Cu nanocomposite food packaging film for extended shelf life of peda

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Development of antimicrobial LDPE/Cu nanocomposite food packaging film for extended shelf life of peda Gayatri B. Lomate, Bhagyashri Dandi, Satyendra Mishra

T

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University Institute of Chemical Technology, North Maharashtra University, Jalgaon, 425001, Maharashtra, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Copper nanoparticles Microwave synthesis Antimicrobial food packaging film Improved shelf life

Low density polyethylene /copper nanocomposites with strong antimicrobial activity and barrier properties were successfully prepared for active food packaging which showed of extended shelf life of Peda (Indian sweet dairy product). Copper nanoparticles (Cu-NPs) were incorporated from 0.5 wt.% to 3.0 wt.% into a low-density polyethylene (LDPE) matrix by using lab scale doctor blade film applicator to prepare 120 μm thin food packaging films. The effect of different weight percent loadings of Cu NPs on to morphology, mechanical properties of LDPE/Cu nanocomposite films was examined. It was found that Cu-NPS uniformly dispersed into a LDPE matrix and provided improved mechanical properties. There was increment in mechanical properties with decrement in water vapour permeability (WVP) with increase in Cu-NPs amount. LDPE/Cu nanocomposites film also shows superior antimicrobial effect averse to gram +ve and gram -ve food deteriorating microorganisms.

1. Introduction Over period, consumers are demanding for process and packed food product with extended shelf life. Packaging of food is the final step in food processing industry. With use of food preservation techniques, finished product can be stored for period however the chance of contamination persists. The use of antimicrobial food packaging films can have a significant impact to minimise recontamination and increase in shelf life (Duncan, 2011). The antimicrobial activity of such films can be achieved by incorporating antimicrobial agents in polymer matrix (Azeredo & Henriette, 2009). Inorganic antimicrobial agents like metal and metal oxides are advantageous over organic agents such as organic acids, bacteriocins, enzymes and spices extract etc since they cannot withstand the harsh processing conditions of polymer films (Othman, 2014). Petrochemical based plastics have been accepted by the food industry since of their obtainability in extents at low price and reason of favourable functional characteristics. These materials are not only flexible and compatible with food stuffs, but are likewise safe, transparent, inexpensive and versatile (Siracusa, Rocculi, Romani, & Dalla Rosa, 2008). Compare to other packaging materials, polymeric films are used in food packaging due to their low cost and valuable properties (Akelah, 2013). To maintain freshness and sensory quality of food, it is important that moisture may not permeate in plastic packaging materials during storage. The presence of moisture in food package provides favourable conditions for microorganisms to grow on food surface.

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Considering all facts, it appears that the suitable material for packaging applications should have sound mechanical properties, high permeability and antibacterial properties. These requirements could be fulfil by embedding appropriate inorganic nanoparticles in a polymeric matrix. (Quintavalla and Vicini, 2002). Application of nanotechnology in packaging film offers a new term nanocomposite (Camargo, Satyanarayana, & Wypych, 2009). Arora & Padua (2010), and Siracusa (2012) reported that nano fillers reinforced polymer films show enhanced barrier properties of nanocomposite food packaging films. The mechanical properties of film can be upgraded with the increasing concentration of nanoparticles in nanocomposites (Grigoriadou et al., 2013; Mishra & Shimpi, 2006; Shimpi, Mali, et al., 2014, Shimpi, Sonawane, et al., 2014, Mishra, Sonawane, Singh, Bendale, & Patil, 2004; Shimpi, Verma, & Mishra, 2010; Sonawane, Mishra, & Shimpi, 2009) Included in various antimicrobial agents, copper has been known for a long time. Studies on antimicrobial mechanism of Cu-NPs are reported in literature (Caroling, Vinodhini, Ranjitham, & Shanthi, 2015; Chatterjee et al., 2012; Grass, Rensing, & Solioz, 2011; Zain, Stapley, & Shama, 2014) Mechanism of inhibition of growth of microorganisms by Cu-NPs was basically depending on particle size and concentration of nanoparticles. By passaging bacterial cell membrane and then destructing their vital enzymes, Cu-NPs show their efficacy against gram +ve and gram −ve bacteria, high stability and antifungal activity (Vincent, Hartemann, & Engels-Deutsch, 2016). Due to rapid oxidation of Cu° on favourable condition the synthesis of Cu-NPs is very

Corresponding author. E-mail addresses: [email protected] (G.B. Lomate), [email protected] (B. Dandi), [email protected] (S. Mishra).

https://doi.org/10.1016/j.fpsl.2018.04.001 Received 28 September 2017; Received in revised form 30 January 2018; Accepted 3 April 2018 2214-2894/ © 2018 Elsevier Ltd. All rights reserved.

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spectrophotometer (agilent technologies, India) in the range of 200 nm to 800 nm. To observe optical properties of Cu-NPs.

challenging. Cu-NPs have been produced by different methods viz., micro-emulsion/reverse micelles (Yallappa et al., 2013), laser irradiation (Longano et al., 2012), thermal decomposition (BetancourtGalindo et al., 2014), chemical reduction (Liu, ZHOU, Yamamoto, Ichino, & Okido, 2012), etc. and many more methods are available in literature. Microwave heating technique is useful for the synthesis of organic compounds, polymers, inorganic materials, and nanomaterials which were introduced in 21st century. By tuning microwave parameters and choice of solvent, nanomaterials can be synthesized using novel green approach of microwave assisted method, which has several advantages over the other like eco-friendly, very short reaction times, mild temperature conditions, one-step synthetic route and low cost are the prime ones (Gawande, Shelke, Zboril, & Varma, 2014). The focus of study is to develop active food packaging film which fulfils demand for good food packaging film. In the present work, LDPE/Cu nanocomposites were prepared for different concentrations of Cu-NPs. With the help of Cu-NPs dual propose could be achieved to act as effective antimicrobial agent and with increased mechanical as well as barrier properties. The performance of the film was examined on Peda which is having limited shelf life for 24 h only.

2.4.2. X-ray diffraction (XRD) For the determination of crystalline structure of Cu-NPs and LDPE/ Cu nanocomposite film, the XRD monochromatic Cu Ka radiation (l 1/4 1.5406 A) at 40 kV° and 40 mA (Model D8 Advance Bruker Limited Germany) was used. The optimization of diffraction patterns was done in an angular range of 5–80 (2θ) with scanning speed of 1° S−1. 2.4.3. Field-emission scanning electron microscopy (FE-SEM) & energy dispersive spectroscopy(EDS) The FE-SEM images of the Cu-NPs and nanocomposites films were recorded on FE-SEM S-4800 Type II Hitachi High Technology Corporation Limited, JAPAN. It was operated at 15.0 KeV. The elemental compositions of the samples were determined by EDS. 2.4.4. Particle size analysis Cu-NPs were dispersed in distilled water to find the particle size using particle size analyzer (Malvern, ZS- 200, Worcestershire UK).

2. Materials and methods

2.4.5. Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of gelatin capped Cu-NPs were recorded on 8400 Shimadzu, Japan FTIR, in the frequency range 400–4000 cm−1 with a resolution of 4 cm−1.

2.1. Materials CuSO4 (Coper sulphate dehaydrated) (Loba Chemical, India), C6H8O6 (Ascorbic acid) (Merk, India), Gelatin (Himedia, India), Low density polyethylene (LDPE) was purchased from (reliance industries), Xylene GR (Merk, India).

2.4.6. Thermal properties Shimadzu TGA 50, Japan Thermogravimetric analyzer and Shimadzu DSC 60, Japan were used to study thermal properties of LDPE/Cu nanocomposite films by following the details given elsewhere (Shimpi, Shirole, & Mishra, 2015, Shimpi et al., 2014a, b)

2.2. Synthesis of Cu-NPs Highly pure ∼50 nm size Cu-NPs were synthesized by microwave method. For the synthesis process 10 mL of 0.1 M CuSO4, 50 mL 1% gelatin solution and 5 mL 0.1 M ascorbic acid were mixed. Colour change is seen at first blue to green on room temperature during mechanical stirring. The contents were then heated to boil in a domestic microwave oven at full power for about 30 s on- off mode of two to three cycles. Green colour of solution changed to yellow, orange and finally wine red coloured Cu-NPs. After centrifugation at 10,000 rpm for 10 min the Cu-NPs were collected in wet form and then vacuum dried at 50 °C for 24 h.

2.4.7. Mechanical properties Universal testing machine (UTM 2302) supplied by, Hi-Tech machines, Mumbai (India) was used for determination mechanical properties of LDPE/Cu nanocomposite films for testing 2 cm × 5 cm samples strips were cut and tested were carried out 5 times for each sample. Also given in (Shimpi et al., 2015). 2.4.8. Cu+ ion release study SL176 double beam atomic absorption spectrometry supplied by Elico science and laboratories. (India) was used to study the quantity of Cu+ ions released from the LDPE/Cu nanocomposites, three rectangular strips of 2 cm × 2 cm and thickness 120 μ samples were taken and immersed in 15 mL of distilled water for 24 h. This experiment was carried out for 30 days (Shameli et al., 2010).

2.3. Preparation of LDPE/Cu nanocomposite film LDPE/Cu nanocomposites containing 0.5, 1, 1.5, 2, 2.5 and 3 wt. % were prepared by using solvent evaporation and thin film of uniform thickness were prepared by doctor blade film applicator. To prepare films, in 45 m l of xylene, 5 g of LDPE polymer was dissolved at a constant temperature 110 °C using oil bath; Prior, Cu-NPs were dispersed in LDPE/ xylene solution by temperature controlled ultrasonic bath sonicator to achieve proper dispersion. Later, composite films were fabricated on glass plates using doctor blade film applicator (120 microns) at 100 °C. LDPE is high crystalline polymer, which get precipitated if temperature of xylene becomes lower than 150 ( ± 5) °C. To fabricate a film, glass plate temperature was maintained at 150 °C (Lock, Walton, & Fernsler, 2008). The prepared films were peeled off from glass plates. The Gas Chromatograms (Figs. S1–S5) and FTIR (Fig. S6) were recorded to confirm the residual concentration of xylene in film (Seo & Shin, 2010; Garcia-Turiel & Jérôme, 2007). The results do not show the presence of xylene in the film.

2.4.9. Water vapour permeability (WVP) Desiccant method or dry cup setup was in accordance with standard ASTM 96 used. Stainless steel permeability cups with an outside diameter of 62 mm and a height of 38 mm (including clamps) and an inner cup diameter of 35 mm were used. Permeability cup was half-filled with 3 g of anhydrous calcium chloride. LDPE/Cu nanocomposite film was placed over the cup and a 2-mm thick Teflon gasket was placed over the film. The top metal ring was then placed on top of the teflon gasket and secured by three screw retained clamps to ensure a tight seal. The permeability cups were placed in the desiccator filled with water. The relative humidity was 95% during these experiments. The weight gain of cup was measured to the nearest 0.0001 mg by using a precision analytical balance after 24 and 48 h. Three samples for each treatment were tested. The WVP of film was calculated by Eq. (1) reported by Rhim, Mohanty, Singh, and Ng (2006) and Kadam et al. (2017) as given below.

2.4. Analysis of Cu-NPs and LDPE/Cu nanocomposite film

WVP = (WVTR × L)/ΔP

2.4.1. Ultraviolet–visible spectroscopy (UV–vis) The UV–vis spectra of Cu-NPs was performed on Cary 60 UV–vis

where, 212

(1)

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stretching vibration of CeN group. The presence of amide group peaks in the spectra indicates the capping of gelatin on Cu-NPs, which have also been reported by Shankar, Teng, Li, and Rhim (2015) and Patel et al. (2013). The peaks at 628 cm−1 and 441 cm−1 attributed to CuNPs.

L is thickness (mm) ΔP was the partial water vapour pressure difference (MPa) to the other two sides of the film. 2.4.10. Antimicrobial study The antimicrobial properties of LDPE/Cu nanocomposite determined by an agar disk diffusion bioassay against Gram −ve bacteria, Escherichia coli (E. coli), and Gram +ve, Staphylococcus aureus (S. aureus). The bacterial suspension (100 μl of 102–104 CFU m/1) was applied homogeneously on the surface of a nutrient agar plate. Then the disk was placed on agar surface (with control) and kept in refrigerator for 2 h for diffusion of disks. The plates were incubated at 37 °C for 24 h. After incubation, the zone of inhibition was measured (Awad et al., 2015). The experiment was carried out in triplicate.

3.4. X-ray diffraction (XRD) To ensure formation of Cu-NPs, XRD was accomplished on powder samples. Pure LDPE, Cu-NPs and its nanocomposites with varying CuNPs are shown in Fig. 2. The characterized peaks at 43º, 50º and 73º angles corresponding to (111), (200) and (220) planes of FCC crystal structure of copper nanoparticles (Dang et al., 2011). There is no identification copper oxide peak which showing oxidations of Cu-NPs. These results are found in a close relation with the work reported by Vaseem, Lee, Kim, and Hahn (2011), conform the formation of pure CuNPs. Moreover, peaks approximately at 2-θ = 20º, 23º and 35º respectively instructive the crystalline structure of LDPE. This Fig. 5 signalize the crystalline structure of LDPE/Cu nanocomposite does not alter after adding together Cu-NPs. However, the characteristic peaks become narrowed and their severity increased with Cu-NPs loadings, which indicates an enhancer of crystallinity of LDPE/Cu nanocomposites. Most remarkable features are experienced in the all characteristic peaks of Cu-NPs present in all wt. % nanocomposite films, which give indication of Cu-NPs dispersion.

2.4.11. Application of developed antimicrobial film as packaging material Peda was purchased from local market and 1 g of sample was packed in the LDPE/Cu nanocomposite films with concentration of 0.5 to 3.0 wt.% of Cu in triplicate. After packaging, the samples were kept at room temperature and analysed after an interval of 48 h. Total viable count (TVC) was performed to detect the colony forming unit CFU by pour plate technique. the plates inoculated with various concentrations, then kept for incubation for 24 h at 37 °C. The untreated films were used as control. Individual samples were taken for determination of CFU after 48 h for the periods of 8 days (Nithya, Murthy, & Halami, 2013).

3.5. Surface morphology and size of Cu-NPs 3. Results and discussion The surface images of Cu (gelatin capped and uncapped) and LDPE/ Cu nanocomposites films for various wt. % of Cu-NPs and pure LDPE is showing in Fig. 3 respectively. The uncapped Cu-NPs were spherical as shown in Fig. 3A with average diameter 120 nm as shown in Fig. 3C. The gelatin capped Cu-NPs (Fig. 3B) were not perfectly spherical but were to be encapsulated in matrix of biopolymer (gelatin). Fig. 3D displays narrow size distribution with average size 50 nm. The drop-in size of gelatin capped Cu-NPs compared to uncapped Cu-NPs indicated that gelatin behaves as a capping agent. EDS spectrum was shown in Fig. 3E, which give evidence of Cu, put forward that the obtained product is composed of pure Cu. Result clearly show 99.15% of copper and only 0.85% of oxygen. The proportions of dispersion of Cu-NPs dispersed in LDPE was observed by FE-SEM. Fig. 3F shows FE-SEM images captured of the nanocomposite films. It is clear from the images that with increase in percentage loading of Cu-NPs, more number of equally dispersed nanoparticles are observed in the nanocomposite films; but up to lower concentration of 2.5 wt.%. At higher concentration (3 wt. %), the nanoparticles were observed in aggregated forms (Fig. 3F).

3.1. Formation of gelatin capped Cu-NPs Ascorbic acid acts as antioxidant agent, which protects Cu-Nps from oxidation as well as it also acts as a reducing agent (Musa, Ahmad, Hussein, Saiman, & Sani, 2016). To control agglomeration and size of Cu-NPs gelatin biopolymer was used as capping agent (Blosi, Albonetti, Dondi, Martelli, & Baldi, 2011) When CuSO4 solution was mixed with gelatin solution it forms Cu2+/gelatin complex by in situ polymerization and it forms a layer or capping over Cu2+ ions. The complex was further reduced by ascorbic acid and microwave heat to finally get gelatin capped Cu-NPs. CuSO4 + Gelatin = (Gelatin + Cu2+) SO4

(2)

2[Gelatin + Cu2+ SO4] + C6H8O6 = 2 [Gelatin + Cu-NP] + 2 SO2 + 4 H2O + 6 CO (3) 3.2. Optical properties Cu-NPsSurface Plasmon resonance (SPR) is a very important characteristic to study size dependant phenomenon of metal nanoparticles. The UV–vis spectra were recorded of the samples collected immediate after reaction for period of every 30 s of microwave heating till colour changes at 1st cycle yellow, at 2nd orange and eventually brick red indicated emergence of Cu-NPs formation (As shown in Fig. 1A). The absorption bands for Cu-NPs obtained in the range of 550–600 nm was found to be in very good agreement of formation of Cu-NPs (Khanna, More, Jawalkar, Patil, & Rao, 2009; Yallappa et al., 2013).

3.6. Thermal analysis The thermal properties of the LDPE/Cu nanocomposite film were determined by DSC and TGA. In Fig. 4A, DSC curves of LDPE/Cu nanocomposite films are displayed. This study gives proof for filler compatibility on the thermal behaviour of LDPE/Cu nanocomposite film. The melting temperature of pure LDPE film was recorded as 124.34 °C where for 0.5, 1, 1.5, 2, 2.5 and 3 wt.% loading of Cu-NPs, the melting temperatures were 124.59, 124.87, 125.10, 125.31, 126.46 and 127.31 °C respectively. Up to 2 wt.% of Cu-NPs loading, there was no drastic change in melting temperature. There was modest addition in melting temperature of nanocomposite film from 2.5 to 3 wt.% loading of Cu-NPs because copper nanoparticles influence movement of polymer chain of LDPE matrix. There is increase in melting peak temperature as increase in Cu-NPs in LDPE/Cu nanocomposite film. Increment in melting point can be due to behaviour of copper nanoparticles obstacles for motion of LDPE polymer chain segments. Fig. 4B shows TGA thermograms of LDPE/Cu nanocomposite films.

3.3. Fourier transform infrared spectroscopy (FTIR) The FTIR spectrum of gelatin capped Cu-NPs is shown in Fig.1B. The peak at 1631 cm−1 corresponds to the amide I which was attached with COO group with C]O stretching/hydrogen bounding. The peak at 1421 cm-1 gives proof of presences of amide-II group come to light from NeH and stretching vibration of CeN group. Presences of Amide III group identified by 1190 cm−1 peak links to CeN and NeH groups and 213

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Fig. 1. (A) UV–vis absorption spectra of gelatin capped CuNPs with number of microwave heating cycles; (B) FTIR of gelatin capped CuNPs.

also more, which increases surface energy of this film ultimately lowering the thermal decomposition temperature of film (Bari, Lanjewar, Hansora, & Mishra, 2016). 3.7. Mechanical properties Influence of Cu-NPs loading on mechanical properties of the LDPE/ Cu nanocomposites is presented in Fig. 5. The tensile strength results, compared to pure LDPE and LDPE/Cu nanocomposites, which were recorded as 9, 11.5, 13.5,15, 16.5 and 16 MPa for 0.5,1, 1.5,2, 2.5 and 3 wt.% respectively, and 6.91 MPa for pure LDPE film. Upgrade in mechanical properties of nanocomposites are due to two factors, consistent dispersion of nanofiller and secondly the intersection tip between nanoparticles and a polymer matrix can carry tension, which is complementary for the modernize in the tensile strength of nanocomposite films (Esthappan, Kuttappan, & Joseph, 2012) However, as nanofiller starts to form agglomerations, the interaction of nanofiller and polymer matrix decreases and hence the interfacial interaction results in formation of defect in nanocomposite diminishing in tensile strength. For elongations at break, same pattern was observed as tensile strength for 0.5 and 1% elongation were increases linearly up to 2.5 wt. % Cu-NPs loading. At 3 wt. % Cu-NPs loading, nanocomposite film decreases its mechanical properties. Due to agglomeration of Cu-NPs in polymer matrix, this decreases interaction between Cu-NPs and LDPE matrix.

Fig. 2. X-ray diffraction patterns of pure gelatin capped Cu, pure LDPE and 0.5–3 wt. % LDPE/Cu nanocomposite films.

Thermal decomposition temperature increases with increase in wt.% of Cu-NPs. Compared to pure LDPE film, thermal decomposition temperature increases for 0.5–2.5 wt.% of Cu-NPs loading; but thermal decomposition decreases as number of nanoparticles increases up to 3 wt.% in nanocomposite films. These results indicate clear adhesion between Cu-NPs and LDPE polymer matrix and diffusion of volatile decomposition product within the LDPE/Cu nanocomposite film delayed Cu-NPs, this gives rise in thermal decomposition temperature. While for 3 wt.% nanocomposite films, there was agglomeration of CuNPs in LDPE. This is also observed in FE-SEM image in Fig. 3F; at 3 wt. % LDPE/Cu nanocomposite film number of copper nanoparticles are

3.8. Release of copper ion from film Fig. 6 shows the ion release curves of LDPE/Cu nanocomposites, filled with 0.5, 1, 1.5,2, 2.5 and 3 wt.% of Cu-NPs. Atomic absorption spectroscopy (AAS) was used to studied quantity of ions liberated from 214

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Fig. 3. FE-SEM image of (A) pure CuNPs (scale bar = 500 nm): (B) gelatin capped CuNPs (scale bar = 1 μm); (C)Particle size distribution of pure CuNPs (scale bar = particle size(nm) vs number%); (D) Particle size distribution of gelatin capped CuNPs(scale bar = particle size(nm) vs number%);(E) EDS of CuNPs ;(F) pure LDPE and LDPE/Cu nanocomposite films (0 wt.% scale bar = 3 μm;0.5 wt.% scale bar = 5 μm; 1.5 wt.% scale bar = 5 μm; 2.0 wt.% scale bar = 3 μm 2.5 wt.% scale bar = 5 μm :3 wt.% scale bar = 5 μm).

Fig. 4. (A) DSC of LDPE/Cu nanocomposites films at 0, 0.5, 1, 1.5, 2, 2.5 and 3 wt. % CuNPs loadings. (B) TGA, A pure LDPE film; B-0.5; C-1; D-1.5; E-2; F-2.5; E-3 wt. % CuNPs loading in LDPE/Cu nanocomposite films. 215

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Fig. 5. Tensile strength and Elongation at break of LDPE/Cu nanocomposite films.

nanoparticles and finally liberates metal ions (Palza, Quijada, & Delgado, 2015) However, the increase in Cu2+ ion release is found to be in a controlled manner and gradually increases with soaking time. It was also being noted that Cu2+ ion release rate increases with increase in quantity of Cu-NPs loading in LDPE nanocomposite films. The maximum Cu2+ ion release was found for the 3 wt.% LDPE/Cu nanocomposite film. As the loading of Cu-NPs in LDPE nanocomposite film increases from 0.5 wt.% to 3 wt.% the ion release rates increased 8 times. Higher Cu2+ ion release rate was observed remarkably greater for 2, 2.5 and 3 wt.% loading of Cu-NPs in nanocomposite films. These results are in virtuous agreement with antimicrobial performance of the films, as shown in Fig. 8(A and B) which was examined by disc diffusion method. The zones of inhibition are large for 2, 2.5 and 3 wt.% loading of Cu-NPs. The ion release rate is an important factor since the antimicrobial efficiency of polymer matrix depends on it. 3.9. Water vapour permeability of film

Fig. 6. Release of copper ion from LDPE/Cu nanocomposite films mg/cm2.

Fig.7 shows water vapour permeability of nanocomposite film. Conferring with (Bodaghi, Mostofi, Oromiehie, Ghanbarzadeh, & Hagh, 2015) a diminish in water vapour permeability apparent since the nanoparticles performance actual hurdle retarding movement for water vapour over the film, hurdle down its speed of flow cover to cross the film by nanoparticles evenly adjusted in the pores of macromolecules structures of polymers. Superior distribution of Cu-NPs into LDPE matrix leads to become hurdler for water vapour to proceed into the film, compared to neat LDPE film. WVP of LDPE/Cu nanocomposite films reduced subsequently as Cu-NPs wt.% loading was increased. It is well known that the permeability depends not only on the Cu-NPs

nanocomposites for 30 days. It was observed that the ion release rate from copper nanoparticles was relatively slow in first week of immersion in distilled water due to the crystalline structure of LDPE polymer matrix. With increase in soaking time, water diffusion begins into LDPE/Cu nanocomposites, water reacts with Cu-NPs and produces Cu2+ ions, which afterwards releases from LDPE/Cu nanocomposites. The Cu2+ ions released LDPE/Cu nanocomposite film were controlled by diffusion process as Cu2+ ions accumulated on surface of film from interior. Metal ions release from polymer nanocomposite is a three-step process. Initially LDPE absorbs the water which reacts with metal 216

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Table 1 Antimicrobial activity, water vapour permeability and total viable count of Sweet (Peda) wrapped in LDPE/Cu nanocomposite film. Wt. % of Cu nanoparticles

0 0.5 1 1.5 2 2.5 3 a

Zone of Inhibition (mm) E coil

S. Aureus

No clear zone ± 16 ± 18 ± 20 ± 21 ± 22 ± 25

No clear zone ± 13 ± 15 ± 18 ± 20 ± 21 ± 22

Total viable count of Pedaa (log10 CFU/g)

1.323 1.230 1.158 1.148 1.130 1.120 1.108

Total viable count of Peda mentioned here was of 7th day.

shown in Fig.8A for Gram +ve and Fig. 8B for Gram −ve bacteria, respectively. However, LDPE/Cu nanocomposite film shows superior antimicrobial activity agents E. coli, and S. aureus mainly food deteriorating and food poisoning microorganisms. In disk diffusion method contact area was used to assess growth inhibition underside film discs in direct contact with target microorganisms in agar. The microbial inhibition specifies release of Cu2+ ion from the LDPE/Cu nanocomposite film and diffusion into the agar layer, avoiding the expansion of microbial colonies in the agar plate. E. coli is a gram −ve microorganism. Its cell wall is relatively thin consisting of a few layers of peptidoglycan. In contrary gram +ve bacterium S. aureus possesses a thick cell wall containing many layers of peptidoglycan. Cu2+ ion releases from film affect relate directly with the bacterial outer membrane, causing the membrane to rupture and killing bacteria (Xue, Jiang, & Liu, 2011). The clear zone shown in Fig. 8(A and B) indicates antimicrobial inhibition efficiency of Cu/LDPE nanocomposite film. The obtained result matches with ion release study diameter of inhibition zone expand with enlarging quantity of Cu-NPs in LDPE. Measurements of clear zone of samples are given in Table 1.

Fig. 7. Water vapours permeability of LDPE/Cu nanocomposite films.

distribution but also on polymer crystallinity. With increase in wt.% of Cu-NPs in Cu/LDPE nanocomposites, the water vapour permeability of the film hurdles. The WVP for pure LDPE film was 1.8 (g/s mPa) × 10 10 , for 2.5 wt. % Cu-NPs loaded LDPE nanocomposite was 0.6 (g/ s mPa) × 10 10 three times slow. Whereas for 3 wt. % loading of Cu-NPs film, it was 0.55(g/s mPa) × 10 10 which shows some stable result. Since this nanocomposite film shows partly agglomeration of nanoparticles in LDPE polymer matrix, seen in (Fig. 3F). Therefore, WVP was positively correlated with proper distribution of filler in polymer matrix and higher crystallinity of nanocomposite. WVP of pristine and LDPE/ Cu nanocomposite films of 0.5–3 wt.% loadings are given in Table 1. 3.10. Antimicrobial study of nanocomposite filmsAntimicrobial efficiency was evaluated by disk diffusion method as

Fig. 8. Antibacterial activity assay of LDPE/Cu nanocomposite films (A) E coli where C- pure LDPE, 1-0.5 wt. % Cu, 2-1 wt. % Cu, 3-1.5 wt.% Cu, 4-2 wt.% Cu, 52.5 wt.% Cu and 6-3 wt.% Cu; (B) S. Aureus where C- pure LDPE, 1- 0.5 wt.% Cu, 2-1 wt. % Cu, 3-1.5 wt. % Cu, 4-2 wt. % Cu, 5-2.5 wt. % Cu and 6-3 wt. % Cu;(C) Number of microorganisms by total viable count as colony forming units (CFU) recovered from Peda. 217

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3.11. Application of developed antimicrobial film as packaging material

Azeredo, De, & Henriette, M. (2009). Nanocomposites for food packaging applications. Food Research International, 42(9), 1240–1253. Bari, P., Lanjewar, S., Hansora, D. P., & Mishra, S. (2016). Influence of the coupling agent and graphene oxide on the thermal and mechanical behavior of tea dust–polypropylene composites. Journal of Applied Polymer Science, 133(4). Betancourt-Galindo, R., Reyes-Rodriguez, P. Y., Puente-Urbina, B. A., Avila-Orta, C. A., Rodríguez-Fernández, O. S., Cadenas-Pliego, G., ... García-Cerda, L. A. (2014). Synthesis of copper nanoparticles by thermal decomposition and their antimicrobial properties. Journal of Nanomaterials, 2014, 10. Blosi, M., Albonetti, S., Dondi, M., Martelli, C., & Baldi, G. (2011). Microwave-assisted polyol synthesis of Cu nanoparticles. Journal of Nanoparticle Research, 13(1), 127–138. Bodaghi, H., Mostofi, Y., Oromiehie, A., Ghanbarzadeh, B., & Hagh, Z. G. (2015). Synthesis of clay–TiO2 nanocomposite thin films with barrier and photocatalytic properties for food packaging application. Journal of Applied Polymer Science, 132(14). Camargo, P. H. C., Satyanarayana, K. G., & Wypych, F. (2009). Nanocomposites: Synthesis, structure, properties and new application opportunities. Materials Research, 12(1), 1–39. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62(3), 373–380. Caroling, G., Vinodhini, E., Ranjitham, A. M., & Shanthi, P. (2015). Biosynthesis of copper nanoparticles using aqueous Phyllanthus embilica (Gooseberry) extract-characterisation and study of antimicrobial effects. Journal of Nanostructure in Chemistry, 1(2), 53–63. Chatterjee, A. K., Sarkar, R. K., Chattopadhyay, A. P., Aich, P., Chakraborty, R., & Basu, T. (2012). A simple robust method for synthesis of metallic copper nanoparticles of high antibacterial potency against E. coli. Nanotechnology, 23(8), 085103. Dang, T. M. D., Le, T. T. T., Fribourg-Blanc, E., & Dang, M. C. (2011). The influence of solvents and surfactants on the preparation of copper nanoparticles by a chemical reduction method. Advances in Natural Sciences: Nanoscience and Nanotechnology, 2(2), 025004. Duncan, T. V. (2011). Applications of nanotechnology in food packaging and food safety: Barrier materials, antimicrobials and sensors. Journal of Colloid and Interface Science, 363(1), 1–24. Esthappan, S. K., Kuttappan, S. K., & Joseph, R. (2012). Effect of titanium dioxide on the thermal ageing of polypropylene. Polymer Degradation and Stability, 97(4), 615–620. Garcia-Turiel, J., & Jérôme, B. (2007). Solvent retention in thin polymer films studied by gas chromatography. Colloid and Polymer Science, 285(14), 1617–1623. Gawande, M. B., Shelke, S. N., Zboril, R., & Varma, R. S. (2014). Microwave-assisted chemistry: Synthetic applications for rapid assembly of nanomaterials and organics. Accounts of Chemical Research, 47(4), 1338–1348. Grass, G., Rensing, C., & Solioz, M. (2011). Metallic copper as an antimicrobial surface. Applied and Environmental Microbiology, 77(5), 1541–1547. Grigoriadou, I., Paraskevopoulos, K. M., Karavasili, M., Karagiannis, G., Vasileiou, A., & Bikiaris, D. (2013). HDPE/Cu-nanofiber nanocomposites with enhanced mechanical and UV stability properties. Composites Part B: Engineering, 55, 407–420. Kadam, D. M., Thunga, M., Srinivasan, G., Wang, S., Kessler, M. R., Grewell, D., ... Lamsal, B. (2017). Effect of TiO2 nanoparticles on thermo-mechanical properties of cast zein protein films. Food Packaging and Shelf Life, 13, 35–43. Khanna, P. K., More, P., Jawalkar, J., Patil, Y., & Rao, N. K. (2009). Synthesis of hydrophilic copper nanoparticles: Effect of reaction temperature. Journal of Nanoparticle Research, 11(4), 793–799. Liu, Q. M., ZHOU, D. B., Yamamoto, Y., Ichino, R., & Okido, M. (2012). Preparation of Cu nanoparticles with NaBH4 by aqueous reduction method. Transactions of Nonferrous Metals Society of China, 22(1), 117–123. Lock, E., Walton, S., & Fernsler, R. (2008). Preparation of ultrathin polystyrene, NRL/MR/ 6750–08-9092). NAVAL RESEARCH LAB WASHINGTON DC PLASMA PHYSICS DIV. Longano, D., Ditaranto, N., Cioffi, N., Di Niso, F., Sibillano, T., Ancona, A., ... Torsi, L. (2012). Analytical characterization of laser-generated copper nanoparticles for antibacterial composite food packaging. Analytical and Bioanalytical Chemistry, 403(4), 1179–1186. Mishra, S., & Shimpi, N. G. (2006). Studies on effect of nano Mg (OH) 2 on mechanical, physical and flame retarding properties of nitrile rubber (NBR). Journal of Nanotechnology and Applications, 1(3), 18–41. Mishra, S., Sonawane, S. H., Singh, R. P., Bendale, A., & Patil, K. (2004). Effect of nanoMg (OH) 2 on the mechanical and flame‐retarding properties of polypropylene composites. Journal of Applied Polymer Science, 94(1), 116–122. Musa, A., Ahmad, M. B., Hussein, M. Z., Saiman, M. I., & Sani, H. A. (2016). Effect of gelatin-stabilized copper nanoparticles on catalytic reduction of methylene blue. Nanoscale Research Letters, 11(1), 438. Nithya, V., Murthy, P. S. K., & Halami, P. M. (2013). Development and application of active films for food packaging using antibacterial peptide of Bacillus licheniformis Me1. Journal of Applied Microbiology, 115(2), 475–483. Othman, S. H. (2014). Bio-nanocomposite materials for food packaging applications: Types of biopolymer and nano-sized filler. Agriculture and Agricultural Science Procedia, 2, 296–303. Palza, H., Quijada, R., & Delgado, K. (2015). Antimicrobial polymer composites with copper micro-and nanoparticles: Effect of particle size and polymer matrix. Journal of Bioactive and Compatible Polymers, 30(4), 366–380. Patel, A. R., Remijn, C., Cabero, A. I. M., Heussen, P., ten Hoorn, J. W., & Velikov, K. P. (2013). Novel all-natural microcapsules from gelatin and shellac for biorelated applications. Advanced Functional Materials, 23(37), 4710–4718. Rhim, J. W., Mohanty, A. K., Singh, S. P., & Ng, P. K. (2006). Effect of the processing methods on the performance of polylactide films: thermocompression versus solvent casting. Journal of Applied Polymer Science, 101(6), 3736–3742.

The Peda was wrapped with the LDPE/Cu nanocomposite films having concentration of 0.5–3.0 wt. % Cu in the films to check the number of viable cells in samples. The result of pour plate shows the slight increase in microorganism. Number of microorganisms by total plate count as colony forming units (CFU) recovered from Peda was shown in Fig. 8C and Table 1. Initially, the effect of the activated films on the colonies of microorganisms was observed just within 48 h of incubation. Initial microbial flora of food sample was 1.001. At 48 h for 0.5, 1, 1.5, 2, 2.5 and 3 wt.% Cu in LDPE/Cu film show 1.011, 1.010, 1.009, 1.008, 1.007 and 1.005 log CFU/g, respectively, where it was around 1.013 log CFU/g for the pure LDPE films (Control). While the LDPE films having concentration of 1.5 to 3.0% Cu show very slight increase in number of microorganisms on the 8th day. The microbial count of neat LDPE film shows 1.323 log CFU/g and in comparison, with this the films of concentration 2.5 and 3 wt. % show very less 1.120 and 1.108 log CFU/g. Though these samples wrapped in various concentrations show slight increase in flora but sweet samples were not degraded up 8 days, since the sample package in Pure LDPE films was started degrading on 7th day which gives foul smell of the samples. Additional incubation did not increase the number of cells as compared to the first day. However, after 8th day, there was no further increase in the number of viable cells. Therefore, the films prepared with concentration 2.5 and 3 wt. % are helpful for packaging of food. From above said results, we can say that more concentration of Cu is inhibitory for the growth of viable cell. 4. Conclusion Antimicrobial food packaging of LDPE/Cu nanocomposites were successfully prepared by using Doctor Blade film fabricator via casting process of the LDPE and gelatin capped Cu-NPs. LDPE/Cu nanocomposites have exhibited primarily exfoliated structures, wherein the Cu-NPs with approximately 50 nm and 99% pure were randomly dispersed. Tensile findings also demonstrated a perfection up to 40% of mechanical properties for the LDPE/Cu nanocomposites film compared to those of the pristine LDPE. Thermal analysis of nanocomposite film highlights the positive influence of Cu-NPs dispersion in LDPE matrix. The quantity of Cu2+ ion release was initiate in a precise manner and progresses with soaking time as it is surface and diffusion phenomenon, in which copper ions must travel from inside of the film to the exterior of food stuff. Water vapor permeability underscore that the LDPE/Cu nanocomposite film addition in Cu-NPs upgrade a notable increase in barrier properties, while, antimicrobial arrangement of the LDPE/Cu nanocomposite films against S. Aureus and E. coli showed absolute growth retardation of bacteria due to the occurrence of enduring antimicrobial copper nanoparticles. Nanocomposite film symbolized possible downsizing in the inhabitants of tested bacteria in Peda, which encourage use of the Cu-Nps in packaging material to jurisdiction microbial spoilage in foodstuff. The developed LDPE/Cu nanocomposite film can be appropriate for food packaging. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fpsl.2018.04.001. References Akelah, A. (2013). Polymers in food packaging and protection. Functionalized polymeric materials in agriculture and the food industry. Springer US293–347. Arora, A., & Padua, G. W. (2010). Nanocomposites in food packaging. Journal of Food Science, 75(1). Awad, M. A., Mekhamer, W. K., Merghani, N. M., Hendi, A. A., Ortashi, K. M., Al-Abbas, F., ... Eisa, N. E. (2015). Green synthesis, characterization, and antibacterial activity of silver/polystyrene nanocomposite. Journal of Nanomaterials, 2015, 5.

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G.B. Lomate et al.

Siracusa, V. (2012). Food packaging permeability behaviour: A report. International Journal of Polymer Science, 2012(2012) Article ID 302029, 11 pages. Siracusa, V., Rocculi, P., Romani, S., & Dalla Rosa, M. (2008). Biodegradable polymers for food packaging: A review. Trends in Food Science & Technology, 19(12), 634–643. Sonawane, S. S., Mishra, S., & Shimpi, N. G. (2009). Effect of nano-CaCO3 on mechanical and thermal properties of polyamide nanocomposites. Polymer-Plastics Technology and Engineering, 49(1), 38–44. Vaseem, M., Lee, K. M., Kim, D. Y., & Hahn, Y. B. (2011). Parametric study of costeffective synthesis of crystalline copper nanoparticles and their crystallographic characterization. Materials Chemistry and Physics, 125(3), 334–341. Vincent, M., Hartemann, P., & Engels-Deutsch, M. (2016). Antimicrobial applications of copper. International Journal of Hygiene and Environmental Health, 219(7), 585–591. Xue, B., Jiang, Y., & Liu, D. (2011). Preparation and characterization of a novel anticorrosion material: Cu/LLDPE nanocomposites. Materials Transactions, 52(1), 96–101. Yallappa, S., Manjanna, J., Sindhe, M. A., Satyanarayan, N. D., Pramod, S. N., & Nagaraja, K. (2013). Microwave assisted rapid synthesis and biological evaluation of stable copper nanoparticles using T. arjuna bark extract. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 110, 108–115. Zain, N. M., Stapley, A. G. F., & Shama, G. (2014). Green synthesis of silver and copper nanoparticles using ascorbic acid and chitosan for antimicrobial applications. Carbohydrate Polymers, 112, 195–202.

Seo, I., & Shin, H. S. (2010). Determination of toluene and other residual solvents in various food packaging materials by gas chromatography/mass spectrometry (GC/ MS). Food Science and Biotechnology, 19(6), 1429–1434. Shameli, K., Ahmad, M. B., Yunus, W. M. Z. W., Ibrahim, N. A., Rahman, R. A., Jokar, M., ... Darroudi, M. (2010). Silver/poly (lactic acid) nanocomposites: Preparation, characterization, and antibacterial activity. International Journal of Nanomedicine, 5, 573. Shankar, S., Teng, X., Li, G., & Rhim, J. W. (2015). Preparation, characterization, and antimicrobial activity of gelatin/ZnO nanocomposite films. Food Hydrocolloids, 45, 264–271. Shimpi, N. G., Mali, A. D., Sonawane, H. A., & Mishra, S. (2014). Effect of nBaCO3 on mechanical, thermal and morphological properties of isotactic PP-EPDM blend. Polymer Bulletin, 71(8), 2067–2080. Shimpi, N. G., Sonawane, H. A., Mali, A. D., & Mishra, S. (2014). Effect of nAl (OH) 3 on thermal, mechanical and morphological properties of millable polyurethane (MPU) rubber. Polymer Bulletin, 71(2), 515–531. Shimpi, N. G., Verma, J., & Mishra, S. (2010). Dispersion of nano CaCO3 on PVC and its influence on mechanical and thermal properties. Journal of Composite Materials, 44(2), 211–219. Shimpi, N., Shirole, S., & Mishra, S. (2015). Polypropylene/nTiO2 nanocomposites using melt mixing and its investigation on mechanical and thermal properties. Polymer Composites, 38(7), 1273–1279.

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