Uv 1700

The activity of the free and immobilized HRP was measured first using the standard method with substrate pyrogallol and H₂O₂ in a reaction medium [50 (link)] consisting of 3 mL of 0.013 M pyrogallol solution in the potassium phosphate buffer (pH 7.0; 0.1 M). Briefly, 2 mg of immobilized enzyme (0.05–10 mg HRP/g_carrier) and 30 µL of H₂O₂ (3% v/v) were placed in the cuvette. By adding 30 µL of H₂O₂ (3% v/v), the reaction was started. The oxidation of pyrogallol (yellow) by HRP-MAB to purpurogallin (dark brown) was monitored at 420 nm absorbance (ε(420 nm) = 12 mM⁻¹ cm⁻¹) using a UV–Vis spectrophotometer (UV Shimadzu 1700, Shimadzu Corporation, Kyoto, Japan). The change in absorbance in the sample, against the reagent blank, was recorded each 30 s for 3 min. One unit of activity was defined as the amount of biocatalyst that formed 1.0 mg of purpurogallin from pyrogallol in 20 s at pH 7.0 and 20 °C [51 ]. The activity of HRP-MABs immobilized under optimal conditions was then compared to the activity of the HRP covalently immobilized on the alginate beads in the absence of magnetite under the same conditions, encapsulating HRP into Mag-alginate beads with the same initial enzyme/carrier ratio and free HRP using the standard method.

Inhibition of biofilm formation of the fungal pathogen CRP1 by EA extract of CNE6 was checked in 24-well polystyrene cell culture plates. Similar to the previous description, fungal conidia were inoculated in 1 mL of ME broth and then added to each well. EA extract of CNE6 dissolved in 10% sterilized DMSO (100 μL) was added at different concentrations (0 to 1,000 μg/mL) to different wells and incubated for 24 h at 28°C. After that, broth was removed from each well and the wells were carefully washed twice with PBS. Biofilms were fixed with 95% ethanol for 15 min at 37°C and finally stained with 1 mL of 0.1% safranin (54 (link)) and left for 5 min. Excessive safranin was eluted from the wells by washing with sterilized distilled water and air dried again. To extract the bound safranin from the fungal cells, 1 mL of 30% glacial acetic acid was added to each well and left at room temperature for 5 min. Color changes of the acetic acid solution were measured at 490 nm with the help of a UV-Vis spectrophotometer (Shimadzu; UV-1700).

To determine total polyphenols and total flavonoids, 3.0 ± 0.01 g of each chrysanthemum tea powder sample was mixed with 120 mL of distilled water in a 250 mL round-bottomed flask and extracted at 90 °C for 40 min in a water bath. After extraction, each extracted solution was centrifuged at 5000 r/min for 5 min, concentrated at 65 °C under reduced pressure for 1 h, and then freeze-dried. After drying, the extracted powder was stored immediately at −20 °C for future analysis. The total polyphenol contents of the chrysanthemum tea samples were measured according to the Folin-Denis method. Briefly, 0.01 g of each extracted chrysanthemum tea powder was mixed with 1.0 mL of Folin-Ciocâlteu reagent (1.0 mol/L) and reacted at room temperature for 1 min. Then, 8 mL of 10% sodium carbonate (100 g/L) was mixed well, and the mixture was left in a dark place for 2 h. The absorbance was measured at 760 nm using a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). The results were expressed as milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g), using gallic acid as a reference standard. The total flavonoid contents of the chrysanthemum tea samples were determined according to the aluminum chloride complex formation method and expressed as milligrams of rutin equivalents per gram of extracted chrysanthemum tea powder in dry weight (mg RE/g).

A photostability study was carried out by dissolving the pigments in acetone and illuminating them continuously with LEDs for 60 min at room temperature. The evolution of the electronic absorption spectrum was recorded periodically using a UV-visible spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan) and examined for any decrease or shift in the absorption peaks or shoulders. Since remnant of plant carotenes might be present, the photostability of pigments was also evaluated using β-carotene as standard reference (Chameleon Reagent, Osaka, Japan).