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Study On The Exopolysaccharides From Arctic Marine Bacterium Polaribacter Sp.SM1127

Posted on:2016-09-30Degree:MasterType:Thesis
Country:ChinaCandidate:M L SunFull Text:PDF
GTID:2310330518989296Subject:Biological engineering
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A flavobacterial strain,SM1127,was isolated from the Arctic brown alga Laminarza which was collected in the intertidal zones of Kongsfjorden(78°55'17.0"N;11°57'0.2”E).The strain SM1127 was identified as Polaribacter.The fermentation conditions of the EPS production of SM1127 were optimized.Moreover,the EPS was isolated and purified,and its glycosyl composition and linkages were further studied.Rheological studies were performed on the aqueous EPS.The potential applications of the EPS in food,cosmetic,pharmaceutical and biomedical fields were also studied.1.Screening and identification of EPS-producing strains from ArcticUsing Congo red agar plates,eight isolates that secreted mucous EPS were screened from 152 Arctic isolates.Isolate SM1127 which had the highest EPS production(2.112±0.13 g/L)from liquid fermentation was chosen for further study.With regard to the 16S rRNA genes,strain SM1127 shared 99.13%sequence similarity with Polaribacter sejongensis KOPRI 21160T,98.67%with Polaribacter butkevichii KMM 3938T and 97.43%with Polaribacter irgensii 23-PT.In the neighbor-joining phylogenetic tree,SM1127 was grouped within the genus Polaribacter.Thus strain SM1127 was phylogenetically affiliated with the genus Polaribacter.2.Optimization of fermentation conditions of the SM1127According to the single factor experiment,a combination of adjusting the concentration of glucose to 30 g/L,pH to 8.0,temperature to 10?,and fermentation time to 5 days would favor maximum EPS production(about 4.01 ± 0.08 g/L).After optimization of fermentation conditions using the response surface methodology(RSM),the optimal combination of the medium constituents for maximum EPS production was determined as 35 g/L glucose,9.43 g/L peptone,5 g/L yeast extract and 30 g/L sea salt.The initial pH was adjusted to 7.5 and the incubation temperature was 11.25?.Under the optimum conditions,the EPS production reached the maximum 6.12 ± 0.08 g/L on the 6th day,which represented the highest EPS yield of the bacteria affiliated with the genus Polaribacter.Then fed-batch fermentation was carried out using a 5 L(working volume)stirred-tank bioreactor.On the 7th day,the maximum EPS production of 17.84 ±0.22 g/L was obtained,which was 2.9-fold of that of batch fermentation.This is the highest one reported for a marine microorganism so far.3.Purification,glycosyl composition and glycosyl linkage analysis of the EPSAfter adding two volumes of chilled absolute ethanol to the fermentation broth,and excluding proteins with compound protease,the crude EPS was obtained.Then the crude EPS was purified by anion-exchange chromatography using a DEAE-Sepharose Fast Flow column,followed by gel-filtration chromatography using a Sepharose 4B column.The molecular mass(MW)of the EPS was 2.2 × 105 Daltons,which was estimated by size-exclusion chromatography.The glycosyl composition and glycosyl linkage analysis of the EPS from strain SM1127 was carried out using combined gas chromatography/mass spectrometry(GC/MS)analysis.The EPS consisted of N-acetyl glucosamine(28%),mannose(23.4%),glucuronic acid(21.4%),galactose(17.4%),fucose(7.4%),glucose(1.6%)and rhamnose(0.8%).Glycosyl linkage analysis of the EPS sample showed that it was mainly contained 4-linked glucuronopyranose(13.2%),2-linked galactopyranose(13.0%),terminally linked galactopyranose(11.5%)and 4-linked glucopyranose(10.2%).This is the first report about the glycosyl linkage of the EPS secreted by the bacteria within Polaribacter.4.Rheological properties of the EPS from SM1127Under same fluid shear stress,the viscosity of the EPS solution increased with elevated concentration of EPS,indicating that the EPS solution was a typical non-Newtonian fluid.When fluid shear stress went up,the viscosity of the EPS solution at the same concentration decreased,revealing that the EPS solution had strong shearing-thinning behavior.Under constant fluid shear stress,the viscosity of the EPS solution decreased gradually.And when the fluid shear stress was removed,the viscosity of the EPS solution returned to the initial value.This revealed that the EPS solution had positive thixotropy.Moreover,the EPS had good stability under the conditions of high temperature,wide range of pH and high concentration of inorganic salts.5.Potential applications of the EPS in biotechnological fieldsAccording to the acute toxicity test on KM mice,the LD50 of the EPS was higher than 5000 mg/kg,revealing the EPS had no toxicity.According to the primary and cumulative cutaneous irritation test on Japan white rabbits,the total score of primary cutaneous irritation index(PII)and cumulative cutaneous irritation index(C?)were both zero,which revealed that the EPS had no irritation to skin.The moisture-absorption rate of the EPS was 10.0 ± 0.5%at 43%relative humidity(RH)and 22.1?0.6%at 81%RH.After dehydration for 72 h in silica gel chamber,the moisture-retention rate of the EPS was 75.79±2.5%.So the EPS had excellent moisture-absorption and retention abilities.To our knowledge,this is the first report about the Arctic microbial EPS that has moisture-retention ability.Particularly,its outstanding moisture-retention ability is found to be superior to those of other EPSs reported and functional ingredients generally used in cosmetics.When the concentration of the EPS was 10.0 mg/ml,its scavenging ratios for DPPH·,·OH and O2-· were 55.40±3%,52.1±2.1%and 28.2 ± 3%,respectively.This showed that the EPS had good antioxidant activity.This was the first report on the antioxidant activity of EPS secreted by an arctic bacterium.In addition,after human dermal fibroblasts were inoculated at 4? for 20 h under different concentrations of the EPS,the percentages of active cells were different.The EPS could protect human dermal fibroblasts from low temperature.This is the first report about low-temperature protection of the human dermal fibroblasts in EPS-containing medium.Thus the EPS from SM1127 has promising potential applications in cosmetic,pharmaceutical and biomedical fields.
Keywords/Search Tags:Arctic marine bacterium, Exopolysaccharide, Response surface methodology, Fed-batch fermentation, Structural characterization, Rheological property, Safety evaluation, Moisture-retention ability, Antioxidant activity, Low-temperature protection
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