Comparison of ISO 14902:2001 with AOCS Ba 12a-2020 for determining trypsin inhibitor activity in protein products

Authors: Liu, Keshun

Submitted to: Journal of the American Oil Chemists' Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2021
Publication Date: 9/29/2021
Citation: Liu, K. 2021. Comparison of ISO 14902:2001 with AOCS Ba 12a-2020 for determining trypsin inhibitor activity in protein products. Journal of the American Oil Chemists' Society. https://doi.org/10.1002/aocs.12542.
DOI: https://doi.org/10.1002/aocs.12542

Interpretive Summary: Trypsin inhibitors are naturally present in seeds of legume crops, such as soybeans and pulses, where they are considered antinutritional. Therefore, trypsin inhibitor activity has been a key quality parameter for products of soybeans and other legumes. At present, there are two major official methods for determining trypsin inhibitor activity: the American Oil Chemists’ Society (AOCS) Method Ba 12a-2020 and the Geneva-based International Organization for Standardization (ISO) Method 14902:2001. The AOCS method was just recently approved, based on a method developed by USDA scientists at Aberdeen, Idaho, two years ago. The AOCS and ISO methods share the same principle where the difference in absorbance at 410 nm between the absence and the presence of an inhibitor with a synthetic trypsin substrate is measured. Yet, the two differ in sample preparation and extraction, colorimetric assay systems, and calculation of the results. Considering that the AOCS method is relatively new and that the ISO method has been extensively used, the same USDA scientists at Aberdeen, Idaho, recently designed and performed new research to systematically compare the two methods. The study provided answers to two important questions raised by the food and feed industries around the world: Is trypsin inhibitor activity measured by the two methods for a given sample comparable? If comparable, what is the conversion factor between the two? Furthermore, the study also determined the relative contribution of sample preparation and extraction, colorimetric assay system and results calculation, towards the observed difference in trypsin inhibitor values measured by the two methods.

Technical Abstract: For measuring trypsin inhibitor activity (TIA) there are two major official methods: American Oil Chemists Society (AOCS) method Ba 12a-2020 and International Organization for Standardization (ISO) 14902:2001. The former was recently approved. The two methods differ in sample preparation, extraction, colorimetric assay systems and TIA calculations. In this study, the two methods were symmetrically compared using three unique sets of samples: assorted protein products of soybeans, pulses, and grains; soybeans boiled for varied durations; and soy white flakes toasted for varied durations. For given samples, significant differences existed in TIA measured by the two methods, resulting from effects related to the assay systems and TIA calculations, not from the difference in sample preparation and extraction. When the same trypsin was used, TIA (in mg trypsin inhibited/g sample) measured by the two methods were highly correlated (r = 0.9973, n = 27), giving an equation of y = 0.5464x 0.4887, where y represents ISO values and x for AOCS values. The line connecting ratios of ISO/AOCS in TIA and AOCS values remained relatively flat around 0.53 but started to curve down when TIA approached the lowest. Furthermore, for the same samples, TIA values measured by the ISO method decreased with increasing specific activity of trypsin used, while AOCS values remained consistent, leading to decreasing ratios of ISO/AOCS. Therefore, accurate and direct comparison of the two methods was impossible. It could not be resolved by simply changing ISO method’s calculations as hypothesized earlier. Regardless, for most samples, ISO values were roughly about 55% of AOCS values.