C18 column

For the identification of polyphenols, all samples were filtered through 0.22 μm membrane filters and injected into the UPLC-ESI-MS/MS (Shimadzu LC–MS/MS 8060, Kyoto, Japan) equipped with a C18 column (10 cm × 3 mm, 3 μm; GL Sciences, Tokyo, Japan) as previously described in the literature [26 (link)]. The flow rate was set at 0.4 mL/min and the injection volume was 10 μL. MQ water with formic acid (1000:1, v/v) and acetonitrile with formic acid (1000:1, v/v) were used as mobile phase A and B, respectively. The gradient was as the following: 0 min, 20% B; 0.0–0.5 min, 20% B, isocratic; 0.5–7.0 min, 20–50% B, linear; 7.0–12.0 min, 50–95% B, linear; 12.0–12.1 min, 95–20% B, linear; 12.1–15.0 min, 20% B, isocratic. The column and autosampler temperatures were maintained at 40 °C and 10 °C, respectively. The instrument parameters were the following: nebulizing gas (N₂) flow at 3.0 L/min, drying gas (N₂) flow at 10.0 L/min, interface voltage set to 4.0 kV, desolvation line temperature maintained at 250 °C, interface temperature set at 300 °C, and the heat block temperature at 400 °C. The MS/MS system was operated in both negative and positive ion modes with multiple reaction monitoring (MRM) using the ESI source.

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.

The chlorophylls and carotenoids were extracted from the kiwifruit according to previous methods [3 (link),4 (link)]. The extracts were quantified using a high performance liquid chromatography (HPLC) system (1260 Infinity II, Agilent Technologies, Palo Alto, CA, USA) equipped with a DAD detector and a C-18 column (250 nm × 4.6 mm, 5.0 μm, GL Sciences Inc., Tokyo, Japan). The injection volume was 20 μL, the post-run time was 5 min, and the flow rate was 0.7 mL min⁻¹ at 40 °C. The solvent consisted of 90% (v/v) acetonitrile/water (A) and ethyl acetate (B). The gradient profile was 100% A (0 min), 20% A (14 min), 20% A (20 min), and 100% A (30 min). Chlorophyll a was simultaneously monitored at 430 nm, while lutein, β-carotene, and chlorophyll b were monitored at 450 nm.