Agilent 6850 gas chromatograph

The residual DCM content in the microspheres was determined with an Agilent 6850 gas chromatograph (GC; Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector, a CombiPal CTC headspace sampler, and a DB-624 column (30 m × 0.53 mm, 3 µm). As carrier gas, helium with a flow of 7 mL/min was used. The split injection mode was used with a split ratio of 1:15. The initial column temperature was 40 °C maintained for 5 min and then raised (10 °C/min) to 100 °C with a hold time of 1 min. Finally, the temperature was raised to 250 °C with 50 °C/min for 4 min. The syringe and incubation temperatures were 140 °C and 120 °C, respectively. For each formulation, 100 mg of microspheres was accurately weighed in duplicate and dissolved in 5 mL DMSO with 9.4 µg/mL octane as internal standard. Then, 2 mL of the headspace layer was injected into the GC for analysis. An eight-point calibration curve was plotted using a linear fit and a 1/X weighting factor to determine the DCM concentration from the peak area.

Polar lipid analysis was carried out by Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) services using the deposited strain V10^T (=DSM 112951^T). In brief, polar lipids were extracted using the modified Bligh and Dyer method (Bligh and Dyer, 1959 (link)) and separated by two-dimensional silica gel thin-layer chromatography. Total lipids were revealed by spraying molybdate-phosphoric acid with specific reagents to detect defined functional groups.
The cellular fatty acid methyl ester profile of strain V10^T was determined at the CECT, Spanish Type Culture Collection (Valencia, Spain), following the protocol recommended by the MIDI Microbial Identification System (Sasser, 1990 ). The biomass of strain V10^T for the fatty acid determination was obtained from a culture on MA after incubation for 72 h at 30°C. The cellular fatty acid content was analyzed by gas chromatography with an Agilent 6850 gas chromatograph and identified according to the TSBA6 method using the Microbial Identification Sherlock software package (MIDI Inc., 2012 ).

For sterol MS analysis, the internal standard α‐cholestanol (0.02 mg) was added in 0.1 ml of lipid extract and the solvent was evaporated under N2 gas stream. A saponification step was performed by adding 1 ml of ethanol and 0.1 ml of 11 N KOH and incubating it for 1 h at 80 °C. After the addition of 1 ml of hexane and 2 mL of water, the sterol-containing upper phase was recovered, and the solvent was evaporated under N2 gas stream. Sterols were trimethylsilylated by N,O‐bis(trimethylsilyl) trifluoroacetamide (BSTFA)-trimethylchlorosilane for 15 min at 100 °C. After complete evaporation of BSTFA under N2 gas, derivatized sterols were dissolved in 0.1 ml of hexane and analyzed by GC-MS. GC-MS was performed using an Agilent 6850 gas chromatograph and coupled MS detector MSD 5975-EI (Agilent). An HP-5MS capillary column (5% phenyl-methyl-siloxane, 30-m, 250-mm, and 0.25-mm film thickness; Agilent) was used with helium carrier gas at 2 ml/min; injection was done in splitless mode; injector and mass spectrometry detector temperatures were set to 250 °C; the oven temperature was held at 50 °C for 1 min, then programmed with a 25 °C/min ramp to 150 °C (2-min hold) and a 10 °C/min ramp to 320 °C (6-min hold).