Structure And Immune Activity Study Of Extracellular Polysaccharides Produced By Marine Microorganisms Under Different Fermentation Conditions

In this paper, the12kinds of extracellular polysaccharides were isolated fromtwo different sources of microbial fermented liquid with6different mediums, withbasic physicochemical properties were analized. And the immunity activities wereanalized furthermore. The main findings are as followes:1. Among the12crude polysaccharides, AF3, AF2, SEK1and SEK3had got ahigher rate, which were10.1%,9.7%,8.0%and7.9%, relatively. Theirmonosaccharide compositions, average molecular weights and chemical propertieswere determined and compared by high performance liquid chromatography (HPLC),high performance gel permeation chromatography (HPGPC), infrared spectroscopy(IR). The experimental results showed that no uronic acid in these12kinds ofextracellular crude polysaccharides; AF4, AF6, SEK4were sulfate-freepolysaccharides, with the rest contained a samll amount of sulfate. AF4, AF6andSEK4had the highest content of Glc, which were85.5%,80.4%,87.0%respectively,AF1, AF3, SEK5and SEK6were mainly consisted of Man and Glc, which were72.5%,80.7%,81.9%and81.3%; AF2, SEK1, SEK2and SEK3were mainlycomposed of Man, which were66.5%,67.5%,52.9%,73.5%..2. Based on AF3which had a higher content of Man and the highest yield, theisolation and purification of AF3were carried out by732#cation-exchangechromatography, Capto Q anion-exchange chromatography and Sepharyl S100gel-permeation chromatography, the polysaccharides fractions named AF3-A1andAF3-A2, respectively. The relative molecular weight of the former one was32kDa,with the sugar content was85.9%, no protein and no acidic groups such as sulfate,uronic etc. AF3-A1was a mannan which was mainly composed of mannose with a content of more than95%. The fine structure of the purified polysaccharide AF3-A1was carried out by GC/MS, NMR and MS etc. The results indicated that AF3-A1wascomposed of (1â†'6)--D-Manp as its main chain, substituted at C-2with β-D-Manpand (1â†'2)--D-Manp as its branches, which the degrees of polymerization werefrom two to five.3. AF3-A2was obtained by0.6M salt wash fraction which was carried out onCapto Q anion-exchange column, with the relative molecular weight was82kDa, thesuagr content was79%, the protein content was15%, no acidic groups, such assulfate, uronic etc, and the molar ratio of mannaose and galactose was8.3:1.7, whichwas a heteropolysaccharide containing mannose, belonging to galactomannanpeptide. On the basis of GC/MS, NMR and MS, the backbone of AF3-A2characterized to be composed of-D-(1â†'6)-Manp which was a mannan core,substituted at C-2with-D-Manp(1â†', β-D-Galf(1â†', and-D-(1â†'2)-Manp--D-(1â†'3)-Manp as its branches with the degreespolymerization which were from two to five.4. The vitro immunomodulatory effects were carried out on mice with twelvekinds of curde extracellular polysaccharides produced by marine microorganismsunder different fermentation conditions, two kinds of purified polysaccharides andtwo kinds of mannan oligosaccharide mixtures. With mouse spleen cells andthymocytes as experimental models in vitro, observations of the metabolic activitiesor the immune cells or the proliferation were carried out with16kinds ofexopolysaccharides. The results showed that the capacity of the microbialpolysaccharides on normal mouse spleen lymphocytes, thymus lymphocyteproliferative was generally higher than the mannantide, and with a does-dependenttrend within a certain range; crude polysaccharides on normal mouse spleenlymphocytes, thymus lymphocyte proliferation were generally higher than purifiedpolysaccharides and mannan oligosaccharide mixture. Samples with positive controlConA and LPS promoted the metabolic activities jointly, which played a synergyrole in normal immune cells; AF3and SEK1promoted the normal mousethymocytes in vitro prominently. In addition, with the anti-H1N1activities experiments on16kinds of polysaccharides, the inhibitions of the crudepolysaccharides were higher than purified polysaccharides and mannanoligosaccharide mixture, AF3had a higher inhibition rate, which was69.6%.Preliminarily to prove AF3was a good immunomodulatory agent.In this thesis, two kinds of novel structures of mananose were gained, which hada good immunomodulatory and anti-influenza virus (H1N1) activities, laying thefoundation of its in-depth research and development of immunomodulatory drugs.