Réticulation enzymatique des protéines de pois pour la formation de microparticules : application à l'encapsulation de la riboflavine

In this work, pea proteins behavior toward enzymatic gelation by microbial transglutaminase (MTGase) was studied at native state and after denaturation (chemical reduction or thermal denaturation). The final application was the formation of protein microparticules to encapsulate riboflavin, chosen as hydrophilic active molecule model. The extraction process of the pea protein fractions has been optimized in such a way to minimize as possible protein denaturation and recover native fractions rich in albumin (Alb) and globulin (Glob) or a mixture of both.The setting up of the enzymatic reaction monitoring methods has brought out their complementarity as well as their limits. Two new monitoring methods of enzymatic cross-linking reaction have been developed. The first one, based on the NMR, allows to the simultaneous determination of the glutamine-lysine isopeptide bond, product of the enzymatic reaction, and the degree of crosslinking; the second method, based on size measuring techniques (SDS-PAGE and DLS), permit to view the intramolecular links. The study of enzymatic treatment applied to pea Alb and Glob at the native and denatured states, as well as thier native mixture showed that the enzymatic reaction is strongly related to the structure and conformation of proteins. Unlike Alb, the Glob fraction is a good substrate to transglutaminase and crosslinking reaction involves different subunits constituting globulins for each treatment condition. However, the Alb can be used as a booster of enzyme reaction which can be an innovative way for improving the proteins susceptibility toward transglutaminase treatment. The mechanism seems to be based on a selective affinity phenomenon. The good mechanical properties and water holding capacity of total pea proteins gel have been exploited to produce microparticles from a water-in-oil emulsion followed by enzymatic gelation. The produced microparticles were practically insoluble in gastrointestinal media in the absence of enzymes and slowly degradable in the presence of enzymes. The release mechanisms of riboflavin in digestive environments are governed by a diffusion phenomenon in the absence of enzymes and by support degradation phenomenon in the presence of enzymes according to kinetics compatible with nutraceutical applications.Dans ce travail, le comportement des protéines de pois vis-à-vis de la gélification enzymatique par la transglutaminase microbienne (MTGase) a été évalué à l’état natif et après dénaturation (réduction chimique ou thermo-dénaturation). L’application finale concernait la formation de microparticules protéiques permettant d’encapsuler la riboflavine, choisie comme molécule active hydrophile modèle. Le procédé d’extraction des fractions protéiques de pois a été optimisé de manière à affecter le moins possible la structure des protéines et de récupérer des fractions natives riches en albumines (Alb) et en globulines (Glob), ou leur mélange. La mise en place des méthodes de suivi de la réaction enzymatique a permis de mettre en évidence leur complémentarité ainsi que leurs limites. Deux nouvelles méthodes de suivi de la réticulation enzymatique ont été développées. L’une basé sur la RMN permet la détermination simultanée de la quantité du fragment glutamine-lysine, produit de la réaction enzymatique, et le degré de réticulation ; l’autre méthode, basée sur les techniques de mesure de taille (SDS-PAGE et DLS), permet de visualiser les liaisons intramoléculaires. L’étude du traitement enzymatique appliqué aux fractions Alb et Glob de pois à l’état natif et dénaturé, ainsi qu’en mélange natif, a montré que la réaction enzymatique est fortement liée à la structure et à la conformation des protéines. Contrairement à la fraction Alb, la fraction Glob constitue un bon substrat pour la MTGase et la réticulation met en jeu des sous-unités constitutives des globulines différentes pour chaque condition de traitement. Néanmoins, la fraction Alb peut être utilisée en tant que booster de réaction enzymatique ce qui peut faire l’objet d’une voie innovante d’amélioration de la susceptibilité des protéines vis-à-vis de la MTGase. Le mécanisme semble basé sur un phénomène d’affinité sélective. Les bonnes propriétés mécaniques et de capacité de rétention d’eau du gel de la fraction protéique de pois totale ont été exploitées pour produire des microparticules à partir de la dispersion de la solution protéique sous forme d’émulsion suivie d’une gélification enzymatique par la MTGase. Les microparticules ont été pratiquement insolubles dans les milieux gastro-intestinaux en absence d’enzymes et lentement dégradable en présence d’enzymes. La libération de la riboflavine est gouvernée par un phénomène de diffusion en absence d’enzyme et de dégradation de support en présence d’enzymes selon des cinétiques compatibles avec des applications nutraceutiques

Enzymatic cross-linking of pea proteins to produce microparticles : application to the encapsulation of riboflavin

Dans ce travail, le comportement des protéines de pois vis-à-vis de la gélification enzymatique par la transglutaminase microbienne (MTGase) a été évalué à l’état natif et après dénaturation (réduction chimique ou thermo-dénaturation). L’application finale concernait la formation de microparticules protéiques permettant d’encapsuler la riboflavine, choisie comme molécule active hydrophile modèle. Le procédé d’extraction des fractions protéiques de pois a été optimisé de manière à affecter le moins possible la structure des protéines et de récupérer des fractions natives riches en albumines (Alb) et en globulines (Glob), ou leur mélange. La mise en place des méthodes de suivi de la réaction enzymatique a permis de mettre en évidence leur complémentarité ainsi que leurs limites. Deux nouvelles méthodes de suivi de la réticulation enzymatique ont été développées. L’une basé sur la RMN permet la détermination simultanée de la quantité du fragment glutamine-lysine, produit de la réaction enzymatique, et le degré de réticulation ; l’autre méthode, basée sur les techniques de mesure de taille (SDS-PAGE et DLS), permet de visualiser les liaisons intramoléculaires. L’étude du traitement enzymatique appliqué aux fractions Alb et Glob de pois à l’état natif et dénaturé, ainsi qu’en mélange natif, a montré que la réaction enzymatique est fortement liée à la structure et à la conformation des protéines. Contrairement à la fraction Alb, la fraction Glob constitue un bon substrat pour la MTGase et la réticulation met en jeu des sous-unités constitutives des globulines différentes pour chaque condition de traitement. Néanmoins, la fraction Alb peut être utilisée en tant que booster de réaction enzymatique ce qui peut faire l’objet d’une voie innovante d’amélioration de la susceptibilité des protéines vis-à-vis de la MTGase. Le mécanisme semble basé sur un phénomène d’affinité sélective. Les bonnes propriétés mécaniques et de capacité de rétention d’eau du gel de la fraction protéique de pois totale ont été exploitées pour produire des microparticules à partir de la dispersion de la solution protéique sous forme d’émulsion suivie d’une gélification enzymatique par la MTGase. Les microparticules ont été pratiquement insolubles dans les milieux gastro-intestinaux en absence d’enzymes et lentement dégradable en présence d’enzymes. La libération de la riboflavine est gouvernée par un phénomène de diffusion en absence d’enzyme et de dégradation de support en présence d’enzymes selon des cinétiques compatibles avec des applications nutraceutiques.In this work, pea proteins behavior toward enzymatic gelation by microbial transglutaminase (MTGase) was studied at native state and after denaturation (chemical reduction or thermal denaturation). The final application was the formation of protein microparticules to encapsulate riboflavin, chosen as hydrophilic active molecule model. The extraction process of the pea protein fractions has been optimized in such a way to minimize as possible protein denaturation and recover native fractions rich in albumin (Alb) and globulin (Glob) or a mixture of both.The setting up of the enzymatic reaction monitoring methods has brought out their complementarity as well as their limits. Two new monitoring methods of enzymatic cross-linking reaction have been developed. The first one, based on the NMR, allows to the simultaneous determination of the glutamine-lysine isopeptide bond, product of the enzymatic reaction, and the degree of crosslinking; the second method, based on size measuring techniques (SDS-PAGE and DLS), permit to view the intramolecular links. The study of enzymatic treatment applied to pea Alb and Glob at the native and denatured states, as well as thier native mixture showed that the enzymatic reaction is strongly related to the structure and conformation of proteins. Unlike Alb, the Glob fraction is a good substrate to transglutaminase and crosslinking reaction involves different subunits constituting globulins for each treatment condition. However, the Alb can be used as a booster of enzyme reaction which can be an innovative way for improving the proteins susceptibility toward transglutaminase treatment. The mechanism seems to be based on a selective affinity phenomenon. The good mechanical properties and water holding capacity of total pea proteins gel have been exploited to produce microparticles from a water-in-oil emulsion followed by enzymatic gelation. The produced microparticles were practically insoluble in gastrointestinal media in the absence of enzymes and slowly degradable in the presence of enzymes. The release mechanisms of riboflavin in digestive environments are governed by a diffusion phenomenon in the absence of enzymes and by support degradation phenomenon in the presence of enzymes according to kinetics compatible with nutraceutical applications

Controlled release of riboflavin encapsulated in pea protein microparticles prepared by emulsion-enzymatic gelation process

International audienceRiboflavin was encapsulated in pea protein microparticles crosslinked by transglutaminase and the release properties of this system was studied in simulated gastric (pH = 1.2) and intestinal (pH = 7.4) fluids. The microparticles were obtained from a water-in-oil emulsion and subsequent enzymatic gelation. In presence of polyglycerol polyricinoleate as hydrophobic surfactant, magnetic agitation was preferred to high-speed homogenization in order to form spherical microparticles of ~150 μm average size. The encapsulation efficiency of crystallized riboflavin (RF) varied from 74% to 84% depending on the amount of loaded RF (0.1–1.0 wt%). According to the kinetic power law model, the release mechanisms of riboflavin under simulated gastrointestinal conditions were governed by diffusion in absence of digestive enzymes and by a support degradation phenomenon in their presence. The designed microparticles represented a potential system for the delivery of riboflavin up to the intestinal digestion phase

Gelation behaviors of denaturated pea albumin and globulin fractions during transglutaminase treatment

International audienceThe behavior of pea albumins (Alb) and globulins (Glob) in a denatured state toward microbial transglutaminase (MTGase) treatment was studied by SDS-PAGE analysis, free amine group determination, dynamic rheology and confocal laser scanning microscopy. The denaturation of pea proteins by chemical reduction with dithiothreitol (DTT) or by thermal treatment at 80 °C enhanced the enzymatic crosslinking degree of both protein isolates with greater crosslinking for the Glob fraction. The chemical denaturation affected preferentially the participation in crosslinking reaction of legumin acid subunits (40 kDa) for Glob sample and albumin polypeptides PA2 (26 kDa) for Alb sample, whereas the heat treatment led to complete polymerization of 55, 35 and 30 kDa vicilin polypeptides for pea globulins as shown by SDS-PAGE analysis. Up to 10 wt% concentration, the Alb fraction was not able to form MTGase crosslinked gels whatever the initial native or denaturated state. Compared to the native state, chemical and thermal denaturation of Glob fraction before enzymatic treatment led to the formation of weaker and stronger viscoelastic gels respectively. These contradictory results indicated that the enzymatic crosslinking reaction is highly related to polypeptides composition and conformation of proteins and the use of denaturation as a strategy to enhance gel forming properties by transglutaminase treatment has to be used with caution in the case of plant proteins

Size measuring techniques as tool to monitor pea proteins intramolecular crosslinking by transglutaminase treatment

International audienceIn this work, techniques for monitoring the intramolecular transglutaminase cross-links of pea proteins, based on protein size determination, were developed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles of transglutaminase-treated low concentration (0.01% w/w) pea albumin samples, compared to the untreated one (control), showed a higher electrophoretic migration of the major albumin fraction band (26 kDa), reflecting a decrease in protein size. This protein size decrease was confirmed, after DEAE column purification, by dynamic light scattering (DLS) where the hydrodynamic radius of treated samples appears to be reduced compared to the control one

Native-state pea albumin and globulin behavior upon transglutaminase treatment

International audienceThe behavior of pea albumin (Alb) and globulin (Glob) in their native state upon microbial transglutaminase (MTGase) treatment was studied. Only Glob was able to form a gel, at up to a 10% (w/w) concentration, with a minimum gelling concentration of 6% (w/w), and with a cross-linking degree of 25%. The most affected Glob subunits were convicilin (71 kDa), vicilins (55, 50, and 35 kDa), and legumin acidic subunit (40 kDa). In contrast, the legumin basic subunit (20 kDa) and vicilins of molecular weight less than 20 kDa remained mostly intact in all studied conditions. The cross-linking degree of Alb was 12%, which was not sufficient to form MTGase-induced gel. Major albumin polypeptide (PA2 26 kDa) was not affected by the MTGase concentration or by pH variation. Pea Alb and Glob in their native state were ranked as poor and moderately good substrates for MTGase, respectively, and unfolding them by thermal or chemical denaturation could be an interesting way to improve the efficiency of cross-linking

1H NMR Spectroscopy As Tool To Study Transglutaminase Crosslinking Of Pea Globulin

Skiathos Island, GREECE - 30 May - 02 June 2013International audienceA new method based on NMR spectroscopy was developed to detect the G-L (GlutamylLysine) isopeptide bonds formed by the enzymatic transglutaminase reaction. Because of thecomplexity of NMR signals of proteins due to their structures, the method was first developedon a model system (glutamine and lysine) to simplify the detection of the G-L residue. Andthen, the results were applied on the real protein matrix (pea globulin). MTG treatment ofmodel system led to the appearance of a new resonance on NMR spectrum which isoriginated probably from the ε-methylene protons of lysine residues covalently linked toglutamine. The comparison between NMR spectra of MTG-treated and untreated pea globulinwith MTG-treated model system permitted to identify the G-L isopeptide signal. The G-Lisopeptide signal is also observed in the NMR spectra of native pea globulin (untreated whichMTG), indicating that the isopeptide is naturally present in pea proteins. However, thesuperimposition of NMR spectra of MTG-treated and untreated pea globulin shows a higherintensity of G-L isopeptide signal for MTG-treated samples. The increased signal intensityevidences the enzymatic reaction and permits to quantify the G-L isopeptide

Monitoring of transglutaminase crosslinking reaction by 1H NMR spectroscopy on model substrates

International audienceA new method based on 1H NMR spectroscopy was developed for monitoring transglutaminase crosslinking reaction with model molecules (CBZ-Gln-Gly and N-α-acetyl-lysine). The transglutaminase reaction led to the appearance of new resonances on NMR spectrum as well as significant decrease in others. The new observed resonances, originated from newly formed ɛ-(γ-glutamyl)lysine isopeptide bonds, evidence the enzymatic reaction and allow to quantify the ɛ-(γ-glutamyl)lysine fragment. Moreover, the decrease in resonance intensity, originated from lysine, permit to determine the crosslinking degree. These results obtained by 1H NMR spectroscopy can be used as an alternative method to LC–MS, reducing drastically the analysis time from 7 days to 2 h