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

Drought or/and Heat-Stress Effects on Seed Filling in Food Crops: Impacts on Functional Biochemistry, Seed Yields, and Nutritional Quality

Drought (water deficits) and heat (high temperatures) stress are the prime abiotic constraints, under the current and climate change scenario in future. Any further increase in the occurrence, and extremity of these stresses, either individually or in combination, would severely reduce the crop productivity and food security, globally. Although, they obstruct productivity at all crop growth stages, the extent of damage at reproductive phase of crop growth, mainly the seed filling phase, is critical and causes considerable yield losses. Drought and heat stress substantially affect the seed yields by reducing seed size and number, eventually affecting the commercial trait ‘100 seed weight’ and seed quality. Seed filling is influenced by various metabolic processes occurring in the leaves, especially production and translocation of photoassimilates, importing precursors for biosynthesis of seed reserves, minerals and other functional constituents. These processes are highly sensitive to drought and heat, due to involvement of array of diverse enzymes and transporters, located in the leaves and seeds. We highlight here the findings in various food crops showing how their seed composition is drastically impacted at various cellular levels due to drought and heat stresses, applied separately, or in combination. The combined stresses are extremely detrimental for seed yield and its quality, and thus need more attention. Understanding the precise target sites regulating seed filling events in leaves and seeds, and how they are affected by abiotic stresses, is imperative to enhance the seed quality. It is vital to know the physiological, biochemical and genetic mechanisms, which govern the various seed filling events under stress environments, to devise strategies to improve stress tolerance. Converging modern advances in physiology, biochemistry and biotechnology, especially the “omics” technologies might provide a strong impetus to research on this aspect. Such application, along with effective agronomic management system would pave the way in developing crop genotypes/varieties with improved productivity under drought and/or heat stresses

Influence of protein state on the phase separation and gelation within an aqueous system made of pea proteins and alginate

Deux systèmes aqueux à 20°C 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-δ-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 20°C 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-δ-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 network

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

Two aqueous systems at 20°C 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-δ-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 networksDeux systèmes aqueux à 20°C 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-δ-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 polyosidiqu

Interactions in casein micelle – Pea protein system (part I): Heat-induced denaturation and aggregation

International audienceThe aim of this work was to investigate the heat-induced interactions between pea proteins (vicilin 7S or legumin 11S enriched-fractions) in admixture with suspended casein micelles (SCM), at weight protein ratio of 1:1 and pH 7.1. The single-protein samples and mixtures thereof were prepared at concentrations of 18 and 36 mg(protein)/g(sample), respectively, then heated from 40 to 85 degrees C and incubated for 0-60 min. As compared to single-protein samples, differential scanning calorimetry (DSC) data indicated that the denaturation temperature of pea proteins increased of about 4 degrees C in the presence of SCM. Heat-induced change in protein composition of the soluble (SP) and micellar (MP) phases from centrifuged SCM pea protein mixture was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry. Likewise SP was analyzed by size-exclusion chromatography (SEC-HPLC). While pea legumin 11S and vicilin 7S oligomers were markedly sedimentable in MP from their respective unheated mixture, thermal denaturation and protein aggregation (>= 75 degrees C) resulted in increasing levels of dissolved pea proteins in SP. Heating of the SCM legumin mixture (85 degrees C, 15-60 min incubation) resulted in the dissociation of the legumin subunits L-alpha beta into acidic Le and basic L-beta polypeptides, yielding in comparable amounts soluble and insoluble disulfide-bonded aggregates, respectively. In contrast in the SCM vicilin mixture, the heat-denatured vicilin polypeptides in a temperature range of 70-80 degrees C produced in majority soluble and non-covalent aggregates. Though the heat-induced interactions between pea proteins were altered in the presence of micelles, caseins would not be involved into pea proteins aggregation

Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and β-Lactoglobulin

International audienceThe present work investigates the formation of protein aggregates (85 degrees C, 60 min incubation) upon heat treatment of beta-lactoglobulin (beta lg) pea globulins (Glob) mixtures at pH 7.2 and 5 mM NaCl from laboratory-prepared protein isolates. Various beta lg/Glob weight ratios were applied, for a total protein concentration of 2 wt % in admixture. Different analytical methods were used to determine the aggregation behavior of "mixed" aggregates, that is, surface hydrophobicity and also sulfhydryl content, protein interactions by means of SDS-PAGE electrophoresis, and molecule size distribution by DLS and gel filtration. The production of "mixed" thermal aggregates would involve both the formation of new disulfide bonds and noncovalent interactions between the denatured beta lg and Glob subunits. The majority of "mixed" soluble aggregates displayed higher molecular weight and smaller diameter than those for Glob heated in isolation. The development of pea whey protein "mixed" aggregates may help to design new ingredients for the control of innovative food textures

Partition of volatile compounds in pea globulin–maltodextrin aqueous two-phase system

International audienceThis study is based on the assumption that the off-flavour of pea proteins might be decreased using the retention of volatile compounds by a mixture with another biopolymer. The partition of volatile compounds in an aqueous system containing pea protein and maltodextrins was followed under thermodynamic incompatibility conditions. Firstly, the phase diagram of the system was established. Then, the partition of aroma compounds between the phase rich in protein and the phase rich in maltodextrin was measured by SPME–GC–MS. There was a transfer of volatile compounds during phase separation. Variations of pH were also used to vary the retention of volatile compounds by proteins. The concentration of volatile compounds in protein solution at pH 2.4 was higher than at pH 7.2. It was possible to increase the transfer of volatile compounds from the phase rich in protein to the phase rich in maltodextrin using the effect of pH on protein denaturation

Thermal Denaturation of Pea Globulins (<i>Pisum sativum</i> L.)Molecular Interactions Leading to Heat-Induced Protein Aggregation

The heat-induced denaturation and aggregation of mixed pea globulins (8%, w/w) were investigated using differential scanning calorimetry (DSC), SDS-PAGE, and size-exclusion chromatography (SEC-HPLC). DSC data showed that the pea proteins denaturation temperature (<i>T</i>_d) was heating-rate dependent. The <i>T</i>_d value decreased by about 4 °C by lowering the heating rate from 10 to 5 °C/min. The SDS-PAGE analysis revealed that protein denaturation upon heating at 90 °C was mainly governed by noncovalent interaction. The SEC-HPLC measurements indicated that low-denatured legumin (≈350–410 kDa) and vicilin/convicilin (≈170 kDa) globulins were heat-denatured and most of their subunits reassociated into high-molecular weight, soluble aggregates (>700 kDa). The addition of <i>N</i>-ethylmaleimide slightly modified the aggregation route of pea globulins. However, partially insoluble macroaggregates were produced in the presence of dithiothreitol, reflecting the stabilizing effect of disulfide bonds within legumin subunits