Centrifuge 5418

The optimized parameters for each factor from the statistical design experiments were implemented for the scaled-up production of the xylanases. The nutrient salt solution was prepared as previously described (“Medium and cultivation”) and supplemented with the optimized wheat bran and ammonium sulphate concentrations. Erlenmeyer flasks (2 l) containing 400 ml of the medium were each inoculated with two 5 mm fungal plugs from a 5-day-old plate culture and incubated at the optimized parameters in a shaker (New Brunswick Innova 44, Germany). Cultured media were removed after the incubation period and the cell-free supernatant was recovered by centrifuging samples at 16,873 × g for 10 min (Eppendorf centrifuge 5418, Germany). Xylanase activity was determined as described in “Xylanase assay”.

The selected xylanase-producing isolates (after primary screening) were inoculated into potato dextrose broth and incubated at 30°C for 7 days at 200 rpm in a shaking incubator (New Brunswick Scientific, incubator shaker series, Innova 44). The cultured media were then centrifuged (Eppendorf Centrifuge 5418, Germany) at 1,6873 × g for 10 min. Using sterile pipette tips, 5 mm wells were made on substrate agar plates as prepared above. The supernatants containing the crude enzymes were dispensed into the wells and the plates incubated at 30°C for 2 to 3 days, after which they were stained, destained, and analysed as described previously in 2.2.1.

GSH-Au NP samples, quenched at 0 s, 2 s and 5 s were analyzed by ESI-DMA-MS. As excess GSH and boric acid would disturb the detection of the Au clusters, the samples were purified prior to ESI-DMA-MS analysis. The dried samples were dispersed in a washing solution consisting of 20 mL methanol, 25 mL 2-propanol and 5 mL of 20 mM aqueous formic acid by bath sonication for 15 min and then four washing steps were performed. In each washing step the samples were centrifuged at 12 000 rpm (RCF: 12 500g) for two minutes using a “Centrifuge 5418” (Eppendorf, Hamburg, Germany). The supernatant was removed and the sediment was dispersed in the washing solution by bath sonication for 5 min. After the last centrifugation step, the sediment was dispersed in 1 mL aqueous solution of 50 mM ammonium acetate and 10 mM ammonia. Hereby, more than 90% of soluble contaminations were removed. These dispersions were finally transferred to gas phase by electrospray ionization, classified by their mass/charge ratio in the electric field and analyzed by MS. The ESI-DMA-MS setup and technique are described in more details by Lübbert et al.^{37,38 (link)}

The encapsulation efficiency (EE) of CUR-LCNPs was determined by comparing the mass of free, non-encapsulated curcumin to the total mass of curcumin [33 (link)]. An equal volume of acetonitrile was added to the CUR-LCNPs to dissolve them and determine the total mass of curcumin. The sample was then vortexed and sonicated. Next, the sample was diluted and analyzed spectrophotometrically (Multiskan Go, Thermo Fisher Scientific, Waltham, MA, USA) at λ = 424 nm. The mass of non-encapsulated curcumin was determined after an appropriate volume of CUR-LCNPs was centrifuged at 2000× g (Centrifuge 5418, Eppendorf AG, Hamburg, Germany) and washed thrice with PBS (pH 7.4). Afterward, the supernatant was removed carefully, and the remaining curcumin was mixed with acetonitrile (1:1 v/v) and analyzed as described above. The following Equation (1) was used to calculate the encapsulation efficiency: $$EE\%=\frac{totalmassofcurcumin\left[mg\right]-massoffreecurcumin\left[mg\right]}{totalmassofcurcumin\left[mg\right]}\%$$

After 5–6 weeks of growth, the tomato plants were inoculated with 10 μL of the sporangial suspension of P. infestans at four different sites, two on each side of the midrib of the leaf. 96 h post inoculation, infected tomato leaves were excised and then immediately macerated in liquid nitrogen. 100 mg of the crushed powder were vortexed (Multi Reax, Heidolph, Germany), mixed with 1 mL of methanol for 10 min at room temperature, and then centrifuged for 5 min at 12,000× g at 20 °C (Centrifuge 5418, Eppendorf, Hamburg, Germany). The supernatants were stored at −20 °C until analysis [29 (link)].