A bioguided approach to elucidate the impact of extraction parameters on the extractability, nutritional, functional, and biological properties of black bean proteins

Abstract: Mainstream adoption of new protein sources requires a holistic understanding of the role of key extraction conditions on the nutritional, functional, and biological properties of the extracted protein. The effects of extraction medium, time, pH, solids-to-liquid ratio (SLR), and type/amount of enzyme on black bean protein extractability, composition, and functionality were evaluated. Black bean flour was sequentially extracted to produce albumin- (56%), globulin- (22%), prolamin- (0.65%), and glutelin-rich (16%) fractions. Although proteomics revealed similar composition in all fractions, secondary structure analysis revealed that albumin- and globulin-rich fractions had the highest α-helix (19%) and β-sheet (36%) contents, respectively, while the glutelin-rich fraction had no α-helix structures. Aqueous and enzymatically extracted proteins (EAEP) had similar composition and molecular weight distributions to albumin- and globulin-rich fractions. The thermal denaturation temperature of AEP/EAEP proteins was significantly lower (92 °C) than that of albumin- and globulin-rich fractions (99-101 °C), suggesting partial protein denaturation due to alkaline extraction conditions. Proteolysis improved protein solubility in acidic conditions (50 vs. 93%) but decreased foaming capacity in acidic and neutral conditions. The efficacy of additional enzymes (Bromelain, Flavourzyme®, Neutral Protease, Papain, FoodPro® Alkaline Protease, and FoodPro® CBL) was assessed, along with the effect of extraction pH (7.0, 9.0, 5.0/9.0 shift, 7.0/9.0 shift), with respect to TPE, solubility, and in vitro protein digestibility. Alkaline extraction increased protein extractability from 66 to 75%% but reduced protein solubility due to aggregation. However, solubility was subsequently improved using a protease to assist the extraction. The use of a carbohydrase pretreatment followed by extraction with alkaline protease did not increase TPE, compared to extraction with alkaline protease alone (81-82% TPE), but all pH 5.0/9.0 extractions had higher in vitro digestibility (80-95 vs. 35-43%), suggesting that acid-induced denaturation followed by enzymatic extraction may have caused protein structural modifications that facilitated proteolysis by digestive enzymes.