Cell Hydrolysis

Filtered cells were hydrolyzed by refluxing for 2 h in 1 N HCl in methanol (MeOH). After cooling, the hydrolysate was adjusted to pH 4 with 2 N KOH in MeOH–water (1:1, v/v) and, following the addition of water (final H₂O–MeOH ratio 1:1, v/v), extracted four times with dichloromethane. The combined extracts were dried over anhydrous Na₂SO₄, concentrated with a rotary evaporator and evaporated to dryness under a gentle stream of N₂.

A known amount of n-tricosanol (n-C₂₃-1-ol) used as an internal standard was added to each hydrolyzed extract. AGEs and the internal standard were silylated by reaction with N,O-bis(trimethylsilyl)trifluoroacetamide in pyridine (1:1 v/v, 50°C, 45 min) before gas chromatography-mass spectrometry (GC-MS) analysis. FAs were trans-esterified during the hydrolysis and analyzed as FAs methyl esters. The analysis of blanks, elaborated by filtering non-inoculated medium on GF/F filters, showed that lipid contamination from the filters and the medium was negligible.