Comparative assessment of ultrasound and enzyme assisted extraction of rice bran protein following sequential pretreatments: yield, functional properties improvement, and potential application

• Rice bran protein yield was increased by 3 to 5 % with sequential pretreatment. • Enzyme-assisted extraction achieved higher protein purity (89.7 %) than ultrasound (84.3 %). • Degree of hydrolysis was improved from 18.3 % (EAE) to 28.4 % (SPT-EAE). • Water-binding (4.8 g/g) and oil-binding (2.7 g/g) were enhanced in EAE proteins. • Rice bran protein fortified bread showed improved moisture retention and texture. Rice bran is an abundant byproduct of rice milling. It is a rich source of high-quality protein yet remains underutilized due to extraction challenges. This study compared ultrasound-assisted extraction (UAE) and enzyme-assisted extraction (EAE) following an optimized sequential pretreatment (SPT) involving thermal stabilization, washing, defatting, and particle size reduction to maximize rice bran protein (RBP) yield and functionality. Pretreatment significantly enhanced extraction efficiency, increasing protein yield by 3–5 % for both methods. EAE produced higher protein purity (89.7 %) than UAE (84.3 %) and improved functional properties, including solubility, water and oil binding capacities, emulsifying activity, and foam stability. Structural analyses using FTIR, sulfhydryl/disulfide content, surface hydrophobicity indicated that pretreatment promoted protein unfolding and enhanced enzymatic accessibility while maintaining structural integrity. Moreover, Degree of hydrolysis increased notably in SPT-EAE (28.4 %) compared to untreated EAE (18.3 %). In bread formulations, RBP fortification increased protein content and moisture retention but also led to reduced loaf volume and higher crumb firmness. Economic assessment showed that despite higher enzyme costs, EAE reduced energy consumption and environmental impact relative to UAE. Overall, integrating SPT with EAE offers a efficient and sustainable pathway for valorizing rice bran into high-quality protein ingredients suitable for functional food applications.