The selective complex behavior between soybean whey proteins and ι-carrageenan and isolation of the major proteins of the soybean whey

Abstract

Proteins in soybean whey were separated into two groups by graded salt precipitation, low and high isoelectric point protein fractions (LIP and HIP), corresponding to the mixture of Kunitz trypsin inhibitor (KTI) and Bowman–Birk protease inhibitor (BBI) as well as the mixture of soybean agglutinin (SBA) and β-amylase, respectively. The complex behavior of LIP and HIP with ι-carrageenan (CG) as a function of pH (7.0–2.0) and protein-polysaccharide mass mixing ratio (1:1 to 20:1, w/w) was studied by turbidimetric titration and SDS-PAGE. During pH titration, pHϕ1 (the initial pH for the formation of insoluble complexes) and pHmax (the pH for maximum optical density) showed mixing ratio dependence so that the choice of mixing ratio determined the order of coacervation. SDS-PAGE results showed that at protein/polysaccharides mass ratio (Rpr/ps) of ≥15:1, just only KTI (or SBA) complex with ι-carrageenan and shift to precipitates after centrifugation, BBI (or β-amylase) was kept in supernatant. This selectivity was also independent of the total biopolymer concentration (Cp) and titration path. After removal of carrageenan, high-purity KTI, BBI and SBA (over 90% by SEC-HPLC) were obtained. Isothermal titration calorimetry (ITC) showed that, the smaller thermodynamic stoichiometry (n) with higher thermodynamic constants (K) was observed when KTI (or SBA) complexing with CG compared to BBI (or β-amylase). The higher surface charge density of KTI (+0.22 × 10−2 C/m2) and SBA (+0.14 × 10−2 C/m2) may contribute partly to their higher CG thermodynamic affinity.