L 7490

One milliliter of the MeOH extract obtained for sugar analysis was evaporated to dryness and dissolved in 0.75 mL of water. The same volume of 1% (w/v) mannitol (as an internal standard) solution was added to the sample solution, and this mixture was used for subsequent sugar analysis. Sugars (glucose, fructose, sucrose, and sorbitol) were quantified by HPLC according to the conditions described below. The analytical conditions of HPLC were as follows: column, Shim-pack SCR101-C column, 70°C; solvent, distilled water; flow rate, 1 mL/min, isocratic; detection, RI detector (HITACHI L-7490). For starch analysis, the residues of sample extracts were used. An alcohol-insoluble residue was prepared using the method described by Murayama et al. [9 ]. For starch quantification, the dried alcohol-insoluble residue was first suspended in distilled water and boiled for 30 min. After cooling, the gelatinized starch was digested with amyloglucosidase (from Aspergillus niger; Roche Applied Science) in 50 mM sodium acetate buffer (pH 4.5). The released glucose was measured using the glucose oxidase–peroxidase method of Barham and Trinder [10 (link)].

Organic acid concentrations were determined by high-performance liquid chromatography (HPLC) using a chromatograph from Waters (Waters Corp., Milford, MA, USA), equipped with a Hitachi (Foster City, CA, USA) differential refractometer detector L-7490. An ICSep ICE-ION-300 column (Transgenomic, Omaha, NE, USA) was used with 8.5 mM of H₂SO₄ (180 µL H₂SO₄/L) as the mobile phase and at a flow rate of 0.4 mL/min. The column temperature was kept constant at 40 °C. Samples were centrifuged for five minutes at 5000 rpm (Allegra™ 25R Centrifuge, Beckman Coulter, Brea, CA, USA) and filtered (0.22 µm nylon; CHROMSPEC Syringe Filter, ON, Canada) before injection (15 µL). A mixture of lactose anhydrous (PHR1025), citric acid (251275), DL-lactic acid (L1750), and acetic acid (338826) (from Sigma-Aldrich, St. Louis, MO, USA) was used as an external standard to perform the quantification in g/L.