| Bachu mushroom,named Helella leucopus,is a rare wild edible fungus growing in Xinjiang.This edible fungus has a unique fragrance and umami taste,and is rich in nutrients.Its nutritional value is considered higher than that of common edible fungi,such as Shiitake mushroom,oyster mushroom,black fungus,etc.Previous studies have shown that the polysaccharide content of Bachu mushroom is rich,which has the effects of promoting immunity,anti-oxidation and anti-tumor,etc.,but there are few studies have reported the regulation effect of Bachu mushroom on lipid metabolism.This thesis is to analysis the structure of the new polysaccharide in the Bachu Mushroom after the extraction and purification;then study the effect of Bachu Mushroom polysaccharides on the regulation of lipid metabolism by using in vitro and animal model;thirdly,study the regulatory effect and mechanism of Bachu Mushroom polysaccharides on the intestinal microbes of mice with high-fat diet.The main results are as follows:1.Extraction,purification and structural analysis of polysaccharides from the Bachu Mushroom: the method of water extraction and alcohol precipitation was used to extract the polysaccharides from Bachu Mushroom.After protein removal,the crude polysaccharides were further separated and purified by DEAE Seplife FF anion exchange column chromatography and Sephacryl S-400 HR gel column chromatography.Then,liquid chromatography,ion chromatography,infrared spectroscopy and nuclear magnetic resonance spectroscopy were used to analyze the structural characteristics of polysaccharide of Bachu Mushroom.The main conclusions are as follows:(1)The total content of crude polysaccharide in Bachu Mushroom is 19.01% with an average molecular weight of 2067 k Da,and the rest components are water(21.67%),protein(8.75%)and ash(46.23%).is indicates that the polysaccharide from Bachu mushroom is rich in nutritional value.(2)The purified polysaccharide HLP prepared from the separation and purification of the crude polysaccharide of Bachu Mushroom has a molecular weight of 1489.104 k Da and is mainly composed of glucose,rhamnose,galactose and mannose with the molar ratio of35.6:19.4:1.91:42.9.The scanning electron microscope results show that the surface of the Bachu Mushroom polysaccharide is a complex and irregular network with a few pores.The results of infrared spectroscopy show that the polysaccharide of Bachu Mushroom has a typical polysaccharide structure.(3)The methylation and two-dimensional nuclear magnetic resonance spectroscopy were used to explore the precise structure of purified polysaccharide HLP of Bachu Mushroom.Combining the results of methylation analysis and NMR analysis,the structure of HLP was Analyzed.The backbones of the HLP is mainly composed of →4)-α-D-Glcp-(1→、→2)-α-DManp-(1→ and a small amount of →6)-α-D-Manp-(1→,the side chains include the α-L-Rhap-(1→,which is connected to the residue of →4,6)-α-D-Glcp-(1→ at position O-6;and α-DGlcp-(1→,which is connected to the residue of →3,6)-α-D-Manp-(1→ at position O-6.The activity of HLP may be related to the composition and proportion of the monosaccharides,glycosidic bond and spatial structure.2.The effects of crude polysaccharide and purified polysaccharide HLP of Bachu mushroom on regulating lipid metabolism in vitro were studied.The results are as follows:(1)The crude polysaccharide and purified polysaccharide HLP of Bachu mushroom have good bile salt binding ability and cholesterol esterase inhibitory activity,results are dose dependent,among which the purified polysaccharide HLP has a better effect.When the concentration of HLP polysaccharide was 0.5 mg/m L,the bile salt binding ability of HLP was89.11%,and the cholesterol esterase inhibitory activity was 95.12%.(2)The 3T3-L1 preadipocyte model was used to study the role of crude polysaccharide and purified polysaccharide HLP of Bachu Mushroom in regulating lipid metabolism.The conclusions are as follows:(1)The intervention of crude polysaccharide and purified polysaccharide of Bachu Mushroom can significantly degrade intracellular triglyceride and cholesterol content,and the purified polysaccharide has a better effect,which is comparable to that of the positive drug.(2)The intervention of crude polysaccharide and purified polysaccharide of Bachu Mushroom can significantly increase the intracellular HDL-C content and reduce the LDL-C content.The regulatory effect of HLP is better,and there is no significant difference between HLP and positive drugs.The results of the in vitro experiment indicated that the polysaccharide is a potential substance with regulatory effects on obesity and lipid metabolism.3.The model of high-fat diet mice was constructed to evaluate the effect of Bachu Mushroom polysaccharide in regulating lipid metabolism in a systematical way,and obtained the following conclusions:(1)Different doses of polysaccharide intervention can reduce the accumulation of visceral fat caused by high-fat diet to a certain extent.(2)Different doses of polysaccharide intervention can reduce the levels of TG,TC,LDLCand FFA,and increase the level of HDL-C in the serum of high-fat diet mice,thereby having certain ability to lower plasma cholesterol.(3)Different doses of polysaccharide intervention can promote the synthesis of liver glycogen by enhancing the activity of GK and G-6-PD,and reducing the activity of G-6-P in the liver,thus reducing the blood sugar level to certain extent.(4)Different doses of Bachu mushroom polysaccharides can up-regulate the relative expression levels of PPARα,ACS and CPT1 a genes,which is beneficial for accelerating fatty acid oxidation.Also bachu Mushroom polysaccharide can inhibit the de novo synthesis of cholesterol by down-regulating the relative expression of AMPKα,SREBP-2 and HMGR genes;and promote the conversion of cholesterol to bile acid by up-regulating the expression of CYP7A1 gene,thereby reducing hepatic cholesterol content.The results of the animal model experiments indicated that the Bachu mushroom polysaccharide can reduce blood lipid levels and improve the body’s lipid metabolism by regulating the relative expression of genes related to lipid metabolism in the liver and regulating the activity of sugar and lipid metabolism related enzymes.4.The regulatory effect of Bachu Mushroom polysaccharide on the intestinal flora of highfat diet mice was studied in depth by 16 s r RNA high-throughput sequencing technology,and the conclusions were as follows:(1)Different doses of polysaccharide intervention can increase the species diversity of flora and the richness of flora in high-fat diet mice.(2)High-fat diet feeding reduces the proportion of Bacterodidetes in the intestinal flora of mice,while the intervention of different doses of Bachu Mushroom polysaccharide can downregulate the abundance of Fimicutes;it can also down-regulate the phylum abundance and Patescibacteria phylum abundance to the levels comparable to that of the normal control group.(3)The relative abundance of norank＿f＿Murbaculaceae and Lactobacillus in the intestinal flora of mice is significantly reduced by feeding mice with a high-fat diet.The relative abundance of Candidatus＿Saccharimonas and norank＿f＿Lachnospiraceae can be reduced and the level of norank＿f＿Murbaculaceae can be increased by feeding mice with Bachu Mushroom polysaccharide.(4)Bachu Mushroom polysaccharide intervention is effective in improving the structural changes of intestinal flora and the intestinal microbial dysbiosis caused by high-fat diet.The experimental results indicated that HLP polysaccharide has a good regulatory effect on the intestinal microbes,which is beneficial to improving lipid metabolism.The regulatory effect on the intestinal microbes may be related to the monosaccharides composition of HLP and the proportion of glucose in the HLP.In conclusion,the structure of the polysacharide HLP was analyzed after extraction and purification from the Bachu Mushroom,which shows that HLP has the typical polysaccharide characteristics.The in vitro experiment showed that HLP has a good regulatory effect on lipid metabolism.The further animal experiment showed that polysaccharide can reduce blood lipid levels and improve lipid metabolism in the body,and the mechanism of the action was analyzed.The regulation effect of polysaccharide on the intestinal microbes of high-fat diet mice was also studied.The results showed that the intervention of polysaccharide has a good regulation effect on the intestinal microbes of the mice,which is also beneficial to improving lipid metabolism.This regulatory effect may be related to the special structure of the polysaccharide,such as the monosaccharides composition. |
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