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.