Differences between the AOCS and ISO assay systems for measuring trypsin inhibitor activity and effects of substrate inhibition

Authors: Liu, Keshun; Woolman, Michael

Submitted to: Sustainable Food Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2025
Publication Date: 7/11/2025
Citation: Liu, K., Woolman, M.J. 2025. Differences between the AOCS and ISO assay systems for measuring trypsin inhibitor activity and effects of substrate inhibition. Sustainable Food Proteins. 3:e70020. https://doi.org/10.1002/sfp2.70020.
DOI: https://doi.org/10.1002/sfp2.70020

Interpretive Summary: Legumes and cereals are major sources of plant proteins for food and animal feed. However, the natural presence of protein proteinase inhibitors in these products exerts antinutritional effects (such as reducing protein digestion, enlarging pancreas, and depressing growth), and therefore, limits their uses. For global trade, two major official methods: American Oil Chemists Society (AOCS) Method Ba 12a-2020 and International Organization for Standardization (ISO) Method 14902:2001, have been used to measure trypsin inhibitor activity (TIA) in various protein products. For achieving accurate and comparable results, USDA-ARS researchers at Aberdeen, Idaho conducted research to compare these two methods. It was found that for most samples TIA by the ISO method is roughly 55% of TIA measured by the AOCS method, resulting from differences between the assay systems and the reference trypsin for TIA standardization. Therefore, the issue of incomparability between the AOCS and ISO methods cannot be resolved by simply applying the same reference trypsin of the AOCS method to the ISO method. Recently, further investigation into the effect of the differences in the assay systems between the two methods showed that among several differences, only the ratio of substrate over enzyme (trypsin) (S/E) has a significant effect on TIA measurements. The AOCS assay has a S/E ratio of 50, but the ISO assay has a S/E ratio of 222. Therefore, in the assay system, the substrate concentration is much higher in ISO method than AOCS method, relative to enzyme concentration. This leads to higher substrate inhibition in the ISO assay system. This effect was particularly noticeable when measuring samples with less dilution and higher presence of sample particles. This new research provides another reason why the ISO method gives a lower TIA than the AOCS method for the same samples and lays out a strategy to make the two methods comparable by recommending replacement of the ISO method directly with the AOCS method, instead of modifying the former.

Technical Abstract: For measuring trypsin inhibitor activity (TIA) in protein products, two official methods: American Oil Chemists Society (AOCS) Method Ba 12a-2020 and International Organization for Standardization (ISO) Method 14902:2001, have been used. Our previous research showed that for most samples, TIA by the ISO method is roughly 55% of TIA measured by the AOCS method resulting from differences in their assay systems and the reference trypsin for TIA standardization. The present study investigated the effects of three major differences in the assay systems between the two methods, using raw and heated soybeans. These included assay volume (5 mL for AOCS vs. 10 mL for ISO), substrate type [DL-Na-benzoyl-arginine-'-nitroanilide (BAPA) for AOCS; L-BAPA for ISO), and concentrations and volumes of substrate and enzyme working solutions which is reflected in the ratio of substrate/enzyme (S/E, w/w) in the assay system (50 for AOCS, 222 for ISO). When expressing TIA as mg trypsin inhibited/g sample, assay volume and BAPA type had no significant effect except for the S/E ratio, with high S/E ratio leading to low TIA. Kinetic evaluation showed that in the absence of inhibitors, the two methods had valid assay systems (zero order velocity). However, in the presence of inhibitors, substrate inhibition exhibited only by plotting % enzyme inhibition against substrate concentration, not by the traditional way of plotting reaction velocity vs. substrate concentration. Since the ISO method has issues of expensive substrate, inaccurate purity assumption for reference trypsin, and prevailing substrate inhibition, its replacement with the AOCS method is recommended.