Water 717 plus autosampler

The runs were carried out in a Waters 600 E HPLC Multisolvent Delivery System equipped with a Water 717 Plus Autosampler and a Waters 996 Photodiode Array Detector (Waters Corporation, Milford, MA, USA). SunFire prep C8, 5 mm ST 10/250 (Waters Corporation) column was equilibrated with solvent A (water:acetonitrile, 98:2 (v/v); 0.065 mL/100 mL Trifluoracetic Acid, TFA in water). Two milliliters of 3 mg/mL MPI-h in 0.0351 mol/L K 2 HPO 4 , pH 8, m 0.1 was injected. Samples were eluted with solvents A and B (water:acetonitrile, 35:65 (v/v); 0.065 mL/100 mL TFA in water) in a linear gradient of B from 0 to 100% in 55 min at a flow rate of 5.2 mL/min. Detection was performed at 210 and 280 nm. Fractions (0.6 min) were collected manually from 5 runs, pooled and freeze-dried.

Each sample of fermented broth was centrifuged at 7,741 × g for 20 min at 4°C. The cell-free supernatant of fermented broth was used for determination of organic acids (lactic acid and acetic acid) by HPLC (Mirsiaghi and Reardon, 2015) with some modifications.
The analysis was carried out using a Waters 2695 separation module with temperature control, a Water 717plus autosampler, Waters 1525 Binary HPLC Pump, and Waters 2414 Refractive Index Detector (Waters, Milford, MA). Equipment and signal were controlled and processed using Waters Empower Pro software. An Aminex HPX-87H (Bio-Rad) column was used with 9 μm particle size and dimensions of 300 × 7.8 mm. Detection of organic acids was carried out at a wavelength of 210 nm. Samples and mobile phases were filtered through a 0.22-μm Millipore filter (Millipore, Bedford, MA) before HPLC injection, and 10 μL of sample was injected. The mobile phase was 5 mmol/mL sulfuric acid with a flow rate of 0.6 mL/min, and the oven temperature was 35°C. Standards for the organic acids were used to identify and quantify the components in samples.