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The blue crab (BC) Portunus segnis is considered an invasive species colonizing Tunisian coasts since 2014. This work aims to explore its associated bacteria potential to produce anionic exopolysaccharides (EPSs) in order to open up new ways of valorization. In this study, different BC samples were collected from the coastal area of Sfax, Tunisia. First, bacterial DNA was extracted from seven different fractions (flesh, gills, viscera, carapace scraping water, and three wastewaters from the production plant) and then sequenced using the metabarcoding approach targeting the V3-V4 region of the 16S rDNA to describe their microbiota composition. Metabarcoding data showed that the dominant bacterial genera were mainly Psychrobacter, Vagococcus, and Vibrio. In parallel, plate counting assays were performed on different culture media, and about 250 bacterial strains were isolated and identified by sequencing the 16S rDNA. EPS production by this new bacterial diversity was assessed to identify new compounds of biotechnological interest. The identification of the bacterial strains in the collection confirmed the dominance of Psychrobacter spp. strains. Among them, 43 were identified as EPS producers, as revealed by Stains-all dye in agarose gel electrophoresis. A Buttiauxella strain produced an EPS rich in both neutral sugars including rare sugars such as rhamnose and fucose and uronic acids. This original composition allows us to assume its potential for biotechnological applications and, more particularly, for developing innovative therapeutics. This study highlights bacterial strains associated with BC; they are a new untapped source for discovering innovative bioactive compounds for health and cosmetic applications, such as anionic EPS.

Bacteria are well-known to synthesize high molecular weight polysaccharides excreted in extracellular domain, which constitute their protective microenvironment. Several bacterial exopolysaccharides (EPS) are commercially available for skincare applications in cosmetic products due to their unique structural features, conferring valuable biological and/or textural properties. This review aims to give an overview of bacterial EPS, an important group of macromolecules used in cosmetics as actives and functional ingredients. For this purpose, the main chemical characteristics of EPS are firstly described, followed by the basics of the development of cosmetic ingredients. Then, a focus on EPS production, including upstream and downstream processes, is provided. The diversity of EPS used in the cosmetic industry, and more specifically of marine-derived EPS is highlighted. Marine bacteria isolated from extreme environments are known to produce EPS. However, their production processes are highly challenging due to high or low temperatures; yield must be improved to reach economically viable ingredients. The biological properties of marine-derived EPS are then reviewed, resulting in the highlight of the challenges in this field.

Blooms of toxic or harmful dinoflagellates in coastal marine environment are frequently observed on a global scale. The massive proliferation of certain species can in particular lead to discoloration of seawater and impact local economic activities such as tourism or shellfish production. This is the case of the non-toxic dinoflagellate Lepidodinium chlorophorum known to produce Green Colored Waters along the French Atlantic coast. Mortalities of marine organisms have been associated with these events without, however, being able to establish a direct cause and effect link. The production of transparent exo-polymeric particles (TEP), viscous and rich in carbon, could represent a detrimental property of this dinoflagellate. The work carried out within the framework of the Lepido-Pen project provides the first elements of understanding of the ecology of L. chlorophorum through a multidisciplinary approach ranging from the cell biology of the species to the impact on the ecosystem., Les efflorescences de dinoflagellés toxiques ou nuisibles en milieu marin côtier sont des phénomènes fréquemment observés à l’échelle mondiale. La prolifération massive de certaines espèces peut notamment engendrer une coloration de l’eau de mer et impacter les activités économiques locales telles que le tourisme ou la production conchylicole. C’est le cas des efflorescences du dinoflagellé non-toxique Lepidodinium chlorophorum connu pour produire des Eaux Colorées Vertes le long de la côte Atlantique Française et plus particulièrement en Loire Atlantique. Des mortalités d’organismes marins ont été associées à ces évènements sans toutefois pouvoir faire directement le lien de cause à effet. La production de Particules Exo-polymériques Transparentes (TEP), visqueuses et riches en carbone, pourrait représenter une propriété nuisible de ce dinoflagellé. Les travaux réalisés dans le cadre du projet Lepido-Pen fournissent de premiers éléments de compréhension sur l’écologie de L. chlorophorum à travers une approche multidisciplinaire allant de la biologie cellulaire de l’espèce à l’impact sur l’écosystème et les espèces exploitées.

Sulfated glycosaminoglycans (GAGs) are fundamental constituents of both the cell surface and extracellular matrix. By playing a key role in cell–cell and cell–matrix interactions, GAGs are involved in many physiological and pathological processes. To design GAG mimetics with similar therapeutic potential as the natural ones, the specific structural features, among them sulfate content, sulfation pattern, and chain length, should be considered. In the present study, we describe a sulfation method based on microwave radiation to obtain highly sulfated derivatives as GAG mimetics. The starting low-molecular-weight (LMW) derivative was prepared from the infernan exopolysaccharide, a highly branched naturally slightly sulfated heteropolysaccharide synthesized by the deep-sea hydrothermal vent bacterium Alteromonas infernus. LMW highly sulfated infernan derivatives obtained by conventional heating sulfation have already been shown to display GAG-mimetic properties. Here, the potential of microwave-assisted sulfation versus that of the conventional method to obtain GAG mimetics was explored. Structural analysis by NMR revealed that highly sulfated derivatives from the two methods shared similar structural features, emphasizing that microwave-assisted sulfation with a 12-fold shorter reaction time is as efficient as the classical one.

Recent advances in glycobiotechnology show that bacterial exopolysaccharides (EPS) presenting glycosaminoglycan (GAG)-like properties can provide a valuable source of bio-active macromolecules for industrial applications. The HE800 EPS, named diabolican, is a marine-derived anionic high-molecular-weight polysaccharide produced by Vibrio diabolicus CNCM I-1629 which displays original structural features close to those of hyaluronic acid. We investigated the impact of carbon and nitrogen substrates on both Vibrio diabolicus growth and diabolican production. Both substrates were screened by a one-factor-at-a-time method, and experimental designs were used to study the effect of glucose, mannitol, and ammonium acetate various concentrations. Results showed that the medium composition affected not only the bacterium growth and EPS yield, but also the EPS molecular weight (MW). EPS yields of 563 and 330 mg L−1 were obtained in the presence of 69.3 g L−1 glucose and 24.6 g L−1 mannitol, respectively, both for 116.6 mM ammonium acetate. MW was the highest, with 69.3 g L−1 glucose and 101.9 mM ammonium acetate (2.3 × 106 g mol−1). In parallel, the bacterial maximum specific growth rate was higher when both carbon and nitrogen substrate concentrations were low. This work paves the way for the optimization of marine exopolysaccharide production of great interest in the fields of human health and cosmetics

As size exclusion chromatography (SEC), Flow Field Flow Fractionation is a separation technique that assesses the size distribution of the molecules via theoretical equations or representative standards. In FFF technique, high-resolution separation is achieved within a very thin flow against which a perpendicular force field is applied. It takes place in an asymmetrical flat channel (A4F) or in a hollow fiber (HF5). Separation depends on the Brownian diffusion coefficient of the molecule / particle, and thus on its size. Multi Angle Light Scattering detector (MALS) is used for the determination of the absolute molar mass and size determination of macromolecules / nanoparticles. In FFF separation, no stationary phase is required that avoids interferences with the samples. Consequently, for very high molar weight polymers, shearing forces do not apply. The entire separation is smooth, fast, and non-destructive without a stationary phase that may interact, degrade, or alter the sample. Field-Flow Fractionation-based techniques are powerful tools for characterizing polysaccharides and for monitoring the potential polydispersity and/or polymodality. They are powerful tools especially in the case of drug delivery. In a Pharmacopeia approach, these techniques must be coupled to complementary techniques for a full characterization of products. Viscosity measurements have been conducted together with A4F-UV-MALS and to study the size and size distribution of exopolysaccharides.

Antimetastatic properties on both murine and human osteosarcoma cell lines (POS-1 and KHOS) have been evidenced using exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium. These derivatives had no significant effect on the cell cycle neither a pro-apoptotic effect on osteosarcoma cells. Based on this observation, these EPSs could be employed as new drug delivery systems for therapeutic uses. A theranostic approach, i.e., combination of a predictive biomarker with a therapeutic agent, has been developed notably by combining with true pair of theranostic radionuclides, such as scandium 47Sc/44Sc. However, it is crucial to ensure that, once complexation is done, the biological properties of the vector remain intact, allowing the molecular tropism of the ligand to recognize its molecular target. It is important to assess if the biological properties of EPS evidenced on osteosarcoma cell lines remain when scandium is complexed to the polymers and can be extended to other cancer cell types. Scandium-EPS complexes were thus tested in vitro on human cell lines: MNNG/HOS osteosarcoma, A375 melanoma, A549 lung adenocarcinoma, U251 glioma, MDA231 breast cancer, and Caco2 colon cancer cells. An xCELLigence Real Cell Time Analysis (RTCA) technology assay was used to monitor for 160 h, the proliferation kinetics of the different cell lines. The tested complexes exhibited an anti-proliferative effect, this effect was more effective compared to EPS alone. This increase of the antiproliferative properties was explained by a change in conformation of EPS complexes due to their polyelectrolyte nature that was induced by complexation. Alterations of both growth factor-receptor signaling, and transmembrane protein interactions could be the principal cause of the antiproliferative effect. These results are very promising and reveal that EPS can be coupled to scandium for improving its biological effects and also suggesting that no major structural modification

Background: Exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium, showed anti-metastatic properties. They may represent a new class of ligands to be combined with theranostic radionuclides, such as 47Sc/44Sc. The goal of this work was to investigate the feasibility of such coupling. (2) Methods: EPSs, as well as heparin used as a drug reference, were characterized in terms of molar mass and dispersity using Asymmetrical Flow Field-Flow Fractionation coupled to Multi-Angle Light Scattering (AF4-MALS). The intrinsic viscosity of EPSs at different ionic strengths were measured in order to establish the conformation. To determine the stability constants of Sc with EPS and heparin, a Free-ion selective radiotracer extraction (FISRE) method has been used. (3) Results: AF4-MALS showed that radical depolymerization produces monodisperse EPSs, suitable for therapeutic use. EPS conformation exhibited a lower hydrodynamic volume for the highest ionic strengths. The resulting random-coiled conformation could affect the complexation with metal for high concentration. The LogK of Sc-EPS complexes have been determined and showing that they are comparable to the Sc-Hep. (4) Conclusions: EPSs are very promising to be coupled with the theranostic pair of scandium for Nuclear Medicine.

In the marine environment, biofilm formation is an important lifestyle for microorganisms. A biofilm is comprised of cells embedded in an extracellular matrix that holds them close together and keeps the biofilm attached to the colonized surface. This predominant lifestyle and its main regulation pathway, namely quorum-sensing (QS), have been shown to induce specific bioactive metabolites. In this study, we investigated the biofilm formation by two marine bacteria belonging to the Vibrio species to discover potentially innovative bioactive compounds. We proposed a protocol to isolate biofilm extracts, to analyze their biochemical composition, and to compare them to planktonic cell extracts. Cells were grown attached to a plastic surface; extracts were prepared in water, NaOH, or in ethyl acetate and analyzed. Extracellular matrix components featured carbohydrates, proteins, lipids, and low amount of DNA. Carbohydrates appeared to be the main constituent of biofilm but also of the planktonic cell supernatant. Moreover, antimicrobial and QS-signaling activities were evidenced in extracts.

Bacteria have developed a unique strategy to survive in extreme environmental conditions through the synthesis of an extracellular polymeric matrix conferring upon the cells a protective microenvironment. The main structural component of this complex network constitutes high-molecular weight hydrophilic macromolecules, namely exopolysaccharides (EPS). EPS composition with the presence of particular chemical features may closely be related to the specific conditions in which bacteria evolve. Deep-sea hydrothermal vent bacteria have already been shown to produce EPS rich in hexosamines and uronic acids, frequently bearing some sulfate groups. Such a particular composition ensures interesting functional properties, including biological activities mimicking those known for glycosaminoglycans (GAG). The aim of the present study was to go further into the exploration of the deep-sea hydrothermal vent IFREMER (French Research Institute for Exploitation of the Sea) collection of bacteria to discover new strains able to excrete EPS endowed with GAG-like structural features. After the screening of our whole collection containing 692 strains, 38 bacteria have been selected for EPS production at the laboratory scale. EPS-producing strains were identified according to 16S rDNA phylogeny. Chemical characterization of the obtained EPS highlighted their high chemical diversity with the presence of atypical compositional patterns. These EPS constitute potential bioactives for a number of biomedical applications, including regenerative medicines and cancer treatment.

Polysaccharides of marine origin are gaining interest as biomaterial components. Bacteria derived from deep-sea hydrothermal vents can produce sulfated exopolysaccharides (EPS), which can influence cell behavior. The use of such polysaccharides as components of organic, collagen fibril-based coatings on biomaterial surfaces remains unexplored. In this study, collagen fibril coatings enriched with HE800 and GY785 EPS derivatives were deposited on titanium alloy (Ti6Al4V) scaffolds produced by rapid prototyping and subjected to physicochemical and cell biological characterization. Coatings were formed by a self-assembly process whereby polysaccharides were added to acidic collagen molecule solution, followed by neutralization to induced self-assembly of collagen fibrils. Fibril formation resulted in collagen hydrogel formation. Hydrogels formed directly on Ti6Al4V surfaces, and fibrils adsorbed onto the surface. Scanning electron microscopy (SEM) analysis of collagen fibril coatings revealed association of polysaccharides with fibrils. Cell biological characterization revealed good cell adhesion and growth on bare Ti6Al4V surfaces, as well as coatings of collagen fibrils only and collagen fibrils enhanced with HE800 and GY785 EPS derivatives. Hence, the use of both EPS derivatives as coating components is feasible. Further work should focus on cell differentiation

Bacteria from deep-sea hydrothermal vents constitute an attractive source of bioactive molecules. In particular, exopolysaccharides (EPS) produced by these bacteria become a renewable source of both biocompatible and biodegradable molecules. The low molecular weight (LMW) derivatives of the GY785 EPS produced by the deep-sea hydrothermal vent strain Alteromonas infernus have previously displayed some biological properties, similar to those of glycosaminoglycans (GAG), explored in cancer and tissue engineering. These GAG-mimetic derivatives are obtained through a free radical depolymerization process, which could, however, affect their structural integrity. In a previous study, we have shown that A. infernus produces depolymerizing enzymes active on its own EPS. In the present study, an enzymatic reaction was optimized to generate LMW derivatives of the GY785 EPS, which could advantageously replace the present bioactive derivatives obtained by a chemical process. Analysis by mass spectrometry of the oligosaccharide fractions released after enzymatic treatment revealed that mainly a lyase activity was responsible for the polysaccharide depolymerization. The repeating unit of the GY785 EPS produced by enzyme cleavage was then fully characterized.