Soybean oil extraction from soybean flour by a neutral metallo-endopeptidase enzyme-assisted aqueous extraction process, optimized for effectiveness in reducing oil content of the solid residue, yields a small fraction of the oil as free oil whereas most is emulsified in a cream layer. Analysis of the cream showed the presence of peptides/proteins and phospholipids, either of which could serve as emulsifiers. Several demulsification treatments targeting these components - protease addition, phospholipase addition, and acidification (pH 45) - were evaluated for effectiveness against the targeted emulsifiers and in conversion of the cream to free oil. Acidification increased the oil yield from 2% to 83%. After a two-stage enzymatic demulsification process with an alkaline endopeptidase, the oil recovery increased to 95%. A lysophospholipase A(1) treatment at pH 4.5 provided complete conversion of emulsified to free oil. The phospholipids (PL) present in the original and post-treatment cream were quantified using phosphorus-31 nuclear magnetic resonance spectroscopy. PL contained phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and their lyso-derivatives. Protein size distribution, PL proportions and fatty acid composition changed after the demulsification treatments. Recycle of the endopeptidase is feasible as more than 90% of its activity was retained. (C) 2009 Elsevier Inc. All rights reserved.