Studies On Extraction, Purification, Chemical Structure And Anti-bacteria Bioactivity Of Polysaccharides From Chlorella Autotrophica

There are novel structure compounds in unique ocean environment that would contribute to new medicines. Chlorella autotrophica, a wide distributed economic alga, was studied in our research, we optimized the extraction of sulfated polysaccharides, explore the process of separation and purification, illustrate the composition and structure and by chemical methods and give a primary discuss about their anti-bacterium activity.Chlorella autotrophica is a widespread marine microalga which classified in Chlorella, Oocysteceae, Chlorococcales, Chlorophyta. In ultrasonic-assist extraction experiments, we take HCl(0.01 M), pure water and NaOH(4 %) as medium, crush for 10 min, repeat 2 times, and the power is 300 W, frequency is 20 kHz, ultrasonic pulse duration time is 9.9 s and latency time is 3.3 s, then the mixture was put into 80℃water bath for 1h, comparing with traditional hot water extraction method we got higher yield rate by ultrasonic-assist method. The highest yield rate is 120.2 mg/g (crude polysaccharide/alga powder) in NaOH(4 %) extracting solution. In order to find out best microwave-assist extraction condition, 4 factors of extraction temperature, time, medium solution pH and microwave power were adopted to build a orthogonal optimization experimental system, and we found that the influence shows to be extraction time>temperature>medium solution pH>microwave power, and results show that the best condition is under 70℃, 30 min, pH=7 and 600 w, the recovery ratio is 291.13 mg/g (crude polysaccharide/dried weight) which is higher than that by traditional hot water extraction(7.85 %). FT-IR results show little difference between crude extractions obtained by ultrasonic-assist, microwave-assist and traditional methods that means both the extraction methods is safe and reliably.Gel chromatogram was adopted to separate and purify the crude polysaccharide that obtained by ultrasonic-assist extraction. Results showed that DEAE-Sepharose Fast Flow has the advantage of larger load and higher separation speed, and is more suitable for primary separation than gel-filtration. Agarose electrophoresis was used to test the homogeneity of PCA1, PCA2, PCA3 that acquire in ion-exchange chromatogram,and we found that only PCA1 has a single point, PCA2 and PCA3 show to be mixtures, and sugar contain of PCA3 is only 33.75 %. We reject PCA3 because it has low obtain rate and complex composization. We separate PCA2 into two homogeneity fractions of PCA2-1 and PCA2-2 with Sephacryl S-400 HR in an AKTA system.PCA1, PCA2-1 and PCA2-2 were further examined use HPGFC, FT-IR, GC-FID, GC-MS and 13C-NMR. Results show the molecular weight of PCA1, PCA2-1 and PCA2-2 were 1.30×105,3.07×105,2.06×105, sulfate content was 0.77 %,13.03 %,7.46 %.α-D-1,4 Glup makes up of PCA1's backbone. PCA2-1 is a complex polysaccharide, methylation results show that it has a embranchment on its backbone, and monosaccharide composition is Xyl:Man:Glu:Gal=1.97:1.49:1.00:1.92, linkage is 1,4-Glc:1,6-Gal:1,4-Man:1-Xyl:2,6-Man:2,4,6-Gal = 0.9:1.0:1.0:0.4:0.4:0.7. Mono- saccharide composition of PCA2-2 is Rha:Xyl:Man:Glu:Gal = 0.15:0.22:0.21:1.00:1.36, we did not found embranchment in its backbone,α-2,6-Galp andα-1,4-Glcp make up its backbone with a ratio of 2:1.Primary anti-bacterium experiment was performed in our lab, the concentration of test samples were all adjust to 0.2 mg/mL, as we got so limited content of sulfated polysaccharides, PCA2-1 and PCA2-2. We picked 4 bacteria as our object which is familiar in marine environment, Gram-negative bacteria of Escherichia coli and Vibrio alginolyticus, Gram-positive bacteria of Staphylococcus epidermidis and Micrococcus lysodeikticus fleming. Data show that PCA2-1 have a weak effect on Escherichia coli and Micrococcus lysodeikticus fleming, and the KI is 30 %, but useless to the other two bacteria. PCA2-2 has a good performance in restraining Micrococcus lysodeikticus Fleming and Vibrio alginolyticus, the KI reaches about 70 %, but also useless to the other two. In general, the anti-bacterium effect of polysaccharides takes a decrease with time. Since PCA2-2 has a steady and excellent killing index to Vibrio alginolyticus, we expect to have a detail study in future.There is no report about polysaccharides of Chlorella autotrophica inside and outside, our research has given a first report about the purification methods, chemical composition and structure and primary bio-activity of it.