Soybean trypsin inhibitor assay: the sequence effect of adding reagents, factors involved, and mechanistic explanations

Authors: Liu, Keshun

Submitted to: Journal of the American Oil Chemists' Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2019
Publication Date: 4/24/2019
Citation: Liu, K. 2019. Soybean trypsin inhibitor assay: the sequence effect of adding reagents, factors involved, and mechanistic explanations. Journal of the American Oil Chemists' Society. 96:619-633. https://doi.org/10.1002/aocs.12216.
DOI: https://doi.org/10.1002/aocs.12216

Interpretive Summary: Soybeans are an important oilseed, providing oil, defatted protein meals and related products to food and feed industries. However, soybeans contain trypsin inhibitors (TI), which are antinutritional and can cause digestive and metabolic diseases and retard growth in animals. Therefore, TI activity has been an important quality parameter for soy products. However, during TI assay, the sequence of adding trypsin, substrate, and inhibitor has been found to have a profound effect on measured values. This study systematically investigated this reagents’ sequence effect with regards to factors involved and mechanistic explanations. Furthermore, based on the observations, the current standard method for measuring TI levels in soy products would tend to give less values than the actual trypsin inhibition. These findings have provided a scientific basis for improving current TI assay methods for food and feed quality assessments, which is reported in a separate communication.

Technical Abstract: For assaying trypsin inhibitor activity (TIA) of protein-based inhibitors, the sequence of adding enzyme, substrate, and inhibitor has profound effects on measured values. This study systematically investigated this reagents’ sequence effect, with regard to factors involved and mechanistic explanations, using raw and toasted soy flours. When the AOCS standard method (Ba 12-75), which features adding substrates last, was used, TIA values measured varied greatly with not only the pH and Ca2+ concentration of the premix (the mixture of the first two reagents) and preincubation time, but also extractant, postextraction workup (addition of Tris assay buffer and/or centrifugation), and extract dilution levels. Raw and toasted soy flours showed different TIA changes with these factors. Yet, when the standard method was slightly modified by adding enzyme last, TIA values obtained were unaffected by the premix pH and Ca2+ concentration, preincubation time, and extract dilution levels, and thus remained constant. Strong interactions of some factors also existed, as exemplified by significant suppression of the reagents’ sequence effect by the Ca2+ presence in the premix when the premix was neutral and alkaline, but not acidic. These observations are explainable by three proposed mechanisms: limited hydrolysis of the trypsin inhibitor by trypsin in acidic medium according to a reactive site model of the inhibitor, trypsin autolysis in neutral or alkaline medium, and trypsin protection by Ca2+. This study provided a strong basis for modifying the standard method. Because the reactive site model of soybean trypsin inhibitors is applicable to many other protein-based proteinase inhibitors, the reagents’ sequence will affect the results when assaying their activities.