KD,iis the distribution coefficient of the oligosaccharide, i. Vc,iis the mean retention elution volume of the oligosaccharide i. Vc,exlis the mean exclusive elution volume probed by dextran (molecular mass 50,000 Da). Vc,permis the mean permeable volume probed by acetone but
determined by UV detector (Aglient 1260, Aglient Technologies, USA) at 280 nm under the same LC condition.
2.4. Separation and purification of oligosaccharides
The oligosaccharide mixture was fractionated by two Superdex Peptide columns operating in series and eluted at a flow rate of 0.4 mL/min with the same elution buffer as for the LC–MS. A refractive index detector (RID) (Aglient 1260, Aglient Technologies, USA) was employed as detector. Four major oligosaccharides in the enzymatic hydrolysate were collected and lyophilised for future structural studies, and their composition was predicted according
to the distribution coefficient of oligosaccharides determined by LC–MS. In order to obtain sufficient samples (up to 15 mg of each oligosaccharide) for structural determination, the purification procedure was performed repeatedly. The purity of each fraction was examined using fluorophore-as-sisted carbohydrate electrophoresis (FACE) according to the method of Oonuki, Yoshida, Uchiyama, and Asari (2005). In brief, each oligosaccharide was labelled by 8 -aminonaphthalene-1,3,6-tri-sul-phonic acid disodium salt. Separation of the labelled oligosaccharides was performed on polyacrylamide gel containing 36% acrylamide in 50 mM Tris–glycine buffer (pH 8.3) at a constant current of 15 mA at 4 °C for 90 min. Detection was performed on a trans-illuminator at 365 nm.