Lipids extraction and fatty acids methyl ester (FAME) analysis

Bacterial aliquots were collected in triplicate at the mid-log phase (A₅₈₀ = 0.4) and pelleted by centrifugation. After removal of supernatant and three washes with sterile saline solution, bacteria were centrifuged at 4 °C (13,000 × g) for 15 minutes. Aliquots were resuspended in deionized water and lyophilized using a Heto PowerDry PL9000-50/HSC500 Freeze Dryer (Thermo Fisher Scientific, Saint-Herblain, France). Fatty acid methyl esters (FAME) were prepared by incubation for 15 min at 95 °C in a mix of 14% boron trifluoride (BF₃)/methanol followed by extraction with hexane as described by Morrison and Smith⁶³. FAMEs were separated and analyzed by gas chromatography (GC) coupled to flame ionization detection using an Agilent Technology, 6890 Network GC System, equipped with a split/splitless 7683 Series Injector. The samples were separated through a CP-Sil 88 capillary column (Chrompack, Middelburg, the Netherlands; length, 50 m; inner diameter, 0.25 mm; 0.25 mm film). FAMEs were identified by co-injection of reference standards obtained from Supelco (Bellefonte, Pennsylvania, USA) and were quantified on basis of their peak areas in total ion counts. The degree of FA saturation was determined as the ratio between the saturated FAs and the unsaturated FAs⁶⁴. All experiments were performed in triplicate.