The microstructure of the milk fat globule membrane (MFGM) is still poorly understood. The aim of this study was to investigate the dynamics of the MFGM at the surface of milk fat globules in relation to temperature and time, and in relation to the respective lipid compositions of the MFGM from bovine, goat and sheep milks. In-situ structural investigations were performed using confocal microscopy. Lipid domains were observed over a wide range of temperatures (4-60°C). We demonstrated that rapid cooling of milk enhances the mechanisms of nucleation and that extended storage induces lipid reorganization within the MFGM with growth, leading to circular lipid domains. Diffusion of the lipid domains, coalescence and reduction in domain size were observed upon heating. Different MFGM features could be related to the respective cholesterol/sphingomyelin molar ratio in the three milk species. These structural changes may affect the interfacial properties of the MFGM, with consequences for the functional properties of fat globules and the mechanisms of their digestion.
The milk sphingomyelin (MSM) is involved in the formation of ordered lipid domains in the biological milk fat globule membrane (MFGM), where it accounts for about 30%wt of the polar lipids. Moreover, MSM exhibits a large variety in saturated acyl chain lengths (from C16:0 to C24:0-SM) compared to other natural sphingomyelins, which may impact the packing of MSM molecular species in the gel phase domains and the topography of the MFGM. To investigate this, supported lipid bilayers of synthetic sphingomyelins or of MSM-containing mixtures, including a MFGM polar lipid extract, were imaged at temperatures below the Tm of MSM (i.e. <34°C for which MSM is in the gel phase) in hydrated conditions using atomic force microscopy. In all compositions containing MSM, the MSM-rich gel phase domains exhibited lower and upper height levels H, interpreted as two distinct gel phases with ∆H~0.5-1.1nm. Two (lower and upper) gel phases were also found for pure C24:0-SM bilayers or for bilayers of a C16:0-SM/C24:0-SM equimolar mixture, while C16:0-SM bilayers were uniformly flat and less thick than C24:0-SM bilayers. The upper gel phase of MSM-containing bilayers was interpreted as mixed interdigitated C24:0-SM molecules, while the lower gel phase was attributed both to fully interdigitated C24:0-SM molecules and non-interdigitated C16:0-SM molecules. These results show that the composition of natural sphingomyelins, inducing a mismatch between the d18:1 sphingosine and the acyl chains, is important in both the internal organization and the topography of biological membranes, especially that of the MFGM. This organization could be involved in specific biological functions, e.g. the insertion of proteins.
The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20°C and 50°C using atomic force microscopy (AFM) and force spectroscopy. At 20°C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10-20 MPa and 4-6 MPa, respectively. Upon heating at 50°C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0-5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50°C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties.
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