An Efficient HPLC Approach to Quantify Kunitz Trypsin Inhibitor in Soybean Seeds

Abstract

Trypsin inhibitor (TI), an important antinutritional factor present in soybean [Glycine max (L.) Merr.] seeds, prevents animal protein digestibility. Accurately determining seed TI concentration is essential for screening soybean breeding lines to select genotypes with low TI. A colorimetric assay is widely used to measure TI activity in soybean seeds. This bioassay is time consuming, expensive, and has repeatability issues. This study developed a high‐performance liquid chromatography (HPLC) method as a high‐throughput, less expensive, and more reliable assay to quantify Kunitz trypsin inhibitor (KTI), the major TI, in soybean seeds. We extracted KTI using sodium acetate buffer, separated on a Poros R2/H perfusion column and detected at 220 nm. The HPLC method was compared with two popular enzymatic bioassays using 100 soybean lines. For the bioassays, TI was extracted using HCl or NaOH extractants and determined according to Kakade et al. (1974). The KTI from the HPLC method ranged from 0.52 to 12.15 mg g−1 with an average of 5.25 mg g−1 and a limit detection of 0.05 mg g−1. The recovery of KTI in spiked soybean samples was 82.33%. The KTI data from HPLC and both bioassays were strongly correlated (r = 0.82 and 0.80, p ≤ 0.0001). The CVs of KTI data (66.20% for HPLC, 18.84 and 32.55% for bioassays) suggest that the HPLC method is capable of detecting a wider range of KTI quantities and providing a better sensitivity for quantifying KTI in soybean seed samples.