| Dunaliella salina is an unicellular green alga growing in salty environment such as sea, lagoon, salt lake and so on. At present, D. salina is used mainly in the extraction ofβ-carotene. D. salina residue after extraction ofβ-carotene contains quantities of polysaccharides which have many bioactivities such as antiviral, antitumor, immune regulation and so on. In this paper, the residue was used to extract polysaccharides and the isolation, purification and structure analysis of D. salina polysaccharides were systematically studied.The orthogonal design of four factors and three levels was performed to get the optimum extraction condition. The condition is as follows:extraction time is 8 hours, extraction temperature is 90℃, solid-liquid ratio is 20:1, pH value is 10. Under the optimum extraction condition, Dunelinella salina polysaccharide was extracted, and then deproteinization was performed as continuation treatment to get the polysaccharide PDS. The sugar content of PDS was 74.4%, protein level was 6.0%. The anti-DPPH free radical activity of PDS was preliminary assessment, and the result showed that PDS had a little anti-DPPH free radical activity.PDS was separated by ion exchange chromatography Q Sepherose 4 Fast Flow and got five products. Three of them were purified by gel permeation chromatography Sepharcryl S300 and got products PDS1, PDS2 and PDS3. They were analyzed by Shodex Ohpak SB-804 HQ collumn HPLC. The result showed that PDS2 and PDS3 have good purity, molecular weight were 19.5kD and 44.2kD, respectively. The suger cotents of PDS1, PDS2 and PDS3 were 67.4%,92.6% and 49.0%. Protein cotents of them were 8.3%, 3.8% and 8.1%, respectively. Uronic acid contents of them were 3.6%, 7.35% and 18.7% respectively. Sulfate contents of PDS1 and PDS3 were 3.1%and 7.7%, respectively, while no sulfate was detected in PDS2.PDS3 has complicated monosaccharide composition. The method of 1-phenyl-3-methyl-5-pyrazolone precolumn-derivatization high performance liquid chromatography (PMP-HPLC) which can detect 11 monosaccharides was set up. The derived condition of three kinds of monosaccharides(uronic acid, aminosaccharide and neutral saccharide) was optimized, and the optimized condition is as follows: reaction time is 60 min, incubation temperature is 70℃, ratio of NaOH and monosaccharide is 6:1, ratio of PMP and monosaccharide is 12:1. The mix monosaccharides were derived under optimized condtion and then analyzed by HPLC. The monosaccharide composition of PDS3 is Gal: Man: Ara: Glc: Rha: Xyl: GlcUA: GalUA: GlcNAc: GlcN =19.3: 10.6: 6.9: 1.6: 1.1: 1.0: 12.9: 6.4: 4.1: 1.6. Otherwise, an unknown saccharide was found by GC and PMP-HPLC respectively, which was supposed to be the same one. With the help of GC-MS, the unknown saccharide was considered as a 2-methoxyl pentose. In order to study the further structure of PDS3, degradation and chromatographic separation were used to get PDS3 fragments with different molecular weight. With the help of PMP-HPLC and ESI-MS, PDS3 was supposed to have a principal chain mainly containing Man, side chains mainly containing Gal and GalUA, meanwhile Ara3 and Glc4 were discovered in PDS3.Different chemical analysis methods and modern instrument analysis techniques were used to determine the main chain structure and linkage ways of PDS2 and find that PDS2 is mainly composed ofα-1,4-glucose containing little-1,3-, and -1,2-Glc branches.The paper ascertained the optimized extract condition and isolation method of D. salina polysaccharide, set up the PMP-HPLC method for analyzing monosaccharide composition of heteropolysaccharide and studied the structure of two polysaccharides. These provide references for comprehensive utilization of D.salina residue after extraction ofβ-carotene, and are helpful for further study of structure and bioactivity of D. salina polysaccharides.
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