Supelco 37 component fame mix

A fatty acid methyl ester mix was used as GC standards (SupelcoTM 37 Component FAME Mix, Sigma-Aldrich). All solvents used for sample preparation were of analytical grade and the solvents used for GC analysis were of HPLC grade.

Extracted lipids were subjected to an acid-catalyzed transesterification process prior to analysis by gas chromatography with a flame ionization detector (GC-FID). Firstly, lipids were dissolved in 4 mL of n-hexane (99.0% Panreac AppliChem, Darmstadt, Germany). For the transesterification step, 1 mL of the diluted sample, 100 μL of heptadecanoic acid (internal standard, 98.0% Sigma Aldrich, Steinheim, Germany), and 1 mL of the mixture 10% MeOH:H₂SO₄ (10:1 (v/v)) were added to glass capped test tubes with a screw cap and heated at 100 °C for 2 h. The organic phase was collected and after residual water removal with Na₂SO₄, fatty acid methyl esters were analyzed by GC-FID (CP-3800 Varian, Agilent, Santa Clara, CA, USA) using a Teknokroma^® TR WAX 30 m × 0.25 mm × 0.25 µm column. GC-FID operational conditions were as follows: carrier gas flow (He) 1 mL min⁻¹; injector—250 °C; detector—280 °C; oven—40 °C with a ramp of 30 °C min⁻¹ up to 150 °C followed by a ramp of 3 °C min⁻¹ up to 250 °C. The volume of the injected sample was 1 µL and the split (1:10) injection technique was applied. For peak identification, a mixture containing saturated, monounsaturated, and polyunsaturated FAME ranging from C4:0 to C24:1 was used (Supelco^TM 37 Component FAME Mix, Sigma Aldrich, Steinheim, Germany). Results were expressed as percentages and as means of triplicates ± standard deviations.

After an appropriate amount of time for consuming nitrogen, strain JSC4 cells were harvested from the culture medium by centrifugation (7,000 rpm for 2 min). The cells were washed twice with deionized water, lyophilized, and weighed. The lipid composition was determined as fatty acid methyl esters (FAMEs) following direct transesterification of lipids according to the method described in Ho et al. [7 (link)]. FAMEs were analyzed by gas chromatography/mass spectrometry (GC/MS) on a GCMS-QP2010 Plus instrument (Shimadzu). Samples were injected onto a DB-23 capillary column (60 m, 0.25 mm internal diameter, 0.15-μm film thickness; Agilent Technologies, Palo Alto, CA, USA). Helium was used as the carrier gas at a flow rate of 2.3 mL min⁻¹. The injector, ion source, and interface source temperatures were set at 230°C, 230°C, and 250°C, respectively. The oven temperature was initially set at 50°C for 1 min, increased from 50°C to 175°C at a rate of 25°C/min, increased from 175°C to 230°C at a rate of 4°C/min, and held at 230°C for 5 min. Purified FAMEs were identified based on retention time and the pattern of fragmentation by electron impact analysis. Supelco 37 Component FAME Mix (Sigma-Aldrich Co., St. Louis, MO, USA) was utilized as a quantitative standard, and pentadecanoic acid (Sigma-Aldrich Co.) was used as an internal standard.

Fatty acid methyl esters extracted were analyzed by a gas chromatography equipment with auto-sampler, a split/splitless injector, FID detector, and a hydrogen gas generator (Thermo Fisher Scientific, Milan, Italy). The analysis was carried out on a BPX 70 capillary column (SGE Analytical Science, P/N SGE054623, 60 m × 0.25 mm ID—BPX70 0.25 µM, SGE Europe Ltd., Milton Keynes, UK). Hydrogen was used as carrier gas, 3.0 mL min⁻¹, constant flow mode; the amount injected was 1 µL in splitless mode (split flow 50 mL min⁻¹, splitless time 1 min). The temperature of the injector and the FID detector were 250 °C and 270 °C, respectively. The initial temperature of the oven was 40 °C, then it increased to 170 °C at 10 °C min⁻¹ for 5 min, then to 200 °C at 4 °C min⁻¹ for another 5 min, and finally the temperature increased to 255 °C at 50 °C min⁻¹ and held for 4.5 min. The identification of each peak was obtained by comparing the retention times with those of a mixture of standards (Supelco 37-Component FAME Mix, Sigma-Aldrich, Milan, Italy). Data were expressed as percentage of each fatty acid calculated on the total amount of fatty acids.

Free fatty acid contents in dry- and wet-aged beef were assessed by the method of Lee et al. [3 (link)]. Briefly, 1 g of lipid was put into the test tube with 1 mL of chloroform and an internal standard (1 mg of triundecanoate/mL isooctane). After removing triglycerides from the samples, free fatty acids were extracted using 2% acetic acid in diethyl ether. The extract was evaporated with nitrogen gas and heated at 85 °C for 10 min. After that, 2 mL of 14% boron trifluoride–methanol was put into the test tube for methylation and heated at the same condition. Then, 2 mL of isooctane and 1 mL of saturated sodium chloride were added into the test tube and centrifuged at 1573× g for 3 min (Continent 512R, Hanil Co. Ltd., Daejeon, Korea). The upper layer containing fatty acid methyl ester (FAME) was dehydrated with anhydrous sodium sulfate. FAME was analyzed using gas chromatography (HP 7890, Agilent Technologies, Santa Clara, CA, USA) with a DB-23 column (60 m × 250 μm × 0.25 μm; length × diameter × thickness) (Supelco, Bellefonte, PA, USA). Each FAME was identified by comparing the retention time of external standards (Supelco^® 37 Component FAME mix, Sigma-Aldrich, St. Louis, MO, USA).

Freeze-dried and homogenized cell material (3 mg) was extracted and transesterfied as detailed in Mudimu et al. [40 ]. The extract was dissolved in DCM to a concentration of 0.5 mg ml^-l and subjected to the analysis of FAMEs using an Agilent 7820A gas chromatograph equipped with a Phenomenex ZB-Wax column (30 m × 0.25 mm i.d.; 0.50 μm film thickness) and connected to a flame ionization detector (FID). Hydrogen was the carrier gas at 1 ml min^-1. The oven temperature was programmed from 50°C (1 min) to 200°C at 25°C min^-1, then to 230°C (held 18 min) at 3°C min^-1. FAMEs were assigned by comparison of retention times with those of the “Supelco 37 Component FAME mix” (Sigma-Aldrich, Germany).
FAMEs not included in the standard mixture were assigned from their mass spectral characteristics using an Agilent 7820A gas chromatograph coupled to an Agilent 5975 MSD mass spectrometer. Analytical conditions were as above. Assignment of FAMEs was accomplished by comparison of mass spectra with those in the “Lipid Library” of the American Oil Chemists’ Society (http://lipidlibrary.aocs.org).