This work investigated the influence of enzymatic tenderization on digestibility changes of beef semimembranosus proteins using peptidomics methods. Hydrolysis by proteinase K and bromelain elevated the average bitterness index of identified peptides by generating high-Q values peptides (1714–1790 Cal/mol), including KDLFDPIIQ, LIDDHFLFDKPVSPL, and QLIDDHFLFDKPVSPLLL. Proteolysis during enzymatic tenderization acted as a “pre-digestion” step and significantly elevated the degree of hydrolysis of beef protein (by 4.5–17.3%) in subsequent simulated gastrointestinal digestion. Peptidomics analysis of digests revealed large variations in the peptide composition, which was positively correlated with the degree of proteolysis during enzymatic tenderization. Enzymatic tenderization with proteinase K- (for 0.5 h) or bromelain-treated samples largely increased the survival rate (by 65.5 or 82.8%) of peptides during simulated digestion, possibly because of the “secondary enzyme–substrate interaction” effect. This work could provide a new sight into the possible influence of enzymatic tenderization on meat nutrition.

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