Hydrothermal cooking (HTC) combined with amylase pretreatment (AP) was used to improve protein extraction from heat-stabilized rice bran. The physicochemical and emulsifying properties of rice bran protein isolate (RPI) were evaluated. Depending on HTC temperature (120 and 150 degrees C), HTC alone significantly increased extraction yield, while protein purity was decreased. In contrast, HTC combined with AP significantly improved both extraction yield and protein purity (about 45-50% and 72-74%, respectively). The AP avoided the co-precipitation of gelatinized starch during the acidic precipitation. Electrophoresis and size exclusion chromatography profiles indicated that HTC led to the dissociation of insoluble protein aggregates in rice bran, with subsequent increase of soluble aggregates in RPI, linked by non-covalent (e.g., hydrophobic interaction) and covalent bonds (disulfide bond). This result was evidenced by the increased disulfide bond contents and surface hydrophobicity of RPI. In addition, HTC-prepared RPI exhibited excellent emulsifying property. (C) 2011 Elsevier Ltd. All rights reserved.