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

Protein composition and nutritional aspects of pea protein fractions obtained by a modified isoelectric precipitation method using fermentation

Pea albumins are promising for their nutritional, biological, and techno-functional properties. However, this fraction is usually discarded in the industry due to its low protein content compared to globulin fraction and the presence of some anti-nutritional compounds. In the present study, we used an alternative method of pea protein extraction based on alkaline solubilization/isoelectric precipitation in which the reduction of pH was achieved by lactic acid fermentation using specific starters instead of mineral acids. Hence, the main objective of this study was to examine the protein profile and the content of anti-nutritional and nutritional active compounds in pea albumin-rich fractions obtained by the isoelectric extraction method without (control) or with fermentation with different lactic acid bacteria (Streptococcus thermophilus, Lactiplantibacillus plantarum, and their co-culture). Different pea cultivars (Cartouche, Ascension, and Assas) were used here for their differences in protein profile. The results revealed a higher total nitrogen content in albumin-rich fraction for fermented samples and, in particular, for co-culture. The majority of total nitrogen was determined as non-protein (~50%), suggesting the degradation of proteins by LAB to small peptides and amino acids, which were solubilized in the soluble fraction (albumin) as confirmed by size exclusion chromatography (SEC-HPLC) analysis. Moreover, the higher antioxidant activity of fermented albumin samples was attributed to the production of small peptides during extraction. Lactic acid fermentation also resulted in a significant reduction of trypsin inhibitor activity, α-galactoside, and phytic acid content of this fraction compared to control

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

Effect of Lactic Acid Fermentation on Legume Protein Properties, a Review

International audienceLegume proteins have a promising future in the food industry due to their nutritional, environmental, and economic benefits. However, their application is still limited due to the presence of antinutritional and allergenic compounds, their poor technological properties, and their unpleasant sensory characteristics. Fermentation has been traditionally applied to counteract these inconveniences. At present, lactic acid fermentation of legumes is attracting the attention of researchers and industry in relation to the development of healthier, tasty, and technologically adapted products. Hence, we aimed to review the literature to shed light on the effect of lactic acid fermentation on legume protein composition and on their nutritional, functional, technological, and sensorial properties. The antimicrobial activity of lactic acid bacteria during legume fermentation was also considered. The heterogenicity of raw material composition (flour, concentrate, and isolate), the diversity of lactic acid bacteria (nutriment requirements, metabolic pathways, and enzyme production), and the numerous possible fermenting conditions (temperature, time, oxygen, and additional nutrients) offer an impressive range of possibilities with regard to fermented legume products. Systematic studies are required in order to determine the specific roles of the different factors. The optimal selection of these criteria will allow one to obtain high-quality fermented legume products. Fermentation is an attractive technology for the development of legume-based products that are able to satisfy consumers’ expectations from a nutritional, functional, technological, and sensory point of view

Pea Protein Extraction Assisted by Lactic Fermentation: Impact on Protein Profile and Thermal Properties

International audienceAlthough pea protein has been widely explored, its consumption is still limited by undesirable sensory characteristics and low solubility. All these properties can be modified during protein extraction process. Besides, previous studies showed that lactic acid bacteria (LAB) have a positive effect on legume protein ingredients in terms of flavor and functional properties. Hence, the objective of this work was to explore an alternative extraction method based on alkaline extraction/isoelectric precipitation (AEIEP) resulting in globulin-rich and residual albumin-rich fractions. Here, the decrease in pH was achieved by lactic fermentation instead of mineral acid addition. Different bacteria strains (Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium lactis) have been used alone or in co-culture, and the results were compared with the usual acidification. The extraction assisted by fermentation led to the increase by 20-30% in protein content/yield of the albumin fraction, meaning that the solubility of the extracted pea protein was increased. This result could be explained by the proteolytic activity of bacteria during lactic fermentation. Therefore, the thermal denaturation properties of the isolated protein fractions measured by differential scanning calorimetry could be mainly ascribed to differences in their polypeptide compositions. In particular, higher denaturation enthalpy in globulin fractions after fermentation compared to AEIEP (~15 J/g protein vs. ~13 J/g protein) revealed the relative enrichment of this fraction in pea legumins; a higher part of 7S globulins seemed to be consumed by lactic acid bacteria

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

Improvement of the techno-functional properties of pea proteins by microfluidization

International audienceThe use of pea (Pisum sativum (L.)) proteins in the food industry is still limited despite their good environmental sustainability, heath-oriented composition, reliable origin and stable price. However, one of the most important limitations is their low solubility which determines a number of their techno-functional properties. Microfluidization is a non-thermal emerging technology that may modify the structure and the techno-functional properties of pea proteins. Microfluidization combines high shear forces due to the stream speed and direction; impact forces from collisions with the walls and with the fluid itself; and turbulence inside the chamber. The objective of this work was to evaluate the effects of dynamic high pressure (microfluidization) on the techno-functional properties of commercial pea protein isolates. Microfluidization was performed by using a Microfluidizer processor model LM10 (Microfluidics, Newton, MA, USA) provided with a Z-type chamber. Solubility was clearly increased when applying microfluidization at 100MPa. Microfluidization also improved the least gelation concentration by thermal and acidic treatments, and the interfacial tension. However no clear effect was observed in the foaming capacity. This work permitted to conclude that the effect of microfluidization depends on the sample characteristics, on the environmental conditions, and on the microfluidization parameters

The Effect of High‐Pressure Microfluidization Treatment on the Foaming Properties of Pea Albumin Aggregates

International audienceThe effect of dynamic high-pressure treatment, also named microfluidization, on the surface properties of thermal pea albumin aggregates (AA) and their foaming ability was investigated at pH 3, 5, and 7. The solubility of albumin particles was not affected by the increase in microfluidization pressure from 70 to 130 MPa. Particle charge depended only on the pH, whereas protein surface hydrophobicity was stable at pH 5, decreased at pH 3, but increased at pH 7 after microfluidization treatment and with the applied pressure. Surface tension of AA measured at air/water interface was favorably affected by the microfluidization treatment at each pH preferentially due to size reduction and increased flexibility of protein particles. The foaming capacity and stability of AA depended on the pH conditions and the microfluidization treatment. The high-pressure treatment had little influence in foaming properties at acidic pHs, probably related to a more compact form of AA at these pHs. At neutral pH, the foaming properties of pea AA were strongly influenced by their surface properties and size associated with significant modifications in AA structure with microfluidization. Changes in albumin aggregate characteristics with pH and microfluidization pressure are also expected to modulate other techno-functional properties, such as emulsifying property. Practical Application Albumins are known for their interesting nutritional values because they are rich in essential amino acids. This fraction is not currently marketed as a protein isolate for human consumption, but can be considered as a potential new vegetable protein ingredient. This document demonstrated that heat treatment or dynamic high-pressure technology can control the foaming properties of this protein for possible use in expanded foods

Evaluation du potentiel bioprotecteur de bactéries lactiques confinées dans une matrice polymérique

Parmi les différentes méthodes de lutte contre les microorganismes pathogènes et/ou altérants en agroalimentaire, l utilisation de bactéries lactiques (LAB) bioprotectrices s'avère être un outil prometteur pour la préservation des aliments. Ce travail de thèse collaboratif, entre l'équipe PAPC (AgroSup Dijon, Université de Bourgogne) et le laboratoire BioDyMIA (Université Lyon1-Isara Lyon), concerne l'étude de systèmes bioprotecteurs immobilisant des cellules entières de LAB dans une matrice polymérique d'alginate de sodium et de caséinate de sodium pour une activité ciblée contre Listeria spp. Dans un premier temps, la méthodologie mise en œuvre a consisté à sélectionner des souches de LAB bioprotectrices sur la base de leur activité antimicrobienne évaluée par la méthode de diffusion en milieu gélosé contre trois souches de Listeria spp. Quatre souches sur 19 ont ainsi été sélectionnées. Une caractérisation partielle des métabolites antimicrobiens produits par ces 4 souches a ensuite été réalisée en appliquant des traitements thermiques et enzymatiques aux surnageants de culture correspondants pour évaluer si ces traitements altéraient l activité des métabolites antimicrobiens présents. Une purification et une identification partielle des actifs antimicrobiens de nature peptidique ont été réalisées uniquement pour la souche d'intérêt principale : Lactococcus lactis LAB3. Dans un second temps, une formulation de la matrice polymérique d immobilisation des LAB sélectionnées a été choisie en réalisant le diagramme de phases du système aqueux alginate de sodium/caséinate de sodium : 1,5 % (m/m) d'alginate de sodium / 4 % (m/m) de caséinate de sodium / 20 % (m/m) bouillon MRS. Cette formulation a permis d'obtenir une matrice composée d une phase continue riche en alginate et d une phase dispersée riche en caséinate dans laquelle les cellules de LAB se localisent préférentiellement d après les observations en microscopie de fluorescence confocale à balayage laser. Suite à l'inclusion des cellules de LAB dans ces matrices liquides et gélifiées d'alginate seul et d'alginate/caséinate, leur cultivabilité et leur activité anti-Listeria ont été suivies à 30C pendant 12 jours. Ceci a révélé que la cultivabilité et l activité antimicrobienne des cellules de LAB se maintiennent à des niveaux plus élevés dans les matrices d'alginate/caséinate que dans celles uniquement à base d alginate. Ces matrices à base d alginate et de caséinate apparaissent donc comme un système prometteur pour l'immobilisation de LAB bioprotectrices. Leur intérêt pour l inclusion de LAB a pu être corrélé à leur viabilité et à la structure composite de cette matrice à base de protéines qui favoriserait la production et la libération des métabolites antimicrobiensAmong the various methods to control foodborne pathogenic and/or food spoilage microorganisms in food chain, bioprotective lactic acid bacteria (LAB) appear to be promising tools for food biopreservation. This collaborative study, between PAPC (Agrosup Dijon, University of Burgundy) and BioDyMIA (University Lyon1-Lyon Isara) laboratories, concerned the development of sodium alginate/sodium caseinate polymeric matrices intended to entrap LAB cells selected for their anti-Listeria spp. activity. First, 4 LAB strains from 19 LAB strains were selected for their anti-Listeria spp. activity: this screening was performed by the method of agar diffusion against three Listeria spp strains. Then, antimicrobial metabolites produced by the selected LAB strains were partially characterized by assessing the effect of various thermal and enzymatic treatments on the anti-Listeria spp. activity of their culture supernatants. A partial purification and identification of antimicrobial active peptides produced by the main strain of interest (Lactococcus lactis LAB3) was also performed. A composition of the polymer matrix has been selected by performing the phase diagram of sodium alginate/sodium caseinate system: 1.5% (w/w) sodium alginate / 4% (w/w) of caseinate sodium / 20% (w/w) MRS broth. This formulation provides a rich alginate continuous phase and a rich caseinate dispersed phase in which LAB cells localize according to the study by confocal microscopy. LAB cells were immobilized in liquid and gelled matrices of alginate and alginate/caseinate. Culturability and anti-Listeria activities were measured during a storage at 30C for 12 days. The alginate/caseinate matrices were more effective in better maintaining LAB cells cultivability and their antimicrobial activity than alginate matrix. This effectiveness seemed correlated with cell viability and the dispersion-like structure of the protein-based system which enhance production and release of antimicrobial metabolites. Thus, this type of polymeric matrix appeared as a promising immobilization system of bioprotective LABDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF