Influence of high intensity ultrasound and calcium chelating agent on structural and physicochemical properties of casein micelles

Introduction: This study investigates the combined effect of ultrasonication time (UST) and disodium phosphate (DSP) - Na₂HPO₄, used as a calcium chelating agent, on the structural and physicochemical properties of casein micelles.

Methods: Micellar casein concentrate (MCC) was prepared at 3 % (w/w) casein. DSP was added at concentrations of 0, 5, and 10 mM, followed by pH adjustment to 7.0. Ultrasonication was applied at 20 kHz and 550 W for 0, 10, and 20 min in pulsed mode (5 s ON/OFF) at 25 °C. Response Surface Methodology (RSM) was used to evaluate independent and combined effects of UST and DSP. The response variables included non-micellar calcium and magnesium, protein solubility index (PSI), hydrodynamic diameter (Dh), ζ-potential, surface hydrophobicity, viscosity and secondary structure.

Results: Increasing both UST and DSP significantly increased non-micellar calcium concentration (from 238.4 to 327.2 mg/kg) and PSI (from 29.0 % to 44.1 %), while surface hydrophobicity decreased significantly (p < 0.0001). Dh decreased from 206.1 to 186.5 nm with increasing UST up to 10 min but increased with increasing DSP at all USTs (p < 0.0001). Increasing DSP led to a more negative ζ-potential, decreasing from –16.1 mV at 0 mM DSP to –20.4 mV at 10 mM DSP in the absence of sonication (p < 0.0001). DSP and UST showed no significant effect on viscosity. FTIR analysis indicated limited effects of the treatments on the secondary structure of caseins. Overall, the combination of ultrasound treatment and calcium chelation altered key physicochemical properties of micellar caseins, which may be explored in future studies to assess their impact on functional properties.