Sphingomyelin-rich domains in bilayer models of the milk fat globule membrane: temperature governs structural and mechanical heterogeneity.
Résumé
The biological membrane enveloping the milk fat globules (MFGM) supplies consumers, especially suckling infants, with bioactive lipids and proteins, and constitutes the interface to digestion or immunity. Bioactive mechanisms depend on biological structure, which itself highly depend on temperature. However, the polar lipid assembly and biophysical properties of the MFGM are yet poorly known, especially in connection with the temperature history that milk can experience upon storage or processing and up to consumption, e.g. in human milk banks or in dietary intake of bovine milk. Noteworthy, in all mammalian milks, polar lipids of the MFGM include a significant proportion of species with a high phase transition temperature (Tm), especially milk sphingomyelin (MSM; ~30% w/w of the total polar lipids; Tm = 34°C – Murthy et al., 2015), that is held responsible for the formation of lipid domains at the surface of the milk fat globule [br/]
The objective of this study was therefore to investigate the polar lipid packing in hydrated bilayers prepared with a MFGM extract, and to follow cooling-induced changes at inter-molecular level using a combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (XRD), atomic force microscopy (AFM) imaging and force spectroscopy. On cooling, a liquid disordered (ld) to solid ordered (so) phase transition of MSM (and other high-Tm polar lipids) in MFGM bilayers started at ~40°C and reached its maximum at 30.3°C. Using temperature-controlled AFM, phase separation was observed for temperatures below 35°C, with formation of so phase domains with a height difference H 1 nm from the continuous ld phase (Fig.2). In complement to AFM topographical images, indentation measurements to map the yield (or breakthrough) force over the imaged surfaces showed that the mechanical stability was significantly higher for the so phase domains than for the ld phase. Also, the mechanical stability of both the domains and the fluid phase increased with decreasing temperature, probably as a result of lower molecular agitation and increased ordering. However, lipid packing, integrity and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle, which may have important consequences in handling milk samples in neonatal or food applications[br/]
For the first time, AFM was successfully used to report correlated structural and mechanical changes in hydrated multi-component bilayer models of a complex biological membrane: that of the milk fat globule. AFM is therefore a promising tool to investigate temperature-induced changes in intermolecular forces within biologically relevant membranes, with nanoscale resolution.