Soybean trypsin inhibitor assay: further improvement of the standard method approved and reapproved by American Oil Chemists’ Society and American Association of Cereal Chemists International

Authors: Liu, Keshun

Submitted to: Journal of the American Oil Chemists' Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2019
Publication Date: 3/25/2019
Citation: Liu, K. 2019. Soybean trypsin inhibitor assay: further improvement of the standard method approved and reapproved by American Oil Chemists’ Society and American Association of Cereal Chemists International. Journal of the American Oil Chemists' Society. 96:635–645. https://doi.org/10.1002/aocs.12205.
DOI: https://doi.org/10.1002/aocs.12205

Interpretive Summary: As an important oilseed, soybeans provide edible oil, defatted protein meals and related products to food and feed industries. However, soybeans contain trypsin inhibitors (TI), which are antinutritional. It is vitally important to have an analytical method that can accurately measure TI activity in soybean products. Currently, there is a standard method, approved and reapproved by American Oil Chemists Society (Method Ba 12-75) and American Association of Cereal Chemists International (Method 22-40.01). Yet, some problems still exist for the official method. To address the problems, we proposed two improved methods and comparing them with the standard method, based on the knowledge gains from our previous study. The new methods give more accurate results with less variation than the standard method. One of them also improves assay sensitivity and reduces reagents usage. The two methods can be used for measuring TI activity not only in soy products but also in many other TI-containing products.

Technical Abstract: For measuring trypsin inhibitor (TI) activities in soybean products, the current standard method, approved and reapproved by American Oil Chemists Society (Method Ba 12-75) and American Association of Cereal Chemists International (Method 22-40.01), features mixing trypsin with a series of inhibitor levels and then adding a substrate to start the colorimetric reaction. Yet, previous studies have shown flaws with the method, particularly with using several inhibitor levels and the sequence of adding the substrate last. The present study showed that with varying levels of dilution and volumes of a dilute sample extract, the pH of the premix (the mixture of a dilute sample extract and trypsin solution) ranged 3.30–3.60 for raw soy flour, and 3.20–6.70 for toasted soy. Within these premix pH ranges, the standard method of adding substrate last would give TI values equal to or less than those measured by the same method except for adding the enzyme last. The standard method was subsequently improved by using a single sample extract level and the enzyme-last sequence. Other modifications included making stock solutions for reagents, adding Ca2+ to the trypsin solution, diluting sample extracts to a level that causes 30–70% of inhibition, and running both reference and sample blanks for better controls. Alternatively, the full volume assay (10 mL total, as in the standard method) was further modified by using half the volume of each reagent with the same concentration. Compared to the standard method, the improved methods gave more consistent results when assaying 11 selected soy products. The half volume (5 mL) and full volume methods gave the same results, but the former could increase assay sensitivity and reduce amounts of reagents used.