*2.6. HPLC Analysis of Organic Acids*

The predominant organic acids found in kombucha tea were determined by high performance liquid chromatography (HPLC). Six organic acids including glucuronic acid (Sigma-Aldich, Darmstadt, Germany), gluconic acid (Merck, Darmstadt, Germany), d-saccharic acid 1,4-lactone (Sigma-Aldich, Germany), acetic acid (Merck, Germany), ascorbic acid (Merck, Germany), and succinic acid (Merck, Germany) in kombucha tea were optimized for detection by isocratic HPLC systems with a conventional C18 column. The kombucha tea samples were filtered through a 0.22 μm sterile microfilter and 50 μL of the filtrate was injected into the HPLC system (Agilent technologies 1200 series, Santa Clara, CA, USA). The C-18 column (4.6 × 150 mm, 5 μm; GL Sciences, Tokyo, Japan) employing a UV detector (210 nm) was used for the analysis. Moreover, the HPLC system was controlled with a flow rate of 0.8 mL per minute and a running time of 40 min at 25 ◦C. Six organic acids in kombucha tea were separated by 20 mM KH2PO4 elution buffer at pH 2.4 and the standards of organic acids were used for comparison with kombucha tea. The peak area of each organic acid was calculated by Agilent ChemStation level-5 program, and then standard graphs of each organic acid were generated. Thus, the content of each organic acid in kombucha tea was calculated from the standard graph of each organic acid.

#### *2.7. DPPH Radical Scavenging Assay of Kombucha Tea*

The radical scavenging activity of kombucha tea was determined against DPPH free radical content [15]. The kombucha tea was diluted with methanol by 10-fold serial dilution. Each concentration of the kombucha tea (0.5 mL) was incubated with 1.5 mL of 0.1 mM DPPH solution (Sigma-Aldich, Germany). Absorbance at 517 nm was measured by spectrophotometer (Genesys 20, Thermo Scientific, Dreieich, Germany) after the solution mixture was kept in the dark at room temperature for 20 min. Methanol was used as a blank solution, while DPPH without kombucha tea was used as a control. The absorbance of the DPPH solution A1, and the absorbance of kombucha tea mixed with the DPPH solution A2, were measured. The percentage of DPPH free radical inhibition was calculated as follows: percentage inhibition = {(A1–A2)/A1} × 100.

The antioxidant activity of kombucha tea was assessed by comparing the samples to standard gallic acid and was expressed as milligrams of gallic acid per milliliter of kombucha tea (mg GAE/mL kombucha tea).
