本文選題：羅漢果 + 內(nèi)生真菌�。� 參考：《廣西師范大學(xué)》2015年碩士論文

【摘要】：羅漢果(Siraitia grosvenori)屬于葫蘆科植物,其食品和藥品價值已被確認(rèn)。羅漢果甜甙是一種從羅漢果果實(shí)里提取的低熱量、高甜度、健康的天然甜味劑,而甜甙V又是其中的主要成分,在羅漢果干果中占比為0.5-1.4%,是一種葫蘆烷三萜糖苷類化合物。同時人們希望找到合適的內(nèi)生真菌能夠產(chǎn)出有用的代謝產(chǎn)物,從而將內(nèi)生真菌可以發(fā)展成一種重要的生物資源。近年來的研究表明,內(nèi)生真菌與宿主協(xié)同進(jìn)化,可以產(chǎn)生與宿主相同或相似的代謝產(chǎn)物,對相應(yīng)的內(nèi)生真菌及其代謝產(chǎn)物的研究已經(jīng)成為尋找重要代謝產(chǎn)物的一種新途經(jīng)。因此,如果從羅漢果中分離出的內(nèi)生真菌能產(chǎn)生與宿主相同的次生代謝物如甜甙V,這將成為一種獲取甜甙V的新途徑。本文通過對采自廣西永福縣羅漢果植株的根、莖、葉和果實(shí)中的內(nèi)生真菌進(jìn)行分離,篩選出可產(chǎn)生甜甙V的真菌、用形態(tài)學(xué)和分子生物學(xué)方法對菌株種屬進(jìn)行鑒定、以及優(yōu)化菌株產(chǎn)甜甙V的發(fā)酵條件,為研究提供了重要理論依據(jù)。現(xiàn)將取得的主要研究成果歸納如下：1、內(nèi)生真菌的分離和培養(yǎng)。通過PDA固體培養(yǎng)基分離培養(yǎng),從羅漢果組織內(nèi)分離出15種內(nèi)生真菌,4株分離自葉,4株分離自莖,2株分離自根,5株分離自果實(shí)。2、產(chǎn)甜甙V內(nèi)生真菌的篩選。使用高效液相色譜分析發(fā)酵液中甜甙V的存在。通過比較標(biāo)準(zhǔn)品和樣品的保留時間來判斷甜甙V的有無。產(chǎn)甙菌株也通過HPLC和LC-MS技術(shù)被篩選出來(編號為LHG-F5和LHG-L4的內(nèi)生真菌,其中LHG-F5甜甙V產(chǎn)量為3.814μg/mL, LHG-L4甜甙V產(chǎn)量為2.675μg/mL)。3、產(chǎn)甜甙V內(nèi)生真菌的鑒定。采用經(jīng)典的形態(tài)學(xué)鑒定和電鏡掃描的方式對編號為LHG-F5和LHG-L4的真菌的菌絲及孢子顯微形態(tài)進(jìn)行觀察,隨后對這兩株產(chǎn)甜甙內(nèi)生真菌的ITS-rDNA序列進(jìn)行系統(tǒng)發(fā)育分析。通過ITS序列分析技術(shù)將LHG-F5菌株鑒定為當(dāng)歸間座殼菌(Diaporthe angelicae),將LHG-L4菌株鑒定為腐皮鐮刀菌(Fusarium solani)。4、產(chǎn)甜甙Ⅴ真菌的發(fā)酵條件優(yōu)化。采用單因素和正交試驗(yàn)方法對甜甙Ⅴ產(chǎn)量較高的LHG-F5菌株生長的營養(yǎng)因素和環(huán)境因素進(jìn)行優(yōu)化。確定了LHG-F5產(chǎn)甜甙Ⅴ的最優(yōu)發(fā)酵條件,LHG-F5的最優(yōu)發(fā)酵方法是在馬鈴薯液體培養(yǎng)基的基礎(chǔ)上,加上10g/L葡萄糖為補(bǔ)充碳源,2.5g/L的硫酸銨為補(bǔ)充氮源,培養(yǎng)基裝液量為250mL三角瓶裝液150 mL,發(fā)酵時間為5天,發(fā)酵溫度為28℃,初始pH值為7。檢測產(chǎn)物中甜甙Ⅴ的含量達(dá)到7.156μg/mL,是進(jìn)行發(fā)酵條件優(yōu)化前甜甙Ⅴ產(chǎn)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，

本文編號：2037893