This study assesses subcritical water hydrolysis (SWH) as a sustainable approach for protein recovery from okara, following isoflavone valorization. The initial extract, obtained at 120 °C, contained over 1200 µg of isoflavones per gram of dry okara. The solid residue underwent a second protein extraction step in both batch and semi-continuous modes. The batch extraction was conducted at 180 °C for 3 h at 5 MPa in a 0.5 L reactor. Under these conditions, total amino acid recovery reached 81 ± 3% (12.6 g/L), with 20 ± 1% of free amino acids, mainly glutamic acid, and a favorable profile of essential amino acids. These results were comparable to protein extraction from untreated okara, indicating that the prior isoflavone extraction did not hinder protein hydrolysis, but rather contributed positively within the cascade framework. Hydrolysis was also performed in a semi-continuous mode in a 2.3 L reactor, with a residence time of 2.75 min, in a two-stage temperature process (180 °C and 270 °C). The second stage yielded a significantly higher extraction than the first one, achieving a total 70.4% protein recovery (1.22 g/L) with 10% of free amino acids, with glutamic acid as the dominant one. Carbohydrate analysis revealed high hydrolysis yields for galactans, arabinans, and xylans, but these exhibited lower thermal stability compared to proteins. Okara emerges as a promising by-product for a bio-cascade process, combining a mild first stage for isoflavone extraction with a more severe second stage for protein extraction/hydrolysis.
