Thermo scientific sl40r

All A. welwitschiae isolates were grown for 96 h on coarse ground triticale grains, at 28 °C and relative humidity (RH) 60% in solid-state fermentation (SSF) in a Thermostat chamber (HPP IPP plus; Memmert USA, LLC; Eagle, WI, USA). Triticale (×Triticosecale sp.) “Odisej” line was obtained from the Institute of Field and Vegetable Crops, Novi Sad, Serbia. After 96 h, the substrates were covered with white mycelium and there was no sporulation. Fermentation extracts for mycotoxin analysis were obtained by extraction with 100 mL of 25 mmol/L acetic buffer, pH=4.5, and shaking for 2 h at 150 rpm, with subsequent centrifugation at 13 257xg (Thermo Scientific SL40R; Thermo Fisher Scientific, Waltham, MA, USA) and microfiltration (glass microfiber filters GF/B, 1.0 μm; Whatman, GE Healthcare Life Sciences, Buckinghamshire, UK). Protein concentrations in fermentation extracts were determined according to Bradford (39 (link)). Inulinase activity was assayed as described above (section Screening of fungal inulinase producers), at 50 °C for 15 min. Specific inulinase activity was defined as U per mg of proteins. Productivity of inulinase during fermentation was defined as U/(mL·h).

C-phycocyanin was obtained by exhaustive extraction from the biomass of cyanobacterium Arthrospira platensis B-12619 (Russian National Collection of Industrial Microorganisms). A. platensis was grown in modified Zarrouk medium [25 (link)] consisting of (g/L): NaHCO₃, 13.61; Na₂CO₃, 4.03; KH₂PO₄, 0.5; NaNO₃, 2.5; K₂SO₄, 1.0; NaCl, 1.0; MgSO₄·7H₂O, 0.2; CaCl₂, 0.03; FeSO₄·7H₂O, 0.01; and Na₂-EDTA·2H₂O, 0.08 in a batch mode. The initial pH was 9.25 ± 0.05. The cultivation was performed in 500 mL Erlenmeyer flasks, containing 200 mL of the Zarrouk medium. Flasks were kept in shaker-incubator Innova 42R (Eppendorf New Brunswick) set at 140 rpm under 30 °C, continuous lighting, and PAR of 16 ± 2 μmol/(m²·s). The optical density of the cyanobacteria suspension at 750 nm (OD₇₅₀) was used to control biomass growth. The initial OD₇₅₀ was 0.05 ± 0.01. The cyanobacteria biomass was harvested at the end of the exponential growth phase by centrifugation at 12,300× g for 15 min at 25 °C (Thermo Scientific SL40R, Thermo Fisher Scientific, Waltham, MA, USA). The biomass was washed twice with distilled water and re-centrifuged to remove adsorbed salts [26 (link),27 (link)] and stored at −20 °C.

Extraction followed the procedure described by Camenzuli et al., 2018 [20 (link)], in accordance with the QuEChERS method. In short, 2.5 g of sample material was weighed, to which 7.5 mL water, 10 mL of extraction solvent (acetonitrile (Biosolve HPLC Supra Gradient)/acetic acid (Merck, Darmstadt, Germany) 99:1 (v/v)) and 25 μL of a 10µg/mL solution of ¹³C-caffeine (Sigma Aldrich, St. Louis, MO, USA) was added. After shaking (Heidolph, Reax 2), 4 mg of dried magnesium sulfate (MgSO₄) was added, and the mixture was vortexed for 1 min and subsequently centrifuged (10 min at 3500 rpm, Thermo Scientific SL 40 R, Thermo Fisher Scientific, Waltham, MA, USA). Finally, 200 μL of sample extract was transferred to polypropylene vials, and 200 μL water was added. Samples were stored at 4 °C until analysis. Quantification took place through standard addition.