New methodology to determine mass composition of protease inhibitors (Kunitz trypsin inhibitor and Bowman-Birk inhibitor) in soybeans

Soybeans contain two types of protease inhibitors of protein nature: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Together, they contribute up to 6% of the total seed proteins. Because of their difference in molecular structures, the two inhibitors show different genetic constitutions, physicochemical properties, and nutritional and health effects. Historically, trypsin inhibitor activity in soybeans and soy products is routinely measured, but the enzymatic measurement cannot distinguish the two inhibitors. Consequently, in the past three decades, several non-enzymatic methods, including immunoassays, polyacrylamide gel electrophoresis, and liquid chromatography, have been developed to quantitate KTI and/or BBI contents in soybeans. These methods have pros and cons. Recently, a brand-new methodology was developed in the USDA laboratory. It has the capacity to measure the activity and content of each inhibitor. The methodology consists of several steps. Key steps include precise measurements of trypsin inhibitor activity and chymotrypsin inhibitor activity of purified inhibitors and soybean samples and determination of KTI and BBI contents by solving a proposed system of linear equations with two variables. The new methodology has enzymatic and algebraic features and serves as a much needed analytical tool for oilseed chemists, plant scientists, and animal nutritionists to easily determine inhibitory activities and contents of both KTI and BBI in soybeans simultaneously. This will greatly facilitate their efforts to modify protease inhibitor content and activity through genetic manipulation and/or processing and to investigate nutritional and health implications of each inhibitor, and thus, achieve optimal nutrition and health benefits of soybeans as food and feed.