Single-, dual-, and triple-frequency ultrasound, alone or combined with subcritical water, governs selective phenolic recovery and isoflavone profiles in purified defatted soybean meal extracts

Abstract Defatted soybean meal is a rich but underutilized source of phenolic compounds, yet conventional extraction gives low recovery and poor selectivity. This study compared single-frequency (SF; 60 kHz), dual-frequency (DF; 28/60 kHz), and triple-frequency (TF; 20/28/60 kHz) ultrasound, applied alone or followed by subcritical water extraction (SWE; 140°C, 25 min) as SUSWE, DUSWE, and TUSWE, with hot-water extraction (HWE) and SWE alone as references. Compared with HWE, TF doubled phenolic recovery (77.69 vs. 38.66 mg GAE/100 g meal), gave the highest total phenolic content (38.45 mg GAE/g), and increased DPPH radical-scavenging activity from 28.93% to 51.06%. SUSWE gave the highest purified extract yield (2.49%) and flavonoid recovery (92.76 mg QE/100 g meal), whereas DF gave the highest flavonoid concentration (41.79 mg QE/g). The benefit of SWE depended on the preceding ultrasound step: it improved recovery after SF, added little after DF, and, after TF, reduced phenolic recovery by 28.7% and DPPH activity by 73.0%. UPLC-ESI-QTRAP-MS/MS profiling showed that TF raised the daidzein/daidzin ion-signal ratio from 0.143 (HWE) to 0.590 and the glycitein/glycitin ratio from 0.365 to 1.173 at only 45°C, whereas TUSWE lowered them to 0.195 and 0.297, respectively, indicating that the aglycone-rich fraction released by TF was thermally degraded or diluted by newly extracted glycosides during the 140°C step. Ultrasound frequency configuration therefore determines both extract composition and the value of a subsequent hydrothermal step, and the sequential process is beneficial only when SWE complements, rather than overrides, the selectivity established by ultrasound. These findings provide a direct process reference and theoretical basis for the green, targeted preparation of phenolic and isoflavone ingredients from soybean meal and other oilseed by-products.