Degradation,Structural Identification,and Activity Analysis Of Shellfish Polysaccharides

In this study,the Lajonkairia lajonkairii cooking liquid polysaccharide(RPCL)and scallop polysaccharide(SP)prepared in the laboratory were used as raw materials.Through the screening of degradation methods and the optimization of conditions,different molecular weight of clam degradation polysaccharide(ARPCL)and scallop degradation polysaccharide(ASP)were obtained.Different molecular weights of clam degradation polysaccharides(ARPCL20,ARPCL40,ARPCL60,ARPCL80,ARPCL95.)and scallop degradation polysaccharides(ASP20,ASP40,ASP60,ASP80,ASP95.)were collected by graded alcohol precipitation at different ethanol concentrations of 20%,40%,60%,80%,and 95%.The basic composition determination,structural analysis,and biological activity research of the above polysaccharide components were conducted,and the main research results are as follows:1.By comparing the molecular weight of polysaccharides after oxidative degradation and acid degradation,acid degradation is chosen as the more optimal degradation method.By changing the conditions of acid degradation(concentration of trifluoroacetic acid,degradation temperature,and time),polysaccharides with different molecular weights were obtained.Finally,the conditions of trifluoroacetic acid concentration of 0.1 mol/L,degradation temperature of 100°C,and time of 30 minutes were selected to degrade clam polysaccharides RPCL and scallop polysaccharides SP.After ethanol fractionation precipitation,the polysaccharide purity of ARPCL40 and ASP40 components was the highest,with 93.74%and 72.19%,respectively.The polysaccharide purity of ARPCL20 and ASP20 components was the lowest,with 16.08%and17.13%,respectively.2.Molecular weight measurement results showed that acid degradation and fractional alcohol precipitation had certain effects on the selection of polysaccharides with different molecular weight.The molecular weight of ARPCL20 was 1.866×10~6 Da-7.15×10~3 Da,the molecular weight of ARPCL40 was 1.932×10~6 Da-1.454×10~5Da,and the molecular weight of ARPCL60 was 2.915×10~4 Da.The molecular weight of ARPCL80 is less than 1.0×10~4 Da,mainly distributed in 7.809×10~3 Da-2.931×10~3 Da.The molecular weight of ARPCL95 was concentrated in 5.052×10~3Da.The molecular weight of ASP20 was 1.777×10~6 Da-4.090×10~3 Da,the molecular weight of ASP40 was 1.839×10~6 Da-6.261×10~4 Da,and the molecular weight of ASP60 was 1.710×10~4 Da.The molecular weight of ASP80 is less than 1.0×10~4 Da and mainly ranges from 4.968×10~3 Da to 1.752×10~3 Da.The molecular weight of ASP95 was concentrated in 4.444×10~3 Da.3.By analyzing the composition of monosaccharides,it can be determined that RPCL and SP before and after degradation are heteropolysaccharides mainly composed of glucose,and the proportion of glucose in polysaccharide is reduced after degradation of graded alcohol.According to the infrared spectrum,the undegraded clam polysaccharide,scallop polysaccharide and the polysaccharide with fractional alcohol precipitation after degradation had little change.Congo Red experiment showed that the two kinds of shellfish polysaccharides had triple helix structure before degradation,and after degradation,ARPCL80,ASP40 and ASP80 had triple helix structure.The triple helix structure of ARPCL40,ARPCL60 and ASP60 disappeared.Uv full-wavelength scanning showed that ARPCL40 did not contain nucleic acid and pigment,and the absorption peaks of RPCL,ARPCL60,ARPCL80,SP,ASP40,ASP60 and ASP80 were detected at 260 nm,indicating that these seven polysaccharides all contain glycoproteins,and with the increase of alcohol concentration,Protein and other impurities in polysaccharides also increase.4.Cell immune activity assay 8 polysaccharides(RPCL,ARPCL40,ARPCL60,ARPCL80,ASP40,ASP60,ASP80)showed no toxicity to macrophages(RAW264.7)at 25-500μg/m L.ARPCL60 has the most significant effect on macrophage proliferation.The neutral red experiment showed that the ability of promoting neutral red phagocytosis of degraded polysaccharide was stronger than that of undegraded polysaccharide.The smaller the molecular weight was,the stronger the phagocytosis ability of macrophages was.The higher the molecular weight of polysaccharides in scallop group,the stronger the phagocytosis,which may be related to the monosaccharide composition and molar ratio of monosaccharides.The experiment of NO content showed that the higher the molecular weight of polysaccharide,the better the immune enhancement ability of cell.The results of in vitro antioxidant experiment showed that with the increase of fractional alcohol precipitation concentration,the smaller the molecular weight of polysaccharide,the stronger the antioxidant activity.The results of in vitro cholate binding experiment showed that the low molecular weight shellfish polysaccharide significantly increased its hypolipidemic activity.