Acidic pretreatment followed by protease-assisted alkaline extraction improves the extractability, solubility, and in vitro digestibility of black bean proteins

Abstract: Plant-based proteins are commonly produced using aqueous (AEP) and enzyme-assisted aqueous extraction processes (EAEP). However, there is limited information on the impact of key extraction parameters (e.g., pH shift, type/amount of enzyme) beyond protein extraction yields. Therefore, this study aimed to holistically evaluate the integrated impact of extraction pH and enzyme use with respect to total protein extractability (TPE), solubility, and in vitro protein digestibility. The effect of pH shifting (5/7 and 5/9) in the AEP (1:10 solids-to-liquid ratio, 50 °C, 60 min) and addition of commercial proteases (Bromelain (B), Flavourzyme® (F), Neutral Protease (NP), and Papain (P) at pH 7.0, and Alkaline Protease (AP) at pH 9.0) in the EAEP (same conditions as AEP with 0.5% w/w enzyme) were evaluated alone or preceded by a carbohydrase (CBL) pretreatment (pH 5.0, 30 min, 50 °C, 0.5% w/w CBL). While neutral enzymatic extractions (B, F, NP, P) did not significantly alter TPE (64-68%) compared to the AEP (66%), alkaline extraction with AP significantly improved TPE (81-82% EAEP vs. 75% AEP) irrespective of carbohydrase pretreatment. Despite high extractability, alkaline extraction decreased protein solubility compared to neutral extraction, potentially due to protein aggregation as evidenced by high-molecular weight bands in SDS-PAGE. However, protease addition significantly improved solubility at pH 4.0 for all EAEP proteins, with AP and CBL/NP achieving 61 and 65% solubility, respectively. Proteins extracted with a carbohydrase pretreatment followed by alkaline extraction exhibited the highest in-vitro protein digestibility (80-95%) regardless of enzyme use. This suggests that the acidic pretreatment may have induced partial denaturation, which following the shift to alkaline conditions, facilitated accessibility to digestive enzymes. Overall, the sequential use of CBL and AP yielded high extractability, solubility, and digestibility, highlighting the role of pH and proteolysis on developing successful extraction strategies to produce more functional black bean protein ingredients.