SPONTANEOUS INTERACTION OF LACTOFERRIN OR LYSOZYME WITH THE CASEIN MICELLES IN MILK

The casein micelles in milk are naturalself-assembled colloidal particles composed of four casein proteins and acalcium phosphate mineral phase. At the natural pH of milk, casein micelles areprimarily stabilized by a surface layer of κ-casein; however, they also possessa net negative charge as the isoelectric point is about pH 4.6. When positivelycharged globular proteins such as lactotransferrin or lysozyme are added tomilk, they spontaneously interact with the casein micelles. When 2% w/w(~0.25mM) lactotransferrin was added to milk, about 1.25% w/w (0.15mM) bound tothe casein micelles and this binding resulted in a decrease in the absolutevalue of the zeta potential and an initial increase in the casein micelle size.On prolonged holding, the casein micelles disintegrated after addition of lactotransferrin.Out of 1% w/w (0.7mM) added lysozyme, about 0.6% (0.4mM) bound to the caseinmicelles; however this binding did not alter the zetapotential of the casein micelles, and the casein micelle size was unaffected ataddition levels up to ~0.5% w/w. Higher addition levels caused the spontaneousand irreversible flocculation of the casein micelle system. Using differentialcentrifugation techniques, the casein micelles were fractionated into differentsize classes. By comparing the lactotransferrin/casein or lysozyme/caseinratios it was possible to determine whether lactotransferrin or lysozyme werepreferentially binding to the surface or interior of the casein micelles. Lactotransferrinpreferentially bound to the surface of the casein micelles whereas lysozymepreferentially bound to the interior. It is suggested that lactotransferrin(~80kDa) is too large (8nm) compared to the mesh size of the casein proteinmatrix (estimated at 4.5nm). In contrast lysozyme (14kDa) is much smaller thanthe mesh size and can enter the interior of the casein micelle. The differentmode of binding accounts for the contrasting effects on the properties (zetapotential, size) and stability (disintegration or flocculation) when lactotransferrinor lysozyme are added to the milk.