Model 6890

For FA quantification, a mixture of the internal standards including 25% of triglycerides (19:0), 25% of cholesteryl esters (17:0), 45.5% of phospholipids (15:0), and 4.5% of the free FAs (24:0) was added to a 200 mL plasma sample. Lipids were extracted using the Folch method [57 (link)], evaporated under N2, and then reconstituted in 200 mL chloroform. A solid-phase extraction method was used to separate and extract phospholipids, as previously described [58 (link)]. Then, phospholipids were methylated by 12% boron trifluoride methanol solution at 90 °C for 30 min to generate fatty acid methyl esters required for their analysis by gas chromatography (model 6890; Agilent, Palo Alto, CA, USA). Fatty acid methyl esters were separated on a BPX-70 fused capillary column (50 m, SGE, Melbourne, Australia) and detected by a flame ionization detector with the same parameter settings described by Chevalier et al. [59 (link)]. The chromatogram analysis was performed using the OpenLab CDS ChemStation. The results were calculated in the absolute concentration of fatty acid methyl esters compared to the internal standard (GLC-569B, Nu-Check Prep, Inc., Elysian, MN, USA).

An aliquot of the erythrocyte lipid fraction of each sample was mildly saponified, and the fatty acids were analysed by HPLC (an Agilent 1100 HPLC system with a diode array detector, Palo Alto, Calif., USA) as previously described^{66 (link)}. Spectra (195–315 nm) of the eluate were obtained every 1.28 s and were electronically stored. These spectra were acquired to confirm the identified HPLC peaks^{67 (link)}.
Because SAFA are transparent to UV, after derivatization, they were measured as fatty acid methyl esters using a gas chromatograph (Agilent, Model 6890, Palo Alto, CA) as described previously^{49 (link)}.

Biomass of strains AD7-25^T and AB-11 for chemotaxonomic analysis was harvested from cultures after incubation on improved Gibbson medium at 33 °C for 18 h. The analysis of the cell-wall peptidoglycan was carried out with O. neutriphilus CGMCC 1.7693^T as a reference according to the method described by Schleifer and Kandler (1972 (link)) and Schleifer (1985 (link)). Cell-wall hydrolysates were separated by one-dimensional chromatography on micro-cellulose thin layers. Menaquinones were analyzed as described previously (Collins 1985 ) using reverse-phase HPLC (Agilent HPLC-1200). Extraction and analysis of polar lipids by two-dimensional TLC was performed according to Ventosa et al. (1993 (link)). Cellular fatty acids were extracted and methylated according to the standard protocol of Sherlock Microbial Identification System version 6.0 (MIDI), analysed by GC (model 6890; Agilent) and identified using the TSBA6 database of the Microbial Identification System (Sasser 1990). The physiological age at the point of harvest of the three bacterial strains tested was the logarithmic growth phase. O. oncorhynchi subsp. oncorhynchi JCM 12661^T was used a reference in chemotaxonomic analysis tests.

Quantitative analysis of the EO was carried out using a gas chromatograph (GC) equipped with a flame ionization detector (FID) (model 6890, Agilent Technologies, Santa Clara, CA, USA). Column: HP-5MS capillary column (60 m × 0.25 mm, 0.25 μm film thickness). The GC settings were as follows: injection volume (1 μL), split ratio (1:20), carrier gas helium (1 mL/min), and oven temperature program (kept at 70 °C for 2 min, increased to 180 °C at 2 °C/min, raised to 310 °C at 10 °C/min, and finally kept at 310 °C for 14 min). The gas chromatograph–mass spectrometer (GC-MS) (model 6890/5975C, Agilent Technologies, Santa Clara, CA, USA) was used for qualitative analysis of the EO. GC parameters and the column were the same as in the GC-FID analysis. The MS was operated as follows: ion source temperature at 230 °C, interface temperature at 280 °C, ionization voltage at 70 eV, and a scan range of m/z 29 to 500 amu. The relative abundance (%) of the chemical constituents was determined using the peak area. Standard n-alkanes (C₉–C₃₀) were used for the calculation of the retention index (RI). Each component of EO was determined by comparing the RI and mass spectra in the Wiley 275 (Wiley, New York, NY, USA) and NIST 2020 (National Institute of Standards and Technology, Gaithersburg, MD, USA) databases.

The solid-phase micro-extraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis was conducted following our previous studies [40 (link),41 (link)] with some modifications. Volatiles were extracted using an SPME fiber (50/30 μm DVB/Carboxen/PDMS; Supelco, Bellefonte, PA, USA). The SPME fiber was put into the headspace vial, and 1 cm of it was exposed from the headspace for 40 min at 50 °C. After extraction, the fiber was inserted into the injector of a GC-MS (Model 6890; Agilent, Santa Clara, CA, USA) to desorb the adsorbed substances for 5 min at 250 °C. At the same time, the instrument data acquisition was performed.
Gas chromatography was performed using the HP-5 column (50 m × 0.32 mm × 1.05 μm, J&W Scientific, Agilent, Santa Clara, CA, USA) with helium as the carrier gas (37 kPa). The column temperature was set at 40 °C for 2 min, then increased to 250 °C at the rate of 5 °C min⁻¹, and finally maintained at 250 °C for the next 2 min. The volatile compounds were matched against the NIST08 library (NIST/EPA/NIH, American), and the retention indexes were compared with the standard volatile compounds. A standard peak area vs. concentration curve was prepared from the serial dilutions of the standard and used for sample quantification.

Gas chromatography was performed on an Agilent gas chromatograph Model 6890 (Agilent, Palo Alto, Ca), equipped with a DB5 MS column (30 m×0.25 mm, 0.25 µm film thickness). Hydrogen was used as carrier gas (flow 1.0 ml/min). Oven temperature program was from 50°C (5 min) to 300°C at 5°C/min, 5 min post run at 300°C. Sample (1 µL) was injected in split mode (1∶60); injector and detector temperatures were 280 and 300°C, respectively.