An international collaborative study on trypsin inhibitor assay for legumes, cereals and related products

Authors: Liu, Keshun; SEEGERS, SUSAN - Bunge, Ltd; CAO, WENMING - Wilmar International; WANASUNDARA, JANITHA - Agriculture And Agri-Food Canada; CHEN, JUXING - Novus International, Inc; DA SILVA, ALESSANDRO - Cbo Analysis Laboratory; ROSS, CHRISTOPHER - Eurofins Scientific, Inc; FRANCO, ALEXANDRA - Italcol Alimentos Concentrados; VRIJENHOEK, THEO - Nutreco; BHOWMIK, PANKAJ - National Research Council - Canada; LI, YONGHUI - Kansas State University; WU, XIN - American Oil Chemists' Society (AOCS); BLOOMER, SCOTT - American Oil Chemists' Society (AOCS)

Submitted to: Journal of the American Oil Chemists' Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2020
Publication Date: 3/18/2021
Citation: Liu, K., Seegers, S., Cao, W., Wanasundara, J., Chen, J., Da Silva, A.E., Ross, C., Franco, A.L., Vrijenhoek, T., Bhowmik, P., Li, Y., Wu, X., Bloomer, S. 2021. An international collaborative study on trypsin inhibitor assay for legumes, cereals and related products. Journal of the American Oil Chemists' Society. 98:375-390. https://doi.org/10.1002/aocs.12459|.
DOI: https://doi.org/10.1002/aocs.12459

Interpretive Summary: Trypsin inhibitors (TI) are naturally occurring proteinaceous substances. They are abundantly present in legume seeds (such as soybeans), cereal grains and related protein products. Over the years, numerous studies have shown that some TI are antinutritional, which is undesirable, while others are bioactive, which is desirable. With increasingly use of plant proteins for feed and food in recent years, it is rather important to have a standard method that can measure TI in various plant protein products with high sensitivity and precision. Currently, the American Oil Chemists’ Society (AOCS) has an official method, Ba 12-75, for TI assay in soy products. More than a year ago, ARS researchers at Aberdeen, Idaho significantly modified the current AOCS method and proposed a new AOCS method, Ba 12a-20, to replace the existing one. Recently, the same ARS researchers at Aberdeen, Idaho, continued their effort by carrying out a collaborative study to evaluate the performance of the proposed new method Ba 12a-20 for determination of TI activity in soybeans, pulses, grains and their processed products (flours, concentrates and isolates). The study was international in scope, involving 12 participating labs to test 11 selected samples with varying TI activities. The participants came from seven countries across four continents, including Brazil (1), Canada (2), China (1), Columbia (1), Indonesia (1), Netherland (1) and United States (5). They represented commercial laboratories (5), academic institutions (1), government agencies (3), and private research and development labs (3). Results show that the proposed method is rather robust, with good repeatability and reproducibility. Therefore, the collaborative study paves a way for AOCS Method Committee to review and approve the proposed method as the new official AOCS method for TI assay, not only in soy but also other protein products.

Technical Abstract: For determining trypsin inhibitor activity (TIA) in soy products, the American Oil Chemists’ Society (AOCS) Method Ba 12-75 has been used. It measures differences in absorbance at 410 nm of bovine trypsin activity toward a synthetic substrate (Na-benzoyl-DL-arginine-p-nitroanilide) in the absence and presence of an inhibitor. Recently, a significantly improved method was developed (JAOCS, 2019, 96:635–645), featuring 5 mL of total assay volume, enzymelast sequence, and single inhibitor level in duplicate. It is proposed as the AOCS Method Ba 12a-2020. As a part of the AOCS method approval process, a collaborative study involving 12 international laboratories was conducted to evaluate the performance of the proposed method. The study involved measuring TIA in 10 selected test samples plus a blind duplicate. They included soybeans, pulses, cereals, and their processed products (flours, concentrates, and isolates). After rigorous statistical treatment of the data, only three outliers were removed from the data of two samples. Repeatability relative standard deviations (RSDr) for the 11 samples ranged from 0.99% to 5.52%. Reproducibility RSD (RSDR) ranged from 7.07% to 22.92%, with seven samples having RSDR around 10% or less. The remaining four samples had very low TIA, and their RSDR values ranged from 13.34% to 22.92%. The study has demonstrated reliable performance of the proposed AOCS method. Several collaborators carried out additional experiments addressing some aspects of the method, leading to further refinements. The proposed method is undergoing evaluation by the AOCS Uniform Methods Committee for adoption as an Official Method for measuring TIA in various legume and grain products.