产活性多糖猴头菌株筛选及其生长过程中代谢产物的研究
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摘要
猴头菌(Hericium erinaceus)是一种食药两用菌,隶属于菌物界担子菌门担子菌纲猴菇目猴菇科猴菇属。在古代,人们把它与鳖鱼、海参、熊掌刺并列为“四大名菜”,并且有“山珍猴头,海味燕窝”的美称。文献报道,猴头菌具有抗溃疡、抗炎症、抗肿瘤、降血糖、抗衰老、抗突变、保肝等多方面的药理作用。本论文从食药用价值两个角度对猴头菌进行研究,主要对不同菌株进行了活性多糖高产菌株的筛选,并对筛选出的菌株的不同生长发育过程中非挥发性呈味代谢产物以及大分子活性代谢产物的含量及体外免疫活性的变化规律进行了研究,然后对发育后期三个阶段的多糖进行分离纯化、理化性质研究及结构鉴定。本论文分为五个部分,主要研究结果如下：
1、对收集来的10个猴头菌株进行了亲缘关系的分析及菌丝体和子实体活性多糖高产菌株的筛选
拮抗实验和ITS序列的分析对10个菌株的聚类分析结果基本一致,分析中发现,0608菌株和920菌株亲缘关系很近,可能是同种异名,不能有效区分；长刺猴头与其余菌株亲缘关系较远,单独聚为一类。
对10个菌株进行液体发酵培养,分别收集菌丝体和胞外液,经水提、70%醇沉,苯酚硫酸法测定得到10株菌株胞内多糖和胞外多糖的产量,其中猴头王的胞内多糖、胞外多糖产量均最高,分别达到0.13g/L和128.76g/L；对10个菌株胞内多糖进行体外免疫试验,结果表明,0627菌株的体外免疫活性最好。
对10个菌株进行子实体栽培,分别收集10个菌株的子实体,烘干的子实体经经水提、70%醇沉,苯酚硫酸法测定子实体中的多糖含量,结果表明0605菌株的子实体多糖含量最高,达到1.74%,其次为华中猴头1.70%,与其余菌株之间有显著性差异；对10个菌株的子实体多糖进行体外免疫试验,结果表明0605菌株不仅子实体多糖含量高而且多糖体外免疫活性最好,所以选择0605菌株作为以下试验栽培取样所用菌株。
2、对猴头菌生长发育过程中非挥发性呈味代谢产物进行了比较分析
对0627、0605菌株进行子实体栽培,并将猴头菌的整个生长发育过程划分为六个时期—现蕾期、无菌刺期、分裂期、小菌刺期、中菌刺期和成熟期,分别对0627、0605菌株的六个发育时期进行取样,对不同发育时期子实体进行非挥发性呈味代谢产物的分析,结果表明不同的菌株在非挥发性呈味物质的含量上表现出一定的差异性,相同菌株生长发育过程中所含的非挥发性呈味物质的量上也是有差异的。0627菌株在成熟期新鲜子实体中的阿糖醇和海藻糖的含量最高,分别达到23.41和1.55 mg/g,小菌刺期甘露醇的含量最高,达到1.13mg/g,而0605菌株在小菌刺期阿糖醇的含量最高,为13.98 mg/g,分裂期的海藻糖和甘露醇的含量是最高的,分别为1.97和0.78μg/g；0627菌株在分裂期新鲜子实体中的5’-CMP、5’-UMP、5’-GMP、5’-IMP和5’-AMP的含量最高,分别为475.58、88.33、24.58、43.83和91.05μg/g,谷氨酸和天冬氨酸的含量也是最高,分别为1.70和0.05μg/g,而0605菌株成熟期的5’-CMP含量最高,达到488.13,分裂期的5’-UMP、5’-GMP、5’-IMP和5’-AMP含量最高,分别为95.81、46.60、41.38和65.35μg/g,成熟期的谷氨酸的含量最高,为1.43μg/g,小菌刺期的天冬氨酸的含量最高,为0.04μg/g；并且经计算0605菌株的小菌刺期的等鲜浓度EUC的值是最高的。
3、猴头菌生长发育过程中大分子活性代谢产物的研究
对生长发育六个时期的子实体进行了粗蛋白含量、β-葡聚糖含量、子实体多糖含量的测定并对热水提取、70%醇沉的六个生长发育期的多糖进行体外免疫活性的研究,结果表明,在猴头菌的整个生长发育过程中,子实体粗蛋白含量总体呈下降的趋势,现蕾期最高26.56%,成熟期最低16.18%,小菌刺期出现了一个反升高的过程；子实体β-葡聚糖含量整体上呈上升的趋势,中菌刺期含量最高33.58%,小菌刺期出现了一个反下降的过程；子实体多糖的含量呈现上升的趋势,成熟期含量最高1.54%,分裂期含量最低仅0.89%；相同条件提取的粗多糖透析之后多糖含量不断的上升,成熟期最高,达到50.67%；现蕾期、分裂期、中菌刺期、成熟期的多糖均能很好地刺激巨噬细胞RAW264.7产生NO,且这四个时期的多糖的体外免疫活性均优于无菌刺期和小菌刺期的多糖。
4、0605菌株的小菌刺期、中菌刺期和成熟期的子实体多糖的分级分离及理化性质研究
分别采用30%、50%和70%的乙醇终浓度对0605菌株3个时期的热水浸提物进行分级醇沉,对分级分离多糖进行理化性质研究,结果如下：三个发育期50%醇沉的多糖的糖含量比30%和70%醇沉的多糖糖含量都高,其中成熟期50%醇沉多糖糖含量最高,达到67.14%；从各个时期每个醇沉浓度所得多糖所占的比例上来看,小菌刺期30%醇沉所得多糖比例远远高于中菌刺期和成熟期对应多糖所占的比例,而小菌刺期70%醇沉多糖远低于中菌刺期和成熟期对应多糖所占的比例；不同时期不同醇沉浓度所得的多糖的分子量分布不同,小菌刺期30%醇沉多糖中大于590万分子量段的所占比例比中菌刺期和成熟期30%醇沉多糖中的比例高很多；3个时期50%和70%醇沉多糖分子量相差不大；3个时期分级分离多糖的单糖种类基本上相同,都含有岩藻糖、半乳糖、葡萄糖、甘露糖,但是单糖的摩尔比例有差异；3个时期分级分离多糖均能刺激巨噬细胞株RAW264.7分泌N0,每个时期30%醇沉的多糖均比50%和70%醇沉多糖组分的活性好,70%醇沉多糖组分活性最差,成熟期分级分离多糖的活性最好,优于其它生长阶段。
5. H5FP4B. H5FP5B. H5FP6B的进一步分离纯化及结构鉴定
0605菌株小菌刺期、中菌刺期和成熟期50%醇沉多糖(H5FP4B.H5FP5B. H5FP6B)经过Sephacryl S300凝胶层析进行进一步分离纯化,共得到九个组分分别为H5FP4B-1、H5FP5B-1、H5FP6B-1、H5FP4B-2、H5FP5B-2、H5FP6B-2、H5FP4B-3、H5FP5B-3、H5FP6B-3,对这九个组分的分子量分布进行研究,发现每个时期的第3部分(即主峰部分)分子量基本相同,分子量分布差异较大的是三个时期的第1组分,在体外免疫活性上也表现出较大差异；第2、3组分体外免疫活性差异不大；H5FP4B-3的分子量为1.59万,由岩藻糖,半乳糖和葡萄糖组成,三者摩尔比为5.2：23.9：1,甲基化分析结果表明,岩藻糖以端基的形式存在,半乳糖主要为1,6、1,2,6两种连接方式存在。经红外光谱、甲基化和核磁共振综合分析,得出H5FP4B-3均一多糖是以α-D-1,6连接的半乳糖和α-D-1,2,6连接的半乳糖构成主链,侧链为a构型的端基岩藻糖,一级结构重复单元为：α-L-Fucp(?) 1(?)↓(?) 2(?)→6)-α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Galp(1→6)-α-D-Galp-(1-6)
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 Productivity
The 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 stages
The 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 stages
The 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 properties
Hot 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)
1、对收集来的10个猴头菌株进行了亲缘关系的分析及菌丝体和子实体活性多糖高产菌株的筛选
拮抗实验和ITS序列的分析对10个菌株的聚类分析结果基本一致,分析中发现,0608菌株和920菌株亲缘关系很近,可能是同种异名,不能有效区分；长刺猴头与其余菌株亲缘关系较远,单独聚为一类。
对10个菌株进行液体发酵培养,分别收集菌丝体和胞外液,经水提、70%醇沉,苯酚硫酸法测定得到10株菌株胞内多糖和胞外多糖的产量,其中猴头王的胞内多糖、胞外多糖产量均最高,分别达到0.13g/L和128.76g/L；对10个菌株胞内多糖进行体外免疫试验,结果表明,0627菌株的体外免疫活性最好。
对10个菌株进行子实体栽培,分别收集10个菌株的子实体,烘干的子实体经经水提、70%醇沉,苯酚硫酸法测定子实体中的多糖含量,结果表明0605菌株的子实体多糖含量最高,达到1.74%,其次为华中猴头1.70%,与其余菌株之间有显著性差异；对10个菌株的子实体多糖进行体外免疫试验,结果表明0605菌株不仅子实体多糖含量高而且多糖体外免疫活性最好,所以选择0605菌株作为以下试验栽培取样所用菌株。
2、对猴头菌生长发育过程中非挥发性呈味代谢产物进行了比较分析
对0627、0605菌株进行子实体栽培,并将猴头菌的整个生长发育过程划分为六个时期—现蕾期、无菌刺期、分裂期、小菌刺期、中菌刺期和成熟期,分别对0627、0605菌株的六个发育时期进行取样,对不同发育时期子实体进行非挥发性呈味代谢产物的分析,结果表明不同的菌株在非挥发性呈味物质的含量上表现出一定的差异性,相同菌株生长发育过程中所含的非挥发性呈味物质的量上也是有差异的。0627菌株在成熟期新鲜子实体中的阿糖醇和海藻糖的含量最高,分别达到23.41和1.55 mg/g,小菌刺期甘露醇的含量最高,达到1.13mg/g,而0605菌株在小菌刺期阿糖醇的含量最高,为13.98 mg/g,分裂期的海藻糖和甘露醇的含量是最高的,分别为1.97和0.78μg/g；0627菌株在分裂期新鲜子实体中的5’-CMP、5’-UMP、5’-GMP、5’-IMP和5’-AMP的含量最高,分别为475.58、88.33、24.58、43.83和91.05μg/g,谷氨酸和天冬氨酸的含量也是最高,分别为1.70和0.05μg/g,而0605菌株成熟期的5’-CMP含量最高,达到488.13,分裂期的5’-UMP、5’-GMP、5’-IMP和5’-AMP含量最高,分别为95.81、46.60、41.38和65.35μg/g,成熟期的谷氨酸的含量最高,为1.43μg/g,小菌刺期的天冬氨酸的含量最高,为0.04μg/g；并且经计算0605菌株的小菌刺期的等鲜浓度EUC的值是最高的。
3、猴头菌生长发育过程中大分子活性代谢产物的研究
对生长发育六个时期的子实体进行了粗蛋白含量、β-葡聚糖含量、子实体多糖含量的测定并对热水提取、70%醇沉的六个生长发育期的多糖进行体外免疫活性的研究,结果表明,在猴头菌的整个生长发育过程中,子实体粗蛋白含量总体呈下降的趋势,现蕾期最高26.56%,成熟期最低16.18%,小菌刺期出现了一个反升高的过程；子实体β-葡聚糖含量整体上呈上升的趋势,中菌刺期含量最高33.58%,小菌刺期出现了一个反下降的过程；子实体多糖的含量呈现上升的趋势,成熟期含量最高1.54%,分裂期含量最低仅0.89%；相同条件提取的粗多糖透析之后多糖含量不断的上升,成熟期最高,达到50.67%；现蕾期、分裂期、中菌刺期、成熟期的多糖均能很好地刺激巨噬细胞RAW264.7产生NO,且这四个时期的多糖的体外免疫活性均优于无菌刺期和小菌刺期的多糖。
4、0605菌株的小菌刺期、中菌刺期和成熟期的子实体多糖的分级分离及理化性质研究
分别采用30%、50%和70%的乙醇终浓度对0605菌株3个时期的热水浸提物进行分级醇沉,对分级分离多糖进行理化性质研究,结果如下：三个发育期50%醇沉的多糖的糖含量比30%和70%醇沉的多糖糖含量都高,其中成熟期50%醇沉多糖糖含量最高,达到67.14%；从各个时期每个醇沉浓度所得多糖所占的比例上来看,小菌刺期30%醇沉所得多糖比例远远高于中菌刺期和成熟期对应多糖所占的比例,而小菌刺期70%醇沉多糖远低于中菌刺期和成熟期对应多糖所占的比例；不同时期不同醇沉浓度所得的多糖的分子量分布不同,小菌刺期30%醇沉多糖中大于590万分子量段的所占比例比中菌刺期和成熟期30%醇沉多糖中的比例高很多；3个时期50%和70%醇沉多糖分子量相差不大；3个时期分级分离多糖的单糖种类基本上相同,都含有岩藻糖、半乳糖、葡萄糖、甘露糖,但是单糖的摩尔比例有差异；3个时期分级分离多糖均能刺激巨噬细胞株RAW264.7分泌N0,每个时期30%醇沉的多糖均比50%和70%醇沉多糖组分的活性好,70%醇沉多糖组分活性最差,成熟期分级分离多糖的活性最好,优于其它生长阶段。
5. H5FP4B. H5FP5B. H5FP6B的进一步分离纯化及结构鉴定
0605菌株小菌刺期、中菌刺期和成熟期50%醇沉多糖(H5FP4B.H5FP5B. H5FP6B)经过Sephacryl S300凝胶层析进行进一步分离纯化,共得到九个组分分别为H5FP4B-1、H5FP5B-1、H5FP6B-1、H5FP4B-2、H5FP5B-2、H5FP6B-2、H5FP4B-3、H5FP5B-3、H5FP6B-3,对这九个组分的分子量分布进行研究,发现每个时期的第3部分(即主峰部分)分子量基本相同,分子量分布差异较大的是三个时期的第1组分,在体外免疫活性上也表现出较大差异；第2、3组分体外免疫活性差异不大；H5FP4B-3的分子量为1.59万,由岩藻糖,半乳糖和葡萄糖组成,三者摩尔比为5.2：23.9：1,甲基化分析结果表明,岩藻糖以端基的形式存在,半乳糖主要为1,6、1,2,6两种连接方式存在。经红外光谱、甲基化和核磁共振综合分析,得出H5FP4B-3均一多糖是以α-D-1,6连接的半乳糖和α-D-1,2,6连接的半乳糖构成主链,侧链为a构型的端基岩藻糖,一级结构重复单元为：α-L-Fucp(?) 1(?)↓(?) 2(?)→6)-α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Galp(1→6)-α-D-Galp-(1-6)
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 Productivity
The 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 stages
The 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 stages
The 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 properties
Hot 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)
引文
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