Molecular weight distributions of lyophilized crude EPS were determined by size exclusion chromatography. In brief, crude EPS powder was suspended in 0.1 M NaNO3 (0.5 mg/mL) and then filtered through a 0.45 μm pore diameter polyvinylidene fluoride membrane (Millipore Corporation, USA). The average molecular weight (MW) was determined by high-performance molecular exclusion chromatography (HPLC-SEC, Agilent 1,100 Series System, Hewlett-Packard, Germany) associated with a refractive index (IR) detector (Ibarburu et al., 2015 (link)). 50 μL of the samples were injected and eluted at a flow rate of 0.95 mL/min (pressure: 120:130 psi) at room temperature using 0.1 M NaNO3 as mobile phase. Dextrans (0.5 mg/mL) with a molecular weight between 103 and 2.106 Da (Sigma-Aldrich, USA) were used as standards.
Once the molecular weight distributions were determined, low and high molecular weight fractions that composed the crude EPS obtained at 20°C were separated. For this purpose, EPS solutions (0.2% w/v) were centrifuged through a Vivaspin™ ultrafiltration spin column 100 KDa MWCO, (Sartorious, Goettingen, Germany) for 20 min at 6000 g, eluting only the low MW fraction. Subsequently, high MW fraction retained in the column was eluted using hot distilled water. The eluted fractions were passed through a Vivaspin column (cut-off 30KDa) in order to separate the middle and low MW fraction of EPS.
Monosaccharide composition of crude EPS and their fractions were determined by gas chromatography as previously described (Notararigo et al., 2013 (link)). Briefly, 1–2 mg of EPS were hydrolyzed in 1 mL of 3 M trifluoroacetic acid (1 h at 120°C). The monosaccharides obtained were converted into alditol acetates by reduction with NaBH4 and subsequent acetylation. The samples were analyzed by gas chromatography in an Agilent 7890A coupled to a 5975C mass detector, using an HP5-MS column with helium as carrier gas at a flow rate of 1 mL/min. For each run, 1 μL of sample was injected (with a Split 1:50) and the following temperature program was performed: the oven was heat to 175°C for 1 min; the temperature was increased to 215°C at a rate of 2.5°C/min and then increased to 225°C at 10°C/min, keeping it constant at this temperature for 1.5 min. Monosaccharides were identified by comparison of retention times with standards (arabinose, xylose, rhamnose, galactose, glucose, mannose, glucosamine and galactosamine) analyzed under the same conditions. Calibration curves were also processed for monosaccharide quantification. Myo-inositol was added to each sample as internal standard.
Once the molecular weight distributions were determined, low and high molecular weight fractions that composed the crude EPS obtained at 20°C were separated. For this purpose, EPS solutions (0.2% w/v) were centrifuged through a Vivaspin™ ultrafiltration spin column 100 KDa MWCO, (Sartorious, Goettingen, Germany) for 20 min at 6000 g, eluting only the low MW fraction. Subsequently, high MW fraction retained in the column was eluted using hot distilled water. The eluted fractions were passed through a Vivaspin column (cut-off 30KDa) in order to separate the middle and low MW fraction of EPS.
Monosaccharide composition of crude EPS and their fractions were determined by gas chromatography as previously described (Notararigo et al., 2013 (link)). Briefly, 1–2 mg of EPS were hydrolyzed in 1 mL of 3 M trifluoroacetic acid (1 h at 120°C). The monosaccharides obtained were converted into alditol acetates by reduction with NaBH4 and subsequent acetylation. The samples were analyzed by gas chromatography in an Agilent 7890A coupled to a 5975C mass detector, using an HP5-MS column with helium as carrier gas at a flow rate of 1 mL/min. For each run, 1 μL of sample was injected (with a Split 1:50) and the following temperature program was performed: the oven was heat to 175°C for 1 min; the temperature was increased to 215°C at a rate of 2.5°C/min and then increased to 225°C at 10°C/min, keeping it constant at this temperature for 1.5 min. Monosaccharides were identified by comparison of retention times with standards (arabinose, xylose, rhamnose, galactose, glucose, mannose, glucosamine and galactosamine) analyzed under the same conditions. Calibration curves were also processed for monosaccharide quantification. Myo-inositol was added to each sample as internal standard.
Free full text: Click here
