Identification and characterization of multifunctional cationic peptides from enzymatic hydrolysates of soybean...

In this study, we used the commercial soybean protein hydrolysate Hinute-DC6 as a novel starting material from which to purify and identify multifunctional cationic peptides. After fractionation, Hinute-DC6 was separated into 20 fractions with varying isoelectric points (pI) by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we purified and identified the cationic peptides from fractions 19 and 20, which had pI values greater than 12, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectrometry. Of the 83 cationic peptides identified, 14 had high pI values and net charges greater than +2, and were chemically synthesized and assayed for various bioactivities, including hemolytic, antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. None of the 14 cationic peptides tested exhibited hemolytic activity toward mammalian red blood cells at concentrations up to 1000 μM. Five of the cationic peptides exhibited antimicrobial activities against at least one of four human-pathogenic microorganisms tested. In addition, in chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, the 50% effective concentrations of these 14 peptides were between 0.069 and 5.2 μM. Tube-formation assays in human umbilical vein endothelial cells showed that each of the 14 cationic peptides exhibited significant angiogenic activities at 10 μM, with values similar to those of the positive control LL-37. Our results demonstrate that the 14 identified cationic peptides have multiple functions with negligible hemolytic activity. These data indicate that the cationic peptides isolated from Hinute-DC6 and fractions containing these cationic peptides have the potential to be used as multifunctional ingredients for healthcare applications.