Study On Physiology And Biochemistry Characteristics Of Selenium Enrichment,selenium Polysaccharide Isolation And Purification And Antioxidant Activity In Porphyridium Cruentum

Porphyridium cruentum,a relatively primitive single-celled red algae,is capable of synthesizing a variety of bioactive substances and has great economic applications in the microalgae bioindustry.Selenium is an essential daily dietary nutrient with anti-cancer,anti-oxidant,anti-tumor and other health functions.Porphyridium cruentum can enrich selenium efficiently,and selenopolysaccharide,as one of the main fugitive forms of selenium in Porphyridium cruentum,has both the commonalities and advantages of selenium and polysaccharide or even superior to both.Therefore,in this paper,Porphyridium cruentum was taken as the study object to investigate its selenium enrichment properties and optimize the extraction of intracellular crude selenium polysaccharide,and to investigate the physicochemical properties,structural characterization and antioxidant activity of the seperated and purified fraction.The research results were concluded as listed as follows:(1)Selenium lower than 1.0 mg/L promoted the growth of Porphyridium cruentum,selenium at 1.0 to 5.0 mg/L inhibited its growth,and higher concentrations caused rapid death of Porphyridium cruentum.In the range of 0to 5.0 mg/L,the amount of selenium enrichment in Porphyridium cruentum increased exponentially.The highest was reached at 5.0 mg/L selenium concentration with 484.47 μg/g of organic selenium,while the rest of the experimental groups at 0.2,0.5,1.0 and 2.0 mg/L selenium had 14.63 μg/g,29.74 μg/g,59.41 μg/g and 123.51 μg/g of organic selenium,respectively.Superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activities of Porphyridium cruentum behaved similarly and both increased with increasing selenium concentration.(2)The extraction of polysaccharide components in selenium-rich Porphyridium cruentum was carried out by ultrasound-assisted hot water extraction,and the extraction process was optimized by using response surface methodology.The main order of the three factors affecting the extraction rate of intracellular crude selenium polysaccharide from Porphyridium cruentum was C liquid to material ratio(m L/g)> B extraction temperature(℃)> A extraction time(h).The optimal extraction process parameters of Porphyridium cruentum crude selenium polysaccharide were as follows:extraction time was 2 hours,extraction temperature was 77.3 ℃ and the liquid to material ratio was 27:1(m L/g),under this condition,the extraction rate of crude selenium polysaccharide from Porphyridium cruentum reached 6.75%.(3)Four fractions were obtained after the separation of crude selenium polysaccharide of Porphyridium cruentum by DEAE-52 cellulose chromatography column.They were named as selenium Porphyridium cruentum polysaccharide-1(SPCP-1),selenium Porphyridium cruentum polysaccharide-2(SPCP-2),selenium Porphyridium cruentum polysaccharide-3(SPCP-3)and selenium Porphyridium cruentum polysaccharide-4(SPCP-4),and then the SPCP-3 with the highest recovery and polysaccharide content,was further purified by Sephadex G-100 gel column to obtain refined selenium Porphyridium cruentum polysaccharide(RSPCP).Chemical methods verified RSPCP as a non-starchy water-soluble acidic selenium polysaccharide.The total selenium content in selenium-enriched Porphyridium cruentum was 497.45 μg/g and the selenium content in RSPCP was 18.87 μg/g as measured by atomic fluorescence photometry.(4)The structure of RSPCP was characterized,UV spectral scan showed that RSPCP was a homogeneous polysaccharide.Infrared spectrum showed that RSPCP had typical polysaccharide characteristic absorption peaks and was a pyranoacid sulfated selenium polysaccharide with β-glycosidic bond.The molecular weight of RSPCP was determined by high performance gel permeation chromatography to be 8033 Da.Ion chromatograph showed that RSPCP was composed of galactose,mannose,glucose,glucuronic acid and galacturonic acid with a molar ratio of 0.426:0.344:0.151:0.044:0.035.(5)Using Vitamin C(Vc)as a control,the antioxidant activity of Porphyridium cruentum selenium polysaccharides at different purification stages was examined.The results showed that the scavenging rate of Porphyridium cruentum selenium polysaccharides on DPPH radicals,hydroxyl radicals and superoxide anion radicals was dose-dependent with concentration,and the purer Porphyridium cruentum selenium polysaccharide had the stronger scavenging ability.The scavenging ability of Porphyridium cruentum selenium polysaccharide for the three free radicals was from strong to weak:DPPH radicals > superoxide anion radicals > hydroxyl radicals.At the concentration of 1.0 mg/m L,the scavenging rate of Porphyridium cruentum refined selenium polysaccharide RSPCP reached 88.05%,79.01% and 85.25%for DPPH radicals,hydroxyl radicals and superoxide anion radicals,respectively.