Study On Extraction,Purification,Physicochemical Properties And Immune Activity Of Carbohydrates From Misgurnus Anguillicaudatus

Misgurnus anguillicaudatus is widely distributed in China as it is delicious meat and high nutritional value.It is popular among consumers due to it is rich in high-quality protein,fatty acids,a variety of mineral elements and B vitamins,etc.Misgurnus anguillicaudatus carbohydrates,which have a variety of biological activities,is a kind of substances extracted from Misgurnus anguillicaudatus.However,the research on Misgurnus anguillicaudatus carbohydrates is still not systematic and comprehensive,which limits its development and utilization.Therefore,the extraction and purification of Misgurnus anguillicaudatus carbohydrates,physical and chemical properties and immune activity are studied in this paper.The main contents are as follows:1.Extraction and separation of Misgurnus anguillicaudatus carbohydratesTaking the yield of Misgurnus anguillicaudatus carbohydrates as the index,the optimal technological conditions for the extraction of Misgurnus anguillicaudatus carbohydrates were determined by single factor experiment and response surface experiment as follows: the material-liquid ratio was 1:5 g/m L,the extraction time was83 min,the extraction temperature was 59℃ and the extraction times were 3 times.The yield of Misgurnus anguillicaudatus carbohydrates was 1.89%.The Misgurnus anguillicaudatus carbohydrates was deproteinized by the traditional Sevage method,and the Misgurnus anguillicaudatus carbohydrates was further ractionated and purified by DEAE-52 cellulose ion exchange chromatography column to obtain MAP(Misgurnus anguillicaudatus polysaccharide)and MAOS(Misgurnus anguillicaudatus oligosaccharides).2.Physical and chemical properties of Misgurnus anguillicaudatus carbohydratesThe molecular weight distribution of MAP and MAOS was determined by high performance liquid gel exclusion chromatography,and its structure was analyzed by chemical method and modern instrumental method.The results showed that MAP was a Misgurnus anguillicaudatus polysaccharide with a weight average molecular mass(Mw)of 3873 Da;MAOS was a Misgurnus anguillicaudatus oligosaccharide with a weight average molecular mass(Mw)of 2854 Da.The monosaccharide composition of MAP was rhamnose,fucose and galactose,and the molar percentage was8.86:87.55:3.59.The monosaccharide composition of MAOS was rhamnose,fucose and arabinose,and the mole percentage was 21.97:0.92:77.11.IR and NMR results showed that MAOS was β-pyranose,MAP was ɑ-pyranose.The results of SEM showed that MAP was composed of spherical particles with tightly bound,smooth surface and regular distribution.MAOS was accumulated as massive particles,tight binding and smooth surface,with weak molecular regularity.Congo red experiment showed that neither MAOS nor MAP contained a triple helix structure.The rheological results showed that the Misgurnus anguillicaudatus carbohydrate solution was a pseudoplastic non-newtonian fluid with the characteristics of shear thinning.Its apparent viscosity can be affected by mass concentration,temperature and metal ions.DSC results showed that MAP has a better thermal stability between room temperature and 109.54 ℃;Meanwhile,the thermal stability of MAOS is better between room temperature and 124.03℃.3.Immune activity of Misgurnus anguillicaudatus carbohydratesIn this study,RAW264.7 cells were used to study the immune activity of Misgurnus anguillicaudatus carbohydrates in vitro.MTT method,neutral red phagocytosis experiment,scanning electron microscope,acridine orange staining method,RT-PCR and Western blot were used to observe and determine the proliferation activity,phagocytic activity,cell morphology and related gene expression of RAW264.7 macrophages.The results of the MTT experiment showed that Misgurnus anguillicaudatus carbohydrates concentration in the range of 2-10μg/m L promoted the proliferation of RAW264.7 cells in a dose-dependent manner.Neutral red experiment showed that Misgurnus anguillicaudatus carbohydrates could enhance the phagocytic ability of RAW264.7 cells.Scanning electron microscopy and acridine orange staining experimental results showed that Misgurnus anguillicaudatus carbohydrates could induce a transition from the resting to the excited state of RAW264.7 cells,while increasing their size and elongating their pseudopods.RT-PCR results showed that compared with the blank control group,different concentrations of Misgurnus anguillicaudatus carbohydrates could significantly increase the expression of IL-6 and TNF-ɑ mRNA(p<0.05).Western blot results showed that in a certain concentration range,Misgurnus anguillicaudatus carbohydrates could increase the expression of cell surface receptor protein TLR4,and promote the expression of downstream proteins My D88,JNK and p38 in a dose-dependent manner.These results suggested that Misgurnus anguillicaudatus carbohydrates can promote immunological status by stimulating immune-related gene and protein expression.