Integrated impact of extraction and fractionation methods on the physicochemical, thermal, and functional properties of black bean proteins

This study investigated the combined effect of extraction and fractionation methods on black bean proteins’ physicochemical, thermal, and functional properties. Black bean flour was processed by two extraction methods: one using enzymes and one without, with a 1:10 solids-to-liquid ratio (50 °C, 60 min). In the aqueous extraction process (AEP), the pH was adjusted to 7, while in the enzyme-assisted aqueous extraction process (EAEP), the pH was set to 9 with 0.5% alkaline protease. Both extracts underwent isoelectric precipitation at pH 4.5, followed by centrifugation and neutralization to obtain whey and curd fractions. Results showed that the EAEP yielded higher protein extraction, 78.1% for the concentrate, 32.0% for the whey, and 45.0% for the curd, compared to the AEP fractions, with 66.6%, 21.1%, and 40.5%, respectively. Additionally, the EAEP fractions exhibited higher total phenolic content compared to the AEP fractions (concentrate: 23.6 vs. 15.0 mg GAE/g powder, whey: 14.0 vs. 10.4 mg GAE/g powder, and curd: 32.0 vs. 16.7 mg GAE/g powder). EAEP resulted in protein fractions with double the antioxidant potential (ORAC and ABTS). Regardless of the extraction method, the curd fraction exhibited higher water retention and oil absorption capacities than the concentrate and whey fractions. The solubility of all protein fractions was highest at pH 7, with minimal differences between AEP and EAEP. Thermal stability was affected by enzymatic extraction, with the EAEP concentrate showing lower T_o (85.4 °C) and T_d (97.3 °C) than AEP (T_o: 93.0 °C, T_d: 98.7 °C). Whey and curd from EAEP had higher denaturation temperatures compared to AEP. Zeta potential analysis revealed that the concentrate and curd fractions had isoelectric points between pH 3-5, whereas the whey fraction exhibited a pI below pH 3. These functional properties highlight their potential for use in functional food applications.