Agilent zorbax nh2 column

HPLC-purified ERG was identified by LC–MS (Agilent 1260/6460LC/Triple Quadrupole MS, Agilent Technologies) with Agilent ZORBAX NH₂ column (4.6 × 250 mm, 5 μm). Analysis was performed with a mobile phase of acetonitrile/4 mmol/L ammonium acetate (70:30, v/v).

For FOS synthesis, a reaction mixture consisting of 20% sucrose (w/v) and fungal cells (6 units of enzyme per g sucrose) in 50 mM citrate phosphate buffer (pH 5.5) was stirred at 50 °C. The mixture was taken at appropriate times and treated in boiling water for 5 min to terminate the reaction. Then the sample was centrifuged at 1, 600 g for 10 min. The supernatant was collected and subjected to TLC to examine the homogeneity of the resulting products. To further analyze the effects of substrate concentration on the transfructosylation activity, different concentrations of sucrose (w/v) (0.2, 2, 5, 20 and 50%) were used for FOS synthesis. Quantitative analysis of the reaction products was carried out using high-performance liquid chromatography (HPLC, LC-6A, Shimadzu, Japan) with a Agilent Zorbax NH₂ column (5 μm, 4.6 mm × 250 mm) (Agilent Technologies, Santa Clara, CA) coupled with a refractive index detector (2414, Waters, USA). The mobile phase was acetonitrile: water (70:30, v/v) at a flow rate of 1.0 mL/min. The identification and quantification of each FOS was carried out by a calibration curve built with authentic standards of kestose, nystose and fructosyl nystose (Sigma Corp., St Louis, MO).

The supernatant of fermentation broth was obtained by centrifugation at 15,777 × g for 10 min and diluted for 5 folds. The concentration of 1 was analyzed by HPLC (Agilent series 1260, Agilent Technologies, USA) with Agilent ZORBAX NH₂ column (4.6 × 250 mm², particle size 5 μm) using an elution buffer of acetonitrile/phosphate buffer (65:35, v/v) at a flow rate of 1 mL min⁻¹ and detected at 210 nm. The titer of 1 was calculated according to the corresponding standard curves and processed by the GraphPad Prism 7 and Origin 8.1 softwares.