Glycerol

Whey protein concentrate (WPC, 85% protein content) and whey protein isolate (WPI, 90% protein content) manufactured from sweet cheese whey using cross-flow membrane filtration were purchased from Volac International Ltd. (Hertfordshire, UK). Calcium chloride, hydrogen peroxide, disodium phosphate, monosodium phosphate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulphate, potassium ferricyanide, trichloroacetic acid, ferric chloride, iron sulphate, tris(hydroxymethyl)aminomethane, pyrogallol, ortophenantroline, L929 murine fibroblasts, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), resazurin, l-glutamine, penicillin, streptomycin, and all other cell culture reagents were purchased from Sigma Aldrich (Darmstad, Germany). Glycerol, ammonia water, hydrochloric acid, sodium hydroxide, chloroform, ethyl acetate, ethanol and methanol were supplied from Chempur (Piekary Śląskie, Poland). Cell culture plasticware was purchased from VWR International (Radnor, PA, USA). All chemicals were of analytical grade.

Portions of 1.00 g of glycerol (Chempur, Karlsruhe, Germania) and 8.63 g of demineralized water were weighed into a glass beaker. The whole mixture was heated to 80 °C. Then, hydroxycellulose (Sigma-Aldrich) was gradually added in the amount of 0.25 g, all the while intensively mixing the obtained formulation with a glass dipstick. After obtaining gel consistency, the system was cooled to 40 °C, then Microcare^® SB (Thor GmbH) was added and the whole mixture was stirred for 5 min. In the next step, once the temperature was below 30 °C, the active ingredient, curcumin (Sigma Aldrich), was added to the hydrogel in an amount of 0.10 g (1 wt.%). The obtained formulation was stirred for 10 min to stabilize the system.

Native potato starch (29 wt% amylose content, 15.5 wt% moisture) as a film-forming material was supplied by Nowamyl S.A., hoary willowherb Epilobium parviflorum (E) was purchased from Dary Natury (Grodzisk, Poland) and glycerol (anhydrous) used as a plasticizer was obtained from Chempur (Piekary Śląskie, Poland). DPPH–(2,2-diphenyl-1-picrylhydrazyl radical) (Tokyo Chemical Industry Co., Tokyo, Japan) and methanol (pure, Chempur) were used for the evaluation of antioxidative properties. Folin-Ciocalteau reagent (Eurochem BGD, Tarnów, Poland), gallic acid (p.a. 97.5–102.5%, Sigma-Aldrich, Milan, Italy) and Na₂CO₃ (anhydrous, reagent grade, Scharlau, Barcelona, Spain) were applied for the measurement of total phenolic compounds content in the extract and the films.

Escherichia coli strains were cultivated in LB medium according to standard protocols¹⁷ . Rich yeast extract peptone glucose (YPD) medium, containing 1% (w/v) yeast extract, 1% (w/v) peptone and 2% (w/v) glucose, was used to obtain yeast biomass for DNA extraction and inoculum preparation. Medium containing YNB without amino acids (Sigma-Aldrich) supplied with 2% (w/v) glucose was used for yeast inoculum preparation.
During shake-flask experiments the cultures were grown in 0.3 L baffled flasks containing 0.03 L or 0.05 L medium on a rotary shaker (CERTOMAT IS, Sartorius Stedim Biotech) at 28 °C and 240 rpm. Erythritol utilization rate was examined on medium containing YNB and 5% (w/v) or 10% (w/v) erythritol (YNB-e medium). Erythritol synthesis was conducted in Erythritol Synthesis Medium (ESM medium) containing 100 g/L glycerol (Chempur, Poland), 2.3 g/L (NH₄)₂SO₄ (Chempur), 1 g/L MgSO₄ × 7H₂O (Chempur), 0.23 g/L KH₂PO₄ (Chempur), 26.4 g/L NaCl (Chempur), 1 g/L yeast extract (Merck, Germany) and 3 g/L CaCO₃, pH 3.0. CaCO₃ was added separately to each flask after establishing pH 3 in order to prevent a fall of pH value.

Glycerol (pure per analysis grade, ppa grade), gelatine (ppa grade), sodium hydroxide 0.1 M analytical weighed amount, sodium acetate trihydrate (ppa grade), potassium hexacyanoferrate (II) trihydrate (ppa grade), and zinc acetate dihydrate (ppa grade) were purchased from Chempur (Piekary Śląskie, Poland). The Phadebas Honey Diastase Test tablets were bought from Magle Life Sciences (Malmö, Sweden) and glacial acetic acid (ppa grade) from Pol-Aura (Zawroty, Poland). Methanol and hydroxymethylfurfural were obtained from J.T. Baker (Gliwice, Poland) and Merck (Darmstadt, Germany), respectively, and were of HPLC grade. For the analyses, distilled or ultrapure water from the Milli-Q system (Merck, Darmstadt, Germany, resistivity 18.3 MΩ cm) was used.

1,1-diphenyl-2-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-acid) sulfonic acid (ABTS), potassium persulfate, sodium carboxymethyl cellulose (Mw = 250,000; DS = 0.7, 0.9, 1.2) were purchased from Sigma-Aldrich (Darmstad, Germany). Glycerol, ammonia water, chloroform, ethanol and methanol were from Chempur (Piekary Śląskie, Poland). Peptone water and MacConkey medium were from Scharlau Chemie (Barcelona, Spain). Silver (III) nitrate was from POCh (Gliwice, Poland). Chapman’s medium and plate count agar were from Merck (Dramstadt, Germany). All reagents were of analytical grade. The microorganisms used to evaluate the antimicrobial properties of the films were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The strains used were Escherichia coli ATCC8739 and Staphylococcus aureus ATCC12600, Candida albicans ATCC14053, Pseudomonas aeruginosa ATCC 39327, Bacillus cereus ATCC13061.

Portions of 1.00 g of glycerol (Chempur, Karlsruhe, Germania) and 8.23 g of demineralized water were weighed into a glass beaker. The whole mixture was heated to 80 °C. Then, hydroxycellulose (Sigma-Aldrich, Poznań, Poland) was gradually added in the amount of 0.25 g, all the while under intensive stirring of the obtained formulation with a glass dipstick. After obtaining the gel consistency, the system was cooled to 40 °C, then Microcare^®SB (Thor GmbH, Speyer, Germany) was added and the contents were stirred for 5 min. In the next step, once the temperature was below 30 °C, the active ingredient curcumin (Sigma-Aldrich, Poznań, Poland) was added to the hydrogel in an amount of 0.50 g (5 wt.%). The obtained formulation was stirred for 10 min to stabilize the system.