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International audience; BACKGROUND. High circulating levels of ceramides (Cer) and sphingomyelins (SM) have been associated with cardiometabolic diseases. The consumption of whole-fat dairy products, which naturally contain such polar lipids (PL), is associated with health benefits, but the impact on sphingolipidome remains unknown. We investigated how milk PL supplementation impacts circulating and intestinal SM and Cer composition in association with improvement of cardiovascular markers.METHODS. In a 4 week-randomized double-blind controlled study, 58 postmenopausal women consumed daily a cream cheese containing 0, 3 or 5 g of milk PL. Postprandial metabolic explorations were performed before and after the supplementation. SM and Cer species were analyzed in serum, intestine-derived chylomicrons and feces. The ileal content of 4 ileostomy patients was also explored after milk PL intake in a crossover double-blind study.RESULTS. Milk PL consumption decreased serum atherogenic C24:1 Cer (Pgroup = 0.033), C16:1 (Pgroup = 0.007) and C18:1 (Pgroup = 0.003) SM species. Changes in serum C16+18 SM species were positively correlated with the reduction of total cholesterol (r = 0.706, P < 0.001), LDL-C (r = 0.666, P < 0.001) and ApoB (r = 0.705, P < 0.001). Milk PL decreased the concentration in chylomicrons of total SM (Pgroup < 0.0001) and of C24:1 Cer (Pgroup = 0.001). Saturated SM and Cer species, which are also the major species found in milk PL-enriched cheeses, increased in ileal efflux and feces. There was a marked increase in total fecal Cer after milk PL supplementation (Pgroup = 0.0002). Milk PL also modulated the abundance of some specific SM and Cer species in ileal efflux and feces, suggesting differential absorption and metabolization processes in the gut.CONCLUSION. These data demonstrate that milk PL supplementation decreases atherogenic SM and Cer species associated with an improvement of cardiovascular risk markers. Our findings bring new insights on sphingolipid metabolism in the gastrointestinal tract, especially Cer as such signaling molecules potentially participating in the beneficial effect of milk PL. ClinicalTrials.gov, NCT02099032, NCT02146339.

Dietary lipids play a major role in infant nutrition, development and health. As an alternative to breast milk, infant formulae (IF) are the manufactured products given to infants. However, many differences exist between breast milk fat globules that are naturally secreted by lactating mothers and the processed lipid droplets formed under pressure during homogenization and found in IF. The lipid composition and structure of the emulsion in IF could be improved to mimick breast milk fat globules. This book chapter i) describes breast milk fat globules covered by their biological membrane (MFGM), including their functions and digestion in the gastrointestinal tract of infants, ii) presents the technological steps involved in IF preparation and the blending of oils with other ingredients leading to the final composition and structure of processed lipid droplets, iii) highlights the health benefits for infants of adding dairy lipids (Fatty acids, TAG, MFGM rich in phospholipids, sphingolipids, cholesterol and glycoproteins) in IF, iv) explains the opportunities to produce food-grade ingredients enriched in bovine MFGM and to prepare processed lipid droplets in IF bio-inspired by breast milk fat globules. The next generation of IF will integrate the advantages provided by dairy lipids to improve the quality of IF and bring nutritional and heath benefits to infants worldwide.

Breast cancer is the second most common cancer in the world based on incidence, reaching more than 2 million new cases in 2018, while continuing to increase. Invasive ductal carcinoma is the most common type of this cancer, making up approximately 70-80% of all breast cancer diagnoses. In particular, the type of breast cancer overexpressing human epidermal growth factor receptor 2 (HER2) has potential of strong proliferation, migration and invasion and early treatment is necessary. The authors identified and studied a single patient displaying complete therapeutic resistance to monoclonal anti-HER2 antibody therapy, chemotherapy and radiotherapy. A patient who exhibited resistance to postoperative adjuvant therapy after mastectomy was selected from HER2-positive breast cancer, and this patient had the grade of T4bN2aM0, Stage IIIB. The patient samples, blood serum and cancer tissue, were analyzed by metabolome and immunostaining technique, respectively. The characteristics of peripheral blood serum and solid tumor were investigated, aiming to find new serum biomarker(s) using the metabolomics technique. A correlation between the appearance of HER2-positive cancer tissue and serum concentration of the sphingomyelin family was found. In addition, HER2-positive tumor tissue in both the primary and recurrent cancer express the sphingomyelinase. These results suggest that sphingomyelins from this cancer tissue leads to therapy resistance, induction of invasion and strong proliferation.

Improving the nutritional and health properties of food products, e.g. infant milk formula, by the addition of functional ingredients is of primary importance. This study focused on bioactive milk polar lipids (PLs) recovered from dietary sources that are of increasing interest. The chemical compositions of buttermilks and butter serums were determined and the modulation of the fatty acid composition of milk PLs was investigated. Butter serums contain a higher amount of milk PLs than buttermilks (88 vs. 13-18g/kg dry matter), with a higher proportion of sphingomyelin (34 vs. 19% of PLs, respectively) interestingly close to human milk PL profile. Butter serums are also interesting sources of choline, an important nutrient for infant brain development. We demonstrated that the unsaturated fatty acid content of milk PLs recovered in the buttermilks and the butter serums, mainly the amount of C18:3n-3 and C22:6n-3 (DHA) that are of nutritional interest, can be increased by dietary strategies. This work opens perspectives for a better valorization of milk PLs in human nutrition (both infants and adults) to benefit their functional, nutritional and health properties.

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 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.

Scope Enhanced adiposity and metabolic inflammation are major features of obesity associated with altered gut microbiota and intestinal barrier. How these metabolic outcomes can be impacted by milk polar lipids (MPL), naturally containing 25% of sphingomyelin, is investigated in mice fed a mixed high-fat (HF) diet . Methods and results Male C57Bl/6 mice receive a HF-diet devoid of MPL (21% fat, mainly palm oil, in chow), or supplemented with 1.1% or 1.6% of MPL (HF-MPL1; HF-MPL2) via a total-lipid extract from butterserum concentrate for 8 weeks. HF-MPL2 mice gain less weight versus HF (p < 0.01). Diets do not impact plasma markers of inflammation but in the liver, HF-MPL2 tends to decrease hepatic gene expression of macrophage marker F4/80 versus HF-MPL1 (p = 0.06). Colonic crypt depth is the maximum in HF-MPL2 (p < 0.05). In cecal microbiota, HF-MPL1 increases Bifidobacterium animalis versus HF (p < 0.05). HF-MPL2 decreases Lactobacillus reuteri (p < 0.05), which correlates negatively with the fecal loss of milk sphingomyelin-specific fatty acids (p < 0.05). Conclusion In mice fed a mixed HF diet, MPL can limit HF-induced body weight gain and modulate gut physiology and the abundance in microbiota of bacteria of metabolic interest. This supports further exploration of how residual unabsorbed lipids reaching the colon can impact HF-induced metabolic disorders.

Objectives: Nutritional strategies can play a major role in the management of cholesterolemia, notably in postmenopausal women at risk of CVD. Interest has recently grown on the potential health benefitsof milk polar lipids (MPL).We showed that isolipidic enrichment of the diet with MPL improved several lipid CV risk factors but underlying mechanisms remained unclear. We hypothesized that MPL reduceintestinal cholesterol absorption in humans.Methods:We performed a double-blind randomized controlled trial in 58 postmenopausal women with fasting HDL-cholesterol < 1.6 mM.They were subjected to a 4-week dietary intervention with daily consumption of a cream-cheese containing 12 g of milk fat including either 0 g (control, n = 19), 3 g (n = 19) or 5 g (n = 20) of MPL. Before and after each intervention, blood lipids were measured in the whole cohortwhereas fecal lipids and coprostanol were analyzed in a subgroup (n = 7–9 per group). A proof-of-concept mechanistic crossover study was also carried out in 4 ileostomized subjects who performed 8hpostprandial tests after consuming 0g-, 3g- or 5g-MPL enriched cheese labelled with 2H-cholesterol tracer. Plasma, chylomicrons and ilealefflux were analyzed.Results: Milk fat enriched with 3 to 5 g MPL induced doseresponse reductions in serum total cholesterol (up to −6.8% in 5 g group, p < 0.05), LDL-cholesterol (−8.7%, p < 0.05) and HDL/total-cholesterol ratio (p < 0.001), compared to the control that had no effect. Fecal excretion of coprostanol increased after MPL supplementation (p < 0.05, 3g- and 5g-MPL vs control), and the fecal coprostanol/cholesterol ratio was inversely correlated with serum total- and LDL-cholesterol after intervention (r = −0.5, p < 0.05). In ileostomized subjects, postprandial accumulation of 2Hcholesterol in plasma and chylomicrons was reduced after 3 to 5 g MPL consumption (p < 0.05, vs control). Both cholesterol and milk sphingomyelin increased in ileal efflux after MPL enriched cheeses (p < 0.05).Conclusions: Present results suggest that milk polar lipids decrease cholesterol absorption in humans through interactions with sphingomyelin and by increasing conversion of cholesterol to coprostanol.Funding Sources: ANR (French National Research Agency, VALOBAB project, ANR-11-ALID-007–01), PHRC-I (French Clinical Research Program, 14–007), CNIEL (French Dairy Interbranch Organization).

Increasing knowledge about the composition and biophysical properties of milk lipids is essential to better understand, modulate and control their properties in food products, to developinnovative functional foods but also to tailor specific lipid structures able to provide nutritional and health benefits. Milk lipids are an important source of energy and bioactive molecules for the optimal growth and development of breast-fed newborns. They are also involved in the functional properties and sensory quality of many dairy products that are consumed by adults[1]. Milk is a natural O/W emulsion in whichfat globules are highly complex and unique entities as regard to the wide diversity of lipid molecules (98% triacylglycerols withmore than 400 fatty acids characterized byvarious chain length and saturation; polar lipidscontaining 30% sphingomyelin, cholesterol) and the multiscale organization of lipids (mean diameter of fat globules 4 µm; crystals of TAG; trilayer of polar lipids in the biological membrane).The structure of milk lipids, both TAG and polar lipids, is affected by temperature and thermal kinetics that induce phase transitions. Dairy processingalso have a major impact on the structure of milk lipids, with consequences on their reactivity in complex dairy products, mainly interactions with milk proteins[1].Thelecturewill provide an overview of the specificities of milk fat globules in terms of composition, structure and functions.Moreover, the recent advances about the structure of the milk fat globule membrane including the dynamics of the lipid domainsrich in sphingomyelin and cholesterol that are responsible for topographical and mechanical heterogeneities will be presented [2,3,4]. The development of processed emulsion droplets bio-inspired by milk fat globules to improve the structure of lipids in infant formulas and narrow the gap with human milk will also be described [5].These recent and new findingswill undoubtadelyopen perspectives in dairy technology and human nutrition(infants and adults).

The biological membrane surrounding fat globules in milk (the MFGM) is poorly known despite its importance in the functional properties of many dairy products,in the mechanisms of milk lipids digestion occurring in the gastrointestinal tract and the increased scientific evidence of its health benefits.Studies recently revealed the formation of lipid domains inthe MFGM that havebeen interpreted as the lateral segregation of high phase transition temperature (Tm) lipids, mainly milk sphingomyelin (MSM; Tm 34°C), and cholesterol[1]. The properties ofthese MSM-rich domains and their functional rolein the MFGM arecurrently unknown and needs further investigation. The aim of this study was to investigate the biophysical properties of hydrated MSM bilayers in absence or presence of increased amount of cholesterol. The experiments have been performed using synchrotron radiation X-ray diffraction and differential scanning calorimetry[2].Also, the interactions between MSM and cholesterol have been investigated in monolayers composed of various MSM/cholesterol molar ratios by the recording of Langmuir isotherms of compression both for temperatures below and above Tm of MSM [3,4]. The role of cholesterol in the topography and mechanical properties of hydrated bilayers composed of MFGM polar lipids has been characterised using atomic force microscopy [5]. This study allowed the characterisation of the gel and liquid crystalline phase of MSM bilayers as well as their structural changes as a function of temperature. The addition of more than 40% mol cholesterol induces the formation of the liquid ordered phaseand dedrease the rigidity oft he membrane as compared to the MSM gel phase. These results improved knowledge about MSM properties and its interactions with cholesterol that areinvolved in the formation of complexes in the MFGM and potentially in the gastrointestinal tractduring digestion of milk fat globules.

The interactions between the milk fat globule membrane (MFGM) and proteins (e.g. enzymes, caseins, whey proteins) are poorly known despite their importance in many interfacial mechanisms occurring at the surface of milk fat globules. The MFGM exhibits polar lipids lateral heterogeneities: the zwitterionic milk sphingomyelin (MSM) molecules form domains in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, that are surrounded by a fluid matrix of phospholipids in the liquid-disordered (Ld) phase composed of the unsaturated phosphatidylcholine (PC) and the anionic phosphatidylserine (PS) and phosphatidylinositol (PI) [1,2].AimThe aim of this study was to determine how the phase state and head-group charge of MFGM polar lipids drive interactions with the negatively charged casein micelles, as the major milk protein assembly.MethodsHydrated supported lipid bilayers with various compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. Real-time atomic force microscopy (AFM) imaging in liquid was performed before and after injection of casein micelles. Zeta-potential and Langmuir isotherms of the different polar lipids offered complementary information.ResultsThe interaction of casein micelles with milk polar lipids is hindered by the high lateral packing density of MSM domains in gel and Lo phases and by the negative charge of PI and PS, certainly due to electrostatic repulsions. But the casein micelles did adsorb onto the Ld phases of DOPC and of MSM above its phase transition temperature (T>Tm=34.3°C[3]), probably as a result of large inter-molecular distances.ConclusionThe absence of interaction between casein micelles and the complex MFGM polar lipids is due to the MSM-rich ordered domains and to the presence of anionic phospholipids in the Ld phase. These results open perspectives for the design of liposomes and emulsions with variable capacity for protein adsorption by the modulation of phase state and charge of the interfacial polar lipids.References: [1] Et-Thakafy et al., Food Chem., 2017, 220, 352–361 [2] Murthy et al., BBA-Biomembranes, 2016, 1858, 2181-2190[3] Murthy et al., BBA-Biomembranes, 2015, 1848, 2308-2316

International audience; Introduction Propionibacterium freudenreichii (PF) is a beneficial bacterium, used as Swiss-type cheeses starter. PF exerts several beneficial effects, including anti-inflammatory modulation. This feature relies on several metabolites and on surface proteins. PF emerges as a suitable candidate to develop fermented functional foods. As it is established, the probiotic properties are strain-dependent, however the delivery vehicle effect is barely explored. Aim In this study, our purpose is to explore how the cheese matrix impact its immunomodulatory properties on model of healthy piglets. Methods Three groups of weaned piglets were fed during 2 weeks with PF in form of a culture or of a fermented cheese and compared to a control group fed with sterile cheese. PF’s metabolic activity was assessed by short chain fatty acids dosage and microbiota modulation. Immune cells from small intestinal mesenteric nodes (MLNC) and blood (PBMC) were extracted. The basal cytokines secretion and T lymphocytes phenotypes were evaluated. Then, the immune cells were ex vivo stimulated by pro-inflammatory and anti-inflammatory stimuli and the cytokine response was determined. Results PF was metabolically active in piglets’ colon and enhanced bifidobacteria population and SCFAs production, without significant difference between the two delivery vehicles. PF decreased the secretion of TNFα and IL-10 secretion only by PBMC. In MLNC, PF cheese enhanced Treg and Th2 populations and sustained Th17 population. PF culture enhanced decreased Th17 population in MLNC. In PBMC, PF ingestion enhanced Treg and Th2 populations, without difference between the two delivery vehicles. Ex vivo stimulation of PBMC and MLNC showed a difference in immunomodulation of PF, according the delivery vehicle. Conclusion These results provide new insights into the cheese matrix effect on P. freudenreichii' probiotic functionalities. Such data showed the relevance to take in account the delivery vehicle to develop functionnal foods.

International audience; The present paper describes biophotonic sensors realized by way of inexpensive processes. As hybrid silica/polymer resonators, they are suited to detect biological molecules in gel/fluid phase transition at infinitesimal concentrations (sphingomyelin lipids). The photonic structure is made of specific amplified deep UV210 photoresist-polymer waveguides coupled by a sub-wavelength gap with racetrack microresonators allowing a minimal dependence in temperature. Then, temperature dependent wavelength shifts characterizing the optical resonances of the device have been evaluated, highlighting a quite low thermal feature of the sensor advantageous for relevant applications. With an appropriate vesicle lipid deposition process, specific in biology, together with an apt experimental bio-thermo-photonic protocol, the dynamic evolution of the sphingomyelin lipid phase transition has been assessed. The ability to detect their gel/fluid transition phase and melting temperature has been demonstrated with a mass product factor value 1.4×10 7 lower than that of classical methods. The equilibrium regimes of the resonators and the scattered part of the light are clearly highlighted as markedly modified by the dynamic of the sphingomyelin during its own phase transition.

Ceramides are minor structural components of membranes involved in biological functions. In the milk fat globule membrane (MFGM), ceramides are susceptible to affect the lateral packing of polar lipids, especially the milk sphingomyelin (MSM). To investigate this, palmitoylceramide (PCer) was added to MSM/DOPC (dioleoylphosphatidylcholine) in order to form hydrated lipid bilayers. Differential scanning calorimetry evidenced interactions of PCer with the MSM in the solid-ordered phase to form MSM/PCer structures with a higher thermostability than MSM. Atomic force microscopy revealed that PCer modified lipid packing in both the liquid-disordered DOPC phase where it increased thickness and mechanical stability, and the solid-ordered MSM phase where it recruited MSM molecules yet initially in the liquid phase at 26 °C and then increased the area of the MSM/PCer domains. The effect of PCer on the mechanical properties of the MSM-rich domains remains to be elucidated. These results bring new insights on the role of ceramides in the control of biophysical and biological properties of the MFGM. They also open perspectives for the design of emulsions and liposomes, using milk polar lipids as food-grade ingredients.

Many food products are oil in water (O/W) emulsions. Milk has the specificity to be a natural O/W emulsion secreted by the lactating cells. The milk fat globules are recognized as being efficient conveyors of energyin the form of triacylglycerols and of bioactive molecules, specifically provided by the biological membrane, the MFGM, which is the interface with the gastrointestinal tract. However, the MFGM is altered in manyprocessed dairy products and is currently absent from standard infant milk formulas. The objectives of this invited article are to evaluate whether the specific composition and structure of the MFGM and milk polar lipidassemblies, associated with their nutritional and health benefits, could be used to tailor functional emulsions bioinspired by the MFGM-coated milk fat globules. Recent knowledge about the structure of the MFGM revealing the heterogeneous distribution of proteins and the formation of ordered lipid domains rich in milk sphingomyelin and cholesterol is presented. The impacts of the unique composition and biophysical properties of milk polar lipids on the structure and potential functions of the MFGM are highlighted. Facing the evidence of both the functional and health benefits provided by the MFGM, the preparation of MFGM-enriched ingredients from dairy products has recently attracted wide attention. The utilization of MFGM components to tailor the surface of processed lipid droplets in food emulsions and to develop new products providing health benefits isdiscussed.

Background: Polar lipid (PL) emulsifiers such as milk PLs (MPLs) may affect digestion and subsequent lipid metabolism, but focused studies on postprandial lipemia are lacking. Objective: We evaluated the impact of MPLs on postprandial lipemia in mice and on lipid digestion in vitro. Methods:Female Swiss mice were gavaged with 150 mL of an oil-in-water emulsion stabilized with 5.7 mg of either MPLs or soybean PLs (SPLs) and killed after 1, 2, or 4 h. Plasma lipids were quantified and in the small intestine, gene expression was analyzed by reverse transcriptase‐quantitative polymerase chain reaction. Emulsions were lipolyzed in vitro using a static human digestion model; triglyceride (TG) disappearance was followed by thin-layer chromatography. Results: In mice, after 1 h, plasma TGs tended to be higher in the MPL group than in the SPL group (141 mg/mL vs. 90 mg/mL; P= 0.07) and nonesterified fatty acids (NEFAs) were significantly higher (64 mg/mL vs. 44 mg/mL; P< 0.05). The opposite was observed after 4 h with lower TGs (21 mg/mL vs. 35 mg/mL; P < 0.01) and NEFAs (20 mg/mL vs. 32 mg/mL; P < 0.01) in the MPL group compared with the SPL group. This was associated at 4 h with a lower gene expression of apolipoprotein B (Apob) and Secretion Associated, Ras related GTPase 1 gene homolog B (Sar1b), in the duodenum of MPL mice compared with SPL mice (P < 0.05). In vitro, during the intestinal phase, TGs were hydrolyzed more in the MPL emulsion than in the SPL emulsion (decremental AUCs were 1750%/min vs. 180%/min; P < 0.01). MPLs enhance lipid intestinal hydrolysis and promote more rapid intestinal lipid absorption and sharper kinetics of lipemia. Conclusions: Postprandial lipemia in mice can be modulated by emulsifying with MPLs compared with SPLs, partly through differences in chylomicron assembly, and TG hydrolysis rate as observed in vitro. MPLs may thereby contribute to the long-term regulation of lipid metabolism. J Nutr 2015;145:1770‐7.

National audience; Nous avons réalisé, par des procédés relativement peu coûteux, un capteur bio-photonique architecturé sur une base de micro-résonateurs (hybrides silice/polymère) capable de détecter la transition de phase gel/fluide de molécules biologiques (sphingolipides) à de très faible concentration. La structure photonique est constituée de guides d'ondes polymère spécifiquement couplés à une succession de résonateurs. La structure complète opto-géométrique (sans lipides) présente une faible dépendance thermique la rendant efficace. Grâce à un procédé spécifique de dépôt de lipides en vésicules associé à un protocole expérimental 'bio-thermo-photonique' de mesures appropriées, l'évolution dynamique de la transition de la phase lipidique de la sphingomyéline a été suivie puis détectée. La capacité à détecter la transition de phase gel/fluide et la température de fusion a ainsi été démontrée avec une quantité de produit, d'un facteur de plus de 10 7 , inférieure à celle utilisée par les méthodes plus classiques comme la méthode de calorimétrie différentielle en balayage. L'équilibre du régime de couplage des résonateurs ainsi que la proportion de lumière diffractée ont ainsi été modifié par la dynamique de la sphingomyéline à sa propre transition de phase.

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.

The miscibility of milk sphingomyelin (milk-SM) and cholesterol was investigated in this study. Theeffect of different physical states of milk-SM on its interactions with cholesterol was determined bythe recording of isotherms of compression of Langmuir films for temperatures above and below thegel to La phase transition of milk-SM (Tm 34 C). For T = 15 C < Tm, the liquid expanded (LE) to liquidcondensed (LC) phase transition of milk-SM monolayers was observed at surface pressures of 10–15 mN/m. For T = 43 C > Tm, the milk-SM molecules were in a LE phase regardless of the surface pressureapplied. A phase diagram pressure – milk-SM/cholesterol composition was established. This studydemonstrated that both temperature and surface pressure affected the miscibility between the milk-SM and cholesterol. The strongest attractive forces (i.e. condensing effect) were identified for 30 mol%cholesterol when the milk-SM was in the LE phase state.

Sphingomyelin (SM) molecules are major lipid components of plasma membranes that are involved in functional domains. Among natural SMs, that found in milk (milk-SM) exhibits important acyl chain heterogeneities in terms of length and saturation, which could affect the biophysical properties and biological functions of the milk fat 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 atomic force 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

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 ~ 0.5–1.1 nm. 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.

IntroductionThe biological membrane surrounding fat globules in milk, i.e. the milk fat globule membrane (MFGM), is a trilayer assembly of polar lipids including 25-40% of sphingomyelin (SM) and glycerophospholipids. It is involved in the mechanical stability of fat globules, in the mechanisms of their digestion, and in physiological functions that need to be further understood. Increasing knowledge about the MFGM is therefore of primary importance both for dairy technology and nutrition. AimThe objectives of this study were to explore the dynamics of the MFGM polar lipids in situ in milk and to describe the biophysical properties of milk polar lipids assemblies as a function of temperature and composition, e.g. presence of cholesterol. MethodsThe biophysical properties and organization of polar lipids in the MFGM were investigated by the combination of techniques: DSC, synchrotron radiation XRD, CLSM, electron microscopy, AFM imaging and force spectroscopy. ResultsThe MFGM is a dynamic patchwork exhibiting phase separation of the high melting temperature (Tm) polar lipids, mainly SM, to form domains in ordered phase dispersed in a continuous fluid phase composed of the unsaturated polar lipids. In absence of cholesterol, the SM-rich domains are formed for T < 35°C and can melt upon heating. These domains protrude by 1 nm and have a higher resistance to rupture than the fluid phase composed of the unsaturated polar lipids. When present, cholesterol exhibits attractive interactions with SM with a condensing effect, and affects the topography of the membrane with a fluidizing effect. ConclusionWe propose an updated model for the heterogeneous organization of polar lipids in the MFGM, highlighting the dynamic changes in the topography and nanomechanical properties that are governed by the temperature and the presence of cholesterol. These recent findings on the assemblies of polar lipids in the MFGM and specific biophysical properties of SM in interaction with cholesterol will undoubtedly contribute in the development of smart biomimetic fat globules, e.g. in infant milk formulas.

La microstructure de la membrane des globules gras du lait, et en particulier la séparation de phase des lipides qui induit la formation de microdomaines, sont peu connues malgré leur importance dans les propriétés fonctionnelles des globules gras et les mécanismes interfaciaux (e.g. interaction avec les enzymes digestives au cours de la digestion). La présence de microdomaines riches en sphingomyéline de lait (SM) a été montrée dans la membrane des globules gras de différents laits (humain, vache, bufflonne), à température ambiante [1]. Or, les globules gras peuvent subir des variations de températures: 4°C (stockage au froid), 37°C (digestion), 60°C (traitements thermiques).L’originalité de ce travail était de caractériser les évolutions structurales de la membrane des globules gras du lait en fonction de la température et de la durée de stockage. La microstructure de la membrane des globules gras a été observée, in situ dans le lait, par microscopie confocale en utilisant un phospholipide exogène comme sonde fluorescente (Rhodamine-DOPE).Les domaines riches en SM ont été observés à la surface des globules gras sur une large plage de températures (T : 4 – 60°C), qui inclue des températures inférieures et supérieures à la température de transition de phase phase gel – liquide des lipides polaires dans la membrane des globules gras (Tm = 36.4°C). Pour T > Tm, les domaines doivent correspondre à des complexes entre la SM et le cholestérol en phase liquide ordonnée Lo alors que pour T < Tm les domaines peuvent être en phase gel ou en phase Lo. Nous avons montré que des refroidissements rapides de T=60°C > Tm à T=20 – 4°C < Tm favorisent les mécanismes de nucléation avec la formation de nombreux petits domaines, et que le stockage favorise la croissance des domaines. Au cours du chauffage des laits, nous avons observé la diffusion des domaines, leur coalescence et leur diminution de taille jusqu’à disparition. Ces modifications de la microstructure de la membrane des globules gras peut avoir des conséquences en technologie laitière et dans les mécanismes de digestion des lipides du lait, notamment chez les nouveaux nés allaités.

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.

The polar lipid assembly and biophysical properties of the biological membrane enveloping the milk fat globules (the MFGM) are yet poorly known, especially in connection with the temperature history that milk can experience after its secretion. However, bioactive mechanisms depend on biological structure, which itself highly depend on temperature. The objectives of this study were to investigate polar lipid packing in hydrated bilayers, models of the MFGM, and to follow at intermolecular level temperature-induced changes in the range 60-6°C, using the combination of differential scanning calorimetry, X-ray diffraction, atomic force microscopy (AFM) imaging and force spectroscopy. MFGM polar lipids, especially sphingomyelin, contain long chain saturated fatty acids with high phase transition temperatures. On cooling, the liquid disordered ld to solid ordered so (gel) phase transition of MFGM polar lipids started at about 40°C, leading to phase separation and formation of so phase domains protruding by about 1nm from the ld phase. Indentation measurements using AFM revealed that the resistance of the so phase domains to rupture was significantly higher than that of the ld phase and that it increased for both the domain and fluid phases with decreasing temperature. However, packing and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle. This study showed that MFGM polar lipid bilayers are dynamic systems. Heterogeneity in the structure and mechanical properties of the membrane was induced by temperature-dependent so/ld phase immiscibility of the lipid components. This could have consequences on the MFGM technological and biological functions (e.g. immunity and milk lipid digestion).

La membrane biologique qui entoure les globules gras du lait est un assemblage complexe de lipides et de protéines peu connu malgré son implication dans des propriétés techno-fonctionnelles et dans les mécanismes de digestion des lipides. Nous y avons révélé la présence de domaines lipidiques riches en sphingomyéline, lipide saturé généralement ordonné, dispersés dans une phase fluide désordonnée. Grâce à la haute résolution de la microscopie à force atomique, nous avons montré que cette hétérogénéité structurale s’accompagne d’une hétérogénéité mécanique des membranes, qui est modulée par la température et la présence de cholestérol. Pour les températures supérieures à 35°C, les bicouches de lipides polaires laitiers sont uniformes. En dessous de 35°C, nous avons observé la formation de domaines de plusieurs μm2 qui dépassent d’environ 0,9 nm au-dessus de la phase fluide. Ces domaines présentent une résistance à la rupture élevée par rapport à la phase fluide et induisent donc une hétérogénéité mécanique dans la membrane. Nous avons également montré que le cholestérol, par son effet condensateur et son rôle fluidifiant, disperse ces domaines, diminue leur taille et augmente leur nombre, abolissant ainsi progressivement l’hétérogénéité structurale et mécanique des membranes. L’ensemble de ces travaux nous a permis de proposer un nouveau modèle d’organisation de la membrane des globules gras, et ouvre la voie à la fabrication de globules gras biomimétiques., The biological membrane enveloping the milk fat globule is a complex assembly of lipids and proteins, barely known in spite of its impacts in the techno-functional properties of dairy foodstuff or in the digestion mechanisms of milk lipids. We have revealed the presence of lipid domains, rich in sphingomyelin as a lipid with high transition temperature and generally ordered, dispersed in a fluid and disordered phase. Thanks to the nanoscale resolution of atomic force microscopy, we have shown that this lateral heterogeneity of the milk fat globule membrane is coupled with heterogeneous mechanical resistance of the membranes and depends on temperature or the presence of cholesterol. For temperatures higher than 35°C, the bilayers of milk polar lipids are uniform. Below that temperature, we have observed the formation of μm2-scale domains that protrude above the fluid phase by about 0.9 nm. These domains exhibit a higher resistance to rupture than that of the fluid phase and thereby account for the mechanical heterogeneity of the membrane. Through its fluidizing and condensing effects, the cholesterol disperses the domains, making them smaller and more numerous, and gradually abolishes the structural and mechanical heterogeneity of the membranes. This research supported the proposition of an updated model for the organization of the milk fat globule membrane and opens perspectives for the design of biomimetic fat globules.

Sphingomyelin-rich microdomains have been observed in the biological membrane surrounding milk fat globules (MFGM). The role played by cholesterol in these domains and in the physical properties and functions of the MFGM remains poorly understood. The objective of this work was therefore to investigate the phase state, topography, and mechanical properties of MFGM polar lipid bilayers as a function of cholesterol concentration, by combining X-ray diffraction, atomic force microscopy imaging, and force spectroscopy. At room temperature, i.e. below the phase transition temperature of the MFGM polar lipids, the bilayers showed the formation of sphingomyelin-rich domains in the solid ordered (so) phase that protruded about 1 nm above the liquid disordered (ld) phase. These so phase domains have a higher mechanical resistance to rupture than the ld phase (30 nN versus 15 nN). Addition of cholesterol in the MFGM polar lipid bilayers (i) induced the formation of liquid ordered (lo) phase for up to 27 mol % in the bilayers, (ii) decreased the height difference between the thicker ordered domains and the surrounding ld phase, (iii) promoted the formation of small sized domains, and (iv) decreased the mechanical resistance to rupture of the sphingomyelin-rich domains down to ∼5 nN. The biological and functional relevance of the lo phase cholesterol/sphingomyelin-rich domains in the membrane surrounding fat globules in milk remains to be elucidated. This study brought new insight about the functional role of cholesterol in milk polar lipid ingredients, which can be used in the preparation of food emulsions, e.g. infant milk formulas.

International audience; The 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?

Plusieurs équipes de recherche rennaises utilisent des expériences aux interfaces fluides planes pour connaître et comprendre les propriétés de biomolécules variées. Lors d’une journée d’échanges, des scientifiques de l'UMR STLO et d’autres équipes contribueront à dresser le panorama des thématiques concernées et montrer le potentiel de ces expériences. Une journée d'échanges scientifiques est organisée par l'Institut de Physique de Rennes, le 22 janvier 2015. Elle vise à illustrer le potentiel et à construire une vision d'ensemble des expériences aux interfaces planes, dans des domaines scientifiques aussi variés que la physique des mousses et émulsions, les technologies de transformation des aliments, la digestion néonatale des lipides laitiers ou l'interaction de protéines avec les membranes biologiques. L'opportunité de montrer que les expériences aux interfaces planes livrent des informations abondantes et pertinentes.Plusieurs équipes de recherche rennaises utilisent des expériences aux interfaces fluides planes pour connaître et comprendre les propriétés de biomolécules variées. Lors d’une journée d’échanges, des scientifiques de l'UMR STLO et d’autres équipes contribueront à dresser le panorama des thématiques concernées et montrer le potentiel de ces expériences.Une journée d'échanges scientifiques est organisée par l'Institut de Physique de Rennes, le 22 janvier 2015. Elle vise à illustrer le potentiel et à construire une vision d'ensemble des expériences aux interfaces planes, dans des domaines scientifiques aussi variés que la physique des mousses et émulsions, les technologies de transformation des aliments, la digestion néonatale des lipides laitiers ou l'interaction de protéines avec les membranes biologiques. L'opportunité de montrer que les expériences aux interfaces planes livrent des informations abondantes et pertinentes.; National audience

International audience; Milk sphingomyelin (MSM) and cholesterol segregate into domains in the outer bilayer membrane surrounding milk fat globules. To elucidate the morphology and mechanical properties of theses domains, supported lipid bilayers with controlled molar proportions of MSM, dioleoylphosphatidylcholine (DOPC) and cholesterol were produced in buffer mimicking conditions of the milk aqueous phase. Atomic force microscopy imaging showed that (i) for T

National audience; Milk sphingomyelin (MSM) and cholesterol segregate into domains in the outer bilayer membrane surroundingmilk fat globules. To elucidate the morphology and mechanical properties of theses domains, supported lipid bilayers with controlled molar proportions of MSM, dioleoylphosphatidylcholine (DOPC) and cholesterol were produced in buffer mimicking conditions of the milk aqueous phase. Atomic force microscopy imaging showed that (i) for T

Minerals found in milk have a complex composition which evolves according to pH and technological processes. Among milk components, polar lipids (phospholipids and sphingomyelin) are amphiphilic molecules with a polar head group highly sensitive to ions. Milk polar lipids are mainly located in the membrane surrounding fat globules. They can also be concentrated from by-products of the dairy industry (e.g. buttermilk) and used for their interesting nutritional and functional properties as well as for encapsulation after formation of liposomes. Although the minerals found in milk and dairy products can affect the functional properties of polar lipids and their structural organisation, scientific data are scarce. The objective of the work was to investigate the properties of milk polar lipids as a function of the pH, the ionic strength and the type of minerals: monovalent or divalent ions. The combination of chemical and biophysical methods (zeta potential, turbidimetry, microscopy techniques) was used to characterise polar lipids properties and organisation. Changes in milk polar lipids properties and organisation will be presented as a function of their mineral environment. Decrease in pH and increase in ionic strength lead to a reduction i) of zeta-potential values, ii) of the electrostatic repulsions between polar lipids particles until their aggregation and phase separation from water. Among milk minerals, calcium highly affects polar lipids properties. This work contributes in a better understanding of the physical stability of milk fat globules and will improve the utilization of milk polar lipids for different food applications (cheeses, infant formulas…).

Milk sphingomyelin (MSM) and cholesterol segregate into domains in the outer bilayer membrane surrounding milk fat globules. To elucidate the morphology and mechanical properties of theses domains, supported lipid bilayers with controlled molar proportions of MSM, dioleoylphosphatidylcholine (DOPC) and cholesterol were produced in buffer mimicking conditions of the milk aqueous phase. Atomic force microscopy imaging showed that (i) for T < 35 °C MSM segregated in gel phase domains protruding above the fluid phase, (ii) the addition of 20 mol % cholesterol resulted in smaller and more elongated l o phase domains than in equimolar MSM/DOPC membranes, (iii) the MSM/cholesterol-enriched lo phase domains were less salient than the MSM gel phase domains. Force spectroscopy measurements furthermore showed that cholesterol reduced the resistance of MSM/DOPC membrane to perforation. The results are discussed with respect to the effect of cholesterol on the biophysical properties of lipid membranes. The combination of AFM imaging and force mapping provides unprecedented insight into the structural and mechanical properties of milk lipid membranes, and opens perspectives for investigation of the functional properties of MSM domains during milk fat processing or digestion. © 2014 American Chemical Society.

Milk lipids are an interesting source of bioactive molecules with functional and nutritional properties. Although the composition of milk lipids is of utmost importance for food processing and human consumption, it is far from being fully known. The objective of this study was to perform a comparative analysis of the chemical composition of lipids from bovine milks produced in French Brittany during spring (fresh pasture based diet) and winter (corn silage based diet). The polar lipid content and relative proportions of the glycerophospholipids and sphingomyelin were determined using HPLC/ELSD. The fatty acid composition of total lipids and polar lipids was determined using GC. The milks collected in spring contained i) a lower amount of total lipids: 39.7 ± 0.8 g/kg vs 41.7 ± 0.5 g/kg in winter, ii) a higher amount of polar lipids: 138 ± 11 vs 112 ± 8 mg/kg milk; 3.5 ± 0.3 vs 2.7 ± 0.4 mg/g fat, which was related to a smaller size of fat globules, and iii) a higher amount of sphingomyelin, 32 mg/kg milk vs 25 mg/kg milk in winter. Interestingly, the polar lipids from the milk fat globule membrane contained a higher concentration of unsaturated fatty acids in spring (C18:1 n − 9, C18:2 n − 6, C18:3 n − 3 and long-chain n − 3 fatty acids). Milk from cows fed a fresh pasture-based diet during spring is an interesting source of dietary long chain polyunsaturated fatty acids for human consumption.

International audience; Protein membrane transduction domains are able to translocate through cell membranes. This capacity resulted in new concepts on cell communication and in the design of vectors for internalization of active molecules into cells. Penetratin crosses the plasma membrane by a receptor and metabolic energy-independent mechanism which is at present unknown. A better knowledge of its interaction with phospholipids will help to understand the molecular mechanisms of cell penetration. Here, we investigated the role of lipid composition on penetratin induced membrane perturbations by X-ray diffraction, microscopy and (31)P-NMR. Penetratin showed the ability to induce phospholipid domain separation, membrane bilayer thickening, formation of vesicles, membrane undulations and tubular pearling. These data demonstrate its capacity to increase membrane curvature and suggest that dynamic phospholipid-penetratin complexes can be organized in different structural arrangements. These properties and their implications in peptide membrane translocation capacity are discussed.

Globule gras du lait : nouveau modèle d'organisation de la membrane biologique. 10. journée de l'animation transversale" glande mammaire"

Non disponible / Not available, L'hyperinsulinémie, conséquence de l'insulino-résistance (IR), fréquemment associée à l'obésité, constitue un des éléments du 'syndrome métabolique, facteur de risque pour le diabète de type 2 et les maladies cardio-vasculaires. Les mécanismes de l'IR ne sont pas totalement élucidés. Une hypothèse membranaire suggère que l'IR pourrait être due à des altérations des propriétés structurales des membranes cellulaires. Des travaux antérieurs ont mis en évidence une relation positive entre le contenu membranaire en SM et l'hyperinsulinémie. Dans ce travail, nous avons approfondi l'étude des relations SM-IR et tenté d'en approcher les mécanismes en utilisant la membrane d'hématie dont la composition en SM reflète celle de l'adipocyte. Dans une étude transversale, les phospholipides de l'hématie ont été déterminés chez des sujets non obèses, des adultes et enfants obèses. Une augmentation de la SM membranaire avec l'IR est observée dans les trois groupes, la SM étant le facteur membranaire le plus significatif expliquant la variance de l'insulinémie.Dans deux études d' intervention, les phospholipides et acides gras ont été déterminés-avant et après perte de poids à la suite d'un régime hypocalorique ou une intervention pharmacologique (Orlistat). A l'état basal, l'hyperinsulinémie est associée à un excès de SM et à un déficit en acide arachidonique. Dans les deux études, l'insulinémie diminue et les paramètres membranaires se normalisent chez les sujets les plus insulino-résistants. Malgré une perte de poids identique, l'insulinémie et la SM ne varient pas chez les sujets les moins insulino-résistants.Dans une étude préliminaire, le rôle éventuel des échanges lipoprotéines-cellules sur les altérations de la composition lipidique membranaire associées à l'IR a été abordé. La composition en phospholipides des HDL et LDL semble importante. En conclusion, un excès de SM membranaire pourrait être un élément dans la constellation de facteurs constituant le syndrome métabolique.