*3.12. Gas chromatography–Mass Spectrometry (GC-MS)*

### 3.12.1. Hydrolysis and Derivatization of EPS

Polysaccharides samples (2 mg) and monosaccharides standards were treated with the same procedure. First, 100 μL of the standard stock solution of 1 mg mL−<sup>1</sup> of each monosaccharide was dried under nitrogen gas flow. Second, the samples of polysaccharides, and a mixture containing the standard monosaccharides included in the IS (Internal Standard), were methanolized in 2-mL methanol/3-M HCl at 80 ◦C during 24 h. The monosaccharides glucose, galactose, rhamnose, fructose, mannose, xylose, apiose and myo-inositol (Internal Standard, IS), as well as pyridine, hexane and methanol/3-M HCl solution, were purchased from Sigma-Aldrich. Then, the saccharides were washed with methanol and dried under nitrogen gas flow. Third, the trimethylsilyl reaction was accomplished with 200 μL of Tri-Sil HTP (Thermo Fisher Scientific, Franklin, MA, USA). Each vial with the sample was heated at 80 ◦C for 1 h. The derivatized sample was cooled to a room temperature and dried under a steam of nitrogen. Forth, the dry residue was extracted with hexane (2 mL) and centrifuged. Finally, the hexane solution containing silylated monosaccharides was concentrated and reconstituted in hexane (200 μL), filtered and transferred to a GC-MS autosampler vial. Sample preparation and analyses were performed in triplicate.

### 3.12.2. Gas Chromatography/Mass Spectrometry (GC-MS) Analysis

GC/MS analyses were carried out using a gas chromatograph Trace GC (Thermo Fisher Scientific, Franklin, MA, USA), an autosampler Triplus RSH (Thermo Fisher Scientific, Franklin, MA, USA) and a DSQ mass spectrometer quadrupole (Thermo Fisher Scientific, Franklin, MA, USA). The GC column was set ZB-5 Zebron, Phenomenex (5% phenyl and 95% dimethylpolysiloxane), 30-m (length) × 0.25-mm (I.D) × 0.25-μm film thickness. Injection volume was 1 μL in split mode, with a split ratio of 40. Helium was used as the carrier gas with a flow rate of 1.2 mL min−1. The injector was set at 250 ◦C in split mode. The initial oven temperature was 80 ◦C for 2 min, then ramped from 10 ◦C min−<sup>1</sup> to 180 ◦C, followed by ramping from 5 ◦C min−<sup>1</sup> to 250 ◦C, remaining constant for 2 min. The electron impact ionization (EI) mode of the mass spectrometer was set at 70 eV. Monitored in full scan mode

with mass range 50-650 *m*/*z* with interface temperature 250 ◦C and ionization source temperature of 230 ◦C. The identification of monosaccharides in polysaccharide samples was carried out by comparing retention time and mass spectra of monosaccharide standards, previously analyzed under identical conditions (glucose, galactose, mannose, arabinose, xylose, rhamnose, ribose, fucose, galacturonic acid and glucuronic acid). The compounds were identified by comparing the mass spectra with those in the National Institute of Standards and Technology (NIST 2014) library.
