Arabinoxylan in Water through SANS: Single-Chain Conformation, Chain Overlap, and Clustering.

Using small-angle neutron scattering (SANS), we examine the structure and conformational behavior of wheat arabinoxylan (AX) prepared at various concentrations in a sodium phosphate aqueous buffer. As for another major hemicellulose, xyloglucan, we observe a small number of large clusters surrounded by AX chains that behave exactly as a polymer in good solvent with a Flory exponent ν = 0.588. The fit of the data at high q-values to a standard worm-like chain model gives the persistence length lp = 45 Å and cross section of the chains 2Rc = 11-12 Å. In addition, using a dedicated modeling approach, we extract from the SANS data at the intermediate q-range the correlation length ξ of the solutions in the semidilute regime. The decay of ξ with concentration follows a scaling law that further confirms the self-avoiding statistical behavior of the AX chains. This first comprehensive study about the properties of water-soluble AX at different length scales may help in the development of products and processes involving AX as a substitute for fossil carbon molecules.

Protein Transport upon Advection at the Air/Water Interface: When Charge Matters.

The formation of dense protein interfacial layers at a free air-water interface is known to result from both diffusion and advection. Furthermore, protein interactions in concentrated phases are strongly dependent on their overall positive or negative net charge, which is controlled by the solution pH. As a consequence, an interesting question is whether the presence of an advection flow of water toward the interface during protein adsorption produces different kinetics and interfacial structure of the adsorbed layer, depending on the net charge of the involved proteins and, possibly, on the sign of this charge. Here we test a combination of the following parameters using ovalbumin and lysozyme as model proteins: positive or negative net charge and the presence or absence of advection flow. The formation and the organization of the interfacial layers are studied by neutron reflectivity and null-ellipsometry measurements. We show that the combined effect of a positive charge of lysozyme and ovalbumin and the presence of advection flow does induce the formation of interfacial multilayers. Conversely, negatively charged ovalbumin forms monolayers, whether advection flow is present or not. We show that an advection/diffusion model cannot correctly describe the adsorption kinetics of multilayers, even in the hypothesis of a concentration-dependent diffusion coefficient as in colloidal filtration, for instance. Still, it is clear that advection is a necessary condition for making multilayers through a mechanism that remains to be determined, which paves the way for future research.

How High Concentrations of Proteins Stabilize the Amorphous State of Calcium Orthophosphate: A Solid-State Nuclear Magnetic Resonance (NMR) Study of the Casein Case.

Understanding how proteins stabilize amorphous calcium ortho-phosphate (ACP) phases is of great importance in biology and for pharmaceutical or food applications. Until now, most of the former investigations about ACP-protein stability and equilibrium were performed under conditions where ACP colloidal nanoclusters are surrounded by low to moderate concentrations of peptides or proteins (15-30 g L-1). As a result, the question of ACP-protein interactions in highly concentrated protein systems has clearly been overlooked, whereas it corresponds to actual industrial conditions such as drying or membrane filtration in the dairy industry for instance. In this study, the structure of an ACP phase is monitored in association with one model phosphorylated protein (casein) using solid-state nuclear magnetic resonance (ssNMR) under two conditions of high protein concentration (300 and 400 g L-1). At both concentrations and at 25 °C, it is found that the caseins maintain the mineral phase in an amorphous form with no detectable influence on its structure or size. Interestingly, and in both cases, a significant amount of the nonphosphorylated side chains interacts with ACP through hydrogen bonds. The number of these interacting side chains is found to be higher at the highest casein concentration. At 45 °C, which is a destabilizing temperature of ACP under protein-free conditions, the amorphous structure of the mineral phase is partially transformed at a casein concentration of 300 g L-1, while it remains almost intact at a casein concentration of 400 g L-1. Therefore, these results clearly indicate that increasing the concentration of proteins favors ACP-protein interactions and stabilizes the ACP clusters more efficiently.

Osmotic pressures of lysozyme solutions from gas-like to crystal states.

We obtained osmotic pressure data of lysozyme solutions, describing their physical states over a wide concentration range, using osmotic stress for pressures between 0.05 bar and about 40 bar and volume fractions between 0.01 and 0.61. The osmotic pressure vs. volume fraction data consist of a dilute, gas-phase regime, a transition regime with a high-compressibility plateau, and a concentrated regime where the system is nearly incompressible. The first two regimes are shifted towards a higher protein volume fraction upon decreasing the strength or the range of electrostatic interactions. We describe this shift and the overall shape of the experimental data in these two regimes through a model accounting for a steric repulsion, a short-range van der Waals attraction and a screened electrostatic repulsion. The transition is caused by crystallization, as shown by small-angle X-ray scattering. We verified that our data points correspond to thermodynamic equilibria, and thus that they consist of the reference experimental counterpart of a thermodynamic equation of state.

Diffusion and partitioning of macromolecules in casein microgels: evidence for size-dependent attractive interactions in a dense protein system.

Understanding the mechanisms that determine the diffusion and interaction of macromolecules (such as proteins and polysaccharides) that disperse through dense media is an important fundamental issue in the development of innovative technological and medical applications. In the current work, the partitioning and diffusion of macromolecules of different sizes (from 4 to 10 nm in diameter) and shapes (linear or spherical) within dispersions of casein micelles (a protein microgel) is studied. The coefficients for diffusion and partition are measured using FRAP (fluorescence recovery after photobleaching) and analyzed with respect to the structural characteristics of the microgel determined by the use of TEM (transmission electron microscopy) tomography. The results show that the casein microgel displays a nonspecific attractive interaction for all macromolecules studied. When the macromolecular probes are spherical, this affinity is clearly size-dependent, with stronger attraction for the larger probes. The current data show that electrostatic effects cannot account for such an attraction. Rather, nonspecific hydration molecular forces appear to explain these results. These findings show how weak nonspecific forces affect the diffusion and partitioning of proteins and polysaccharides in a dense protein environment. These results could be useful to better understand the mechanisms of diffusion and partitioning in other media such as cells and tissues. Furthermore, there arises the possibility of using the casein micelle as a size-selective molecular device.

Structural heterogeneity of milk casein micelles: a SANS contrast variation study.

We examine the internal structure of milk casein micelles using the contrast variation method in Small-Angle Neutron Scattering (SANS). Experiments were performed with casein dispersions of different origins (i.e., milk powder or fresh milk) and extended to very low q-values (∼9 × 10(-4) Å(-1)), thus making it possible to precisely determine the apparent gyration radius Rg at each contrast. From the variation of I(q → 0) with contrast, we determine the distribution of composition of all the particles in the dispersions. As expected, most of these particles are micelles, made of casein and calcium phosphate, with a narrow distribution in compositions. These micelles always coexist with a very small fraction of fat droplets, with sizes in the range of 20-400 nm. For the dispersions prepared from fresh milk, which were purified under particularly stringent conditions, the number ratio of fat droplets to casein micelles is as low as 1 to 10(6). In that case, we are able to subtract from the total intensity the contribution of the fat droplets and in this way obtain the contribution of the micelles only. We then analyze the variation of this contribution with contrast using the approach pioneered by H. B. Stuhrmann. We model the casein micelle as a core-shell spherical object, in which the local scattering length density is determined by the ratio of calcium phosphate nanoclusters to proteins. We find that models in which the shell has a lower concentration of calcium phosphate than the core give a better agreement than models in which the shell has a higher density than the core.

Correction: Structural heterogeneity of milk casein micelles: a SANS contrast variation study.

Correction for 'Structural heterogeneity of milk casein micelles: a SANS contrast variation study' by Antoine Bouchoux et al., Soft Matter, 2015, DOI: 10.1039/c4sm01705f.

A general approach for predicting the filtration of soft and permeable colloids: the milk example.

Membrane filtration operations (ultra-, microfiltration) are now extensively used for concentrating or separating an ever-growing variety of colloidal dispersions. However, the phenomena that determine the efficiency of these operations are not yet fully understood. This is especially the case when dealing with colloids that are soft, deformable, and permeable. In this paper, we propose a methodology for building a model that is able to predict the performance (flux, concentration profiles) of the filtration of such objects in relation with the operating conditions. This is done by focusing on the case of milk filtration, all experiments being performed with dispersions of milk casein micelles, which are sort of ″natural″ colloidal microgels. Using this example, we develop the general idea that a filtration model can always be built for a given colloidal dispersion as long as this dispersion has been characterized in terms of osmotic pressure Π and hydraulic permeability k. For soft and permeable colloids, the major issue is that the permeability k cannot be assessed in a trivial way like in the case for hard-sphere colloids. To get around this difficulty, we follow two distinct approaches to actually measure k: a direct approach, involving osmotic stress experiments, and a reverse-calculation approach, that consists of estimating k through well-controlled filtration experiments. The resulting filtration model is then validated against experimental measurements obtained from combined milk filtration/SAXS experiments. We also give precise examples of how the model can be used, as well as a brief discussion on the possible universality of the approach presented here.

Contribution of surface ß-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-ß-D-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of ß-glucan. To evaluate this hypothesis, gtfF genes of three ß-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of ß-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of ß-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of ß-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, ß-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.

AFM imaging of milk casein micelles: evidence for structural rearrangement upon acidification.

Milk casein micelles are natural association colloids that we all encounter in everyday life, yet we still lack an accurate description of their internal structure and the interactions that stabilize it. In this letter, we provide for the first time detailed images of intact casein micelles as obtained through atomic force microscopy under liquid conditions close to physiological. The micelles appear as heterogeneous raspberry-like particles, which is consistent with a hierarchical/spongelike structure made of connected 10-40 nm dense casein regions. Upon in situ acidification to pH 5, the micelles decrease in size and lose their surface heterogeneities, indicating that this structure is highly sensitive to variations in mineral content and caseins net charge.

How to squeeze a sponge: casein micelles under osmotic stress, a SAXS study.

By combining the osmotic stress technique with small-angle x-ray scattering measurements, we followed the structural response of the casein micelle to an overall increase in concentration. When the aqueous phase that separates the micelles is extracted, they behave as polydisperse repelling spheres and their internal structure is not affected. When they are compressed, the micelles lose water and shrink to a smaller volume. Our results indicate that this compression is nonaffine, i.e., some parts of the micelle collapse, whereas other parts resist deformation. We suggest that this behavior is consistent with a spongelike casein micelle having a triple hierarchical structure. The lowest level of the structure consists of the CaP nanoclusters that serve as anchors for the casein molecules. The intermediate level consists of 10- to 40-nm hard regions that resist compression and contain the nanoclusters. Those regions are connected and/or partially merged with each other, thus forming a continuous and porous material. The third level of structure is the casein micelle itself, with an average size of 100 nm. In our view, such a structure is consistent with the observation of 10- to 20-nm casein particles in the Golgi vesicles of lactating cells: upon aggregation, those particles would rearrange, fuse, and/or swell to form the spongelike micelle.

Casein micelle dispersions under osmotic stress.

Casein micelles dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis. This technique measured an equation of state of the dispersions over a wide range of pressures and concentrations and at different ionic strengths. Three regimes were found. i), A dilute regime in which the osmotic pressure is proportional to the casein concentration. In this regime, the casein micelles are well separated and rarely interact, whereas the osmotic pressure is dominated by the contribution from small residual peptides that are dissolved in the aqueous phase. ii), A transition range that starts when the casein micelles begin to interact through their kappa-casein brushes and ends when the micelles are forced to get into contact with each other. At the end of this regime, the dispersions behave as coherent solids that do not fully redisperse when osmotic stress is released. iii), A concentrated regime in which compression removes water from within the micelles, and increases the fraction of micelles that are irreversibly linked to each other. In this regime the osmotic pressure profile is a power law of the residual free volume. It is well described by a simple model that considers the micelle to be made of dense regions separated by a continuous phase. The amount of water in the dense regions matches the usual hydration of proteins.

Microfluidic model systems used to emulate processes occurring during soft particle filtration.

Cake layer formation in membrane processes is an inevitable phenomenon. For hard particles, especially cake porosity and thickness determine the membrane flux, but when the particles forming the cake are soft, the variables one has to take into account in the prediction of cake behavior increase considerably. In this work we investigate the behavior of soft polyacrylamide microgels in microfluidic model membranes through optical microscopy for in situ observation both under regular flow and under enhanced gravity conditions. Particles larger than the pore are able to pass through deformation and deswelling. We find that membrane clogging time and cake formation is not dependent on the applied pressure but rather on particle and membrane pore properties. Furthermore, we found that particle deposits subjected to low pressures and low g forces deform in a totally reversible fashion. Particle deposits subjected to higher pressures only deform reversibly if they can re-swell due to capillary forces, otherwise irreversible compression is observed. For membrane processes this implies that when using deformable particles, the pore size is not a good indicator for membrane performance, and cake formation can have much more severe consequences compared to hard particles due to the sometimes-irreversible nature of soft particle compression.

Conformational changes influence clogging behavior of micrometer-sized microgels in idealized multiple constrictions.

Clogging of porous media by soft particles has become a subject of extensive research in the last years and the understanding of the clogging mechanisms is of great importance for process optimization. The rise in the utilization of microfluidic devices brought the possibility to simulate membrane filtration and perform in situ observations of the pore clogging mechanisms with the aid of high speed cameras. In this work, we use microfluidic devices composed by an array of parallel channels to observe the clogging behavior of micrometer sized microgels. It is important to note that the microgels are larger than the pores/constrictions. We quantify the clog propensity in relation to the clogging position and particle size and find that the majority of the microgels clog at the first constriction independently of particle size and constriction entrance angle. We also quantify the variations in shape and volume (2D projection) of the microgels in relation to particle size and constriction entrance angle. We find that the degree of deformation increases with particle size and is dependent of constriction entrance angle, whereas, changes in volume do not depend on entrance angle.

Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides.

Enzymes are involved in various types of biological processes. In many cases, they are part of multi-component machineries where enzymes are localized in close proximity to each-other. In such situations, it is still not clear whether inter-enzyme spacing actually plays a role or if the colocalization of complementary activities is sufficient to explain the efficiency of the system. Here, we focus on the effect of spatial proximity when identical enzymes are immobilized onto a surface. By using an innovative grafting procedure based on the use of two engineered protein fragments, Jo and In, we produce model systems in which enzymes are immobilized at surface densities that can be controlled precisely. The enzyme used is a xylanase that participates to the hydrolysis of plant cell wall polymers. By using a small chromogenic substrate, we first show that the intrinsic activity of the enzymes is fully preserved upon immobilization and does not depend on surface density. However, when using beechwood xylan, a naturally occurring polysaccharide, as substrate, we find that the enzymatic efficiency decreases by 10-60% with the density of grafting. This unexpected result is probably explained through steric hindrance effects at the nanoscale that hinder proper interaction between the enzymes and the polymer. A second effect of enzyme immobilization at high densities is the clear tendency for the system to release preferentially shorter oligosaccharides from beechwood xylan as compared to enzymes in solution.

Soft-Matter Approaches for Controlling Food Protein Interactions and Assembly.

Animal- and plant-based proteins are present in a wide variety of raw and processed foods. They play an important role in determining the final structure of food matrices. Food proteins are diverse in terms of their biological origin, molecular structure, and supramolecular assembly. This diversity has led to segmented experimental studies that typically focus on one or two proteins but hinder a more general understanding of food protein structuring as a whole. In this review, we propose a unified view of how soft-matter physics can be used to control food protein assembly. We discuss physical models from polymer and colloidal science that best describe and predict the phase behavior of proteins. We explore the occurrence of phase transitions along two axes: increasing protein concentration and increasing molecular attraction. This review provides new perspectives on the link between the interactions, phase transitions, and assembly of proteins that can help in designing new food products and innovative food processing operations.

Major Role of Voluminosity in the Compressibility and Sol-Gel Transition of Casein Micelle Dispersions Concentrated at 7 °C and 20 °C.

The objective of this work is to bring new information about the influence of temperatures (7 °C and 20 °C) on the equation of state and sol-gel transition behavior of casein micelle dispersions. Casein micelle dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis at 7 °C and 20 °C. The osmotic stress technique measured the osmotic pressures of the dispersions over a wide range of concentrations. Rheological properties of concentrated dispersions were then characterized, respectively at 7 °C and at 20 °C. The essential result is that casein micelle dispersions are less compressible at 7 °C than at 20 °C and that concentration of sol-gel transition is lower at 7 °C than at 20 °C, with compressibility defined as the inverse to the resistance to the compression, and that is proportional to the cost to remove water from structure. From our interpretations, these two features were fully consistent with a release of soluble ß-casein and nanoclusters CaP and an increased casein micelle hydration and apparent voluminosity at 7 °C as compared with 20 °C.

Molecular mobility in dense protein systems: an investigation through 1H NMR relaxometry and diffusometry.

Understanding how proteins behave in highly concentrated systems is a major issue in many fields of research, including biology, biophysics, and chemical engineering. In this paper, we provide a comprehensive (1)H NMR study of molecular mobility in dilute to highly concentrated dispersions of the exact same protein (casein) but organized in two distinct supramolecular forms: spongelike casein micelles or soft casein aggregates. Both relaxometry and diffusometry experiments were performed, so that three different parameters are reported: spin-spin relaxation rates of non-water protons (1/T(2,ne)), spin-spin relaxation rates of water protons (1/T(2,e+w)), and water self-diffusion coefficients (D(w)). The results are discussed in an effort to understand the respective effects of protein crowding and protein supramolecular organization on each mobility indicator. We also examine if connections exist between the observed changes in molecular mobility and the already documented changes in rheological and osmotic properties of casein dispersions as concentration is increased.