Screening Of Hypoglycemic Active Components Of Polysaccharides From Fermented Mycelium Of Inonotus Obliquus And Study Of Their Hypoglycemic Effect

Inonotus obliquus is a rare edible and medicinal fungus mainly distributed in the cold zone of the northern hemisphere,with a long history of use.Inonotus obliquus polysaccharide（IOP）is the main component of Inonotus obliquus,which can be obtained from fruiting body,mycelium and fermentation broth.The harsh survival conditions and excessive collection of wild Inonotus obliquus fruiting bodies as well as the immature artificial cultivation technology limit the research and development of Inonotus obliquus.At present,the research of Inonotus obliquus polysaccharide mainly focuses on the polysaccharides of fruiting body,and less on the polysaccharides of liquid fermentation mycelium.Taking Inonotus obliquus as the research object,the mycelium was obtained by liquid fermentation,and the extraction,separation and purification process of mycelium polysaccharides were optimized to obtain a single component polysaccharide.The structure of polysaccharides were characterized by instrumental analysis and chemical methods,and the best hypoglycemic active components were explored through enzyme,cell and animal experiments.The aim is to provide some reference materials for further research and comprehensive development of polysaccharides from Inonotus obliquus mycelium.The main research results of this paper are as follows:（1）Screening of polysaccharides from Inonotus obliquus mycelium strains with high yield and high hypoglycemic activity and study on the extraction process of mycelial polysaccharide.The mycelium biomass（14.96±0.11 g/L）,the growth rate（6.039±0.34 mm/d）and the extraction rate of mycelium polysaccharide（8.02±0.45%）of strain H6 were higher than those of the other 6 strains,and the inhibition ofα-glucosidase was also higher than that of the other 6 strains.With extraction time,liquid-material ratio and extraction temperature as influencing factors and extraction rate of crude polysaccharide as evaluation index,Box-Behnken design was used to optimize the extraction process of polysaccharide from mycelium of strain H6.The results showed that extraction rate of polysaccharides from Inonotus obliquus mycelium reached 8.02±0.45%when the extraction was carried out at 97℃and a liquid-material ratio of 51:1（m L/g）for 1.9 h and three times of extraction.（2）Isolation and purification of crude polysaccharides from Inonotus obliquus mycelium.Enzyme+TCA-n-butanol method was superior to other methods,the protein removal rate was 75.93±0.89%,and the polysaccharide retention rate was71.88±5.32%.Orthogonal experiments optimized the decolorization conditions of S-8macroporous resin method.The optimal decolorization conditions were resin addition amount of 2 g,decolorization temperature of 30℃,decolorization time of 6 h,decolorization rate of polysaccharide was 78.31±1.34%,and polysaccharide retention rate was 90.79±5.46%,which was superior to activated carbon method,hydrogen peroxide method and other three macroporous adsorption resins.Three components,IOP40,IOP60 and IOP80,were obtained by ethanol fractionation.Four polysaccharide components,IOP40-a,IOP40-b,IOP60-a and IOP80-a,were finally obtained by DEAE-52 cellulose and Sephacryl S-200 HR chromatography.Chemical composition analysis showed that the content of total polysaccharide was significantly higher than that of ethanol fractions after column chromatography,and the content of protein was significantly reduced,which further indicated that the purification effect was good.（3）Structural characterization of polysaccharides from Inonotus obliquus mycelium by column chromatography.The results of ultraviolet visible spectroscopy scanning and Congo red experiment showed that the four polysaccharide components had high purity and did not have triple helix structure;The results of Fourier transform infrared spectroscopy,¹H-NMR and ¹³C-NMR nuclear magnetic resonance showed that IOP40-a,IOP40-b and IOP60-a wereα-pyranose andβ-pyranose,while IOP80-a wasα-pyranose;High performance liquid chromatography was used to analyze the monosaccharide composition.IOP40-a,IOP40-b and IOP80-a were mainly composed of glucose,while IOP60-a was mainly composed of mannose,galactose and glucose.The content ratio of the three was 39.34:32.96:23.26;The permeation molecular weights of the four polysaccharide components by gel permeation chromatography were 165.455 k Da,192.121 k Da,3.924 k Da and 1.066k Da,respectively;Scanning electron microscope was used to analyze the surface morphology of polysaccharides.The results showed that IOP40-a was wavy;IOP40-b was a lamellar structure;IOP60-a was a raised flocculent gully;IOP80-a was a dense honeycomb shape.（4）Study on hypoglycemic activity of polysaccharides from Inonotus obliquus mycelium.Crude polysaccharide（IOP）,ethanol-graded polysaccharides（IOP40,IOP60 and IOP80）and column chromatography polysaccharides（IOP40-a,IOP40-b,IOP60-a and IOP80-a）were used as test materials.When the polysaccharide concentration was 10 mg/m L,IOP showed the strongest inhibitory effect onα-glucosidase andα-amylase,followed by IOP80-a（inhibition rates were 62.88±1.77%and 37.48±1.55%,respectively）.The results of glucose consumption test in human normal liver cells（L-02 cells）showed that IOP80 and IOP80-a significantly promoted glucose uptake,showing a dose-effect relationship.In the range of 0-640μg/m L,compared with the control group,the difference was not statistically significant（P>0.05）,indicating no toxic effect.In a T2DM mouse model,IOP80 was found to lower fasting blood glucose（31.28%lower than before administration）and had little effect on body weight.In conclusion,IOP80 and IOP80-a are potential hypoglycemic active components.