Lichenase

The ratio of cellulosic oligomers in the chain of β-glucans was determined by lichenase and β-glucosidase treatment (1 ). β-Glucan powders were dissolved in buffer solution (25 mg/mL, 20 mM sodium phosphate buffer, pH 6.5) and incubated with lichenase (10 U/mL, Megazyme International Ireland Ltd.) for 1 h at 50°C. Then 5.0 mL of 200 mM sodium acetate buffer (pH 4.0) was added to terminate the enzyme reaction and tubes were centrifuged at 13,000 rpm for 15 min. The supernatants (1.0 mL) were incubated with exo-(1→3)-glucanse (1 U/mL, Sigma-Aldrich Co.) for 1 h at 50°C. The digests were heated to 95°C (15 min) to inactivate the enzyme and the supernatants were reacted with GOPOD determining reagent and measured at 510 nm for analysis of total glucose contents from the β-glucans. In another experimental, 1.0 mL of the supernatant was transferred to three separate test tubes. Then 0.1 mL of 50 mM sodium acetate buffer (pH 4.0) was added to the first tube (reaction blank). In the other two tubes, β-glucosidase (5 U/mL) was added and tubes were incubated at 50°C for 20 min. After incubation, 3.0 mL of GOPOD determining reagent was added to all tubes and incubated at 50°C for 20 min. A spectrophotometer was used to measure absorbance at 510 nm for analysis of glucose contents from β-(1→3)-glucans.

Nata de coco (Morinaga nata de coco plain) was purchased from Morinaga Milk Industry Co., Ltd., Tokyo, Japan. BGL (P-BGH, high viscosity, Megazyme), lichenase (endo-1,3:1,4-β-D-glucanase: E-LICHN, Megazyme; 273 U/mg protein), and xylanase (endo-1,4-β-xylanase: E-XYNACJ, Megazyme; 38 U/mg protein) were purchased from Biocon (Japan) Ltd., Nagoya, Japan. Barley grain was purchased from Hakubaku Ltd., Chuo, Japan. Potato powders were purchased from Syunsai-Kobo KOKORO, Yokkaichi, Japan. Potato starch and turmeric powders were purchased at a domestic supermarket. Other chemicals were of the reagent grade.