Two Optimized Cysteine Protease-Aided Protein Hydrolysates of Soybean Tempeh Inhibit Angiotensin-Converting Enzyme Activity <i>In Vitro</i>

Peptides produced from soybean tempeh that inhibit angiotensin-converting enzyme (ACE) provide a promising source of novel antihypertensive agents. This study utilized two cysteine proteases (papain and bromelain) to generate ACE inhibitory peptides from the protein hydrolysate of soybean tempeh. The trials were arranged using a Box-Behnken design to achieve optimal hydrolysis conditions. The optimal conditions, which were determined by response surface methodology optimization, were as follows: papain: pH 9, temperature 62.88°C, enzyme/substrate ratio 0.54%, and hydrolysis duration 14.94 h; bromelain: pH 9, temperature 60°C, enzyme/substrate ratio 1%, and hydrolysis duration 1 h. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry analysis, the protein hydrolysates displayed peptide bands within the 23－30 kDa range, comprising various peptides from monopeptides to nonapeptides. Moreover, these protein hydrolysates exhibited a more pronounced ACE inhibition than the crude protein extract, with the lowest IC₅₀ value obtained from the protein hydrolysate with papain. These findings offer a conceptual basis for the development of food-derived ACE inhibitory peptides and the highly valued application of soybean tempeh.