Shno N Hassan, Dlzar Dlshad Ghafoor
Enzymatic hydrolysis can release bioactive peptides from spirulina proteins, but the influence of protease type and molecular-weight fraction on antioxidant and cell viability endpoints remains incompletely defined. This study compared papain- and trypsin-derived fractions from a food-grade, C-phycocyanin-rich spirulina preparation. Hydrolysis was monitored by UV‒visible spectroscopy and quantified using the o-phthaldialdehyde degree-of-hydrolysis assay, after which six membrane-derived fractions (P1, P5, P10, T1, T5, and T10) were evaluated by ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assays. Fractions below 5 kDa (P1, P5, T1, and T5) were additionally screened in MCF-7 estrogen receptor-positive breast adenocarcinoma cells, a widely used model for preliminary assessment of food-derived antiproliferative activity, and in MCF-10A nontumorigenic breast epithelial cells. Papain at pH 6.5 produced the highest measured degree of hydrolysis (21.4 ± 1.8%). At 500 µg/mL for 48 h, P1, P5, T1, and T5 reduced MCF-7 metabolic viability to 20.83 ± 4.33%, 28.43 ± 4.32%, 21.14 ± 4.33%, and 29.00 ± 4.95%, respectively. Screening-level IC50 estimates were 135.8, 154.0, 265.2, and 326.3 µg/mL, respectively. MCF-10A viability remained above 90% at 500 µg/mL. Antioxidant activity was endpoint- and fraction-dependent: P5 showed the highest FRAP value (1.534 mmol FeSO4 equivalents/L at 405 µg/mL) and DPPH scavenging (86.24% at 8.89 mg/mL). The results identify P1 and P5 as distinct lead fractions for subsequent peptide identification and independent biological validation, while supporting the broader potential of controlled spirulina hydrolysis for the development of functional food peptide ingredients.