Mops buffer

The activities of four different enzymes were monitored at time points 0, 24, 48, and 72 h. Substrates for β-glucosidase (BG) (EC. 3.2.1.37), cellobiohydrolase (CBH) (EC. 3.2.1.91), β-mannosidase (BM) (3.2.1.25), and β-xylosidase (BX) (EC. 3.2.1.37) activities were used. The first two substrates report on the degradation of cellulose and the last two on that of the hemicellulose part of wheat straw. The activities were quantified on the basis of the (enzyme-specific) substrate label 4-methylumbelliferone (MUB): 4-MUB-β-glucosidase, 4-MUB-β-cellobiosidase, 4-MUB-β-mannosidase, and 4-MUB-β-xylosidase (Sigma–Aldrich, Darmstadt, Germany). The reaction mixtures consisted of 150 μL diluted supernatant (usually 1/4) in MOPS buffer (50 mM, pH 6.5; Sigma–Aldrich, Darmstadt, Germany) and 2 mM of MUB substrate in black 96-well plates. The reactions were incubated 1 h at 28°C in the dark, after which 30 μL of NaOH (1 M) was added. Fluorescence was measured at an excitation wave length of 365 nm with emission at 445 nm. The enzymatic activities were then calculated from the fluorescence units using a standard calibration curve. Supernatant recovered from the NC was also tested, and thus served as the NC. The enzymatic activities are reported as the rate of MUB production (nmol MUB per h at 28°C, pH 6.8). All assays were done in triplicate.

The blueberry anthocyanins were extracted from Rabbiteye blueberries (Lishui Town, Nanjing, China) [53 (link)] and stored at −18 °C at the Institute of Agricultural Products Processing, Chinese Academy of Agricultural Sciences. High-performance liquid chromatography (HPLC) solvents potassium chloride and acetonitrile were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and TEDIA (Fairfield, OH, USA), respectively. Ferritin (from horse spleen), MOPS buffer, 2,2-bipyridine, and bovine serum protein (BSA) were procured from Sigma-Aldrich (Shanghai, China). The bicinchoninic acid (BCA) and protein-assay kits were purchased from Beijing Solarbio Company (Beijing, China). The reagents used in the experiment are all of analytical grade.

The screening of plant extracts for antifungal activity was investigated using a modified broth microdilution method. The plant extract/fungal isolate microtiter trays were stored at 35°C and assessed for growth after 48 h. The antifungal agents were prepared for minimum inhibitory concentration (MIC), according to Clinical and Laboratory Standards Institute (CLSI-formerly NCCLS) [16 , 17 ] with RPMI-MOPS (RPMI 1640 medium containing l-glutamine, without sodium bicarbonate (Sigma-Aldrich Co., St. Louis, USA) buffered to pH 7.0 with 0.165 mol/L MOPS buffer-Sigma). The SDB media were used as negative control, and fluconazole (1 mg/mL) was used as positive control. MIC was represented as the lowest concentration of compounds at which the microorganism tested did not demonstrate visible growth. The experiments were carried out in triplicate. Inhibition (%) was calculated as follows:
$\begin{array}{c}\text{Inhibition}\mathrm{\hspace{0.17em}\hspace{0.17em}}\left(\%\right)=\left[\frac{\left(\text{DC}-\text{DE}\right)}{\text{DC}}\right]\times \mathrm{100},\end{array}$
where DE is the diameter of growth zone in experimental disc (cm) and DC is the diameter of the growth zone in the control disc (cm).