【作者】 张鑫；

【导师】 刘选明；

【作者基本信息】 湖南大学 ， 生物医学工程， 2015， 硕士

【摘要】 香茅草是禾本科香茅属多年生草本植物,是一种重要的热带香料作物和药用植物,其挥发性产物主要以萜烯化合物为主,是香料生产的主要原料之一。然而,香茅草种植规模深受农耕面积,环境气候和病害的制约,而且常规提取手段获得的萜烯化合物含量较低,难以满足市场需求,生物合成方法(主要是微生物合成)逐步成为萜烯化合物合成的研究热点。药用植物能够合成大量丰富的天然代谢产物,内生菌长期内生于其中,具有独特的新陈代谢过程,很可能会具备与宿主植株相似次生代谢的潜能。开发药用植物内生菌资源,将会为天然产物的合成提供新方案。通过植物表面消毒结合四种分离培养基(WA、TWYE、MS、TSA)的方法,从成熟香茅草中分离出一批内生细菌,根据感官评定初步筛选出36株产香内生细菌,从中选择一株具有明显清新愉悦香味的内生细菌CcSh-1。根据形态学、生理生化和16S rRNA序列分析,鉴定该菌株为菠萝泛菌(Pantoea ananatis)。利用顶空气相色谱质谱联用仪技术,对香茅草和内生细菌CcSh-1挥发性成分进行鉴定,得出香茅草挥发性成分为α-蒎烯、1s-α-蒎烯、3-蒈烯、艾蒿醚、2,2-二甲基-3,4-辛二烯、橙花醛和香叶醛;CcSh-1的挥发性成分为莰烯,α-蒎烯,檀香三烯,橙花醛,香叶醛和白檀油烯醇,其中以柠檬醛(橙花醛和香叶醛)含量较高,为2.1μg/mL。对香茅草及CcSh-1共有的挥发性成分为:α-蒎烯、橙花醛和香叶醛,并且柠檬醛(橙花醛和香叶醛)的含量均相对较高。文章初步探究了CcSh-1产香的最佳培养条件。综合考察CcSh-1在液体培养条件下的产香效果和生长状况,初步确定CcSh-1的最适宜的培养条件为:以牛肉膏为碳源,蛋白胨为氮源,温度30℃,pH7,此时CcSh-1的生长状况最好,产香最优。通过在培养基中加入1%(w/v)香茅草粉末,发现CcSh-1的挥发性成分种类不会发生变化,但其柠檬醛含量有明显提高。总之,本文筛选得到了一株具有明显清新愉悦香味的内生细菌CcSh-1,经鉴定和分析,确定其挥发性组分包含以柠檬醛为主的萜类化合物,初步建立了CcSh-1的最优产香条件,本文还探究了香茅草对CcSh-1产香的影响,体现了内生菌与其宿主间存在相互作用。更多还原

【Abstract】 Cymbopogon citrates(DC.) Stapf is a perennial herbaceous plant, which belongs to Cymbopogon of ramineae.As an important tropical spice crop and medicinal plant, the volatile products of Cymbopogon citrates(DC.) Stapf are mainly terpene compounds which are kinds of spice source. However, the planting scale was restricted by the farming area, the climate of environment and the disease. Moreover, it is hard to meet the market demand by using the conventional extraction method of terpene compound content because of the low productivity. Therefore, the method has gradually become the focus especially in the microbial synthesis. Endophytes have the potential for secondary metabolite production which similar to the host, when live in the medicinal plants that synthesize a large number of natural metabolites. Endophytes isolated from medicinal plants have the higher possibility than the other and have specific physiological and biochemical functio under the unique living environment. Development and utilization of plant endophytes resource will be a new solution for the synthesis of natural medicine resources.Through using four kinds of separation medium(WA, TWYE, MS, TSA) after plant surface disinfection, a number of endophytic bacterias were isolated from mature Cymbopogon citrates(DC.) Stapf.Then 36 endophytic bacterias which can produce fragrance were screened according to sensory evaluation. One endophytic bacterium which have obvious and pleasant fragrance was chosen and named CcSh-1. Furthermore, according to the morphological, physiological and biochemical and 16 s rRNA sequence analysis, CcSh-1 was identified as Pantoea ananatis.Using headspace gas chromatography mass spectrometry, we identified volatile components from Cymbopogon citrates(DC.) Stapf and Pantoea ananatis CcSh-1. Volatile components from Cymbopogon citrates(DC.) Stapf were α-pinene, 1s-α-pinene, 3-carene, mugwort-ether, 2, 2-dimethyl-3, 4-symplectic diene, neral and geranial and the volatile components in CcSh-1 were camphene, α-pinene, sandalwood triene, neral, enol geranial and sandalwood oil. The citral(neral and geranial) content was the highest, which reached 2.1 μg/m L. They had the same volatile components which were: α-pinene, neral and geranial. And citral(neral and geranial) content was relatively higher.We preliminarily explored the best condition of CcSh-1 for flavor producing. The factors involved in the flavor productivity and the growth status of CcSh-1 were investigated, and the optimal liquid culture conditions for CsSh-1 were confirmed: beef extract as carbon source, peptone as nitrogen source, 30℃ and pH 7. In addition, we discovered that adding 1%(w/v) abrasive of host plant as substrate could promote flavor production of CcSh-1(particularly citral content), and flavor species had no change.In conclusion, we successfully screened an endophytic bacteria strain CcSh-1 that produces obvious fragrance. After identification analysis, we found that the volatile constituents predominantly include citral of terpenoids. We preliminarily determine the optimal flavor production conditions of CcSh-1; In addition, we researched the flavor production of CcSh-1 with the affection by Cymbopogon citrates(DC.) Stapf and this influence reflected the interaction between endophytes and their host.更多还原