Screening Of Hericium Erinaceus Strains With High Active Polysaccharide Productivity And Research On Its Metabolites Harvested At Different Developmental Stages

Hericium erinaceus is an edible and medicinal fungi belonging to Basidiomycota. In ancient times, people took it and Trionyx sinensis, sea cucumbers and bear’s paw as "four famous dishes". And we have a saying"Hericium delicacies, seafood bird’s nest". Reported in the literature, Hericium erinaceus has anti-ulcer, anti-inflammatory, antitumor, hypoglycemic, anti-aging, anti-mutation, hepatoprotective, and many other pharmacological effects. Based on the edible and medicinal value, the paper attempted to Screen of Hericium erinaceus Strains with High Active Polysaccharide Productivity and Researched on its non-volatile flavor metabolites and the variation of the macromolecular active metabolite content and in vitro immune activity Harvested at Different Developmental. And then polysaccharides of the later three stages were isolated and purified and studied the physicochemical properties and structural identification. This thesis is divided into five parts, the main findings are as follows:1、Mycelium comparison and Screening of ten Hericium erinaceus Strains with High Active Polysaccharide ProductivityThe researches of antagonistic test and ITS sequence analysis was basically the same, the analysis showed that phylogenetic relationship of 0608 and 920 strains was closed and could not effectively distinguish. But the long thorned strain and other strains distantly related, separately clustered into one group.Mycelium and extracellular fluid of ten strains were collected by Liquid fermentation. By water extraction,70% alcohol precipitation, intracellular polysaccharides and extracellular polysaccharide were obtained,and polysaccharides yields were determined by phenol sulfuric acid method. Intracellular polysaccharides, extracellular polysaccharide production of H9 strain were the highest, respectively, 0.13g/L and 128.76g/L. Immune activity in vitro of H1 strain was the best.Fruiting bodies of ten strains were collected.By the determination of the polysaccharide content in the fruiting body, results showed that polysaccharide content of the 0605 strain was the highest and reached 1.74%, followed by 1.70% for the Huazhong strain, and there were significant differences with other strains. Immune test results in vitro of polysaccharide of ten strains showed that the 0605 strain was not only polysaccharide content was high but also immune activity in vitro of was the best, so 0605 strain was used as the following experiment. 2、Comparative analysis of non-volatile flavor metabolites of Hericium erinaceus harvested at different developmental stagesThe whole developmental stages were divided into six stages, including button formation、without fungal spine age、split age、small fungal spine age、mid-fungal spine age、maturing stage.Samples were collected by industrialized cultivation. Then non-volatile flavor analysis of the metabolites of different developmental stages of fresh fruiting bodies, the results showed that the contents of non-volatile flavor substances of different strains were different, the contents of different developmental stages of the same strain were also different.The contents of arabitol and trehalose from maturing stage of 0627 strain were highest, respectively,23.41 and 1.55 mg/ g.The content of the mannitol was the highest at small fungal spine age, up to 1.13mg/ g.While the content of arabitol of the 0605 strain was the highest at small fungal spine age,13.98 mg/g. The content of trehalose and mannitol was the highest at split age, respectively,1.97 and 0.78μg/g. The content of 5’-CMP,5’-UMP,5’-GMP,5’-IMP and 5’-AMP of 0627 strain were the highest at split age, respectively for 475.58,88.33,24.58,43.83, and 91.05μg/g And glutamate and aspartate content were also the highest, respectively,1.70 and 0.05μg/g.While 5’-CMP content of the 0605 strain was the highest at maturing stage, to reach 488.13, the 5’-UMP,5’-GMP,5’-IMP and 5’-AMP content were the highest at split age respectively for 95.81,46.60,41.38 and 65.35μg/g. The glutamate content was the highest at maturing stage, for 1.43μg/ g,while aspartate content the highest at small fungal spine age,for 0.04μg/g.And the EUC value of 0605 strain was the highest at small fungal spine age. 3、Research on macromolecular active metabolite of Hericium erinaceus harvested at different developmental stagesThe analysis determined the crude protein content,β-glucan content, polysaccharide content and immune activity in vitro of Hericium erinaceus harvested at different developmental stages.The results showed that in the whole developmental stages of Hericium erinaceus, crude protein content of the fruiting body presented overall downward trend, up to 26.56% at button formation,only 16.18% at maturing stage.β-glucan content in fruiting bodies presented the overall rising trend,the highest content of 33.58% at mid-fungal spine age and had an anti-drop process at small fungal spine age.Polysaccharide content of fruiting bodies showed an upward trend, up to 1.54% at maturing stage, only 0.89% at split age. Polysaccharide content of crude polysaccharide extracted from the same conditions showed continuous rising process, the maximum 50.67% at maturing stage. Immune activity in vitro of the polysaccharides of the button formation, split age, mid-fungal spine age and maturing stage were better than that of without fungal spine age and small fungal spine age.4、Fractionation of polysaccharide from small fungal spine age, mid-fungal spine age and maturing stage of the 0605 strain and research of physicochemical propertiesHot water extracts of the three periods of the 0605 strain were graded alcohol precipitation by 30%,50% and 70% ethanol final concentration. Then physicochemical properties of polysaccharide were studied, the results were as follows:sugar content of polysaccharides obtained by 50% alcohol precipitation were higher than that of 30% and 70% alcohol precipitation, in which sugar content of H5FP6B was the highest,up to 67.14%. The total polysaccharide from small fungal spine age is the most. The proportion of polysaccharide derived from the different concentration of alcohol was different.Yield of H5FP4A was far higher than the yield of H5FP5A and H5FP6A,but the proportion of H5FP4C far lower than that of H5FP5C and H5FP6C. Molecular weight distribution of the different polysaccharides were different. HPLC atlas of polysaccharide from 30% alcohol precipitation was divided into three molecular weight segment,including 5.9 million, more than 1.4 million less than 5.9 million and less than 12,000, where the main peak was less than 12000 parts.5.9 million molecular weight segment of H5FP4A in proportion was bigger than that of H5FP5A and H5FP6A. Polysaccharide molecular weight of 50% and 70% alcohol precipitation with three periods was basically the same.These polysaccharides all contained fucose, galactose,glucose, mannose, but the molar ratio of each monosaccharide differed. Polysaccharides obtained by fractionation could stimulate macrophage cell RAW264.7 secret NO, and immune activity of polysaccharides of 30% alcohol precipitation was better than that of 50% and 70% alcohol precipitation. Immune activity of polysaccharide by 70% alcohol was the lowest. Immune activity of polysaccharide from maturating stage was the best.5、Further separation, purification and structural identification of H5FP4B, H5FP5B, H5FP6B H5FP4B, H5FP5B, H5FP6B for further separation and purification Sephacryl S300 gel filtration chromatography, a total of nine components were H5FP4B-1 H5FP5B-1, H5FP6B-1, H5FP4B-2, H5FP5B-2, H5FP6B-2, H5FP4B-3, H5FP5B-3, H5FP6B-3, these nine components of the molecular weight distribution of research, found in part 3 of each period (ie, the molecular weight spectrum of the peak part) share essentially the same area, the molecular weight distribution the big difference between a component of the three periods; 2,3 components of the three periods in vitro immune activity differences are not found in vitro immune activity of a component of the three periods of in vitro immune activity show very different; H5FP4B-3 molecular weight of 15,900, composed of fucose, galactose, glucose, three molar ratio 5.2:23.9:1, methylation analysis results show that H5FP4B-3 was terminal fucose linkage and 1,6、1,2,6 galactose linkage. NMR analysis further showed that main chain was composed of a-D-1,6 linkeda-D-galactan backbone and branches composed of terminala-L-fucose.Its structure was: a-L-Fucpv 1(?)↓(?) 2(?) 6)-α-D-Galp-(1→6)-α-D-Gaip-(1→6)-α-D-Galp-(1→6)-α-D-Galp-(1→6)...