Enzymatic modification of pumpkin and groundnut seed proteins for enhanced digestibility: BCAA-enriched peptide...

The increasing demand for sustainable plant-based protein sources has highlighted oilseed sources like pumpkin and groundnut seeds for their protein content. However, poor digestibility and compact structure limit their absorption and bioavailability, particularly for aging population with compromised digestive efficiency. This study investigates the effect of sequential protein hydrolysis using alcalase and thermolysin on protein digestibility, peptide profile and structural-functional profiles of hydrolysates. Using these two enzymes in sequence allows to utilize their complementary specificities to generate BCAA-rich peptides. Proteins abundant in BCAAs - leucine, isoleucine and valine are crucial for muscle protein synthesis and preserving muscle mass. Hydrolysis improved IVPD from 78.53% in PSI to 94.57% in PSH and from 75.36% in GSI to 90.16% in GSH. SDS-PAGE confirmed the degradation of high molecular weight proteins into peptides. The appearance of smear below 10 kDa in PSH and GSH suggests diverse range of oligopeptides. The proteomic characterization using LC-MS/MS analysis of PSH and GSH provided insights into the predominant proteins of pumpkin and groundnut seeds. The majority of proteins in PSH were identified as 11S globulin family namely, beta and beta-like subunits and seed storage protein 2-like isoforms. In case of GSH, the most abundant proteins belong to cupin-type storage proteins and allergenic proteins, particularly Ara h 1. Several peptides were identified with confidence score ranging from 70 to 99%. Both hydrolysates, PSH and GSH showed substantial presence of BCAA-containing peptides (Leucine, Isoleucine and Valine). These BCAAs residues were either part of the peptide core or present at the terminals significant for their bioavailability and physiological function. PSH predominantly contained short to medium chain peptides, ranging from 8 to 18 amino acids. GSH displayed a broader diversity of peptides, including shorter as well as longer peptides, extending upto 20-25 amino acids. This size distribution of peptides showed effective enzymatic hydrolysis that are favourable for intestinal absorption and supporting improved digestibility and bioavailability. FTIR and CD spectroscopy revealed reduced α-helix and increased β-sheets, depicting protein unfolding. SEM showed microstructural changes and XRD highlighted formation of semi-crystalline peptide aggregates. Hydrolysis significantly improved functional properties, exhibiting high solubility, foaming capacity and emulsification. These findings demonstrate the efficacy of dual enzyme on improving the nutritional profile of hydrolysates in enhancing nutritional quality of seed proteins for potential application in functional foods to support muscle health, particularly for aging people or protein-deficient population.