槐糖脂产生菌的筛选及槐糖脂抑菌和抗肿瘤作用的研究

【作者】 陈静；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 微生物学， 2006， 博士

【摘要】 表面活性剂在工农业生产和人类日常生活中的应用越来越广泛，但是通过化学法合成的表面活性剂在合成和使用的过程中会对环境造成污染和破坏。而生物表面活性剂是微生物在一定条件下代谢产生的或者通过酶法合成的，与化学法合成的表面活性剂相比有相同特性又有很多优点，特别是无毒、微生物可降解这一优点是化学法合成的表面活性剂所不可比拟的，因此在石油、环境、食品、农业及医药等方面具有极其重要的应用价值。 微生物产生的生物表面活性剂种类很多，槐糖脂由于结构特殊、无毒、生物可降解，容易获得并且产量较高，是目前研究较多、也是最具有应用前途的一种。对于槐糖脂的研究，目前主要集中在两个方面：一是提高槐糖脂的产量；二是槐糖脂的应用，集中在石油、环境保护、洗涤剂、化妆品等方面。槐糖脂在医药方面的应用研究不多，特别是槐糖脂的抗肿瘤作用机理研究国内外尚无报道。因此，有必要筛选新型的槐糖脂产生菌，并对槐糖脂的药效作用及其作用机理进行探讨，这将为抗癌新药的发现和开发提供可能和指导。 主要研究内容和结果如下： 1．新型槐糖脂产生菌的筛选。以菜籽油为唯一底物，通过富集培养、平板划线等方法筛选到200多株菌；利用油膜破裂法和测定发酵液的表面张力得到5株产表面活性剂的菌株；最后通过色谱技术，包括TLC（thin layerchromatography）、IR（infrared）spectra、HPLC、NMR（nuclear magnetic resonance）及MS（mass spectroscopy），进一步确定了产槐糖脂的菌株为Y_2A。该菌株能将发酵液的表面张力降为37.3mN／m，产生的产物是糖脂的混合物，含量较高的主要有三种成分，其保留时间分别为33.28，34.62和38.15min，命名为化合物Ⅰ、化合物Ⅱ、化合物Ⅲ，化合物Ⅲ是内酯型双乙酰槐糖脂。 通过形态学观察、生理生化实验及BIOLOG菌种鉴定系统对Y_2A菌株进行种属鉴定，确定Y_2A菌株为拟威克酵母(Wickerhamiella domercqiae Y_2A)。这是更多还原

【Abstract】 It is well-known that surfactants can be used in pharmaceutic, energetic, cosmetic industries, etc. Biosurfactants are the metabolic products of some microorganisms and have many advantages including low toxicity, biodegradability and biocompatibility compared with synthetic surfactants by chemical methods. Therefore, biosurfactants have promising applications in petroleum, environment, food, pharmaceutical industries.Sophorolipids are the most important and promising ones of many kinds of biosurfactants due to their biodegradability, low toxicity, effectiveness and high yields.In the past decades, the optimization of fermentation yields and applications of sophorolipids were studied extensively. Only a few of papers were related to the pharmacological function of sophorolipids, Very little information was available concerning mechanism of antitumor activities of sophorolipids. The main research aspects and results are as following:1. A new strain producing sophorolipids was isolated from oil-containing wastewater. First, using rapeseed oil as sole carbon source, more than 200 strains were isolated from soil and wastewater samples by enrichment culture and plate streaking. Second, these strains were cultured in fermentation media, 5 strains producing biosurfactant were isolated by measuring the oil film-collapsing activity and surface tension of the culture broth. Finally, it is demonstrated that the yeast strain Y_2A can produce sophorolipids by some spectral methods, including TLC, IR, HPLC, NMR and MS. The surface tension of Y_2A culture broth was decreased to 37.3 mN/m. The product is a mixture containing more than six glycolipids. After HPLC analysis, there are three main products, compound Ⅰ , compound Ⅱ and compound Ⅲ, occurring at retention time 33.28, 34.62 and 38.15min. Compound Ⅲ was 17-L-（ [2 ’-O-β-D-glucopyranosyl-1"-β-D-glucopyranosyl]-oxy）-octadecanoic acid 1, 4"-lactone 6’, 6"-diacetate, that is lactonic diacetyl-sophorolipid.By using BIOLOG system and conventional methods for yeast identification,更多还原