Search

International audience; Phase diagrams are widely used to map and control the assembly states of biopolymers. However, these studies are highly time and material consuming. Here, we present the optimization of a simple tool based on millifluidics to screen phase diagram of biopolymers, pure or in mixtures with other biopolymers. It is used (i) to generate a homogeneous mixture of biopolymers/buffer and/or biopolymers 1/ biopolymers 2 in a short time (similar to s), (ii) to vary the composition of the mixtures by adjusting the flow rates, and (iii) to determine the turbidity of the drop by grey level analysis. The mixing efficiency and the calibration turbidity vs. grey level were performed using colloidal titanium dioxide dispersions. The use of millifluidics reduced the amount of material of ten-fold and the experimentation time by a factor five compared to a conventional bulk approach. This set-up was used to explore functional synergies between proteins and polysaccharides as an example of application.

Oppositely charged proteins can form soluble assemblies that under specific physical chemical conditions lead to liquid-liquid phase separation, also called heteroprotein coacervation. Increasing evidence suggests that surface charge anisotropy plays a key role in heteroprotein complexation, and coacervation. Here, we investigated complexation of an acidic protein, β-lactoglobulin (BLG), with two basic proteins, rapeseed napin (NAP) and lysozyme (LYS), of similar net charge and size but differing in surface charge distribution. Using turbidity measurements and isothermal titration calorimetry, we confirmed that LYS binds BLG as expected from previous studies. This interaction leads to two types of phase separation phenomena, depending on pH: liquid-solid phase separation in the case of strong electrostatic attraction and liquid-liquid phase separation for weaker attraction. More interestingly, we showed using dynamic light scattering that NAP interacts with BLG, resulting in formation of assemblies in the nanometer size range. The formation of assemblies was also evident when modeling the interactions using Brownian dynamics for both BLG + NAP and BLG + LYS. Similarly, to DLS, BLG and NAP formed smaller assemblies than BLG with LYS. The molecular details rather than the net charge of BLG and NAP may therefore play a role in their assembly. Furthermore, simulated BLG + NAP assemblies were larger than those experimentally detected by DLS. We discuss the discrepancy between experiments and simulations in relation to the limitations of modelling precisely the molecular characteristics of proteins.

International audience; Droplet microfluidics revolutionizes the way experiments and analyses are conducted in many fields of science, based on decades of basic research. Applied sciences are also impacted, opening new perspectives on how we look at complex matter. In particular, food and nutritional sciences still have many research questions unsolved, and conventional laboratory methods are not always suitable to answer them. In this review, we present how microfluidics have been used in these fields to produce and investigate various droplet-based systems, namely simple and double emulsions, microgels, microparticles, and microcapsules with food-grade compositions. We show that droplet microfluidic devices enable unprecedented control over their production and properties, and can be integrated in lab-on-chip platforms for in situ and time-resolved analyses. This approach is illustrated for on-chip measurements of droplet interfacial properties, droplet–droplet coalescence, phase behavior of biopolymer mixtures, and reaction kinetics related to food digestion and nutrient absorption. As a perspective, we present promising developments in the adjacent fields of biochemistry and microbiology, as well as advanced microfluidics–analytical instrument coupling, all of which could be applied to solve research questions at the interface of food and nutritional sciences.

International audience; The partial enzymatic hydrolysis of wheat gliadins constitutes an interesting tool to unravel their structural specificity. In this work, the structure and conformation of γ-gliadin were investigated through its limited chymotrypsic digestion. Using a combination of computational, biochemical and biophysical tools, we studied each of its N and C terminal domains. Our results reveal that γ-gliadin is a partially disordered protein with an unfolded N-terminal domain surprisingly resistant to chymotrypsin and a folded C-terminal domain. Using spectroscopic tools, we showed that structural transitions occured over the disordered N-terminal domain for decreasing ethanol/water ratios. Using SAXS measurements, low-resolution 3D structures of γ-gliadin were proposed. To relate the repeated motifs of the N-terminal domain of γ-gliadin to its structure, engineered peptide models PQQPY/F were also studied. Overall results demonstrated similarities between the N-terminal domain and its derived model peptides. Our findings support the use of these peptides as general templates for understanding the wheat protein assembly and dynamics.

International audience; Understanding the dynamics of macromolecular assemblies in solution, such as Liquid-Liquid Phase Separation (LLPS), represents technologic and fundamental challenges in many fields. In cell biology, such dynamics are of great interest, because of their involvement in subcellular processes. In our study, we aimed to control the assembly of macromolecules in aqueous semi-permeable vesicles, that we named osmosomes, using microfluidics. We developed a microfluidic chip that allows for producting and trapping Giant Unilamellar Vesicles (GUVs) encapsulating macromolecules. This device also allows for modification of the composition of the inner phase and of the membranes of the trapped GUVs. The vesicles are produced from water-in-oil-in-water (w/o/w) double emulsions in less than 20 min after discarding the oil phase. They are highly monodisperse and their diameter can be modulated between 20 and 110 mu m by tuning the flow rates of fluid phases. Their unilamellarity is proofed by two techniques: (1) fluorescence quenching experiments and (2) the insertion of the alpha-hemolysin membrane protein pore. We demonstrate that the internal pH of osmosomes can be tuned in less than 1 min by controlling solvent exchanges through the alpha-hemolysin pores. The detailed analysis of the exchange kinetics suggests that the microfluidic chip provides an efficient pore formation due to the physical trapping of vesicles and the constant flow rate. Finally, we show a proof of concept for macromolecular assembly within osmosomes by pH-triggered LLPS of wheat proteins within a few minutes. (C) 2020 Elsevier Inc. All rights reserved.

Liquid-liquid phase separation is a phenomenon occurring spontaneously upon changing the physicochemical conditions of (bio-)polymers mixtures such as pH, ionic strength, total concentration, mixing ratio or temperature. The establishment of a phase diagram is a useful approach to highlight interactions and phase separation conditions of a mixture. Such approach generally performed in bulk is highly time and raw material consuming. In the present work, we developed a droplets-based millifluidic device for rapid phase diagram determination of liquid-liquid phase separated system. The proof of concept was first performed using a colloidal suspension of titanium dioxide (TiO2). The developed millifluidic device was then applied to a model protein-polysaccharide mixture made of beta-lactoglobulin (BLG) and Gum Arabic (GA) in water and at pH 4.2. In this model, a specific type of liquid-liquid phase separation occurs: complex coacervation. Biopolymers droplets of about 2.5 mu L of various compositions were generated to investigate the impact of total biopolymers concentration (0.1-5 wt %) and protein-polysaccharide mixing ratio (1:8 to 8:1). Initiation and suppression of phase separation were then evidenced through turbidity measurements within the droplets using image analysis. High similarities between cloud points probed using the millifluidic device and the binodal curve determined in bulk were found in the total biopolymers concentration range explored. Droplets-based millifluidic represents an efficient and highly versatile tool to probe liquid-liquid phase separation of various biopolymers mixtures.

Various studies have shown that food structure has an impact on digestion kinetics. We focus here on the effects of gastric and intestinal enzymes (in-vitro digestion) on two canola seed storage proteins, napin and cruciferin. To monitor structure effect we conducted experiments on gels of these proteins at different pHs, yielding different structures and elastic modulus. What is new is to get information on the mechanisms at the lowest scales, using imaging and radiation scattering at large facilities: Synchrotron fluorescence microscopy, X-Ray scattering, at SOLEIL synchrotron, and Small-Angle Neutron Scattering, at Laboratoire Leon Brillouin reactor. We can identify the mechanisms at each step and in two distinct scale ranges, observed simultaneously, the one of the individual protein scale and the one of the structure connectivity: • during gelation individual canola proteins are not deeply modified in comparison with their state in solution ; larger scale gel heterogeneity appears due to connectivity or aggregation • in the gastric step (up to 40 min): ○ at short scale (large q) we see that the proteins disintegration is much slowed down in gels than in solutions, particularly in the gastric phase; ○ at larger scales (low q), we see that the gel structure is also self-resistant to the action of the enzyme (pepsin). • in the intestinal step, such kinetics differences hold until major disintegration after no more than 15 min.

International audience; 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.

In this Article, we investigated the interaction properties of wheat gliadins, properties-that are at the basis of:their functionality in wheat grain and in food matrixes: We established the equation of state of our isolate by osmotic compression and characterized the concentration-induced structural transitions, from the secondary structure of proteins to the rheological properties. We evidenced three thermodynamical regimes corresponding to several structuring regimes. First, for Phi < 0.03, gliadins behave as repulsive colloids, with a positive second virial coefficient, arising presumably from their surface charge density and/or their, steric repulsion: No intermolecular interaction was detected by FT-IR, suggesting that proteins form a stable dispersion. In the second regime, the system becomes more easily compressible, i.e., less repulsive and/or more attractive.,It is associated with beta-the disappearance of beta-sheet intraramolecular structures of the proteins in favor of random coils/alpha-helix and intermolecular beta-sheet interactions. This coincides with the appearance of elasticity and the increase of the apparent viscosity. Finally, in the last regime, for Phi > 0.16, FT-IR spectra show that proteins are strongly interacting via intermolecular interactions. A correlation peak develops in SAXS, revealing a global order in the dispersion. Interestingly, the osmotic pressure applied to extract the solvent is higher than expected from a hard-sphere-like protein and we highlighted a liquid-like state at very high concentration (>450 g L-1) which:is in contrast with most proteins that form gel or glass at such concentration. In the discussion, we questioned the existence of supramolecular assemblies and the role of the solvation that would lead to this specific behavior.

During wheat seeds development, storage proteins are synthetized and subsequently form dense protein phases, also called Protein Bodies (PBs). The mechanisms of PBs formation and the supramolecular assembly of storage proteins in PBs remain unclear. In particular, there is an apparent contradiction between the low solubility in water of storage proteins and their high local dynamics in dense PBs. Here, we probe the interplay between short-range attraction and long-range repulsion of a wheat gliadin isolate by investigating the dynamics of liquid-liquid phase separation after temperature quench. We do so using time-resolved small angle light scattering, phase contrast microscopy and rheology. We show that gliadins undergo liquid-liquid phase separation through Nucleation and Growth or Spinodal Decomposition depending on the quench depth. They assemble into dense phases but remain in a liquid-like state over an extended range of temperatures and concentrations. The analysis of phase separation kinetics reveals that the attraction strength of gliadins is in the same order of magnitude as other proteins. We discuss the respective role of competing interactions, protein intrinsic disorder, hydration and polydispersity in promoting local dynamics and providing this liquid-like behavior despite attractive forces. ispartof: SCIENTIFIC REPORTS vol:8 issue:1 ispartof: location:England status: published

• Protein interactions play a key role in determining the final structure and properties of products.

We investigated the liquid-liquid phase separation of wheat storage proteins with molecular weight (MW) ranging from 25 kg mol(-1) to 300 kg mol(-1). We characterized the onset of the phase separation upon a temperature decrease by turbidity. The protein compositions at equilibrium after temperature quenches were additionally determined by size-exclusion-high performance liquid chromatography. We found that wheat proteins can be classified into two classes according to their phase behaviour. The partition of the proteins between the two phases depends on their MW above a critical size of 45 kg mol(-1). For those high MW proteins, we observed that the behaviour is similar to the one of neutral, linear polymers. Their partition and critical parameters are well described by Flory-Huggins theory. Proteins below 45 kg mol(-1), namely alpha-, beta- and gamma-gliadins, have similar interaction potentials under the physicochemical conditions tested. Their phase diagrams, characterized by a low value of critical volume fraction, are characteristic of long-range interactions. Wheat protein behaviour can resultantly be rationalized in a much simpler way than expected from their complex composition.