Breeze system

The isolation and purification of succinoglycan from S. meliloti Rm1021 was performed as previously described [18 (link)]. Bacteria were cultured in medium comprised of d-mannitol (10 g/L), glutamic acid (1.5 g/L), K₂HPO₄ (5 g/L), KH₂PO₄ (5 g/L), MgSO₄·7H₂O (0.2 g/L), and CaCl₂·2H₂O (0.04 g/L), which was adjusted to a pH of 7.00 at 30 °C for 7 days with shaking (180 rpm). After, cells were centrifuged at 8000× g for 15 min at 4 °C and the supernatant was collected. To obtain succinoglycan, three volumes of ethanol were added to the supernatant. Furthermore, the precipitated succinoglycan was dissolved in distilled water and dialyzed (MWCO 12–14 kDa, distilled water for 3 days). After collection, succinoglycan purified via dialysis was lyophilized for later use. The molecular weights of succinoglycan were estimated via gel permeation chromatography (GPC) analysis. GPC was performed using a Waters Breeze System equipped with a Waters 1525 Binary pump and a Waters 2414 refractive index detector and was performed at 30 °C with a flow rate of 0.8 mL min⁻¹ using 0.02 N sodium nitrate as a solvent. The molecular weight (Mw) of succinoglycan, as estimated via GPC, is 1.8 × 105 Da.

The molecular weight of copolymers was determined by GPC (Breeze System, Waters, Milford, MA, USA). Copolymers were permeated through columns (Waters Ultrahydrogel Linear, 500, 250, and 120) and detected by a refractive index detector (Waters 2414). Pullulans (6100~642,000 gmol⁻¹) were used as a standard polymer. The columns were eluted with NaNO₃ solution (0.02 N) flowing at rate of 0.08 mL/min.