Bacterial extracellular proteases from six strains of marine bacteria and seven strains of terrestrial bacteria were prepared through fermentation. Proteases were analyzed through substrate immersing zymography and used to hydrolyze the collagen and muscle proteins from a salmon skin byproduct, respectively. Collagen could be degraded much more easily than muscle protein, but it commonly showed weaker antioxidant capability. The hydrolysate of muscle proteins was prepared with crude enzymes from Pseudoalteromonas sp. SQN1 displayed the strongest activity of antioxidant in DPPH and hydroxyl radical scavenging assays (74.06% ± 1.14% and 69.71% ± 1.97%), but did not perform well in Fe²⁺ chelating assay. The antioxidant fractions were purified through ultrafiltration, cation exchange chromatography, and size exclusion chromatography gradually, and the final purified fraction U2-S2-I displayed strong activity of antioxidant in DPPH, hydroxyl radical scavenging assays (IC₅₀ = 0.263 ± 0.018 mg/mL and 0.512 ± 0.055 mg/mL), and oxygen radical absorption capability assay (1.960 ± 0.381 mmol·TE/g). The final purified fraction U2-S2-I possessed the capability to protect plasmid DNA against the damage of hydroxyl radical and its effect was similar to that of the original hydrolysis product. It indicated that U2-S2-I might be the major active fraction of the hydrolysate. This study proved that bacterial extracellular proteases could be utilized in hydrolysis of a salmon byproduct. Compared with collagen, muscle proteins was an ideal material used as an enzymatic substrate to prepare antioxidant peptides.

Purposes: This study aimed to develop a sustainable strategy for valorizing protein-rich industrial by-products into functional antioxidants using halophilic biocatalysts, addressing environmental challenges and the demand for bioactive compounds.Methods: A moderately halophilic bacterium, Salinivibrio sp. YH4, was isolated from Yuncheng Salt Lake and identified as S. costicola (99% 16S rRNA homology). The extracellular protease EYHIII? was purified and biochemically characterized for thermal/pH stability, halotolerance, and substrate specificity. Fish collagen hydrolysates generated by EYHIII were evaluated for antioxidant capacity via 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and peroxyl radical scavenging assays. Cellular bioactivity was validated in high glucose-stressed human umbilical vein endothelial cells (HUVECs), analyzing ROS levels and antioxidant enzyme activity.Results: EYHIII was a thermostable (5060°C) and alkaliphilic (pH 7.59.5) M4-family metalloprotease. The enzyme retained >80% activity under high salinity conditions (1 M NaCl) and exhibited strict substrate specificity for hydrophobic residues (Phe/Leu) at the P1’ position. It efficiently hydrolyzed both soluble and insoluble collagens. Fish collagen hydrolysates generated by EYHIII demonstrated potent antioxidant activity, scavenging 33.53 ± 3.30% of DPPH radicals and 45.55 ± 3.00% of hydroxyl radicals at 3 mg/mL, with a peroxyl radical absorbance capacity of 1.69 ± 0.07 mmol TE/g. In human umbilical vein endothelial cells (HUVECs), the hydrolysate reduced high glucose-induced reactive oxygen species (ROS) to baseline levels at 200 μg/mL. It also significantly upregulated antioxidant enzymes compared to damaged controls: superoxide dismutase (SOD, 103.55%), catalase (CAT, 110.96%), and glutathione peroxidase (GSH-Px, 135.79%) (all P < 0.05).Conclusions: This study highlighted Salinivibrio sp. YH4 and its protease EYHIII as a sustainable platform for converting collagen waste into high-value antioxidants. These findings addressed both environmental pollution and the growing demand for functional bioactive compounds. The results underscored the potential of halophilic biocatalysts in advancing circular economy strategies for protein resource utilization.