Quality determination

2.3. Quality determination

2.3.1. Moisture determination

Moisture content was analyzed by weighed the biomass of

E. cottonii used analytical balance. Wet biomass determined as W 0 and dried biomass was determined as W 1 . Then the percentage of moisture content in seaweed calculated with following formula.

Sept ember 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At ma Jaya Yogyakart a

2.3.2. Determination of protein

E. cottonii powder were grinded in mortar with 5 mL of phosphate buffer (pH 7). The extract was transferred into centrifuge tube then phosphate buffer (pH 7) was added to 10 mL. The homogenate was centrifuged at 8000 rpm for 20 minutes. After extraction, 0.1 mL of different samples were taken out in separate test tubes. 5 mL of protein reagent (Coomasie Brilliant Blue G-250) were added to the test tube and the contents mixed by vortexing. The absorbance at 595 nm was measured after 2 min and before 1 h in cuvettes against a reagent blank. A standard curve was made using Bovine Serum Albumin in varies concentrations. Protein content in the extracted samples was determined from the standard curve and the amount of protein in mg/ml was calculated (Bradford, 1976; Pandey and Budhathoki, 2007).

2 g of

2.3.3. Determination of vitamin C

E. cottonii coarse powder and 2 mL glacial acetic acid was mixed. The mixture was stirred about 20 min and rapidly filtrate. The volume of sample is made up to 10 ml with distilled water. Then 50

2.5 g of

μL of 0.4 mmoll -1 Methylene Blue solution and diluted up to 10 mL with distilled water. Decrease of absorption was measured at

max = 665 nm. Results are expressed in mg of ascorbic acid per 100 g of dry sample (Tahirovic et al ., 2012).

2.3.4. Determination of β -carotene

E. cottonii powder sample (1 g) was weighed in a test tube. Then 5 mL of chilled acetone was added and the tube was held for 15 min with occasional shaking at 4 ± 1°C, vortexed at high speed for 10 min, and centrifuged at 1370 × g for 10 min. Supernatant was collected into a separate test tube and the compound was re-extracted with 5 mL of an acetone followed by centrifugation once again as above. Both of supernatant were pooled together and then passed through the Whatman filter paper No. 42. The absorbance of the extract was determined at

A representative portion of

49 nm wavelength in UV-Vis 10 Genesys spectrophotometer (Biswas et al ., 2011).

2.3.5. Determination of carrageenan

10 g of dried material was washed with tap water to remove sand and salt and then incubated in 6% KOH solution in an 80°C water bath for 3 h. Ratio between samples and solution is 1:3 (g/mL). The samples were washed overnight in slowly tap water. The samples were then stirred tightly in 1 L of distilled water and boiled for at least 1

h. Carrageenan was precipitated in ethanol with the ratio between filtrate and solution is 1:3. Precipitates carrageenan was then soaked in chloroform and dried at room temperature for 2 days.

2.3.6. Phytochemical determinations

2.3.6.1. Determination of flavonoids

E. cottonii powder was extracted repeatedly with 50 mL of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No. 42 (125 mm). The filtrate then evaporated into dryness over a water bath and weighed to a constant weight (Biradar and Rachetti, 2013).

5 g of

Sept ember 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At ma Jaya Yogyakart a

2.3.6.2. Determination of saponins

5 g of samples powder was put into conical flask and 25 mL of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 3 h with continuous stirring at about 55°C. The mixture was filtered and the residue re-extracted with another 50 mL 20% ethanol. The combined extracts were reduced to 20 mL over water bath at about 90°C. The concentrate was transferred into a 250 mL separating funnel and 5 mL of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. Purification process was repeated. 30 mL of n-butanol was added. The combined n-butanol extracts were washed twice with 5 mL of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the sample were dried in the oven to a constant weight (Biradar and Rachetti, 2013).

2.3.6.3. Determination of terpenoids

5 g of seaweed powder were taken separately and macerated in alcohol for 24 h then filtered. The filtrate was extracted with petroleum ether. Then the ether extract was treated as total terpenoids (Biradar and Rachetti, 2013).

2.3.6.4. Determination of tannins

500 mg of the sample was weighed into 50 mL plastic bottle then distilled water was added and shaken for 1 h in a mechanical shaker. This was filtered into volumetric flask then 5 ml of the filtrate was pipette out into a test tube and then mixed with 2 mL

of 0.1 M FeCl 3 in 01 N HCL and 0.008 M potassium ferricyanide. The absorbance was measured at 700 nm within 10 minutes using UV-Vis 10 Genesys spectrophotometer.