Acetic anhydride

HiCrome Bacillus agar was purchased from Sigma-Aldrich (Seelze, Germany), and a physiological solution (0.9% NaCl) was obtained from Panreac AppliChem (Darmstadt, Germany). The glacial acetic acid from Merck (Darmstadt, Germany), acetic anhydride from Panreac (EU), sulphuric acid from JMGS (José Manuel Gomes dos Santos, Lisbon, Portugal), ethanol from Fisher Scientific (Loughborough, UK), and glycerin from VWR chemicals (Fontenay-sous-Bois, France) were used for the pollen analysis.

The honey samples’ pollen was analysed using the methodology described by Erdtman, (1969) [29 ]. Ten grams of the sample was dissolved in 20 mL of distilled water. The obtained mixture was centrifuged at 2000 rpm for 5 min (Eppendorf Centrifuge 5804R, Hamburg, Germany) and the supernatant was discarded. After that, 5 mL of glacial acetic acid (Merck, Darmstadt, Germany) was added and centrifuged at 2000 rpm for 5 min. Then, acetolysis of the pollen sediments was carried out, using a mixture of 9:1 of acetic anhydride (Panreac Applichem, Barcelona, Spain) and sulphuric acid (José Manuel Gomes dos Santos, Lisbon, Portugal), in a water bath at 100 °C for 2 min. The mixture was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. After carefully washing the sediment with 5 mL of water containing 3 drops of ethanol (Fisher Scientific, Loughborough, UK) and centrifuging it, 5 mL of glycerin–water (50%) was added, and the sediment was mounted in gelatin-glycerinate (VWR chemicals, Fontenay-sous-Bois, France). The frequency of the appearance of the different pollen types was divided into the following classes: dominant pollen (>45% of the pollen spectrum); accompanying pollen (15–45%); important pollen (3–15%); minor pollen (1–3%); and other pollen (≤1%).

Acetic anhydride, bismuth nitrate, and sodium nitrite were obtained from Panreac^® (Barcelona, Spain). Diphenylboric acid-β-ethylamino ester, fast blue B salt, gallic acid, iodine, oleanolic acid, polyethylene glycol-4000, protocatechuic acid, and vanillin were obtained from Sigma-Aldrich^® (Steinheim, Germany). Ethanol was obtained from Aga^® (Prior Velho, Portugal). Glacial acetic acid and sulfuric acid were obtained from Chem-Lab^® (Zedelgem, Belgium). Acetone, Folin–Ciocalteu reagent, hydrochloric acid 37% extra pure, iron trichloride, perchloric acid, potassium iodate GR, sodium carbonate, sodium hydroxide, and TLC cellulose plates (catalog number 105552) were obtained from Merck^® (Darmstadt, Germany). Butanol-1 and methanol were obtained from Fisher Chemicals^® (Leicestershire, UK). Ferrous sulfate was obtained from May and Baker LTD^® (Dagenham, UK). The (+)-Catechin (hydrate) was obtained from Cayman Chemical Company^® (Ann Arbor, MI, USA). Aluminum chloride was obtained from Fluka^® (Buchs, Switzerland). Acetonitrile was obtained from Honeywell Riedel-de Haën™ (Seelze, Germany). Formic acid 99–100% was obtained from VWR Chemicals (Fontenay-sous-Bois, France). All used reagents were of analytical grade.