Walnut (Juglans regia L.) kernel oil bodies recovered by aqueous extraction for utilization as ingredient in food emulsions: Exploration of their microstructure, composition and the effects of homogenization, pH, and salt ions on their physical stability.

Natural oil-in-water emulsions containing plant oil bodies (OBs), also called oleosomes, rich in health-promoting omega-3 polyunsaturated fatty acids (ω3 PUFA) are of increasing interest for food applications. In this study, we focused on walnut kernel OBs (WK-OBs) and explored their microstructure, composition and physical stability in ionic environments as well as the impact of homogenization. A green process involving aqueous extraction by grinding of WK allowed the co-extraction of OBs and proteins, and centrifugation was used to recover the WK-OBs. Confocal laser scanning microscopy images showed the spherical shape of WK-OBs with an oil core envelopped by a layer of phospholipids (0.16 % of lipids) and embedded proteins. Their mean diameter was 5.1 ± 0.3 µm. The WK-OBs contained 70.1 % PUFA with 57.8 % ω6 linoleic acid and 12.3 % ω3 α-linolenic acid representing 68 % and 11.6 % of the total fatty acids in the sn-2 position of the triacylglycerols (TAG), respectively. Trilinolein was the main TAG (23.1 %). The WK-OBs also contained sterols (1223 ± 33 mg/kg lipids; 86 % ß-sitosterol), carotenoids (0.62 ± 0.01 mg/kg lipids; 49.2 % ß-carotene), and tocopherols (322.7 ± 7.7 mg/kg lipids; 89 % γ-tocopherol), confirming their interest as health-promoting ingredients. The decrease in the size of WK-OBs under high-pressure homogenization avoided phase separation upon storage. The anionic WK-OB surface at neutral pH was affected by stressful ionic environments (pH, NaCl, CaCl2), that induced aggregation of WK-OBs and decreased the physical stability of the emulsions. Emulsions containing WK-OBs are promising to diversify the market of the ω3-rich plant-based food products and beverages.

Surface proteins of Shiga toxin-producing Escherichia coli mediate association with milk fat globules in raw milk.

Introduction: By adhering to host cells and colonizing tissues, bacterial pathogens can successfully establish infection. Adhesion is considered the first step of the infection process and bacterial adhesion to anti-adhesive compounds is now seen as a promising strategy to prevent infectious diseases. Among the natural sources of anti-adhesive molecules, the membrane of milk fat globules (MFGs) is of interest because of its compositional diversity of proteins and glycoconjugates. However, few studies have focused on the bacterial molecules involved in MFG- mediated inhibition of bacterial adhesion to enterocytes. Methods: We used three pathogenic Shiga toxin-producing Escherichia coli (STEC) strains (O26:H11 str. 21765, O157:H7 str. EDL933, and O103:H3 str. PMK5) as models to evaluate whether STEC surface proteins are involved in the affinity of STEC for MFG membrane proteins (MFGMPs). The affinity of STEC for MFGMPs was assessed both indirectly by a natural raw milk creaming test and directly by an adhesion test. Mass spectrometry was used to identify enriched STEC proteins within the protein fraction of MFGMs. Bacterial mutants were constructed and their affinity to MFGs were measured to confirm the role of the identified proteins. Results: We found that free STEC surface proteins inhibit the concentration of the pathogen in the MFG-enriched cream in a strain-dependent manner. Moreover, the OmpA and FliC proteins were identified within the protein fraction of MFGMs. Our results suggest that FliC protein participates in STEC adhesion to MFGMPs but other STEC molecules may also participate. Discussion: For the first time, this study highlighted, the involvement of STEC surface proteins in the affinity for MFGs. The mechanism of STEC-MFG association is still not fully understood but our results confirm the existence of receptor/ligand type interactions between the bacteria and MFGs. Further studies are needed to identify and specify the molecules involved in this interaction. These studies should consider the likely involvement of several factors, including adhesion molecules, and the diversity of each STEC strain.

Solubilization of free ß-sitosterol in milk sphingomyelin and polar lipid vesicles as carriers: Structural characterization of the membranes and sphingosome morphology.

High consumption of plant sterols reduces the risk of cardiovascular diseases in humans and provides health benefits. Increasing the amount of plant sterols in the diet is therefore necessary to reach the recommended daily dietary intake. However, food supplementation with free plant sterols is challenging because of their low solubility in fats and water. The objectives of this study were to investigate the capacity of milk-sphingomyelin (milk-SM) and milk polar lipids to solubilise ß-sitosterol molecules in bilayer membranes organised as vesicles called sphingosomes. The thermal and structural properties of milk-SM containing bilayers composed of various amounts of ß-sitosterol were examined by differential scanning calorimetry (DSC) and temperature-controlled X-ray diffraction (XRD), the molecular interactions were studied using the Langmuir film technique, the morphologies of sphingosomes and ß-sitosterol crystals were observed by microscopy. We showed that the milk-SM bilayers devoid of ß-sitosterol exhibited a gel to fluid Lα phase transition for Tm = 34.5 °C and formed facetted spherical sphingosomes below Tm. The solubilisation of ß-sitosterol within milk-SM bilayers induced a liquid-ordered Lo phaseabove 25 %mol (1.7 %wt) ß-sitosterol and a softening of the membranes leading to the formation of elongated sphingosomes. Attractive molecular interactions revealed a condensing effect of ß-sitosterol on milk-SM Langmuir monolayers. Above 40 %mol (25.7 %wt) ß-sitosterol, partitioning occured with the formation of ß-sitosterol microcrystals in the aqueous phase. Similar results were obtained with the solubilization of ß-sitosterol within milk polar lipid vesicles. For the first time, this study highlighted the efficient solubilization of free ß-sitosterol within milk-SM based vesicles, which opens new market opportunities for the formulation of functional foods enriched in non-crystalline free plant sterols.

Oil Bodies from Chia (Salvia hispanica L.) and Camelina (Camelina sativa L.) Seeds for Innovative Food Applications: Microstructure, Composition and Physical Stability.

Exploring and deciphering the biodiversity of oil bodies (OBs) recovered from oilseeds are of growing interest in the preparation of sustainable, natural and healthy plant-based food products. This study focused on chia (Salvia hispanica L.) and camelina (Camelina sativa L.) seed OBs. A green refinery process including ultrasound to remove mucilage, aqueous extraction by grinding and centrifugation to recover OBs from the seeds was used. The microstructure, composition and physical stability of the OBs were examined. Confocal laser scanning microscopy images showed that chia and camelina seed OBs are spherical assemblies coated by a layer of phospholipids and proteins, which have been identified by gel electrophoresis. The mean diameters determined by laser light scattering measurements were 2.3 and 1.6 µm for chia and camelina seed OBs, respectively. The chia and camelina seed OBs were rich in lipids and other bioactive components with, respectively, 64% and 30% α-linolenic acid representing 70% and 53% of the total fatty acids in the sn-2 position of the triacylglycerols, 0.23% and 0.26% phospholipids, 3069 and 2674 mg/kg oil of ß-sitosterol, and lipophilic antioxidants: 400 and 670 mg/kg oil of γ-tocopherol. Phenolic compounds were recovered from the aqueous extracts, such as rutin from camelina and caffeic acid from chia. Zeta-potential measurements showed changes from about -40 mV (pH 9) to values that were positive below the isoelectric points of pH 5.1 and 3.6 for chia and camelina seed OBs, respectively. Below pH 6.5, physical instability of the natural oil-in-water emulsions with aggregation and phase separation was found. This study will contribute to the development of innovative and sustainable food products based on natural oil-in-water emulsions containing chia and camelina seed OBs for their nutritional and health benefits.

Milk sphingosomes as lipid carriers for tocopherols in aqueous foods: Thermotropic phase behaviour and morphology.

Foods containing polyunsaturated lipids are prone to oxidation. Designing food-grade hydrocolloidal encapsulation systems able to load lipophilic antioxidant molecules, such as tocopherols (vitamin E), is necessary to prevent oxidation and its deleterous consequences. In this study, we hypothesised that α-tocopherol molecules could incorporate in a host membrane composed of milk sphingomyelin (milk-SM) and performed a multi-scale biophysical study. The thermal properties of milk-SM bilayers with various molar proportions of α-tocopherol were characterised by differential scanning calorimetry (DSC), their structural properties were examined by X-ray diffraction (XRD). The miscibility between milk-SM and α-tocopherol was investigated in mixed Langmuir monolayers. The morphology of milk-SM sphingosomes was observed by confocal laser scanning microscopy (CLSM). We found that molecules of α-tocopherol inserted into the milk-SM bilayers and induced a physical desorganisation in the membrane packing, both in the ordered and fluid states. In the presence of α-tocopherol, the bilayers were no longer in a gel phase below the phase transition temperature Tm, but in the liquid ordered Lo phase. Furthermore, the sphingosomes formed elongated structures in presence of α-tocopherol as a result of membrane softening and changes in the bilayer curvature associated to membrane fusion. The findings of this work contribute in a better understanding of the capacity of milk-SM bilayers to incorporate guest molecules. Milk-SM sphingosomes loaded with tocopherols could be used to prevent oxidation in aqueous foods containing polyunsaturated lipids such as oil-in-water emulsions.

Serotype-dependent adhesion of Shiga toxin-producing Escherichia coli to bovine milk fat globule membrane proteins.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms for humans. Raw milk products are often incriminated as vehicule for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: (i) evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs), (ii) highlight STEC surface proteins associated with adhesion and (iii) evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the ζ-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The ζ-potential values were between -3.7 and - 2.9 mV for the strains and between -12.2 ± 0.14 mV for MFGs. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21,765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21,765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human gastrointestinal tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.

Deciphering the properties of hemp seed oil bodies for food applications: Lipid composition, microstructure, surface properties and physical stability.

Hemp seed oil bodies (HSOBs) are of growing interest in response to the demand of consumers for healthy and natural plant-based food formulations. In this study, we used minimal processing including aqueous extraction by grinding and centrifugation to obtain HSOBs. We determined the lipid composition of HSBOs, their microstructure, and the impact of the homogenization pressure, pH and minerals on their surface properties and the physical stability of the emulsions. HSOBs contain high levels of well-balanced PUFA with LA/ALA = 2.9, γ-tocopherol, lutein and phytosterols. The mean diameter of HSOBs was 2.3 ± 0.1 µm with an isoelectric point in the range of pH 4.4 to 4.6. Homogenization of hemp seed extracts induced a decrease in the size of HSOBs but did not eliminate the sedimentation of the protein bodies composed of the globulin edestin. By changing the surface properties of HSOBs, pH values below 6 and NaCl induced the aggregation of HSOBs, while CaCl2 induced both aggregation and membrane-fusion mediated coalescence of HSOBs by involving probably the anionic phospholipids together with membrane proteins. This study will contribute to extend the range of novel food products and designed emulsions containing hemp seed proteins and oil bodies.

Tempering governs the milk fat crystallisation and viscoelastic behaviour of unprocessed and homogenised creams.

The crystallisation behaviour of milk fat plays an important role in the functionality and sensory properties of fat-rich dairy products. In this study, we investigated the impact of tempering to 25 °C on the viscoelastic properties, particle size and thermal behaviour of 20% w/w unprocessed and homogenised creams prepared from bovine milk. The crystallisation properties were examined by synchrotron X-ray diffraction (XRD) at small (SAXS) and wide angle (WAXS) and differential scanning calorimetry (DSC). Oscillation rheology was performed to characterise the cream's viscoelastic properties. Homogenisation (35 MPa) reduced the average droplet size from 4.4 to 1.3 µm. After 24 h storage at 4 °C, milk fat structures showed triacylglycerol (TAG) 2L and 3L(001, 002, 003, 005) lamellar stacking orders associated predominantly with the α and ß' polymorphic forms. Tempering to 25 °C induced the complete melting of the 3L crystals and led to an irreversible loss in the elastic modulus (G') and a reduction in the viscous modulus (G'') once returned to refrigerated conditions, due to changes in the particle-particle interactions and structure of the reformed milk fat crystals. The results demonstrate that crystallisation behaviour of milk fat is influenced by droplet size and the rearrangement of triacylglycerol (TAG) upon tempering, and lead to changes in the viscoelastic behaviour of dairy products containing a high level of milk fat.

Physicochemical, techno-functional, and fat melting properties of spray-dried camel and bovine milk powders.

In this study, three skimmed and one whole-fat spray-dried camel milk powders were produced and their characteristics were compared to those of bovine milk powders. The physicochemical analysis of the produced powders indicated that camel milk powders (whether skimmed or not) presented higher ash and whey protein contents as compared to those of bovine milk powders. Our results indicated that the investigated camel and bovine milk powders exhibited a high solubility index (>99%) with poor dispersibility and wettability indexes due to their small particles size (d50 ≤ 12 µm) and their narrow size distribution (span ≤ 2). In addition, although camel and bovine milk powders presented the same total fat content, lower free fat content was measured for camel milk powders. Besides, the whey protein nitrogen index and the SDS-PAGE electrophoresis underlined that camel and bovine milk proteins remained intact after drying with low denaturation extent. It is worth noticed that camel milk proteins were less denaturized due to the absence of the heat-sensitive ß-lactoglobulin in camel milk. Moreover, the low denaturation extent participated in the enhancing of the foaming capacity and stability of camel and bovine milk powders. Finally, the calorimetric analysis showed that higher fat melting temperatures were recorded in whole-fat camel milk powder and in their anhydrous form as compared to those of bovine milk. PRACTICAL APPLICATION: Camel milk powder is an emerging non-bovine dairy product. Understanding its rehydration ability and evaluating the impact of spray drying on its protein quality are promising approaches to obtain high-quality camel milk powder with high reconstitution ability. Findings of this study indicated that spray drying is a suitable technique to produce highly soluble camel milk powders with low denaturation extent. These results will benefit the research and development department of food industry (especially those producing camel milk powder) as well as the direct consumers.

Loading of lutein in egg-sphingomyelin vesicles as lipid carriers: Thermotropic phase behaviour, structure of sphingosome membranes and lutein crystals.

Lutein is a xanthophyll carotenoid provided exclusively by the diet, that has protective functions and beneficial effects on human health. Supplementation in lutein is necessary to reach the recommended daily dietary intake. However, the introduction of lutein into foods and beverages is a real challenge since this lipophilic nutrient has a poor aqueous solubility and a low bioavailability. In this study, we investigated the capacity of egg-sphingomyelin (ESM) vesicles called sphingosomes to solubilise lutein into the bilayers. The thermal and structural properties of ESM bilayers were examined in presence of various amounts of lutein by differential scanning calorimetry (DSC) and temperature-controlled X-ray diffraction (XRD), the structures of sphingosomes and lutein crystals were observed by microscopic techniques. ESM bilayers were in the fluid Lα phase above the phase transition temperature Tm = 39.6 °C and in the lamellar ripple Pß' phase below Tm where ESM sphingosomes exhibited ondulations and were facetted. Lutein molecules were successfully incorporated into the ESM bilayers where they induced a structural disorganisation. For ESM/lutein 90/10 %mol (91.8/8.2 %wt; 89 mg lutein / g ESM), lutein partitioning occured with the formation of lutein crystals in the aqueous phase together with lutein-loaded ESM vesicles. This study highlighted the capacity of new lipid carriers such as egg-sphingosomes to solubilise lutein and opens perspectives for the formulation of effective lutein-fortified functionnal foods and beverages providing health benefits.

The adhesion of homogenized fat globules to proteins is increased by milk heat treatment and acidic pH: Quantitative insights provided by AFM force spectroscopy.

The rheological properties and microstructure of dairy gels involve the connectivity between milk fat globules (MFG) and casein micelles that is affected by technological processes such as milk homogenization and heat treatment. The underlying mechanisms require further quantification of the interactions at the nanoscale level to be fully understood and controlled. In this study, we examined the adhesion of homogenized MFG to milk proteins and evaluated the role of ultra-high temperature (UHT) heat treatment and pH. The combination of physico-chemical analysis, rheology and microscopy observations at different scale levels associated to atomic force microscopy (AFM) force spectroscopy were used. AFM experiments performed at the particle scale level showed that adhesion of individual homogenized MFG to milk proteins (1) is increased upon acidification at pH 4.5: 1.4 fold for unheated samples and 3.5 fold for UHT samples, and (2) is enhanced by about 1.7 fold at pH 4.5 after UHT heat treatment of milk, from 176 pN to 296 pN, thanks to highly-reactive heat-denatured whey proteins located at the surface of MFG and caseins. The increased inter-particle adhesion forces accounted for more connected structures and stiffer UHT milk acid gels, compared to unheated-milk gels. Using a multiscale approach, this study showed that heat treatment of milk markedly affected the interactions occurring at the particle's surface level with consequences on the bulk structural and rheological properties of acid gels. Such findings will be useful for manufacturers to modulate the texture of fermented dairy products through the tailoring of heat-induced complexation of proteins and the connectivity of homogenized MFG with the protein network. This work will also contribute in a better understanding of the impact of process-induced changes on the digestibility and metabolic fate of proteins and lipids.

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 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.

The surface properties of milk fat globules govern their interactions with the caseins: Role of homogenization and pH probed by AFM force spectroscopy.

The surface of milk fat globules consists of a biological membrane rich in polar lipids and glycoproteins. However, high shear stress applied upon homogenization disrupts the membrane and leads to the adsorption of casein micelles, as the major protein fraction of milk. These changes in the interface properties could affect the interactions between native or homogenized milk fat globules and the surrounding protein matrix, at neutral pH and upon acidification. In this study, macroscale rheometry, microscopic observations, nanoscale AFM-based force spectroscopy and physico-chemical analysis were combined to examine the interfacial composition and structure of milk fat globules and to evaluate their interactions with casein micelles. We showed that the surface properties of milk fat globules (biological membrane vs. caseins) and pH govern their interactions with casein micelles. The adhesion between individual fat globules and casein micelles was higher upon homogenization, especially at acid pH where the work of adhesion increased from 3.3 x 10-18 to 14 x 10-18 J for native and homogenized fat globules, respectively. Consequently, casein-coated homogenized fat globules yield stiffer milk acid gels. These findings cast light on the importance of colloidal particle's surface properties and pH on their connectivity with the surrounding matrix, which modulates the bulk microstructure and rheological properties with potential functional consequences, such as milk lipid digestion.

The phase and charge of milk polar lipid membrane bilayers govern their selective interactions with proteins as demonstrated with casein micelles.

The biological membrane surrounding fat globules in milk (milk fat globule membrane; MFGM) is an interface involved in many biological functions and interactions with the surrounding proteins or lipolytic enzymes in the gastro-intestinal tract during digestion. The MFGM exhibits lateral heterogeneities resulting from the different phase states and/or head-group charge of the polar lipids, which were both hypothesized to drive interaction with the casein micelles that is the major milk protein assembly. Atomic force microscopy (AFM) imaging was used to track the interactions of casein micelles with hydrated supported lipid bilayers of different composition, phase state and charge. Zeta-potential and Langmuir isotherms of the different polar lipids offered additional information necessary to interpret AFM observations. We showed that the negatively-charged casein micelles did not interact with milk sphingomyelin in the gel or liquid-ordered phases but did interact with polar lipids in the liquid-disordered phase (unsaturated polar lipids and milk sphingomyelin above its melting point). A wide intermolecular distance between polar lipids allowed protein adsorption on the membranes. However, the presence of the anionic polar lipids phosphatidylserine and phosphatidylinositol prevented any interaction with the casein micelles, probably due to electrostatic repulsion. These results open perspectives for the preparation of tailored emulsions covered by polar lipids able to modulate the interfacial interactions with proteins.

Casein interaction with lipid membranes: Are the phase state or charge density of the phospholipids affecting protein adsorption?

Casein micelles are ~200 nm electronegative particles that constitute 80 wt% of the milk proteins. During synthesis in the lactating mammary cells, caseins are thought to interact in the form of ~20 nm assemblies, directly with the biological membranes of the endoplasmic reticulum and/or the Golgi apparatus. However, conditions that drive this interaction are not yet known. Atomic force microscopy imaging and force spectroscopy were used to directly observe the adsorption of casein particles on supported phospholipid bilayers with controlled compositions to vary their phase state and surface charge density, as verified by X-ray diffraction and zetametry. At pH 6.7, the casein particles adsorbed onto bilayer phases with zwitterionic and liquid-disordered phospholipid molecules, but not on phases with anionic or ordered phospholipids. Furthermore, the presence of adsorbed caseins altered the stability of the yet exposed bilayer. Considering their respective compositions and symmetry/asymmetry, these results cast light on the possible interactions of casein assemblies with the organelles' membranes of the lactating mammary cells.

Thermotropic phase behavior of milk sphingomyelin and role of cholesterol in the formation of the liquid ordered phase examined using SR-XRD and DSC.

Sphingomyelin (SM) and cholesterol are major lipid components of biological membranes involved in the formation of ordered domains. In this study, we investigated the biophysical properties of milk-SM bilayers and determined the effect of cholesterol. The thermotropic phase behaviours of milk-SM and milk-SM/cholesterol mixtures were characterized using differential scanning calorimetry (DSC) and high flux synchrotron radiation X-ray diffraction (SR-XRD). The melting phase transition temperature determined for fully hydrated milk-SM bilayers was Tm = 34.3 ± 0.1 °C. The thermotropic phase behavior of milk-SM is complex, reflecting the mixture of different molecular species. Structural reorganisations successively occurring on heating of fully hydrated milk-SM bilayers were interpreted as follows: i) melting of C16:0-SM, ii) conversion of long and saturated SM species from fully interdigitated (Lß1) to mixed interdigitated (Lß2) lamellar structures evidencing gel phase polymorphism and then iii) transition to the fluid liquid-crystal Lα phase. We demonstrated that cholesterol modulates the physical properties of milk-SM bilayers and that building up of the lamellar liquid-ordered Lo phase is completed for 33 mol% of cholesterol. The ordering effect of cholesterol on milk-SM bilayers and the temperature-independent behavior of the Lo phase formed by milk-SM/cholesterol complexes were characterized. The findings of this work will contribute in a better understanding of the biological functions exerted by the milk-SM as a function of its phase state and interactions with cholesterol (e.g. hypocholesterolemic effect).

Dairy curd coagulated by a plant extract of Calotropis procera: Role of fat structure on the chemical and textural characteristics.

Milk is often subjected to technological treatments which have impacts on the structure of milk constituents and the characteristics of rennet curds. In this paper, the influence of the dairy fat structure on the biochemical and textural characteristics of curds coagulated by an extract of Calotropis procera leaves was studied. Standardized milks were reconstituted with the same contents in protein (35g·kg-1) and fat (35g·kg-1) but with different structures of fat i.e. homogenized anhydrous milk fat (HAMF), homogenized cream (HC) and non-homogenized cream (NHC). As expected, the size distributions of fat globules in the different milks were different. After their coagulations by the plant extract, the physico-chemical characteristics of the curds and respective wheys were determined. No difference was observed in the coagulation time between the three milks but the whey removed more quickly from HAMF and HC curds than NHC-curd. The biochemical analyses of curds revealed a lower content in dry matter and fat in the NHC-curd compared to HAMF- and HC-curds. Otherwise, the NHC-whey exhibited the highest amount of fat. Observations by confocal microscopy showed that the fat globules were homogenously distributed and well trapped in the protein networks of HAMF- and HC-curds. In the NHC-curd, the fat globules were located in whey pockets, with less connectivity with the protein network. The textural analysis showed that the NHC-curd was more elastic, soft and adhesive than HAMF- and HC-curds. Homogenization significantly reduced the loss of fat during cheese manufacturing and conferred specific textural characteristics to the curds coagulated by an extract of Calotropis procera.

Palmitoyl ceramide promotes milk sphingomyelin gel phase domains formation and affects the mechanical properties of the fluid phase in milk-SM/DOPC supported membranes.

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.

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 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<200nm) membranes in the gel phase, e.g. the Young modulus E and the bending rigidity kC. This study increases our knowledge about the key role of naturally complex lipid compositions in tailoring the physical properties of biological membranes. It could be also used in liposomes development e.g. to select the suitable lipid composition according to usage.

Microstructure and physicochemical properties reveal differences between high moisture buffalo and bovine Mozzarella cheeses.

Mozzarella cheese is a classical dairy product but most research to date has focused on low moisture products. In this study, the microstructure and physicochemical properties of both laboratory and commercially produced high moisture buffalo Mozzarella cheeses were investigated and compared to high moisture bovine products. Buffalo and bovine Mozzarella cheeses were found to significantly differ in their microstructure, chemical composition, organic acid and proteolytic profiles but had similar hardness and meltability. The buffalo cheeses exhibited a significantly higher ratio of fat to protein and a microstructure containing larger fat patches and a less dense protein network. Liquid chromatography mass spectrometry detected the presence of only ß-casein variant A2 and a single ß-lactoglobulin variant in buffalo products compared to the presence of both ß-casein variants A1 and A2 and ß-lactoglobulin variants A and B in bovine cheese. These differences arise from the different milk composition and processing conditions. The differences in microstructure and physicochemical properties observed here offer a new approach to identify the sources of milk used in commercial cheese products.