Cereal Chem 67:296-302   |  VIEW ARTICLE

Measuring Trypsin Inhibitor in Soy Meal: Suggested Improvements in the Standard Method.

C. E. Stauffer. Copyright 1990 by the American Association of Cereal Chemists, Inc. 

The parameters involved in the Trypsin Inhibitor Assay (AACC method 71-10) were examined. The peak absorbance of the difference spectrum is at 385 nm rather than 410 nm as presently used; absorbance is 40% greater at the lower wavelength. The formation of product during the incubation of N-benzoyl-DL- arginine rho-nitroanilide was not linear with time. The effedt this has on the numbers derived from the analysis was explored, and it is shown that the inhibitor concentration in a soy extract is underestimated by about 5%. A better method of treating the data derived from the assay is given, namely making a direct linear fit to the absorbance found in the presence of varying amount of inhibitor. Michaelis-Menten parameters for the reaction of porcine trypsin with substrate at 25 C, pH 8.2, 10 mM CaCl2 are determined. For the reaction with the L-isomer (substrate)KM = 2.20mM and k(cat) = 8.92 sec-1. The D-isomer is a competitive inhibitor with Ki = 2.33 mM. Methods for expressing the amount of trypsin used per assay tube in SI units by performing a titration assay on stock trypsin and by a rate assay uising N-benzoyl-L-arginine ethyl ester are presented. Certain modifications in the present standard method of trypsin inhibitor assay are suggested that allow determination of inhibitor concentration in soy samples in SI units (moles per gram) rather than the present arbitrary TI units.