Preparation, identification, structure, and in vitro anti-obesity effects of protease inhibitors isolated from potato fruit juice

Abstract

In this study, potato protease inhibitors (PPI) were obtained from industrial potato fruit juice (PFJ) using membrane separation method and ion exchange chromatography. The molecular weight distribution, families, structural characterization, and in vitro anti-proliferative activity of PPI were evaluated in this paper. The molecular weight distribution of PPI was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI–TOF-MS). The inhibitor families were identified using high-performance liquid chromatography/mass spectrometry. The structures of the isolated PPI were studied by circular dichroism and fluorescence spectroscopy. Anti-proliferative activity of PPI on 3T3-L1 preadipocytes was investigated by MTT method. The results suggest that ultrafiltration (UF) was more suitable for the isolation of crude proteins from PFJ than nanofiltration. The PPI purified from UF freeze-dried powder were mainly focused on proteins in the region of relative molecular masses (Mr) 20 × 103 to 22 × 103 with a few low molecular weight proteins, containing protease inhibitor, protease inhibitor II, aspartic protease inhibitor, and other serine protease inhibitor families. The major conformation of PPI was β-structure, and the tryptophans were mainly located in hydrophobic environments. Furthermore, the structure changed significantly when the temperature reached 80 °C. Moreover, PPI were able to inhibit the proliferation of 3T3-L1 preadipocytes with a 50 % inhibitory concentration of 343.51 μg/mL. Therefore, PPI may have a potential implication in anti-obesity foods in the future.