4.7. Analytical Methods

For the measurement of crude violacein, cells were first collected by centrifugation. After removing the supernatant, violacein was extracted by mixing the collected cells with ethanol in an ultrasonic water bath (Cpx5800h-e, Branson Ultrasonics Corporation, Danbury, CT, USA) at 60 °C until the cells were fully bleached. All ethanol extracts were collected, and the absorbance was measured at 570 nm to quantify the violacein using a Hidex Sense 425-301 microplate reader (HIDEX, Turku, Finland). The concentration of violacein was calculated based on a standard curve prepared using the purchased violacein (Sigma-Aldrich). In order to accurately quantify violacein in the flask fermentation, we used analytical high-performance liquid chromatography (HPLC) system (UltiMate^TM 3000 analytical HPLC system, Dionex, Sunnyvale, CA, USA) equipped with Acclaim 120 C18 reverse-phase column (Dionex). The mobile phases were acetonitrile with 0.1% formic acid (A) and water containing 0.1% formic acid (B). The following gradient was carried out at a flow rate of 1 mL min⁻¹: 0 min, 5% A; 1 min, 5% A; 5 min, 45% A; 7 min, 55% A; 9 min, 95% A; 10 min, 5% A; 12 min, 5% A. The signal was detected using an ultraviolet-visible (UV-Vis) diode array detector. Ultraviolet-visible (UV-Vis) diode array detector was used to detect signals. Galactose was analyzed with an Aminex HPX-87H column (Bio-Rad Laboratories, Richmond, CA, USA) maintained at 65 °C. The mobile phase was 5 mM H₂SO₄ at a flow rate of 0.6 mL min⁻¹. The signals were monitored using a Shodex RI-101 refractive index detector (Shodex, Klokkerfaldet, Denmark).