A Strategy for Sustainable Production of Isoflavones from Black Soybean Okara via Solid-State Co-Fermentation

Okara, the soybean residue generated during soymilk and tofu production, is nutrient-rich but underutilized due to its high moisture content and perishability. This study established a sustainable solid-state co-fermentation strategy using Rhizopus oligosporus (BCRC 31631) and Yarrowia lipolytica (BCRC 21252) to enhance the bioactive value of black soybean okara from two Taiwanese cultivars—Tainan No. 3 (TN.3) and Tainan No. 5 (TN.5). Co-fermentation markedly enhanced β-glucosidase activity, reaching 0.75 U/g DW at 30 °C after 48 h in TN.3 and 0.68 U/g DW after 24 h in TN.5, approximately 3.5-fold higher than single-strain fermentation. Near-complete (97–100%) hydrolysis of daidzin, glycitin, and genistin occurred within 24–48 h, producing 672.9 µg/g DM of total aglycone-type isoflavones—an ~11-fold increase compared with unfermented okara (61.5 µg/g DM), where most isoflavones (~740 µg/g DM) remained glycosylated. Varietal structure affected conversion efficiency: TN.3 exhibited stronger enzymatic responsiveness and higher aglycone yield, whereas TN.5 showed faster but less extensive transformation. The process also revealed a bioactive–pigment trade-off, as enhanced isoflavone activation coincided with anthocyanin degradation. These findings demonstrate that ambient-temperature solid-state co-fermentation effectively boosts β-glucosidase activity and isoflavone bioconversion, transforming low-value okara into a high-value functional ingredient consistent with circular bioeconomy and sustainable food system goals.