Fanny Guyomarc’h

Lactosylation of milk proteins during the manufacture and storage of skim milk powders

Abstract Extensive lactosylation of milk proteins in standard skim milk powder dried against air between 185 and 90°C (inlet and outlet temperatures of the air) was detected by capillary electrophoresis. Optimisation of the drying conditions included keeping the outlet temperature low (preferably

Milk sphingomyelin domains in biomimetic membranes and the role of cholesterol: morphology and nanomechanical properties investigated using AFM and force spectroscopy.

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 l(o) 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.

Cholesterol Decreases the Size and the Mechanical Resistance to Rupture of Sphingomyelin Rich Domains, in Lipid Bilayers Studied as a Model of the Milk Fat Globule Membrane

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.

Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation

The enzymatic lipolysis of complex natural lipoproteic assemblies such as milk fat globules is central in neonatal digestion. This process first requires the rapid adsorption of a lipolytic enzyme, gastric lipase, onto the membrane enveloping the triglyceride substrate before the onset of catalytic activity. The interactions governing lipase adsorption onto this complex lipid/water interface are not fully elucidated. This study was designed to unravel the interactions of recombinant dog gastric lipase (rDGL) with model monolayers presenting liquid-liquid phase coexistence and mimicking the outer leaflet of the milk fat globule membrane. Combining biophysical tools (ellipsometry, tensiometry and atomic force microscopy), it was evidenced that rDGL partitions toward liquid expanded phase and at phase boundaries. rDGL gets adsorbed at several levels of insertion suggesting molecular cooperation that may favor insertion and strongly impacts on the lipid phase lateral organization. The addition of phosphatidylserine, negatively charged, reinforced adsorption; hence besides hydrophobic interactions and as further investigated through surface potential modeling, rDGL adsorption is favored by electrostatic interactions.

Effects of Adding Low Levels of a Disulfide Reducing Agent on the Disulfide Interactions of β-Lactoglobulin and κ-Casein in Skim Milk

Low concentrations of a disulfide reducing agent were added to unheated and heated (80 °C for 30 min) skim milk, with and without added whey protein. The reduction of the β-lactoglobulin and κ-casein disulfide bonds was monitored over time using electrophoresis. The distribution of the proteins between the colloidal and serum phases was also investigated. κ-Casein disulfide bonds were reduced in preference to those of β-lactoglobulin in both unheated and heated skim milk (with or without added whey protein). In addition, in heated skim milk, while the serum κ-casein was reduced more readily than the colloidal κ-casein, the distribution of κ-casein between the two phases was not affected.

The protein interactions and rheological properties of skim milk heated in the presence of low levels of reducing agent

Skim milk with low levels of added β-mercaptoethanol (SM-ME) and untreated skim milk (SM) were heated and then made into acid gels. Acid gels prepared from heated SM-ME had markedly higher firmness and contained more protein connections than acid gels prepared from heated SM. Electrophoretic analyses of the milks showed that the levels of β-lactoglobulin and α-lactalbumin associated with the casein micelles increased with increasing β-ME concentration. The levels of disulphide-linked whey proteins were higher in SM-ME than in SM. This suggested that there may be higher levels of initiators for thiol-disulphide exchange reactions, resulting in an increase in the rate of the reactions and the formation of greater numbers of small aggregates, in SM-ME than in SM. Consequently, acid gels made from SM-ME may have more bonds and more particles participating in the network, resulting in firmer gels, than acid gels made from SM.

Mechanical properties of membranes composed of gel-phase or fluid-phase phospholipids probed on liposomes by atomic force spectroscopy

In many liposome applications, the nanomechanical properties of the membrane envelope are essential to ensure, e.g., physical stability, protection, or penetration into tissues. Of all factors, the lipid composition and its phase behavior are susceptible to tune the mechanical properties of membranes. To investigate this, small unilamellar vesicles (SUV; diameter < 200 nm), referred to as liposomes, were produced using either unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in aqueous buffer at pH 6.7. The respective melting temperatures of these phospholipids were -20 and 41 °C. X-ray diffraction analysis confirmed that at 20 °C DOPC was in the fluid phase and DPPC was in the gel phase. After adsorption of the liposomes onto flat silicon substrates, atomic force microscopy (AFM) was used to image and probe the mechanical properties of the liposome membrane. The resulting force-distance curves were treated using an analytical model based on the shell theory to yield the Youngs modulus (E) and the bending rigidity (kC) of the curved membranes. The mechanical investigation showed that DPPC membranes were much stiffer (E = 116 ± 45 MPa) than those of DOPC (E = 13 ± 9 MPa) at 20 °C. The study demonstrates that the employed methodology allows discrimination of the respective properties of gel- or fluid-phase membranes when in the shape of liposomes. It opens perspectives to map the mechanical properties of liposomes containing both fluid and gel phases or of biological systems.

Recrystallized S-Layer Protein of a Probiotic Propionibacterium: Structural and Nanomechanical Changes upon Temperature or pH Shifts Probed by Solid-State NMR and AFM.

Surface protein layers (S layers) are common constituents of the bacterial cell wall and originate from the assembly of strain-dependent surface layer proteins (Slps). These proteins are thought to play important roles in the bacterias biology and to have very promising technological applications as biomaterials or as part of cell-host cross-talk in probiotic mechanism. The SlpA from Propionibacterium freudenreichii PFCIRM 118 strain was isolated and recrystallized to investigate organization and assembly of the protein using atomic force microscopy and solid-state (1)H and (13)C-nuclear magnetic resonance. SlpA was found to form hexagonal p1 monolayer lattices where the protein exhibited high proportions of disordered regions and of bound water. The lattice structure was maintained, but softened, upon mild heating or acidification, probably in relation with the increasing mobilities of the disordered protein regions. These results gave structural insights on the mobile protein regions exposed by S layer films, upon physiologically relevant changes of their environmental conditions.