Complementary gastric protein hydrolysis by pepsin and an actinidin-enriched kiwifruit concentrate (Kwd+®)

This study investigated the complementarity between Kwd+®, an actinidin-enriched kiwifruit concentrate, and pepsin in the hydrolysis of major dietary protein isolates under simulated gastric pH conditions (pH 3–5) to evaluate the potential of actinidin-based preparations as natural aids to support gastric protein digestion in humans. In silico analyses showed that pepsin generated from 3 to 5-fold more predicted cleavage sites than Kwd+® across all tested substrates, and a symmetric comparison revealed that the two enzymes shared only ∼13–16% of all unique sites. This limited intersection indicates that actinidin and pepsin act on largely distinct sequence environments, supporting their mechanistic complementarity. Experimental hydrolysis results demonstrated that Kwd+® retained stable proteolytic activity across all pH values, whereas pepsin showed a strong pH-dependent decline. Protein digestion was quantified as the experimental degree of hydrolysis (DHe), determined from the release of free amino groups during enzymatic hydrolysis. Across all substrates, the combined hydrolysis with Kwd+® and pepsin produced the most extensive protein fragmentation and generally higher DHe values than pepsin over the tested pH range (3–5). SDS-PAGE analyses confirmed these findings, showing near-complete disappearance of legume 7S/11S globulins, caseins, β-lactoglobulin, and α-lactalbumin under the mixed treatment. Kwd+® alone effectively hydrolyzed compact plant globulins even at pH 5, while pepsin alone produced minimal degradation except at pH 3. Overall, the results demonstrate that Kwd+® expands the hydrolytic window of pepsin toward higher gastric pH, providing a robust complementary mechanism capable of enhancing protein digestion independently of protein origin or structural class, thereby supporting its potential application as a functional digestive aid for improving gastric protein hydrolysis.