Influence of enzymatic hydrolysis conditions on antimicrobial activities and peptide profiles of milk protein-derived hydrolysates from white wastewater

The focus of our investigation lies in the hydrolysis of milk proteins found in the substantial wastewater generated by the dairy industry, particularly in the white and cleaning wastewater resulting from rinsing and cleaning-in-place processes in order to enhance the value of dairy constituents by producing a diverse population of peptides, including potential antimicrobial peptides generated by 4 different enzymes: pepsin, trypsin, pronase E, and thermolysin. The protein/peptide content was influenced by the degree of hydrolysis values ranging from 2 % to 13 %, and UPLC-MS/MS characterization reveals distinct peptide sequences in enzymatic hydrolysates. The impact of hydrolysis time was also examined, revealing significant differences between 30 minutes and 240 minutes, with 555 peptides identified at 30 minutes, increasing to 693 at 240 minutes. After 4 hours, grouping showed variations: 181 peptides (thermolysin), 153 (pepsin), 126 (trypsin), and 83 (pronase E). A comparative analysis of the characterized sequences with antimicrobial peptides databases identified 17 antimicrobial peptides after 240 minutes pronase E and thermolysin hydrolysis, though they were insufficient to inhibit strains like Clostridium tyrobutyricum and Pseudomonas aeruginosa. Antimicrobial assays also revealed that peptides from Pronase T30, Pronase T240, and Thermolysine T240 exhibited antifungal activity against Mucor racemosus (MIC 2.5 mg/mL), but none against Penicillium commune. The effet of enzymatic hydrolysis demonstrated in this study highlights, for the first time, the potential for valorizing dairy white wastewater within the context of a circular economy framework.