Enzymatic Hydrolysis With Pepsin Enhanced the Nutrient Compositions of Unfractionated Soy Protein Hydrolysate and Its Cell Viability and Nitric Oxide Activities

Abstract

ABSTRACT The crude soy protein isolate (SPI), a by‐product of soybean processing, is a valuable source of plant proteins but is limited by poor digestibility, low solubility, and restricted functionality due to its high molecular weight. Enzymatic hydrolysis offers an effective way to overcome these limitations. This study aimed to develop soy protein hydrolysate (SPH) using pepsin and evaluate its nutrient composition, antioxidant, and anti‐inflammatory activities. SPI was pre‐incubated and hydrolyzed with pepsin at an enzyme‐to‐substrate ratio of 1.5% (w/w) for 4 h. The molecular weights of SPI and SPH were determined by SDS‐PAGE. The nutrient composition, antioxidant (DPPH, FRAP, ORAC), cell viability, and the inhibitory effect of the SPH on nitric oxide‐mediated inflammation were evaluated using RAW‐264.7 cells. SDS‐PAGE analysis revealed that SPH had lower molecular weight peptides (< 20 kDa) compared to crude SPI (> 65 kDa). The hydrolysate showed a high protein recovery yield (89.70%) and consisted of 71.18% protein, 13.66% carbohydrates, 13.14% dietary fiber, 10.79% ash, 2.45% moisture, 1.92% fat, sodium (5496.63 mg), calcium (85.53 mg), and iron (6.54 mg), providing 356.64 kcal/100 g energy. Amino acid profiling indicated 45.36% essential and 54.70% non‐essential amino acids, with glutamate being predominant (18.35%). SPH exhibited significant ( p < 0.05) radical scavenging activity and inhibited nitric oxide production in LPS‐stimulated RAW 264.7 cells at 10 mg/mL. It also enhanced cell viability by over 80% at 1, 2.5, and 10 mg/mL. These results suggest that pepsin‐derived SPH has enhanced nutritional quality and biological activity and could serve as a functional food ingredient to promote human health.