Isolation and Characterization of Trypsin Inhibitors (Kunitz Soybean Trypsin Inhibitor, Bowman-birk Inhibitor) in Soybean

Soybean (Glycine max (L.) Merr.) has emerged as one of the most economical and nutritious foods. Kunitz Soybean Trypsin Inhibitor (KSTI) and Bowman -Birk Inhibitor (BBI) are the two major trypsin inhibitors in soybeans. The ingested soybean trypsin inhibitors cause growth depression as demonstrated in different animal species and human. The main part of the present study was devoted to isolation, purification and characterization of trypsin inhibitors in soybean. Thus soybean seeds were grounded by food processor grinder and defating was performed by use of supercritical fluid extraction (SFE). After aqueous extraction resulting in a crude extract containing the inhibitors, further separation were performed by means of other techniques such as sephadex G-75 gel filtration, flash chromatography (F.C.) and fast polymer liquid chromatography (FPLC). The results of these inhibitor separations were investigated by use of isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) as electrophoretical separation methods. Finally, purity control of the inhibitor in all samples was checked by high performance capillary electrophoresis (HPCE). Protein content and inhibitor activity for each separated sample were measured. We found that peak 2 G-75, peak 3 G-75 and peak 2 F.C. had the highest amount of trypsin inhibitor, respectively.