Total Trypsin Inhibitor Activity Research Articles

Kunitz trypsin inhibitor in soybean: contribution to total trypsin inhibitor activity as a function of genotype and fate during processing

Detrimental effects of trypsin inhibitor activity (TIA) in soybean on human health have been attributed to Kunitz trypsin inhibitor (KTI) polypeptide. Literature is replete with reports pertaining to TIA in soybean seeds, however, the studies on the contribution of KTI polypeptide to total TIA have not been carried out. Further, very limited information is available on the quantitative changes occurring in this polypeptide due to different processing methods. In the present investigation, KTI polypeptide in soybean seeds was resolved by native polyacrylamide gel electrophoresis (PAGE) and substantiated by western blotting. Densitometry was employed to quantify KTI polypeptide in 102 soybean genotypes and assess the inactivation of this polypeptide due to boiling, autoclaving, microwave irradiation and sprouting. KTI concentration exhibited wide genetic variation, ranging from 0.07 to 15.9 mg/g soy flour, which corresponded to 1.0–79.8% of total TIA. Boiling and autoclaving for 15 min both resulted in complete inactivation of KTI. Microwave irradiation induced significantly higher reduction for KTI in soaked than dry seeds. Sprouting for 4 days caused 71.4% inactivation of KTI. The study showed that KTI contribution to total TIA was genotype-dependent and the inactivation of this polypeptide was a function of processing methods.

Comparative Assessment of Trypsin Inhibitor vis-à-vis Kunitz Trypsin Inhibitor and Bowman-Birk Inhibitor Activities in Soybean

Trypsin inhibitor activity (TIA) in soybean is attributed to two polypeptides, namely, Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Standard spectrophotometric protocol widely followed for estimation of TIA is cumbersome and does not distinguish KTI from BBI. In the present investigation, extraction conditions for KTI were optimized and different forms of this polypeptide were resolved in 180 soybean genotypes of Indian and exotic origin through native PAGE. This led to the identification of three KTI alleles, namely, Tia, Tib, and Tic, with Tia occurring in most of the Indian genotypes. Trypsin-KTI complex assay exhibited binding of Tia polypeptide with 2.51 fold concentration of trypsin. Subsequently, seeds of selected genotypes were subjected to estimation of KTI and BBI activity through densitometry and enzyme-linked immunosorbent assay (ELISA), respectively; and total TIA through standard spectrophotometric protocol. Summation of KTI and BBI was significantly (P < 0.05) lower than that of TIA determined through the spectrophotometric method.

Trypsin inhibitors of fababeans (Vicia faba L. var. minor). 1. Preliminary studies.

Trypsin-inhibitor activity (TIA) of three cultivars of fababeans was compared with that of soybean. A 50 mM phosphate buffer, pH 7.6, extract of soybean contained 35 times more specific TIA than similarly prepared extracts of fababeans. The fababean trypsin inhibitors (TI), like those of soybean, were highly thermostable. The crude extract possessed 13 to 20% of the original inhibitor activity after heating in a boiling water bath for 60 min. The total TIA was higher in extracts of phosphate (pH 7.6) and of ammonium formate (pH 3.2) than of acetate (pH 4.6). However, the specific TIA of the lower pH extracts was higher as a result of a reduced solubility of the fababean proteins at these pH values.