【作者】 伦志明；

【导师】 池玉杰；

【作者基本信息】 东北林业大学 ， 森林保护， 2014， 博士

【摘要】 花脸香蘑(Lepista sordida)香味浓郁,味道鲜美,是一种具有较高食用与药用价值的珍贵食用菌,能够进行人工栽培,但由于技术不成熟,产量不高,尚未进行大规模商业化生产。多糖是食用菌中最广为人知、最有效的抗肿瘤和免疫调节活性物质,国内外学者已经对700多种担子菌的多糖进行了研究,相比之下,花脸香蘑多糖的研究才刚刚开始。本文探索了花脸香蘑高产栽培模式,筛选出高产配方,为早日实现规模化生产提供理论依据；进行多糖提取工艺研究,优化出最佳提取工艺条件,并对分离纯化后得到的子实体多糖进行初步结构分析,以期为花脸香蘑多糖资源的开发利用打下良好的基础。主要研究内容如下：1、首先对野生香蘑产量较大的齐齐哈尔市甘南县双河农场、牡丹江市镜泊湖自然风景区和牡丹江三道关自然保护区等三地的生态环境进行了调查和香蘑子实体采集,并对采集的子实体进行了菌种分离,获得3个菌株,编号分别为S1(齐齐哈尔)、J1(镜泊湖)和M1(三道关)。在对这3个菌株依据子实体形态特征进行初步分类鉴定的基础上,从基因的GeneBank中选取了15个香蘑属菌株及1个栓菌属菌株,与3个菌株的ITS序列进行比对并建立系统发育树。结果显示,包括J1和M1在内的9个紫丁香蘑菌株聚为一类,且彼此之间遗传距离较短,而包括S1在内的9个花脸香蘑菌株聚为一类,且遗传距离也很接近,由此确定,J1和M1为紫丁香蘑,S1是花脸香蘑。2、进行了花脸香蘑大棚覆土与仿野生两种出菇方式对比的栽培试验研究,结果显示,两者覆土后至现蕾时间相同,出菇时温度条件也接近；大棚内产出的花脸香蘑比较粗壮,没有香味,仿野生栽培的子实体比较单薄,与野生采集的相近,有的具备其特有的香气；仿野生栽培产量高于大棚覆土栽培,前者生物学转化率为36.8%,后者仅为17.38%。然后进行了配方对比试验研究,结果表明,随着玉米芯添加比例的增加,菌丝生长速度呈上升趋势,添加比例最大的配方六菌丝生长速度最快,达到了3.95mm/d,而配方三(木屑48%,玉米芯32%,麦麸18%,石膏1%,石灰1%)的生物学转化率最高,为28.4%。3、以人工栽培的花脸香蘑分离获得的菌株为研究对象,对菌丝体进行碳源和氮源利用能力试验,通过比较菌丝体麦角固醇含量可以发现,果糖、甘露糖、纤维二糖、蔗糖、葡萄糖和麦芽糖都可以作为花脸香蘑菌丝体生长的碳源,但相比之下,果糖、甘露糖和纤维二糖是更为理想的碳源；比较多糖产量结果发现,选择果糖作为碳源,多糖产量最高；丙氨酸、甘氨酸、黄豆粉和蛋白胨都可以作为氮源使用,添加丙氨酸的培养基中,菌丝生长效果最好；而含有黄豆粉的培养基中,则是多糖产量最高；尿素则不能被花脸香蘑菌丝所利用。4、以齐齐哈尔双河农场采集的野生花脸香蘑子实体为材料,进行了热水浸提法提取多糖试验,通过单因素试验研究了浸提时间、浸提温度、料水比、提取次数对多糖得率的影响,并在单因素试验的基础上,进行正交试验获得最佳提取工艺。结果显示,影响花脸香蘑多糖提取率各因素大小顺序为,提取温度>料水比>提取时间,提取温度影响最大,其次是料水比,且差异性显著(P<0.01)。同时优化出热水提取花脸香蘑子实体多糖最佳工艺条件为：浸提温度650C、浸提时间5h、料水比1：40(g/mL),在该条件下多糖得率高达10.39%,RSD仅为1.33%,证明优化实验获得的工艺条件重现性好,稳定可行。5、以驯化栽培的花脸香蘑菌丝体为原料,进行了热水浸提法提取多糖试验,通过单因素试验研究了浸提时间、浸提温度、液料比、提取次数对多糖得率的影响,并在单因素试验的基础上,进行正交试验获得最佳提取工艺。结果表明,影响花脸香蘑多糖提取率各因素大小顺序为,提取温度>提取时间>液料比,提取温度对多糖提取率影响最大,且差异显著,提取时间影响在其次。同时优化出热水提取花脸香蘑菌丝体多糖最佳工艺条件为：浸提温度95℃、浸提时间3h、料液比1：40(g/mL),在该条件下提取多糖,得率高达9.41%,RSD仅为1.45%,证明优化实验获得的工艺条件重现性好,稳定可行。6、采用热水浸提、乙醇浓度分别为30%、60%和80%情况下分步进行醇沉的方法提取野生花脸香蘑子实体粗多糖,得到3种多糖组分,分别命名为LSP-30、LSP-60和LSP-80,它们的总糖含量分别为64.0%,41.4%和17.2%。进一步研究显示,多糖LSP-30主要由甘露糖(Man),葡萄糖(Glc)和半乳糖(Gal)三种单糖组成,含量分别为23.0%、49.2%和27.8%；用Sephrose CL-6B层析柱根据分子量不同对LSP-30进行分级纯化,得到LSP-30-a和LSP-30-b两个级分,分子量分别约为600KDa和10KDa。LSP-60主要由Man、Glc、和Gal三种单糖组成,它们的含量分别为31.6%、46.4%和22.0%；用Sephadex G-75层析柱对LSP-60进行纯化,结果表明LSP-60分子量分布范围较广且不均一,分子量约为1KDa。LSP-80主要由Man、Glc、Gal和Rha(鼠李糖)四种单糖组成,含量分别为17.4%、68.0%、11.6%和3.0%；用Bio-Gel P-2层析柱根据分子量不同对LSP-80进行分级纯化,得到LSP-80-a、LSP-80-b和LSP-80-c三个级分,分子量分别为1KDa,700Da和400Da。对LSP-80-a, LSP-80-b, LSP-80-c的单糖组成进行分别测定,结果表明,LSP-80-a主要由Man, Glc, Gal组成,含量分别为44.9%、34.4%和20.7%；LSP-80-b主要由Man, Glc, Gal, Rha组成,含量分别为20.5%、60.3%、16.4%和2.8%；LSP-80-c由Man, Glc, Gal和Rha组成,含量分别为12.2%、50.7%、10.1%和27.0%。更多还原

【Abstract】 Lepista sordida is a kind of delicious edible fungi which owns higher diatery and medicinal value. Though it can be cultivated artificially, the technology is not developed and lower yield prevented this precious mushroom from commercial production. Polysaccharides are the best known and most potent mushroom-derived substances with antitumor and immunomodulating properties. The data on mushroom polysaccharides were summarized for approximately700species of higher basidiomycetes. In contrast, the research on L. sordida polysaccharides has just begun. In the present thesis, high production cultivating pattern of L. sordida was explored and high yield formula was found, which provided theory basis for cultivating commercially. In the meantime, the extracting technique of L. sordida polysaccharides was studied and the optimal extraction parameter was obtained. Then polysaccharides were extracted and fractioned from the fruiting bodies of wild L. sordida, and the preliminary analysis of the polysaccharides structure was made. The contents are as follows:Firstly, the ecology of wild Lepista spp. growing in the Shuanghe farm of Qiqihaer city, Jingpo-lake natural park and Sandaoguan natural park of Mudanjiang city, which are major production areas of Lepista spp. in Heilongjiang province, was investigated and sporocarps were collected. Subsequently, S1(Qiqihaer), M1(Sandaoguan) and J1(Jingpo-lake) strains were isolated. Based on the initial identification by morphological characteristics of the three unknown strains, the sequence alignment was made and a phylogenetic tree was established with15Lepista spp. strains and one Trametes spp. strain coming from Genebank by using the internal transcribed spacer (ITS) region. The result showed that S1was nearer to L, sordida in genetic distance and clustered together with them, whereas M1and J1were nearer to L. nuda and clustered together with them. So, the strain S1was L. sordida, and the other two strains were L. nuda.Secondly, comparasion test of bionics wild cultivation and greenhouse cultivation of L. sordida were carried out. The result showed that the budding time was the same after covering the soil, and the temperature of fruiting was near to each other. The fruiting bodies were thick, strong and had no sweet smell in greenhouse. But bionics wild sporocarps were thin and similar to wild ones in appearance, and some had special fragrant odor. Comparing with greenhouse cultivation, the bionics wild cultivation appeared higher yield and its biological efficiency was36.8%, yet the former is only17.38%. In the meantime, the effect of formulation on L. sordida mycelia growth and output was investigated. The data showed that the hypha growing rate is positively correlated with the addition of corncobs. The mycelia grew fast in the formula6with the maximum supplementation of corncobs (3.95mm/d), however, the formula3(sawdust48%, corncob32%, wheat bran18%, gypsum powder1%, lime dust1%) appeared the highest biological efficiency,28.4%.Thirdly, the strain of artificial L. sordida was investigated for carbon and nitrogen nutrition by ergosterol contents and the production of polysaccharide in liquid media. The result showed that fructose, mannose, cellobiose, sucrose, glucose and maltose can be used as a carbon source for L. sordida strain by analyzing ergosterol contents. And the former three carbon materials were better for mycelia growth in contrast with sucrose, glucose and maltose. The highest polysaccharide yield could be obtained with fructose as a carbon source. Alanine, glycine, soybean powder and peptone can be used as a nitrogen source. Alanine is best for mycelia growth and bean powder can produce the highest polysaccharide.Forthly, the polysaccharide was extracted from wild L. sordida sporocarps by using hot water extraction method. Effects of four single factors-, different extraction temperature, the ratio of raw material and water, different extraction time, and extraction times, on the yield of polysaccharide were determined. And then, one orthogonal test was conducted so as to obtain the optimal extraction technique by evaluating the yield. The influence of three factors on extraction efficiency decreased in the following order:reflux temperature>material to water ratio> extraction time. Based on2extraction times, the optimal extraction technique for polysaccharides from wild L. sordida sporocarps was extraction temperature65℃, material to water ratio1:40(g/mL) and extraction time was five hours. And under these conditions, the extraction rate of wild L. sordida sporocarp polysaccharides could reach10.39%, and RSD is only1.45%. So, this technique is stable and reliable.Fifthly the polysaccharide was extracted from artificial L. sordida mycelia by using hot water extraction method. Effects of four single factors-, different extraction temperature, the ratio of raw material and water, different extraction time, and extraction times, on the yield of polysaccharide were determined. And then, one orthogonal test was conducted so as to obtain the optimal extraction technique by evaluating the yield. The influence of three factors on extraction efficiency decreased in the following order:reflux temperature> extraction time>. material to water ratio. Based on2extraction times, the optimal extraction technique for polysaccharides from L. sordida mycelia was extraction temperature95℃, material to water ratio1:40(g/mL) and extraction time was three hours. Under these conditions, the extraction rate of artificial L. sordida mycelia polysaccharides could reach9.41%, and RSD is only1.33%. So, this technique is stable and reliable.Sixthly, the crude polysaccharides were obatained from the fruiting bodies of wild L. sordida by hot water extraction method. The different ethanol concentrations were used step-by-step(30%→60%→80%) to precipitate carbohydrates and got three extracting samples of LSP-30, LSP-60and LSP-80. Their total sugar contents were64.0%,41.4%and17.2% respectively. LSP-30was composed of glucose (Glu), mannose (Man) and galactose (Gal), their contents were23.0%,49.2%and27.8%. LSP-30was then purified on a column of Sephrose CL-6B, resulting in producting two purified polysaccharides (LSP-30-a and LSP-30-b) and their average molecular weight were approximately600KDa and lOKDa. LSP-60was mainly composed of Glc, Man and Gal, and their contents were31.6%,46.4%and22.0%respectively. The molecular weight range of LSP-60was wide and not well-distributed, approximately1KDa. LSP-80was mainly composed of Glc, Man, Gal, and Rha (rhamnose), and their contents were17.4%,68.0%,11.6%and3.0%respectively. LSP-80was then purified on a column of Bio-Gel P-2, resulting in producting three purified carbohydrates (LSP-80-a, LSP-80-b and LSP-80-c) and their average molecular weight were approximately1KDa,700Da and400Da. LSP-80-a was mainly composed of Glc, Man and Gal in a ratio of44.9%,34.4%and20.7%, LSP-80-b was mainly composed of Glc, Man, Gal and Rha in a ratio of20.5%,60.3%,16.4%and2.8%. And LSP-80-c was mainly composed of Glc, Man, Gal and Rha with a ratio of12.2%,50.7%,10.1%and27.0%respectively.更多还原