Applying RSM for optimization of Ferula assafoetida protein hydrolysis to enhance bioactivity and functional properties

Plant-based protein hydrolysates are gaining attention due to their potential health benefits, particularly their antioxidant properties. While various methods have been explored for optimizing protein hydrolysis, achieving a high yield of bioactive peptides with enhanced antioxidant activity remains challenging. This study aimed to optimize the enzymatic hydrolysis of Ferula assafoetida proteins using pepsin at enzyme to substrate ratios of 1–2%, temperature 32–42 °C, time 60–120 min, and pH 1.5–2.5 to enhance antioxidant properties, employing Response Surface Methodology (RSM). Protein extraction was most efficient at pH 10, while pH 4.5 caused significant precipitation, yielding a recovery rate of 37.1 ± 0.14 mg/g of seed. The hydrolysis process was optimized by varying pH, temperature, time, and enzyme-to-substrate (E/S) ratio, revealing optimal conditions for maximizing bioactivity: 38 °C, 120 min, pH 1.5, E/S 1.9% for hydrolysis degree; 37 °C, 88 min, pH 2.0, E/S 1.6% for DPPH radical scavenging; and 34 °C, 109 min, pH 2.1, E/S 1.2% for cupric-ion reducing power. SDS-PAGE analysis confirmed the removal of 70–90 kDa bands, yielding peptides <10–15 kDa. Hydrolysis enhanced Glu and Lys content, with hydrophobic amino acids at 78.23 mg/g and an essential amino acid ratio of 46%. Structural analysis showed reduced particle size (2042.5 to 374.9 nm) and ζ-potential shift (-18.2 to -3.2 mV). These findings provide a refined approach for producing bioactive peptides with enhanced antioxidant properties, supporting their application in functional foods and nutraceuticals. This study advances the understanding of plant-based protein hydrolysates and offers scalable solutions for industrial use.