Smaller and smaller organelles may be sendimented by successive centrifugation at increasing speed. 1ml of the homogenate (H) was pipetted into a clean microfuge tube labelled P1. The tube labelled ‘P1’ was centrifuged at 1,000g for 10 minutes to sediment the first pellet (P1). The supernatant was carefully removed with an automatic pipette and placed in a micro centrifuge tube labelled ‘P2’. The P2 tube was then placed in the refrigerated centrifuge at a speed of 15,000g for 30-60 minutes at 4°C.
If this happens, the mixture boils and it would be necessary to start the experiment all over again. After obtaining an homogeneous mixture, the flask was placed in an ice bath during five minutes next to a graduated cylinder containing 5.0 mL of concentrated sulfuric acid. The temperature of the ice bath was recorded to be 1.1 °C. Likewise, a second graduated cylinder containing 1.8 mL of nitric acid and 2.5 mL of sulfuric acid was immersed in the cold ice bath to keep the three different solutions at the same temperature. Thereafter, the cold 5.0 mL of H2SO4 were added to the erlenmeyer flask containing the acetanilide solution, which remained in the cold water for approximately another 4 minutes.
An equivalent volume of saline solution was added (NaCl 0.16 M; pH = 7) and it was mixed completely. The resultant solution was centrifugated for 30 minutes at 9000 rpm at 4ºC in a rotor Beckman JA-20, eliminating the pellet. The supernatant was collected to obtain the lipoproteins by gradient density ultracentrifugation: 10ml of the yolk solution were taken and then 0.9 g of potassium bromide (KBr) was added. The bromide was dissolved with a smooth agitation, with care not to denature the proteins. Straight afterwards, saline solution was added (NaCl 0.16 M, pH=7) to the 10ml of the yolk solution with a Pasteur pipette, avoiding the sample diffusion, forming two phases and filling the tube completely.
Subsequently extracted by Microwave at power level 70 for 16 minutes and then filtered. The filtrate obtained, was added 10% HCl (until pH 2-3). Then do the bleaching with NaOCl diluted with water 1: 1 to white. Then converted to sodium alginate by adding 20 g of Na2CO3 and stirred in a mixer. The resulting solution is then etched with ethanol to form sodium alginate fibers.
200ml of water was then added to the filtrate in a 500ml beaker with constant stirring. White solid was formed in the process of addition and the solution was then left undisturbed in an ice bath for 10minutes. Once most of the solids had settled at the base of the beaker, the solution was decanted. 10ml of ethanol was added to the remaining suspension and was transferred in a clean centrifuge and centrifuged for 2minutes at 6000rpm. After the first centrifugation, the supernatant was discarded and the residue was washed by adding 14ml of ethanol.
The absorbance of the supernatant was read in spectrophotometer (UV-1601, SHIMADZU) at 540 nm against blank. Blank consisted of 1 mL distilled water, 0.5 mL of 30% w/v TCA and 0.5 mL of 0.8% w/v TBA. The results were expressed as nM of MDA (malondialdehyde) formed /hr/ mg of
Chapter 3: Methodology 3.0 Preparation of Polyaniline 1 0.2 M Aniline hydrochloride was oxidized with 0.25M ammonium peroxydisulfate in an aqueous medium. Firstly, the Aniline hydrochloride was dissolved in distilled water in a volumetric flask to obtain 50mL of the solution. The same step was repeated with Ammonium peroxydisulfate. Both solutions were stored for 1 hour at room temperature and then mixed in a beaker, stirred and left to polymerize. A day later, the PANI precipitate was collected on a filter and washed with three portions of 100mL of 0.2M HCl, and then with the same amount of acetone.
The substrate was hydrolyzed separately using various concentrations (10, 25, and 50 mg/mL) of Alcalase, Flavourzyme, Neutrase and papain. The samples were activated with the individual enzyme conditions listed in Table 1. After the hydrolysis process, the SBP were immediately heated at 80°C for 20 min to inactivate the enzyme, followed by centrifugation at 5,000×g for 15 min and collection of the supernatants for further use. DPPH radical scavenging assay The antioxidant activity of SBP was measured with DPPH radicals according to the method of Chantaranothai et al.  Appropriate concentrations of SBP hydrolysate were mixed 1: 4 (v/v) with 200 M DPPH solution in anhydrous methanol for 30 min in the dark.
The sodium salt of p-nitrophenol is treated with concentrated sulphuric acid at 35-45°C. p-Nitrophenol is filtered. With proper filtration and water wash, alkaline liquor trapped into sodium salt of p-nitrophenol is minimized and consumption of sulphuric acid for neutralization is reduced. The dissolved salts content in the acidic effluent is decreased. p-Nitrophenol is reduced with iron at 90-100°C temperature in wooden vat.