| Hericium erinaceus is a famous medicinal and edible fungus belonging to Basidiomycota, and commonly used in the treatment of neurasthenia, tumors, and digestive diseases like gastritis and ulcers. It tastes sweet natured with the function of benefiting viscera and helps digestion. The development of fungus is a complex process of life, containing multiple metabolic pathways of metabolism, hormonal regulation, synthesis and transport of nutrients, cell division and differentiation, signal transduction of coordinately regulated at different growth stages. The bioactive of polysaccharides is related to their physico- chemical properties like polysaccharide contents, molecular weight distribution and chemical composition, etc, meanwhile these physical and chemical properties were closely related to the development of the fruiting body. Based on the the physical and chemical characteristics and immune activity of polysaccharides, the paper mainly studied the dynamic formation of extracellular and cell wall polysaccharides during the development of Hericium erinaceus in the cultivation proces, providing a theoretical basis for the picking and cultivation of Hericium erinaceus. Then the structural identification and immunomodulatory effects of the macromolecular homogeneous polysaccharide at the mature stage were studied, laing the foundation for study on the relationship between structure and activity of Hericium polysaccharide. Specific studies were divided into the following sections:1. Physicochemical properties and immune activities in vitro of extracellular polysaccharides from Hericium erinaceus at different developmental stages.The whole developmental stages were divided into seven stages, including bud-forming stage, without fungal spine stage, split stag, small fungal spine stage,mid-fungal spine stage, mature stage, post-mature stage and the fruiting bodies of0605 strains were collected by industrialized cultivation. Then the physicochemical properties and immune activities in vitro of polysaccharides were analyzed with graded precipitated by ethanol and controlling contents of soluble solid, indicates that polysaccharides with excellent biocompatibility can be prepared by controlling the content of soluble solids and low concentration of ethanol in the actual production.Fruiting bodies of Hericium erinaceus harvested at seven different growth stages were extracted by hot water and graded ethanol precipitation to final concentration of20%, 50% and 70%, and 21 crude polysaccharide fractions were obtained. Then structural properties and immunity activities in vitro of polysaccharides were studied.Results showed that in the whole developmental stages of H. erinaceus fruiting bodies,the overall yield of polysaccharides appeared an increasing trend at first then decreasing after the fifth stage. At fifth stage the maximum yield was 0.92%.Polysaccharide content of 20% ethanol precipitation also showed an increasing trend at first then decreasing, and the maximum polysaccharide content was 42.87% at the fifth phase, in which high molecular weight polysaccharides(1 000~5 000 k Da) had the highest proportion. Polysaccharide molecular weights of 50% and 70% alcohol precipitation were all small, ranging from 10 to 40 k Da. Most of the H. erinaceus polysaccharides obtained consisted of fucose, galactose, glucose and mannose, while some differences in the relative proportions existed. The polysaccharides of 20%,70% ethanol precipitations contained a small amount of ribose at the first stage and20% ethanol precipitation contained some rhamnose at the seventh stage. All stages of50% alcohol precipitation polysaccharides contained a certain amount of glucuronic acid. All the 21 fractions of polysaccharide could significantly stimulate macrophages to release NO in vitro, and activity of polysaccharides of 20% ethanol precipitation was better than those of 50% and 70% ethanol precipitation, which showed good immune activity in vitro at 50μg/m L. Results showed that the activities of polysaccharides at the fifth stage were best, indicating of high activity polysaccharides could get at this stage. Our findings provided a theoretical basis for the study of polysaccharide dynamic formation during the development of Hericium erinaceus.2. Physicochemical properties and immune activities in vitro of cell wall polysaccharides from Hericium erinaceus at different developmental stages.Splintered time and extraction solvents were studied for extraction yields and structural characteristics of cell wall polysaccharides from Hericium erinaceus residues. Results showed that the yields of water-soluble cell wall polysaccharides would increase with splintered time prolonging, while the polysaccharide contents decreased. Splintered time had major impact on the molecular distribution patterns and monosaccharide composition of the polysaccharides extracted, and the optimal time was 15 min. With Na OH concentration increasing, yield and content of alkali-soluble cell wall polysaccharides increased, but their immune activity reduced,and the optimum Na OH concentration was 0.5mol/L. Both of polysaccharides extracted by water and alkaline could significantly stimulate murine macrophages to release NO, showing a better immune activity in vitro. Comparison of structural differences indicated that the molecular weigh of water-soluble cell wall polysaccharides was much larger than that of alkali-soluble ones. The later contained glucose only, while the former also contained a certain amount of galactose and the ratio of glucose and galactose was approximately 4:1. This study obtained a superior preparation method of cell wall polysaccharide from Hericium erinaceus, and pointed out the difference of structural features between water-soluble and alkali-soluble cell wall polysaccharides.Obtain water-soluble and alkali-soluble cell wall polysaccharides by Splintered and water extraction and alkaline extraction, precipitated by 70% ethanol, dialysis from Hericium erinaceus residues at seven stages. Then structural properties and immunity activities in vitro of polysaccharides were studied. Results showed that in the whole developmental stages of the water-soluble cell wall polysaccharides, the yield and content of polysaccharides appeared an a decreasing trend after the first increase. the seventh stage the lowest yield was 2.04%, the largest polysaccharide content was 73.73% at the third phase. The molecular weight distribution in the100 000 k Da, The proportion of high molecular weight polysaccharide first increases and then decreases after stage 5 and molecular weight of between 300~900k Da, the proportion of phase 1, 2, 6, 7 was significantly higher than other phases.Results also showed that in the whole developmental stages of the Alkali-soluble cell wall polysaccharides, the yield also showed increased and then decreased after the first stage. At sixth highest stage of 3.50 percent, the seventh stage of the lowest rate of polysaccharide was 2.20%, Phase 3 polysaccharide content of up to 74.19%. In addition, the molecular weight mainly in the 2 000 k Da, the proportion of polysaccharides after the first increased and then decreased, the highest was stage 5.Monosaccharide composition of alkali-soluble cell wall polysaccharides containing only glucose, and water-soluble cell wall polysaccharides mainly composed of glucose and galactose, while some differences in the relative proportions existed.Water-soluble and alkali-soluble cell wall polysaccharides have better biological activity. the former activity was better at low concentration 50μg/m L, Conversely, the later activity was better at high concentration 500μg/m L. Overall, the activities of cell wall polysaccharides at the fourth stage(small fungal spine stage) were best.3. Separation and purification, structure identification and immunomodulatory effects of complex polysaccharide from Hericium erinaceus fruiting bodies.H6PC20 is a complex polysaccharide, isolated and purified by boiling water,standing at low temperatures, high-speed centrifugation and washing with the low concentration of ethanol repeatedly. Polysaccharide yield was 0.25%, the polysaccharide content was 90.99% and its Mw were 2.39×106Da. The results show that monosaccharide composition of H6PC20 is a dextran, methylation analysis results show that H6PC20 was terminal glucose linkage and 1,3、1,3,6 glucose linkage.NMR analysis further showed that main chain was composed of β-1,3-linked D-glucose, with 1,6-β-linked D-glucose as side chain. The repetitive structure unit was:H6PC20 can significantly stimulate NO release from RAW264.7 cells, stimulate the proliferation of mouse spleen lymphocytes. The results showed that H6PC20 have T lymphocyte mitogen nature, can significantly promote the proliferation of T lymphocytes, modulation of immune responses by combined effects of H6PC20 and mitogen. |
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