Study On The Substance Of Polysaccharides And Proteins In Dictyosphaerium Sp. 1A10

Microalgae are diverse population of microorganisms capable of photosynthesis.Compared with other organisms,microalgae have the advantages of adaptability,rapid growth and low cultivation cost,which makes the accumulate a large amount of valuable biomass in microalgae including polysaccharides,proteins,lipids,pigments and vitamins,etc.In this paper,Dictyosphaerium sp.1A10 was researched for the basic nutrient components,the optimization of cultivation conditions,the comparation and optimization of extraction for polysaccharides and proteins,and the antioxidant capacity of polysaccharides and proteins in vitro.The main results of the trial are as follows:（1）The basic nutrient contents of Dictyosphaerium sp.1A10 were:moisture（8.60±0.01%）,ash content（4.13±0.01%）,polysaccharide（2.71±0.23%）,protein（56.88±0.33%）,lipid（27.11±0.52%）,and crude fiber（12.73±0.26%）.Twelve fatty acids were detected in Dictyosphaerium sp.1A10,with polyunsaturated fatty acids accounting for 65.72%.For the polyunsaturated fatty acids,which composed with linoleic acid andα-linolenic acid.Nineteen amino acids were detected in Dictyosphaerium sp.1A10,and the essential amino acids accounted for 25.03%.Twenty-two mineral elements and six vitamins（VB₂,VB₃,VB₆,VA,VD₃and VE）were detected in Dictyosphaerium sp.1A10.（2）To optimize the cultivation conditions of Dictyosphaerium sp.1A10,seven factors were selected for single-factor experiments,and then four factors（p H,nitrogen concentration,phosphorus concentration and carbon concentration）were selected for respond surface design base on the results of the single-factor experiment The optimum cultivation conditions were p H 8.10,nitrogen concentration 2.47 g/L,phosphorus concentration 0.06 g/L,carbon concentration 0.06 g/L,and the optimized respond value of biomass,the extraction rate of polysaccharides and the extraction rate of protein isolates were 2.14±0.08 g/L,3.19±0.11%and 18.60±0.21%respectively,which was significantly improved compared with unoptimized（P<0.05）.（3）The extraction methods of crude polysaccharide and protein isolates from Dictyosphaerium sp.1A10 were compared and optimized.For the extraction of crude polysaccharide,the ultrasonic extraction of green and convenient was chose,and the method for crude polysaccharide rate was 2.71±0.43%.Through the single-factor experiment and the orthogonal optimization test,the optimum extraction conditions were obtained as ultrasonic time of 35 min,ultrasonic temperature of 40℃,the ultrasonic power of 300 W for 3.59±0.05%of the extraction rate of crude polysaccharide,which was32.47%higher than that of the unoptimized.For the extraction of protein isolates,it was determined that the extraction rate of alkali extraction with ultrasonic wave was the highest,which was 15.61±0.30%.Through the single-factor experiment and the orthogonal optimization test,and the optimal conditions were ultrasonic time of 35 min,ultrasonic temperature of 40℃,the p H of 10 and the extraction rate of the protein isolates was18.97±0.06%,which was 21.53%higher than that of the unoptimized.（4）The crude polysaccharide of Dictyosphaerium sp.1A10 was precipitated with80%ethanol and was purified with DEAE-52 column chromatography and four polysaccharide components were obtained.The content of polysaccharide and uronic acid in the four polysaccharide components were different.The antioxidant capacity in vitro of the four polysaccharide components was compared,and the polysaccharide component PC-4 with higher antioxidant capacity was chose for the second purification with agarose gel CL-6B to obtain the purified polysaccharide component of PCP-4.It was confirmed that PCP-4 did not contain protein and nucleic acid through ultraviolet and infrared spectroscopy,PCP-4 was a carbohydrate compound containing uronic acid and sulfuric acid group,and it could be considered that PCP-4 was an acidic polysaccharide.The relative molecular weight of the PCP-4 was 121762 Da,which is composed of mannose,rhamnose,glucuronic acid,galacturonic acid,glucose and galactose with the molar ratio of9.26:40.62:0.93:5.98:2.01:1.7 respectively.The sodium periodate oxidation showed that the proportion of（1→）or（1→6）glycosidic bonds in PCP-4 was low accounting for7.79%,（1→2）,（1→2,6）,（1→4）or（1→4,6）glycosidic bonds accounted for 54.55%,（1→3）,（1→2,3）,（1→2,3,4）,（1→3,4）or（1→3,6）glycosidic bonds accounted for37.66%.In addition,the polysaccharide component PCP-4 had a certain antioxidant capacity in vitro.（5）The isoelectric point method was used to precipitate the protein isolates of Dictyosphaerium sp.1A10,the isoelectric point was 3.5,and the protein precipitation rate was 66.73±0.81%.In addition,the albumin,prolamin and glutenin were extracted by the Osborne fractional extraction method.The solubility,water and oil holding capacity of the four proteins were compared.The ultraviolet and infrared spectra of the four proteins all showed the characteristic absorption peaks of protein.The thermal denaturation temperature of the four proteins was different,among which albumin had the highest thermal denaturation temperature 95.54℃,indicating that the thermal stability of albumin was highest.The SDS-PAGE gel electrophoresis of the four proteins showed multiple bands,indicating that the four proteins contained many subunits and were supposed as mixed proteins,among which the gluten had two distinct bands near 100 k Da.The in vitro antioxidant capacity of the four proteins was determined.The prolamin and the protein isolates had better antioxidant capacity in vitro compared with two other proteins,which lower than Vc at the same concentration.