Plant protein in vitro gastric and intestinal digestion: A comparison between pH-stat and ortho-phthalaldehyde (OPA) methods

Variations in reported values for protein degree of hydrolysis (DH) often arise from different assessment methods. This study compared the pH-stat and o -phthalaldehyde (OPA) assays to analyse the in vitro digestion of pea, wheat, and soy proteins. The pH-stat method consistently yielded significantly lower DH values (20 % to 45 % depending on the protein source) than the OPA assay. OPA analysis revealed that soluble oligopeptides, rather than free amino acids, were the dominant hydrolysis products. Despite the discrepancy, a strong linear correlation ( p < 0.05, r > 0.99 ) was discovered between pH-stat DH values and the free amino acids fraction quantified by OPA. These findings demonstrate that while the pH-stat method tracks with free amino acids release, it may underestimate the extent of hydrolysis compared to OPA, which accounts for significant oligopeptide accumulation. The OPA assay provides a more comprehensive assessment by differentiating between these key hydrolytic components. • pH-stat showed lower protein hydrolysis values than OPA method. • pH-stat estimated peptide bond cleavage, but was influenced by side reactions. • OPA assay distinguished free amino acids from peptides. • pH-stat values and OPA's free amino acids release strongly correlated ( r > 0.99 ).