*4.3. Exopolysaccharides Characterization*

4.3.1. Total Sugars, Neutral Sugars, Uronic Acids, Sulfate and Proteins Content

Total sugar content of samples was evaluated by the resorcinol method using glucose as standard [59]. Uronic acids and neutral sugar contents of EPS extracts were assayed with meta-hydroxyldiphenyl (m-HDP) and resorcinol as described by, respectively, [60] and [59] using glucose and glucuronic acid as standards. In these assays, glycosidic linkages are broken during high temperature sulfuric acid treatment and dehydration of sugar units leads to furfural compounds formation. These latter interact by condensation with phenolic compounds (resorcinol or m-HDP depending on the assay), giving colored compounds that can be measured respectively at 450 or 520 nm. Briefly, for the m-HDP assay, 200 μL of sample are mixed with 1 mL of a 0.12 M borax (Na2B4O7,10H2O) prepared in 96% ( *w*/*v*) sulfuric acid. After a 1 h incubation at 90 ◦C, 200 μL of m-HDP solution are added and absorbance measured at 520 nm. The m-HDP solution is prepared just before assay by diluting 102 μL of m-HDP stock solution (100 mg of m-HDP solubilized in 1 mL DMSO, kept at 4 ◦C) with 5 mL of 80% ( *w*/*v*) sulfuric acid. The results were expressed in mg/g of D-glucuronic acid equivalent (GlcAEq). For resorcinol assay, 200 μL of sample are mixed with 200 μL of a 6 g/L resorcinol solution and 1 mL of 80% ( *w*/*v*) sulfuric acid. After 30 min at 90 ◦C, followed by 30 min at room temperature, 1.4 mL of water are added and absorbance at 450 nm is measured. The results directly expressed in mg/g of D-glucose equivalent (GlcEq) correspond to the total sugars amount. As the uronic acids interfere with the assay, a glucuronic acid standard curve was performed following the same procedure, and the corrective formula described by [61] was applied, in order to access the neutral sugars amount.

Sulfur content was determined by the turbidimetric method [62] using K2SO4 as a standard. Results were expressed in mg/g of SO4 equivalent. Briefly, 120 mg of samples were hydrolyzed with 3 mL of 2 M chlorhydric acid for 2 h at 100 ◦C. After centrifugation (10,000× *g*, 30 min), 1 mL of supernatant is added to 9 mL of milliQ water, 1 mL of 0.5 M HCl, and 0.5 mL of BaCl2/gelatin reagen<sup>t</sup> (150 mg of gelatin dissolved in 50 mL of water at 70 ◦C, kept 16 h at 4 ◦C, and finally mixed with 0.5 g of BaCl2). After 30 min incubation at room temperature, sulfate groups released during the hydrolysis of polysaccharides form a precipitate of barium sulfate, which can be detected at 550 nm. Protein content was evaluated by the Lowry method [63] using bovine serum albumin (BSA) as a standard.

#### 4.3.2. Monosaccharide Composition of EPS

The identification and quantification of the constitutive monosaccharides of *P. marinum* EPS was conducted by high performance anion exchange chromatography (HPAEC) on an ICS 3000 (Dionex, Sunnyvale, CA, USA). Exopolysaccharides were hydrolyzed with 2 M TFA at 120 ◦C for 1 h 30 min, neutralized with 35% ammonia and then centrifuged at 14,000× *g* for 15 min. The recovered supernatant was filtered at 0.22 μm before injection. The elution was carried out with a pre-column and a CarboPac PA1 column (4 × 50 mm and 4 × 250 mm, respectively) whose stationary phase is an anion exchange resin. Samples were eluted isocratically with 18 mM NaOH for 25 min, followed by a linear gradient between 0 and 0.5 M sodium acetate in 200 mM NaOH for 20 min to elute acidic monosaccharides. Run was followed by 15 min washing with 200 mM NaOH. The eluent flow rate was kept constant at 1 mL/min. A range of external standards (Fucose, Arabinose, Galactose, Glucose, Rhamnose, Xylose, Mannose, Fructose, Ribose, Galactosamine, Glucosamine, N-AcetylGlucosamine, N-AcetylGalactosamine, Galacturonic acid and Glucuronic acid) with concentrations ranging from 0.001 to 0.01g/L, and then the addition of internal standards in the samples enabled qualitative and quantitative analysis of the composition of polysaccharides.

#### 4.3.3. Molar Masses Determination

Molar masses and polydispersity index of EPS were analyzed by a size exclusion chromatography coupled to a multiangle laser light scattering detector, a viscometric detector and a differential refractive index detector (SEC/MALLS/Visco/DRI). The polysaccharide solutions were prepared at 0.5 mg/mL (dilute regime) in the chromatographic eluent (i.e., LiNO3 at 0.1 M, filtered on 0.1 μm) under soft magnetic agitation at 60 ◦C during 4 h and then filtrated on a 0.45 μm filter unit (regenerated cellulose). An automatic sample injector (SIL 20A, Shimadzu, Kyoto, Japan) equipped with a 500 μL injection loop was used for injection. The solvent was driven by a pump (LC 10Ai, Shimadzu, Kyoto, Japan) at a flow rate of 0.5 mL/min. The sample was first drawn into a pre-column (Shodex OHPAK SB-G) before being directed to two standard steric exclusion columns (Shodex OHPAK SB 804 and 806 HQ). At the outlet of the column, the compounds were quantified by a differential refractometer (DRI 10A, Shimadzu, Kyoto, Japan), a multi-angle laser light scattering detector (Dawn® heleos, Wyatt technology Corp., Santa Barbara, CA, USA) equipped with a K5 cell of 50 μL, a red source (Ga-As 690 nm) of 5 mW, 18 measurement diodes and a viscosity detector (Viscostar 2, Wyatt Technology Corp., Goleta, CA, USA). The data obtained was processed with the Astra 6.1.1 software which provided the weight and number average molar masses (Mn and M w, respectively) and also the gyration (Rg) radius and both intrinsic viscosities ([η]) and hydrodynamic (Rh) based on the viscosity detector.

#### 4.3.4. Infrared Spectrometry Footprint (FTIR)

Infrared measurements were made using a VERTEX 70 (Bruker, Billerica, MA, USA) spectrometer. The samples were analyzed on an ATR A225 diamond module. IR spectra (50 scans) were obtained at room temperature (referenced against air) over the 500–4000 c m<sup>−</sup><sup>1</sup> range. The spectra were analyzed with the OPUS 7.2 software.

#### 4.3.5. Shear Flow Behavior of EPS Samples

The shear flow behavior of samples (viscosity measurements as function of shear rate) was determined using an AR-G2 rheometer (TA Instruments Ltd., Hertfordshire, UK). Measurements were conducted using a steel cone-plate (40 mm radius, 2◦, gap 50 μm) geometry. The temperature was set at 30 ◦C, with a circulating bath or a Peltier system depending on geometry. The viscosity measurements were made over a shear rate range of 1 to 10<sup>3</sup> s<sup>−</sup>1.
