Pressure-induced aggregation of β-lactoglobulin in ph 7.0 buffers

Solutions of a β-lactoglobulin isolate in water, in potassium phosphate buffer (20 or 50 mmol/L), and in pressure-resistant buffers, at a protein concentration of 25 g/kg and at pH 7.0, were processed at 150, 250, 350 or 450 MPa and 25 °C, for 15 min, then stored at 4 °C, usually for 24 h, before analysis. Bis-Tris (20 or 50 mmol/L) and bis-Tris-propane (10, 20 or 50 mmol/L) were selected as pressure-resistant buffers, while the pH of water or phosphate buffer is known to decrease reversibly by 0.2–0.3 pH unit per 100 MPa. After pressurization, nitrogen solubility at pH 4.7 and 7.0, and aggregation patterns by polyacrylamide gel electrophoresis were determined. Both indicated that aggregation of β-lactoglobulin was more extensive in pressure-resistant buffers than in phosphate buffer or in water. Electrophoretic patterns also revealed the progressive formation of dimers to hexamers and of higher polymers of β-lactoglobulin as a function of the type and molarity of buffer and of the pressure level. All high molecular weight aggregates and most oligomers disappeared when pressurized solutions were treated with β-mercaptoethanol (15 mL/L) prior to electrophoresis. Thus pressure induced the formation of intermolecular S-S bonds, especially when the pH was maintained close to neutral. Previous studies with thermally-induced β-lactoglobulin gels had shown that SH/ S-S interchange reactions were enhanced at or above neutrality.