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
• Injection volume: 20μl • Injection concentration: 1mg /ml • Detection wavelength: 280nm 2.3.6-Isolation and purification of Flavonoids (Genistien, Rutin, Quercetin) by: 2.3.6.1- Preparative TLC plate: Isolation and purification of genistein,rutin, quercetin were carried out by preparative TLC. On a glass plates (20 cm x20 cm) a slurry of 60 gm of silica gel GF 254 suspended in 120 ml of distilled water was applied in 0.75 mm thickness manually by using Jobling laboratory division plate coater. The freshly coated plates were left until the transparency of the layer disappears. After 10 minutes, the plates heated for 1 hour at 110ºC. The completely dried and activated plates are kept in a dry place for use.
The sterile medium was inoculated with known volume of inoculum and incubated at different temperatures (25, 30 and 350C) for different periods (48,72 and 96h) of fermentation. After fermentation the mouldy substrate was mixed with distilled water (1:4 w/v), agitated at through cheese cloth followed by centrifugation at 20,000 rpm for 20 minutes. The clear supernatant was used as crude enzyme. Assay of glucoamylase The assay of glucoamylase was carried out according to the method of Shazia Malik, Tehreema Iftikhar and Ikram Ul Haq13. One unit (U) of glucoamylase is defined as the amount that liberates 1 µmol of reducing sugar as glucose/ml/min under the assay condition.
After 10 minutes, the solution was deleaded by adding potassium oxalate crystals in excess and the volume was made up to a known amount with distilled water and filtered through whatman No. 1 filter paper. The filtrate was taken in a burette and titrated against boiling Fehling’s mixture (5 ml of Fehling’s solution A + Fehling’s solution 5 ml of B) till the blue colour faded. Then one ml of methylene blue indicator (1 per cent) was added and the titration was continued till the contents attained a brick red colour and titre value was noted. The percentage of reducing sugar was calculated according to the following
The dried mushroom samples were further heated at 105 ºC overnight until constant weight obtained for moisture content determination. The samples were incinerated at 550 ºC for 24 h and reweighed to determine ash content. MicroKjeldahl method was employed for the crude protein content (N × 4.38). The crude fat was determined by extracting a known weight of sample in Soxhlet apparatus using petroleum ether as a solvent. Total carbohydrates calculated by the difference.
Plasmid DNA was precipitated from the supernatant by adding 0.7 volumes of isopropanol and centrifugation at 13,000rpm for 30min and washed with 70% ethanol. The pellet was air dried, resuspended in TE buffer (pH 8.0) with RNase 10μg/ml and allowed to dissolve completely. The isolated plasmid DNA was electrophoresed on 1 % agarose gel (containing Ethidium Bromide) at 75 volts and visualized under UV light in order to check its
This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate. After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min).
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.
First step was the emulsification of curcumin and wall material using an emulsifier, followed by spray drying of the resultant emulsion. Curcumin-egg albumin emulsion was prepared by dissolving egg albumin (%w/v based on the total solution) in distilled water and subsequent addition of curcumin (%w/v based on total solution) into it according to the experimental design (Table 3). Emulsion has a continuous phase containing the film–forming carrier materials, and a discontinuous phase containing the curcumin. Prepared emulsion was stirred at 700 rpm for 10 min using magnetic stirrer. Two drops of Tween-80 was added to aid the emulsification process.
The culture was grown in a shaker incubator (S1-600R, Lab Companion) for 24 hours at 30°C. Lipid production: For lipid production, 200 ml of mineral medium in glass bottle was inoculated with 10 ml of overnight culture in YPD to a final optical density at λ = 600 nm (OD600) of 0.2 and incubated on a shaker at 150 rpm and 30°C for 72 hours. Three mineral media were used (listed in materials section, Table 3). 6.2.2 Lipid droplet staining and microscopic observation The stock solution was prepared by adding 1g of Oil Red O powder to 100 ml of isopropanol, in glass bottle. Prior to use, the working solution was prepared by diluting the stock solution with Mili Q water