Application of high-resolution ultrasonic spectroscopy for detection of the plasmin activity toward β-casein

Abstract

High-resolution ultrasonic spectroscopy (HR-US) was applied for precise detection of plasmin activity towards β-casein in buffer at pH 7.8 and 37 °C. The evolution of ultrasonic velocity and ultrasonic attenuation measured at 15.5 MHz is related to the concentration of peptide bonds hydrolyzed and loss of β-casein aggregates, respectively. The ultrasonic assay presents sensitive and direct activity-based quantification of plasmin levels in milk. The variation in plasmin concentration between HR-US and ELISA method owed to the differing detection principles. The real-time ultrasonic profiles of hydrolysis were utilized to describe the kinetic aspect of plasmin activity. The non-linear activity curve was fitted with classic and inverse Michaelis–Menten type models. Within 1–8.6 mg·mL−1 β-casein, the Vmax and KM obtained were (6.30 ± 2.21) × 10−5 mol.kg−1·min−1 and 10.33 ± 3.50 mg·mL−1, respectively. The maximum peptide bond cleaved was 5–6 (2.7% degree of hydrolysis) achieved at 1 mg·mL−1 β-casein.