Structural Characterization And Antioxidant Properties Of Exopolysaccharides Produced By4Marine Microbe With Different Sources

Microbial polysaccharides are of growing interest in application and large-scaleproduction for the advantages of novel functions, constant chemical, physical propertiesand possibility to easily control the growth conditions in a bioreactor. It contains richmicrobial resources widely distributed in the oceans which are important sources ofvarious exopolysaccharides (EPSs). For the demonstrated biochemical and physiologicaldiversity, it is of great significance to find new EPSs with unique bioactivities frommarine microorganisms.In this paper, the exopolysaccharides were derived from the fermented liquid of14ocean microorganisms with different sources. Through contents analysis,4species of themarine microorganisms, including the mangrove endophytic fungus in the south Chinasea (Aspergillus sp. Y16), coral symbiotic fungus in the south China sea (Aspergillusversicolor LCJ-5-4), fungus from the sediment of deep sea (Penicillium griseofulvum)and sponge endogenous fungus in the south China sea (Alternaria sp. SP32) werechoosed to futherly separate and purify their exopolysaccharides, study the structures andevaluate their antioxidant activities. The research results were as follows:1. Two exopolysaccharides As1-1and As2-1were isolated from the broth ofmangrove endophytic fungus strain Aspergillus sp.Y16and purified by Q–Sepharose FastFlow anion-exchange chromatography and Sephadex G150, Superdex75gel-permeationchromatography.The structures of the exopolysaccharides were investigated by GC,HPLC, methylation and1D,2D–NMR analysis. The results showed that As1-1was agalactomannan composed of mainly mannose(88.9%) and galactose(11.1%) with themolecular weight of15kDa. The backbone consisted of (1→2)-linked α-Manp units,substituted mainly at O-6by units of β-Manp, β-Galf and small amounts of (1→6)-linkedα-Manp. As2-1was mostly composed of mannose with the molecular weight of about6kDa. It was nearly a linear polysaccharide with the main chain of (1→6)-linked α-Manp and small quantity of side chains composed of α-Manp and (1→3)-linked α-Manp whichwere linked to O-3of the main chain. The antioxidant properties in vitro were evaluatedby DPPH, hydroxyl and superoxide radicals scavenging abilities, lipid peroxidateinhibition assays. From the results, the two polysaccharides possessed good antioxidantproperties, especially on superoxide radicals and DPPH radicals. As1-1showed strongDPPH and superoxide radicals scavenging ability as evidenced by its low EC50value(1.45mg/mL for DPPH radicals and3.4mg/mL for superoxide radicals). In this study, anew galactomannan with a backbone of (1→2)-α-D-Manp was gained from theexopolysaccharides produced by mangrove endophytic fungus Aspergillus sp.Y16, whichpossess good antioxidant properties and have the value to further study.2. Two exopolysaccharides AV-1and AVP were isolated and purfied from thefermented broth of coral-associated fungus Aspergillus versicolor LCJ-5-4byQ–Sepharose Fast Flow anion-exchange chromatography and Sephacryal S400, Superdex75gel-permeation chromatography. AV-1was mainly composed of glucose with smallamounts of mannose in the molar ratio of8.6:1.0with molecular weight of about500kDa. AV-1was a slightly branched polysaccharide, and its backbone was composed of(1→6)-α-D-glucopyranose residues (90%), branching of single non-reducingα-D-mannopyranose (10%) occurred at O-3position of (1→6)-α-D-glucopyranoseresidues. AVP was a mannoglucan with molecular weight of about7kDa, and the molarratio of glucose and mannose was1.7:1.0. The backbone of AVP was characterized to becomposed of (1→6)-linked α-D-glucopyranose and (1→2)-linked α-D-mannopyranoseunits. The mannopyranose residues in the backbone were substituted mainly at C-6by theside chain of (1→2)-linked α-D-mannopyranose trisaccharides units. The antioxidantactivity of AVP was evaluated with the scavenging abilities on DPPH, superoxide andhydroxyl radicals in vitro, and the results indicated that AVP had good antioxidantactivity, especially scavenging ability on superoxide radicals with EC50value about1.5mg/mL. AVP was a novel mannoglucan with antioxidant activity produced bycoral-associated fungus Aspergillus versicolor, indicating that symbiotic fungi of marineorganisms were important sources of novel active exopolysaccharides.3. A galactofuranomannans Ps1-1, was isolated from the Deep-Ocean sedimentderived fungus Penicillium griseofulvum, using ethanol precipitation and anion-exchange and size-exclusion chromatography. The average molecular weight of Ps1-1wasestimated to be20kDa. Ps1-1consisted of Man and Gal in a molar ratio of1.1:1.0.According to the data obtained, Ps1-1was a galactomannan composed of5-O-linkedgalactofuranosyl chains linked to a mannan core. The mannan core consists of a mainchain of α-(1→6)-linked mannopyranosyl residues, substituted at O-2with either a singlea-mannopyranosyl unit or (1→2)-linked di-, trisaccharides. The galactofuranosyl chainhas a few additional branching at6-O-position of the chain by a terminal galactofuranoseand phosphate esters. Through the partial acid hydrolysis and purification by Bio-GelP4, oligosaccharides composed of β-(1→5)-Galf with the DP2-5and phosphorylatedoligosaccharides with DP3-5were obtained. MS/MS was used to analyse the structuresof the oligosaccharides. The ion fragmentation of the ring and the mass losses of theβ-(1→5)-Galf was observed and could be evidence for the diagnose of theoligosaccharides with the similar glycoside linkage. This study supplied novelgalacto-oligosaccharides with β-(1→5)-Galf linkage for the “marine specialoligosaccharides database”.4. A galactoglucomannan, SP-S was isolated from sponge endogeneous fungiAlternaria sp. SP32in south China sea. The average molecular weight of SP-S was about27kDa. The monosaccharide composition of SP-S was Mannose, Glucose and Galactosein the ratio of3:2:1. Through the gradual acid hydrolysis, methylation,1D,2D–NMR andoligosaccharides analysis derived from partial acid hydrolysis, the structure of SP-S waselucidated. The results showed that, SP-S was a galactoglucomannan with core mannosestructure. The core of the main chain was composed of (1→6)-α-Manp linkage with lotsof branches occured at O-2with (1→2)-α-Manp. The galactose residues of SP-S at thelinkages of→2)-β-Galf-(1→，→2,6)-β-Galf-(1→and β-Galf-(1→together with(1→2)-α-Glcp and (1→6)-α-Glcp also constituted the branch part of SP-S. Thisexopolysaccharide was novel in fungus genus Alternaria. From preliminary antioxidantand antitumor activity screening in vitro, it was found that SP-S had certain free radicalsscavenging abilities. The EC50value of scavenging DPPH radicals was about5mg/mL.This study provided a novel source for the bioactivites research of galactoglucomannan.The data in this paper supplied novel polysacharides and special oligosaccharides to“marine polysaccharides database”, provided promising sources of novel polysaccharides with good antioxidant activity, had potential value and important reference meanings forfurther research and development of marine microbe exocellular polysaccharideresources. And this study clearly indicated that marine microorganisms were exciting andpromising sources of novel polysaccharides with good bioactivity.