An International Collaborative Study on Trypsin Inhibitor Assay for Legumes, Cereals, and Related Products

Abstract

AbstractFor 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 (Nα‐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, enzyme‐last 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.