Integrated enzymatic and sonication strategy for sustainable soybean processing: from cell wall deconstruction to product separation

Conventional soybean processing relies on hexane extraction and high-temperature treatments, which cause protein denaturation and hinder the separate recovery of oil, protein, and carbohydrate. To address this limitation, this study aimed to establish a sustainable enzymatic soybean processing (ESP) strategy for separate collection of all three major components: intact oil bodies, undenatured protein, and monomerized carbohydrate. This is the first demonstration that ESP efficiency can be significantly enhanced by integrating pulsed sonication. Multi-enzyme systems were produced via solid-state fermentation (SSF) of soyhull by Aspergillus niger and applied to cracked soybean particles. Screening of 15 SSF enzyme extracts revealed that pectinase, polygalacturonase, and invertase were the limiting carbohydrase activities for cell wall degradation. Effects of processing variables including protease activity, reaction media, and reaction time were evaluated to minimize protein loss. Using water instead of citrate buffer as reaction medium and limiting processing time to ≤ 48 h reduced protein dissolution to below 20%. ESP was further enhanced through pulsed probe-sonication (12 W/mL, 1 s on/23 s off), which reduced processing time to 24 h while increasing carbohydrate solubilization to up to approximately 90% depending on enzyme loading. Simple centrifugation enabled efficient fractionation into intact oil bodies (100%), native proteins (≈70%), and hydrolysates containing soluble proteins (≈30%) and monomerized carbohydrate (≈90%). These findings demonstrate an integrated enzymatic-sonication approach that enables hexane-free, low-temperature soybean processing with minimally denatured, high-value products and offers a pathway for sustainable soybean biorefinery.