Influence of pH and lipid membrane on the liquid-liquid phase separation of wheat γ-gliadin in aqueous conditions.

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

Trends in the Synthesis of Polymer Nano- and Microscale Materials for Bio-Related Applications.

Polymeric nano- and microscale materials bear significant potential in manifold applications related to biomedicine. This is owed not only to the large chemical diversity of the constituent polymers, but also to the various morphologies these materials can achieve, ranging from simple particles to intricate self-assembled structures. Modern synthetic polymer chemistry permits the tuning of many physicochemical parameters affecting the behavior of polymeric nano- and microscale materials in the biological context. In this Perspective, an overview of the synthetic principles underlying the modern preparation of these materials is provided, aiming to demonstrate how advances in and ingenious implementations of polymer chemistry fuel a range of applications, both present and prospective.

Droplet Microfluidics for Food and Nutrition Applications.

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.

Semi-permeable vesicles produced by microfluidics to tune the phase behaviour of encapsulated macromolecules.

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 µm 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 α-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 α-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.

Heat-induced gelation of mixtures of micellar caseins and plant proteins in aqueous solution.

The aim of this work was to investigate how the heat-induced gelation of micellar casein (MC)-plant protein mixtures in aqueous solution is affected by protein composition (MC/plant proteins = 100/0 to 0/100) and total protein content (4%, 6% and 8% w/w) at pH 5.8 and 6.0. Two types of plant proteins were used: soy proteins (SP) and pea proteins (PP). Storage moduli (G') were measured during heating ramps from 20 to 90 °C and heat-induced gelation was characterised by a sharp increase in G' at a critical temperature (Tc). The gel stiffness (Gel) was determined after 1 h at 90 °C and the microstructure before and after heating was investigated by confocal laser scanning microscopy (CLSM). Tc was found to increase with increasing the fraction of MC replaced by SP or PP, due to binding of calcium to the plant proteins. The effect was stronger for SP, which bound calcium more efficiently than PP. Tc decreased with decreasing pH, possibly caused by decreased electrostatic repulsion and increased calcium release from MC. Gel increased with increasing total protein content and did not depend significantly on the pH. Interestingly, Gel showed a minimum as a function of the plant protein fraction (40% for SP and 70% for PP) in the mixtures. It is concluded that MC and plant proteins did not co-aggregate in the mixtures during heating, and that each type of protein formed networks independently.

Water-In-Water Emulsion Gels Stabilized by Cellulose Nanocrystals.

Particle-stabilized water-in-water emulsions were prepared by mixing dextran and poly(ethylene oxide) (PEO) in water and adding cellulose nanocrystals (CNC). The CNC formed a layer at the surface of the dispersed droplets formed by the PEO-rich phase. Excess CNC partitioned to the continuous dextran phase. Aggregation of CNC at different rates was induced by adding NaCl between 10 and 100 mM. In the presence of more than 2 g/L CNC, fast aggregation led to the formation of an emulsion gel showing no signs of creaming. Confocal laser scanning microscopy showed that the emulgels were formed by a continuous network of CNC in which the randomly distributed droplets were embedded. The gel stiffness was measured with oscillatory shear rheology and found to increase strongly with increasing CNC concentration ( C). The dispersed droplets were elastically active and increased the gel stiffness at low C. However, up to C = 10 g/L, the yield stress was too small to inhibit the flow when the gels were tilted. At C < 2 g/L, creaming was observed until the network of connected droplets became sufficiently dense to be strong enough to resist buoyancy.