Study On Structure Of Abalone Gonad(Haliotis Discus Hannai Ino) Polysaccharide And Its Interactions With Metal Ions

In the processing of abalone,a large number of by-products were produced,causing waste of resources.It was found that polysaccharide extracted from abalone gonad had good biological activity,and the bioactivity of polysaccharides was closely related to their conformation.However,the conformation of abalone gonad polysaccharide had not been investgated.In addition,abalone gonad polysaccharide product contains minerals and trace elements,and metal ions could affect the conformation of polysaccharides.Therefore,the purpose of this experiment was to analyze the conformation of abalone gonad polysaccharide and the effects of four metal ions such as Na⁺,K⁺,Ca²⁺and Mg²⁺on the conformation and properties of abalone gonad polysaccharide,which will provide references for the development of abalone polysaccharide products.Abalone gonad sulfated polysaccharide?AGSP?was extracted from abalone gonad using enzyme-assisted water-extraction and alcohol precipitation and anion exchange resin adsorption methods.Phenol sulfuric acid method,Folin-phenol method,barium chloride gelatin turbidimetry,1,9-dimethylmethylene blue method and m-hydroxyldiphenyl colorimetric method were used to determine basic composition contents of AGSP,and results showed that the total sugar content of AGSP was 49.5±1.9%,protein content was 5.4±0.3%,the sulfate content was 10.4±0.1%,the content of sulfated polysaccharide was 67.1±2.3%,uronic acid content was 17.2±0.2%.The purity and monosaccharide composition of polysaccharides were identified by high performance liquid chromatography.It was found that the polysaccharides were homogeneity,and AGSP contained nine kinds of monosaccharides,and galactose,glucuronic acid and mannose were the main monosaccharides.The two sugar fragments of AGSP were analyzed by high performance liquid chromatography-mass spectrometry.The results showed that AGSP contained two kinds of disaccharide fragments,of which Gal-GlcA was the main fragment.The ¹H and ¹³C spectra of AGSP were measured by NMR spectroscopy.It was found that AGSP contained two kinds of polysaccharide configurations of alpha and beta.The presence of sulfated group and uronic in polysaccharides was confirmed by infrared spectroscopy.Congo red experiment results showed that the polysaccharide in aqueous solution was a helical conformation and the microstructure of AGSP determined by atomic force microscopy was spherical.The helical conformation of AGSP was verified by the molecular dynamics simulation.The result of X-ray diffraction showed that the freeze-dried AGSP was an amorphous state and the apparent morphology of AGSP was flaky structures with a smooth surface observed by scanning electron microscope.The particle size of the polysaccharide in aqueous solution was determined to be about 190 nm by dynamic light scattering.The melting temperature of the polysaccharide was determined to be about 135.57°C and the degradation temperature was232.27°C,indicating that AGSP had good thermal stability.The variation of apparent viscosity of polysaccharide with shear rates was measured by a rheometer.It was proved that the AGSP aqueous solution was a pseudoplastic fluid with shear thinning characteristics.The effects of four metal cations of Na⁺,K⁺,Ca²⁺and Mg²⁺on the conformation and properties of AGSP were studied by means of dynamic light scattering,congo red test,X-ray diffraction,scanning electron microscope and infrared spectroscopy.The results of dynamic light scattering showed that the particle size of AGSP could be reduced by added Na⁺.With the increase of the concentration of salt cations,the size of polysaccharides was decreased first and then increased by added K⁺.After added Ca²⁺,the particle size of AGSP was gradually increased.Mg²⁺could cause reduction of particle size below the pure AGSP,but it also increased slightly with the increase of the concentration of Mg²⁺.The results of congo red test showed that Na⁺and K⁺couldn't change the maximum absorption wavelength of complex of AGSP with congo red.With the increase of salt concentrations,the maximum absorption wavelength of complex gradually decreased after added Ca²⁺and fluctuated slightly by added Mg²⁺.By scanning electron microscope observation,it was found that the monovalent metal cations Na⁺and K⁺caused changes of apparent morphology of AGSP from flaky structures with a smooth surface to meshed flaky structures,and when salts concentration was higher than 0.05 mol/L,crystal precipitation was observed.After added divalent metal cations Ca²⁺and Mg²⁺,most polysaccharides were aggregated into blocks and the surface of these blocks was smooth with no crystal precipitation was observed.The results of X-ray diffraction indicated that with the increase of salt cations concentrations,the“bun-shaped”diffraction peaks of AGSP gradually decreased,and it was found sharp peaks when the concentrations of Na⁺and K⁺were higher than 0.05mol/L,but there was no sharp peak after added Ca²⁺and Mg²⁺.After added four metal cations,the infrared spectra of AGSP produced vibration peaks by OH deformation vibration and stretching vibrations of C-O-C,>S=O and carboxyl group.