Euglena Metabolite Analysis by LC/MS

Liquid chromatography-mass spectrometry (LC/MS) measurement and analysis were performed according to a previously described methods [10 (link),11 (link)]. The carbohydrate concentration was measured following the method reported by Suzuki et al. [12 (link)]. Euglena extract was treated with exo-1,3-β-D-glucanase (Neogen Chemicals Ltd., MI, USA.) or β-1,3-glucanase (Thermo Stable Enzyme Laboratory Co., Ltd., Kobe, Japan). Polyvinylpolypyrrolidone (PVPP; Sigma-Aldrich, St. Louis, MO, USA) treatment was used to remove polyphenols from Euglena extract. The chelating agents ethylenediaminetetraacetic acid (EDTA; FUJIFILM Wako, Osaka, Japan) and ethylene glycol tetraacetic acid (EGTA; FUJIFILM Wako, Osaka, Japan) were added to trap metal ions. A cation exchange resin (CER; GL sciences Inc., Tokyo, Japan) was used to remove metal ions from Euglena extract. The samples were treated twice to remove approximately 99.4% zinc. The zinc concentration was determined using a zinc assay kit (Metallogenics, Tokyo, Japan), according to the manufacturer’s instructions.