Organic Acid Profiling of TBE-8 Culture

Organic acids in the TBE-8 culture were analyzed using Chinese National Standard methods (CNS12635-N6224) and methods reported by Han et al.^{51 (link)} with some modifications. TBE-8 cultures were collected from different incubation time points (i.e., 24, 48, and 72 h) and centrifuged at 5000 rpm for 10 min. The resulting supernatants were filtered through a 0.22-μm-pore-size membrane filter for further chromatographic analysis. A total of 20 μL of the filtered sample was loaded into the Cosmosil 5 C18-AR-II column (4.6 × 250 mm, 5 µm, Cosmosil, Japan) with 0.01 M KH₂PO₄ aqueous solution (pH 2.5; including 0.06 mmol tetra-butylammonium phosphate) as the mobile phase and a flow rate of 1.0 mL/min. The UV detection wavelength was set at 210 nm (Waters TaperSlit, USA). The specific fingerprint chromatograms of eight organic acids, namely oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid, were identified through comparison with the reference standards. The standard curves of the eight organic acids were prepared using six concentrations (1%, 0.5%, 0.25%, 0.125%, 0.0625%, and 0.03125%) to calculate the organic acid content of the culture. Each experiment was performed in three replicates.