Hexadecane

Overnight M. xanthus cultures (50 mL CYE in 250 mL flasks) were inoculated at an initial OD₆₀₀ of 0.05 and grown at 32°C with shaking (220 rpm) to saturation (OD₆₀₀, approximately 5.0–7.0). Cultures were transferred to a 50-mL conical tube and sedimented at 7,000g (25 minutes, 22°C, JA-17 rotor). Supernatants were decanted into a syringe, passed through a 0.22- μm filter to remove remaining cells, and transferred (4 mL) to a quartz cuvette, followed by addition of 300 μL hexadecane (Sigma) colored with Sudan Black dye (0.1 g of Sudan Black powder per 50 mL of hexadecane). Each cell-free supernatant sample was vigorously mixed with the colored hexadecane 250 times over 2 minutes (via aspiration/ejection with a p1000 micropipette). Cuvettes were then immediately inserted into a spectrophotometer, with continual, rapid manual attempts made to obtain an initial OD₆₀₀ reading, with this time recorded. After obtaining an initial OD₆₀₀ reading, subsequent OD₆₀₀ readings were manually carried out at 20-second intervals over 10 minutes to monitor the rate of emulsion clearance. All OD₆₀₀ readings for each time course were normalized with respect to the initial OD₆₀₀ value detected for each sample.

For determination of the retention indices (RI), the following hydrocarbons were used: octane (≥99%), nonane (99%), decane (≥99%), undecane (≥99%), dodecane (99%), tridecane (≥99%), tetradecane (≥99%), hexadecane (≥99%), heptadecane (99%), octadecane (99%), nonadecane (99%), eicosane (99%), heneicosane (≥99.5%), docosane (99%), tricosane (99%), tetracosane (99%), pentacosane (99%), hexacosane (99%), octacosane (99%), and triacontane (99%) purchased from Merck KGaA (Darmstadt, Germany). Hexane (GC grade) purchased from Thermo Fisher Scientific GmbH (Bremen, Germany) was used for dilution of the EOs.

To determine the retention indices (RI), the following hydrocarbons were used: nonane (≥99%), decane (≥99%), undecane (≥99%), dodecane (99%), tridecane (≥99%), tetradecane (≥99%), and hexadecane (≥99%), purchased from Merck KGaA (Darmstadt, Germany). Dichloromethane (Sigma Aldrich, Steinheim, Germany) was used for the dilution of the EO.

For the analysis, LC-MS-grade solvents and reagents were used. Formic acid, ammonium formate, tert-Butyl methyl ether, BSTFA, methoxyamine, Hexadecane, and tridecanoic acid were procured from Merck (Darmstad, Germany), pyridine, water and acetonitrile were from VWR (Milano, Italy), and methanol and isopropanol were from Scharlab (Barcelona, Spain). As internal standards for lipidomics analysis we employed the SPLASH Lipidomix^® [PC 15:0-18:1(d7); PE 15:0-18:1(d7); PS 15:0-18:1(d7); PG 15:0-18:1(d7); PG 15:0-18:1(d7); PA 15:0-18:1(d7); LysoPC 18:1(d7); LysoPE 18:1(d7); Chol Ester 18:1 (d7); 18:1(d7) MG; DG 15:0-18:1(d7); TG 15:0-18:1(d7)-15:0; SM 18:1(d9); Cholesterol (d7)]; DG 12:0-12:0 (Avanti Polar Lipids, Alabaster, AL, USA); and 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA) (Cayman Chemicals, Ann Arbor, MI, USA).

Methanol, acetonitrile and hexadecane (HD) were obtained from Merck chemicals, Darmstadt, Germany; orthophosphoric acid from Riedel-de-Haen Chemical, Seelze, Germany; potassium hydroxide from Scharlau Chemical, Barcelona, Spain; methyl paraben (MP), butyl paraben (BP), caffeine (CF) and triethylamine from Sigma Chemical Co., Gillingham, UK; oleic acid (OA) and liquid paraffin (LP) from Sigma, Taufkirchen, Germany; isopropanol from BDH Laboratory Supplies, Poole, UK; isopropylmyristate (IPM) and isohexadecane (IHD) from Uniqema, Klang, Malaysia; and silicone membrane from Samco Ltd., Nuneaton, UK. Whatman no. 1 filter paper was purchased from Whatman International Ltd, Maidstone, UK. High-density polyethylene (HDPE) membrane (pharmaceutical grade package material) and polyurethane (PU) membrane were donated by TQ pharma, Amman, Jordan and Exopack, Wrexham, UK, respectively.

Flaxseed oil cake (FOC) was purchased from ACS Sp. z o.o. (Bydgoszcz, Poland). The proximate composition of FOC (based on supplier information) was: solids—80.50%, including: proteins—41.97%; carbohydrates—27.99%; fiber—6.29%; fat—6.11%; ash—4.50%. Lacticaseibacillus rhamnosus GG (ATCC53103), was procured from ATCC (Manassas, VA, USA). Buffered peptone water, microbiological agar, MRS agar and broth were purchased from Oxoid (Basingstoke, UK). Potassium chloride, sodium chloride, potassium thiocyanate, disodium hydrogen phosphate, monosodium dihydrogen orthophosphate, calcium chloride, sodium hydrogen carbonate, hydrochloric acid, ammonium chloride, sodium peroxide, urea, α-amylase, uric acid, mucin from porcine stomach (type II), glucose, glucuronic acid, glucosamine hydrochloride, bovine serum albumin (BSA), pepsin, pancreatin, oxgall, Triton X-100, cholesterol, ethanol, ninhydrin, glacial acetic acid, cadmium chloride and hexadecane were purchased from Merck Chemical (Saint Louis, MI, USA). All reagents were of analytical grade.

Acetonitrile (≥ 99.9%), methanol (99.9%), fenofibrate, warfarin, porcine pancreatin (8 x USP specifications), bovine serum albumin, dimethyl sulfoxide (DMSO, ≥ 99.9%) D-α-Tocopherol polyethylene glycol succinate (TPGS), hexadecane (anhydrous, 95%), Tris-maleate, 4-bromophenol boronic acid, olive oil, Kolliphor EL (macrogolglycerol ricinoleate), Kolliphor RH40 (macrogolglycerol hydroxystearate), Tween 85, and Carbitol (diethylene glycol monoethyl ether) were purchased from Merck (Darmstadt, Germany). Felodipine was kindly donated by Lundbeck Pharma (Valby, Denmark). Captex 355 and Capmul MCM EP (Abitec, Janesville, WI, USA) were kindly donated by Barentz (Odense, Denmark). Miglyol 812 N was obtained from IOI Oleo (Wittenberge, Germany). FaSSIF/FeSSIF/FaSSGF powder were bought from Biorelevant.com (Croydon, UK). Lucifer Yellow CH dilithium salt was obtained from Biotium (Fremont, CA, USA). Lecithin 20% soy PC extract was obtained from Avanti Polar Lipids (Alabaster, AL, USA). GIT-0 lipid solution and Acceptor Sink Buffer were purchased from Pion (Billerica, MA, USA). N-dodecane (≥ 99%) was obtained from Alfa Aesar (Lancashire, UK). Ethanol (99.5%, denatured with 0.4% isopropyl alcohol) was obtained from Solveco (Rosersberg, Sweden). All water used was of grade I from a Milli-Q lab water purification system (Merck).