Rezex roa organic acid h 8 guard column

The collected fermentation broth was treated with 6 M HCl, heated at 80 °C for 30 min, and then filtered through a 0.22 μm filter membrane to obtain the supernatant samples. Organic acids were quantified by high-performance liquid chromatography using a standard high-performance liquid chromatography (HPLC) device (Agilent 1100 Series, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a Rezex ROA organic acid H+(8%) column (300 by 7.8 mm, 8 m; Phenomenex) and a Rezex ROA organic acid H⁺(8%) guard column (50 by 7.8 mm). The samples were analyzed at 35 °C with 0.5 mM H₂SO₄ as the mobile phase and a flow of 0.6 mL/min at a wavelength of 210 nm, according to a method described previously.

The concentrations of glucose and acetate in the cultures without PAC were determined by reverse-phase HPLC (Agilent 1100 Series, Agilent, Waldbronn, Germany) using a Rezex ROA organic acid H+ (8%) column (300 × 7.8 mm, 8 µm particle size; Phenomenex) and a Rezex ROA organic acid H+ (8%) guard column (50 × 7.8 mm). For the quantification of the glucose content, the oven temperature was set at 50 °C, and 5 mM H₂SO₄ was used as eluent, while 60 °C and 3 mM H₂SO₄ were chosen for the determination of the acetate concentration. In both cases, elution was performed isocratically at a flow rate of 0.5 mL/min. Glucose was detected using a refractive index detector, while a UV detector with a wavelength of 220 nm was used for the organic acid. The actual quantification was performed via calibration curves in the range of 0.1–5 g/L.

The water content of honey and agave syrup was measured via Karl Fischer titration (TitroLine 7500 KF trace, SI Analytics, Germany). Before measuring, the titrator was tested with a water standard (Merck Millipore, Germany).
The water activity a_w of honey and agave syrup was determined with an AquaLab CX-2 at 22°C (Decagon Devices, USA).
After calibrating, the pH was directly measured in honey and agave syrup and as a 10% dilution (w/v) (SenTix® Mic, Xylem Analytics, Germany).
The two main carbohydrates, fructose and glucose, were quantified by HPLC (Agilent 1100 Series, Agilent Technology, Germany) with a Rezex ROA organic acid H⁺ (8%) column (300 mm length, 7.8 mm diameter) and a Rezex ROA organic acid H⁺ (8%) guard column (50 mm length, 7.8 mm diameter) from Phenomenex (Phenomenex Ltd., Germany) as described in Dörsam et al. (2017 (link)). Separation was performed with 5 mM H₂SO₄ for 45 min and a flow rate of 0.5 ml/min under isocratic conditions at 50°C column temperature. Carbohydrates were detected via a refractive index detector (Agilent 1200 series, Agilent Technology, Germany).
Quantification of the sugars were performed by using three different dilutions of honey or agave syrup (0.3–1 mg/ml) and an external 10-point calibration curve for each component from 10 to 500 mg/l.
All measurements were made in triplicates.