Enzymatic hydrolysis of soy and chickpea protein with Alcalase and Flavourzyme and formation of hydrogen bond mediated insoluble aggregates

Food applications involving plant proteins require modification of their functionality to mimic the unique properties of animal proteins. Enzymatic hydrolysis is commonly used to alter the functionality of plant proteins, particularly to improve their solubility near the isoelectric point. Current methodological approaches mostly indicate improved solubility upon hydrolysis. However, published methods include the removal of insoluble material before analysis, and calculations are based on only the solubilized material as a percentage of the filtered protein. This approach artificially increases solubility estimation and gives an incorrect assessment of the efficacy of hydrolysis. By using the total amount of protein, this study aims to determine the effect of two mi-crobial proteases, Flavourzyme and Alcalase, on the solubility and structural and thermal properties of soy and chickpea proteins. Protein isolates were first extracted from soy and chickpea flour and hydrolyzed from 0 to 3 h. Then, their degree of hydrolysis and solubility at a range of pHs were determined using the o-phthaldialdehyde (OPA) and Lowry methods, respectively. Proteins' electrophoretic mobility, protein-protein interactions, thermal properties, and protein secondary structures were also determined. Solubility decreased over time though the solubility of the hydrolysate improved near the isoelectric point. Soy Flavourzyme hydrolysates remained the most soluble and chickpea Flavourzyme hydrolysates showed the least solubility. Thermal data suggested that Alcalase reduced the protein denaturation temperature, leading to a loss of solubility upon thermal enzyme inactivation. The loss of solubility of hydrolysates was strongly associated with hydrogen bonding, which may result from the formation of polar peptide termini. These results challenge commonly accepted beliefs that hydrolysis inevitably improves solubility of plant proteins. Instead, it is shown that hydrolysis causes structural changes that result in aggregation, thus potentially limiting the application of enzymatic hydrolysis without the addition of further processing methods.