Extraction and Purification of Low Molecular Weight Fucoidan

LMW fucoidan extracted from Laminaria japonica were collaboratively manufactured by Hi-Q Marine Biotech International Ltd. (New Taipei City, Taiwan). Seaweed samples were ground to flour with a miniblender, and then dried with a dryer at 50 °C. The 100 g dried seaweed was treated with 5 L of distilled water and boiled at 100 °C for 30 min, and the extract was centrifuged at 10,000× g for 20 min. The supernatant was added with 4 M CaCl₂ incubated for 1 h to separate alginic acid, and re-centrifuged at 10,000× g for 20 min. All polysaccharides were dialyzed (cut off 10,000 Da) using deionized water for 48 h, and precipitated by the addition of ethanol at the ratio of 1:3 (V/V) and left overnight to give the crude fucoidan. The fucoidan so obtained was fractionated by anion-exchange chromatography using DEAE-Sephadex A-25 (Cl⁻ form, Pharmacia, Uppsala, Sweden) using gradually increasing concentrations of sodium chloride (0–2.5 M) at a flow rate of 1 mL/min, and eluents (5 mL/tube) were separately collected. Each fraction was analyzed for sulfate content (Section 3.3) and monosaccharide composition (Section 3.4). The fraction of rich sulfate and fucose content (fraction 3) was extracted and hydrolyzed with a mixture of crude glycolytic enzymes, isolated from Bacillus subtilis by our lab (Section 3.5). The reaction mixture was passed through a filter (3000 Da cut off) by using Amicon Stirred Cells (Merck Germany) to collect the LMW fucoidan with a molecular weight lower than 3000 Da. The hydrolysis reaction of the fucoidan by the crude glycolytic enzymes was performed at pH 6.5, 37 °C for 24 h and heated at 95 °C for 20 min for inactivation of the enzyme.