Christine Gardarin

New Method Showing the Influence of Matrix Components in Leuconostoc mesenteroides Biofilm Formation

Studying biofilm formation and influence of the matrix composition was heavy because only old and long methods were employed up to now: confocal microscopy, fluorescent chemical markers, and/or dying techniques. In this context, an innovative tool, the BioFilm Ring Test®, was here employed to explore the role of exopolysaccharides, proteins, and nucleic acids in the formation of biofilm by Leuconostoc mesenteroides. The principle is to add magnetic particles in the culture medium. When a biofilm is formed, particles are unable to migrate in the media to form a ring when a magnet is brought nearer to the well. Therefore, culture media supplemented with proteases, glycanases, and/or nucleases allowed us to identify the involvement of these substances in L. mesenteroides biofilm formation. The results permitted to demonstrate that dextran, proteins, and nucleic acids are implied in biofilm formation.

Enzymatic degradation and bioactivity evaluation of C-6 oxidized chitosan

C-6 oxidized chitosan was produced from chitosan by performing selective oxidation with NaOCl and NaBr using 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO) as catalyst. Endocellulase, Celluclast 1.5 L, Glucanex(®), Macerozyme R-10, hyaluronidase, hyaluronate lyase, red scorpionfish chitinase, glucuronan lyase and a protein mix from Trichoderma reesei were used to degrade the C-6 oxidized chitosan. Glucanex(®), the crude extract from T. reesei IHEM 4122 and Macerozyme R-10 validated the enzymatic degradation through final hydrolysis yields of the derivative respectively close to 36.4, 20.3 and 12.9% (w/w). The best initial reaction velocity (2.41 U/mL) was observed for Glucanex(®). The antileishmanial activity of the derivative was evaluated against Leishmania infantum LIPA 137. The antibacterial activities against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were also tested. Results showed an antileishmanial activity (IC50: 125 μg/mL) of the obtained derivatives against L. infantum LIPA 137.

Dextranase immobilization on epoxy CIM® disk for the production of isomaltooligosaccharides from dextran

Endodextranase D8144 from Penicillium sp. (EC 3.2.1.2.) was immobilized on an epoxy-activated monolithic Convective Interaction Media (CIM(®)) disk in order to produce isomaltooligosaccharides (IMOS) from Dextran T40 in a continuous IMmobilized Enzymes Reactor (IMER). Enzymatic parameters and structure of IMOS were studied for free and immobilized enzymes. The immobilization efficiency of endodextranase D8144 was about 15.9% (w/w) and the real specific activity was close to 6.5 U mg enz(-1). The Km values (4.8 ± 0.2 g L(-1)) for free and immobilized enzymes were the same, showing the absence of diffusional limitation. Moreover, specific patterns of DPs (Degrees of Polymerization) distributions were observed during the enzymatic hydrolysis by HPAEC-PAD (High Pressure Anion Exchange Chromatography-Pulsed Amperometric Detection). Thus, sought-after sizes of IMOS (DPs 8-10) were generated all over the hydrolysis. Finally, the results showed the high stability of this IMER since a relative enzymatic activity about 78% was measured after 5400 volumes column.

A new tool to detect high viscous exopolymers from microalgae.

Microalgae are microorganisms often surrounded by a slime layer made of secreted polymeric substances sometimes including polysaccharides. These polysaccharides, weakly described in the literature, can constitute value-added molecules in several industrial areas. The aim of this article is to show that a new tool, the BioFilm Ring Test®, can be used to detect viscous microalgal exopolymers. Two red microalgal strains (Rhodella violacea and Porphyridium purpureum), one cyanobacterium (Arthrospira platensis) and their excreted polymeric fractions were studied. R. violacea and P. purpureum induced a positive response with the BioFilm Ring Test® contrary to A. platensis. Finally, the understanding of the fractions viscosity involvement in the BRT response was performed by a rheological study.

A new method to screen polysaccharide cleavage enzymes

The activity of polysaccharide cleavage enzymes has usually been evaluated by qualitative plate screening methods and quantitative colorimetric or chromatographic assays. The recent development of protein engineering has shown the limits of these techniques when applied to high throughput screening. Here we propose a microplate method to measure the activity of polysaccharide cleavage enzymes through small variations in viscosity. Polysaccharide solutions are co-incubated with magnetic particles in enzyme buffers. The cleavage action of polymer-degrading enzymes increases the mobility of the particles in a magnetic field, even at low levels of enzyme activities. This reproducible, sensitive technique was used to evaluate enzymatic specificity towards substrates. BioFilm indices (BFI) determined by associated software were used to follow enzyme kinetics and measure the usual variables.

Improvement of exopolysaccharide production by Porphyridium marinum

With the aim to optimize the production of exopolysaccharide (EPS) by Porphyridium marinum, cultures in photobioreactors were conducted on a modified Provasoli medium (P) and compared to a new medium (Pm) with an elemental composition of N0.0205S0.0597P0.005. Cultivation on this medium allowed the increase of EPS concentration up to 2.5gL(-1), without modification of the EPS productivity (0.096gL(-1)) and EPS structure. In a second time, photosynthetic activity of the strain was monitored as a function of irradiance and temperature, allowing improvement of kinetic parameters of growth and EPS production. A semi-continuous culture, carried out with the Pm medium, an optimal irradiance and temperature of respectively 360μmolphotonsm(-2)s(-1) and 28°C led to an EPS process productivity of 0.031gh(-1) instead of 0.020gh(-1) in batch culture.

Structural characterization and rheological behavior of a heteroxylan extracted from Plantago notata Lagasca (Plantaginaceae) seeds

Plantago notata (Plantaginaceae) is a spontaneous plant from Septentrional Algerian Sahara currently used by traditional healers to treat stomach disorders, inflammations or wound healing. A water-soluble polysaccharide, called PSPN (PolySaccharide fraction from Plantago Notata), was extracted and purified from the seeds of this semi-arid plant. The structural features of this mucilage were evaluated by colorimetric assays, Fourier transformed infrared spectroscopy (FT-IR), gas chromatography coupled to mass spectrometry (GC/MS) and 1H/13C Nuclear Magnetic Resonance (NMR) spectroscopy. PSPN is a heteroxylan with a backbone composed of β-(1,3)-d-Xylp and β-(1,4)-d-Xylp highly branched, through (O)-2 and (O)-3 positions of β-(1,4)-d-Xylp by various side chains and terminal monosaccharides such as α-l-Araf-(1,3)-β-d-Xylp, β-d-Xylp-(1,2)-β-d-Xylp, terminal Xylp or terminal Araf. The physico-chemical and rheological analysis of this polysaccharide in dilute and semi diluted regimes showed that PSPN exhibites a molecular weight of 2.3×106g/mol and a pseudoplastic behavior.

Structural characterization and rheological properties of a galactomannan from Astragalus gombo Bunge seeds harvested in Algerian Sahara

A water soluble polysaccharide (WSP) was extracted and purified from Astragalus gombo seeds (Fabaceae) harvested in Septentrional Sahara (Ouargla, Algeria) with a yield of 6.8% (w/w of the dry seed ground). It was characterized by gas chromatography coupled to the mass spectrometry (GC-MS), size exclusion chromatography with Multi-Angle Light Scattering analysis (SEC-MALLS), high-resolution 1H and 13C NMR, and rheological measurements. The structural characterization indicated that this WSP fraction is a galactomannan with a mannose/galactose ratio of 1.7 formed by a backbone of β-(1,4)-d-mannopyranosyl residues (63%) substituted at O-6 position by a single α-galactopyranose residue (37%). SEC-MALLS analysis revealed that this galactomannan has an average molecular mass (Mw) of 1.1×106g/mol, an intrinsic viscosity of 860mL/g and, a random coil conformation structure. Rheological analysis in semi diluted regimes shown pseudo-plastic and viscoelastic behaviour.

The red microalga Flintiella sanguinaria as a new exopolysaccharide producer

Flintiella sanguinaria is a red unicellular microalgae belonging to Porphyridiophyceae with a high potential as an exopolysaccharide producer. A new culture medium was developed by a stoichiometry approach based on elemental analysis of microalgae, and culture conditions were improved after measurements of photosynthetic activity as a function of irradiance, temperature and pH. These optimal conditions were thus validated in a photobioreactor for the production of biomass and exopolysaccharide (EPS). After extraction and purification of the EPS, it was characterised by HPAEC-PAD, HPLC and FT-IR as a methylated and acetylated galactoxylan including a significant amount of rhamnose and glucuronic acid in its structure. Macromolecular conformation in dilute solution of native, deproteinised and desubstituted EPS from F. sanguinaria revealed that this polysaccharide had a strong associative behaviour in which hydrophobic interactions or hydrogen bonding but also proteins were implicated.

Rheological and functional properties of asafoetida gum

The asafoetida gum was extracted and purified from oleo-gum-resin of Ferula assa foetida root and characterized by high pressure anions exchange chromatography after acidic hydrolysis. It was composed of Gal:Ara:Rha:GlcA with the ratio 11.5:5.0:2.1:1.0. This monosaccharide composition was found similar to that of a commercial Arabic gum which exhibited a Gal:Ara:Rha:GlcA ratio of 11.7:5.4:3.2:1.0. As the Arabic gum is currently used for its emulsifying properties, the two gums were evaluated for their functional and rheological behaviors. Surface and interfacial tensions values were lower for asafoetida gum compared to Arabic gum. Critical micelle concentration was achieved at concentrations of 0.5% w/w and 1% w/w for asafoetida and Arabic gums, respectively. Values of emulsion capacity, emulsion stability and foaming properties were considerably higher for asafoetida gum in contrast to emulsion activity index that was lower than that of Arabic gum. As those of Arabic gum, solutions of asafoetida gum (2-30% w/w) exhibited Newtonian flow behavior at shear rates between 1 and 500 s-1. Apparent viscosities of Arabic and asafoetida gums were close and logically decreased by increasing temperature (10-80 °C). Higher viscosities were achieved at higher pH and CaCl2 concentrations.