Cow blood hydrolysis using pineapple waste: Optimization for amino acid release and potential antioxidant bioactive production

Enzymatic hydrolysis of slaughterhouse blood is constrained by the high cost and limited availability of purified enzymes, hindering large-scale waste valorization. This study aims to develop a low-cost and sustainable hydrolysis strategy by directly utilizing raw pineapple waste (PW) as a natural protease source for cow blood (CB), without enzyme purification or supplementation. A central composite design (CCD) was employed to optimize hydrolysis time, PW concentration, and CB concentration, while protein reduction (P red ), free amino acid (FAA) release, and volatile solids reduction (VS red ) were evaluated as key responses. The developed models were statistically significant (p < 0.0001) with R 2 values exceeding 0.90, demonstrating strong predictive reliability. Under optimized conditions (desirability = 0.98), P red and VS red reached 96% and 30%, respectively, at PW:CB concentrations of 10:4 g VS L −1 , while FAA concentration increased to 1321 μg mL −1 at PW:CB concentration of 10:8 g VS L −1 after 72 h. Extending the hydrolysis period to 168 h further increased FAA concentration to 2731 μg mL −1 and enhanced antioxidant activity by 4.2-fold, achieving 51% 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed peptide bond cleavage and disruption of protein secondary structures, indicating the formation of low-molecular-weight peptides. Compared to purified enzyme approaches, this study demonstrates, for the first time, the direct waste-to-waste use of untreated PW as the sole protease source for CB hydrolysis, eliminating enzyme processing costs. The process provides an eco-friendly and cost-effective pathway for CB valorization, supporting bioenergy applications, specifically anaerobic digestion and antioxidant-rich hydrolysate production for food, nutraceutical, and pharmaceutical industries.