Organic acid composition analysisThe organic acid composition of Shiikuwasha juice, particularlycitric and malic acids, was determined using an HPLC systemwith electroconductivity detection.10 Briefly, a sample (1mL) wascentrifuged at 8000×g for 5 min and filtered through anAdvantec0.45 μm membrane filter (Toyo Roshi Kaisha, Ltd, Tokyo, Japan)and diluted before injection. The column used was a Shim-packSCR-102H column (300mm×8mm i.d., Shimadzu Corp., Kyoto,Japan) connected to a guard column (50mm×6mm i.d.). TwoShimadzu LC-10 AD pumps were used to flow the mobile phasecontaining 5 mmol L−1 p-toluenesulfonic acid and post-columndetection reagent containing 5 mmol L−1 p-toluenesulfonic acid,100 μmol L−1 ethylenediaminetetraacetic acid disodium salt, and20 mmol L−1 Bis–Tris buffer in isocratic mode at a flow rateof 0.7mLmin−1. The mobile phase and post-column detectionsolvent were streamed to a post-column reactor and mixed at aratio of 1:1 before detectionwith a Shimadzu CDD-6A conductivitydetector. The column, guard column, and post-column reactorwere maintained at a constant temperature of 40 ◦C using aShimadzu CTO-10 AC oven, and the injection volume was 10 μL.The concentrations of citric and malic acids were calibrated byplotting peak area against concentration for the respective acidstandards and expressed as grams per liter of juice. All assays wereperformed in triplicate.