Influence de l'état protéique sur la dynamique de séparation de phase et de gélification dans un système ternaire aqueux à base de protéines de pois et d'alginate

Deux systèmes aqueux à 20C constitués de protéines globulaires de pois et d alginate de sodium ont été considérés au cours de cette étude, dans des conditions de solvant fixées à pH 7,2 et 0,1 M NaCl. Dans un premier temps, le comportement de phase de globulines faiblement dénaturées (i) ou pré-agrégées thermiquement (ii) en mélange avec de l alginate a été comparé à différentes échelles d observation, en termes de diagrammes de phase et de microstructure analysée par microscopie confocale. Attribuée à un phénomène général d incompatibilité thermodynamique, la séparation de phase a été décrite tout particulièrement sous des aspects morphologiques et cinétiques à l échelle microscopique, selon la composition de départ en biopolymères et le mode de préparation des globulines. Par la suite, une gélification de chacun des deux systèmes a été opérée à froid, par libération de calcium ionique in situ à partir d un sel de calcium de carbonate peu soluble au-dessus de pH 7, sous l effet acidifiant d une hydrolyse lente de la glucono- -lactone (GDL). L intérêt d un tel procédé reposait sur l obtention de gels remplis à mixtes lorsque l alginate seul ou l alginate et la phase protéique pouvaient gélifier en présence de calcium. Des corrélations entre propriétés rhéologiques mesurées en régime dynamique (modules G et G ) et données de microstructure ont été effectuées, par l intermédiaire de l analyse de texture d image selon la méthode de cooccurrence. Chaque mélange témoignait d une séparation de phase bloquée cinétiquement par sa gélification. Par rapport aux gels d alginate seul ou gels remplis où l alginate seul pouvait gélifier via le calcium, les gels mixtes témoignaient d un effet de synergie remarquable d un point de vue élasticité finale des gels. Dans le même temps, les globulines pré-agrégées ne montraient pas d aptitude à la gélification selon le procédé appliqué ici. En outre, des effets ségrégatifs induisaient un enrichissement des protéines et du polyoside dans deux phases coexistantes, renforçant de ce fait des interactions entre biopolymères du même type. Les gels mixtes les plus élastiques présentaient une structure enchevêtrée avec un réseau protéique prédominant. Les observations en microscopie électronique à transmission effectuées par un marquage différentiel des deux biopolymères suggèreraient qu il puisse se former localement des interactions attractives inter-biopolymères, probablement via le calcium, à l interface des deux phases initialement immiscibles. Ce pontage consoliderait globalement la cohésion entre les deux réseaux protéique et polyosidiqueTwo aqueous systems at 20C in 0.1 M NaCl and pH 7.2 containing globular pea proteins and sodium alginate were investigated in this study. First, phase behavior of (i) either low-denatured mixed globulins or (ii) their thermally pre-aggregated counterparts - alginate mixtures was compared using a multi-scale approach, by means of phase diagram and microstructure analysis by confocal microscopy. Thermodynamic incompatibility was the main driving force leading to phase separation within the mixtures, which presented according to their initial biopolymer composition both different morphological and time-evolution features of coexisting phases. Thereafter, a cold-set gelation for each system was performed, as the slow hydrolysis of glucono- -lactone (GDL) acidified the media and mediated the release in situ of calcium ions from calcium carbonate, practically insoluble at pH higher than 7. Such procedure would allow gelation via calcium of alginate only or both alginate and the protein phase, giving rise to filled and mixed gels, respectively. An attempt to correlate rheological measurements (G , G dynamic moduli) with microstructural data was carried out according to image texture analysis by the cooccurrence method. Phase separation was kinetically entrapped by gelation. Compared to single-alginate gels or native globulins-alginate filled gels where alginate was the only gelling agent via calcium, mixed gels reflected in fact great synergism effect regarding final gel elasticity. Meanwhile, pre-aggregated pea globulins could not form a gel with the gelation procedure of choice here. Besides, stronger segregative effects were evidenced by increasing initial biopolymer composition thus enhancing self-biopolymer interaction in their respective enriched-coexisting phases. The strongest mixed gels displayed entangled structure. According to a differential labelling of each incompatible biopolymer, observations with transmission electron microscopy suggested inter-biopolymer attractive interaction at the interface of coexisting phases, probably via calcium cations. Salt-bridging would reinforce cohesiveness between both protein and alginate networksDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Impact of Glucose on the Nanostructure and Mechanical Properties of Calcium-Alginate Hydrogels

Alginate is a polysaccharide obtained from brown seaweed that is widely used in food, pharmaceutical, and biotechnological applications due to its versatility as a viscosifier and gelling agent. Here, we investigated the influence of the addition of glucose on the structure and mechanical properties of alginate solutions and calcium-alginate hydrogels produced by internal gelation through crosslinking with Ca2+ . Using1H low-field nuclear magnetic resonance (NMR) and small angle neutron scattering (SANS), we showed that alginate solutions at 1 wt % present structural hetero-geneities at local scale whose size increases with glucose concentration (15–45 wt %). Remarkably, the molecular conformation of alginate in the gels obtained from internal gelation by Ca2+ crosslinking is similar to that found in solution. The mechanical properties of the gels evidence an increase in gel strength and elasticity upon the addition of glucose. The fitting of mechanical properties to a poroelastic model shows that structural changes within solutions prior to gelation and the increase in solvent viscosity contribute to the gel strength. The nanostructure of the gels (at local scale, i.e., up to few hundreds of \uc5) remains unaltered by the presence of glucose up to 30 wt %. At 45 wt %, the permeability obtained by the poroelastic model decreases, and the Young’s modulus increases. We suggest that macro (rather than micro) structural changes lead to this behavior due to the creation of a network of denser zones of chains at 45 wt % glucose. Our study paves the way for the design of calcium-alginate hydrogels with controlled structure for food and pharmaceutical applications in which interactions with glucose are of relevance

Release of coumarin encapsulated in chitosan-gelatin irradiated films

Chitosan and fish gelatin were used to formulate active biobased films containing an antioxidant (coumarin). After drying, the films were irradiated at 40 and 60 kGy using an electron beam accelerator. The effect of irradiation on the film properties as well as the coumarin release mechanism were investigated and compared with the control. Electron Spin Resonance (ESR) revealed free radical formation during irradiation in films containing coumarin. Antioxidant addition and/or irradiation treatment at a dose of 60 kGy resulted in a shift of amide A and amide B peaks. Furthermore a shift of amide II band was only observed for the control film at the same dose. Irradiation allowed improving the thermal stability of the control films. Both irradiation process and addition of coumarin increased the surface wettability (increase of the polar component of the surface tension). From the water barrier analysis, neither irradiation nor coumarin addition influenced the permeability at the lower RH gradient used (0e30% RH). Using the higher RH gradient (30e84%) induced a rise of the WVP of all films (containing or not coumarin) after irradiation treatment. At 60 kGy, the tensile strength of only the control films increased significantly. Considering coumarin release from the film in aqueous medium, the apparent diffusion coefficient of coumarin is two times reduced after irradiation. Irradiation also allowed to better protect the incorporated antioxidant. Indeed, the amount of coumarin in the non-irradiated film was significantly lowered compared to the initial quantity, which is probably due to chemical reactivity

Combined hydrogels of starch and β-lactoglobulin as matrices for the preservation of C-phycocyanin

The color of food products is an important aspect in food industry, and its preservation remains a big challenge. We aim to preserve the natural blue dye of C-phycocyanin (C-PC) phycobiliprotein from Spirulina microalgae. For this purpose, we incorporated C-PC in combined starch and β-lactoglobulin (BLG) hydrogels by using a high-pressure (HP) process. Indeed, in thermal treatment, the color derived from C-PC is entirely lost. We characterized the obtained HP gels by both rheology and small-angle X-ray scattering (SAXS). Various formulations of binary (BLG/C-PC) and ternary (starch/BLG/C-PC) systems were tested under HP up to 4,500 bar. A good preservation of the C-PC pigment was established by mixing BLG and starch with C-PC at pH 7, with concentrations of 180, 5, and 10 mg/mL, respectively. Identical component concentrations were maintained in the binary systems. Structure of gels was characterized by SAXS providing insight of C-PC interactions with BLG and starch after HP process which leads to the formation of solid gels with larger mesh compared to two-component systems. This results in enhanced mechanical properties, which were determined by amplitude and frequency sweep measurements using a rheometer with applied plane/plane geometry. Therefore, adding starch, even at small concentration, significantly improves gel visual appearance and mechanical properties. Our study reveals that preservation through HP treatment is more effective than high temperature treatment, as visually observed through the sustained color integrity of C-PC blue dye

The use of starch and β-lactoglobulin composite hydrogels as frameworks for preserving c-phycocyanin

Our study aimed to preserve the natural blue dye of C-phycocyanin (C-PC) phycobiliprotein from Spirulina microalgae due to its importance in the food industry. We incorporated C-PC into hydrogels formed by combining starch and β-lactoglobulin (BLG) using high-pressure (HP) processing to achieve this objective. Notably, thermal treatment resulted in the complete loss of colour derived from C-PC. We performed a comprehensive characterization of the resulting HP gels by rheology measurements, texture profile analysis (TPA), small-angle X-ray scattering (SAXS), and scanning electron microscopy (SEM). Different compositions of binary (BLG/C-PC) and ternary (starch/BLG/C-PC) systems were processed under high-pressure (HP) conditions reaching up to 4,500 bar. The C-PC pigment was effectively preserved by mixing BLG and starch with C-PC at pH 7, maintaining concentrations of 180, 5, and 10 mg/mL, respectively. The same concentrations of components were retained in the binary systems. Rheological properties of the gels were determined using a rheometer with plane/plane geometry, and texture analysis was conducted through TPA. These findings enabled the assessment of food gel's properties, such as hardness, springiness, chewiness, and cohesiveness. The structural characteristics of the gels were determined by SAXS, offering insights into the interactions between C-PC, BLG, and starch after HP processing. Adding CPC and starch formed solid gels with a larger mesh than the pure BLG gels. SEM scans of the gel surface revealed that all components influenced the overall morphology of gels. Even at low concentrations, the addition of starch notably influenced the gels' visual appearance and mechanical properties. Our investigation highlights the superior effectiveness of HP treatment in the preservation of C-PC compared to high-temperature treatment, evident in the sustained colour integrity of the C-PC blue dye

Polysaccharide-based hydrogels: structure and function

Biopolymer-based hydrogels with a tuneable structure have drawn significant attention due to their wide range of applications in the food, pharmaceutical, and biomedical fields. This can be attributed to their remarkable features such as desirable mechanical properties, high water content, biodegradability and biocompatibility. At first order, hydrogels can be described as a network containing a crosslinked polymer and open spaces (meshes) between polymers chains enabling the diffusion of liquids and entrapped molecules such as proteins, peptides, and vitamins.In this work, we prepared polygalacturonate-based hydrogels by diffusing divalent cations X2+ (eg., Ca2+, Zn2+, Fe2+) from an external reservoir, through a dialysis membrane, to the polygalacturonate (polyGalA) solution. These hydrogels exhibited various degrees of inhomogeneity and showed a significant gradient of both polyGalA and divalent cation concentrations, ranging from the part of the gel formed near the dialysis membrane (i.e., the initial stage of the gelation process) to the furthest part of the gel. We demonstrated that Fe2+ cations keep their +II oxidation sate in the entire gel, making them suitable for iron fortification purposes.1 Furthermore, we observed that the mesh size () in the regions of the gel closer the dialysis membrane remained close to 7.5 nm, regarless of the divalent cation used (Ca2+, Zn2+, Fe2+).2 However, in the farthest parts of the gel, the mesoscopic structure depended on the nature of the cation, particularly on the different modes of interaction known to occur between the cation and the GalA unit, such as bidentate versus “egg-box” geometry.AcknowledgementsLaboratoire Léon Brillouin, Synchrotron SOLEIL and Région Bourgogne Franche-Comté are acknowledged for their financial support.References1.Maire du Poset A, Lerbret A, Zitolo A, Cousin F, Assifaoui A, Design of polygalacturonate hydrogels using iron(II) as crosslinkers: A promising route to protect bioavailable iron againstoxidation. Carbohydr. Polym. 2018, 188, 276−283.2.Maire du Poset A, Lerbret A, Boué F, Zitolo A, Assifaoui A, Cousin F, Tuning the Structure of Galacturonate Hydrogels: External Gelation by Ca, Zn, or Fe Cationic Cross-Linkers. Biomacromolecules 2019, 20 (7), 2864−2872

How Accurate Is the Egg-Box Model in Describing the Binding of Calcium to Polygalacturonate? A Molecular Dynamics Simulation Study

International audienceWe performed molecular dynamics (MD) simulations of octameric galacturonate, GalA8, chains in the presence of Ca2+ in a ratio of R = [Ca2+]/[GalA] = 0.25 in order to determine to which extent the popular "egg-box model" (EBM) is able to describe the association between Ca2+ cations and polygalacturonate (polyGalA) chains. To this aim, we slig h t l y revised the empirical parameters for the interaction between Ca2+ and the carboxylate oxygen atoms of GalA units so as to reproduce the experimental Ca2+-GalA association constant. We also defined an ad hoc order parameter, referred to as the egg-box score (EBS), that quantifies any deviation of the local coordination geometry of calcium cations with respect to an "ideal" EBM coordination geometry. The results reveal that the local coordination geometry of Ca2+ cations bound to polyGalA chains differs from that of the EBM. Moreover, polyGalA chains exhibit significant conformational disorder, and the cross-l i n k angles formed between polyGalA chain axes are broadly distributed . Overall, the present study suggests that the EBM fails to describe accurately the association modes between calcium and polyGalA chains at a molar ratio R of 0.25

How Accurate Is the Egg-Box Model in Describing the Binding of Calcium to Polygalacturonate? A Molecular Dynamics Simulation Study

We performed molecular dynamics (MD) simulations of octameric galacturonate, GalA8, chains in the presence of Ca2+ in a ratio of R = [Ca2+]/[GalA] = 0.25 in order to determine to which extent the popular “egg-box model” (EBM) is able to describe the association between Ca2+ cations and polygalacturonate (polyGalA) chains. To this aim, we slightly revised the empirical parameters for the interaction between Ca2+ and the carboxylate oxygen atoms of GalA units so as to reproduce the experimental Ca2+–GalA association constant. We also defined an ad hoc order parameter, referred to as the egg-box score (EBS), that quantifies any deviation of the local coordination geometry of calcium cations with respect to an “ideal” EBM coordination geometry. The results reveal that the local coordination geometry of Ca2+ cations bound to polyGalA chains differs from that of the EBM. Moreover, polyGalA chains exhibit significant conformational disorder, and the cross-link angles formed between polyGalA chain axes are broadly distributed. Overall, the present study suggests that the EBM fails to describe accurately the association modes between calcium and polyGalA chains at a molar ratio R of 0.25

Insights into gelation kinetics and gel front migration in cation-induced polysaccharide hydrogels by viscoelastic and turbidity measurements: Effect of the nature of divalent cations

International audiencePolysaccharide-based hydrogels were prepared by the diffusion of various divalent cations (X2+) into the polygalacturonate (polyGal) solution through a dialysis membrane. The diffusion of various divalent cations (Mg2+, Ca2+, Zn2+ and Ba2+) was investigated. The polyGal gel growth was studied as a function of the initial cation concentration by both viscoelastic and turbidity measurements. We have demonstrated for the first time that the determination of the spatiotemporal variation of turbidity during the gelation process allowed to study the gel front migration. For Ca-polyGal, Zn-polyGal and Ba-polyGal, the gel front migration was characterized by the presence of a peak at the sol/gel interface. This peak was not observed in the case of Mg-polyGal where the gel was not formed. The apparent diffusion coefficient of the gel front (D-app) which was calculated from the evolution of this peak increased when the initial cation concentration was increased. Moreover, we have suggested a gelation mechanism based on the presence of a threshold molar ratio R* (=[X2+]/[Galacturonic unit]) in which some point-like crosslinks are precursors of the formation of dimers and multimers inducing the contraction of the gel and thus the formation of the gel front

How Accurate Is the Egg-Box Model in Describing the Binding of Calcium to Polygalacturonate? A Molecular Dynamics Simulation Study

We performed molecular dynamics (MD) simulations of octameric galacturonate, GalA8, chains in the presence of Ca2+ in a ratio of R = [Ca2+]/[GalA] = 0.25 in order to determine to which extent the popular “egg-box model” (EBM) is able to describe the association between Ca2+ cations and polygalacturonate (polyGalA) chains. To this aim, we slightly revised the empirical parameters for the interaction between Ca2+ and the carboxylate oxygen atoms of GalA units so as to reproduce the experimental Ca2+–GalA association constant. We also defined an ad hoc order parameter, referred to as the egg-box score (EBS), that quantifies any deviation of the local coordination geometry of calcium cations with respect to an “ideal” EBM coordination geometry. The results reveal that the local coordination geometry of Ca2+ cations bound to polyGalA chains differs from that of the EBM. Moreover, polyGalA chains exhibit significant conformational disorder, and the cross-link angles formed between polyGalA chain axes are broadly distributed. Overall, the present study suggests that the EBM fails to describe accurately the association modes between calcium and polyGalA chains at a molar ratio R of 0.25