In the present study, characterization and destabilization of the emulsions formed during two alternative processes, traditional aqueous extraction and enzyme-assisted aqueous extraction, for oil extractability from soybean flour were investigated. The emulsions were collected as cream layers and these were subjected to various single and combined treatments, including thermal, chemical and enzymatic treatments, aimed at recovery of free oil.;The soybean oil emulsion formed during the aqueous extraction processing (AEP) contained high molecular weight glycinin and beta-conglycinin proteins and smaller oleosin proteins which formed a multilayer interface. This interface provided protection against droplet aggregation before demulsification treatment and centrifugation. Treatment with Protex 7L (protease) increased the free oil recovery from 3% to 23%. When enzymatic treatment was followed by a freeze-thaw step, the oil recovery increased to 46%. The increase can be attributed to the combined effect of film degradation by Protex 7L and partial coalescence of the oil droplets in the frozen emulsion.;The cream formed during the alternative enzyme-assisted aqueous extraction process (EA-AEP) with Protex 7L also contained polypeptides and phospholipids as emulsifiers. The SDS-PAGE profile of the polypeptides in the cream layer showed that the subunits of beta-conglycinin and glycinin were fully degraded after 2 h of extraction. Only small residual polypeptides remained but these were still sufficient to form a multilayer interface capable of providing stability to the cream. Chemical demulsification by adjusting the pH to 4.5 increased the oil yield from 2% to 83%. A two-stage enzymatic demulsification process with Protex 6L (protease) increased to 95% the oil recovery. When enzymatic demulsification with G-ZYME (A1 lyso-phospholipase) was combined with pH 4.5, the oil recovery increased to 100%. The mean droplet size increased significantly in each demulsification treatment facilitating the coalescence/aggregation of the oil droplets. The increase in free oil recovery can be attributed to the disruption of the interfacial film due to enzymatic hydrolysis, partial coalescence during incubation, and coalescence after centrifugation. Protex 6L retained more than 90% of its activity after a single demulsification treatment offering the potential for recycle in the process. Despite the reduction in emulsion stability and the high recovery of free oil from the cream further improvements in the extraction step are necessary to release more oil to the cream. Modification of the extraction step will require additional demulsification studies as the demulsification behavior depends on how the cream is obtained.