Preparation, Characterization and Anti-Bacterial Activity of Zinc Oxide-Gelatin Nanocomposite Film for Food Packaging Applications

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1 Preparation, Characterization and Anti-Bacterial Activity of Zinc Oxide-Gelatin Nanocomposite Film for Food Packaging Applications Preparation, Characterization and Anti-Bacterial Activity of Zinc Oxide-Gelatin Nanocomposite Film for Food Packaging Applications G. Umamaheswari a, S. Sanuja a, Vino Arul John a, Swarna V. Kanth b, and M.J. Umapathy a a Corresponding author: Department of Chemistry, College of Engineering Guindy, Anna University, Chennai, India b Centre for Human and Organizational Resources Development, Central Leather Research Institute, Adyar, Chennai, India Received: 23 October 2013, Accepted: 12 May 2014 SUMMARY Gelatin based composite films were prepared using various crosslinking agents for possible application in the food packaging industry. In the present study, gelatin films were formed by the incorporation of zinc oxide (ZnO) nanoparticles. The ZnO nanoparticles were synthesized by a wet chemical method using zinc nitrate as precursor and starch as stabilizing agent. It was characterized by FTIR. Surface morphology and thermal studies were performed by SEM and TGA respectively. The average particle size was calculated from the X-ray diffraction pattern using Scherrer s equation. Mechanical studies were done with a Universal Testing Machine. The distribution of particles was clearly seen by SEM and thermal studies showed that the decomposition temperature of gelatin was increases by the incorporation nano ZnO. The mechanical properties were also improved with increase in concentration of nano ZnO. Antibacterial activities of the films were also studied against S. aureus, V. cholera and E. coli by the disc diffusion method. Keywords: Gelatin, Zinc oxide, Wet chemical method, Nanocomposites films, Mechanical property 1. INTRODUCTION Synthetic plastics are quite popular in packaging industries across the world due to their high durability, resistance to degradation, cheapness and effectiveness. However, plastic packaging has two major challenges such as decline in fossil fuel and problem in waste disposal. These polymers stay long within the environment after the final use creating significant environmental pollution. This demands to explore new biodegradable packaging materials from renewable sources and it became a topic of great interest in the recent past 1-3. Gelatin a natural polymer obtained by the partial hydrolysis of collagen extracted from the skin, boiled crushed bones, connective tissues, organs and some intestines of animals such Smithers Information Ltd., 2015 as domesticated cattle, chicken and pigs. It has widespread applications in the field of drug delivery, cosmetics, health and nutrition and food packaging due to its unique physicochemical properties such as oxygen impermeability, film-forming capability and biocompatibility. The demand for biodegradable packaging materials involves short-term usage finding it as a potential candidate to replace other non-degradable packaging material in food industry. It exhibits good mechanical properties and is non-toxic, but it is highly brittle, hydrophilic and easily undergoes microbial attack. These drawbacks can be overcome by blending with synthetic polymers, by chemical modification or by incorporating nanoparticles into the gelatin matrix 4-9. The properties can be improved by crosslinking, blending, incorporating Corresponding author: Tel.: , mj_umapathy@yahoo.co.in materials like metal oxides the interaction of metal oxide and gelatin was explained in the case of iron oxide and it has been used for the removal of Cr (IV) 10. In this study, gelatin nanocomposite films were formed by incorporating various weight percentages of ZnO nanoparticles. The films showed good thermal stability with improved mechanical properties, and their greater resistance to microbial attack compared to the native gelatin make them suitable for food packaging applications. 2. MATERIALS AND METHODS 2.1 Materials Gelatin was obtained from the Sd. Fine Chem. (Chennai), zinc nitrate hexahydrate was purchased from the Qualigens Fine Chemicals (Chennai) and starch was obtained from Thomas Baker Chemical Limited (Chennai). Polymers & Polymer Composites, Vol. 23, No. 3,

2 G. Umamaheswari, S. Sanuja, Vino Arul John, Swarna V. Kanth, and M.J. Umapathy 2.2 Preparation of ZnO Nanoparticles Zinc nitrate hexahydrate, g (0.1 mol) was dissolved in 500 ml of 0.5% soluble starch solution. After complete dissolution, 500 ml of sodium hydroxide solution (0.2 mol) was added drop-wise under constant stirring. The aqueous clear solution turned into a milky white colloid without any precipitation. The reaction was allowed to proceed for 2 hours after the complete addition of sodium hydroxide. The solution was centrifuged at 1500 g for 10 minutes, and the settled nano-zno was washed thrice using distilled water to remove the by-products and the excess soluble starch. After complete washing, the nano-zno was dried at 110 C for 3 h for complete conversion of Zn(OH) 2 to ZnO Preparation of Gelatin/ ZnO Nanocomposite Films 1 gram of gelatin was first dissolved in 50ml of water and kept under magnetic stirring for 3 hours. The prepared nano ZnO were dispersed into the gelatin accordingly and sonicated for 5 minutes. The mixture solution was cast onto Petri plates and dried at room temperature for 48 h to obtain nanocomposite films with various percentages of nano ZnO FTIR FT-IR measurements of synthesized nano ZnO, gelatin film and ZnO/gelatin films were recorded by Perkin-Elmer RX1 infrared spectrometer using NaCl to confirm their structure. 2.5 XRD XRD analysis of synthesized nano ZnO was carried out with a Panalytical Expert Pro instrument to investigate crystallinity. 2.6 SEM The morphologies of the gelatin and ZnO/gelatin nanocomposite film were examined by a scanning electron microscopy (DXS-10ACKT). 2.7 TGA Thermogravimetric analysis (TGA, TG-2009, and Netzsch) was used to study the thermal stability of gelatin powder, nano ZnO powder and gelatin- ZnO nanocomposite films (1:0.5%). The heating rate was 100 C/min in N 2 with a flow rate of 20 ml/min. 2.8 Mechanical Property Tensile strength and elongation at break were determined using a Universal Testing Machine (TNUM-5900). The films were cut into a dumbbell shape with thickness of 1 mm and subjected to tensile and elongation testing. 2.9 Swelling Property Test The specimens were dried in an oven for a specified time and temperature and then placed in a desiccator to cool. Immediately upon cooling, they were weighed. The material was then immersed in water for 24 hours or until equilibrium at room temperature. Specimens are removed, patted dry with a lint free cloth, and weighed. Water absorption is expressed as increase in weight percent. Swelling property (%) = [(Wet weight - Dry weight)/ Dry weight] x Antibacterial Test The in vitro antibacterial activities of polymers were determined by disc diffusion method. Muller Hinton agar (MHA) medium was used for preparation of plates. The medium was poured into sterile Petri plates of 90 mm diam. The agar was allowed to set at ambient temperature. Fresh bacterial cultures of Gram positive bacterium, Staphylococcus aureus and two Gram negative, Escherichia coli and Vibrio choleae were spread on surface of the MHA plate with the swab. After incubation, the MHA plates were allowed for the pre-incubation for 10 min. The concentrated polymers were weighted corresponding to 25 µg, 50 µg and 75 µg of these polymers. The inoculated plates were initially incubated for 15 min at room temperature, and then they were incubated at 37 C for 24 h. Then, the plates were examined for any zone of growth inhibition. Inhibition zones were recorded as the diameter of growth free zones including the diameter of the well in mm at the end of incubation period 22. Percentage of inhibition was calculated by the formula: % of inhibition = I(Diameter of the Inhibition zone) x100 90(Diameter of the petri plate in mm) 3.0 RESULTS AND DISCUSSION 3.1 FTIR Analysis FTIR spectra of gelatin powder and synthesized nano-zno are shown in Figure 1. The FT-IR of gelatin, Figure 1a, shows the N-H stretching frequency coupled with the hydrogen bonding present in the aminoacid at 3285 cm -1. The aliphatic substitution present in the aminoacid shows a stretching band at 2956 cm -1. The bending vibration of bound water molecules present in the film coupled with the acid carbonyl stretching of aminoacid is seen at 1631 cm -1. The bending vibrations of N-H and CH 2 are seen at 1240 and 1444 cm -1, and the C-O stretching vibrations at 1078 cm -1. The FT-IR spectrum of ZnO nanoparticles in Figure 1b shows a peak around 514 cm -1 which indicates the formation of ZnO. The peak around 3477 cm -1 may be due to the O-H stretching vibrations of starch present in the ZnO. The FT-IR of gelatin and ZnO nanoparticles incorporated in gelatin films are shown in Figure 2. The films show the O-H stretching frequency at 3430 cm -1. and the bending vibration of water molecules at 1650 cm -1. Gelatin film of all concentrations show a peak around cm -1 which indicates the uptake of ZnO by the gelatin. 200 Polymers & Polymer Composites, Vol. 23, No. 3, 2015

3 Preparation, Characterization and Anti-Bacterial Activity of Zinc Oxide-Gelatin Nanocomposite Film for Food Packaging Applications 3.2 XRD Analysis The XRD diffractogram of nano ZnO (Figure 3) shows that the synthesized ZnO was highly crystalline and the particle size was found to be 70 nm using the Scherrer equation. 3.3 TGA Thermograms of gelatin powder, nano ZnO powder and gelatin- ZnO nanocomposite films (1:0.5%) are compared in Figure 4. The decomposition temperature of gelatin was 284 C; at this temperature major weight loss takes place and the compound completely decomposed by 700 C. The thermogram of ZnO nanoparticles shows a weight loss around 301 C that corresponds to the decomposition of starch; the same has been seen in the FT-IR and the 85% of the material that remains stable corresponds to the ZnO. The gelatin/ ZnO films shows the same pattern as gelatin but the film does not completely degrade; about 30% of the material remains stable after 800 C, indicating the presence of ZnO in the gelatin films. 3.4 SEM SEM images of native gelatin film and gelatin/zno are shown in Figure 5. Figures 5a and b show the morphology of gelatin at different magnifications. The films are seen to be clear without any particles. The SEM images of gelatin/zno shown in Figure 5c and d show the dispersion of ZnO nanoparticles, and the penetration of the particles into the gelatin matrix is revealed under higher magnification. The incorporation of nano ZnO into the gelatin matrix affects the morphology of the films. The apparent size of the particles was found to be 10.3 microns, which may be due to the agglomeration of particles during dispersion. The increase in concentration of ZnO leads to the agglomeration of particles, as may be clearly seen in Figure 6. Figure 1. FTIR spectra of (a) gelatin powder, (b) nano-zno powder Figure 2. FTIR spectra of (a) gelatin film, (b) gelatin-zno 0.1%, (c) gelatin-zno 0.2%, (d) gelatin-zno 0.3%, (e) gelatin-zno 0.4%, (f) gelatin-zno 0.5% Figure 3. XRD of nano-zno powder Polymers & Polymer Composites, Vol. 23, No. 3,

4 G. Umamaheswari, S. Sanuja, Vino Arul John, Swarna V. Kanth, and M.J. Umapathy Figure 4. TGA of (a) gelatin film, (b) nano-zno powder, (c) gelatin-zno nanocomposite films Figure 5. SEM images of gelatin film (a) and (b), ZnO/gelatin films, (c) and (d) at various magnifications Figure 6. SEM of 0.5% ZnO/gelatin film 3.5 Tensile Strength and Elongation The tensile strength and elongation of the films are shown in Figure 7 and Figure 8 respectively. Both the tensile strength and the elongation of the ZnO-gelatin nanocomposites film were higher when compared to the native gelatin film. This is likely to be due to the good interaction between the metal oxide and the gelatin matrix which enhances the film flexibility. 3.6 Swelling Property The swelling behaviour of the film is illustrated in Figure 9. Generally gelatin films are hydrophilic in nature due to presence of hydrogen bonding. An increase in the concentration of ZnO nanoparticles decreases the intake of water and it shows a decrease in swelling percentage that may be due to the stabilization of gelatin matrix by ZnO nanoparticles. 3.7 Antibacterial Activity The antibacterial activity of films was studied with regard to possible packaging applications. The films showed activity against both gram positive and gram negative bacteria. The results show increased activity of gelatin/nano ZnO blends when compared to raw gelatin film. The gelatin/ nano ZnO blend showed good activity for S. aureus, followed by V. cholera and E. coli. Tetracycline antibiotic was used as positive control. The results were presented in Table CONCLUSIONs The synthesized ZnO nanoparticles were characterized by FT-IR, XRD and thermal studies. Their properties were in accordance with those reported previously. The thermogram of ZnO-gelatin film showed that 85% of the material remains stable at elevated temperature. The residue that degraded at 301 C corresponded to the starch present even after 202 Polymers & Polymer Composites, Vol. 23, No. 3, 2015

5 Preparation, Characterization and Anti-Bacterial Activity of Zinc Oxide-Gelatin Nanocomposite Film for Food Packaging Applications Figure 7. Tensile strength of gelatin and gelatin/zno films Figure 8. Elongation of gelatin and gelatin/zno films calcination. The demerits of native gelatin can be overcome by the incorporation of ZnO nanoparticles in various concentrations, which helps to optimize the film properties. The morphology at varying magnifications shows the distribution of metal oxide on the surface and the deep penetration of the material into the matrix at higher magnification levels. The studies of the antibacterial activity showed that the ZnO nanoparticles incorporated films inhibited the growth of both gram positive and gram negative bacteria. The improved tensile and elongation showed that it was not damaged when force was applied, and the reduced intake of water was endorsed by the change in swelling properties. Hence the present preliminary study makes clear that this system could be useful for food packaging applications. ACKNOWLEDGEMENTS We thank the Department of Chemistry, College of Engineering, Guindy, Anna University, Chennai for providing laboratory facilities and chemicals to carry out this research work. Table 1. Antibacterial activity of gelatin (G) and gelatin/zno (GZ) nanocomposite film Microbial Disc diffusion method (Zone of inhibition (dia. in mm) strains +ve control 25 µg 50 µg 75 µg G GZ G GZ G GZ I I% I I% I I% I I% I I% I I% I I% S. aureus V. cholera E. coli Polymers & Polymer Composites, Vol. 23, No. 3,

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