| Chlorella sorokiniana is a kind of unicellular green algae,which is rich in oil,protein,polysaccharide,amino acids,vitamins and minerals.At present,the research on C.sorokiniana is mainly in oil,while its research on polysaccharides and protein?isolation and purification,composition structure,biological activity,etc.?is relatively less and lacks systemicity.The residue of C.sorokiniana after oil extraction is rich in polysaccharides and protein.As one of the active ingredients of C.sorokiniana,polysaccharides has many biological activities,such as anti-oxidation,anti-tumor and bacteriostasis.C.sorokiniana is rich in protein and can be used as a raw material for single-cell proteins.Therefore,the systematic study of C.sorokiniana polysaccharides and protein is helpful to further explore and utilize algae resources,provide a basis for the development of microalgae with biological activities,and provide support for the industrial production of C.sorokiniana with high yield of polysaccharides and proteins.In this paper,the strains of algae isolated from freshwater or seawater areas of Hainan Province were screened to determine the experimental algae?C.sorokiniana C74?,and its nutritional components,polysaccharides and protein extraction methods,and the culture conditions of the algae strain were studied systematically.On this basis,the separation,purification,physicochemical properties and biological activities of polysaccharides and proteins were studied.The results are as follows:?1?Based on the growth rate,biomass,polysaccharides and protein yields and antioxidant activity,24 freshwater or seawater algae were compared.C.sorokiniana C74was identified as a further research alga whose nutritional composition is:protein?28.17%?,fat?20.05%?,total sugar?28.96%?,ash?7.28%?,moisture?4.65%?,crude fiber?8.25%?,polysaccharide?5.90%?,amino acid?1.24%?.Its fatty acids and amino acids are all 18 types,of which 11 are unsaturated fatty acids,accounting for 74.74%.Essential amino acids accounted for 45.61%of the total amino acids.C.sorokiniana C74 contains 25 different trace elements,and the contents of Cr,As,Cd,Hg,and Pb are all lower than the limit of pollutants.?2?The extraction rate of polysaccharides by freeze-thaw ultrasonic method was the highest?5.90±0.32%?.By optimizing the response surface design of the process parameters in the method?liquid-material ratio,number of freeze-thaw ultrasonic,ultrasonic time,and ultrasonic power?was performed.The best extraction process conditions were obtained:liquid-to-material ratio of 27:1,five freeze-thaw cycles,21 minutes of ultrasound time,and325 W of ultrasound power.At this time,the extraction rate of C.sorokiniana C74polysaccharide was 9.28±0.11%,which was 17.02%higher than that before optimization?7.93±0.26%?.The microwave-assisted alkaline method is the best method for protein extraction?50.07±1.21%?.The optimal process parameters obtained through orthogonal experiments are sodium hydroxide concentration 0.05 mol L-1,microwave temperature 55?,microwave time 15 min,material-liquid ratio 1:25.The protein extraction rate was 62.50±0.52%,which was 24.83%higher than before optimization?50.07±1.21%?.?3?BG11 medium was used for C.sorokiniana C74 culture,and the biomass,polysaccharide and protein yield of alga powder were 1.05±0.06 g L-1,0.15±0.01 g L-1,0.11±0.01 g L-1,respectively.Based on the BG11 medium,a four-factor and three-level response surface design was performed with nitrogen,carbon,ventilation and magnesium concentrations as independent variables,biomass,polysaccharides and protein yields as response values.The optimal processes were nitrogen(15.80 mmol L-1),carbon(0.36 mmol L-1),ventilation(40 L h-1)and magnesium(0.33 mmol L-1).The optimized biomass,polysaccharide and protein yield of C.sorokiniana C74 were 3.58±0.07 g L-1,0.52±0.02g L-1,and 0.46±0.02 g L-1,respectively.The three parameter values of the algal flour obtained by outdoor scale-up experiments were increased by 150.63%,211.11%,and187.50%,respectively.?4?In this experiment,C.sorokiniana C74 polysaccharide was purified by DEAE-52 and agarose gel CL-6B column chromatography,and four purified polysaccharide components were obtained.The results of high-performance gel chromatography showed that CM1-1,CM2-1,CM3-1 and CM4-1 are homogeneous components.The results of UV scanning,anthrone-sulfuric acid reaction,Coomassie brilliant blue reaction,iodine-potassium iodide reaction,Fehling reagent reaction and infrared spectrum analysis showed that the four components are polysaccharides without protein,starch and reducing sugar.Analysis of relative molecular weight and monosaccharide composition showed that the relative molecular weights of CM1-1,CM2-1,CM3-1,and CM4-1 were 1544 D,31533 D,92900 D,98536 D,respectively.They consist of mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose,and fucose in varying amounts.The results of differential scanning calorimetry show that CM1-1 has the highest thermal stability and CM2-1 has the worst thermal stability.The analysis of periodate oxidation and Smith degradation showed that the four purified polysaccharides contain 1?2,1?3,1?4,1?6 glycoside bonds or non-reducing end groups.Among them,CM1-1 contains more 1?4 glycoside bonds.CM2-1 contains more 1?6 glycoside bonds or non-reducing end groups;CM3-1 contains more 1?2 or 1?4 glycoside bonds.CM4-1 contains more 1?4 glycoside bonds.The biological activity analysis showed that CM3-1 and CM4-1 had stronger scavenging ability to DPPH,ABTS,and hydroxyl radicals than CM1-1 and CM2-1.?5?Based on a comparison of three methods of protein precipitation?alcohol precipitation method,salting-out method,isoelectric point method?,DEAE-52 column chromatography was used to purify the protein precipitated by salting-out method,and five were obtained.Protein component.The UV and IR spectra of each component have protein-specific absorption peaks.The anthrone-sulfuric acid and Coomassie brilliant blue reactions indicated that each protein component was sugar-free.Differential calorimetry results showed that YM2 and YM5 were the two components with the worst thermal stability and the highest thermal stability among the five protein components.The analysis of SDS-PAGE showed that all the protein components are heterogeneous proteins,among which YM1,YM2 and YM3 are mainly concentrated in the range of 25-35 KD,while the molecular weight of YM4 is relatively small,and YM5 is mainly distributed in the range of 17-25 KD.Baseline correction,deconvolution,and second-order derivative fitting analysis were performed on the amide?band of each protein component.Each protein component contains?-sheet,random coil,?-helix,and?-turn structure,and?-turn structure is the main structure?accounting for 38-42%?.Biological activity analysis showed that YM2 has a strong ability to scavenge ABTS free radicals,while YM3 has a strong ability to scavenge DPPH and hydroxyl radicals.The results show that C.sorokiniana C74 is rich in a variety of nutrients in addition to polysaccharides and proteins with a high proportion.Its rich nutritional components and high nutritional value of protein indicate that it has value for development and utilization as feed,and its relatively excess tryptophan and valine can be considered as nutritional supplements for research and utilization.The optimal method for extracting C.sorokiniana C74polysaccharides and proteins was obtained through experiments.The freeze-thaw ultrasonic method improved the polysaccharide extraction rate while its relatively mild conditions reduced the damage to the polysaccharide structure to a certain extent.The optimized design of the cultivation process significantly increased the yield of C.sorokiniana C74 biomass,polysaccharides,and proteins,and provided a large amount of raw materials for the use of C.sorokiniana C74 algal flour and the research of polysaccharides and proteins,which was helpful to the development of algae resources.The experiment analyzed the structure and properties of C.sorokiniana C74 polysaccharides and proteins,and provided multiple perspectives to understand C.sorokiniana C74 polysaccharides and proteins,which are helpful for the comprehensive utilization of algae resources.At the same time,the determination of polysaccharide and protein biological activity has increased its potential for development as feed. |
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