Fatty Acids Analysis

Fatty acid methyl esters (FAME) were obtained according to Leone et al. [27] using boron trifluoride (BF3), as follows. The total lipid extract in hexane (200 μL) was saponified at 90 ◦C for 20 min with 0.5 M KOH in methanol (3 mL) with a known quantity of internal standard (methyl-tricosanoate). Fatty acids were methylated with 2 mL of BF3 in methanol (14%), and the samples were evaporated under a stream of nitrogen and dissolved in 50 μL of hexane, and 1 μL was analyzed by gas chromatography–mass spectrometry (GC-MS). GC–MS analyses were performed using an AGILENT 5977E gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) on a VF-WAXms (60 m, 0.25 mm i.d., 0.25 mm film thickness, Agilent) with the following parameters: the column temperature was maintained at 160 ◦C for 1 min, programmed at 4 ◦C/min to 240 ◦C for 30 min. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The mass spectrometer was operated in the electron impact mode with a scan range of 50–700 m/z. The temperature of the MS source and quadrupole were set at 230 ◦C and 150 ◦C, respectively. Analyses were performed in the full-scan mode. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic standards (F.A.M.E. Mix C8- C24, Sigma-Aldrich Corporation, St. Louis, MO, USA) analyzed under the same conditions. The MS fragmentation patterns were compared with those of pure compounds, and a mass spectrum database search was performed using the National Institute of Standards and Technology (NIST) MS 98 spectral database.

### *2.10. Amino Acid Analysis*

The amino acid profile in lyophilized samples was analyzed with an HPLC system (Agilent Infinity 1260, Agilent Technologies) coupled with an online post-column derivatization module (Pinnacle PCX, Pickering Laboratories, Mountain View, CA, USA), using ninhydrin (Trione) as a derivatizing reagen<sup>t</sup> and a Na+ ion-exchange column (4.6 × 110 mm, 5 μm). Eighteen standard amino acids, ammonia, and taurine were quantified from standard curves measured with the amino acid standards. Prior to the analysis, the samples were hydrolyzed in 6 M HCl containing 0.4% mercaptoethanol for 24 h at 110 ◦C (HCl hydrolysis). Glutamine and asparagine were converted to glutamic and aspartic acid, respectively.

Cysteine (Cys) was quantified as cystin (Cys-Cys). The samples were filtered via a micro filter, the pH was adjusted to 2.2, and the samples were further diluted with a citrate buffer (pH 2.2) for HPLC analysis.
