Response surface methodology (RSM) was used to evaluation the effects of Ɛ-polylysine (Ɛ-PL: 0–5 wt%) and zinc oxide nanoparticle (ZnO: 0–5 wt%) on the physico-mechanical properties of the gelatin/polyvinyl alcohol (G/PVA) based nanocomposite packaging films. Using Design-Expert 7.0.0 software, numerical and graphical improvements were also carried out. The optimization was focused on increasing the values of the ultimate tensile strength (UTS), strain at break (SAB), Young's modulus (YM), and lightness (L), and reducing the values of the water vapor permeability (WVP), yellowness index (YI), and overall color difference (∆E). The overall optimal concentrations of ZnO and Ɛ-PL were 1.00 (wt%) and 4.30 (wt%), respectively, with a maximum desirability of 75 %. Additionally, the microstructure, surface topography, interactions, and distribution quality of ZnO nanoparticles and Ɛ-PL in the biopolymer matrix were analyzed using Scanning Electron Microscopy, Atomic Force Microscopy, Fourier Transform Infrared Spectroscopy, and X-ray diffraction measurements. The result of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) confirmed improved thermal stability of nanocomposite films containing ZnO nanoparticles and Ɛ-PL. MIC test showed that ZnO and Ɛ-PL were effective against E. coli, S. aureus, P. fluorescence, and S. typhimurium at the concentration of 2–3 and 10–12 mg/mL, respectively. The antimicrobial activity of G/PVA nanocomposites containing ZnO, Ɛ-PL, and ZnO/Ɛ-PL against E. coli was more than S. aureus.
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