The phosphoprotein was hydrolysed by HCl digestion. Samples of the phosphoprotein residue were hydrolysed in 2N HCl in vacuum-sealed tubes at 100C for 10 hr. The resulting digests were evaporated in vacuo and dissolved in 0.5 ml of water. This was then applied to a Dowex 50x8 mm H+ columns. The phosphoamino acids were eluted with water and dried in vacuo.
X 1000 / wt of sample. 220.127.116.11 Iodine value: Principle: Iodine value (I.V) is the nomber of g of iodine absorbed per 100 g of the oil or fat, when determined using Wijs solution.The material is treated in carbon tetrachloride medium with excess of iodine monochloride in glacial acetic acid. The excess of iodine monochloride is treated with potassium iodide, and the liberated iodide is estimated by titrating with standard Na2S2O3. Reagents: 1. Wijs solution : Dissolve 8g of iodine trichloride in 200 ml of glacial acetic acid and mix with 9g of iodine dissolved in 400 ml of glacial acetic acid.
Introduction: The oxalic acid is an organic compound with the formula C₂H₂O₄. It is a colorless crystalline solid that forms a colorless solution in water. The condensed formula is HOOCCOOH, reflecting its classification as the simplest dicarboxylic acid. Reaction: Procedure: In a 500 ml flat bottom flask , take 15g of cane sugar( sucrose) and keep the flask in a fume cupboard. Add 75 Ml Conc.HNO3 and heat the flask on a boiling water bath.
Apparatus- Chromatography column: C18 (10 microns particle size), with Guard column Flow rate: 1.2ml/min Pressure: 30-40kgf Wavelength: 326nm Mobile phase: methanol : water (95:5 v/v) Internal standard: retinyl acetate Injection volume: 20µl Procedure for Retinol extraction from serum samples- 1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins. 2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec. 3) Centrifuged at 2500 rpm for 12 mins.
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.
5-aminotetrazole monohydrate: In a 250 ml round-bottom flask equipped with a condenser for refluxing (90 °C) and a magnetic stirring bar, 5.00 g (5.95 mmol) dicyandiamide (three times crystallized), 7.47 g (11.9 mmol) sodium azide and 11.00 g (17.8 mmol) boric acid and 100 ml of water is added and allowed to reflux for 24 hours, after the completion of the reaction, until the solution pH to about 2 to 3 as hydrochloric acid 37% is added (about 12 ml) Then the reaction mixture was cooled in a refrigerator for 18 hours and the white crystals formed. The mixture was filtered and washed three times with 10 ml of water and and dried in 60 °C for 5 hours and finally 45.8 g of product by it will be obtained. 5-Aminotetrazol monohydrate: Yield:,
Step-III: Synthesis of Cr(II) Complexes: The Schiff's base complexes were synthesized by mixing the Schiff's base (1.5 g) in ethanolic solution of Chromium chloride [CrCl2]. This reaction is refluxed in a waterbath for two hours and their volume were reduced to 70% of it’s original volume and residue was obtained. The coloured product obtained was filtered under suction, washed with ethanol. The product were recrystallized from ethanol. Their yields ranges from 50-55%, the product obtained were light green colour and melting point was 2100C.
First the solution of 1 mL of extract was added to deionizer water (10 mL), then Folin–Ciocalteu phenol reagents (1.0 mL) added to the mixture. The mixture was left for 5 minutes, and solution of 20% sodium carbonate (2.0 mL) was added to the mixture. This mixture diluted until 50 mL with deionizer water. And after that kept in total darkness room for 1 hour. The mixture absorbance was measured at 750 nm using a spectrophotometer.
Effect of pH To check the pH stability of the bacteriocins, 5 ml aliquots of CFS was taken in sterile tubes and were adjusted to pH values of 2, 4, 7 and 9 busing 1N HCl or 1 N NaOH. After incubation at room temperature for 30 minutes, pH in each tube was readjusted to 6.5 and the antimicrobial activity was determined by agar well diffusion method17. 6.3. Effect of proteolytic enzyme 5 ml of CFS was taken in a test tube and was treated with trypsin-chymotrypsin enzyme (10AU/ml) at pH 7. The test tubes with and without enzyme (control) were incubated at 370C for 1 hour.