Response surface optimization of cost-effective enzymatic hydrolysis of slaughterhouse blood using kiwi waste for antioxidant peptide production

Cost is a major barrier in treating protein-rich wastes such as slaughterhouse blood, as conventional hydrolysis relies on purified commercial proteases that are economically impractical at scale. This study aimed to develop and optimize, for the first time, a low-cost, waste-to-waste enzymatic hydrolysis strategy using kiwi waste (KW) as a direct, non-extracted protease source for cow blood (CB) hydrolysis under ambient conditions (21 °C) without chemical reagents or pH control. The effects of hydrolysis time and KW and CB concentrations on protein reduction (P red ), free amino acids concentration (FAA), and volatile solids reduction (VS red ) were evaluated using a central composite design (CCD). All response models were significant (p < 0.0001) with R² > 0.95. Across the RSM design space, P red approached 90%, FAA concentration reached approximately 2300 µg mL⁻¹, and VS red reached approximately 40%. Numerical optimization identified the selected conditions (168 h, 12 g VS L⁻¹ KW, 6 g VS L⁻¹ CB), with predicted values of 57.83% P red and 2193.72 µg mL⁻¹ FAA and an overall desirability of 0.928. Antioxidant activity increased markedly, with DPPH scavenging rising from 7.4% to 71.9% and total antioxidant capacity from 12.36 to 33.44 µM TE. Increased total ammonia nitrogen (TAN) reflected nitrogen release during hydrolysis, while FTIR analysis supported peptide bond cleavage and structural changes consistent with protein hydrolysis. Compared with enzyme-based pretreatments that require extraction, heating, or chemical inputs, this approach offers a simplified, scalable alternative that reduces process costs and energy demand. The proposed waste-to-waste strategy enables simultaneous valorization of KW and CB, producing antioxidant-rich hydrolysates with potential applications in food, nutraceutical, pharmaceutical, and anaerobic digestion-based bioenergy systems.