Purification of Candida krusei mannan by using Benanomicin A

Preparation of mannan using Benanomicin A was carried out following Fig. 2A. First, 25 mL of 0.2% (w/v) Benanomicin A in 0.2 m CaCl₂ was added to the solution in which 500 mg of crude extract was dissolved, and under vigorous stirring. After 2 h, the resultant red precipitate was collected by centrifugation at 1450 g for 10 min, and the residue was rinsed with 25 mL of 0.2 m CaCl₂ under vigorous stirring. The precipitate (Benanomicin A–mannan complex) was transferred to a 100‐mL beaker. Next, 20 mL of the aqueous solution of 0.2 m EDTA•2Na and mixed with 20 mL of 0.01 m HCl was mixed with the precipitate. After 10 min, the precipitin (Benanomicin A and EDTA‐Ca•2Na chelate) was removed by centrifugation at 1450 g for 10 min. The supernatant was neutralized with 0.1 m NaOH, dialyzed against running tap water for 48 h, concentrated in vacuo to 5 mL, and then lyophilized. The above‐mentioned method is referred to as the Benanomicin method, and C. krusei mannan purified by this method is abbreviated as ‘Fr. K‐B’.

Procedure of Benanomicin method (A) and Fehling method (B).