Chitosan–oligosaccharide preparation and TLC analysis

Chitosan (1.0 g) was dissolved in 100 mL of 0.1% (v/v) acetic acid solution. The pH of the obtained chitosan solution was adjusted to 5.0 with 1.0 M NaOH, and chitosanase (30 mg) was added, mixed well, and incubated at 45 °C with shaking for 12 h. After hydrolysis, the chitosan hydrolysate solution was adjusted to pH 7.0 with 1.0 M NaOH and terminated in boiling water for 10 min. The chitosan hydrolysate solution was then immediately cooled in an ice bath and centrifuged at 4000 g for 15 min. The obtained supernatant was concentrated to approximately 10 mL and added with tenfold volume of ethanol for chitosan precipitation. The precipitation was converted to chitosan–oligosaccharide powder by drying at 40 °C. The composition of chitosan–oligosaccharide was analyzed via silica gel TLC in accordance with the method developed by Cabrera and Cutsem⁴¹. The sample and standard were applied onto a TLC plate in a solvent system composed of n-propanol:water:ammonia water (7:2:1, v/v/v), and the obtained plate was developed by spraying methanol containing 0.5% (w/v) ninhydrin.