Methyl linoleate

All the reagents were acquired from Sigma-Aldrich Química S.A. (Madrid, Spain). Anhydrous sodium carbonate (Na₂CO₃), Folin–Ciocalteu reagent, DPPH (2,2-diphenyl-1-picrylhydrazyl), methanol (CH₃-OH), ethanol (C₂H5OH), glacial acetic acid (CH₃-COOH), aluminum trichloride hexahydrate (AlCl₃·6H₂O), sodium acetate trihydrate (CH₃COONa·3H₂O), hydrochloric acid 37% (HCl), iron chloride (III) hexahydrate (FeCl₃ 6H₂O, TPTZ (2,4,6-tripyridyl-s-triazine), ABTS (2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), potassium persulfate (K₂S₂O₈), PBS (phosphate-buffered saline), AAPH (2,2-Azobis(2-methylpropionamidine) dihidrochloride 97%)), fluorescein sodium salt (C₂₀H₁₀O₅Na₂), Trolox 97% (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonsaeure 97%), Tween-20, methyl linoleate (C₁₉H₃₄O₂), iron (II) chloride (FeCl₂), and ammonium thiocyanate (NH4SCN).
The flowing standards epicatechin (C₁₅H₁₄O₆), arbutin (C₁₂H₁₆O₇), ferulic acid (C₁₀H₁₀O₄), sinapic acid (C₁₁H₁₂O₅), p-Coumaric acid (C₉H₈O₃), cinnamic acid (C₉H₈O₂), myricetine (C₁₅H₁₀O₈), kaempferol (C₁₅H₁₀O₆), vanillic acid (C₈H₈O₄), and gallic acid (C₇H₆O₅) were also acquired from Sigma-Aldrich Quimica S.A. (Madrid, Spain).

The analytical-grade methyl esters (Methyl Stearate, Methyl Oleate, and Methyl Linoleate) used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) without further purification. Aluminum coupons (>99.99%) were used as test specimens or working electrodes.

The chemical reagents used for VOO characterization were of GC or HPLC grade. Methanol, acetonitrile, o-phosphoric acid and the Folin-Ciocateu (F-C) reagent were provided from Sigma-Aldrich (Steinheim, Germany), n-hexane from Hipersolv Chromanorm (Pennsylvania, USA), Na₂CO₃ anhydrous from Acros organics (New Jersey, USA) and KOH from Fisher Scientific (Loughborough, UK). Regarding the commercial standards, methyl palmitate, methyl palmitoleate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate, hydroxytyrosol, tyrosol, p-coumaric acid, luteolin, and apigenin were purchased from Sigma-Aldrich (Steinheim, Germany) and Fluka (Steinheim, Germany), vanillic and syringic acid standards were obtained from Acros organics (New Jersey, USA), gallic acid from Fisher Scientific (Loughborough, UK), and oleacein and oleocanthal were supplied by Prof. Prokopios Magiatis (Athens, Greece). Finally, deionized water (18 MΩ cm) from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare the mobile phases for HPLC analysis.

Protamex and Alcalase L 2.4 FG were purchased from Novozymes (Bagsvaerd, Denmark). Sodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), extran neutral MA02, (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic) acid [ABTS]), and potassium persulfate (K₂S₂O₈) were purchased from Merck (Darmstadt, Germany). Iron (II) sulfate (FeSO₄) and 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (Ferrozine) were purchased from Sigma-Aldrich (St. Louis, MO). 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), sodium ascorbate, hydrochloric acid, methyl linoleate, and tween-20 were purchased from Sigma-Aldrich (Steinheim, Germany). Hemin was purchased from Acros Organics (Geel, Belgium). Ethanol (99.9%) was purchased from Chemethyl A/S (Køge, Denmark), and sodium hydroxide was purchased from Sigma-Aldrich (Renningen, Germany).

To dissect the function of PvFAD2 and PvFAD3 in yeast, we constructed yeast expression vectors using homologous recombination strategy. Briefly, the full-length CDS was amplified by PCR using Phanta Max Super-Fidelity DNA polymerase (Vazyme, Nanjing, China). The primers used in this step were listed in Table S1. The PCR products were purified and then ligated into P426-GAP vector (digested with BamH I and EcoR I) using ClonExpress Entry One Step Cloning Kit (Vazyme, Nanjing, China). Finally, the plasmid harbored the PvFAD2 or PvFAD3 was transformed into S. cerevisiae (INVSc1) using Frozen-EZ Yeast Transformation II Kit (Zymo, USA). An empty vector was also transformed into S. cerevisiae as a negative control. Positive clones were obtained via selection on plates of SD medium lacking uracil at 30 °C. Recombinant strains of S. cerevisiae were then transferred into lipid SD medium lacking uracil, and shaken at 30 °C/230 rpm. To test PvFAD3 function, we exogenously added methyl linoleate (Sigma, USA) into media for the supply of C18:2 fatty acid substrates. When cells reached the stationary phase, cells were collected for subsequent analysis.

Millet was purchased from the Jianjun agricultural and agricultural products processing plant (Shijiazhuang, China). High-temperature-resistant amylase was purchased from Novozyme (Denmark). Anhydrous glucose was purchased from Sigma (China). Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate were obtained from Sigma (China). Calcium, zinc, manganese, copper, iron, and magnesium standard stock solutions (10 mg/mL) were purchased from the national center of analysis and testing for nonferrous metals and electronic materials. Amino acids were purchased from Hitachi (Japan). Boron trifluoride-methanol solution (14% BF₃-methanol) was purchased from CNW Technologies (Dusseldorf, Germany). N-hexane (chromatographic purity) was purchased from Fisher Chemical.