The Extraction, Separation And Purification Of Agaricus Bisporus Polysaccharide

With the rapid development of social economy and the improvement of people’sliving standards, the focus on nutrition and health care is an important topic in modernlife. Our health care and eating habits have been a thousand years of history. Especiallyin recent years, health-care food gradually become a major highlight of the dailyconsumption. Fungal polysaccharides play an increasingly important role as animportant part of functional products. The main subjects of the thesis are extraction,separation, purification, and identification of Agaricus bisporus Polysaccharide. Thedetails and results were as follows:(1) In this experiment, the measure of moisture content was87.93%using the highquality Agaricus bisporus as raw material. The UV scanning spectrum of Agaricusbisporus Polysaccharide showed that the maximum absorption peak was489nm.Agaricus bisporus homogenate could be generated after color protection andpreprocessing using1%citric acid.(2) The optimum process conditions were determined by extraction ofpolysaccharides using ultrasonics assisted complex enzyme method and single factorexperiments for the ultrasonics and as follows: extraction time was controlled in about30min, the best ratio of feed liquid was1:20, the optimum temperature was30°C, andthe best ultrasonics extraction power was600W.The optimum technological conditions of the cellulase were determined by singlefactor and L9(34) orthogonal experiment and as follows: the enzyme content was1.2%,pH value was4.0, enzymolysis temperature was50℃, enzymolysis time was100minutes; and the best conditions of the neutral protease were as follows: the enzymecontent was1.0%, pH value was7.5, enzymolysis temperature was45℃, enzymolysistime was100min.(3) On the basis of the single factor experiment, orthogonal experiment wasexecuted and indicated by decolorization rate and polysaccharide loss rate. The optimaldecolorization conditions were determined by activated carbon bleaching temperature,time, dosage and pH value and as follows: adding1.5%active carbon to polysaccharidesolution at50℃, with pH at5.0and the decoloring time for60minutes.(4) Sevag method separates the protein by using the theory of protein denaturationand precipitation causing by organic solvents, under the mild conditions and with high removal efficiency. UV full wavelength scanning results of liquid glucose afterremoving protein by Sevag method showed that the absorption peaks were not at260nmand280nm, indicating that the high protein removal effectiveness and efficiency of theSevag method.(5) The study of the relationship between alcohol precipitation with differentvolume of ethanol and polysaccharide yield showed that, polysaccharide yield washigher and the color was lighter fawn when the ethanol volume fraction was75%.Therefore, in order to process the polysaccharide and alcohol precipitation, theconcentration of ethanol was75%and the temperature was4℃.(6) Through displacement chromatography of DEAE-cellulose52, two objectivepeak were obtained from Agaricus bisporus polysaccharide by different adsorptioncapacity to each liquid glucose component, which were PR-I and PR-II, as well asimpurities. Both peak materials were easy to dissolve in the water. PR-I appeared at thebeginning of elution with phosphate buffer under the conditions of0.02mol/L density、pH=6.8. PR-Ⅱ was eluted with NaCl-phosphate buffer under the conditions of0.2mol/L density.(7) Gel filtration method is one of indispensable media in separation andpurification of biological macromolecules technology. Single and symmetrical completepeak shapes were obtained after the G-100gel filtration chromatography of PR-I andPR–II components which were acquired by ion exchange. The results indicated that PR-I and PR-II was homogeneous polysaccharides, and high purity Agaricus bisporusPolysaccharide was isolated from the experiment.(8) After freeze drying of Agaricus bisporus Polysaccharide components PR-I andPR-II, faint yellow powder was obtained, that was soluble in water and especially inwarm water but insoluble in other organic solvents. The experimental results alsoshowed that it didn’t contain amino acid, starch and other substances.