Isa 07 s2504 column

Sake meter value, sake acidity, and amino acid levels were measured as previously described⁶ . Organic acids were quantified using HPLC with a Shim-pack SCR-102H column (Shimadzu, Kyoto, Japan). Aroma components were quantified using headspace GC with a J & W DB-WAX capillary column (30 m × 0.32 mm internal diameter × 0.50 μm film thickness; Agilent Technologies, Santa Clara, California, USA). Ethanol concentration was measured using an ethanol analyser (RIKEN KEIKI, Tokyo, Japan). Sugars were determined by monitoring post-column derivative reducing sugars separated using a Prominence reducing-sugar HPLC analytical system (Shimadzu) equipped with a fluorescence detector. Sake fermented with MC87-46 and K901 was separated on a Shim-pack 4.0 × 250-mm ISA-07/S2504 column (Shimadzu) with a linear gradient of 0.1 M potassium borate buffer (pH 8.0) and 0.4 M potassium borate buffer (pH 9.0) for 140 min at a flow rate of 0.6 mL/min^{37 (link),38 (link)}. Sugars were also lyophilized, trimethylsilylated, and analysed using a GCMS-QP2010 (Shimadzu) equipped with a J & W DB-5MS capillary column (30 m × 0.25 mm internal diameter × 0.25 μm film thickness; Agilent Technologies)^{39 (link)}.

Test substances were desalted using ultrafiltration via an ultrafiltration filter unit, and 20 μg aliquots were hydrolyzed with 2.5 mol/L trifluoroacetic acid at 100°C for 3 h. The samples were then cooled, dried, and reconstituted in L-rhamnose solution. Neutral monosaccharide was analyzed using a Shin-pack ISA-07/S2504 column (0.7 μm ID and 250 mm in length, Shimadzu) with a 50-min linear gradient of 0.1–0.4 mol/L potassium buffer (pH 8.0–9.0) by the Reducing Sugar Analysis System (Shimadzu). For amino sugar determination, test substances were hydrolyzed with 4 mol/L HCl at 100°C for 3 h, cooled, dried, and reconstituted in 0.02 mol/L HCl. An amino acid analyzer JLC-500/V2 (JEOL, Tokyo, Japan) was used for the analysis. For sialic acid analysis, test substances were treated with 0.1 mol/L NaOH at 37°C for 30 min, neutralized with HCl, and then reacted with 0.035 mol/L HCl at 70°C for 140 min to release sialic acids. The released sialic acids were fluorescently labeled with 1,2-diamino-4,5-methylenedioxybenzene at 50°C for 150 min. The solutions were analyzed by RP-HPLC using a COSMOSIL 5C18-PAQ column (4.6 mm ID and 150 mm in length, Nacalai Tesque, Kyoto, Japan) with a methanol/acetonitrile/water solvent system and fluorescence detection (Ex: 373 nm, Em: 448 nm). Quantification was performed relative to the appropriate monosaccharide standard curve.