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International audience; Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depends greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. It is responsible for the effective hydrolysis of the main lipids composing plant photosynthetic membranes, which represent an excellent source of omega-3 for human nutrition. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and physical state of its substrate at the nanoscopic and microscopic scales. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipid, and/or phospholipid, and/or phytosterol, presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their digestion kinetics by gPLRP2 were investigated in the presence or absence of bile salts (NaTDC) during static in vitro, so called “bulk”, digestion. The enzymatic activity of gPLRP2 onto mixed monolayer of galactolipids and/or phospholipids, and/or phytosterols was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. Additionally, gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids, but the compositional complexity in these systems induced a lag phase before the initiation of lipolysis, and a slowdown in enzymatic activity. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes. The understanding of PLRP2 enzymatic activity at the level of natural plant-based assemblies open the way to new strategies to vectorize bioaccessible omega-3 and rebalance human and animal diets.; La lipase pancréatique apparentée à la protéine 2 (PLRP2) présente des activités galactolipase et phospholipase A1 remarquables, qui dépendent fortement de l'organisation supramoléculaire des substrats et de la présence de molécules tensioactives telles que les sels biliaires. Elle est responsable de l'hydrolyse efficace des principaux lipides composant les membranes photosynthétiques des plantes, qui représentent une excellente source d'oméga-3 pour la nutrition humaine. L'objectif de l'étude était de comprendre la modulation des mécanismes d'adsorption et de l'activité enzymatique de la PLRP2 de cobaye (gPLRP2), par l'environnement physique de l'enzyme et l'état physique de son substrat à l'échelle nanoscopique et microscopique. Des monocouches de Langmuir ont été utilisées pour reproduire des membranes modèles photosynthétiques homogènes et hétérogènes contenant des galactolipides, et/ou des phospholipides, et/ou des phytostérols, présentant des interfaces chargées ou non. Les mêmes mélanges lipidiques ont également été utilisés pour former des liposomes micrométriques, et leur cinétique de digestion par la gPLRP2 a été étudiée en présence ou en absence de sels biliaires (NaTDC) au cours d'une digestion statique in vitro, dite "bulk". L'activité enzymatique de la gPLRP2 sur une monocouche mixte de galactolipides et/ou phospholipides, et/ou phytostérols a été caractérisée avec une activité optimale à 15 mN/m, en l'absence de sels biliaires. De plus, la gPLRP2 a montré une meilleure adsorption sur des monocouches modèles biomimétiques contenant des lipides chargés négativement, mais la complexité de la composition de ces systèmes a induit une phase de latence avant l'initiation de la lipolyse, et un ralentissement de l'activité enzymatique. En bulk, aucune activité enzymatique n'a pu être démontrée sur les liposomes à base de GL en l'absence de sels biliaires, probablement en raison de la pression latérale élevée des bicouches lipidiques. En présence de NaTDC (4 mM), cependant, la gPLRP2 a montré des activités galactolipase élevées et phospholipase A1 modérées sur les liposomes, probablement en raison d'une diminution du packing et de la pression latérale lors de l'adsorption du NaTDC, et de la rupture ultérieure des liposomes. La compréhension de l'activité enzymatique de la PLRP2 au niveau des assemblages naturels à base de plantes ouvre la voie à de nouvelles stratégies pour vectoriser des oméga-3 bioaccessibles et rééquilibrer les régimes alimentaires humains et animaux.

International audience; The microalgae Chlorella vulgaris represent a good model for the study of photosynthetic membranes and are rich in photosynthetic proteins, galactolipids, and pigments (Demarco et al., 2022). These photosynthetic organisms have been consumed in some parts of the world for centuries and their nutritional potential has been highlighted in many reports, explaining a growing interest in their use in new food applications (Torres-Tiji et al., 2020). However, their digestibility by gastrointestinal enzymes has not been extensively studied. So we used the Pancreatic lipase related-protein 2 (PLRP2) to get more information on the digestive mechanisms. Indeed, it has been shown to be responsible for the effective hydrolysis of galactolipids, the main lipids composing plant photosynthetic membranes. Guinea pig pancreatic lipase related-protein 2 (gPLRP2) is a model enzyme close to its human analogue, and that has been extensively studied for its ability to hydrolyze a wide range of acylglycerol substrates, exhibiting remarkable galactolipase and phospholipase A1 activity. In this study, Langmuir model monolayers were used to fellow the interfacial behavior of gPLRP2 at the level of natural plant lipid monolayers from Chlorella microalgae extracts, and to understand the impact of the chemical and physical heterogeneity of natural photosynthetic plant membranes on the enzymatic activity of gPLRP2. We compared also with data obtained on lipid extracts from nuts and almonds for which the composition is mainly composed of phospholipids. The results have shown that the enzymatic activity of gPLRP2 is with an optimum activity obtained at 15 mN/m. Interfacial studies showed enhanced adsorption of gPLRP2 onto natural monolayers containing negatively charged lipids, and that the compositional complexity in these systems induced a lag phase before the initiation of lipolysis, and a slowdown in enzymatic activity.; Les microalgues Chlorella vulgaris représentent un bon modèle pour l'étude des membranes photosynthétiques et sont riches en protéines photosynthétiques, galactolipides et pigments (Demarco et al., 2022). Ces organismes photosynthétiques sont consommés dans certaines régions du monde depuis des siècles et leur potentiel nutritionnel a été mis en évidence dans de nombreux rapports, expliquant un intérêt croissant pour leur utilisation dans de nouvelles applications alimentaires (Torres-Tiji et al., 2020). Cependant, leur digestibilité par les enzymes gastro-intestinales n'a pas été étudiée de manière approfondie. Nous avons donc utilisé la lipase pancréatique apparentée à la protéine de type 2 (PLRP2) pour obtenir plus d'informations sur les mécanismes digestifs. En effet, il a été démontré qu'elle est responsable de l'hydrolyse efficace des galactolipides, les principaux lipides composant les membranes photosynthétiques des plantes. La lipase pancréatique de cochon d'Inde (gPLRP2) est une enzyme modèle proche de son analogue humain, et qui a été largement étudiée pour sa capacité à hydrolyser une large gamme de substrats d'acylglycérol, présentant une remarquable activité galactolipase et phospholipase A1. Dans cette étude, des monocouches modèles de Langmuir ont été utilisées pour étudier le comportement interfacial de la gPLRP2 au niveau de monocouches lipidiques végétales naturelles provenant d'extraits de microalgues Chlorella, et pour comprendre l'impact de l'hétérogénéité chimique et physique des membranes végétales photosynthétiques naturelles sur l'activité enzymatique de la gPLRP2. Nous avons également comparé avec les données obtenues sur des extraits lipidiques de noix et d'amandes dont la composition est principalement composée de phospholipides. Les résultats ont montré que la gPLRP2 présente une activité optimale à 15 mN/m. Les études interfaciales ont montré une adsorption accrue de la gPLRP2 sur des monocouches naturelles contenant des lipides chargés négativement, mais également que la complexité de la composition de ces systèmes induisait une phase de latence avant l'initiation de la lipolyse, et un ralentissement de l'activité enzymatique.

Health organizations now recommend diversifying and increasing our dietary intake of polyunsaturated fatty acids (PUFA) omega-3, which are essential for the proper functions of our cells. To meet these recommendations, plant membranes, and in particular photosynthetic membranes, represent a real potential, as they contain specific lipids, and in particular galactolipids, whose degradation by enzymatic reactions provides PUFA omega-3. Due to their particular composition, these systems present phase coexistences and thus lateral heterogeneity impacting their digestibility. In order to optimally exploit plant lipid sources, it is necessary to understand their fate in the human gastrointestinal tract. The results obtained in this research work provide an in-depth understanding of the mechanisms of interaction between digestive enzymes and plant membrane assemblies. In particular, this work is the first to investigate the digestion of heterogeneous monolayers of galactolipids and phospholipids by close analogues of the main enzymes responsible for the gastrointestinal degradation of plant lipids in humans. The originality of this study is based on a thorough characterization of the interfacial behavior of plant lipid systems, presenting a chemical heterogeneity inducing a physical heterogeneity. The digestibility of these heterogeneous plant lipid assemblies was studied at the molecular scale, but also at the micronic object scale. This work allowed to report the substrate specificity of the studied lipases and phospholipases (rDGL, sPLA2-IB, gPLRP2) on plant polar lipids. In particular, the galactolipase, but also phospholipase A1 activities of gPLRP2 were observed in heterogeneous galactolipid and phospholipid systems. The genericity of action of these enzymes with respect to the physical state of the membranes has also been demonstrated. We demonstrate here that at the lipid interfaces, a high content of polyunsaturated acyl groups can amplify the local compressibility and be an asset for the insertion of digestive lipases. This physical aspect remains to be confronted with the chemical specificity of the enzyme. Overall, plant lipid assemblies are rich in polar lipids and particularly galactolipids, which concentrate significant quantities of omega-3 PUFA, and are bioaccessible under model conditions under the action of gPLRP2. These assemblies also exhibit surface-active and oxidative stability properties characterized in this work. The galactolipase activity of PLRP2 at the level of modulable model membrane monolayers, but also at the level of natural membrane assemblies, opens the way to the development of plant-based dietary alternatives to vectorize omega-3 PUFA, and contribute to rebalancing human and animal diets.; Les organismes de santé recommandent aujourd’hui de diversifier et d'augmenter nos apports alimentaires en acides gras polyinsaturés (AGPI) oméga-3, essentiels au bon fonctionnement de nos cellules. Pour répondre à ces recommandations, les membranes végétales, et en particulier les membranes photosynthétiques, représentent un réel potentiel, car elles contiennent des lipides spécifiques, et notamment des galactolipides, dont la dégradation par des réactions enzymatiques fournit des AGPI oméga-3. De par leur composition particulière, ces systèmes présentent des coexistences de phases et donc une hétérogénéité latérale influant sur leur digestibilité. Or, afin d’exploiter de façon optimale les sources lipidiques végétales, il est nécessaire de comprendre leur devenir dans le tractus gastro-intestinal humain. Les résultats obtenus dans ce travail de recherche viennent apporter une compréhension approfondie des mécanismes d’interactions entre les enzymes digestives et les assemblages membranaires végétaux. En particulier, ce travail est pionnier sur la digestion des monocouches hétérogènes de galactolipides et de phospholipides par de proches analogues des principales enzymes responsables de la dégradation gastro-intestinale des lipides végétaux chez l’Homme. L’originalité de cette étude repose sur une caractérisation poussée du comportement interfacial de systèmes lipidiques végétaux, présentant une hétérogénéité chimique induisant une hétérogénéité physique. La digestibilité de ces assemblages lipidiques végétaux hétérogènes a été étudiée à l’échelle moléculaire, mais également à l’échelle de l’objet micronique. Ce travail a permis de rendre compte de la spécificité de substrat des lipases et phospholipase étudiées (rDGL, sPLA2-IB, gPLRP2) sur les lipides polaires végétaux. En particulier, les activités galactolipase, mais également phospholipase A1 de la gPLRP2 ont été observées au niveau des systèmes hétérogènes de galactolipides et de phospholipides. La généricité d’action de ces enzymes par rapport à l’état physique des membranes a également été prouvée. Nous démontrons ici qu’au niveau des interfaces lipidiques, une forte teneur en groupements acyls polyinsaturés peut amplifier la compressibilité locale et être un atout pour l’insertion des lipases digestives. Cet aspect physique reste à confronter avec la spécificité chimique de l’enzyme. Globalement, les assemblages lipidiques végétaux sont riches en lipides polaires et particulièrement en galactolipides, qui concentrent des quantités importantes d’AGPI oméga-3, et sont bioaccessibles en conditions modèles sous l’action de la gPLRP2. Ces assemblages présentent en outre des propriétés tensioactives et de stabilité à l’oxydation caractérisées dans ce travail. L’activité galactolipase de la PLRP2 au niveau des monocouches membranaires modèles modulables, mais également au niveau des assemblages membranaires naturels, ouvre la voie vers le développement d’alternatives alimentaires végétales pour vectoriser des AGPI oméga-3, et contribuer à rééquilibrer les régimes alimentaires humains et animaux.

International audience; Gastrointestinal lipases are crucial for lipid hydrolysis and must get adsorbed onto the substrate interface prior lipolysis. Such adsorption had not been deeply investigated on biomimetic vegetal membranes. Our objective was thus to characterize such adsorption and lipolysis using complementary biophysical tools (tensiometry, ellipsometry, atomic force microscopy) and in vitro digestion.Heterogenous monolayers based on galactolipids, phospholipids, and phytosterols were used. Four lipases were studied: i) gastric lipase (GL), ii) pancreatic lipase 2 (PLRP2), (iii) pancreatic triacylglycerol lipase and its cofactor, colipase (PTL/coPTL), and (iv) pancreatic secreted phospholipase A2 (sPLA2-IB).A strong surfactant property of GL and its preferential adsorption onto expanded lipid phase and at the phase boundary were observed, in line with previous results on milk fat globule membrane. With PLRP2, changes in surface pressure indicated a lipolytic activity. Such variation was not observed upon PTL/coPTL adsorption, and may be related to the absence of activity of PTL on polar lipids. The injection of sPLA2-IB did not indicate a clear trend of lipolysis on the lipid film but changed the morphology of condensed domains.This study is a step forward to understand the interactions of gastrointestinal lipases with plant lipid membranes, an overlooked aspect of lipid digestion

International audience

Oil bodies (OB) are the natural form of energy storage in oilseeds. They consist of a triacylglyceride core stabilized by proteins (mainly oleosins) embedded in a phospholipid membrane. In some tree nut species, OB contain an important amount of polyunsaturated fatty acids that make them interesting food ingredients but prone to oxidation. Due to the growing interest in minimally processed nut-based beverages, the understanding and the preservation of the physico-chemical properties of these lipoprotein assemblies are of great interest. In this context, previous inte rfacial studies have led to the proposition of a new model of adsorption for minimally processed OB that is useful to design functional emulsion or foam in which OB act as emulsifiers. The objective of the present study was to determine the impact of processing and fat content on the physico-chemical behavior of tree nut-based matrixes. The interfacial and oxidative behaviors of minimally processed isolated OB were compared with processed complex nut-based matrixes (i.e., beverages) of different fat content. Processing operations (heat-treatment, homogenization) affected OB interfacial properties and oxidative stability but fat content had even stronger impact on this last parameter, with more concentrated systems more stable to oxidation. The results underline the good physico-chemical stability of tree nut OB and raise the interest of their use in the minimally processed form to fully exploit their beneficial properties. (Texte integral)

Langmuir films and their analysis by tensiometry, ellipsometry and Langmuir–Blodgett transfer coupled to atomic force microscopy are suitable tools to study the interactions of various lipases with model membranes. The objective of our study was to characterize the adsorption and lipolysis behavior of different gastrointestinal enzymes onto biomimetic vegetal membranes. An heterogenous monolayer based on a mixture of unsaturated galactolipids (MGDG, DGDG), saturated phospholipids (DPPC), and phytosterols was used. Four enzymes were studied, (i) gastric lipase (GL), which has no lipolytic activity on polar lipids but whose adsorption may modulate the subsequent adsorption and activity of other lipases, and three pancreatic enzymes: (ii) pancreatic lipase 2 (PLRP2) presenting both galactolipase and phospholipase A1 activities, (iii) pancreatic triacylglycerol lipase and its cofactor, colipase (PTL/coPTL), and (iv) pancreatic secreted phospholipase A2 (sPLA2-IB). A strong surfactant property of GL and its preferential adsorption at both the expanded lipid phase and at the boundary phase were observed, in line with previous results obtained with biomimetic milk fat globule mem- brane. With PLRP2, changes in surface pressure indicated a lipolytic activity in addition to lipase adsorption, with a higher rate at surface pressure below 15 mN/m. A drastic decrease in film thickness was also observed by ellipsometry, suggesting the removal of some lipolysis products. Such variation was not observed upon PTL/coPTL adsorption and may be related to the absence of activity of PTL on polar lipids. Finally, the evolution of adsorption kinetic after injection of sPLA2-IB did not indicate a clear trend of lipolysis on the lipid film. However, a change in the morphology of condensed domains was observed by AFM, suggesting a phase separation of lipids induced by binding of sPLA2-IB. This study is a step forward in the understanding of interaction of gastrointestinal lipases with plant lipid membranes, an overlooked aspect of lipid digestion. (Texte intégral)

International audience

supervisors Véronique Vié (department of soft matter) and Claire Bourlieu, sous la direction de Véronique Vié dans le département matière molle et de Claire Bourlieu

International audience; Langmuir films and their analysis by tensiometry, ellipsometry and Langmuir-Blodgett transfer coupled to atomic force microscopy are suitable tools to study the interactions of various lipases with model membranes. The objective of our study was to characterize the adsorption and lipolysis behavior of different gastrointestinal enzymes onto biomimetic vegetal membranes. An heterogenous monolayer based on a mixture of unsaturated galactolipids (MGDG, DGDG), saturated phospholipids (DPPC), and phytosterols was used. Four enzymes were studied, i) gastric lipase (GL), which has no lipolytic activity on polar lipids but whose adsorption may modulate the subsequent adsorption and activity of other lipases, and three pancreatic enzymes: ii) pancreatic lipase 2 (PLRP2) presenting both galactolipase and phospholipase A1 activities, (iii) pancreatic triacylglycerol lipase and its cofactor, colipase (PTL/coPTL), and (iv) pancreatic secreted phospholipase A2 (sPLA2-IB). A strong surfactant property of GL and its preferential adsorption at both the expanded lipid phase and at the boundary phase were observed, in line with previous results obtained with biomimetic milk fat globule membrane. With PLRP2, changes in surface pressure indicated a lipolytic activity in addition to lipase adsorption, with a higher rate at surface pressure below 15 mN/m. A drastic decrease in film thickness was also observed by ellipsometry, suggesting the removal of some lipolysis products. Such variation was not observed upon PTL/coPTL adsorption, and may be related to the absence of activity of PTL on polar lipids. Finally, the evolution of adsorption kinetic after injection of sPLA2-IB did not indicate a clear trend of lipolysis on the lipid film. However, a change in the morphology of condensed domains was observed by AFM, suggesting a phase separation of lipids induced by binding of sPLA2-IB.This study is a step forward in the understanding of interaction of gastrointestinal lipases with plant lipid membranes, an overlooked aspect of lipid digestion.

International audience

National audience

International audience; Oil bodies (OB), the form of triacylglycerol storage in seeds, are interesting natural assemblies for nutritional applications. In walnuts, OB contain an important amount of polyunsaturated fatty acids that could be interesting food ingredients but may be prone to oxidation. The oxidative and interfacial behavior of walnut OB, either minimally-processed or after processing, were compared with processed complex walnut juice. The good oxidative stability of minimally-processed OB over 10 days (PV ≤ 8.4 meq O2/kg, TBARS = 1.4 mmol eq MDA/kg) and of processed walnut complex matrixes over 20 days (PV ≤ 4.8 meq O2/kg, TBARS = 1.4 mmol eq MDA/kg) was evidenced. In comparison, processing of OB promoted their oxidation. The interfacial studies led to the proposition of a new model of adsorption for minimally-processed OB that will be useful to design functional emulsion or foam in which OB act as emulsifiers.