Pullulan standard set

The Mw distributions were estimated following the method described by Muñoz-Almagro et al. (2018). Samples (0.1% w/v, 50 µL) were eluted using a TSK-Gel guard column (6.0 mm × 400 mm) and two TSK-Gel columns connected in series G5000 PWXL (7.8 mm × 300 mm, 10 μm) and G2500 PWXL (7.8 mm × 300 mm, 6 μm) (Tosoh Bioscience, Stuttgart, Germany). The mobile phase was 0.01-M NH₄Ac as the mobile phase and pectin samples were separated at 0.5 mL/min and 30 °C during 50 min. Dextran blue was used for the establishment of the exclusion volume of the columns (1000 kDa). For the calibrations, a Pullulan Standard Set (Sigma, St. Louis, MO, USA) was used. The average molecular weight (Mw) of the different pullulans were 0.342 kDa; 1.32 kDa, 6.20 kDa, and 10 kDa; 21.7 kDa; 48.8 kDa; 113 kDa; 200 kDa; and 348 kDa and 805 kDa.

The molecular weight of chitosan in acetic acid solutions on samples after 24 h for each temperature was determined by gel permeation chromatography (GPC) with an Agilent 1100 HPLC Pump (Agilent Technologies, Santa Clara, CA, USA) and an Agilent 1200 Refractive Index Detector (Agilent Technologies, Santa Clara, CA, USA). The column used was PL-aquagel-OH Mixed-H (8 μm, 300 × 7.5 mm²) (Agilent Technologies, Santa Clara, CA, USA) and the eluent used was a buffer solution at pH 4.6 (0.5 M CH3COOH/0.5 M CH3COONa) with a flow rate 1 mL/min. The equipment was calibrated using Pullulan Standard Set (Sigma Aldrich, Milan, Italy). Results show the elution volumes of each sample, which are inversely proportional to the molecular weight.

The molecular weight distributions of the polysaccharides were measured using a HPSEC system (JASCO PU-2089 Plus, Jasco, Tokyo, Japan) with a RI detector (JASCO RI-2031 Plus, Jasco, Tokyo, Japan). Asahipak GS-620, GS-520, and GS-320 columns (0.76 × 30 cm each; Showa Denko Co., Tokyo, Japan) were connected and used in the series. The column temperature maintained 40 °C, and the sample was eluted with 50 mm ammonium formate (pH 5.5 with formic acid) at a flow rate of 0.4 mL/min. A pullulan standard set (P-805, 336, 113, 48.8, 21.7, 10, 6, 1.32, 0.342 kDa; Sigma, St. Louis, MO, USA) was used to calibrate the standard curve, and the equation was as follows: log Mw = −0.0876 RT + 9.1538 (R² = 0.9952).