Method for determining chymotrypsin inhibitor activity: investigations into sample blank measurements, factors involved, and further improvement

Authors: Liu, Keshun; Woolman, Michael

Submitted to: Sustainable Food Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/16/2025
Publication Date: 3/12/2025
Citation: Liu, K., Woolman, M.J. 2025. Method for determining chymotrypsin inhibitor activity: investigations into sample blank measurements, factors involved, and further improvement. Sustainable Food Proteins. 3,e70001. https://doi.org/10.1002/sfp2.70001.
DOI: https://doi.org/10.1002/sfp2.70001

Interpretive Summary: Protease inhibitors, including inhibitors of trypsin, chymotrypsin and other proteases, are naturally present in seeds of legumes and cereals. Since trypsin and chymotrypsin are the two major proteolytic enzymes in mammalian digestive tracts, their inhibitors block absorption of key nutrients. For example, soybeans are rich in protease inhibitors which provide up to 6% of the total seed proteins or 2.5% of the seed weight. Their presence affects nutritional values and utilization of soybean products as food and feed. For many years, trypsin inhibitor activity has been measured as a key quality parameter for soybeans and other legume products. Yet, with increasing use of plant proteins in diets and feed, there is a growing interest in measuring these products for chymotrypsin inhibitor activity (CIA) as well. To meet this demand, three years ago, USDA-ARS scientists at Aberdeen, Idaho, developed an optimized method for measuring CIA in soybean and other plant protein products. During the development and optimization of the CIA assay, the sequence of adding the chemical reagents for the lab test was found to affect not only sample readings, but also sample blank readings (calibrator). Due to the significant effect a sample blank has on accurate CIA measurement, USDA-ARS scientists most recently conducted a follow-up study, and found that for measuring sample blanks during the CIA assay, not only are there more than two sequences of adding reagents, but also many other factors are involved. They proposed several hypotheses about the sequence effects on sample blanks and validated them by conducting a series of experiments. Consequently, a further improved method for accurately measuring CIA in legume and other protein products was described, which can be rather useful for food and feed industries and scientific communities for measuring protease inhibitors activities in legume and cereal protein products.

Technical Abstract: An optimized method for determining chymotrypsin inhibitor activity (CIA) for legume and cereal products was recently developed. As a follow-up, the present study focused on sample blank (SB) measurement and further method improvement for the previously developed CIA assay. It started with an observation that the reagent addition sequence (RAS) had profound effects on SB with soybean samples but not with purified inhibitors. Several hypotheses were proposed, including that the effect of RAS on SB results from differential interactions between other components (including other proteins) in sample extracts with those in the substrate working solution in the presence and absence of acetic acid. Several factors were investigated for their effects and interactions with RAS on SB measurements. These included the duration between adding the second and third reagents, the absorbance at each step of the measurement, the effects of presence and absence of calcium (Ca2+), Ca2+ concentration, method of sample extraction and post-extraction storage, post-reaction centrifugation, wavelength for absorbance measurement, sample type, and volumes of diluted sample extracts. The experiments validated the hypotheses and showed that among the different RAS, the sequence of adding Inhibitor, Acid, Substrate and Enzyme (the IASE sequence) was least affected by most factors and overnight refrigeration of sample extracts had little effect. It was concluded that the RAS effect on SB was unavoidable. Recommendations include using an assay buffer containing 20 mM Ca2+, using the IASE sequence with 0.8 mL sample volume for SB measurements, and centrifuging all reaction mixtures before absorbance measurements.