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Structural Characteristics Of Microbial Exopolysaccharides And Their Antioxidant Activities

Posted on:2011-11-03Degree:DoctorType:Dissertation
Country:ChinaCandidate:S D GuoFull Text:PDF
GTID:1100330332465066Subject:Medicinal chemistry
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The exopolysaccharides(EPSs) produced by marine and Antarctic derived microorganisms of special environments become a new research front due to their novel structural characteristics, particular chemical properties, various bioactivities as well as commercial opportunities in recent years. In this paper, the yield, chemical properties, monosaccharide component and antioxidant activity of the EPSs obtained from 16 marine and Antarctic microorganisms were analyzed, based on the analysis results the EPSs produced by marine bacterium Edwardsiella tarda, Antarctic fungi Oidiodendron truncatum and Dichotella gemmacea epiphytic fungi Aspergillus ochraceus were further purified, their structural characteristics and antioxidant activities were investigated. The research results are as follows:1. The yields of the EPSs produced by 16 marine and Antarctic microorganisms were 0.03-0.45g/L, and their content of sugar, protein, GlcA and GlcN were 57.20%-94.71%,3.41%-15.50%,0.75%-6.03% and 0.3%-12.4%, respectively, while their sulfate content were all below the detection limit. The two EPSs obtained from Antarctic fungi were mainly composed of Man, Glc and Gal; Ten EPSs produced by marine microorganisms were mainly contained Man with various contents of Glc and Gal; two EPSs mainly consisted of Glc and Gal, while the rest two contained majority of Glc. The results showed that the chemical properties, monosaccharide component and DPPH radicals scavenging abilities of the EPSs obtained from microorganisms varying with their living environments. Finally, the EPSs with better antioxidant activities produced by marine bacterium E. tarda, Antarctic fungi O. truncatum and epiphytic fungi A. ochraceus were screened out for further investigation. 2. Two purified EPSs, ETW1 and ETW2 with molecular weights of 29.0 kDa and 70.0 kDa, respectively, were isolated from the crude EPS of E. tarda using Q-Sepharose F.F. anion exchange chromatography and Sephadex G150 gel permeation chromatography. The results indicated that ETW1 and ETW2 had a similar structure with [β-D-Manp(1'], ['2)-α-D-Manp(1'],['3)-α-D-Manp(1'], ['2,3)-α-D-Manp(1'] and ['3,6)-α-D-Manp(1'] in a molar ratio of 2:1:2:1:1. Both of them had a heptasaccharide repeating unit with a ['3)-α-D-Manp(1'] backbone chain, which were substituted by Man(β1'2)Man(α1') and Man(β1') at the C-2 and C-6 positions, respectively. ETW1 and ETW2 were composed of 26 and 62 the repeating units, respectively. The two mannans with novel structures separated from the fermentation broth of the unusual E. tarda are firstly reported in this paper.3. Five purified EPSs AFW1, AFW2, AFS1, AFS2 and AFS3 were isolated from the crude EPS of O. truncatum, their molecular weights were 60.0 kDa,7.5 kDa,64.5 kDa,19.0 kDa and 9.0 kDa, respectively. The protein content of AFS1, AFS2 and AFS3 were 1.8%,1.9% and 5.1%, respectively. The results indicated that AFW2 was a linear glucan with ['6)-α-D-Glcp(1'] glycosyls as its backbone chain; AFS2 also had a ['6)-α-D-Glcp(1'] glycosyl backbone chain with an average branch of [α-D-Glcp(1'] at the C-3 position of every six ['6)-α-D-Glcp(1'] glycosyls; AFS3 had a linear structure with ['6)-α-D-Glcp(1'] and ['2)-a-D-Glc(1'] in a molar ratio of 6:1; AFW1 was composed of [Galp(1'], ['4,6)-α-D-Glcp(1'], ['2,4,6)-a-D-Manp(1'] and ['6)-α-D-Glcp(1'] glycosyls in a molar ratio of 3: 1:1:1; AFS1 consisted of [Galp(1'], ['6)-α-D-Glcp(1'], ['4,6)-a-D-Glcp(1'], ['2,4,6)-a-D-Manp(1'], and ['2)-α-D-Manp(1'] in a molar ratio of 3:1:1:1:1. The branches of AFW1 and AFS1 were majority of [α-D-Galp(1'] and [β-D-Galp(1'] glycosyls. Among them, AFW2 belongs to the dextran series linear glucan with wide applications.4. Two novel galactomannans, AW1 and AW2 with molecular weights of 29.0 kDa and 7.4 kDa, respectively, were obtained from the crude EPS of A. ochraceus. Both of them were subjected to depolymerization by controllable acid hydrolysis with optimized conditions, and nine oligosaccharides were obtained by Bio-Gel P4 chromatography. The results indicated that galacto-oligosaccharides with consecutive degrees of polymerization from one to nine were in the uncommon form of ['5)-β-D-Galf(1'], the mannopyranosyls in AW1 and AW2 were in the form of [a-D-Manp(1'], [β-D-Manp(1'], ['2)-α-D-Manp(1'],['2,6)-a-D-Manp(1'], while the galactofuranosyls were in the form of [β-D-Galf(1'] and ['5)-β-D-Galf(1']; After depolymerization the molar ratio of ['2)-α-D-Manp(1'] glycosyls increased observably in AW1 and AW2, while [-+2,6)-a-D-Manp(1'] and ['5)-β-D-Galf(1'] glycosyls decreased remarkably, all of which suggested that the galacto-oligosaccharides should be linked to the C-6 positions of the mannan core, whose backbone chain was ['2)-α-D-Manp(1'] glycosyls. This study provides a new way to obtain uncommon ['5)-β-D-Galf(1'] linked oligosaccharides from marine microorganisms; and supplies novel characteristics of galacto-oligosaccharide for the "marine carbohydrate database".5. All of the seven purified EPSs exhibited various degrees of antioxidant activities in vitro, which were evaluated by DPPH radicals, hydroxyl radicals and superoxide radicals scavenging abilities, lipid peroxidation inhibition and BSA oxidation damage SDS-PAGE assays. Their antioxidant activities were in the order of AFS3>AFS2> AFS1>ETW1>AFW2>ETW2>AFW1. The EC50 values of AFS3 on DPPH radicals, superoxide radicals were 0.46 mg/mL and 0.55 mg/mL, respectively. AFS3 also showed antioxidant activity in D-Gal induced aging mice by improving the activities of antioxidant enzymes (SOD, GPx and CAT), it restored the high levels of lipid peroxidation product MDA and inhibited the "oxidative stress" in D-Gal induced aging mice. In addition, its antioxidant activity may also exerted by enhancing the immune function of the aging mice or scavenging the ROS directly by the tested models in vitro. The molecular weights of EPSs may be related to their antioxidant activities, and 20-30 kDa seems to be good for their antioxidant effects; while the proteins bounded to the EPSs may also play roles in their antioxidant activities.
Keywords/Search Tags:Exopolysaccharide, Structural characteristic, Antioxidant activity, Marine microorganism, Antarctic microorganism
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