Bioactivity of multifunctional peptides from goat casein using a serine protease from Mucor subtilissimus URM 4133

This study specifically explored the potential of a novel proteolytic enzyme to produce peptides with antihypertensive, antimicrobial, and antioxidant properties from goat casein. Goat casein was hydrolyzed using a protease purified from the fungus Mucor subtilissimus URM 4133, and the biological activities of the resulting hydrolysates with a molecular weight < 3 kDa were evaluated. Among the activities studied, antihypertensive potential was selected for further investigation, including in vitro gastrointestinal digestion and separation by HPLC. The hydrolysate produced using an enzyme/substrate ratio of 1:5 for 12 h (H12 < 3 kDa) at 40 °C demonstrated the highest ACE inhibition, reaching 92.57 ± 0.63%, which was statistically different from the other hydrolysates. Post-digestion, this activity was the most significantly affected, with a 13.88% reduction. In contrast, antimicrobial activity increased for all tested microorganisms, most notably against methicillin-resistant Staphylococcus aureus (10.63%). Antioxidant activity showed a varied response, increasing for ABTS•+, DPPH•, and ORAC-FL assays, but decreasing for copper and iron chelating activity. These results indicate that the peptides were partially resistant to gastrointestinal digestion, with enhanced activity observed in some cases. The H12 < 3 kDa hydrolysate was separated into five fractions by size exclusion chromatography, from which 53 peptides were identified by MALDI-TOF/TOF spectrometry. The P5 fraction was the most potent, with an ACE inhibition of 89.88 ± 0.79% and an IC50 of 35.42 ± 1.03 µg/mL. Many are unpublished and with high rankings (> 0.8 - PeptideRanker) for biological activity (YQEPVLGPVRGPFPIL, IFTCLL, FYPQLF, PSGAWYY, and SGAWYYL) representing a novelty with potential for pharmaceutical or food industrial use.