Co-acervates of lactoferrin and caseins

On mixing positively charged lactoferrin (LF) with negatively charged caseins (*CN) it is observed that complexes are formed. The * stands for α, β, κ or Na. The size of the complex co-acervates appears to grow indefinitely and asymptotically near the point of charge equivalency. Away from the charge equivalent ratio it seems that build-up of (surface) charges limits complex size. We proposed a simple scaling law so as to predict the size of the complex. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1 − x/xcrit)|−3 where x is the mole (mass) fraction of the cationic protein and xcrit the value of the mole (mass) fraction at the charge equivalent ratio. Both scattering intensity and particle size obey this simple assumption. We investigated three different caseins, all of which formed co-acervate complexes with LF, but at different molar ratios. Critical composition varied inversely with pH, showing that charge neutrality is the determining factor. Sodium caseinate formed complexes as well but the growth was limited, presumably due to the intrinsic surfactant properties of whole casein. Adding NaCl diminishes the interaction and above 0.4 mol L−1 of NaCl no β-CN–LF complexes are formed. The charge neutral composition shifts to the LF side on adding NaCl, probably because the casein can wrap around the LF more effectively

Lactoferrin binding to transglutaminase cross-linked casein micelles

Casein micelles in skim milk were either untreated (untreated milk) or were cross-linked using transglutaminase (TGA-milk). Added lactoferrin (LF) bound to the casein micelles and followed Langmuir adsorption isotherms. The adsorption level was the same in both milks and decreased the micellar zeta potential, indicating charge neutralization and the formation of complex coacervate-type interactions. For the untreated milks, the adsorption of LF was initially accompanied by an increase in turbidity of the milk and in the size of the casein micelles; however, after several hours, the turbidity and the casein micelle size of these milks decreased markedly. For the TGA-milks, no change in casein micelle size was observed on adsorption of LF, but the turbidity increased due to the increased mass of the casein micelles, and remained constant on holding. These results indicate that the cross-linking of the casein micelles prior to adding LF prevents disintegration of the casein micelles

Coacervates of lysozyme and β-casein

Complexes are formed when positively charged lysozyme (LYZ) is mixed with negatively charged caseins. Adding b-casein (BCN) to LYZ leads to flocculation even at low addition levels. Titrating LYZ into BCN shows that complexes are formed up to a critical composition (x = [LYZ]/([LYZ] + [BCN]). The formation of these complex coacervates increases asymptotically toward the molar charge equivalent ratio (xcrit), where the size of the complexes also seems to grow asymptotically. At xcrit, insoluble precipitates of charge-neutral complexes are formed. The precipitates can be re-dispersed by adding NaCl. The value of xcrit shifts to higher values on the LYZ side with increasing salt concentration and pH. Increasing the pH, de-protonates the BCN and protonates the LYZ, and therefore, charge neutrality will shift toward the LYZ side. xcrit increases linearly from 0.2 at no salt to 0.5 at 0.5 M NaCl. It ends abruptly at a salt concentration of 0.5 M after which a clear mixed solution remains. Away from the charge equivalent ratio, it seems that the buildup of charges limits the complex size. A simple scaling law to predict the size of the complex is proposed. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1 x/xcrit)| 3 where x is the mole fraction of one protein and xcrit the value of the mole fraction at the charge equivalent ratio. Both scattering intensity and particle size seem to obey this simple assumption. For BCN–LYZ, the buildup occurs only at the LYZside in contrast to lactoferrin which forms stable complexes on either side of xcrit. The reason that the complexes are formed at the BCN side only may be due to the small size of LYZ, which induces a bending energy in the BCN on adsorption

Coacervates of lysozyme and β-casein

Complexes are formed when positively charged lysozyme (LYZ) is mixed with negatively charged caseins. Adding b-casein (BCN) to LYZ leads to flocculation even at low addition levels. Titrating LYZ into BCN shows that complexes are formed up to a critical composition (x = [LYZ]/([LYZ] + [BCN]). The formation of these complex coacervates increases asymptotically toward the molar charge equivalent ratio (xcrit), where the size of the complexes also seems to grow asymptotically. At xcrit, insoluble precipitates of charge-neutral complexes are formed. The precipitates can be re-dispersed by adding NaCl. The value of xcrit shifts to higher values on the LYZ side with increasing salt concentration and pH. Increasing the pH, de-protonates the BCN and protonates the LYZ, and therefore, charge neutrality will shift toward the LYZ side. xcrit increases linearly from 0.2 at no salt to 0.5 at 0.5 M NaCl. It ends abruptly at a salt concentration of 0.5 M after which a clear mixed solution remains. Away from the charge equivalent ratio, it seems that the buildup of charges limits the complex size. A simple scaling law to predict the size of the complex is proposed. By assuming that surface charge density is constant or can reach only a maximum value, it follows that scattering intensity is proportional to |(1 x/xcrit)| 3 where x is the mole fraction of one protein and xcrit the value of the mole fraction at the charge equivalent ratio. Both scattering intensity and particle size seem to obey this simple assumption. For BCN–LYZ, the buildup occurs only at the LYZside in contrast to lactoferrin which forms stable complexes on either side of xcrit. The reason that the complexes are formed at the BCN side only may be due to the small size of LYZ, which induces a bending energy in the BCN on adsorption