Soybean Whey Protein/Chitosan Complex Behavior and Selective Recovery of Kunitz Trypsin Inhibitor

Proteins in soybean whey were separated by Tricine-SDS-PAGE and identified by MALDI-TOF/TOF-MS. In addition to beta-amylase, soybean agglutinin (SBA), and Kunitz trypsin inhibitor (KTI), a 12 kDa band was found to have an amino acid sequence similar to that of Bowman Birk protease inhibitor (BBI) and showed both trypsin and chymotrypsin inhibitor activities. The complex behavior of soybean whey proteins (SWP) with chitosan (Ch) as a function of pH and protein to polysaccharide ratio (R-SWP/Ch) was studied by turbidimetric titration and SDS-PAGE. During pH titration, the ratio of zeta potentials (absolute values) for proteins to chitosan (vertical bar Z(SWP)vertical bar/Z(Ch)) at the initial point of phase separation (pH(phi 1)) was equal to the reciprocal of their mass ratio (SWP/Ch), revealing that the electric neutrality conditions were fulfilled. The maximum protein recovery (3296) was obtained at R-SWP/Ch = 4:1 and pH 6.3, whereas at R-SWP/Ch = 20:1 and pH 5.5, chitosan consumption was the lowest (0.196 g Ch/g recovered proteins). In the protein-chitosan complex, KTI and the 12 kDa protein were higher in content than SBA and beta-amylase. However, if soybean whey was precentrifuged to remove aggregated proteins and interacted with chitosan at the conditions of SWP/Ch = 100:1, pH 4.8, and low ionic strength, KTI was found to be selectively complexed. After removal of chitosan at pH 10, a high-purity KTI (90% by SEC-HPLC) could be obtained.