Yeast extract

D(+)-Glucose anhydrous for biochemistry (M_w = 180.16 g/mol, C₆H₁₂O₆), disodium hydrogen phosphate (M_w = 141.96 g/mol, Na₂HPO₄), acetic acid (glacial) (100% purity, M_w = 60.05 g/mol, CH₃COOH), sodium hydroxide (M_w = 40.00 g/mol, NaOH), sulfuric acid (95–97% purity, M_w = 98.08 g/mol, H₂SO₄), formic acid (98–100% purity, M_w = 46.03 g/mol molecular, CH₂O₂), and glycerol (M_w = 92.1 g/mol, C₃H₈O₃) were purchased from Merck KGaA, Darmstadt, Germany. Peptone from animal tissue from meat with suitability for cell and plant culture, citric acid (99% purity, M_w = 192.12 g/mol, C₆H₈O₇), gelatin from porcine skin (powder, gel strength ~300 g Bloom, Type A, BioReagent, for electrophoresis, suitable for cell culture), polycaprolactone (PCL) with 80,000 average M_n were bought from Sigma-Aldrich, St. Louis, MO, USA. Yeast extract and agar bacteriological were purchased from Biolife, Italia. Hydroxyapatite (M_w = 502.31 g/mol) from was bought from Oerlikon Metco, Wohlen, Switzerland.

Sterile, non-coated PTFE membranes and saliva-coated membranes were used to test bacterial adhesion. Membranes were placed in wells of 96-well microtiter plates, part of the membrane was conditioned for 4 h at 30 °C in 50% artificial saliva with composition as described elsewhere [78 (link)]. Briefly, artificial saliva included porcine stomach mucins (Sigma-Aldrich, Burlington MA USA) (0.25% w/v), sodium chloride (Chem-Lab NV, Zedelgem, Belgium) (0.35 w/v), potassium chloride p.a. (Kemika, Zagreb, Croatia) (0.02 w/v), calcium chloride dihydrate p.a. (Kemika, Zagreb, Croatia) (0.02 w/v), yeast extract (Biolife, Milan, Italia) (0.2 w/v), lab lemco powder (Oxoid, Basingstoke, UK) (0.1 w/v), proteose peptone (Biolife, Milan, Italia) (0.5 w/v) in ddH₂O (Sigma-Aldrich, Burlington MA USA), and urea p.a. (Kemika, Zagreb, Croatia) 0.05% (v/v). Artificial saliva was removed and 200 µL of individual bacterial suspension in protein-rich, BHI liquid medium was added in a concentration of 10⁷ CFU/mL. After incubation under anaerobic conditions for 4 h, the planktonic bacteria in the medium were removed and membranes were washed 3× in sterile saline solution. Adhered bacteria were detached by treatment in an ultrasonic bath (Bactosonic, Bandelin, Germany) at 40 kHz for 1 min. In order to quantify the adherent bacteria, ten-fold dilutions were plated on blood agar and CFU/mL was determined.

All chemicals were of reagent-grade quality or higher and used without further purification; solvents were used as received. Ethyl acetate (EtOAc), hydrogen peroxide (H₂O₂), dimethyl sulfoxide (DMSO), HBr, cholesterol, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), NaHCO_3, KH₂PO₄, Na₂HPO₄, and other salts were purchased from Sigma Aldrich, Steinheim, Germany. Yeast extract, NaCl, and agar were purchased from Biolife, Milan, Italy. Methanol (MeOH), glycerol, Nutrient Broth (NB), and Na₂SO₄ were purchased from Fisher Scientific, Loughborough, UK. Tryptone, peptone, Tryptic Soy Broth (TSB) were purchased from Torlak, Belgrade, Serbia. KCl and beef extract were purchased from Becton Dickinson, New Jersey, US. Hexane was purchased from J. T. Baker, Deventer, The Netherlands. MgSO₄∙7H₂O was purchased from Acros Organics, Geel, Belgium. Instant Ocean^® Salt was purchased from Instant Ocean, Blacksburg, US.

The Z. tritici strain 60.2, characterized and kindly provided by the Department of Agri-Food Science and Technology, University of Bologna, Italy, was used in this study. The fungus, stored at −80 °C, was preliminarily grown on a potato dextrose agar (PDA, Biolife Italiana, Milan, Italy) for 30 days at 22 °C in the dark to produce mycelium. Mycelium plugs for in vitro experiments were obtained from solid media as described in Section 2.3. Conidia for bread wheat artificial inoculation were obtained by using a liquid yeast–glucose (GY) medium (30 g/L glucose (Sigma-Aldrich, St. Louis, MO, USA), 10 g/L yeast extract (Biolife Italiana, Milan, Italy)) inoculated with a mycelium plug (5 mm diameter) and grown in an orbital shaker (250 rpm) for 14 days at 21 °C under a photoperiod of 10 h. Conidia were collected by centrifugation at 3000 rpm for 15 min at 4 °C; their concentration was calculated by using a hemocytometer, and they were immediately used for artificial inoculations.