**2.13 Thin layer chromatography (TLC) analysis of the digestion products**

The digestion products of xylan and xylooligosaccharides (Wako Pure Chemical Industries, Osaka, Japan) by xylanase were analyzed by thin layer chromatography (TLC) according to the method previously reported (Kiyohara et al., 2005) with some modifications.

As a substrate solution, 0.5 % xylan or 0.5 % xylooligosaccharides in 100 mM sodium phosphate buffer (pH 6.5) was used. In a test tube (15 mmΦ × 10.5 cm), 0.5 mL of substrate solution and 0.5 mL of enzyme solution containing 0.5 U of xylanase in 100 mM sodium phosphate buffer (pH 6.5) were mixed and the reaction mixture was incubated at 120 spm at 37°C. After 1, 3, and 16 h of incubation, 100 μL of reaction mixture was sampled to microtube, and mixed with 200 μL of ethanol. Then, the mixture was centrifuged at 18,000×*g* for 10 min and the supernatant obtained was evaporated with a centrifugal concentrator (VC-36N; Taitec, Saitama, Japan). The dried material was dissolved in distilled water and spotted on a Silica Gel 60 TLC plate (Merck, Tokyo, Japan), which was then developed with *n*-butanol/acetic acid/ water (10:5:1, by vol.). After development, the TLC plate was sprayed with aniline hydrogen phthalate reagent. The reagent consisted of 0.93 g of aniline, 1.48 g of phthalic anhydride, 84.5 mL of *n*-butanol and 15.5 mL of distilled water (Partridge, 1949), and heated at 100°C to visualize the digestion products.
