A total of 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50mM phosphate buffer (pH 7). The reaction was started by the addition of 1 ml freshly prepared 30mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm. Catalase values were expressed as n moles H2O2 consumed/min/mg protein. Measurement of lipid peroxidation TBARS, a measure of lipid per oxidation, was measured as described by Ohkawa .
Decomposition of Aspirin Studied with UV/Visible Absorption Spectroscopy Aims: To determine the concentration of salicylic acid, formed from the hydrolysis of Aspirin, at regular intervals using the UV/Visible Absorption Spectroscopy From the concentration of salicylic acid, concentration of Aspirin to be determined using an equation Calculate the rate constant of this reaction and its order from a plot of graph of ln(aspirin) vs time Discuss the overall flaws and improvements to the experiment Results: As per schedule1, 0.212g of aspirin was added to 50 ml boiling water to form salicylic acid in a 100 ml flask, of which 1 ml was then pipetted to a 50 ml volumetric flask at the 5th min. Following an ice bath, the solution was mixed
Referring to Table 1, the reactants for each run were transferred to an Erlenmeyer Flask (250 mL) via a buret. Using a precision pipette, the volume of I3- required for each run was carefully extracted and poured into the flask containing all of the reactants. Immediately after the Iodine solution was placed in the flask, the LabQuest began collection data. Meanwhile, a small portion of the solution, was used to rinse the cuvette, then using a disposable pipette a small amount of the solution was transferred to the cuvette (approx. ¾).
After the finalization of alkali formulation to be used for extraction of cornhusk fibres, the fibres were treated with Pulpzyme HC to increase their fineness. Pulpzyme HC is a xylanase enzyme (EC.184.108.40.206) produced by submerged fermentation of a genetically modified Bacillus microorganism, and was obtained from Novozymes. The enzyme has an activity of 1000 AXU per gram. (http://fzfz.nbdl.gov.cn:81/files/20130815/1376527490502_36.pdf) Effect of concentration of Pulpzyme HC and Treatment Time The extracted fibres were treated with three different concentrations of enzyme i.e. 1%, 3%, 5% (owf).The time taken for treatment was 30 minutes, 60 minutes and 90 minutes.
Linoleic acid peroxidation was initiated by the addition of 4 mM FeSO4.7H2O, incubated for 60 min at 37oC and terminated by the addition of 2 mL of ice cold trichloroacetic acid (10% v/v). An amount of 1 mL of thiobarbituric acid (1% w/v in 50 mM NaOH) was added to 1 mL of the reaction mixture, followed by heating at 95oC for 60 min. The reaction sample was read at 532 nm.7 The percentage of linoleic acid peroxidation inhibition activity was calculated using the following equation: % Inhibition = [(AB - AA)/AB] x 100, where AB, absorption of blank sample, AA, absorption of test sample. 2.5.4. 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 alkalized membrane was then washed with de-ionized water several times and soaked in de-ionized water with numerous washings for 24 h prior to next step of operation. The ion exchange capacity (IEC), hydroxyl conductivity, water absorption percentage, oxygen and substrate crossover and thickness of the membranes were determined and the obtained values are tabulated (Table 1). [Table 1] 2.3. Characterization of membranes 2.3.1. Structural characterization of membranes The chloromethylation and quarterization of poly sulphone (PSU) were confirmed with an FTIR Alpha Bruker spectrometer in transmittance mode and 1H NMR Bruker Advance 500 MHz multinuclear FT-NMR spectrometer.
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
MATERIALS AND METHODS Bacterial strains and culture conditions Two S. aureus strains were used in the present study; S. aureus 8325-4 (SigB-) and SH1000 representing a SigB+.strain. Overnight cultures were grown in Luria Broth (LB) at 37°Cwith shaking at 150 rpm. Exposure to antibiotics was carried out as detailed below. Antibiotics Ciprofloxacin were purchased from Sigma-Aldrich CO. 10 mg/ml stock solution of antibiotic were prepared freshly with 0.1N HCl and stored at -20°C. During the experiment we diluted with sterile water 1:10 and 1:100 depending on the different drug concentration.
Quantification and trypsin digestion of polypeptides Protein concentration was estimated by Bradford assay, and 100µg of total protein from each sample was subjected to in-solution trypsin digestion to generate peptides. Initially, treating the sample with 5µl of 100mM dithiothreitol in 50 mM ammonium bicarbonate for 30 min at 60ºC and alkylation with 200mM iodoacetamide in 50 mM ammonium bicarbonate at room temperature for 30 minutes reduced the protein disulphide bonds. Proteins were then digested with 4µg of sequencing grade-modified trypsin (Sigma) in 50 mM ammonium bicarbonate by incubating overnight at 37ºC. The trypsin digestion reaction was stopped by 1µl of 100% formic acid. The digested peptide solutions were centrifuged at 14000
.4 Agar Disk Diffusion The antimicrobial tests were carried out according to disc diffusion tests (Lennette et al., 1985, Kim et al., 1995). Cells were streaked on MHA agar plate to obtain colonies at 37°C for 16 hrs after which they were resuspended in sterile saline solution to give an O.D of 0.1 at 600nm. On solidified 2% MHA plate, the bacterial culture is swabbed and sterile disc impression was made, 10 µl plant extracts was loaded on the agar plate. After 10 minutes the plate is incubated at 370C The diameter of the clear zone was measured. Fig 9: Agar disk diffusion 5.5 Time Kill Assay The basic concept of the Time-Kill Kinetic study is establishment of the rate at which a microorganism is killed by a product as a function of survival
The dried roots of Inula racemosa were pulverized and sieved with 100 ~ 200 mesh. The herb powder was placed into a glass bottle. Ultrasound-assisted extraction was carried out in an ultrasonic cleaner RK102H (Bandelin sonorex, Germany). The powder of Inula racemosa was extracted three times under the following conditions: the ratio of material to solvent was 10:1, undergoing ultrasonic treatment 30 minutes at 25 °C, 100 kHz /450 W.31 Before large extraction, a small-scale extraction experiments were carried out: 95% ethanol and ethyl acetate as the extractive solutions was investigated, respectively. The extraction efficiency was evaluated according to the percent content of AL and IS contained in the dried roots of Inula racemosa and calculated