Ri 71

During sample preparation of alcoholic content, 1 g of each mash sample into 250 mL Erlenmeyer flask and diluted it with 20 mL of high-purity water (ZeneerPower 1, Human Corporations, Korea) was measured, then stored for 1 h on a rotary shaker (Elphan 358S, Bohemia) for extraction at room temperature (24 °C). Extracted samples were centrifuged for 30 min at 6000 RCF (Labnet Hermle Z206A, USA) in a 15 mL tube, then 1.5 mL of each supernatant was centrifuged for an additional 20 min 14 500 RCF (Biosan Micro spin 12, Latvia) in 2 mL Eppendorf tube. Subsequently, the samples were simply filtered through a membrane filter (polyvinylidene difluoride [PVDF nylon] 0.22 µm, Filter Bio) into screw topped HPLC vials of 2 mL with an inner septum (Berrytec, Germany). Ethanol was measured with an HPLC-UV system. Before analyses, the measuring system was calibrated with solutions of 0.05–10 mg/mL concentration made from the measurand (Ethanol 96 % a.r. – Merck, Germany). Measuring solutions were diluted to the graduation mark with the eluent (high purity water). The separation of ethanol was also carried out using an ion-exchange HPLC system (LC 900, Jasco, Japan); components were identified with a refractive index detector (RI 71, Merck, Germany). The column (Supelcogel H, Sigma Aldrich, USA) was kept at room temperature, and the flow rate of the eluent was 0.5 mL/min.