The supernatant was assayed for SOD activity by following the inhibition of epinephrine auto-oxidation. 0.5ml of sample was diluted with 0.5 ml of distilled water, to this 0.25 ml ethanol, 0.5 ml of chloroform (all reagents chilled) was added. The mixture was shaken for 1 min and centrifuged at 2000 rpm for 20 min. The enzymatic activity in supernatant was determined. To 0.05 ml of carbonate buffer (0.05 M, pH 10.2) and 0.5 ml of EDTA (0.49 M) was added.
After the specified time, the solutions were completed to mark by using distilled water to achieve a final concentration of 20 µg/mL each, filtered and injected into the HPLC system. Dry Heat Degradation For thermal stress, 10 mg portions of each of VAL and SAC dry powder were placed in porcelain dish in a controlled-temperature oven at 100˚C for 4 hours. After the specified time, the content of the porcelain dish was transferred quantitatively with HPLC-grade methanol into a 10 mL volumetric flask and the volume was made to the mark by using the same solvent. Then, an aliquot of this methanolic stock was diluted to volume with distilled water to obtain a final concentration of 20 µg mL-1. After the preceding treatments, solutions were filtered and injected into the HPLC
100ul of Folin reagent was added to the wells and incubated for another 20 minutes. The absorbance was measured at 630-700nm. A standard curve of absorbance vs. protein concentration was plotted and the protein concentration in the diluted sample and the total percentage of activity were
2.2.Yeast cell preconditioning and inoculum preparation 1 g dry weight of yeast was resuspended in 100 mL of deionized water in an Erlenmeyer flask of 250 mL volume, at 30–35°C, for 30 min with NaCl 6% w/v. Inoculum for experimental fermentations was prepared
The sample was transferred to a 250 ml conical flask kept in water bath for alkali treatment. 75 ml of 17.5% caustic soda was measured using a measuring cylinder at 20°C. 15 ml of 17.5% NaOH was added and fibres were macerated gently with a flattened glass rod for 1 minute. 10 ml more NaOH was added and the solution was mixed for 45 seconds. 10 ml NaOH was again added and mixed for 15 seconds to make lump free slurry.
Fe3O4 nanoparticles were synthesized according to our previously reported method by chemical co-precipitation of Fe2+ and Fe3+ ions with a molar ratio of 1:2 . Briefly, 2.4 g of FeCl3.6H2O and 0.8 g of FeCl2.4H2O were dissolved in 30 mL of deionized water under using continuous N2 purge at 70 °C. and Under vigorous stirring, followed by dropwise addition of 10 mL of NH3.H2O was dropwise added to the reaction mixture until the color of mixture turned to black and kept reacting for 90 min to complete the reaction. At the end, the synthesized Fe3O4 nanoparticles were separated by a magnet and washed by using water and ethanol for further three times with 89.3%
The volumetric flask was then filled up to its 100 mL mark with deionized water. The buret was washed out with dionized water and then with the strong base NaOH before being filled up with NaOH. About 20 mL of the unknown weak acid was pipetted into a beaker. The starting volume of the NaOH in the buret was recorded before about 4 mL of the strong base was titrated into the weak acid solution. The final volume was recorded.
H2SO4 • Catalyst: potassium sulphate (K2SO4), copper (II) sulphate pentahydrate (CuSO4.5H2O) • NaOH (40%) • 0.1 N HCl solution • Boric acid (4%) • Indicator: methyl red (200 mg make up to 100 ml with %95 ethanol ) 1 gram Feta Cheese was used as a sample in this experiment. First of all, the experiment was started with the digestion phase which was performed by mixing 10g potassium sulphate and 0.5 g CuSO4 solution that acts as catalyst, and added 20ml conc. H2SO4. The digestion unit temperature was set to 420±10°C and preheated for 15 minutes. Then, it was heated continuously at least 2 hours till a clear colorless solution was obtained.
Metal chelating activity Briefly, 2 mM FeCl2 was added to different concentrations of test sample and reaction was initiated by the addition of 5 mM ferrozine. The mixture was vigorously shaken and left to stand at room temperature for 10 min. Absorbance was measured at 562 nm after 10 min.8 % Inhibition = [(AB - AA)/AB] x 100, where AB, absorption of blank sample, AA, absorption of test sample. 2.6. Antibacterial
Alginate sample (30 mg) was hydrolyzed in 10 mL HCl (0.3 M) at 100 ºC for 2 h. After cooling, the mixture was centrifuged (6000 rpm, 45 min), and the supernatant solution was separated and neutralized with 1 M NaOH and referred to as fraction A. The insoluble material was dissolved in 1 M NaOH and the pH was decreased to 2.85 by the addition of 1 M HCl. The suspension was recentrifuged and the supernatant was separated and referred to as fraction B. The insoluble fraction was dissolved by neutralization with 1 M NaOH and referred to as fraction C. The fractions A, B, and C are enriched in MG, MM, and GG blocks
Once the gel hardened, .5X TBE (44.5 mM Tris base, 44.5 mM boric acid, and 1.0 mM EDTA) was added just until the gel was covered with the TBE buffer. Each sample was loaded into the gel as well as 10 μL of DNA size markers (1kb ladder, New England Biolabs) into a separate lane. The gel was allowed to harden at room temperature and then electrophoresed at 100 volts for 75 minutes. Using a UV imager, a photo was taken of the resulting traveled DNA fragments in the gel. Results Table 1.
The silver ion TLC was prepared through the following procedure: Silver nitrate was dissolved in 10 ml of distilled water. This aqueous solution of silver nitrate was absolutely mixed with 9 g of silica gel (10 ~ 40 μm particles). Then, a 10 × 5 cm TLC plate was coated with the above slurry and activated for 1 h at 90 °C before use. They were immediately transferred into a desiccator in dark for storage after cooling. 32 100 μL of afore-prepared sample solution and the mixed reference standard were diluted 100 times with ethyl acetate.
The absorbance was read at 0 seconds, at 30 seconds, at 60 seconds, at 90 seconds and at 120 seconds. All the absorbances were remained 0 for the blank. After 120 seconds, the blank was then removed, and the appropriate amount of enzyme Tyrosinase (0.40 mL) was measured and added into the blank (cuvette #1) using the micropipette P-1000 according to the table 2. The final volume in the cuvette was 3mL. The cuvette contained the enzyme sample was wiped off with a KimWipe and was placed into the sample compartment of the machine.