Sequential Fractionation as a Tool for Understanding the Physicochemical and Thermal Properties of Aqueous and Enzyme-assisted Aqueous Extracted Black Bean Proteins

Abstract

Black bean proteins were sequentially extracted and characterized to understand the contribution of different protein classes to the physicochemical and thermal properties of black bean protein extracts produced with commercially-feasible extraction processes (i.e., aqueous extraction process, AEP, and enzyme-assisted aqueous extraction process, EAEP). The Osborne fractionation method was used to produce protein fractions rich in albumins (56%), globulins (22%), prolamins (0.65%), and glutelins (16%). AEP (pH 9.0, 50 °C, 1:10 solids-to-liquid ratio, 60 min) and EAEP (same conditions as AEP, except by the addition of 0.5% (w/w) alkaline protease) enabled the extraction of 75 and 81% of the bean flour protein, respectively. The protein molecular weight distribution showed that the AEP generates a mixture of albumins, globulins, and high-molecular weight glutelins while the EAEP hydrolyzed a majority of the ~42-48 kDa phaseolin into ~26.5 kDa fragments. EAEP proteins exhibited decreased surface hydrophobicity and increased absolute zeta potential values in relation to AEP proteins. The thermal stability of AEP and EAEP proteins decreased compared to that of the albumin- and globulin-rich fractions, likely due to partial denaturation in alkaline extraction conditions. However, the nearly identical thermal transition behavior of the AEP (To = 85.2 °C, Td = 92.0 °C) and EAEP (To = 84.1 °C, Td = 92.3 °C) proteins suggests that enzymatic hydrolysis did not significantly affect the thermal properties of bean proteins. Furthermore, the physicochemical modifications observed when enzymes were used during the extraction resulted in proteins with higher solubility at pH 4.0 (27% for AEP vs. 60% for EAEP). The results of this study demonstrate that aqueous extraction methods are effective in extracting albumins, globulins, and glutelins from black beans. Moreover, the use of commercial enzymes to assist protein extraction is a feasible method to improve bean protein extractability and functionality while retaining their thermal stability.