Hétérogénéité des membranes lipidiques et propriétés mécaniques : des bicouches modèles aux membranes des globules gras du lait

ProdInra 422327The milk fat globules are enveloped by a biological membrane, called MFGM, of highly complex composition and structure. Investigation of this membrane, in situ in milk, using confocal microscopy suggested that polar lipids with high transition temperature (Tm) form domains in gel or liquid-ordered phase, dispersed in a continuous fluid phase. From this observation, the aim of this project was to understand how the composition and organization of dairy polar lipids can modulate the elastic properties of the MFGM, in order to better control stability of the fat globules in the dairy industry. The mechanical heterogeneity created by the coexistence of phases was then characterized by AFM force spectroscopy using lipid bilayers models at low (TTm).In order to closely analyze the factors that direct membrane elasticity, force spectroscopy measurements were undertaken on curved liposome membranes, in combination with structural characterization by TEM and SAXS. We showed, in particular, that heterogeneity in acyl chain length and unsaturation made gel-phase milk sphingomyelin membranes less rigid than expected, although more rigid than a fluid phase membrane. This approach was finally applied to native milk fat globules, where mechanical heterogeneity was visible. However, elasticity values were somewhat different from those calculated on model systems, probably because of the presence of membrane proteins.Les globules gras du lait sont entourés d’une membrane biologique extrêmement complexe en composition et en structure, appelée MFGM (milk fat globule membrane). L’investigation de cette membrane, in situ dans le lait, par microscopie confocale nous suggère que les lipides polaires à haute température de transition de phase (Tm) forment des domaines en phase gel ou liquide ordonné, dispersés dans une phase continue fluide. Sur la base de cette observation, ce projet vise à comprendre en quoi la composition en lipides polaires laitiers et leur état de phase peuvent moduler les propriétés élastiques de la MFGM, en vue d’une meilleure maîtrise de la stabilité des globules gras en industrie laitière.L’hétérogénéité mécanique générée par la coexistence de différents types de phase a ainsi été caractérisée par spectroscopie de force AFM en utilisant des bicouches de lipides modèles de la membrane réelle, à basse (TTm). Pour analyser finement les déterminants de l’élasticité de la membrane, et tenir compte de la courbure, une étude approfondie des effets de l’état de phase et de la composition hétérogène en lipides polaires a été entreprise par spectroscopie de force atomique, en complément d’une analyse structurale par microscopie électronique ou diffraction des rayons X. Nous y avons montré, en particulier, que la présence de molécules de longueur de chaîne acyles et d’insaturation variables rend les membranes de sphingomyéline de lait en phase gel moins rigides qu’attendu, bien que significativement plus rigide qu’une membrane fluide. Cette appro

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel, fluid or liquid ordered phase, determined using AFM force spectroscopy

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 continuouslipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-richdomains induced by temperature (T Tm) or cholesterol affected the Young modulus of the lipidmembrane. 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 Youngmodulus values of 10–20 MPa and 4–6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains inMFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membranewith similar Young modulus values to that of a fluid phase (0–5 MPa). Upon addition of cholesterol to the milkSM 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 theYoung modulus was weakly affected by the temperature. These results open perspectives for the development ofmilk polar lipid based vesicles with modulated mechanical properties

Lipid domains in the milk fat globule membrane: Dynamics investigated in situ in milk in relation to temperature and time

The microstructure of the milk fat globule membrane (MFGM) is still poorly understood. The aim of thisstudy was to investigate the dynamics of the MFGM at the surface of milk fat globules in relation totemperature and time, and in relation to the respective lipid compositions of the MFGM from bovine, goatand sheep milks. In-situ structural investigations were performed using confocal microscopy. Lipiddomains were observed over a wide range of temperatures (4–60 C). We demonstrated that rapid coolingof milk enhances the mechanisms of nucleation and that extended storage induces lipid reorganizationwithin 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 couldbe related to the respective cholesterol/sphingomyelin molar ratio in the three milk species. These structuralchanges may affect the interfacial properties of the MFGM, with consequences for the functionalproperties of fat globules and the mechanisms of their digestion

Lipid domains in the milk fat globule membrane: the dynamics investigated in situ in milk in relation to temperature and time

International audienceMilk is an emulsion of fat droplets stabilized by a biological membrane called the milk fat globule membrane (MFGM; Figure 1). The MFGM is mainly composed of polar lipids that have different melting temperatures (Tm), cholesterol, glycoproteins, and enzymes. The microstructure of the MFGM depends on the packing of polar lipids in the outer bilayer of the membrane that can lead to phase separation with formation of ordered domains (Figure 2). Milk is usually submitted to changes in temperature e.g. storage (4-7°C), churning (10-12°C) and heat treatments (e.g. pasteurization). Do these temperature variations and thermal history of milk affect the packing of polar lipids in the plane of the MFGM ? Experiments were performed by confocal laser scanning microscopy (CLSM) under temperature controlled conditions, using the head-labelled phospholipid fluorescent probe Rh-DOPE. Only the fluid phase of MFGM was labelled

The temperature and storage effect on the microstructure of the milk fat globule membrane

Dans les pays en développement, quels que soient le climat et lesespèces animales domestiquées, le lait et ses dérivés occupent leplus souvent une place importante dans la vie quotidienne et constituentun vecteur indéniable de développement. La diversité des pratiques,des outils et des produits nis est considérable et implique unensemble d’acteurs le long des chaînes d’approvisionnement. Parfoismenacés par les dynamiques en cours de la mondialisation, le lait etses dérivés représentent néanmoins une valeur patrimoniale inestimable.Outre leur rôle nutritif incontesté, ces produits ont souvent unrôle clé dans la microéconomie et le développement territorial, avecde multiples modèles selon les contraintes et les opportunités locales.L’objectif de ces rencontres, qui font suite à la 1re édition organisée àRennes (France) en 2014 (colloque6.inra.fr/lait2014), est de favoriser leséchanges d’expériences autour de la diversité des modèles de développementlaitier observés à l’échelle de la planète, de rassembler toutes lesinitiatives et réseaux impliqués, de mesurer l’importance des femmes etdes jeunes dans ces dynamiques et de m@ieux cerner le rôle des politiquespubliques pour accompagner cet essor.Aboutir ensemble à une meilleure coordination des actions autour dulait dans les pays en voie de développement, et notamment à des projetsconjoints de recherche, de formation ou d’appui est la principaleambition de ces rencontres.Milk is an emulsion of fat droplets stabilized by a biological membrane called the milk fat globule membrane. The MFGM is mainly composed of polar lipids that have different melting temperatures (Tm), cholesterol, glycoproteins, and enzymes. The microstructure of the MFGM depends on the packing of polar lipids in the outer bilayer of the membrane that can lead to phase separation with formation of ordered domains [1]. This microstructure and the phase separation of lipids induced by the formation of domains are still poorly known despite its importance in dairy processing and in nutrition. Milk is usually submitted to changes in temperature e.g. storage (4-7°C), churning (10-12°C) and heat treatments (e.g. pasteurization). Do these temperature variations and thermal history of milk affect the packing of polar lipids in the plane of the MFGM?In this work,we characterized the evolution of the MFGM microstructure in situ, in milk as function of temperature and time by using confocal microscopy and Rh-DOPE as fluorescent probe.The dynamic of lipid domains was investigated on wide range of temperature (4-60°C) which includes the temperature of MFGM phase transition (gel/fluid; Tm = 36.4°C). The cooling kinetics from 60°C and storage duration at low temperature (4 or 20°C) affect the shape, the size and the number of lipid domains, through the mechanisms of nucleation and growth. On the other hand, heating kinetics show several responses of lipid domains such as diffusion, coalescence, reduction in size or disappearance at the surface of the fat globules [2]. We suggest also that the reaction of lipid domains to temperature changes can indicate its phase state (gel or ordered liquid) and the composition (presence of cholesterol). These structural changes can affect the interfacial properties of the MFGM with consequences on the functional properties of fat globules and on the digestion mechanisms

Lipid domains in the milk fat globule membrane: the dynamics investigated in situ in milk in relation to temperature and time

Milk is an emulsion of fat droplets stabilized by a biological membrane called the milk fat globule membrane (MFGM; Figure 1). The MFGM is mainly composed of polar lipids that have different melting temperatures (Tm), cholesterol, glycoproteins, and enzymes. The microstructure of the MFGM depends on the packing of polar lipids in the outer bilayer of the membrane that can lead to phase separation with formation of ordered domains (Figure 2). Milk is usually submitted to changes in temperature e.g. storage (4-7°C), churning (10-12°C) and heat treatments (e.g. pasteurization). Do these temperature variations and thermal history of milk affect the packing of polar lipids in the plane of the MFGM ? Experiments were performed by confocal laser scanning microscopy (CLSM) under temperature controlled conditions, using the head-labelled phospholipid fluorescent probe Rh-DOPE. Only the fluid phase of MFGM was labelled

Mechanical properties of milk sphingomyelin bilayer membranes in the gel phase: Effects of naturally complex heterogeneity, saturation and acyl chain length investigated on liposomes using AFM

Sphingomyelin (SM) molecules are major lipid components of plasma membranes that are involved in functionaldomains. Among natural SMs, that found in milk (milk-SM) exhibits important acyl chain heterogeneities interms of length and saturation, which could affect the biophysical properties and biological functions of the milkfat globule membrane or of liposome carriers. In this study, the thermotropic and mechanical properties of milk-SM, synthetic C16:0-SM, C24:0-SM and the binary mixtures C16:0-SM/C24:0-SM (50:50% mol) and C24:0-SM/C24:1-SM (95:5% mol) bilayer membranes were investigated using differential scanning calorimetry and atomicforce microscopy, respectively. Results showed that acyl chain length, heterogeneity and unsaturation affected i)the temperature of phase transition of SM bilayers, and ii) the mechanical properties of liposome (diameter< 200 nm) membranes in the gel phase, e.g. the Young modulus E and the bending rigidity kC. This studyincreases our knowledge about the key role of naturally complex lipid compositions in tailoring the physicalproperties of biological membranes. It could be also used in liposomes development e.g. to select the suitablelipid composition according to usage

Key role of lipid composition and phase on the mechanical properties of membranes probed by AFM on liposomes

Polar lipids from milk are valuable sources to design affordable and biocompatible liposomes for food or medical applications. In particular, the milk sphingomyelin (MSM) that accounts for about 30 % wt of milk polar lipids exhibits a complex composition with over 95 % mol. of saturated acyl chains (C16:0, C22:0, C23:0 and C24:0) and a melting temperature (Tm) of 35°C. The mechanical properties of the liposome’s bilayer are important to ensure e.g. physical stability. The objective of this study was to investigate the mechanical properties of liposomes as a function of their chemical composition and physical state. Liposomes (diameter < 200 nm) were produced using controlled compositions of polar lipids and immobilized onto flat silicium substrate in aqueous environment. The elastic modulus E and bending rigidity kc of their bilayer were measured using atomic force microscopy (AFM). As references, liposomes made of dipalmitoylphosphatidylcholine in the gel phase (DPPC; C16:0 ; Tm= 41°C) was found to be about 9 times more elastic at 20°C than that of dioleoylphosphatidylcholine in the fluid phase (DOPC; C18:1n-9 ; Tm=-20°C). Surprisingly, MSM liposome bilayers exhibited intermediate mechanical properties to those of DPPC and DOPC, in spite of the presence of long and saturated acyl chains. Experiments performed with liposomes of synthetic C16:0-SM, C24:0-SM and C24:1-SM showed that chain length heterogeneity or unsaturation both contribute to decrease the overall elasticity of MSM liposomes. The results demonstrate the potential of AFM indentation to assess the mechanical properties of curved and soft nano-objects such as liposomes. They also show that biologically relevant liposomes with complex lipid compositions, such as MSM, exhibit overall softer elasticity than expected, probably because of altered lateral packing of the molecules in the gel phase. This study contributes in a better understanding of the mechanical properties of membranes as a function of their composition and physical state

Mechanical properties of membranes composed of milk polar lipids in the gel or fluid-phase probed by AFM on liposomes

International audienceThe milk fat globule membrane ( has recently gained a lot of attention, due to the growing interest in its nutritional and technological properties Its composition, rich in polar lipids, has great potential for new product applications with unique physical properties such as liposomes Milk polar lipids might be advantageous for both the stability and the biological acceptance of the formulation Milk polar lipids possess different structures of molecules saturated with high melting temperatures (Tm),in particular the milk sphingomyelin SM,Tm 35 C) or unsaturated with low Tm Does this difference in the molecular structure affect the mechanical properties of the membranes

Lipid domains in the biological membrane surrounding milk fat globules: role of temperature and cholesterol on their morphology and nanomechanical properties

The biological membrane surrounding fat globules in milk, the MFGM, is poorly known despite its importance in the functional properties of many dairy products and in the mechanisms of milk lipids digestion occurring in the gastrointestinal tract of mammal newborns. Studies recently revealed the formation of lipid domains in the MFGM that have been interpreted as the lateral segregation of high phase transition temperature (Tm) lipids, mainly milk sphingomyelin (MSM; Tm 34°C) [1]. The role played by these MSM-rich domains in the MFGM is currently unknown and needs further investigation. In this context, the aim of this study was to investigate the role of temperature and cholesterol in the morphology and nanomechanical properties of the MFGM. Accessing the biophysical properties of biological membranes at nanoscale is possible using Atomic Force Microscopy (AFM). In the complex biomimetic membranes formed using a MFGM lipid extract, topographical AFM images revealed for T < 35°C the formation of µm-large domains that protruded above the continuous phase by ~1 nm [2]. The resistance of the MSM-rich gel phase domains to rupture was significantly higher than that of the liquid disordered continuous phase, i.e. breakthrough force FB of 23 nN vs. 6 nN at 25°C, respectively. The key role played by cholesterol in the lateral segregation of high Tm lipids, by decreasing the size of the lipid domains, their height and by fluidizing the MFGM has been demonstrated [3]. Such work on the heterogeneous distribution of polar lipids in the MFGM may open perspectives in dairy technology and infant nutrition, e.g. optimisation of the interface of processed lipid droplets in infant milk formulas to mimick the biological MFGM in breast milk