Understanding the Structure of the Bovine Milk Fat Globule and its Membrane by Means of Microscopic Techniques and Model Systems
Gallier, Sophie Yvette Fabienne Christine
The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied but its structure, especially the lateral organization of its components, still remains unclear. The aim of this project was to improve the knowledge about the structure of the bovine milk fat globule and, in particular, its membrane by means of microscopic techniques and model systems. Phospholipids are the backbone of the MFGM structure. Different techniques of phospholipid extraction were carried out on buttermilk powder to determine the technique most reproducible and giving the highest recovery of sphingomyelin. Natural complex mixtures of phospholipids from raw milk, raw cream, homogenized and pasteurized milk, and buttermilk powder were recovered through total lipid extraction following the Folch method and solid-phase extraction using the Bitman method. All mixtures were analyzed using electronspray-ionization tandem mass spectrometry to determine their phospholipid profile and their fatty acid distribution, and also to reveal the effect of milk processing on the phospholipid composition. Confocal-Raman microspectroscopy was used to investigate the lipids of the milk fat globules. A comparison of the fat globule composition between two breeds of cows and between globules of different size was carried out and revealed differences in lipid content and fatty acid distribution. Confocal-Raman microspectroscopy provided information on both the lipid core and the milk fat globule membrane. Milk fat globules from raw milk, raw cream and processed milk and reconstituted buttermilk powder were stained with fluorescent probes and observed with a confocal laser scanning microscope (CLSM). Domains, which are thought to be rich in sphingomyelin and cholesterol, were observed on the surface of the native globules. These domains, also called lipid rafts, are a liquid-ordered, lo, phase coexisting with a liquid-disordered, ld, phase. Phase separation, distribution of glycoproteins and glycolipids and association of milk proteins with the MFGM were determined in all samples. Lipid microdomains, analogous to lipid rafts in cell membranes, were observed at the surface of the globules. Glycoproteins and glycolipids were heterogeneously distributed within the MFGM and located outside of the lipid microdomains. Proteins were found associated in a higher amount with the fat globule membrane after processing the milk. The temperature effect on phase separation was also assessed. Temperature induced a change in the size, shape and number of the lipid microdomains at the surface of the globules. A comparative study with the milk fat globule membrane from mares’ milk revealed similar phase coexistence at the surface of the globules. The observation in situ with non-invasive techniques provides further progress in the understanding of the lateral heterogeneities existing within the MFGM. Over the past few years, increasing attention has been given to the study of the functions and properties of lateral microdomains in biological membranes. However the environmental conditions of the native membrane system, such as temperature and pressure, cannot be readily modified and their effects on the MFGM monitored. Therefore the use of model systems is a very promising tool to investigate the effect of temperature and pressure on this system. The physical behavior of the phospholipid monolayers from each dairy product was investigated with a Langmuir trough mounted on an epifluorescence microscope to probe the impact of milk processing. Phase separation was observed as a function of varying pressure (0mN/m to 60mN/m) and temperature (16, 20, 24 and 27°C). The surface pressure (π)-specific area (A) isotherms showed a similar trend, however the phase coexistence under epifluorescence microscopy revealed different patterns with different phospholipid sources. The surface pressure, the temperature, the phospholipid composition, the degree of saturation and the fatty acid chain length were factors determining the shape and number of the liquid-ordered domains. Atomic force microscopy (AFM) was used as a complementary method to reveal topographic nanometer-size height differences between the liquid-ordered phase and the liquid-disordered phase. To reach a closer model system to mimic the MFGM, an abundant milk protein, β-casein, was added sub-phase to the phospholipid monolayer and the effects on the compression isotherms and the shape of the liquid-ordered domains were followed at different pressures and temperatures for each phospholipid monolayer film. AFM was used to locate the protein within the film as the detection of the protein location was not possible within Langmuir phospholipid-protein films by epifluorescence microscopy. AFM imaging revealed the branching of β-casein molecules, clustering three to four liquid-ordered domains together. Even though the composition of the MFGM is well established, there are still unanswered fundamental questions concerning the structure and dynamics of the MFGM and the existence of rafts within this unique trilayer membrane. The aim of this thesis was to study the native intact and processed MFGM, as well as complementary investigations of model monolayer systems composed of natural complex mixtures of phospholipids present in the MFGM and milk proteins. Model system studies gave critical insights into the role of lipid-lipid and lipid-protein interactions in raft formation and function in the MFGM.
Advisor: Everett, David W.; Jiménez-Flores, Rafael
Degree Name: Doctor of Philosophy
Degree Discipline: Food Science
Publisher: University of Otago
Keywords: milk fat globule membrane; microscopy; model systems