| Phycocyanin is an intracellular protein of cyanobacteria,which has good anti-oxidation,anti-inflammatory,anti-cancer and fluorescence properties,and it is valuable in the application in food industry,cosmetics and medicine.Phycocyanin in accordance with the purity can be divided into food grade,pharmaceutical grade and reagent grade,the higher the purity,the greater the value.At present,there are few studies on the extraction and purification of reagent-grade phycocyanin from the cyanobacteria in Chaohu Lake,and the studies basically stay in the laboratory stage,the research on the process technology combination mode of extraction and purification of reagent grade phycocyanin is not systematic.Therefore,the basic idea of this paper is to take the fresh algae in Chaohu Lake as the object,use the UV-Vis absorption spectroscopy,explore and analyze the technology and mechanism of extraction and purification of phycocyanin by experiment,establish the process technology combination mode of extraction and purification of reagent-grade phycocyanin,set up a pilot production line of a certain scale and master the basic operational parameters of scale-up production,and provide the basis for the study of high value-added resources of cyanobacteria in Chaohu Lake.The conclusions are:(1)Investigation on the reclamation of bloom-forming cyanobacteria in Chaohu Lake,comprehensive analyzed and compared the reclamation of bloom-forming cyanobacteria in several directions,the reclamation of bloom-forming cyanobacteria can be divided into three directions including general additional value,higher additional value and high additional value.It is concluded that the extraction and purification of phycocyanin is an important aspect of cyanobacteria in high additional value reclamation research.Furthermore,combined with theoretical analysis and the actual situation,the phycocyanin extraction and purification technology were systematically compared,the basic technical line of phycocyanin extraction and purification was pre-selected,which provided the preliminary guidance for the experimental research.(2)In the experiment of extracting phycocyanin by freeze-thaw method,the results showed that the optimum parameters were determined as follows: 96.5% water content,–20 °C freezing temperature,0.75%(w/v)phosphate buffer solution(used as freezing and thawing medium).Through two freezing and thawing experiments,the purity and yield of phycocyanin in crude extract reached the maximum,0.451 and 3.73%,respectively.According to the actual situation of laboratory study and pilot,the fresh algae whose water content was about 96% was selected as the treatment target.The freezing-thawing temperature was below –15 °C,freezing-thawing times was 1,the deionized water was selected as the freeze-thaw medium for pilot-scale experiment,the operation procedure was simplified and the cost was reduced.The yield of phycocyanin was more than 3% and the purity was more than 0.35.(3)In the experimental study of general purification of phycocyanin,a six-step salting-out was carried out.The results showed that the optimal concentration of(NH4)2SO4 in odd number of salting-out was 1.0 mol/L,and that of(NH4)2SO4 in even-number of salting-out was 1.8 mol/L.The purity of phycocyanin increased to 2.26 by two-step salting-out,the purity increased to 3.48 by four-step salting-out,and the purity rose to 3.71 by six-step salting-out.Moreover,the recovery of phycocyanin was 77.38% after two-step salting-out,the recovery was 54.92% after four-step salting-out and the recovery was 45.98% after six-step salting-out.The results of UV-visible absorption spectrum indicated that,the effect of the first salting-out might be that it removed a small amount of cell wall residues which were not completely removed,a small amount of nucleic acid substances and vitamins and other light yellow substances,the second salting-out could remove a large number of nucleic acids and vitamins,a small amount of nucleic acids and some other proteins could be removed by the third salting-out,the influence of other proteins on phycocyanin could be further removed by the fourth salting-out,the absorption spectroscopic analysis of the fifth and sixth salting-out showed that a certain amount of phycocyanin and allophycocyanin were lost simultaneously,but the two proteins with similar properties could not be separated at the same time.(4)In the experiment on obtaining phycocyanin from refined purification,the comparison of the two-step fractional salting-out combined with aqueous two-phase extraction and two-step fractional salting-out combined column chromatography was conducted.The two-step fractional salting-out combined Cellufine A-500 column chromatography,followed by HA column chromatography was finally determined.Salting-out was followed by Cellufine A-500 column chromatography,the sample volume accounted for 2/3 of the bed volume,the isocratic elution was performed with 20 mmol/L phosphate buffer at pH 6.5,and then gradient elution was carried out by adding 0.1,0.3 and 1.5 mol/L NaCl to 20 mmol/L,pH = 6.5 phosphate buffer solution,the elution flow rate was 6 m L/min.Cellufine A-500 column chromatography was followed by HA column chromatography,coloring 1/3-2/3 of column bed,the isocratic elution was performed with 10 mmol/L phosphate buffer at pH 7.0,and then gradient elution was carried out by adding 0.1 mol/L NaCl to 20,50,100 mmol/L,pH = 7.0 phosphate buffer solution,the elution flow rate was 4 m L/min.Finally,phycocyanin and allophycocyanin with purity of more than 4.0 could be obtained at the same time.(5)The phycocyanin and the removed or separated components obtained during the experiment were analyzed by ultraviolet-visible absorption spectroscopy.The main role of the first Cellufine A-500 chromatography was to isolate phycoerythrin,and the second HA column chromatography was to isolate and obtain phycocyanin from allophycocyanin.After four-step purification,the absorption peak of phycocyanin in the ultraviolet absorption region redshifted gradually from 260 nm,269 nm,277 nm to 280 nm.In the absorption band of the visible spectrum,the absorption peak redshifted gradually from 617 nm,618 nm to 620 nm and the purity of phycocyanin solution was increased gradually.(6)The process combination of C-PC extraction and purification in pilot-scale was finally as follows: one-step freezing-thawing to obtain the C-PC crude extract-one-step secondary membrane to obtain food-grade C-PC solution-two-step fractional salting-out to obtain a pharmaceutical grade C-PC solution-two-step column chromatography to obtain reagent grade C-PC solution.And finally C-PC and C-APC with purity of more than 4.0 were obtained at the same time.The purified phycocyanin solution was characterized by UV-Vis spectrum,electrophoresis identification and fluorescence analysis.The results showed that phycocyanin with purity of more than 4.0 have good spectral absorption characteristics and fluorescence. |
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