Bstfa tmcs 99 1

Monosaccharide composition was determined according to Kamerling et al. [48 (link)] method, modified by Montreuil et al. [49 ]. EPS produced were firstly hydrolyzed for 4 h at 100 °C in MeOH/HCl 3N (Merck). The obtained methyl glycosides were then converted to trimethylsilyl derivatives using N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA:TMCS) 99:1 (Merck). Quantification of the per-O-trimethylsilyl methyl glycosides formed was performed using gas chromatography (GC-FID, Agilent Technologies 6890N, Santa Clara, CA, USA). Myo-inositol was used as internal standard.

Monosaccharide composition was determined, according to Kamerling et al. [42 (link)] method, modified by Montreuil et al. [43 ]. Samples were hydrolyzed for 4 h at 100 °C by 3M MeOH/HCl with myo-inositol used as the internal standard. After re-N-acetylation with acetic anhydride overnight at room temperature, the methyl glycosides were converted into their corresponding trimethylsilyl derivatives using N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA:TMCS) 99:1 (Merck, Darmstadt, Germany). Separation and quantification of the per-O-trimethylsilyl methyl glycosides were performed by gas chromatography (GC-FID, Agilent Technologies 6890N).

Plant material for the analysis of the root metabolome was collected in the same experiment as that earlier used for pea leaf metabolome analysis [5 (link)]. The samples of plant material (0.2–0.3 g) were ground as described by Puzanskiy et al. [55 (link)] and subjected to a single-stage extraction with 2 mL methanol: chloroform: water (5:2:1) mixture. Tissue debris was removed by centrifugation at 12,000× g for 10 min at −5 °C. The supernatant was collected and evaporated in a vacuum evaporator (Eppendorf, Germany). The dried material was dissolved in pyridine with the internal tricosane standard (nC23, tricosane). The samples were then supplied with the sylilating agent BSTFA:TMCS 99:1 (Sigma-Aldrich) and derivatizated at 90 °C for 20 min.