In situ digestion of canola protein gel observed by synchrotron X-Ray Scattering

We address the issue of structure changes of a canola protein gel (as a solid food model) during gastrointestinal digestion. We present a method for synchrotron Small-Angle X-ray Scattering analysis of the digestion of a gel in a capillary. Scanning the capillary allows tracking the digestion under diffusion of enzymatic juices. The fitting parameters characterizing the sizes, scattering intensities and structures allow to distinguish the compact, unfolded or aggregated states of proteins. The evolutions of these parameters enable to detail the complex changes of proteins during gel digestion, involving back-and-forth evolutions with proteins unfolding (1 st and 3 rd steps), re-compaction (2 nd step) due to gastrointestinal pH and enzyme actions, before final protein scissions (4 th step) resulting in small peptides. This complexity is related to the wide ranges of successive pH and enzyme activity acting on large and charged protein assemblies. Digestion is therefore impacted by the conditions of food preparation

Stability of food proteins at high pressure conditions

High pressure (HP) is particularly suited to study protein folding/unfolding, revealing subtle structural rearrangements not accessible by other types of denaturation. HP also has many industrial-scale advantages over heat treatments, including “greener” processing and preservation of nutritional values, colors, and flavors of foods. We have combined in situ HP with small-angle (X-ray and neutron) scattering (SAS) and spectrophotometry to follow the structure in solution of proteins of interest for the food industry. SAS is an essential technique for obtaining structural, but low-resolution, information about proteins, when conventional high-resolution structural biology methods are not possible. I will illustrate this approach with two studies on proteins of food interest: (i) bovine β-lactoglobulin (BLG), a whey protein with a high propensity to bind to various bioactive molecules. We probed by SANS1 and absorbance the effects on pressure stability and reversibility of BLG of the binding of retinol (vitamin A), resveratrol (polyphenol), and biliverdin (linear tetrapyrrole chromophore) to different sites on the protein2, 3. (ii) C-phycocyanin (CPC), a phycobiliprotein from cyanobacteria, to which tetrapyrrole chromophores are covalently attached and which can be used as a natural blue dye in the food industry. We studied by SAXS and absorbance HP-induced CPC unfolding and reversibility from two oligomeric states of the protein as a function of pH. Acknowledgements LLB, SOLEIL, and I2BC facilities are acknowledged for beamtime and proteomic expertise. This work was also supported by ANSO Project No. ANSOCR-PP-2021- 01. References 1. Annighöfer B, et al. High pressure cell to investigate protein unfolding up to 600 MPa by small-angle neutron scattering. Rev Sci Instr 2019;90:025106. 2. Minic S, et al. Effect of ligands on HP-induced unfolding and oligomerization of β-lactoglobulin. Biophysical J 2020;119:2262-74. 3. Minic S, et al. Structure of proteins under pressure: covalent binding effects of biliverdin on beta-lactoglobulin. Biophysical J 2022;121:2514-25

Field dependence of the electronic phase separation in Pr0.67Ca0.33MnO3 by small angle magnetic neutron scattering

We have studied by small angle neutron scattering the evolution induced by the application of magnetic field of the coexistence of ferromagnetism (F) and antiferromagnetism (AF) in a crystal of Pr$_{0.67}$Ca$_{0.33}$MnO$_3$. The results are compared to magnetic measurements which provide the evolution of the ferromagnetic fraction. These results show that the growth of the ferromagnetic phase corresponds to an increase of the thickness of the ferromagnetic ''cabbage'' sheets

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

Etudes structurales et dynamiques par diffusion de neutrons aux petits angles de polymères cristaux liquides

Membres du jury d'habilitation: J.P. Cotton, F. Hardouin, B. Pansu, J. Prost, M. Rawiso, D. Richter.The conformation and the dynamics of liquid crystal (LC) polymers are studied by small angle neutron scattering in the isotropic and nematic phases. In the isotropic phase, linear and comb like LC polymers adopt a gaussian conformation. In the nematic phase, linear and side on grafted polymers are confined in extremely long cylinders. For the linear ones this raises to a conformation with abrupt contour reversals (as hairpins) predicted by P.G. de Gennes. The conformation of comb-like polymers is weakly anisotropic. Dynamics studies of these non entangled polymers reveal the existence of clusters due to living interactions between mesogens. The cluster size lies in between the chain size and the sample one. The relaxation of the chain conformation is described in both phases.Nous avons déterminé, par diffusion de neutrons aux petits angles, la conformation et la dynamique de polymères cristaux liquides dans la phase nématique ou isotrope. En phase isotrope les polymères, linéaires ou en peigne adoptent une conformation gaussienne. En phase nématique les polymères linéaires ou greffés en haltère sont confinés dans de très longs cylindres. Pour les linéaires cela implique les repliements en épingle à cheveux prévus par P.G. de Gennes. La conformation des polymères en peigne est faiblement anisotrope. L'étude de la dynamique à l'état fondu de ces polymères non enchevêtrés a révélé l'existence d'amas dus à des interactions temporaires entre les mésogènes. La taille de ces amas est supérieure aux dimensions d'une chaîne mais inférieure à celle de l'échantillon. La relaxation de la conformation est décrite en détail

How necklace pearls evolve in hydrophobic polyelectrolyte chains under good solvent addition: A sans study of the conformation

International audienceThe chain conformation in sulfonated polystyrene PSSNa of a degree of sulfonation 0.34 <= f <= 1, i.e., of various hydrophobicity, is followed in mixtures of water and increasing content of tetrahydrofuran (THF), a good solvent of the hydrophobic polystyrene moieties (which improves the solvent quality of the mixture). This is achieved by measuring the chain form factor by small-angle neutron scattering using the zero average contrast method (ZAC). Polymer concentrations 0.17 and 0.34 M correspond in our case to the semidilute regime or its limit with dilute regime depending on the chain conformation. The main result is the monitoring with added THF of the pearl necklace conformation. This heterogeneous structure, made of wormlike chain parts (strings) and pearls, was observed formerly in water: when f decreases, the string contribution decreases, and the pearls size, characterized by a maximum in Kratky q(2)S(1)(q) representation, slightly increases. Here we see that in the presence of increasing content of added THF (i) the pearls contribution decreases, as expected, and (ii) their size does not change by more than 10% in most cases (30% at the most). Among different modeling, the most complete has been done following the pearl necklace models of Schweins, Huber et al. and Lages, Huber et al.; beyond the size and distribution of pearls, it addresses the radius of gyration, the correlation distance between spheres, weakly visible, and, importantly their number N. The values of R-g, as well as the modeling, suggest that while the sphere size varies a few, N decreases clearly with added good solvent. A link with the simulation of Liao et al. can be found. A second result, important in practice, is that all modeling of the scattering establishes that THF addition makes vanish an excess of low q scattering due to large compact spheres present in water. A third result is obtained after a THF treatment procedure, consisting of adding THF and then removing it by evaporation: (i) the spherical aggregates are washed out, and (ii) the pearl necklace conformation of the chain returns to the one in water solution before treatment. Therefore, the pearl necklace conformation of the hydrophobic polyelectrolyte in aqueous solution appears to be in an annealed equilibrium state resulting from hydrophobic attraction and electrostatic repulsion

Molecular structure of poly methyl methacrylate thermoreversible gels

The molecular structure of PMMA gels and aggregates in bromobenzene and ortho-xylene has been investigated by small-angle neutron scattering. It is found that in bromobenzene the gels are constituted of connected, prolate cylinder-like structures. A large proportion of these structures (about 90 %) possess a radius of about 1.5 nm. The remaining possess larger radii (from 3.0 to 5.3 nm). On heating, the melting process seems to proceed via two-steps : melting of the junction domains while the cylinder-like structures remain virtually unaffected. Scattering curves that differ significantly are obtained in ortho-xylene. They are also interpreted with cylinder-like objects of larger radii (up to 10 nm). As with bromobenzene, melting seems to take place in two steps. These results prove to be relevant for interpreting rheological data that will be presented in a forthcoming paper

In situ digestion of canola protein gel observed by synchrotron X-Ray Scattering

We address the issue of structure changes of a canola protein gel (as a solid food model) during gastrointestinal digestion. We present a method for synchrotron Small-Angle X-ray Scattering analysis of the digestion of a gel in a capillary. Scanning the capillary allows tracking the digestion under diffusion of enzymatic juices. The fitting parameters characterizing the sizes, scattering intensities and structures allow to distinguish the compact, unfolded or aggregated states of proteins. The evolutions of these parameters enable to detail the complex changes of proteins during gel digestion, involving back-and-forth evolutions with proteins unfolding (1 st and 3 rd steps), re-compaction (2 nd step) due to gastrointestinal pH and enzyme actions, before final protein scissions (4 th step) resulting in small peptides. This complexity is related to the wide ranges of successive pH and enzyme activity acting on large and charged protein assemblies. Digestion is therefore impacted by the conditions of food preparation