Abstract: A two-step enzymatic method was presented for soybean protein extraction by sequential hydrolysis with multiple enzymes (simultaneously added) and alcalase. The multiple-enzyme hydrolysis process was optimized by using mixed design to modeling the effects of cellulase amount, hemicellulase amount, pectin amount and hydrolysis time on total protein extraction efficiency. The optimal hydrolysis conditions for soybean protein extraction were hydrolysis at pH 5, 37 ℃ and a material/liquid ratio of 1:6 (g/mL) for 0.75 h with 0.64% cellulase and 0.56% hemicellulase and for another 3.6 h at 50 ℃ and pH 9.26 with 1.85% alcalase. Under these conditions, a maximum total protein extraction efficiency of 85.78% was observed. Multiple-enzyme hydrolysis caused an increase in total protein extraction efficiency by nearly 10% when compared with traditional heat-moisture treatment. We speculated that the reason for this was due to complete disruption of the soybean cellular structure after multiple-enzyme hydrolysis. However, the detailed mechanisms remain to be clarified. 

Key words: aqueous enzymatic method, multiple-enzyme hydrolysis, soybean protein, mixed design