Toasted soybean flour components with trypsin inhibitor activity

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Abstract
Compounds in toasted soybean flour having trypsin inhibitor (TI) activity were isolated and characterized. Sodium hydroxide (0.01N) extracts of toasted soybean flour had an average of 2.59 mg TI/g sample. These extracts, after trichloroacetic acid (TCA) precipitation and dialysis, yielded supernatant and precipitate fractions. Addition of polyvinylpyrrolidone to eliminate free tannins and phenolics in the extracts, which may lead to overestimation of TI activity, was unnecessary. Material balance studies revealed 91% protein recovery and 92% recovery of TI activity in the TCA supernatant (1.1% protein, 2.0% TI) and precipitate (89.8% protein, 90.0% TI) fractions. Column chromatography and electrophoresis showed the TCA supernatant and precipitate fractions to contain proteins, including those having TI activity. Kunitz type TI and Bowman‐Birk type protease inhibitors accounted for most residual TI activity of toasted soybean flour, as verified by column chromatography, isoelectric focusing, sodium dodecyl sulfate polyacrylamide electrophoresis, and size‐exclusion high performance liquid chromatography, using the two similarly treated protease inhibitors as standards. Immunoblotting was also used to detect and identify Kunitz type TI’s in toasted soybean meal extracts. This study established the proteinaceous nature of residual trypsin inhibitor activity in toasted soybean flour and the presence of both Kunitz and Bowman‐Birk inhibitors.