A Taguchi Approach for Optimization of Antimicrobial Effect of Whey Protein Based Edible Film Fermented by Bacillus clausii

<i>Bacillus clausii</i>, an antagonistic bacterium, was utilized to develop antimicrobial edible films based on whey protein concentrate. This study employed a Taguchi test (3 × 3) to evaluate the impact of temperature, pH, and protein concentration on film properties. Optimal growth of <i>B. clausii</i> occurred at 6% (<i>w</i>/<i>v</i>) protein and pH 9.5. The resulting film solutions demonstrated antimicrobial activity, exhibiting inhibition zones against <i>Aspergillus niger</i>, <i>Penicillium expansum</i>, <i>Staphylococcus aureus</i>, and <i>Escherichia coli</i>, with inhibition zone diameters of 13.68 mm, 16.88 mm, 11.38 mm, and 17.15 mm, respectively. The optimum antimicrobial property of the films was observed when the incubation condition of pH 8.5, 35 °C and 6% (<i>w</i>/<i>v</i>) protein. Survival rates of <i>B. clausii</i> in the dry film were 86% at 4 °C and 87% at 25 °C over 14 days. Additionally, the highest tensile strength (TS) and percent elongation at break (%E) for the films were recorded at 3.14 MPa (pH = 9.5, 37 °C, 8% protein) and 27.63% (pH = 9.0, 35 °C, 10% protein), respectively. These findings demonstrate the potential for developing effective antimicrobial films through 24-h fermentation of <i>B. clausii</i> in the film solution. This antimicrobial film shows potential for use in wound dressings or food packaging applications.