Addition of citrate to casein micelles: Modifications of their physico-chemistry and acid gelation.

The organization and functional properties of casein micelles (CMs) are influenced by several factors such as temperature, pH, and ionic environment. In this study, different CMs suspensions were obtained by addition of citrate to milk at final added concentrations: 8.5, 14.5 and 18.7 mM and readjustment of pH to 6.75. Modified milks were concentrated by ultrafiltration at ~10°C and some samples were then diafiltered against milk ultrafiltrate and standardized at ~45 g.kg-1 total proteins. The analyses revealed that CMs were demineralized in Ca and P and dissociated. After acidification to pH 4.6 by HCl, set-style gels from samples containing 8.5 and 18.7 mM added citrate have a firmness reduced to 75 and 56% of that of the gel from the control sample, respectively (without citrate). In the opposite, the firmness obtained after addition of 14.5 mM of citrate increased to 116% of the value of the control sample. Same trends were observed with the viscosity values determined on the different stirred acid gels. For both rheological analyses, the comparison of diafiltered samples against undiafiltered samples did not show significant differences. These changes in the rheological properties of acid gels were probably related to the modifications of the micellar calcium phosphate responsible for the structure of CMs

Addition of citrate to casein micelles: Modifications of their physico-chemistry and acid gelation.

The organization and functional properties of casein micelles (CMs) are influenced by several factors such as temperature, pH, and ionic environment. In this study, different CMs suspensions were obtained by addition of citrate to milk at final added concentrations: 8.5, 14.5 and 18.7 mM and readjustment of pH to 6.75. Modified milks were concentrated by ultrafiltration at ~10°C and some samples were then diafiltered against milk ultrafiltrate and standardized at ~45 g.kg-1 total proteins. The analyses revealed that CMs were demineralized in Ca and P and dissociated. After acidification to pH 4.6 by HCl, set-style gels from samples containing 8.5 and 18.7 mM added citrate have a firmness reduced to 75 and 56% of that of the gel from the control sample, respectively (without citrate). In the opposite, the firmness obtained after addition of 14.5 mM of citrate increased to 116% of the value of the control sample. Same trends were observed with the viscosity values determined on the different stirred acid gels. For both rheological analyses, the comparison of diafiltered samples against undiafiltered samples did not show significant differences. These changes in the rheological properties of acid gels were probably related to the modifications of the micellar calcium phosphate responsible for the structure of CMs

Gastric lipase interacting with lipid membrane model: atomic force microscopy visualization

International audienceIn the context of the neonatal digestion of milk fat globule, we studied the interaction between a gastric lipase and lipid membrane models in order to identify key parameters in the first step of enzyme anchoring to the membrane before the onset of the catalytic activity. Indeed, little is known about the effect of the lipid nature constituting the external membrane of the milk fat globule on the digestion process. Lipid Langmuir films were used to mimic the outer leaflet of the external membrane of the milk fat globule. Different lipid mixtures with variable physical phases, surface charge and lateral packing to identify the nature of the enzyme/membrane interactions. Recombinant dog gastric lipase (rDGL) was used as model of the human gastric lipase.A combination of different techniques including tensiometry, ellipsometry, atomic force microscopy gave an insight on the ability to the enzyme to access and insert in the lipid membrane. Indeed, tensiometry is related to the ability of the molecule to insert in the lipid film, ellipsometry gives information on the amount of enzyme interacting with the membrane (with or without penetration) while atomic force microscopy allows visualization at the nanoscale the lateral organization of the enzyme embedded in the membrane. The modulation of the surface charge of the film, lateral packing and lipid physical phase were tested. In addition natural lipid mixtures (with and without enzyme substrate) extracted from cow buttermilkwere used to complete the set of membrane lipids. Our funding show that the large amount of rDGL molecule is located close to the lipid membrane, however little insertion exclusively in the l.e. phase is noticed. Moreover, the molecular interactions are clearly impacted by the nature of the lipid membrane

Deux acteurs de la digestion néonatale et leurs interactions : la lipase gastrique et le globule gras

Plusieurs équipes de recherche rennaises utilisent des expériences aux interfaces fluides planes pour connaître et comprendre les propriétés de biomolécules variées. Lors d’une journée d’échanges, des scientifiques de l'UMR STLO et d’autres équipes contribueront à dresser le panorama des thématiques concernées et montrer le potentiel de ces expériences.Une journée d'échanges scientifiques est organisée par l'Institut de Physique de Rennes, le 22 janvier 2015. Elle vise à illustrer le potentiel et à construire une vision d'ensemble des expériences aux interfaces planes, dans des domaines scientifiques aussi variés que la physique des mousses et émulsions, les technologies de transformation des aliments, la digestion néonatale des lipides laitiers ou l'interaction de protéines avec les membranes biologiques. L'opportunité de montrer que les expériences aux interfaces planes livrent des informations abondantes et pertinentes.National audienceLa lipolyse d’assemblages lipoprotéiques complexes tels que le globule gras laitier est très fréquente notamment durant la phase néonatale1. Cette lipolyse est initiée dans l’estomac par la lipase gastrique humaine qui agit à pH acide et possède des propriétés biochimiques et biophysiques uniques lui permettant d’initier l’attaque du globule gras, ce qui favorise ensuite l’action de la lipase pancréatique. La lipolyse est une réaction interfaciale qui nécessite avant la phase catalytique stricte une étape d’adsorption de la lipase gastrique sur cette interface2. Les interactions qui gouvernent l’adsorption de la lipase gastrique sur la membrane du globule gras ne sont pas totalement élucidées. L’objectif de ce travail était donc d’élucider ces interactions ainsi que la distribution latérale de la lipase gastrique dans des membranes modèles présentant une coexistence de phases identique à celle observée dans la membrane du globule gras. Une lipase gastrique recombinante de chien (rDGL) a été utilisée comme modèle à la lipase gastrique humaine. Une combinaison d’outils biophysiques de la plateforme BioMIF (ellipsométrie, tensiométrie et microscopie de force atomique) a été mise en œuvre pour caractériser l’adsorption de la rDGL dans les membranes modèles3. Des films de Langmuir ont été utilisés pour imiter le feuillet extérieur de la membrane du globule gras. Des mélanges de glycérophospholipides et extraits de babeurre présentant différentes phases physiques, charges de surface et densités moléculaires ont permis d’identifier les interactions enzyme/membrane gouvernant l’adsorption. Nous avons montré que: 1.La rDGL présente une forte affinité pour les interfaces lipide/liquide. Néanmoins, si une grande partie des molécules est proche de l’interface un nombre restreint de molécules s’insère à l’interface. 2. La rDGL s’adsorbe préférentiellement dans la phase liquide expansée et aux frontières des deux phases, l’adsorption se fait à trois niveaux de profondeur dans la membrane (h1~5.5 nm, h2<4 nm, and h3<2 nm) et impacte fortement l’organisation latérale de la monocouche de lipide (Figure); 3. L’addition de charges négatives via l’addition de phosphatidylsérine favorise l’adsorption; ainsi en plus des interactions hydrophobes, l’adsorption de la rDGL est favorisée par des interactions électrostatiques qui ont été approchée par la modélisation du potentiel de surface de la rDGL.Une montée massive de la rDGL à proximité du substrat peut favoriser une prélipolyse rapide avant la vidange gastrique. L’application de ces outils biophysiques dans des multicouches supportées pourra permettre de mieux approcher la complexité de la membrane du globule gras. Ces outils pourront également être appliqués à d’autres lipases digestives d’intérêt pour la digestion néonatale

Gastric lipase: interactions governing adsorption and distribution in model membranes

Lipolysis of complex lipoproteic assemblies such as milk fat globules is particularly frequent during neonatal digestion1. Such lipolysis proceeds at acid pH and requires a rapid adsorption of the enzyme onto the external membrane enveloping the substrate before the onset of catalytic activity2. The interactions governing this adsorption are not fully elucidated. Our objective was thus to unravel the involved interactions and precise gastric lipase lateral distribution in model membranes of milk fat globules presenting liquid phase separation. Recombinant dog gastric lipase (rDGL) was used as model of the human gastric lipase. Ellipsometry, tensiometry and atomic force microscopy were used to get an insight on the ability of rDGL to get inserted into the lipid membrane3. Lipid Langmuir films were used to mimic the outer leaflet of the external membrane of the milk fat globule. Different lipid mixtures and natural extracts of cow’s buttermilk with variable physical phases, surface charge and lateral packing were tailored to identify the nature of the enzyme/membrane interactions.We showed that: 1.rDGL is characterized by a high affinity for the lipid/liquid interface as revealed by adsorption equilibrium coefficients. Nevertheless, a large amount of molecules is located close to the interface but a limited number is inserted; 2. rDGL partitions toward liquid expansed phase and at phase boundaries, gets adsorbed at three levels of insertion (h1~5.5 nm, h2<4 nm, and h3<2 nm) and strongly impacts on lipid phase lateral organization (Figure); 3. Addition of local negative charges using phosphatidylserine reinforced adsorption; hence besides hydrophobic interactions, rDGL adsorption is favored by electrostatic interactions which were investigated through surface potential modelling.The massive amount of gastric lipase located close to the substrate may favor rapid prelipolysis before gastric emptying and subsequent action of pancreatic lipases. Generalization of this biophysical approach to other digestive lipases to precise their interactions with complex food substrate is a promising research direction

Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation

International audienceThe enzymatic lipolysis of complex natural lipoproteic assemblies such as milk fat globules is central in neonatal digestion. This process first requires the rapid adsorption of a lipolytic enzyme, gastric lipase, onto the membrane enveloping the triglyceride substrate before the onset of catalytic activity. The interactions governing lipase adsorption onto this complex lipid/water interface are not fully elucidated. This study was designed to unravel the interactions of recombinant dog gastric lipase (rDGL) with model monolayers presenting liquid-liquid phase coexistence and mimicking the outer leaflet of the milk fat globule membrane. Combining biophysical tools (ellipsometry, tensiometry and atomic force microscopy), it was evidenced that rDGL partitions toward liquid expanded phase and at phase boundaries. rDGL gets adsorbed at several levels of insertion suggesting molecular cooperation that may favor insertion and strongly impacts on the lipid phase lateral organization. The addition of phosphatidylserine, negatively charged, reinforced adsorption; hence besides hydrophobic interactions and as further investigated through surface potential modeling, rDGL adsorption is favored by electrostatic interaction