Heptadecanoic acid

For fatty acid methyl esters (FAMEs) analysis in M. gaditana, approximately 1 × 10⁸ total cells were hydrolyzed and methyl-esterified in 300 μl of 2% H₂SO₄ in methanol for 2 h at 80°C. Prior to the reaction, 50 μg of heptadecanoic acid (Alfa Aesar, USA) was added as internal standard. After esterification step, 300 μl of 0.9% (w/v) NaCl solution and 300 μl of hexane were added and mixed thoroughly for 20 s. To separate both the phases, samples were centrifuged at 3,000 xg for 3 min. One microlitre of hexane layer was injected into a 7890A gas chromatography (Kaczur et al.) - mass spectrometry (MS) system (Agilent 7000 GC/MS triple quadrupole system) (Schuhmann et al., 2014 (link); Duong et al., 2015 (link)). The running conditions for GC-MS were described by Agilent’s RTL DBWax method (Brown, 1991 (link); Shaikh et al., 2019 (link)).

Three kinds of Japanese soy sauce were purchased online (Taobao) and divided into three groups. Koikuchi Shoyu was named S1, Usukuchi Shoyu S2, and Amakuchi Shoyu S3. GC-grade of 2-octanol and N, O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA + 1% TMCS, 99:1) were purchased from Sigma (Sigma Aldrich Co., St. Louis, MO, United States). N, N-dimethylformamide (DMF) (99%) was purchased from damas-beta (Adamas Reagent, Ltd.). Heptadecanoic acid (98%) was purchased from Alfa Aesar (Ward-Hill, MA, United States).