japan) with an accuracy of 0.001 mm. Five different points of the sample were selected randomly, and thickness measurements were performed. The mean value was used in mechanical properties calculation. 2.3.5. Thermal analysis The thermal stability of the film samples was studied using thermogravimetric analysis (Linseis STA PT-1000, German). For this purpose, 5 mg of each sample was heated from room temperature up to 600 ◦ C at a heating rate of 10 ◦ C/min under a nitrogen flow (50 cm3 /min). A derivative form of TGA (DTG) was calculated using the central finite difference for the first derivate based on the TGA values as below: DTG =

Wt+t − Wt−t 2t

(1)

Where,Wt+t and Wt−t are the residual weight of the sample at time t + t and t − t, respectively. t is the time interval for reading the sample weight. 2.3.6. Water solubility Water solubility of the films was determined according to the method described by Casariego et al. [35]. This parameter is defined as a percentage of dry matter solubilized in water after 24 h immersion. Briefly, two disks of the sample with 2 cm diameter were cut and then, placed in an oven at 105 ◦ C. After 24 h, the films were taken out, weighed (Mi ), and immersed in closed beakers including 50 ml deionized water. All samples were kept for 24hr in a shaker incubator at 250 rpm at 20 ◦ C. The insolubilized film pieces were then, separated using filter paper and again placed in an oven at 105 ◦ C for 24 h. The WS of the film was calculated according to the following equation: M − Mf WS% = i × 100 Mi

(2)

Where, Mi and Mf are the initial and final mass of the film, respectively. 2.3.7. Water vapor permeability Water vapor permeability (WVP) of the films was determined at 25 ◦ C and 97% relative humidity (RH) according to the ASTM E96 gravimetric method with some modification [22]. The circular test cups containing anhydrous calcium sulfate (0% RH, assay cup) were sealed by the test films with 0.001586 m2 film area. The cups were placed inside a glass desiccator containing potassium sulfate saturation solution. The difference in RH corresponded to a driving force of 3169 Pa, expressed as water vapor partial pressure. Weight gain of the test cups (the transferred water through the film and adsorbed by the desiccant) was determined at intervals of 2 h and the water vapor transmission rate (WVTR) and WVP were calculated according to Eqs. (3) and (4): WVRT = WVP =

m t×A

WPTR × X P

(3) (4)

where m is the slope of the straight line in the diagram moisture gain versus time (gr/hr), A is the area of the exposed film surface (m2 ), X is the average film thickness (mm) and p is the water vapor pressure difference between the two sides of the film (Pa). WVP was measured in three replicated samples for each type of film.

1221

2.3.8. Oxygen permeability (OP) Oxygen permeability (OP) of all film samples were determined based on the ASTM standard method D3985-05 (ASTM, 2005) via an oxygen permeation analyzer (Coesfeld, model GDP-C). A sample film was placed in a gas permeation cell with 5 cm2 open testing area and two gas inlet and outlet chambers. The oxygen as permeation gas and nitrogen as a sweep gas were passed continuously through the film in the form of counter current. All tests were performed at 25 ◦ C with no moisture in the environment (0% RH). Oxygen transmission rate (OTR) was recorded and oxygen permeability (OP) values were calculated by the following equation:





OP Cm3 ␮mm−2 day−1 KPa−1 =

OTR × X p

(5)

Where x is the sample thickness and p is the difference in partial pressure of oxygen at the two sides of the sample film that in this study was 1 atm. 2.3.9. Mechanical properties The mechanical properties of film samples, including elongation at break (E) and tensile strength (TS), were measured at 25 ◦ C with a tensile testing machine with a 1000 N load cell (SANTAM, STM-20, Iran) following the guidelines of ASTM Standard Method D882. Sample films were cut into strips (15 mm × 70 mm) and conditioned for 24 h at 25 ◦ C and 50 ± 5% RH before measurement. The initial grip separation and cross-head speed were set to 30 mm and 5 mm m−1 in, respectively. A computer was used to record the stress-strain curves. The TS (MPa) was obtained by dividing the maximum load (N) by the initial cross-sectional area (m2 ) of the film sample, and the EB (%) was calculated by dividing the extension at rupture of the film by the initial length of the film (70 mm) multiplied by 100. Three to five samples of each were tested, and the mean value was reported in this study. 2.4. Antibacterial study The antibacterial activity of the MMT-CuO nanocomposites and chitosan based films were studied by viable cell colony count method (CFU) against four foodborne pathogenic bacteria, the Gram- negative bacterium (E.coli PTCC 1270, P.aeruginosa (PTCC 1430) and the Gram-positive bacterium (S.aureus PTCC1112, B.cereus PTCC- 1015). Firstly, all sample of films were washed with 75% ethanol to kill bacteria, and 0.2 gr of each sample were immersed in 4 ml nutrient broth containing ∼ 106 -107 colony forming units per mL (CFU/mL). Then, the solution was shaken at 37 ◦ C. A flask containing bacteria, and no sample was used as control. After 24 h incubation, 0.2 ml of bacterial culture was taken out from the flask, and serial dilutions were repeated with each initial sample. 100 ␮l diluent of the sample was then spread onto nutrient agar plates and incubated at 37 ◦ C for 24 h. The number of viable microorganism colonies was counted manually using a pen and a click-counter and multiplied by the dilution factor. The percentage of inhibition of each film was calculated with the following equation (ASTM E2149-01): Mortality (%) =

B−A × 100 B

(6)

Where B and A are the mean number of bacteria in the control samples (CFU/sample) and the treated samples after 24 h incubation (CFU/sample), respectively. Besides, agar diffusion method was used for the assessment of antimicrobial activities chitosan film-forming solution. Firstly, bacterial strains of E.coli, P.aeruginosa, S.aureus, and B.cereus were cultured in Müller Hinton Broth at 37 ◦ C for 24 h and then, chitosan film-forming solutions were cast into Mueller Hinton agar (MHA) wells with 5 mm diameter. 100 ␮l of mentioned bacteria (106 –107

1222

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

Fig 1. XRD patterns of MMT, MMT-CuO nanocomposite, Cs, CSG, CSG3MMT, CSG3MMT-CuO-20, CSG3MMT-CuO-90 films (a–d).

CFU/ml) was inoculated by swab was spread on plates containing MHA and films samples and incubated at 37◦C for 24 h. The inhibition zone of chitosan film-forming solution was measured via diameters of the zone surrounding the wells. 3. Results and discussion 3.1. Optical properties The diffuse reflectance spectroscopy (DRS) is a preliminary technique that was used to confirm CuO NPs formation in MMT’s layered and identify the effect of different alkaline ion exchange time on the size and size distribution of CuO nanoparticles (Fig.S1). Copper sulfate shows two absorption bands around 247 and 310 nm, while MMT revealed an original band around 310 nm. In MMTCuO-20 nanocomposite the appearance of a characteristic peak around 313 nm indicates the presence of copper oxide in MMT microstructure. This unique absorption peak is due to the collective oscillation of the free conduction band electrons of CuO nanoparticles under electromagnetic radiation which is called localized surface plasmon resonance (LSPR) [36]. By increasing alkaline ion exchange time, this characteristic peak showed about 12 nm bathochromic shift with decreasing intensity of the LSPR peaks, which may be related to an increase CuO nanoparticles size as LSPR is sensitive to particles size, shapes and dielectric value of the reaction medium [37].According our previous report [34], the red shift of CuO nanoparticles has occurred rapidly in initial of alkaline exchange time (20 min), which might be due to two reasons; 1) At the beginning of the process, the diffusion phenomenon occurs more quickly, and as the system approaches the equilibrium, the mass transfer rate slows down. 2) Montmorillonite or any other silicate layer has a maximum cation exchange capacity. As a result, it seems that ion exchange capasity of MMT was almost

completed after 20 min alkine ion exchange time and afterward, more duffusion of copper ions lead to coalescence of particles to each other. Therefore, in the MMT-CuO-90 nancomposite, optical absorption spectra of CuO nanoparticle show lower intensity with slight red shift of maximum absorbance peak. Similar trend has been observed for Ag NPs where by increasing size of NPs, the SPR band has red-shift and the intensity of SPR decreased [38]. Fig. 1 The protection of foodstuffs from the light, especially from UV radiation, is one of the major functions required for food packaging films [1] because UV light may lead to some undesirable reaction such as lipid oxidation, discoloration, the formation of off-flavor, and loss of nutrients from the packaged food [39,40]. Fig. S2 shows the UV–visible absorption spectra of chitosan-based films. As can be seen in Fig. S2a, the neat CS and CSG films did not show any absorption peak in the wavelength scanned range and have the same light absorption spectra. However, CSMMT nanocomposite films showed a peak at 310 nm that intensity of this peak increased by increasing the MMT content in the samples which this results could be confirmed by the obtained DRS analysis results (See Fig. S2a). In CsGMMT-CuO nanocomposite films (Fig. S2b and S2c), MMT’s peak gradually weakened by increasing the nanofiller concentration whereas another peak appeared in 250 nm that is related to copper oxide nanoparticle[41]. The percentage transmittance values of chitosan-based films are presented in Table 1. The visible light transmittance (T660nm ) and the UV light transmittance (T280nm ) of the neat chitosan film were 27.1 ± 4.0% and 86.7 ± 1.3%, respectively. As can be seen, the addition of glycerol did not affect T280nm and T660nm of the film, while the introduction of MMT into the CSG led to decrease in the visible light and UV transmittance of samples. The T280nm and T660nm of three CSGMMT films decreased with the increase in MMT content. Compared with MMT, MMT-CuO nanocomposite reduced T280nm

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

1223

Table 1 Thickness, Percentage of UV and Visible light transmittance (T280nm and T660nm ), mechanical (TS and E) and water solubility of chitosan- based films incorporated with MMT and MMT-CuO nanocomposite Sample code

Film thickness (␮m)

T280nm

T660nm

TS (MPa)

E (%)

WS (%)

Cs CsG CsG1MMT CsG3MMT CsG5MMT CsG1MMT-CuO-20 CsG3MMT-CuO-20 CsG5MMT-CuO-20 CsG1MMT-CuO-90 CsG3MMT-CuO-90 CsG5MMT-CuO-90

52.2 ± 9.9 64.6 ± 9.7 70.2 ± 6.8 76.4 ± 8.5 81.0 ± 11.7 66.6 ± 7.5 68.4 ± 7.5 71.0 ± 6.8 68.2 ± 4.1 70.8 ± 16.8 73.4 ± 6.3

27.1 ± 4.0 25.1 ± 6.5 21.9 ± 5.7 15.1 ± 5.2 11.7 ± 4.7 17.3 ± 1.2 7.9 ± 2.0 3.8 ± 1.0 13.1 ± 4.5 4.1 ± 2.0 2.6 ± 0.7

86.4 ± 1.3 85.7 ± 1.9 82.0 ± 1.6 72.4 ± 2.7 57.1 ± 9.7 83.5 ± 0.7 73.2 ± 1.0 55.1 ± 9.2 81.0 ± 2.6 70.2 ± 4.7 58.8 ± 5.9

46.2 ± 0.6 20.5 ± 1.4 23.8 ± 1.6 27.2 ± 4.2 20.2 ± 6.7 28.4 ± 2.0 29.4 ± 4.7 24.6 ± 2.6 23.7 ± 4.6 32.5 ± 4.0 24.0 ± 3.0

8.2 ± 4.2 38.5 ± 11.3 52.6 ± 9.5 58.5 ± 15.2 42.7 ± 16.9 73.6 ± 3.6 81.8 ± 7.2 28.4 ± 8.8 59.3 ± 10.7 58.7 ± 7.7 50.3 ± 7.5

13.4 ± 2.4 19.0 ± 0.8 18.5 ± 0.8 17.4 ± 1.1 17.2 ± 1.1 17.5 ± 1.1 17.4 ± 0.6 15.5 ± 0.3 17.2 ± 1.0 16.5 ± 1.6 15.5 ± 2.8

significantly but did not affect T660nm . On the other hand, T280nm is directly proportional to alkaline ion exchange time, so that the presence of 5% MMT/CuO-90 nanocomposite reduced the UV light transmittance about 90.4%. These results are consistent with the data reported by Cardenas et al. [35]. Therefore, MMT-CuO containing films can be applied as a food packing for products which are susceptible to oxidation of UV-light. 3.2. Structural analysis The XRD patterns of MMT and MMT-CuO nanocomposite were prepared to investigate the effect of alkaline ion exchanged time on the variations in the d001 spacing of MMT (Fig. S3). The XRD pattern of the MMT indicates a characteristic peak at 2␪ = 5.83◦ which according to Bragg equation (2dsin =n␭, ␭ = 0.15406 nm) corresponding to a d001 =1.51 nm. After 20 and 90 min heating of MMT-Copper sulfate mixture at 550 ◦ C, this diffraction peak shifted to 2␪ = 8.91◦ and 9.24◦ (corresponding to d001 = 0.99 and 0.95 nm, respectively). The reduction in d-spacing value could be due to replacement of Ca2+ with Cu2+ and formation of CuO nanoparticle in MMT interlayer and the complex of the three square micropores from the six silicon–oxygen tetrahedron, as well as the micropore of aluminum oxide octahedral [42,43]. Also, the dehydration of the interlayer cation may be reduced d001 value [44]. Hence, the d-spacing reduction for the sample with 90 min alkalineexchange was only 3.4% more than that with 20 min (34.4% vs. 37.8%). Result shows that the evaporation of water between the 2:1 layers happened faster for short alkaline exchange time; however, the evaporation rate decrease for longer alkaline exchange time. This result has good agreement with the reported data by Sarikayad et al. [44] and Mosser et al. [45]. The XRD can offer valuable information on the intercalation and exfoliation processes in a biopolymer/clay nanocomposite. In general, three main types of composites could be obtained when a silicate layer is associated with a biopolymer; i) Flocculated structure or micro composites, where the polymer chains are unable to intercalate between the silicate layers and filler diffraction peak stay at the same position. ii) Intercalated structure, where one or more than one polymer chain is intercalated between the silicate layers causing to a well-ordered multi-layer morphology built up with alternating polymer and inorganic layers. Also, diffraction peaks would be shifted to lower values. iii) Exfoliated or delaminated structure, where the silicate layers are completely dispersed homogeneously in the polymer matrix, and diffraction peaks totally disappear [46,47]. Fig. 1 shows Wide-Angel X-ray Diffraction spectra (WAXD) of MMT, MMT-CuO nanocomposite and chitosan based films. In Fig. 1a, it is evident that the neat chitosan film shows characteristic crystallinity peaks at 2␪=8.1◦ and 11.3◦ [15]. The crystalline structure of chitosan strongly depends on some parameters including processing treatment method, origin and molecular constitution

(molecular weight and the degree of deacetylation) [27]. The data reported in Fig. 1a shows that the crystallinity of chitosan was not affected by the presence of the glycerol as a plasticizer. The findings are consistent with Lavorgna et al. [27]. In contrast, Grigoriadi et al. [48] have reported that addition of glycerol to the chitosan-NaMMT nanocomposite films prepared through solution casting method could improve the crystal structure. This difference between the results could be attributed to the using different preparation method that might lead to the crystalline or amorphous structure. The effect of the addition of MMT and MMT-CuO nanocomposite on the XRD pattern of the chitosan-based film is illustrated in Fig. 1b–d. As can be seen, the MMT has a characteristic diffraction peak at 2␪ = 5.83◦ which corresponds to a d001 spacing of 1.516 nm, according to the Bragg equation. After addition of 3% MMT to CSG film, the MMT characteristic peak disappeared, and a new peak appears at 2␪ = 5.8◦ (d001 = 1.73 nm) due to the formation of intercalated structure. However, the addition of 3 wt% MMT-CuO-20 to the CsG film-forming solutions resulted in the disappearance of the MMT-CuO distinctive peak at 8.91◦ (d001 -spacing of 0.99 nm), showing the formation of an exfoliated structure of MMT-CuO nanofiber which was disordered and not detectable by XRD. Similarly, the same behavior was observed in a CSG film containing 3 wt% of MMT-CuO with 90 min ion exchange time. In all cases, chitosan- based nanocomposite showed a decrease in crystalline phase compared with chitosan film. 3.3. Surface morphology analysis The surface morphology of MMT and MMT-CuO nanocomposite powder was investigated by FESEM image with two magnifications (Fig. 2). It is clear in Fig. 2 that the MMT has a sheet like structure with many nano-sized flakes with 15.29 nm thickness. After alkaline ion exchange at 550 ◦ C, more flakes appeared which this phenomenon related to copper diffusion into the inner layer of MMT and opening crack on its surface. Also, no CuO nanoparticle formation could be seen on the surface of MMT while it has been reported in the silver-MMT system [49]. Energy dispersive X-ray spectroscopy (EDS) analysis was conducted to prove the presence of elemental copper in the MMT-CuO nanocomposite (Fig. 2). According to EDX spectrum of MMT-CuO20, a new peak appeared at 8 KeV which indicates that Cu2+ has exchanged with Ca2+ . The Atomic weight percentage of Cu and O and Ca after 20 min alkaline ion exchange was 2.49%, 50.25%, and 1.72%, respectively. Also, EDX analysis of MMT-CuO-90 represented a slight increase in Cu elemental and decrease of Ca in MMT interlayers; which was 2.55% and 0.06%, respectively. These results confirm that the copper ions have been loaded into the montmorillonite inter layer. Surface morphology of chitosan based films was investigation using FESEM analysis. Fig. 3 Shows the SEM images of CS, CSG and

1224

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

Fig. 2. FE-SEM micrographs in low and high magnification and EDX pattern of a) MMT, b) MMT-CuO-20, and c) MMT-CuO-90.

chitosan-based nanocomposite with loading three 3% w/w of MMT, MMT-CuO-20, and MMT-CuO-90. As presented in Fig. 3a–c, CS, and CSG, as well as CSG3MMT, exhibited a flat and smooth appearance without any cracks or pores on their surface. Similarly, it seems that the MMT-CuO nanofiller was dispersed homogeneously in the chitosan matrix (Fig. 3d and e). However, the MMT-CuO-90 had better distribution than MMT-CuO-20. According to MMT-CuO FESEM analysis, we demonstrated that with increasing alkali-ion exchange time, more copper diffused into the interlayer of MMT and caused to the opening of flakes. This phenomenon increased the available surface for interaction between nanofiller and polymer. 3.4. Film thickness The thicknesses of CS and CS- based nanocomposite films were presented in Table 1. According to the results, films thicknesses for

all samples were in the range of 52.2–81 ␮m. The addition of glycerol into chitosan matrix increased the control film thicknesses to 64.4 ␮m. Since glycerol is a hydrophilic plasticizer, edible chitosan films with higher concentrations of glycerol adsorbed more moisture. Hence, these films swelled to a larger extent, and this led to an increase in the film thickness[50]. Compared to other films, Chitosan −MMT films showed the highest thickness value, and their thicknesses were increased significantly (p < 0.05) with the MMT concentration increasing. Similarly, there was a slight increase in CS-MMT-CuO nanocomposite film thickness when the alkali ion exchange time rose from 20 to 90 min. Therefore, the thicknesses of all samples were increased by adding any type of filler because a greater amount of filler was distributed compared with a fixed weight of the polymer. However, the CsGMMT samples were slightly thicker than samples with the same amount of MMT-CuO as filler. In general,

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

Fig. 3. FE-SEM micrographs of (a) Cs, (b) CsG, (c) CsG3MMT, (d) CsG3MMT-CuO-20 and (e) CsG3MMT-CuO-90 films.

1225

1226

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

incorporation of MMT-CuO to form covalent bonds with hydroxyl and amino groups of chitosan chains leads to a decrease in the availability of chitosan functional groups and consequently, limits the polysaccharide-water interactions by hydrogen bonding. This results in a reduction of moisture content and thickness of CsGMMT-CuO nanocomposite film compared to Cs-MMT films [51]. 3.5. Mechanical properties Packing materials should have good self-supporting properties to be resistant against handling damages. Hence, fillers, especially inorganic nanofillers, could be added to the biopolymers to enhance their mechanical properties such as stiffness and strength [52,53]. Tensile strength (TS) and elongation at break (EB) values of all films were shown in Table 1. The tensile strength and the elongation percentage of the neat chitosan film were 46.2 MPa and 8.2%, respectively. Also, tensile strength showed 55.6% decrease and flexibility of the films increased dramatically by the addition of glycerol into the CS matrix. Incorporation of a plasticizer in the polymer matrix increases the mobility of polymer chains by reducing intermolecular attraction that may lead to decrease in tensile strength but increases in elongation [3,54]. Similar trend reported in other literature [50,54,55]. However, some differences are evident between the reported data for TS and E in this study and other works that might be due to the type of chitosan (with different degree of deacetylation and molecular weight), glycerol weight percent, as well as film preparation method. In the present study, we used a sonicator to degas the final film solution which it might induce polymer’s chain scission, and the films were ripped easily [48,50]. The TS and E of CSG films increased with increasing MMT content up to 3 wt%, followed by a drop with further increase in MMT up to 5 wt%. A similar trend in the TS and E parameter has been observed with the MMT-CuO-20 and MMT-CuO-90 incorporation. The highest TS values for CSG3MMT, CSG3MMTCuO-20, and CSG3MMT-CuO-90 were 27.2, 29.4 and 32.5 MPa, respectively. Hence, the TS values were improved 46% and 59% by the only incorporation of 3% wt% of MMT-CuO-20, and MMT-CuO90, respectively, where revealed improvement compared with CSG blend. The improvement of TS could be ascribed to the uniform dispersion of nanofiller in the polymer matrix which might cause to decrease the stress concentration sites and also the formation of exfoliated structure. Moreover, these results are consistent with XRD analysis. 3.6. Water solubility Food packages barrier properties to gasses, vapors, and organic compounds are other critical parameters in packaging applications. Moisture and oxygen transition to internal or external packing’s environment causes to continuous change in shelf life, and food quality or even can cause spoilage. Hence, nanofillers usually are added to the polymer matrix to improve its barrier properties against gasses or moisture [53,56]. In this study, water resistance of chitosan film was indicated by measuring the water solubility (WS). As can be seen in Table 1, the neat chitosan film has a low WS about 13.4 ± 2.4% due to its firm backbone and highly crystallized structure. However, the WS of CS film increased from 13.4% to 19% by addition of 25 wt% glycerol into the CS matrix which this result could be ascribed to highly hydrophilic nature of glycerol. The effects of type and amount of nanofiller on the WS of the CS Film are also displayed in Table 1. The results showed that the water resistance of the film increased as the clay concentration increased where such a decrease is very substantial for films containing 5% w/w MMT [57]. On the other hand, CSG-MMT-CuO nanocomposites films showed more reduction in WS than CSGMMT film. As it was previously mentioned, alkaline ion exchange increases MMT-

Table 2 WVP and OP of chitosan- based films incorporated with MMT and MMT-CuO nanocomposite. Sample code

WVP × 10−10 (gr m−1 s−1 Pa−1 )

OP (cm3 ␮m m−2 day KPa)

Cs CsG CsG1MMT CsG3MMT CsG5MMT CsG1MMT-CuO-20 CsG3MMT-CuO-20 CsG5MMT-CuO-20 CsG1MMT-CuO-90 CsG3MMT-CuO-90 CsG5MMT-CuO-90

2.26 ± 0.09 4.52 ± 0.01 3.21 ± 0.05 2.82 ± 0.03 3.75 ± 0.04 3.32 ± 0.01 2.72 ± 0.03 2.90 ± 0.07 1.25 ± 0.09 1.01 ± 0.03 1.08 ± 0.06

7.5 ± 2.0 25.1 ± 6.5 21.9 ± 5.7 11.1 ± 5.2 15.7 ± 4.7 17.3 ± 1.2 9.9 ± 2.0 12.8 ± 1.0 13.1 ± 4.5 5.1 ± 2.0 11.2 ± 0.7

CuO interactions with polymer matrix significantly and therefore, reduces the number of hydrophilic groups existed in the chitosan. Consequently, this resulted compact structure would have more resistance against dissolving in water.

3.7. Water vapor permeability (WVP) Water vapor permeability is an important characteristic of a food packing film which utilizes to estimate its ability to reduce or prevent moisture transfer between both side of packing (food and surrounding environment)[58]. Some of factors such as chemical and structural properties of polymeric matrix, type and concentration of the additive and also degree of hydrophobic interaction in the film network have been taken into account on the WVP value [59]. Table 2 shows the effect of glycerol as a plasticizer, MMT and MMT-CuO nanocomposite on the barrier properties of chitosanbased films. The WVP value for chitosan film with no additive was 2.26 × 10−10 gr m−1 s−1 Pa−1 which increases to 4.52 × 1010 gr m−1 s−1 Pa−1 for chitosan film containing 25 wt% glycerol. Addition of hydrophilic plasticizer into the polymer matrix leads to increasing WVP of the film by reducing molecular interaction and increasing the molecular spaces in polymer chains[25]. The increase in WVP properties in edible films as a result of hydrophilic plasticizer was reported by other researchers [15,25,60].From Table 2, it is clear that WVP of CsG film decreases to 3.21 × 10-10, 2.82 × 10-10 and 3.75 × 10-10gr m-1 s-1 Pa-1 after addition 1, 3 and 5 wt% of MMT into film matrix, respectively. According pervious lectures, the presence of an ordered dispersed layered silicate with large aspect ratios into a polymer make a tortuous path which acts as an obstruction against water vapor transmission [19,28]. Also, after incorporation of MMT-CuO nanocomposites into chitosan matrix, the water permeability of plasticized films was significantly reduced, so that the film containing 3% MMT-CuO-90 shows the lowest WVP value compare than MMT and MMT-CuO-20 nanocomposite. This decrease in WVP values might be attributed to the increment hydrogen bonding interaction between CSG and MMTCu, which resulting in reduction the available hydroxyl groups of chitosan in CsG3MMT-CuO-90 film [35]. On other hand, in all chitosan-based nanocomposite films, the WVP of films containing 5 wt% of nanofiller is greater than of films with 3 wt% nanofiller because of the influence of clay on WVP depends on the amounts of nanoclay used and type of composites formed (tactoids, intercalation or exfoliation) [19]. In the present study, it is possible that MMT and MMT-CuO nanocomposite were aggregated in chitosan films which facilitate water vapor permeation through the matrix. These results have good agreement with mechanical and WS analyses.

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

3.8. Oxygen permeability (OP) Oxygen transmission through a packaging materials is one of major characteristics that indicates the oxidation of food which afterwards cusses changing to odor, flavor, color, and nutrient deterioration. A nanocomposite film with proper oxygen barrier property can be a good vehicle for extending food self-life and food quality improvement [58]. Oxygen permeability (OP) values of chitosan based nanocomposite films with various loads of MMT and MMT-CuO nanocomposite as nanofiller are presented in Table 2. As can be seen from this table, Cs film showed OP 7.5 ± 2.0 cm3 ␮m m−2 day−1 KPa−1 , while CsG film displayed OP in the range of 25.1 ± 6.5 cm3 ␮m m−2 day−1 KPa−1 . These results indicate that the addition of glycerol into chitosan film lead to promotion oxygen barrier property. Similar to the WVP results, glycerol increased the chitosan polymer chains mobility that makes oxygen molecules transformation through the film easier [61]. CsG films with 3 wt% MMT-CuO showed excellent oxygen barrier properties. According to XRD and TS analysis, CsG3MMT-CuO films formed intercalated composites which may be due to, chitosan functional groups make an strong inter-molecular attraction with MMT-CuO nanocomposite thus prevents gas from permeating through the nanocomposite film. In all chitosan- based nanocomposite OP was greater than CS film which related to higher weight fraction of plasticize compare to nanofiller. 3.9. Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) is often used to characterize different polymeric materials. Thermal properties of polymers and their composites or blends provide valuable information regarding stiffness, toughness, stability, and miscibility with other compounds [62]. The TGA thermograms, exhibit the weight decreasing pattern of the films during thermal decompositions and the DTG curves clearly show the maximum decomposition temperature at each step of thermal decomposition [40]. Fig. 4 displayed TGA and DTG of weight loss of chitosan-based nanocomposite film, with chitosan and chitosan-glycerol films as a control in the N2 atmosphere. As shown in the TGA thermograms, all chitosan-based films have three distinctive stages of thermal degradation. The first stage, in the temperature range 30–200 ◦ C, is mainly attributed to the evaporation absorbed and bound water and residue of acetic acid. The second one, at around 200–450 ◦ C, corresponds to the chemical degradation and deacetylation of chitosan, as well as decomposition of glycerol. The third step, in the temperature range 450–600◦C, could be due to the oxidative degradation of the carbonaceous residue formed during the second step [27]. Comparing the residual weight of the films exhibit CS film has the highest thermal stability and presence of other components, especially glycerol, decrease the thermal stability. In CSG film, it can be observed that the first step degradation temperature decreased about 20 ◦ C which backs to glycerol hydrophilic nature. However, the second step shows a slight increase (∼ = 5 ◦ C). The addition of MMT and MMT-CuO nanocomposite improved thermal stability and decreased the weight loss rather than CSG film. It seems, after pyrolysis, the nanocomposite forms char with a multilayered carbonaceous silicate structure, which may keep its multilayered structure in the polymer matrix even at 600 ◦ C. This high-performance carbonaceous-silicate char builds up on the surface during burning, thus insulating the underlying material and slowing the escape of the volatile products generated during decomposition. While glycerol has higher weight percentage (25 wt%) compared with nanofillers, the thermal stability of the composite films are lower than that of the Cs film. Also, as can be seen in Fig. 4, CsGMMT-CuO film nanocomposite has shown lower initial degradation temperature compared to the CsGMMT film. The

1227

decrease in the degradation temperature of the nanocomposites may be related to the catalytic properties of copper [63]. 3.10. Antibacterial activity The antibacterial activity of MMT-CuO nanocomposite was investigated against Gram-negative (E.coli, P. aeruginosa) and Gram- positive bacterias (B.cereus, S.aureus) with the colony count method (CFU), and results were presented in Table 2. As can be seen, the numbers of viable cells of both bacteria tested were decreased with respect to the control. Both nanocomposites showed approximately up to 99% mortality against both Grampositive bacteria after 24 h incubation time which this effective inhibitory is come to the presence of CuO in the MMT structure. In addition, MMT-CuO-20 nanocomposite have more antibacterial activity than MMT-CuO-90 against all bacteria tested which related to increasing the size of the CuO particles as result of longer ion exchange time. Applerot et. al [64] and Yalcinkaya and Lubasova [65] were found antibacterial activities of CuO NPs related to their size so that the small CuO NPs having the higher inhibiting or killing activity than big NPs. Both of MMT-CuO nanocomposites did not have the same effect on all microorganisms and showed the highest antibacterial activity against Gram-positive B.cereus bacterias. As mentioned in earlier literature, bacterial strain, nanoparticle type, and size, nature of growth media and initial cell concentration are some of the factors that affect on antibacterial activity of nanoparticle [66]. It was found that Gram-positive bacteria with thicker peptidoglycan layer and diverse negatively functional groups such as carboxylic, amino acids are the more susceptible inhibiting effect of copper ions or nanoparticles [66]. Table 3 shows chitosan- based films qualitative antibacterial activities against foodborne pathogenic bacteria using disc diffusion method. According to disc diffusion assay results, all chitosan and chitosan-based nanocomposite films show clear microbial inhibition zones against all four test bacterias, but no significant difference was seen between Gram-negative and Gram- positive bacteria inhibitory. As it mentioned in the literature, chitosan has antibacterial against a wide range of bacteria which is related to its polycationic nature [67]. While disc diffusion method is a qualitative method and antibacterial activity is only tested based inhibitory effects of the sample, using the quantitative CFU counting method, because of coinciding incorporation of adsorption and inhibition of bacterial cells properties is more appropriate. Hence, the antibacterial activities of the chitosan- based films were also tested based on the CFU counting method to confirm the results obtained by the disc diffusion method. A culture media without any sample was chosen as a control. The neat chitosan shows significant antibacterial activity against B.Cereus and S. Aureus compared to those against E.Coli and P. Aeruginosa. The presence of impermeable lipid in the surface of the Gram-negative bacterial outer membrane is the main reason to more resistance of Gram-negative bacteria than Gram- positive ones [68]. The antibacterial mechanism is related to electrostatic interactions between cationic amino groups (NH3+) in chitosan molecules and anionic teichoic acids backbone in Gram-positive cell wall cause to disruption of the cell functioning and cell death [67,69]. Also antibacterial activity of chitosan film against Gram-negative bacteria may be come to the interaction between ammonium groups of chitosan and anionic lipopolysaccharides of the Gram-negative cell that caused the cell wall destruction, leakage of intracellular components or inhibiting the transport of nutrients into the cells and finally leads to cell death [70]. Other chitosan-based films, including CsG and CSGMMT and CSG3MMT-CuO nanocomposite films, showed antimicrobial activity. As regards, CsG3MMT-CuO-90 nanocomposite film exhibited significantly lower levels of viable CFU in

1228

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

Fig. 4. a) TGA thermograms and b) DTG curves of chitosan- based films.

Table 3 Antimicrobial activity MMT-CuO nanocomposite and chitosan based film against S.aureus, B.cereus, E.coli; P. aeruginosa measured as inhibition zone and colony count. Sample code

Method/Unit

S. aureus

B.cereus

E.coli

P. aeruginosa

Control

Inhibition zone (mm) CFU/mL CFU/mL CFU/mL Inhibition zone (mm) CFU/mL Inhibition zone (mm) CFU/mL Inhibition zone (mm) CFU/mL Inhibition zone (mm) CFU/mL Inhibition zone (mm) CFU/mL

0 4.8 × 1012 1.8 × 1010 4.7 × 1010 8.10 3.9 × 104 10.12 300 1.8 × 1010 50 8.10 20 10.00 0

0 1.1 × 1014 1.2 × 106 4.20 × 106 7.8 800 5.7 440 1.5 × 107 2000 7.10 220 7.00 200

0 4.3 × 1014 1.5 × 107 4.20 × 109 10.12 1.1 × 1011 8.10 1.1 × 104 1.2 × 106 1.3 × 104 7.10 5.4 × 103 8.10 120

0 5.6 × 1013 1.0 × 109 2.40 × 1010 10.00 3.1 × 108 8.10 3.8 × 103 1.0 × 109 2.7 × 104 10.12 7 × 103 8.00 20

MMT-CuO-20 MMT-CuO 90 Cs CsG CsG3MMT CsG3MMT-CuO-20 CsG3MMT-CuO-90

both gram-positive and gram-negative pathogenic bacteria and almost all inoculated bacteria were killed.This intrinsic antibacterial action of the Cs2G3MMT-CuO-90 nanocomposite is related to higher adsorption capacity of this nanocomposite and presence

of CuO with intrinsic antibacterial activity [32]. The well-known mechanism for antibacterial properties of CuO NPs is adhesion to the cell membrane and also generating the reactive oxygen species (ROS) which will lead to increase in cell permeability and leakage of

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

1229

Table 4 A comparison of antimicrobial activity of different bio-nanocomposite with MMT-CuO on various bacteria. Biopolymer type

Test microorganism

Nanofiller content (%w/w)

Initial bacteria concentration (CFU/ml)

Final bacteria concentration (CFU/ml) or inhibition zone (mm)

Method

Reference

Chitosan

E.coli P. aeruginosa B.cereus S.aureus E. coli L. innocua E. coli E. coli L.monocytogenes B.cereus S.aureus E.coli P. aeruginosa

3

107

Colony forming units

Current Research

5

107

Colony forming units

[71]

5 5

5.34 × 10 105 –106 105 –106 107

120 20 200 0 15 2.9 × 104 1 × 109 8.5 22 10.12 6.1 10.12 8

Colony forming units Disc diffusion

[72] [33]

Disc diffusion

Current Research

Polylactic acid Cellulose acetate Gelatin Chitosan

3

8

cell content and, consequently reduction in adenosine triphosphate (ATP) levels. Hence, CuO NPs could transport to the cytoplasmic membrane and cause cell death [36,64,71] In addition, another possible mechanism for antibacterial activity of composites containing CuO could be due to leaching the Cu2+ ions. Copper ions can interact with the negatively charged surface of the bacterial cell and lead to protein denaturation and cell death. Moreover, Cu2+ ions uptake by the bacterial cells can damage some biochemical process, such as cross-linking within/between the nucleic acid strands due to binding to deoxyribonucleic acid molecules[72]. In our previous work [34], we showed that the amount of Cu ions release from CuO-MMT composite was well below the WHO standard[73] (our reasrech = 0.38 mg/L, WHO = 2 mg/L) that could be used for food packing. Antibacterial activity of Cu2+ MMT in other polymeric matrix reported by other researchers is listed in Table 4. It is clear that in our study, the higher antibacterial effect was seen against both groups of bacteria with a lower level of MMT-CuO nanocomposite (3% w/w) in polymer matrix. The presence of CuO in the interlayer sites, hexagonal and octahedral cavities increase electrostatic forces between bacteria negatively charged and chitosan film which destroys cell wall, leakage of intracellular components and microbial deaths [74–88]. These results demonstrated that CSGMMT-CuO film with intense antimicrobial activity is a good offer for the production of active food packing

4. Conclusion In this investigation, biodegradable active films based on chitosan polymer, glycerol and MMT-CuO were successfully prepared using a simple eco- friendly casting method. Based on our research, it can be concluded that incorporation of only 3% of MMT-CuO nanocomposite increased the antibacterial activity of chitosan film against both Gram-positive and Gram-negative bacteria, significantly. Also, other results showed tensile strength, elongation at break, water solubility, microstructure UV light and water barrier properties were enhanced compared with the control film. Thus, chitosan-MMT-CuO nanocomposite film could be a promising novel active food packing. However, further studies are required to identify the exact antibacterial mechanism of MMT-CuO nanocomposite in the polymer matrix.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ijbiomac.2017.11. 119.

References [1] S. Shankar, X. Teng, J.-W. Rhim, Properties and characterization of agar/CuNP bionanocomposite films prepared with different copper salts and reducing agents, Carbohydr. Polym. 114 (2014) 484–492. [2] A.R.V. Ferreira, C.A.V. Torres, F. Freitas, C. Sevrin, C. Grandfils, M.A.M. Reis, V.D. Alves, I.M. Coelhoso, Development and characterization of bilayer films of FucoPol and chitosan, Carbohydr. Polym. 147 (2016) 8–15. [3] N.R. Saha, G. Sarkar, I. Roy, A. Bhattacharyya, D. Rana, G. Dhanarajan, R. Banerjee, R. Sen, R. Mishra, D. Chattopadhyay, Nanocomposite films based on cellulose acetate/polyethylene glycol/modified montmorillonite as nontoxic active packaging material, RSC Adv. 6 (2016) 92569–92578. [4] S. Sahraee, J.M. Milani, B. Ghanbarzadeh, H. Hamishehkar, Effect of corn oil on physical, thermal, and antifungal properties of gelatin-based nanocomposite films containing nano chitin, LWT − Food Sci.Technol. 76 (Part A) (2017) 33–39. [5] B. Priya, V.K. Gupta, D. Pathania, A.S. Singha, Synthesis, characterization and antibacterial activity of biodegradable starch/PVA composite films reinforced with cellulosic fibre, Carbohydr. Polym. 109 (2014) 171–179. [6] J. Sundaram, J. Pant, M.J. Goudie, S. Mani, H. Handa, Antimicrobial and physicochemical characterization of biodegradable, nitric oxide-Releasing nanocellulose-Chitosan packaging membranes, J. Agric. Food Chem. 64 (2016) 5260–5266. [7] M.M. Reddy, S. Vivekanandhan, M. Misra, S.K. Bhatia, A.K. Mohanty, Biobased plastics and bionanocomposites: current status and future opportunities, Prog. Polym. Sci. 38 (2013) 1653–1689. [8] P.K. Dutta, S. Tripathi, G.K. Mehrotra, J. Dutta, Perspectives for chitosan based antimicrobial films in food applications, Food Chem. 114 (2009) 1173–1182. [9] A.I. Bourbon, A.C. Pinheiro, M.A. Cerqueira, C.M.R. Rocha, M.C. Avides, M.A.C. Quintas, A.A. Vicente, Physico-chemical characterization of chitosan-based edible films incorporating bioactive compounds of different molecular weight, J. Food Eng. 106 (2011) 111–118. [10] M.Z. Elsabee, E.S. Abdou, Chitosan based edible films and coatings: a review, Mater. Sci. Eng.: C 33 (2013) 1819–1841. [11] F. Kara, E.A. Aksoy, Z. Yuksekdag, S. Aksoy, N. Hasirci, Enhancement of antibacterial properties of polyurethanes by chitosan and heparin immobilization, Appl. Surf. Sci. 357 (2015) 1692–1702. [12] J. Cai, W. Ye, X. Wang, W. Lin, Q. Lin, Q. Zhang, F. Wu, Preparation of copper-chelate quaternized carboxymethyl chitosan/organic rectorite nanocomposites for algae inhibition, Carbohydr. Polym. 151 (2016) 130–134. [13] M. Shahbazi, G. Rajabzadeh, S.J. Ahmadi, Characterization of nanocomposite film based on chitosan intercalated in clay platelets by electron beam irradiation, Carbohydr. Polym. 157 (2017) 226–235. [14] D. Hu, H. Wang, L. Wang, Physical properties and antibacterial activity of quaternized chitosan/carboxymethyl cellulose blend films, LWT − Food Sci. Technol. 65 (2016) 398–405. [15] S. Ahmed, S. Ikram, Chitosan and gelatin based biodegradable packaging films with UV-light protection, Journal of photochemistry and photobiology. B, Biology 163 (2016) 115–124. [16] M.G.N. Campos, N. Satsangi, H.R. Rawls, L.H.I. Mei, Chitosan cross-Linked films for drug delivery application, Macromol. Symp. 279 (2009) 169–174. [17] S. Sanuja, A. Agalya, M.J. Umapathy, Synthesis and characterization of zinc oxide–neem oil–chitosan bionanocomposite for food packaging application, Int. J. Biol. Macromol. 74 (2015) 76–84. [18] E. Jahed, M.A. Khaledabad, H. Almasi, R. Hasanzadeh, Physicochemical properties of Carum copticum essential oil loaded chitosan films containing organic nanoreinforcements, Carbohydr. Polym. 164 (2017) 325–338. [19] A. Casariego, B.W.S. Souza, M.A. Cerqueira, J.A. Teixeira, L. Cruz, R. Díaz, A.A. Vicente, Chitosan/clay films’ properties as affected by biopolymer and clay micro/nanoparticles’ concentrations, Food Hydrocolloids 23 (2009) 1895–1902.

1230

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231

[20] A.M. Youssef, H. Abou-Yousef, S.M. El-Sayed, S. Kamel, Mechanical and antibacterial properties of novel high performance chitosan/nanocomposite films, Int. J. Biol. Macromol. 76 (2015) 25–32. [21] B.S. Rathore, G. Sharma, D. Pathania, V.K. Gupta, Synthesis, characterization and antibacterial activity of cellulose acetate-tin (IV) phosphate nanocomposite, Carbohydr. Polym. 103 (2014) 221–227. [22] A.S. Shekarabi, A.R. Oromiehie, A. Vaziri, M. Ardjmand, A.A. Safekordi, Investigation of the effect of nanoclay on the properties of quince seed mucilage edible films, Food Sci. Nutr. 2 (2014) 821–827. [23] C.N. Cheaburu-Yilmaz, O. Yilmaz, C. Vasile, Eco-Friendly chitosan-Based nanocomposites: chemistry and applications, in: V.K. Thakur, M.K. Thakur (Eds.), Eco-friendly Polymer Nanocomposites: Chemistry and Applications, Springer, India, New Delhi, 2015, pp. 341–386. [24] Y. Ling, Y. Luo, J. Luo, X. Wang, R. Sun, Novel antibacterial paper based on quaternized carboxymethyl chitosan/organic montmorillonite/Ag NP nanocomposites, Ind. Crops Prod. 51 (2013) 470–479. [25] A. Giannakas, K. Grigoriadi, A. Leontiou, N.-M. Barkoula, A. Ladavos, Preparation, characterization, mechanical and barrier properties investigation of chitosan?clay nanocomposites, Carbohydr. Polym. 108 (2014) 103–111. [26] Y. Xu, X. Ren, M.A. Hanna, Chitosan/clay nanocomposite film preparation and characterization, J. Appl. Polym. Sci. 99 (2006) 1684–1691. [27] M. Lavorgna, F. Piscitelli, P. Mangiacapra, G.G. Buonocore, Study of the combined effect of both clay and glycerol plasticizer on the properties of chitosan films, Carbohydr. Polym. 82 (2010) 291–298. [28] J.-W. Rhim, S.-I. Hong, H.-M. Park, P.K.W. Ng, Preparation and characterization of chitosan-Based nanocomposite films with antimicrobial activity, J. Agric. Food Chem. 54 (2006) 5814–5822. [29] J. Luo, M. Xie, X. Wang, Green fabrication of quaternized chitosan/rectorite/Ag NP nanocomposites with antimicrobial activity, Biomed. Mater. 9 (2014) 011001. [30] N. Ekthammathat, T. Thongtem, S. Thongtem, Antimicrobial activities of CuO films deposited on Cu foils by solution chemistry, Appl. Surf. Sci. 277 (2013) 211–217. [31] B. Liu, S. Shen, J. Luo, X. Wang, R. Sun, One-pot green synthesis and antimicrobial activity of exfoliated Ag NP-loaded quaternized chitosan/clay nanocomposites, RSC Adv. 3 (2013) 9714–9722. [32] J.F. Martucci, R.A. Ruseckaite, Antibacterial activity of gelatin/copper (II)-exchanged montmorillonite films, Food Hydrocolloids 64 (2017) 70–77. [33] J. Drelich, B. Li, P. Bowen, J.-Y. Hwang, O. Mills, D. Hoffman, Vermiculite decorated with copper nanoparticles: novel antibacterial hybrid material, Appl. Surf. Sci. 257 (2011) 9435–9443. [34] H. Pourabolghasem, M. Ghorbanpour, R. Shayegh, Antibacterial activity of copper-doped montmorillonite nanocomposites prepared by alkaline ion exchange method, J. Phys. Sci. 27 (2016) 1. [35] G. Cárdenas, J. Díaz V, M.F. Meléndrez, C. Cruzat C, A. García Cancino, Colloidal Cu nanoparticles/chitosan composite film obtained by microwave heating for food package applications, Polym. Bull. 62 (2009) 511–524. [36] D. Das, B.C. Nath, P. Phukon, S.K. Dolui, Synthesis and evaluation of antioxidant and antibacterial behavior of CuO nanoparticles, Colloids Surf. B: Biointerfaces 101 (2013) 430–433. [37] A. Ahmad, Y. Wei, F. Syed, K. Tahir, A.U. Rehman, A. Khan, S. Ullah, Q. Yuan, The effects of bacteria-nanoparticles interface on the antibacterial activity of green synthesized silver nanoparticles, Microb. Pathog. 102 (2017) 133–142. [38] Y. Cao, R. Zheng, X. Ji, H. Liu, R. Xie, W. Yang, Syntheses and characterization of nearly monodispersed, size-tunable silver nanoparticles over a wide size range of 7–200 nm by tannic acid reduction, Langmuir: ACS J. Surf. Colloids 30 (2014) 3876–3882. [39] P. Kanmani, J.-W. Rhim, Physical, mechanical and antimicrobial properties of gelatin based active nanocomposite films containing AgNPs and nanoclay, Food Hydrocolloids 35 (2014) 644–652. [40] J.-W. Rhim, L.-F. Wang, Preparation and characterization of carrageenan-based nanocomposite films reinforced with clay mineral and silver nanoparticles, Appl. Clay Sci. 97–98 (2014) 174–181. [41] M. Nasrollahzadeh, S.M. Sajadi, A. Rostami-Vartooni, S.M. Hussin, Green synthesis of CuO nanoparticles using aqueous extract of Thymus vulgaris L. leaves and their catalytic performance for N-arylation of indoles and amines, J. Colloid Interface Sci. 466 (2016) 113–119. [42] S. Sohrabnezhad, M.J. Mehdipour, Moghaddam, T. Salavatiyan, Synthesis and characterization of CuO–montmorillonite nanocomposite by thermal decomposition method and antibacterial activity of nanocomposite, Spectrochim. Acta A Mol. Biomol. Spectrosc. 125 (2014) 73–78. [43] Z. Ding, R.L. Frost, Study of copper adsorption on montmorillonites using thermal analysis methods, J. Colloid Interface Sci. 269 (2004) 296–302. [44] Y. Sarikaya, M. Önal, B. Baran, T. Alemdaro˘glu, The effect of thermal treatment on some of the physicochemical properties of a bentonite, Clays Clay Miner. 48 (2000) 557–562. [45] C. Mosser, L. Michot, F. Villieras, M. Romeo, Migration of cations in copper (II)-exchanged montmorillonite and laponite upon heating, Clays Clay Miner. 45 (1997) 789–802. [46] S. Shojaee-Aliabadi, H. Hosseini, M.A. Mohammadifar, A. Mohammadi, M. Ghasemlou, S.M. Ojagh, S.M. Hosseini, R. Khaksar, Characterization of antioxidant-antimicrobial ␬-carrageenan films containing Satureja hortensis essential oil, Int. J. Biol. Macromol. 52 (2013) 116–124. [47] S.F. Wang, L. Shen, Y.J. Tong, L. Chen, I.Y. Phang, P.Q. Lim, T.X. Liu, Biopolymer chitosan/montmorillonite nanocomposites: preparation and characterization, Polym. Degrad. Stab. 90 (2005) 123–131.

[48] K. Grigoriadi, A. Giannakas, A.K. Ladavos, N.-M. Barkoula, Interplay between processing and performance in chitosan-based clay nanocomposite films, Polym. Bull. 72 (2015) 1145–1161. [49] K. Shameli, M.B. Ahmad, M. Zargar, W.M.Z.W. Yunus, A. Rustaiyan, N.A. Ibrahim, Synthesis of silver nanoparticles in montmorillonite and their antibacterial behavior, Int. J. Nanomed. 6 (2011) 581–590. [50] W. Thakhiew, S. Devahastin, S. Soponronnarit, Effects of drying methods and plasticizer concentration on some physical and mechanical properties of edible chitosan films, J. Food Eng. 99 (2010) 216–224. [51] S.I. Park, Y. Zhao, Incorporation of a high concentration of mineral or vitamin into chitosan-based films, J. Agric. Food Chem. 52 (2004) 1933–1939. [52] M.Z. Elsabee, E.S. Abdou, Chitosan based edible films and coatings: a review, Mater. Sci. Eng. C Mater. Biol. Appl. 33 (2013) 1819–1841. [53] A.M. Díez-Pascual, A.L. Díez-Vicente, ZnO-Reinforced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) bionanocomposites with antimicrobial function for food packaging, ACS Appl. Mater. Interfaces 6 (2014) 9822–9834. [54] K. Ziani, J. Oses, V. Coma, J.I. Maté, Effect of the presence of glycerol and Tween 20 on the chemical and physical properties of films based on chitosan with different degree of deacetylation, LWT − Food Sci.Technol. 41 (2008) 2159–2165. [55] N.E. Suyatma, L. Tighzert, A. Copinet, V. Coma, Effects of hydrophilic plasticizers on mechanical, thermal, and surface properties of chitosan films, J. Agric. Food Chem. 53 (2005) 3950–3957. [56] T. Bourtoom, M.S. Chinnan, Preparation and properties of rice starch–chitosan blend biodegradable film, LWT – Food Sci. Technol. 41 (2008) 1633–1641. [57] M. Alboofetileh, M. Rezaei, H. Hosseini, M. Abdollahi, Effect of montmorillonite clay and biopolymer concentration on the physical and mechanical properties of alginate nanocomposite films, J. Food Eng. 117 (2013) 26–33. [58] B. Soni, B. Hassan el, M.W. Schilling, B. Mahmoud, Transparent bionanocomposite films based on chitosan and TEMPO-oxidized cellulose nanofibers with enhanced mechanical and barrier properties, Carbohydr. Polym. 151 (2016) 779–789. [59] Y. Shahbazi, The properties of chitosan and gelatin films incorporated with ethanolic red grape seed extract and Ziziphora clinopodioides essential oil as biodegradable materials for active food packaging, Int. J. Biol. Macromol. 99 (2017) 746–753. [60] A. Farhan, N.M. Hani, Characterization of edible packaging films based on semi-refined kappa-carrageenan plasticized with glycerol and sorbitol, Food Hydrocolloids 64 (2017) 48–58. [61] I. Leceta, P. Guerrero, K. de la Caba, Functional properties of chitosan-based films, Carbohydr. Polym. 93 (2013) 339–346. [62] K. Lewandowska, A. Sionkowska, B. Kaczmarek, G. Furtos, Characterization of chitosan composites with various clays, Int. J. Biol. Macromol. 65 (2014) 534–541. [63] S. Mallakpour, A. Jarahiyan, Utilization of ultrasonic irradiation as a green and effective strategy to prepare poly(N-vinyl-2-pyrrolidone)/modified nano-copper (II) oxide nanocomposites, Ultrason. Sonochem. 37 (2017) 128–135. [64] G. Applerot, J. Lellouche, A. Lipovsky, Y. Nitzan, R. Lubart, A. Gedanken, E. Banin, Understanding the antibacterial mechanism of CuO nanoparticles: revealing the route of induced oxidative stress, Small 8 (2012) 3326–3337. [65] F. Yalcinkaya, D. Lubasova, Quantitative evaluation of antibacterial activities of nanoparticles (ZnO, TiO2, ZnO/TiO2, SnO2, CuO, ZrO2, and AgNO3) incorporated into polyvinyl butyral nanofibers, Polym. Adv. Technol. 28 (2017) 137–140. [66] B. Bagchi, S. Kar, S.K. Dey, S. Bhandary, D. Roy, T.K. Mukhopadhyay, S. Das, P. Nandy, In situ synthesis and antibacterial activity of copper nanoparticle loaded natural montmorillonite clay based on contact inhibition and ion release, Colloids and surfaces. B, Biointerfaces 108 (2013) 358–365. [67] A. Verlee, S. Mincke, C.V. Stevens, Recent developments in antibacterial and antifungal chitosan and its derivatives, Carbohydr. Polym. 164 (2017) 268–283. [68] Z. Shariatinia, M. Fazli, Mechanical properties and antibacterial activities of novel nanobiocomposite films of chitosan and starch, Food Hydrocolloids 46 (2015) 112–124. [69] Z. Kalaycıo˘glu, E. Torlak, G. Akın-Evingür, I˙ . Özen, F.B. Erim, Antimicrobial and physical properties of chitosan films incorporated with turmeric extract, Int. J. Biol. Macromol. 101 (2017) 882–888. [70] S.C.M. Fernandes, P. Sadocco, J. Causio, A.J.D. Silvestre, I. Mondragon, C.S.R. Freire, Antimicrobial pullulan derivative prepared by grafting with 3-aminopropyltrimethoxysilane: characterization and ability to form transparent films, Food Hydrocolloids 35 (2014) 247–252. [71] M. Abdollahi, M. Rezaei, G. Farzi, Improvement of active chitosan film properties with rosemary essential oil for food packaging, Int. J. Food Sci. Technol. 47 (2012) 847–853. [72] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, A. Memic, Size-dependent antimicrobial properties of CuO nanoparticles against Gram-positive and −negative bacterial strains, Int. J. Nanomed. 7 (2012) 3527–3535. [73] D.J. Fitzgerald, Safety guidelines for copper in water, Am. J. Clin. Nutr. 67 (1998) 1098S–1102S. [74] Y. Zhou, M. Xia, Y. Ye, C. Hu, Antimicrobial ability of Cu2+-montmorillonite, Appl. Clay Sci. 27 (2004) 215–218.

A. Nouri et al. / International Journal of Biological Macromolecules 109 (2018) 1219–1231 [75] V.K. Gupta, S. Kumar, R. Singh, L.P. Singh, S.K. Shoora, B. Sethi, Cadmium (II) ion Sensing through p-tert-butyl calix[6]arene based potentiometric sensor, J. Mol. Liq. 195 (2014) 65–68. [76] S. Karthikeyan, V.K. Gupta, R. Boopathy, A. Titus, G. Sekaran, A new approach for the degradation of aniline by mesoporous activated carbon as a heterogeneous catalyst: kinetic and spectroscopic studies, J. Mol. Liquids 173 (2012) 153–163. [77] V.K. Gupta, A.K. Singh, L.K. Kumawat, Thiazole Schiff base Turn-On Fluorescent Chemosensor for Al3+ Ion, Sens. Actuators: B Chem. 195 (2014) 98–108. [78] T.A. Saleh, V.K. Gupta, Synthesis and characterization of alumina nano-particles polyamide membrane with enhanced flux rejection performance, Sep. Purf. Technol. 89 (2012) 245–251. [79] N. Mohammadi, H. Khani, Shilpi Agarwal, V.K. Gupta, Adsorption process of methyl orange dye onto mesoporous carbon material-kinetic and thermodynamic studies, J. Colloids Interface Sci. (2011) 457–462. [80] V.K. Gupta, B. Sethi, R.A. Sharma, Shilpi Agarwal, Arvind Bharti, Mercury selective potentiometric sensor based on low rim functionalized thiacalix [4] arene as a cationic receptor, J. Mol. Liq. 177 (2013) 114–118. [81] T.A. Saleh, V.K. Gupta, Processing methods and characteristics of porous carbons derived from waste rubbertires: a review, Adv. Colloid Interface Sci. 211 (2014) 92–100. [82] A. Tawfik Saleh, Shilpi Agarwal, V.K. Gupta, Synthesis of MWCNT/MnO2 Composites and their application for simultaneous oxidation of arsenite and sorption of arsenate, Appl. Catal. B: Env. 106 (2011) 46–53.

1231

[83] V.K. Gupta, A. Nayak, S. Agarwal, Bioadsorbents for remediation of heavy metals: Current status and their future prospects, Environ. Eng. Res. 20 (1) (2015) 001–018. [84] V.K. Gupta, M.R. Ganjali, P. Norouzi, H. Khani, A. Nayak, Shilpi Agarwal, Electrochemical analysis of some toxic metals and drugs by ion selective electrodes, Crit. Rev. Anal. Chem. 41 (2011) 282–313. [85] V.K. Gupta, Necip Atar, M.L. Yola, Zafer Üstünda˘g, Lokman Uzun, A novel magnetic [email protected] core-shell nanoparticles anchored graphene oxide recyclable nanocatalyst for the reduction of nitrophenol compounds, Water Res. 48 (2014) 210–217. [86] N. Mohammadi, H. Khani, Shilpi Agarwal, V.K. Gupta, Adsorption process of methyl orange dye onto mesoporous carbon material- kinetic and thermodynamic studies, J. Colloids Interface Sci. 362 (2011) 457–462. [87] M. Ahmaruzzaman, V.K. Gupta, Rice husk and its ash as low-cost adsorbents in water and wastewater treatment, Ind. Eng. Chem. Res. 50 (2011) 13589–13613. [88] Arash Asfaram, Mehrorang Ghaedi, Shilpi Agarwal, Inderjeet Tyagi, V.K. Gupta, Adsorption of basic dye Auramine-O by ZnS: Cu nanoparticles loaded on activated carbon using response surface methodology with central composite design, RSC Adv. 5 (2015) 18438–18450.