The main chemical and physical features of the Acacia senegal exudate gum and its molecular fract... more The main chemical and physical features of the Acacia senegal exudate gum and its molecular fractions isolated by chromatographies were determined using a wide variety of methods. Three main molecular fractions were isolated after hydrophobic interaction chromatography (HIC) and biochemical analyses confirmed the presence of an arabinogalactan-peptide (FI), an arabinogalactan-protein (FII), and a glycoprotein (FIII) fraction as described commonly in the literature. Further purification of FIII using size exclusion chromatography revealed three distinct populations. A wide molecular weight distribution within each population with the presence of at least two distinct molecular species per population was identified by high performance size exclusion chromatography coupled to on line multi-angle laser light scattering (HPSEC-MALLS). In addition, both sugars content (neutral and uronic acids) and UV profiles revealed that FIII was composed of a continuum of molecular species differing both by their protein-to-sugar ratio and molecular weight. FI and FII had average molecular weight M(w) of 2.86 x 10(5) and 1.86 x 10(6) g.mol(-1), respectively, and a low polydispersity index (M(w)()/M(n) approximately 1.3). The three populations identified in FIII after HIC separation had M(w) of 2.67 x 10(6), 7.76 x 10(5), and 2.95 x 10(5) g.mol(-1) and very low polydispersity indexes (1.13, 1.04, and 1.01). Estimation of the polypeptide backbone length in the three fractions gave 43, 2253, and 4443 amino acid residues, respectively, hydroxyproline (Hyp) and serine being the most prominent residues within FI and FII, Hyp and Asx (asparagine + aspartic acid) within FIII. Secondary structure prediction from circular dichroism data resulted in polyproline II, beta-sheet, and random coil structures for FII and FIII, whereas no secondary structure was identified in FI. The existence of exposed tryptophanyl residues to the solvent was noticed by fluorescence in FII and FIII, tryptophan residues being absent from FI. In addition, 8-5' non cyclic diferulic acid was identified to be covalently linked to carbohydrate moieties of FII. Infrared spectroscopy identified the different vibrations of saccharidic and peptidic bonds with absorbance amplitudes in agreement with sugar and protein elementary analyses. Titration measurements in order to evaluate the number of charges on total Acacia gum and its molecular fractions revealed that 100% of charges came from polysaccharidic moieties (i.e., glucuronic acids) in FI. Charges coming from polysaccharidic moieties were of 91.3% and 37.9% for FII and FIII, respectively, the remaining 8.7% and 62.1% charges in FII and FIII molecular fractions coming from the polypeptidic backbone.
A promising technique for oil encapsulation in Ca-alginate capsules by inverse gelation was propo... more A promising technique for oil encapsulation in Ca-alginate capsules by inverse gelation was proposed by Abang et al. This method consists of emulsifying calcium chloride solution in oil and then adding it dropwise in an alginate solution to produce Ca-alginate capsules. Spherical capsules with diameters around 3 mm were produced by this technique, however the production of smaller capsules was not demonstrated. The objective of this study is to propose a new method of oil encapsulation in a Ca-alginate membrane by inverse gelation. The optimisation of the method leads to microcapsules with diameters around 500 mm. In a search of microcapsules with improved diffusion characteristics, the size reduction is an essential factor to broaden the applications in food, cosmetics and pharmaceuticals areas. This work contributes to a better understanding of the inverse gelation technique and allows the production of microcapsules with a well-defined shell-core structure.
We used a droplet-based microfluidics technique to produce monodisperse responsive alginate-block... more We used a droplet-based microfluidics technique to produce monodisperse responsive alginate-block-polyetheramine copolymer microgels. The polyetheramine group (PEA), corresponding to a propylene oxide /ethylene oxide ratio (PO/EO) of 29/6 (Jeffamine ® M2005), was condensed, via the amine link, to alginates with various mannuronic/guluronic acids ratios and using two alginate:jeffamine mass ratios. The size of the grafted-alginate microgels varied from 60 to 80 m depending on the type of alginate used and the degree of substitution. The droplet-based microfluidics technique offered exquisite control of both the dimension and physical chemical properties of the grafted-alginate microgels. These micro-gels were therefore comparable to isolated grafted-alginate chains in retaining both their amphiphilic and thermo-sensitive properties. Amphiphilicity was demonstrated at the oil–water interface where grafted-alginate microgels were found to decrease interfacial tension by ∼50%. The thermo-sensitivity of microgels was clearly demonstrated and a 10 to 20% reduction in size between was evidenced on increasing the temperature above the lower critical solution temperature (T LCST) of Jeffamine. In addition, the reversibility of thermo-sensitivity was demonstrated by studying the oil–water affinity of microgels with temperature after Congo red labeling. Finally, droplet-based microfluidics was found to be a good and promising tool for generating responsive biobased hydrogels for drug delivery applications and potential new colloidal stabilizers for dispersed systems such as Pickering emulsions.
Capillary flow-based approach such as microfluidic devices offer a number of advantages over conv... more Capillary flow-based approach such as microfluidic devices offer a number of advantages over conventional flow control technology because they ensure highly versatile geometry and can be used to produce monodisperse spherical and non-spherical polymeric microparticles. Based on the principle of a flow-focusing device to emulsify the coflow of aqueous solutions in an organic phase, we were able to produce the following innovative polysaccharide hydrogel microparticles:-Janus hydrogel microparticles made of pectin–pectin (homo Janus) and pectin–alginate (hetero Janus) were produced. The efficiency of separation of the two hemispheres was investigated by confocal scanning laser microscopy (CSLM) of previously labelled biopolymers. The Janus structure was confirmed by subjecting each microparticle hemisphere to specific enzymatic degradation. As a proof of concept, free BSA or BSA grafted with dextran, were encapsulated in each hemisphere of the hetero Janus hydrogel microparticles. While BSA, free or grafted with dextran, was always confined in the alginate hemisphere, a fraction of BSA diffused from the pectin to the alginate hemisphere. Methoxy groups along the pectin chain will be responsible of the decrease of the number of attractive electrostatic interactions occurring between amino groups of BSA and carboxylic groups of pectin.-Pectin hydrogel microparticles of complex shapes were successfully produced by combining on-chip the phenomenon of gelation and water diffusion induced self-assembly, using dimethyl carbonate as continuous phase, or by deformation of the pre-gelled droplets off-chip at a fluid–fluid interface. Sphere, oblate ellipsoid, torus or mushroom-type morphologies were thus obtained. Moreover, it was established that after crossing the interface during their collect, mushroom-type microparticles did not migrate in the calcium or DMC phase but stayed at the liquid–liquid interface. These new and original hydrogel microparticles will open up opportunities for studying relationships between combined enzymatic hydrolysis and active release for Janus particles and relationships between shape and swelling behaviour for anisotropic pectin microparticles.
We describe a pendant drop method that allows one to produce double emulsions in a controllable w... more We describe a pendant drop method that allows one to produce double emulsions in a controllable way. Using a co-flowing drop-maker, we generate a periodic train made of monodisperse droplets that is directed toward the end of a capillary tube where a pendant drop forms. When this drop detaches from the tip of the capillary under the influence of gravity, it may or may not encapsulate one or several droplets depending on experimental conditions. We discuss the advantages of this method when compared with other techniques described in the literature and we present a simple model that predicts well the mean volume of the outer drops and the mean number of encapsulated droplets per drop as a function of the various physical parameters at play in experiments. We illustrate the high potentiality of this simple method by producing well-calibrated emulsion gels of large sizes and we discuss possible applications.
Arabinogalactan (AG) and arabinogalactan-protein (AGP) fractions were treated enzymatically using... more Arabinogalactan (AG) and arabinogalactan-protein (AGP) fractions were treated enzymatically using several proteases in acidic (pH 4) and alkaline (pH 7) conditions in order to go deeper insight into the structure and conformations of the two main fractions of Acacia senegal gum. Endoproteinase Glu-C, pepsin and phosphatase acid were thus used in acidic conditions while subtilisin A, pronase, trypsin, papain and proteinase K were used in alkaline conditions to cleave protein moieties of the two fractions. Structures of AG and AGP were probed using HPSEC-MALLS, small angle neutron scattering and far-UV circular dichroism. Enzymes did not affect AG fraction structure whatever the pH conditions used, highlighting the inac-cessibility of the peptide backbone and the remarkable stability of this fraction in acidic and alkaline conditions. This result was in agreement with the thin oblate ellipsoid model we previously identified for the AG fraction where the 43 amino-acid residues peptide sequence was supposed, based on spectroscopic methods, to be totally buried. Contrary to AG fraction, AGP protein component was therefore cleaved using enzymes in alkaline conditions, the absence of enzymatic efficiency in acidic conditions being probably ascribed to long range electrostatic repulsions occurring between negatively charged AGP and enzymes at pH 4. The decrease of AGP molecular weight after hydrolysis in alkaline conditions went from 1.79 × 10 6 g mol −1 for control AGP to as low as 1.68 × 10 5 g mol −1 for papain-treated AGP. The overall structure of the enzyme-treated AGPs was found to be surprisingly quite similar whatever the enzyme used and close, with however some subtle differences, to AG unit. A tri-axial ellipsoid conformation was found in enzyme-treated AGPs and the two main preferential distances identified in the pair distance distribution function would claim in favor of rod-like or elongated particles or alternatively would indicate the presence of two particles differing in dimensions. The secondary structures content of control and enzyme-treated AGPs were similar, highlighting both the high rigidity of the protein backbone and the overall symmetry of AGP. This conclusion was reinforced by the more compact structures found when AGP was intact compare to the more elongated structures found when AGP was enzymatically cleaved. Finally, the structural similarities found in enzyme-treated AGP together with the theoretical calculations to analytically probe the type of branching would suggest that AGP would be made of a self-similar assembly of two types of building blocks, the second being a five-fold repetition of the first one, for which palindromic amino acid sequence would ensure a self-ordering of carbohydrate moieties along the polypeptide chains. The cleavage would therefore lead to hydrolysed building blocks with similar secondary structures and conformations whatever the enzyme used.
We demonstrated the generation of pectin hydrogel microparticles having complex shapes either by ... more We demonstrated the generation of pectin hydrogel microparticles having complex shapes either by combining the phenomenon of gelation and water diffusion-induced self-assembly in microfluidic channels (on-chip) or by the deformation of the pregelled droplets outside the channels (off-chip) at a fluid−fluid interface. We proved that by tuning the mode of pectin crosslinking (CaCl 2 vs CaCO 3 ) and the degree of shrinking (water content in the dimethyl carbonate (DMC) organic continuous phase) we can control the shape of the final particle. Sphere, doughnut, oblate ellipsoid, or mushroom-type morphologies were thus produced, demonstrating the ability to control the formation of anisotropic biopolymer-based hydrogel microparticles using microfluidics. Shape changes were explained by the redistribution of calcium ions in combination with the local Peclet number experienced by the microdroplets during the on-chip process. Moreover, during the off-chip process, the interplay between elastic and viscous forces for microdroplets entering the CaCl 2 −DMC interface caused deformation of the pregelled droplets to occur and therefore resulted in the formation of microparticles with a mushroom-like morphology.
Phloem sieve elements are highly differentiated cells involved in the long-distance transport of ... more Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translo-cated by mass flow. We used liquid chromatography– tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.
We describe a microfluidic approach for generating Janus microbeads from biopolymer hydrogels. A ... more We describe a microfluidic approach for generating Janus microbeads from biopolymer hydrogels. A flow-focusing device was used to emulsify the coflow of aqueous solutions of one or two different biopolymers in an organic phase to synthesize homo or hetero Janus microbeads. Biopolymer gelation was initiated, in the chip, by diffusion-controlled ionic cross-linking of the biopolymers. Pectin−pectin (homo Janus) and, for the first time, pectin−alginate (hetero Janus) microbeads were produced. The efficiency of separation of the two hemispheres, which reflected mixing and convection phenomena, was investigated by confocal scanning laser microscopy (CSLM) of previously labeled biopoly-mers. The interface of the hetero Janus structure was clearly defined, whereas that of the homo Janus microbeads was poorly defined. The Janus structure was confirmed by subjecting each microbead hemisphere to specific enzymatic degradation. These new and original microbeads from renewable resources will open up opportunities for studying relationships between combined enzymatic hydrolysis and active compound release.
International journal of pharmaceutics, Jan 21, 2002
According to our results concerning the behavior of lysozyme at interfaces, its secondary structu... more According to our results concerning the behavior of lysozyme at interfaces, its secondary structure and its enzymatic activity, successful protein encapsulation would need to maintain a pH value far from the enzyme isoelectric point value during the formulation to reduce, in particular, the adsorption of lysozyme molecules at the created interfaces. Moreover, buffers or salt solution must be used in order to keep intact the native secondary conformation of lysozyme, and preserve its enzymatic activity.
ABSTRACT Microparticulated whey proteins (MWP) in suspension or included in a globular protein ne... more ABSTRACT Microparticulated whey proteins (MWP) in suspension or included in a globular protein network revealed that the degree of flocculation was important in the final structure of the material. The MWP dispersions developed an antithixotropic character when low shear rate was applied to the system even at low temperature. Similar results were obtained on globular protein solutions and could be interpreted in terms of an orthokinetic aggregation. MWP participated to the formation and reinforcement of heat-induced β-lactoglobulin (BLG) gels. The mechanical properties of mixed gels demonstrated synergestic effects at low MWP massic fraction (BLG:MWP ratio higher than 2:1). A possible interpretation would be that both interaction of MWP with the network and flocculation of the MWP inside the pores could occur. However, increasing the fraction of MWP induced heterogeneities in the protein network. In particular, micro-phase separation due to steric hindrance effects or thermodynamic incompatibility between the two components could occur and decrease viscoelastic properties of mixed gels.
The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arab... more The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using high-performance size exclusion chromatography-multi angle laser light scattering (HPSEC-MALLS), small angle X-ray scattering (SAXS), synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) observations. In solution, GP would be a mixture of spheroidal monomers and more anisotropic oligomers as suggested by the two exponent values found in the R g vs. M w relationship and TEM observations. The GP conformation probed by SAXS was ascribed to a thin object with a triaxial ellipsoid morphology, certainly attributed to GP oligomers. A 9 nm diameter particle was also identified by SAXS in agreement with the dimensions identified by TEM on single isolated ring-like structures. The GP oligomerization process, as probed by TEM, would be the result of ring-like subunits self-association. This self-association would lead to more linear or, sometimes, cyclised assembly. At the molecular level, GP fraction was found to have secondary structures mainly made of -sheets and turns (64%) but also, to a lesser extent, made of polyproline II (PPII) and ␣-helices (19%). These features were characteristic of hydroxyprolin-rich glycoproteins with arabinosylated and arabinogalactan polysaccharide side chains grafted to the polypeptide backbone. The GP molecular fraction structure from Acacia gum would be an assembly of ring-like glycoproteins modules. These ring-like structures were certainly due to hydroxyproline (Hyp)-arabinogalactan (AG) subunits.
The structure of the arabinogalactan-protein (AGP) fraction of the gum exudate of Acacia senegal ... more The structure of the arabinogalactan-protein (AGP) fraction of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using HPSEC-MALLS, small angle neutron scattering and TEM observations. Literature reported that the AGP structure of gum Arabic adopts a very compact conformation in solution due to the attachment of short arabinoside side chains and much larger blocks of carbohydrate to the polypeptidic backbone. The present study revealed that AGP in solution had a weight average molecular weight M w of 1.86 × 10 6 g mol −1 and a radius of gyration R g of 30 nm. In addition, two exponent values were identified in the R g , [Á], R h and vs. M w relationships highlighting two types of conformations depending on the molecular weight range considered: a low molar mass population with long-chain branching and a compact conformation and a high molar mass population with short-chain branching and an elongated conformation. AGP would behave in solution as a branched or hyper-branched polymer with conformations ranging from globular to elongated shape depending on the size of the carbohydrate branches. Small angle scattering form factor revealed an elongated average conformation corresponding to a triaxial ellipsoid while inverse Fourier transform of the scattering form factor gave a maximum dimension for AGP of 64 nm. Transmission electron microscopy highlighted the existence of two types of flat objects with thicknesses below 3-5 nm, single particles with a more or less anisotropic spheroidal shape and aggregated structures with a more elongated shape. A remarkable feature of all particle morphologies was the presence of an outer structure combined to an inner more or less porous network of interspersed chains or interacting structural blocks, as previously found for the arabinogalactan (AG) main molecular fraction of Acacia gum. However, clear differences were observed in the density and morphology of the inner porous network, probably highlighting differences in the degree of branching. The existence of assembled AG as part of the AGP family was confirmed using TEM micrographs at high resolution. Fused AGP dimers, trimers, tetramers and multimers were also identified. These molecular assemblies questioned about the nature of interactions involved.
The main chemical and physical features of the Acacia senegal exudate gum and its molecular fract... more The main chemical and physical features of the Acacia senegal exudate gum and its molecular fractions isolated by chromatographies were determined using a wide variety of methods. Three main molecular fractions were isolated after hydrophobic interaction chromatography (HIC) and biochemical analyses confirmed the presence of an arabinogalactan-peptide (FI), an arabinogalactan-protein (FII), and a glycoprotein (FIII) fraction as described commonly in the literature. Further purification of FIII using size exclusion chromatography revealed three distinct populations. A wide molecular weight distribution within each population with the presence of at least two distinct molecular species per population was identified by high performance size exclusion chromatography coupled to on line multi-angle laser light scattering (HPSEC-MALLS). In addition, both sugars content (neutral and uronic acids) and UV profiles revealed that FIII was composed of a continuum of molecular species differing both by their protein-to-sugar ratio and molecular weight. FI and FII had average molecular weight M(w) of 2.86 x 10(5) and 1.86 x 10(6) g.mol(-1), respectively, and a low polydispersity index (M(w)()/M(n) approximately 1.3). The three populations identified in FIII after HIC separation had M(w) of 2.67 x 10(6), 7.76 x 10(5), and 2.95 x 10(5) g.mol(-1) and very low polydispersity indexes (1.13, 1.04, and 1.01). Estimation of the polypeptide backbone length in the three fractions gave 43, 2253, and 4443 amino acid residues, respectively, hydroxyproline (Hyp) and serine being the most prominent residues within FI and FII, Hyp and Asx (asparagine + aspartic acid) within FIII. Secondary structure prediction from circular dichroism data resulted in polyproline II, beta-sheet, and random coil structures for FII and FIII, whereas no secondary structure was identified in FI. The existence of exposed tryptophanyl residues to the solvent was noticed by fluorescence in FII and FIII, tryptophan residues being absent from FI. In addition, 8-5' non cyclic diferulic acid was identified to be covalently linked to carbohydrate moieties of FII. Infrared spectroscopy identified the different vibrations of saccharidic and peptidic bonds with absorbance amplitudes in agreement with sugar and protein elementary analyses. Titration measurements in order to evaluate the number of charges on total Acacia gum and its molecular fractions revealed that 100% of charges came from polysaccharidic moieties (i.e., glucuronic acids) in FI. Charges coming from polysaccharidic moieties were of 91.3% and 37.9% for FII and FIII, respectively, the remaining 8.7% and 62.1% charges in FII and FIII molecular fractions coming from the polypeptidic backbone.
A promising technique for oil encapsulation in Ca-alginate capsules by inverse gelation was propo... more A promising technique for oil encapsulation in Ca-alginate capsules by inverse gelation was proposed by Abang et al. This method consists of emulsifying calcium chloride solution in oil and then adding it dropwise in an alginate solution to produce Ca-alginate capsules. Spherical capsules with diameters around 3 mm were produced by this technique, however the production of smaller capsules was not demonstrated. The objective of this study is to propose a new method of oil encapsulation in a Ca-alginate membrane by inverse gelation. The optimisation of the method leads to microcapsules with diameters around 500 mm. In a search of microcapsules with improved diffusion characteristics, the size reduction is an essential factor to broaden the applications in food, cosmetics and pharmaceuticals areas. This work contributes to a better understanding of the inverse gelation technique and allows the production of microcapsules with a well-defined shell-core structure.
We used a droplet-based microfluidics technique to produce monodisperse responsive alginate-block... more We used a droplet-based microfluidics technique to produce monodisperse responsive alginate-block-polyetheramine copolymer microgels. The polyetheramine group (PEA), corresponding to a propylene oxide /ethylene oxide ratio (PO/EO) of 29/6 (Jeffamine ® M2005), was condensed, via the amine link, to alginates with various mannuronic/guluronic acids ratios and using two alginate:jeffamine mass ratios. The size of the grafted-alginate microgels varied from 60 to 80 m depending on the type of alginate used and the degree of substitution. The droplet-based microfluidics technique offered exquisite control of both the dimension and physical chemical properties of the grafted-alginate microgels. These micro-gels were therefore comparable to isolated grafted-alginate chains in retaining both their amphiphilic and thermo-sensitive properties. Amphiphilicity was demonstrated at the oil–water interface where grafted-alginate microgels were found to decrease interfacial tension by ∼50%. The thermo-sensitivity of microgels was clearly demonstrated and a 10 to 20% reduction in size between was evidenced on increasing the temperature above the lower critical solution temperature (T LCST) of Jeffamine. In addition, the reversibility of thermo-sensitivity was demonstrated by studying the oil–water affinity of microgels with temperature after Congo red labeling. Finally, droplet-based microfluidics was found to be a good and promising tool for generating responsive biobased hydrogels for drug delivery applications and potential new colloidal stabilizers for dispersed systems such as Pickering emulsions.
Capillary flow-based approach such as microfluidic devices offer a number of advantages over conv... more Capillary flow-based approach such as microfluidic devices offer a number of advantages over conventional flow control technology because they ensure highly versatile geometry and can be used to produce monodisperse spherical and non-spherical polymeric microparticles. Based on the principle of a flow-focusing device to emulsify the coflow of aqueous solutions in an organic phase, we were able to produce the following innovative polysaccharide hydrogel microparticles:-Janus hydrogel microparticles made of pectin–pectin (homo Janus) and pectin–alginate (hetero Janus) were produced. The efficiency of separation of the two hemispheres was investigated by confocal scanning laser microscopy (CSLM) of previously labelled biopolymers. The Janus structure was confirmed by subjecting each microparticle hemisphere to specific enzymatic degradation. As a proof of concept, free BSA or BSA grafted with dextran, were encapsulated in each hemisphere of the hetero Janus hydrogel microparticles. While BSA, free or grafted with dextran, was always confined in the alginate hemisphere, a fraction of BSA diffused from the pectin to the alginate hemisphere. Methoxy groups along the pectin chain will be responsible of the decrease of the number of attractive electrostatic interactions occurring between amino groups of BSA and carboxylic groups of pectin.-Pectin hydrogel microparticles of complex shapes were successfully produced by combining on-chip the phenomenon of gelation and water diffusion induced self-assembly, using dimethyl carbonate as continuous phase, or by deformation of the pre-gelled droplets off-chip at a fluid–fluid interface. Sphere, oblate ellipsoid, torus or mushroom-type morphologies were thus obtained. Moreover, it was established that after crossing the interface during their collect, mushroom-type microparticles did not migrate in the calcium or DMC phase but stayed at the liquid–liquid interface. These new and original hydrogel microparticles will open up opportunities for studying relationships between combined enzymatic hydrolysis and active release for Janus particles and relationships between shape and swelling behaviour for anisotropic pectin microparticles.
We describe a pendant drop method that allows one to produce double emulsions in a controllable w... more We describe a pendant drop method that allows one to produce double emulsions in a controllable way. Using a co-flowing drop-maker, we generate a periodic train made of monodisperse droplets that is directed toward the end of a capillary tube where a pendant drop forms. When this drop detaches from the tip of the capillary under the influence of gravity, it may or may not encapsulate one or several droplets depending on experimental conditions. We discuss the advantages of this method when compared with other techniques described in the literature and we present a simple model that predicts well the mean volume of the outer drops and the mean number of encapsulated droplets per drop as a function of the various physical parameters at play in experiments. We illustrate the high potentiality of this simple method by producing well-calibrated emulsion gels of large sizes and we discuss possible applications.
Arabinogalactan (AG) and arabinogalactan-protein (AGP) fractions were treated enzymatically using... more Arabinogalactan (AG) and arabinogalactan-protein (AGP) fractions were treated enzymatically using several proteases in acidic (pH 4) and alkaline (pH 7) conditions in order to go deeper insight into the structure and conformations of the two main fractions of Acacia senegal gum. Endoproteinase Glu-C, pepsin and phosphatase acid were thus used in acidic conditions while subtilisin A, pronase, trypsin, papain and proteinase K were used in alkaline conditions to cleave protein moieties of the two fractions. Structures of AG and AGP were probed using HPSEC-MALLS, small angle neutron scattering and far-UV circular dichroism. Enzymes did not affect AG fraction structure whatever the pH conditions used, highlighting the inac-cessibility of the peptide backbone and the remarkable stability of this fraction in acidic and alkaline conditions. This result was in agreement with the thin oblate ellipsoid model we previously identified for the AG fraction where the 43 amino-acid residues peptide sequence was supposed, based on spectroscopic methods, to be totally buried. Contrary to AG fraction, AGP protein component was therefore cleaved using enzymes in alkaline conditions, the absence of enzymatic efficiency in acidic conditions being probably ascribed to long range electrostatic repulsions occurring between negatively charged AGP and enzymes at pH 4. The decrease of AGP molecular weight after hydrolysis in alkaline conditions went from 1.79 × 10 6 g mol −1 for control AGP to as low as 1.68 × 10 5 g mol −1 for papain-treated AGP. The overall structure of the enzyme-treated AGPs was found to be surprisingly quite similar whatever the enzyme used and close, with however some subtle differences, to AG unit. A tri-axial ellipsoid conformation was found in enzyme-treated AGPs and the two main preferential distances identified in the pair distance distribution function would claim in favor of rod-like or elongated particles or alternatively would indicate the presence of two particles differing in dimensions. The secondary structures content of control and enzyme-treated AGPs were similar, highlighting both the high rigidity of the protein backbone and the overall symmetry of AGP. This conclusion was reinforced by the more compact structures found when AGP was intact compare to the more elongated structures found when AGP was enzymatically cleaved. Finally, the structural similarities found in enzyme-treated AGP together with the theoretical calculations to analytically probe the type of branching would suggest that AGP would be made of a self-similar assembly of two types of building blocks, the second being a five-fold repetition of the first one, for which palindromic amino acid sequence would ensure a self-ordering of carbohydrate moieties along the polypeptide chains. The cleavage would therefore lead to hydrolysed building blocks with similar secondary structures and conformations whatever the enzyme used.
We demonstrated the generation of pectin hydrogel microparticles having complex shapes either by ... more We demonstrated the generation of pectin hydrogel microparticles having complex shapes either by combining the phenomenon of gelation and water diffusion-induced self-assembly in microfluidic channels (on-chip) or by the deformation of the pregelled droplets outside the channels (off-chip) at a fluid−fluid interface. We proved that by tuning the mode of pectin crosslinking (CaCl 2 vs CaCO 3 ) and the degree of shrinking (water content in the dimethyl carbonate (DMC) organic continuous phase) we can control the shape of the final particle. Sphere, doughnut, oblate ellipsoid, or mushroom-type morphologies were thus produced, demonstrating the ability to control the formation of anisotropic biopolymer-based hydrogel microparticles using microfluidics. Shape changes were explained by the redistribution of calcium ions in combination with the local Peclet number experienced by the microdroplets during the on-chip process. Moreover, during the off-chip process, the interplay between elastic and viscous forces for microdroplets entering the CaCl 2 −DMC interface caused deformation of the pregelled droplets to occur and therefore resulted in the formation of microparticles with a mushroom-like morphology.
Phloem sieve elements are highly differentiated cells involved in the long-distance transport of ... more Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translo-cated by mass flow. We used liquid chromatography– tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.
We describe a microfluidic approach for generating Janus microbeads from biopolymer hydrogels. A ... more We describe a microfluidic approach for generating Janus microbeads from biopolymer hydrogels. A flow-focusing device was used to emulsify the coflow of aqueous solutions of one or two different biopolymers in an organic phase to synthesize homo or hetero Janus microbeads. Biopolymer gelation was initiated, in the chip, by diffusion-controlled ionic cross-linking of the biopolymers. Pectin−pectin (homo Janus) and, for the first time, pectin−alginate (hetero Janus) microbeads were produced. The efficiency of separation of the two hemispheres, which reflected mixing and convection phenomena, was investigated by confocal scanning laser microscopy (CSLM) of previously labeled biopoly-mers. The interface of the hetero Janus structure was clearly defined, whereas that of the homo Janus microbeads was poorly defined. The Janus structure was confirmed by subjecting each microbead hemisphere to specific enzymatic degradation. These new and original microbeads from renewable resources will open up opportunities for studying relationships between combined enzymatic hydrolysis and active compound release.
International journal of pharmaceutics, Jan 21, 2002
According to our results concerning the behavior of lysozyme at interfaces, its secondary structu... more According to our results concerning the behavior of lysozyme at interfaces, its secondary structure and its enzymatic activity, successful protein encapsulation would need to maintain a pH value far from the enzyme isoelectric point value during the formulation to reduce, in particular, the adsorption of lysozyme molecules at the created interfaces. Moreover, buffers or salt solution must be used in order to keep intact the native secondary conformation of lysozyme, and preserve its enzymatic activity.
ABSTRACT Microparticulated whey proteins (MWP) in suspension or included in a globular protein ne... more ABSTRACT Microparticulated whey proteins (MWP) in suspension or included in a globular protein network revealed that the degree of flocculation was important in the final structure of the material. The MWP dispersions developed an antithixotropic character when low shear rate was applied to the system even at low temperature. Similar results were obtained on globular protein solutions and could be interpreted in terms of an orthokinetic aggregation. MWP participated to the formation and reinforcement of heat-induced β-lactoglobulin (BLG) gels. The mechanical properties of mixed gels demonstrated synergestic effects at low MWP massic fraction (BLG:MWP ratio higher than 2:1). A possible interpretation would be that both interaction of MWP with the network and flocculation of the MWP inside the pores could occur. However, increasing the fraction of MWP induced heterogeneities in the protein network. In particular, micro-phase separation due to steric hindrance effects or thermodynamic incompatibility between the two components could occur and decrease viscoelastic properties of mixed gels.
The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arab... more The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using high-performance size exclusion chromatography-multi angle laser light scattering (HPSEC-MALLS), small angle X-ray scattering (SAXS), synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) observations. In solution, GP would be a mixture of spheroidal monomers and more anisotropic oligomers as suggested by the two exponent values found in the R g vs. M w relationship and TEM observations. The GP conformation probed by SAXS was ascribed to a thin object with a triaxial ellipsoid morphology, certainly attributed to GP oligomers. A 9 nm diameter particle was also identified by SAXS in agreement with the dimensions identified by TEM on single isolated ring-like structures. The GP oligomerization process, as probed by TEM, would be the result of ring-like subunits self-association. This self-association would lead to more linear or, sometimes, cyclised assembly. At the molecular level, GP fraction was found to have secondary structures mainly made of -sheets and turns (64%) but also, to a lesser extent, made of polyproline II (PPII) and ␣-helices (19%). These features were characteristic of hydroxyprolin-rich glycoproteins with arabinosylated and arabinogalactan polysaccharide side chains grafted to the polypeptide backbone. The GP molecular fraction structure from Acacia gum would be an assembly of ring-like glycoproteins modules. These ring-like structures were certainly due to hydroxyproline (Hyp)-arabinogalactan (AG) subunits.
The structure of the arabinogalactan-protein (AGP) fraction of the gum exudate of Acacia senegal ... more The structure of the arabinogalactan-protein (AGP) fraction of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using HPSEC-MALLS, small angle neutron scattering and TEM observations. Literature reported that the AGP structure of gum Arabic adopts a very compact conformation in solution due to the attachment of short arabinoside side chains and much larger blocks of carbohydrate to the polypeptidic backbone. The present study revealed that AGP in solution had a weight average molecular weight M w of 1.86 × 10 6 g mol −1 and a radius of gyration R g of 30 nm. In addition, two exponent values were identified in the R g , [Á], R h and vs. M w relationships highlighting two types of conformations depending on the molecular weight range considered: a low molar mass population with long-chain branching and a compact conformation and a high molar mass population with short-chain branching and an elongated conformation. AGP would behave in solution as a branched or hyper-branched polymer with conformations ranging from globular to elongated shape depending on the size of the carbohydrate branches. Small angle scattering form factor revealed an elongated average conformation corresponding to a triaxial ellipsoid while inverse Fourier transform of the scattering form factor gave a maximum dimension for AGP of 64 nm. Transmission electron microscopy highlighted the existence of two types of flat objects with thicknesses below 3-5 nm, single particles with a more or less anisotropic spheroidal shape and aggregated structures with a more elongated shape. A remarkable feature of all particle morphologies was the presence of an outer structure combined to an inner more or less porous network of interspersed chains or interacting structural blocks, as previously found for the arabinogalactan (AG) main molecular fraction of Acacia gum. However, clear differences were observed in the density and morphology of the inner porous network, probably highlighting differences in the degree of branching. The existence of assembled AG as part of the AGP family was confirmed using TEM micrographs at high resolution. Fused AGP dimers, trimers, tetramers and multimers were also identified. These molecular assemblies questioned about the nature of interactions involved.
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Papers by D. Renard