Fractionation, characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus

► Exopolysaccharides (EPS) were fractionated by gradient ethanol precipitation. ► Large PS with negligible protein content was precipitated with small ethanol volume. ► Smaller PS–protein complex fractions were attained at larger ethanol volume. ► Protein constituents of EPS were significant for antioxidant activities. Exopolysaccharide (EPS) fractions P1/5, P2/5, P1, P2 and P5 were isolated from the fermentation medium of a medicinal fungus Cordyceps sinensis by gradient precipitation with ethanol at 1/5, 2/5, 1, 2, and 5 volume ratios to the liquid medium. P1/5 and P2/5 were mainly composed of polysaccharides with negligible protein content and a large molecular or particle size (intrinsic viscosity [η] 2025mL/g; hydrodynamic radius Rh 905nm). The fraction attained at a higher ethanol volume ratio had a much higher protein and lower carbohydrate content, and a smaller molecular size. In particular, P5 was composed of mainly protein with an average molecular weight 16kDa, [η] 4.3mL/g and Rh 23.5nm. The antioxidant activities of EPS fractions showed a significant dependence on the protein content, being negligible of P1/5 and P2/5, low to moderate of P1 and P2, and very strong of P5. Gradient ethanol precipitation was proven a simple and workable method for initial fractionation of polysaccharides, proteins and their complexes with different molecular sizes and for further identification of the bioactive components.