0.2 m filter

Consumption of glucose, mannitol, and production of lactic acid, acetic acid, and ethanol by strain AK17 and the derived mutant strains were analysed by high performance liquid chromatography (HPLC). The samples for HPLC analysis were filtered through a 0.2 µm filter (Phenomenex) prior injection. Glucose, mannitol, acetic acid, lactic acid, and ethanol were quantified using a Dionex 2000 HPLC system (Dionex, Idstein, Germany) with a Rezex ROA-Organic Acid H + (8%, Phenomenex, Aschaffenburg, Germany) and a RI-101 detector (Shodex, München, Germany). Separation was performed at a column temperature of 60 °C with 0,2 mM sulfuric acid (Carl Roth, Karlsruhe, Germany) as eluent at a flow rate of 600 μl/min for 30 min. Quantification was carried out using external standards with HPLC grade (Merck, Darmstadt, Germany; Sigma-Aldrich, St. Louis, USA) and the Chromeleon Evaluation Software version 6.80 (Dionex, Idstein, Germany).

Concentrations of sucrose, fructose, and glucose were measured with high-performance liquid chromatography (HPLC) (Shimadzu, Columbia, MD, USA). The HPLC was equipped with a SIL-20AHT autosampler, a CTO-20A oven, an LC-6AD pump, a CBM-20A controller, and a RID-10A refractive index detector. The syrup sample (~0.5 g) was weighed into a 15 mL centrifuge tube and diluted with (~7 mL) HPLC grade water. The actual weights of syrup and water were recorded. The mixture was vortexed for 40 sec and was filtered through the 0.2 µm filter (Phenomenex^®, Torrance, CA, USA), and then filled into an HPLC vial. Isolated sugars were segregated by a Rezex RCM-Monosaccharide Ca+ 300 × 7.8 mm column (Phenomenex^®). Sugars were eluted under the isocratic condition at 80 °C, using HPLC grade water as a mobile phase at a 1 mL/min flow rate for 20 min. LC Solutions software (Version 3.0, Shimadzu, Columbia, MD, USA) was used to integrate chromatograms automatically. The standard curve with concentration ranges from 10 to 50 mg/mL (>99% purity, Fisher Scientific, Fair Lawn, NJ, USA) was plotted to calculate each sugar content.

For terpenoid analyses by GC–MS, 10 ± 2 mg dried samples were extracted in n-heptane (99%, Carl Roth, Karlsruhe, Germany) containing 1-bromodecane as an internal standard (97%, Sigma Aldrich, Taufkirchen, Germany). Samples were sonicated for 5 min, centrifuged and supernatants were used for GC–MS analyses^{53 (link)}. For analyses of metabolic fingerprints by UHPLC-QTOF-MS/MS, dried leaf material was homogenised and 8 ± 2 mg samples were extracted in methanol 90% (v:v) containing hydrocortisone as internal standard (Sigma-Aldrich, Steinheim, Germany) by sonicating in an ice bath for 15 min. Supernatants were collected, centrifuged for 10 min and filtered using 0.2 µm filters (Phenomenex, Torrance, CA, USA) for LC-MS analyses as previously described^{54 (link)}.