Substrate Concentration Modulates Peptide Profile and Bioactivity in Alcalase Hydrolysis of Bovine Plasma Proteins at Fixed Enzyme-to-Substrate Ratio

Many studies have been conducted testing different enzymes and protein combinations to produce peptides with different types and levels of bioactivities; however, their effect on peptide sequence, peptide size distribution, and bioactivity, while maintaining a constant enzyme–substrate ratio, has been less frequently addressed. In this research, bovine plasma has been hydrolysed by Alcalase at several substrate concentrations (5, 10, 15, and 20 g/L) and at a fixed enzyme–substrate ratio of 1:25 (w/w, Alcalase mass per plasma protein mass). For each substrate concentration, the in vitro antioxidant and antihypertensive properties of the hydrolysates produced were assessed. In addition, the molecular weight distribution of the peptides and their sequence were also analysed by SE-HPLC and RP-UPLC-MS/MS, respectively. According to the results obtained, with the lowest substrate loading producing the highest proportion of small peptides: at DH 19%, peptides below 3 kDa represented 45% of the chromatographic area at 5 g/L and 35% at 20 g/L. The hydrolysate obtained at 20 g/L showed the highest antioxidant activity, with ABTS and ORAC values of 1833.98 and 1461.36 μmol TE/g protein, respectively, compared with 1493.13 and 1071.63 μmol TE/g protein for the hydrolysate obtained at 5 g/L. In contrast, the 5 g/L hydrolysate showed the highest ACE inhibitory activity (53.73 ± 3.08%) and the lowest IC50 value (0.41 mg/mL), whereas the 20 g/L hydrolysate showed 35.96 ± 1.55% inhibition and an IC50 of 0.86 mg/mL. These results indicate a trade-off between antioxidant and antihypertensive performance and demonstrate that substrate loading, evaluated under a fixed enzyme-to-substrate ratio, is an effective variable for modulating peptide size distribution, the peptide profile and the resulting bioactivity of bovine plasma hydrolysates.