Studies On Extraction, Purification And Primary Structure Of Polysaccharides RSPS1-3 From Rapeseeds Meal And Cake

In recent years, as going deeply into the studies on chemical function of membrane, immune substances and new medication resources, people found that besides being the energy resources and structural stuff in organisms, polysaccharides plays an more important role in taking part in the various activities of cells and have variety of bio-functions. The bio-functions of polysaccharides have close relation to its primary and senior structure. Rapeseeds cake and meal, which was of great amount in the world, was studied in this paper. Rapeseeds polysaccharides (RSPS) was isolated and purified from double-zero rapeseeds cake and meal. Then the physicochemical properties and the primary structure of rapeseeds polysaccharides fraction (RSPS 1-3) were also studied here, with the advanced methods of isolation and purification technique, chemical and instrumental analysis technique of bio-macromolecular. The main results are as follows:1. RSPS was extracted with water and then precipitated with ethanol, while the protein, the polyphenol and the phytic acid was also isolated respectively from the rapeseed cake and meal simultaneously. The optimized technical condition of isolating RSPS was studied through orthogonal test and the result was following: the rapeseeds cake and meal was marinated with 25 times of hot water at 100°C for 4 hours. The yield of rapeseeds polysaccharides was 2.8% under this condition.2. RSPS 1-3, which was a relative homogeneous fraction, was obtained after two different chromatography techniques, which were ion exchange chromatography and Sephadex chromatography. The purity of RSPS 1-3 was tested on the Sephadex chromatography and PAGE electrophoresis. And it was found that RSPS 1-3 was a polysaccharide-protein conjunct. The content of neutral sugar, uronic acid and protein was respectively 51.4%, 39.2% and 4.45%. The gel permeation chromatography (GPC) analysis indicated that the molecular average-weight of RSPS 1-3 was 2.24 × 105Dal.3. RSPS 1-3 was also proved to be a kind of polysaccharide-protein conjunct by using the method of UV and FT-IR analysis. The results of the TLC and GC analysis showed that RSPS 1-3 consisted of Rha, Man, Fru, Gal, Glu and an unknown sugar with molar ratios of 1.0:2.32:1.34:4.95:2.91:2.05. Two different deviration methods ofmonosaccharides and polysaccharides before injected into GC were compared, however, the results didn't follow the existing references. In contrast, between 3 to 4 peaks were obtained by GC for each monosaccharide after being alditol acetate derived and the retention time (RT) was close to each other, while only one characteristic peak for each monosaccharide after being trimethylsilyl methyl glycoside derived (TMS) and the RT differentiated obviousl. The result of the latter method was much better than the former one.4. The protein in RSPSl-3 was detected to be composed of 15 kinds of amino acids with Glu, Lys and Gly taking a great measure. The results of P-elimination reaction indicated that the chain of polysaccharides and protein was connected by O-linked glucosidic bond. And the backbone of sugar residues chain in RSPSl-3 was connected mainly by 1—>6-linked glucosidic bond, and partly by 1—>3, 1—*2 or 1—>4-linked glucosidic bond, which was detected by the means of periodate oxidation.5. The IR spectrum showed that the chain of RSPSl-3 contained pyranse with a-linked glucosidic bond, while the Congo red test indicated that RSPSl-3 could react with Congo red, which seemed conflicting with the existing references. Only the polysaccharides with p-helix conformation could react with Congo red to form Congo red-sugar complex, which was recorded in the references. Whether there isP-helix conformation in RSPSl-3, is waiting for further studies.The innovation point of this paper was that RSPS was firstly isolated from rapeseeds cake and meal, while the protein, the polyphenol and the phytic acid was also extracted respectively simultaneously. And the primary structure of the relative homogeneous fraction was studied. The preparatory structural information on the RSPS supplied a gap between the rapeseed and polysaccharides, and established a base for the deep study on the relationship between the structure and function of RSPS.