Ammonium iron 3 citrate

50 μM iron solution was prepared by dissolving ammonium iron (III) citrate (Sigma-Aldrich, St. Louis, MO, USA) into serum- and antibiotic-free DMEM. A pre-treatment of 500 μM SDG (kindly provided by Dr. K Prasad) was prepared by dissolving SDG into serum- and antibiotic-free DMEM. For each treatment cells were incubated for 24 hours at 37°C, 5% CO₂ and 100% humidity.

All chemicals used in the synthesis of compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA) were used as received without any prior purification. Penicillin/streptomycin (5000 U/mL), Trypsin-EDTA (0.25%), phenol red, and Eagle’s Minimum Essential Medium (EMEM) were purchased from Gibco, Life Technologies (Paisley, UK). The Caco-2 (ATCC HTB-37; Manassas, VA, USA) and HT-29 (ATCC HHTB-38) cell lines were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Chemicals used for the in vitro culture of G. lamblia were sourced as follows: glucose and L-cysteine (ACROS organics, Thermo Fisher Scientific, Scoresby, VIC, Australia), ammonium iron (III) citrate, ascorbic acid (Sigma-Aldrich, Castle Hill, NSW, Australia), potassium dihydrogen orthophosphate (UNIVAR, Ingleburn, NSW, Australia), bovine bile (Fluka analytical (BD)), and di-potassium hydrogen orthophosphate (Fronine Laboratories and Supplies, Riverstone, NSW, Australia).

Recombinant Bet v 1a was incubated with an equimolar concentration of iron (ammonium iron (III) citrate, Sigma) and a 3-fold molar concentration of catechol because three catechols are necessary for hexadental binding of iron in the LCN2 pocket against deionized water.

Methanol (HPLC grade) was purchased from J. T. Baker (Deventer, The Netherlands); toluene, phosphoric acid (85%) and hydrochloric acid (37%) were obtained from Honeywell (Charlotte, NC, USA); 2, 2, 2-trimethyl pentane (≥99%, ACS reagent), 1-butanol, sodium chloride, sodium hydroxide, ammonium iron (III) citrate, carboxymethyl cellulose (CMC), alpha-pinene (98%), beta-pinene (99%), myrcene (99%), limonene (99%), linalool (97%), alpha-terpineol (99%), beta-cutronellol (99%), nerol (98%), geraniol (98%), geranyl acetate (98%), beta-caryophyllene (99%), alpha-humulene (CRM) and caryophyllene oxide (95%) were purchased from Sigma Aldrich (St. Louis, MO, USA); ethylenediaminetetraacetic acid (EDTA) was obtained from Merck (Kenilworth, NJ, USA); international calibration extract 4 (ICE4), DCHA-Iso and DCHA-Humulinones were obtained from Labort Veritas (Zürich, Switzerland). Light Malt Extract was procured from Muntons (Lombard, Chicago, IL, USA).

p-Nitrophenol (pNP), p-nitrophenyl- α-D-glucopyranoside (pNPαG), p-nitrophenyl- β-D-glucopyranoside (pNPβG), p-nitrophenyl-β-D-cellobioside (pNPβC), p-nitrophenyl- α-L-rhamnopyranoside (pNPαR), p-nitrophenyl- β-D-xylopyranoside (pNPβX), p-nitrophenyl-β-D-galactopyranoside (pNPβGal), o-nitropheyl-β-D-galactopyranoside (o NPβGal), 4-methylumbelliferyl- β-D-glucopyranoside (MUβG), isoflavone glycoside standards (genistin and daidzin), isoflavone aglycone standards (genistein and daidzein), arbutin, avicel, esculin, ammonium iron (III) citrate, cellobiose, maltose, lactose, sucrose, and xylan were obtained from Sigma (St. Louis, MO, USA). All the different chemicals and reagents were used at analytical grade.