Uncertainty of Trypsin Inhibitor Activity Measurement of Legume Crops Using Microtiter Plate Method

Abstract

Trypsin inhibitors could limit utilization of legumes in human nutrition, but they could also have beneficial health effects. The objective of this study was to measure trypsin inhibitor activity (TIA) of different legumes using microtiter plate method and to identify factors that contribute to uncertainty of TIA measurement. TIA measurements were performed on seeds of faba bean, pea, common vetch, soybean, and common bean cultivars. The significant effect of legume crop on TIA measurement uncertainty was confirmed with P = 0.045. Certain sources of measurement uncertainty were related with the content of trypsin inhibitors (Tis) in legume seeds. In respect to that, significant effect of level of sample dilution (P ˂ 0.001) was confirmed. Significant influence of the repeated absorbance measurement of sample reaction mixture on uncertainty of TIA measurement was identified (P ˂ 0.001), and it took 60% of overall TIA measurement uncertainty for soybean cultivars. TIA of soybean cultivars exceeded 90 TUI/mg. Repeated absorbance measurement of positive control reaction mixture took 70% of TIA measurement uncertainty of cultivars with TIA lesser than 4.5 TUI/mg. Graduated cylinder used for preparation of the final sample solutions took the range from 45 to 90% of overall TIA measurement uncertainty of the cultivars whose TIA were in the middle of previously mentioned. The uncertainty of TIA measurement of legume crops was not studied before; thus, this study pointed out that acquiring insight into factors contributing to uncertainty of TIA measurement could give directions for improvement of TIA testing if microtiter plate method is used.