Concept on self-assembly and structure of globular protein fluids

Globular proteins are ubiquitous in our daily life. Not only they are naturally present in the biological matter, they also offer many possibilities to adjust the nutritional and flow properties of fluids or to design drug vehicles [1]. Globular protein systems interact through short range attractive forces; and the interaction between them may lead the system to form aggregates through self-assembling process. Since such biological monomers are complex systems, their aggregation process is most of the time out of control. The current main conceptual framework to describe that process is based on the idea that the monomers may self-assemble through a diffusion and reaction mechanism known as DLA for diffusion limited aggregation, and RLA for reaction limited aggregation respectively [2]. Beta-lactoglobulin (blg) solution gives, after heat-induced denaturation, a suspension of polydisperse aggregates as predicted by the random aggregation concept. Therefore, the transition from native blg to denatured blg aggregate suspension leads to complex correlation with the flow behavior [3]. Although the dependency of the aggregation process to physicochemical factors like, ionic strength, pH, temperature and concentration has been intensively investigated, it still remains much to do to control the aggregate polydispersity via self-assembling process. The composition of the raw product, thermal processing, pH and entropy instability during the aggregation process, are some of the factors influencing the polydispersity of the aggregates. We use different techniques such as SAXS/USAXS, LS, SEM, CSLM and image analysis methods to characterize thoroughly the structure of globular protein aggregates formed after heat-induced denaturation at different experimental conditions [4]. Whether these aggregates are in solution or entrapped by gelation, we do think that investigating their structure will provide us with relevant information to solve the issue related to their formation. Please click Additional Files below to see the full abstract

The EU Center of Excellence for Exascale in Solid Earth (ChEESE): Implementation, results, and roadmap for the second phase

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Agrégation, gélification et filage hydrodynamique des protéines du lactosérum

Whey protein isolates have received increasing interest due to their high nutritional value and their growing availability on the market as a co-product of cheese production. Whey protein isolates (WPI) can be aggregated upon heating to create new functional properties which depend on aggregate size and structural properties. Based on the fractal properties of these aggregates, one major application is to texture food products by two different ways: by forming a stable and thick suspension of aggregates or by forming a space filling network, through a gelation process. Fractal aggregate size generally ranges from a few hundred nanometres to a few microns at most. However, it would be interesting if their size could reach at least 30 microns (the limit of consumer perception) to increase their thickening power. Up to now, technological routes to create thickening particles were based mainly on the physico-chemical conditions of aggregation.The objective of the PhD is to study new aggregation and gelation processes with the aim to produce novel aggregate structures to enhance their texturising ability.Firstly, a good understanding of molecular self-assembly was required, taking into account the industrial process parameters. Indeed, in industrial conditions, aggregates are obtained under flow conditions at high temperature (≥75◦C) in few minutes. We developed a down scaling approach to study both the kinetics of aggregation after few seconds and its dependence with the mean shear rate in which heat transfer does not limit aggregation. The size and mass of aggregates and protein conformation were characterized by small-angle X-ray scattering and resonant mass measurement. We showed that for fractal aggregates formed at low protein and salt concentration, WPI aggregation at 92°C was limited by a step of nucleation, shear rate had no significant effect on the size of the aggregates, or on the aggregation kinetics and slower thermalization lead smaller aggregates size.Secondly, standard characterization methods of aggregates structure being limited to sub-micrometric aggregates, we developed a new method for aggregates of several tens of microns based on a covalent labelling of WPI and fluorescent microscopy. We have shown that, depending on the physico-chemical and heating conditions, the range of size where fractal aggregates exhibits a fractal dimension equal to 2 can be extended from 10 to 60 µm.Thirdly, we developed a new structuration process to spin gels of fractal aggregates by a combination of microflows and Ca2+-induced gelation. The WPI fibers presented a core-shell structure. Moreover, the size and the stability of the fiber was due to a complex interplay between different phenomena: hydrodynamics stresses, gelation kinetics, local pH changes during gelation and osmotic stresses.Finally, characterization of gelation of fractal aggregates by Ca2+ was studied in a bi-dimensional geometry in order to gain insights on the different phenomena that govern the formation and structure of the fiber. Especially, we showed the presence of osmotic flux which concentrates the aggregates in a front and form the shell of the fiber.Les isolats de protéines de lactosérum ont suscité un intérêt croissant en raison de leur haute valeur nutritionnelle et de leur disponibilité croissante sur le marché, étant un coproduit de la production de fromage. Les isolats de protéines de lactosérum peuvent être agrégés par chauffage pour créer de nouvelles propriétés fonctionnelles qui dépendent de la taille des agrégats et de leurs propriétés structurelles.Grâce aux propriétés fractales de ces agrégats, l'une des principales applications consiste à texturer les produits alimentaires et ce de deux manières différentes : en formant une suspension stable et épaisse d'agrégats ou en formant un réseau remplissant l'espace, par un processus de gélification. La taille des agrégats fractals varie généralement de quelques centaines de nanomètres à quelques microns tout au plus. Cependant, il serait intéressant que leur taille atteigne au moins 30 microns (limite de la perception du consommateur) pour augmenter leur pouvoir épaississant. Jusqu'à présent, les voies technologiques pour créer des particules aux propriétés épaississantes étaient principalement basées sur la modulation des conditions physico-chimiques lors de l'agrégation.L'objectif de la thèse est d'étudier de nouveaux processus d'agrégation et de gélification dans le but de produire de nouvelles structures d'agrégats pour améliorer leur capacité de texturation.Tout d'abord, une bonne compréhension de l'auto-assemblage moléculaire était nécessaire, en tenant compte des paramètres des processus industriels. En effet, dans des conditions industrielles, les agrégats sont obtenus dans des procédés en continu (taux de cisaillement) à une température élevée (≥75◦C) en quelques minutes. Nous avons développé un procédé d'agrégation couplée à une réduction d'échelle pour étudier à la fois la cinétique d'agrégation après quelques secondes et sa relation avec le taux de cisaillement moyen, dans lequel le transfert de chaleur ne limite pas l'agrégation. La taille et la masse des agrégats ainsi que la conformation des protéines ont été caractérisées par diffusion des rayons X aux petits angles et la mesure de masse par résonance. Nous avons montré que pour les agrégats fractals formés à faible concentration et de sel, l'agrégation des WPI à 92°C était limitée par une étape de nucléation, que le taux de cisaillement n'avait pas d'effet significatif sur la taille des agrégats et sur la cinétique d'agrégation et qu'une thermalisation plus lente conduisait à des agrégats de plus petite taille.Deuxièmement, les méthodes standards de caractérisation de la structure des agrégats étant limitées aux agrégats sub-micrométriques, nous avons développé une nouvelle méthode pour les agrégats de plusieurs dizaines de microns, basée sur un marquage covalent des WPI et la microscopie fluorescente. Nous avons montré que, selon les conditions physico-chimiques et de chauffage, la gamme de taille où les agrégats fractals présentent une dimension fractale égale à 2 peut être étendue de 10 à 60 µm.Troisièmement, nous avons développé un nouveau processus de structuration pour filer des gels d'agrégats fractals par une combinaison de micro écoulement et de gélification induite par le Ca2+. Nous avons montré que les fibres ainsi formées présentaient une structure cœur-écorce. De plus, la taille et la stabilité de la fibre étaient dues à une interaction complexe entre différents phénomènes : contraintes hydrodynamiques, cinétique de gélification, changements locaux de pH pendant la gélification et contraintes osmotiques.Enfin, la caractérisation de la gélification d'agrégats fractals par le Ca2+ a été étudiée dans une géométrie bi-dimensionnelle afin d'avoir un aperçu des différents phénomènes qui gouvernent la formation et la structure de la fibre. En particulier, nous avons montré la présence d'un flux osmotique qui concentre les agrégats dans un front et forme l'enveloppe de la fibre

Hydrodynamic spinning of protein fractal aggregates into core-shell fibers

International audienceUsing fractal protein aggregates as building blocks, porous fibers were produced. The suspension of aggregates was co-injected with a solution of calcium chloride. Sol-gel transition of the suspension was induced by diffusion of calcium ions in the jet. The production of these fibers required a precise control of both hydrodynamic and physicochemical conditions as hydrodynamic instabilities competed with the gelation kinetics. By increasing the calcium concentration, several regimes were observed: swollen, dispersed and shrunk fibers. In the first regime, homogeneous fibers were obtained. In the last one, osmotic phenomena led to a spontaneous core-shell structure with a dense shell

Kinetic and structural characterization of whey protein aggregation in a millifluidic continuous process

International audienceWhey protein isolates (WPI) can be aggregated upon heating to create new functional properties (e.g. texture), which depend on aggregate size and structural properties. In industrial conditions, aggregates are obtained in continuous processes at high temperature (≥ 75 • C) in few minutes. When studying the kinetics of WPI aggregation at high temperature and under flow, one major issue is to develop a process in which heat transfer does not limit aggregation. To this end, we used a down-scaling approach in which a WPI solution flows in a heated capillary tube. We show that this process makes it possible to study both the kinetics of aggregation after few seconds and its dependence with the mean shear rate in isothermal conditions. The size and mass of aggregates and protein conformation were characterized by small-angle X-ray scattering and resonant mass measurement for a single physico-chemical condition (pH 7.0, 10 mM NaCl, 92 • C, 4 % w/w WPI) which led to sub-micrometric aggregates. Firstly, we report that the size of aggregates were three times larger than when produced in a test tube. Secondly, the size and mass of aggregates reached a steady-state value in a few seconds, whereas the kinetics of aggregation and denaturation had a characteristic time of few minutes. Thirdly, the shear rate had no significant effect on the size of the aggregates, or on the aggregation kinetics. We concluded that WPI aggregation at 92 • C is limited by a step of nucleation, and that the fact that aggregates produced in test tube were smaller is due to a slower thermalization

Formation and stability of fibers obtained by cold gelation of pea protein isolate aggregates in a hydrodynamic spinning process

International audienceUsing plant protein sources in the formulation of food products is an important option to reduce the overall carbon footprint of the human diet. However, the ability to convert plant proteins into functional ingredients can hamper their use by the food industry. Our objective was to assemble pea protein isolate (PPI) aggregates into edible fibers. They were obtained by co-injection of a suspension of PPI aggregates with a solution of calcium chloride by means of physico-chemical bonds, i.e. in the absence of thermal treatment or chemical reaction. As soon as specific hydrodynamic conditions were met, homogeneous fibers of few hundreds µm diameter and few cm length were obtained. Small angle X-ray scattering showed that the building blocks of these fibers were dense aggregates of 400 nm radius. Calcium and PPI concentrations required for the processing of fibers were roughly given by the sol-gel state diagram of PPI aggregates. Compared to their dairy protein based equivalent, PPI fibers were obtained with a low mass fraction of protein (3% w/w) and were stable regardless of the concentration of calcium chloride used. We concluded that the robustness of the hydrodynamic spinning process was attributed to the strong reactivity of PPI aggregates with calcium ions and the low tendency of PPI gels to syneresis