Sodium benzoate

The following materials were used: Lutein (pharmaceutical secondary standard certified reference material; PHR1699; Sigma Aldrich, St. Louis, MO, USA); powdered marigold (Tagetes erecta L.) flower lutein extract (≥20.0% lutein content; henceforth as ‘lutein extract’; Gonmisol, Barcelona, Spain); lutein esters extract (≥20.0% lutein esters content; henceforth as ‘lutein esters extract’; Parry Nutraceuticals, Chennai, Tamil Nadu, India); PSM (59924 [Tween 80]; Sigma Aldrich); MCT oil (Miglyol 812N [F]; IOI Oleochemical Nutrition, Hamburg, Germany); Lipoid H20 (fat-free sunflower lecithin with 20% phosphatidylcholine; Lipoid, Ludwigshafen am Rhein, Germany); xylitol (≥99.0%; X3375; Sigma Aldrich); sodium benzoate (≥99.0%; 71300; Sigma Aldrich); orange concentrate (14.66.001; Presad, Mirna, Slovenia); orange aroma (02 940; Frutarom Etol, Škofja vas, Slovenia); α-tocopherol (vitamin E; ≥87.25%; Shanghai Freemen, Shanghai, China); KOH (Itrij d.o.o., Kropa, Slovenia); NaCl (≥99.5%; 71380; Fluka, Seelze, Germany); acetonitrile (HPLC grade, ≥99.9%; 34851; Honeywell, Seelze, Germany); methanol (HPLC grade, ≥99.8%; 106018; Merck, Darmstadt, Germany); ethanol (absolute; 111727; Merck); ethyl acetate (≥99.5%; 109623; Merck); and n-hexane (≥99.0%; 104367; Merck). Ultrapure water (18.2 MΩ cm, at 25 °C) was used to prepare all of the aqueous solutions, and for all of the other requirements.

We used acid-soluble chitosan (CAS) (food and medical grade with a deacetylation degree of >90%, made of crab shell or shrimp shell, Bio Chitosan, Indonesia), water-soluble chitosan (CWS) (food and medical grade with a deacetylation degree of >90%, made of crab shell or shrimp shell, Bio Chitosan, Indonesia), hEGF obtained from recombinant results from previous studies [40 ], soy lecithin (food and medical grade, Lansida, PT. Saraswanti Indo Genetech, Indonesia), cholesterol monohydrate (sigma grade with purity of ≥99%, made of sheep wool, Sigma Aldrich, St. Louis, MO, USA), liposome KIT (sigma grade, made of lyophilized egg yolk, L4395, Sigma Aldrich, USA), propylene glycol (PG) (PT. Brataco, Indonesia), glycerin (PT. Brataco, Indonesia), tween 80 (Merck, Kenilworth, NJ, USA), phosphate-buffered solution (PBS) (PT. Brataco, Indonesia), potassium bromide (Merck, Kenilworth, NJ, USA), methylparaben (Sigma Aldrich, St. Louis, MO, USA), acetic acid (Sigma Aldrich, St. Louis, MO, USA), benzoic acid (Sigma Aldrich, St. Louis, MO, USA), and sodium benzoate (Sigma Aldrich, St. Louis, MO, USA).

DMEM (Dulbecco’s Modified Eagle Medium), FBS (fetal bovine serum), Collagenase Type I were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Restriction enzyme BglII and HindIII were bought from New England Biolabs (Ipswich, MA, USA). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). Antibodies for C/EBPα, PPARγ and FABP4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for HSL, p-HSL, ERK, p-ERK, S6 and p-S6 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Methylisobutylxanthine, dexamethasone, Insulin, Oil Red O, Quinine hydrochloride dehydrate, Caffeine, Salicin, 6-propyl-2-thiouracil (PROP), Sucrose octaacetate, Hesperidin and Sodium Benzoate were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). All tested bitter compounds were selected based on common knowledge and previous publications mentioned above. The bitter agonists were either dissolved in DPBS or DMSO, and final DMSO concentration did not exceed 0.1% (v/v) to avoid toxic effect on cells.

3-Mercaptopropyltrimethoxysilane (95%), tetraethyl
orthosilicate (98%), (3-glycidyloxypropyl)trimethoxysilane (98%),
chitosan (low molecular weight), 3-aminophenylboronic acid, (3-maleimido)propyl-functionalized
silica gel, ethanol (99%), fluorescein (free acid), triethylamine
(99.7%), xanthan gum, sodium benzoate (99%), sorbitol (99%), and glycerol
were obtained from Sigma-Aldrich (UK). (3-Acryloxypropyl)trimethoxysilane
(96%) was obtained from Gelest (Morrisville, USA). Ammonium solution
(S.G. 0.88, 35%) was obtained from Fisher Scientific. Aerosil R972
Pharma was obtained from Laurence Industries (UK).

The chemical compounds tested in this study and their main properties are listed in Table S4 in the supplemental material. Sodium benzoate, phenol, and 1-octanol were purchased from Sigma-Aldrich at a purity of more than 95%. Methyl jasmonate, musk xylene, and myrcene were provided by Firmenich SA, Geneva, Switzerland. To avoid introducing additional carbon from solvents, we dosed all test chemicals either directly from the pure compound to the assays or from a 10- to 100-fold concentrated aqueous solution in artificial lake water medium (ALW; see Table S2 in the supplemental material). Direct dosing of pure chemicals in liquid form (i.e., methyl jasmonate, myrcene, and 1-octanol) to the test bottles was achieved with an ultra-accurate glass displacement microinjection pipet (Drummond Scientific, USA) delivering volumes as low as 30 nl. Musk xylene was dosed directly into the test bottles after being weighed on an analytical balance. Benzoate, phenol, and 1-octanol were prepared in aqueous stock solutions of up to 1 g C · liter⁻¹ and then volumetrically diluted to the final initial compound concentration (depending on the experiment, between 0.1 mg C · liter⁻¹ and 1 g C · liter⁻¹) in the test bottles.