Pullulan standards

Size-exclusion chromatography (SEC) was performed in a LC-20AD liquid chromatography instrument with refractive index detection (RID; Shimadzu, Japan). Between 3 and 8 mg of sample were initially washed at room temperature in water (3 × 30 min) and methanol (3 ×3 0 min) to swell and open up the structure. After filtering, 1 ml N,N-dimethylacetamide (DMAc) with 8 % (w/w) lithium chloride (LiCl) was added to the silk and left under agitation at room temperature until the silk was completely dissolved. The silk solution was further diluted to achieve injection conditions (DMAc/0.5 % (w/w) LiCl). Separation was performed using a PL gel 20 mm Mixed-A column (Agilent, USA) at 40 °C using DMAc/0.5 % (w/w) LiCl as a mobile phase with a flow of 0.5 ml min⁻¹. Relative calibration was performed using Pullulan standards (Agilent, USA).

The molecular properties (average molecular weight (M_w), the number for average molecular weight (M_n), and the polydispersity index (PDI)) of F. virsoides and C. barbata PS extracted with CE, MAE, and PLE at optimal conditions were detected and measured using high-performance size exclusion chromatography with a refraction index detector (HPSEC-RID). The 1260 Infinity II LC system (Agilent Technologies) consisted of a quaternary gradient pump G7111B, an autosampler G4767A, a multicolumn thermostat G7116A, and a refraction index detector G7162A. HPSEC analysis was performed using PL aquagel-OH guard column (8 µm, 50 × 7.5 mm; Agilent Technologies) and PL aquagel-OH MIXED-M column (8 μm, 300 × 7.5 mm; Agilent Technologies). The mobile phase was H₂O, and the temperatures of the column and RID, the flow rate, and the injection volume were set to 30 °C, 40 °C, 0.5 mL/min, and 50 μL, respectively. Data were collected and processed using OpenLAB CDS ChemStation Edition (Agilent Technologies). A molecular weight calibration curve was constructed with known pullulan standards (Agilent Technologies) with M_w in a range from 180 to 700 kDa.