Antioxidant peptide nanohybrid: a new perspective to immobilize bioactive peptides from milk industry wastewater

In this study, dairy industry wastewater was collected and used as a protein source. The proteins were converted into powder form using lyophilization. The proteins were digested using Bacillus subtilis ( B. subtilis ) NCIM 2724 . The maximum degree of hydrolysis (DH) of protein was observed at pH of 7, 30 °C incubation temperature, 120 rpm shaking speed, and 96 h incubation. The tris-glycine sodium dodecyl sulfate-polyacrylamide (tris-glycine-SDS) gel electrophoresis showed the disappearance of large molecular weight proteins due to the proteolytic action of B. subtilis . The resulting digest was fractionated using a 3 kDa membrane filter. The antioxidant activity of the obtained fractions was evaluated. Antioxidant activity of digest and filtrate was found to be 12.78% (±0.040) and 49% (±0.025), respectively, at a concentration of 50 mg/mL. The 3 kDa filtrate was subjected to liquid chromatography-mass spectrometry (LCMS) analysis. Bioinformatics tools were used to predict the sequences of antioxidant peptides. Furthermore, the 3 kDa filtrate was used for the synthesis of antioxidant nanohybrid. Scanning electron microscopy (SEM)-energy dispersive spectroscopy (EDS) confirmed the nanohybrid formation and encapsulation of peptides. The antioxidant nanohybrid showed enhanced antioxidant activity compared to the free peptide solution. The dairy industry has a significant environmental impact due to high water use and waste generation. This study addresses an important issue of recycling protein-containing wastewater and the potential to be used for converting these proteins into antioxidant peptides. Such practices will help to reduce environmental impact and sustainably operate the industry.