Optimization of Sequential Enzymatic Hydrolysis in Porcine Blood and the Influence on Peptide Profile and Bioactivity of Prepared Hydrolysates.

The search for new alternatives for the revalorization of porcine blood is crucial due to the large quantities that are annually generated in slaughterhouses. In this study, a sequential enzymatic hydrolysis of pig blood was optimized using different combinations of the enzymes, namely, Alcalase 4.0 L and Protana™ Prime, Flavourzyme 1000 L, and Protamex(®), as a sustainable method for obtaining extracts rich in bioactive peptides. All the assayed hydrolysates exhibited different peptide profiles and showed in vitro antioxidant, hypoglycemic, and anti-inflammatory activity, although their values differed significantly depending on the type of hydrolysis in ABTS, FRAP, and ORAC assays, as well as in the determination of the inhibitory activity of DPP-IV, NEP, TACE, and MGL enzymes. The hydrolysate obtained by the combination of Alcalase 4.0 L, Flavourzyme 1000 L, and Protana™ Prime (AFPP) resulted in the highest hydrolysis degree (33.39 ± 0.98%), and its peptide profile reflected a higher amount of peptides < 3 kDa. This hydrolysate also obtained significantly higher values for ABTS and the inhibition of TACE and MGL. However, APP2 stood out in NEP inhibition (79.39 ± 3.91%), while APPP was notable for DPP-IV inhibition (43.02 ± 1.39%). The analysis of the hydrolysates using mass spectrometry in tandem allowed for the identification of those sequences that are potentially responsible for the biological activities determined, which were characterized using in silico bioinformatic tools. The results show the potential of using sequential enzymatic hydrolysis in porcine blood to obtain multifunctional peptides.