产甜甙V罗汉果内生真菌的研究

发布时间：2018-06-19 02:16

  本文选题：罗汉果 + 内生真菌　； 参考：《广西师范大学》2015年硕士论文

【摘要】：罗汉果(Siraitia grosvenori)属于葫芦科植物,其食品和药品价值已被确认。罗汉果甜甙是一种从罗汉果果实里提取的低热量、高甜度、健康的天然甜味剂,而甜甙V又是其中的主要成分,在罗汉果干果中占比为0.5-1.4%,是一种葫芦烷三萜糖苷类化合物。同时人们希望找到合适的内生真菌能够产出有用的代谢产物,从而将内生真菌可以发展成一种重要的生物资源。近年来的研究表明,内生真菌与宿主协同进化,可以产生与宿主相同或相似的代谢产物,对相应的内生真菌及其代谢产物的研究已经成为寻找重要代谢产物的一种新途经。因此,如果从罗汉果中分离出的内生真菌能产生与宿主相同的次生代谢物如甜甙V,这将成为一种获取甜甙V的新途径。本文通过对采自广西永福县罗汉果植株的根、茎、叶和果实中的内生真菌进行分离,筛选出可产生甜甙V的真菌、用形态学和分子生物学方法对菌株种属进行鉴定、以及优化菌株产甜甙V的发酵条件,为研究提供了重要理论依据。现将取得的主要研究成果归纳如下：1、内生真菌的分离和培养。通过PDA固体培养基分离培养,从罗汉果组织内分离出15种内生真菌,4株分离自叶,4株分离自茎,2株分离自根,5株分离自果实。2、产甜甙V内生真菌的筛选。使用高效液相色谱分析发酵液中甜甙V的存在。通过比较标准品和样品的保留时间来判断甜甙V的有无。产甙菌株也通过HPLC和LC-MS技术被筛选出来(编号为LHG-F5和LHG-L4的内生真菌,其中LHG-F5甜甙V产量为3.814μg/mL, LHG-L4甜甙V产量为2.675μg/mL)。3、产甜甙V内生真菌的鉴定。采用经典的形态学鉴定和电镜扫描的方式对编号为LHG-F5和LHG-L4的真菌的菌丝及孢子显微形态进行观察,随后对这两株产甜甙内生真菌的ITS-rDNA序列进行系统发育分析。通过ITS序列分析技术将LHG-F5菌株鉴定为当归间座壳菌(Diaporthe angelicae),将LHG-L4菌株鉴定为腐皮镰刀菌(Fusarium solani)。4、产甜甙Ⅴ真菌的发酵条件优化。采用单因素和正交试验方法对甜甙Ⅴ产量较高的LHG-F5菌株生长的营养因素和环境因素进行优化。确定了LHG-F5产甜甙Ⅴ的最优发酵条件,LHG-F5的最优发酵方法是在马铃薯液体培养基的基础上,加上10g/L葡萄糖为补充碳源,2.5g/L的硫酸铵为补充氮源,培养基装液量为250mL三角瓶装液150 mL,发酵时间为5天,发酵温度为28℃,初始pH值为7。检测产物中甜甙Ⅴ的含量达到7.156μg/mL,是进行发酵条件优化前甜甙Ⅴ产量的1.87倍。
[Abstract]:Siraitia grosvenorii belongs to the family Cucurbitaceae, and its food and drug value has been confirmed. Dioscoside is a kind of natural sweetener with low calorie, high sweetness and health extracted from the fruit of Siraitia grosvenorii. The main component is glucoside V, which accounts for 0.5-1.4% of the dried fruit of Siraitia grosvenori, and is a kind of glucoside of gourd triterpenoid. At the same time, people hope to find suitable endophytic fungi to produce useful metabolites, so endophytic fungi can be developed into an important biological resource. Recent studies have shown that the co-evolution of endophytic fungi and hosts can produce the same or similar metabolites as the host. The study of the corresponding endophytic fungi and their metabolites has become a new way to search for important metabolites. Therefore, if the endophytic fungi isolated from Siraitia grosvenori can produce the same secondary metabolites as the host, such as glucoside V, this will become a new way to obtain glucoside V. In this paper, endophytic fungi from roots, stems, leaves and fruits of Luohanguo were isolated from Yongfu County, Guangxi, and the fungi producing glucoside V were selected. The species of the strains were identified by morphological and molecular biological methods. The optimization of fermentation conditions for the production of glucoside V provides an important theoretical basis for the study. The main research results are summarized as follows: 1, isolation and culture of endophytic fungi. In this paper, 15 endophytic fungi were isolated from the tissue of Siraitia grosvenorii on PDA solid medium. Four strains of endophytic fungi were isolated from leaves, 4 strains were isolated from stem, 2 strains were isolated from root and 5 strains were isolated from fruit. 2. The screening of endophytic fungi producing glucoside V. The presence of glucoside V in fermentation broth was analyzed by high performance liquid chromatography (HPLC). The existence or absence of glucoside V was determined by comparing the retention time of the standard and the sample. The endophytic fungi named LHG-F5 and LHG-L4 were also screened by HPLC and LC-MS. The yield of LHG-F5 and LHG-L4 was 3.814 渭 g / mL and 2.675 渭 g / mL 路3, respectively. The endophytic fungi of glucoside V were identified. The micromorphology of hyphae and spores of fungi numbered LHG-F5 and LHG-L4 were observed by means of classical morphological identification and scanning electron microscope. The ITS-rDNA sequences of these two endophytic fungi were analyzed by phylogenetic analysis. The strain LHG-F5 was identified as Diaporthe angelicaeae by its sequence analysis, and the strain LHG-L4 was identified as Fusarium solani.4. the fermentation conditions of glucoside 鈪，

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