2.5. Organic Acid and Secondary Metabolite Analysis by HPLC

After fermentation extraction and quantification of organic acids and secondary metabolites was possible with the help of high-performance liquid chromatography (HPLC-Agilent 1200 series, Santa Clara, CA, USA) equipped with solvent degasser, quaternary pumps, DAD detector coupled with a mass detector, column thermostat, and automatic injector (Agilent Technologies, Santa Clara, CA, USA). The separation of organic acids could be realized on reversed-phase chromatographic column Acclaim OA (5 µm, 4 mm × 150 mm Dionex), eluted for 10 min with monosodium phosphate solution (NaH₂PO₄) 50 mM concentration, pH 2.8, and a flow rate of 0.5 mL/min, at a temperature of 20 °C. The measurement of chromatograms was possible at the wavelength λ = 210 nm.

Sample preparation for the HPLC consisted of the addition of 2 mL distilled H₂O to 1 g of sample, which was vortexed 30 sec, sonicated 15 min, and centrifuged at 8000 rpm for 10 min at 4 °C. After these steps, the supernatant was filtered with a Millipore membrane filter of 0.45 µm pore size [44]. A volume of 20 µL of sample was injected in the column, with a flow rate of 0.5 mL/min, and detection conducted at 280 and 340 nm [45].