【作者】 邵凌健；

【导师】 曲音波； 宋欣；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 生物表面活性剂是微生物在一定条件下培养时,在其代谢过程中分泌出的具有一定表面活性的代谢产物。与化学合成的表面活性剂相比,生物表面活性剂除具有降低表面张力、稳定乳化液和增加泡沫等相同作用外,还具有一般化学合成的表面活性剂所不具备的无毒、生物可降解性、无污染、良好的选择性、专一性和生物相容性等优点。随着人类社会的进步与发展和人们环保意识的增强,生物表面活性剂部分取代化学合成表面活性剂,在人类生产、生活中发挥重要作用将成为必然趋势。槐糖脂是一种由酵母产生的糖酯类生物表面活性剂,由于其生物可降解性,低毒性,良好的环境兼容性,以及产量高等很多优点,在石油工业、环境保护、化妆品、洗涤剂及药学领域都得到了广泛的应用。近年来,槐糖脂在医药领域的应用尤其得到了研究者的关注,很多报道证明了槐糖脂具有很好的抗微生物、抗肿瘤甚至抗艾滋病毒的活性。本文的主要研究内容及结果如下：1.补料发酵提高槐糖脂的产量在本实验室前期筛选到一株高产槐糖脂拟威克酵母菌株,并开展了部分发酵条件优化研究的基础上,本文通过补料发酵的方法,在发酵过程中向培养基中补加菜籽油提高了槐糖脂的产量。300 mL摇瓶补料发酵,在发酵过程中补加三次5%菜籽油后,槐糖脂产量为43.1 g/L,比分批发酵的槐糖脂产量提高了32%。在5 L发酵罐中扩大生产槐糖脂,补料发酵288 h后,槐糖脂产量为68.2 g/L,比摇瓶分批发酵提高了109.2%,比摇瓶补料发酵产量提高了58.2%。将5L发酵罐的转速从400 rpm提高到500 rpm,发酵196 h后,槐糖脂产量达到71.1g/L,与提高转速之前相比,槐糖脂的产量略高一点,但是发酵周期大大缩短,由288 h缩短到196 h,提前了96 h,槐糖脂的容积生产率由0.24 g/L/h提高到0.37g/L/h。2.槐糖脂对细菌抑制作用的研究研究了槐糖脂对几种常见细菌大肠杆菌(Escherichia. coli)、蜡状芽孢杆菌(Bacillus. cereus)、金黄色葡萄球菌(Staphylococcus. aureus)以及能引起龋齿的变形链球菌(Streptococcus mutans)的抑制作用。结果发现槐糖脂对革兰氏阳性菌有明显的抑制作用,当槐糖脂浓度达到15 mg/L时就能够完全抑制蜡状芽孢杆菌的生长。与对金黄色葡萄球菌的最低抑制浓度40 mg/L相比,槐糖脂对蜡状芽孢杆菌的最低抑制浓度更小。槐糖脂对大肠杆菌几乎没有抑制作用。首次报道了槐糖脂对引起龋齿的变形链球菌具有明显的抑制作用。当槐糖脂浓度达到50mg/L时就能够完全抑制变形链球菌的生长,将其添加到牙膏或漱口水中可以有效的防治龋齿的发生。3.槐糖脂对皮肤癣菌抑制作用的研究研究了槐糖脂对红色毛癣菌、石膏样毛癣菌和犬小孢子菌这三种常见的皮肤病致病菌的抑制作用,结果证明槐糖脂对这三种皮肤癣菌都具有抑制作用。培养5天时,内酯型槐糖脂完全抑制红色毛癣菌、石膏样毛癣菌和犬小孢子菌的浓度分别为0.5 g/L、0.1 g/L和0.1 g/L；酸型槐糖脂完全抑制红色毛癣菌、石膏样毛癣菌和犬小孢子菌的浓度分别为0.5 g/L、0.25 g/L和0.5 g/L。比较了培养7天后内酯型槐糖脂和酸型槐糖脂对这三种皮肤癣菌的作用效果的不同,发现对红色毛癣菌来说,内酯型槐糖脂的抑制效果比酸型槐糖脂的抑制效果好一点；对石膏样毛癣菌来说,在低浓度的条件下,内酯型槐糖脂的抑制作用要好于酸型槐糖脂,在高浓度的条件下,酸型槐糖脂的抑制作用要好于内酯型槐糖脂；对犬小孢子菌来说,内酯型槐糖脂的抑制效果比酸型槐糖脂的抑制效果好很多。内酯型槐糖脂比酸型槐糖脂能更好的抑制三种皮肤癣菌的菌丝延伸。当内酯型槐糖脂浓度为0.5 g/L时,红色毛癣菌、石膏样毛癣菌和犬小孢子菌的菌丝延伸抑制率分别为53.8%,62.5%和68.2%。内酯型槐糖脂对红色毛癣菌、石膏样毛癣菌和犬小孢子菌的MIC5o值分别是0.0625,0.125,0.0625 mg/mL,MIC90值分别是0.125,0.25,0.125 mg/mL, MFC值分别是0.5,0.5,0.25 mg/mL。通过透射电镜观察,发现经过内酯型槐糖脂处理后,红色毛癣菌、石膏样毛癣菌和犬小孢子菌的细胞的显微结构都发生了明显改变。细胞壁变厚而且松散,细胞质凝集,细胞器的膜消失,细胞质中没有完整的细胞器和核区。4.对槐糖脂粗品进行分离纯化,并鉴定槐糖脂纯品的结构将拟威克酵母利用菜籽油为第二碳源发酵得到的槐糖脂粗品经HPLC分析,发现槐糖脂粗品中含有多达十几种以上的组分。其中含量较高的10种组分用制备型HPLC进行了制备,并用质谱分析这10种槐糖脂组分的结构。结果发现我们分离得到的槐糖脂组分的脂肪酸部分都是18碳脂肪酸,而它们的结构的区别在于脂肪酸部分的不饱和程度不同(无双键、一个双键或两个双键),槐糖脂部分的乙酰化程度不同(单乙酰基、双乙酰基、非乙酰化),以及是否存在内酯化(内酯型、酸型)。5.不同结构的槐糖脂组分对食管癌细胞抑制作用的研究研究了不同结构的天然槐糖脂组分对食管癌细胞KYSE 109和KYSE 450的抗肿瘤活性,发现槐糖脂分子的乙酰化程度、脂肪酸部分饱和程度和是否内酯化都对其抗肿瘤活性有重要的影响。(1)内酯型槐糖脂的抗肿瘤作用与槐糖部分的乙酰基的数量有关,双乙酰基内酯型槐糖脂对食管癌细胞的抑制效果(30μg/mL的浓度就可以完全杀灭细胞)好于单乙酰基内酯型槐糖脂(60μg/mL的浓度能够完全杀死细胞)。(2)首次报道了内酯型槐糖脂的抗肿瘤作用与脂肪酸部分的不饱和程度有关。脂肪酸部分含有1个双键的内酯型槐糖脂分子对食管癌细胞具有最强的细胞毒性(30μg/mL的浓度就可以完全杀死细胞),脂肪酸部分含有2个双键的内酯型槐糖脂分子对食管癌细胞的抑制作用稍弱(60μg/mL的浓度能够完全杀死细胞),脂肪酸部分没有双键的内酯型槐糖脂分子对食管癌细胞的细胞毒性最弱(60μg/mL的浓度只能够抑制20%的细胞生长)。(3)酸型槐糖脂无论槐糖部分是双乙酰基还是单乙酰基,脂肪酸部分是一个双键还是两个双键,其对食管癌细胞都基本没有抑制作用。6.不同结构槐糖脂对于食管癌细胞KYSE 450抗肿瘤作用机制的研究实验室之前的研究证明,脂肪酸部分为C18：1的双乙酰内酯型槐糖脂抑制人肝癌细胞H7402的机制是引起细胞的凋亡。本文选取了C18:1 MLSL和C18：1DLSL两种槐糖脂,以食管癌细胞KYSE 450作为研究对象,采用多种检测方法,细胞形态学观察(如光学显微镜观察、电子显微镜观察、荧光显微镜观察),流式细胞技术检测细胞周期分布及凋亡率以及DNA裂解的原位检测对不同结构槐糖脂对于食管癌细胞KYSE 450的抗肿瘤作用和机制进行了研究。通过形态学观察,槐糖脂作用后,细胞形态发生了变化,细胞收缩、变圆、体积变小、膜泡突出并且出现一些小体结构,有些细胞从培养板壁脱离下来。细胞核染色质凝集,凝聚于核膜内侧,发生边缘化,有的细胞核碎裂成多个块状,DNA片断化,并出现膜包绕的凋亡小体。通过流式细胞仪分析说明细胞周期的分布发生了变化,并且出现了非常明显的亚二倍体峰。以上都表明,这两种结构的槐糖脂都能够在一定浓度范围内引起食管癌细胞的凋亡,说明槐糖脂抑制肿瘤细胞的机制对于不同的肿瘤细胞具有一致性,都是引起细胞凋亡。另外,不同结构的槐糖脂在相同浓度下引起肿瘤细胞凋亡的水平也不相同,无论从细胞形态观察还是从细胞周期的变化以及凋亡率的大小,都可以说明,相同浓度的C18:1 DLSL比C18:1 MLSL能诱导食管癌细胞KYSE 450更大程度的发生凋亡。这一点也与双乙酰基的内酯型槐糖脂对食管癌细胞的抑制效果优于单乙酰基的内酯型槐糖脂的结果相符合。更多还原

【Abstract】 Biosurfactants are the metabolic products of some microorganisms and have many advantages including low toxicity, good biodegradability and biocompatibility compared with synthetic surfactants by chemical methods. In order to meet the demands for environment protection, biosurfactants will play more and more role than chemically synthetic surfactants in our daily life.Sophorolipids are glycolipid biosurfactants produced by several selected variety of yeast strains. Due to their properties of low toxicity, high biodegradability biocompatibility and high yields, sophorolipids have great application prospects in petroleum industry, environmental industry, cosmetics, food, detergent industries and pharmaceutical sector. Recently, sophorolipids have been proved to have good antimicrobial, anticancer activities and even the anti-HIV activity, which will broaden the applications of sophorolipids in pharmaceutical sector.The main research aspects and results are as following:1. Sophorolipid production by fed-batch fermentationAfter feeding 5% rapeseed oil three times, the yield of sophorolipids in fed-batch fermentation was increased by 32% than batch fermentation in 300 mL flask. In 5 L fermentor, after 288 h of fed-batch fermentation, the yield of sophorolipids was 68.2 g/L and was increased by 109.2% more than batch fermentation in 300 mL flask and 58.2% than fed-batch fermentation in 300 mL flask. After the rotatory speed was improved from 400 rpm to 500 rpm, the yield of sophorolipids 5 L fermentor reached 71.1 g/L at 196 h, the fermentation period shortened 96h, the productivity of sophorolipids was increased from 0.24 g/L/h to 0.37 g/L/h.2. Inhibition of sophorolipids to bacteriumThe inhibition effects of sophorolipids to some bacterium including Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Streptococcus mutans were studied. It was found that sophorolipids showed strong antibacterial activities against gram-positive bacteria.15 mg/L sophrolipids could fully inhibit the growth of B. cereus, and 40 mg/L sophrolipids could fully inhibit the growth of S. aureus. Sophorolipids showed no antibacterial activities against gram-negative bacteria E. coli. This is the first report that sophorolipid had strong inhibition effect to Streptococcus mutans which could cause decayed tooth.50 mg/L sophrolipids could fully inhibit the growth of Streptococcus mutans.3. Inhibition of sophorolipids to DermatophytesThe inhibition of sophorolipids to three common clinical dermatophytes, Trichophyton rubrum, Trichophyton gypseum, and Microsporum canis were investigated. We compared the inhibition of acidic sophorolipids with that of lactonic ones to the three dermatophytes. Lactonic or acidic sophorolipids showed inhibition on the growth of all the three dermatophytes and showed different inhibition towards each of them. To Trichophyton rubrum, the inhibition of lactonic sophorolipids was a little better than acidic ones. Toward Trichophyton gypseum, when at low concentrations, the inhibition of lactonic sophorolipids was much better than that of acidic ones. However, when at high concentrations, the inhibition of acidic sophorolipids was much better than lactonic ones. Toward Microsporum canis, the inhibition of lactonic sophorolipids was much better than acidic ones.For the inhibition of hypha extension, lactonic sophorolipids could inhibit the extension of hypha much better than the acidic ones. When lactonic sophorolipids concentration was 0.5 mg/mL, the inhibition ratio on hypha extension of Trichophyton rubrum, Trichophyton gypseum and Microsporum canis was 53.8%,62.5% and 68.2%, respectively. The MIC50 of lactonic sophorolipids to the three dermatophytes was 0.0625,0.125,0.0625 mg/mL, respectively. MIC90 of lactonic sophorolipids to Trichophyton rubrum, Trichophyton gypseum and Microsporum canis was 0.125,0.25, 0.125 mg/mL respectively. MFC of lactonic sophorolipids to Trichophyton rubrum, Trichophyton gypseum and Microsporum canis was 0.5,0.5,0.25 mg/mL respectively.The TEM observation results indicated that, after being treated by lactonic sophorolipids, three dermatophytes have some obvious changes in their microstructures. The cell wall became thicker and loose, the cytoplasm agglomerated, the membranes of organelles were disappearing, and no integrated organelles and clear nuclear zone were found in the cytoplasm.4. Purification and Structure elucidation of sophorolipidAfter being analysed by HPLC, it was found that the crude sophorolipids produced by Wickerhamiella domercqiae var. sophorolipid are a mixture composed of more than ten molecules. Ten sophorolipid molecules were separately collected by preparative HPLC. The structures of the ten sophorolipid molecules were elucidated by MS analysis. It was found that all the sophorolipid molecules are sophorolipids with C18 fatty acid. Their structures differ in acetylation degree of sophorose, unsaturation degree of hydroxyl fatty acid and lactonization or ring opening.5. The anticancer effects of sophorolipids with different structures on human esophageal cancer cellThe anticancer effects of sophorolipids with different structures on human esophageal cancer cell KYSE 109 and KYSE 450 were investigated. It was found that the differences of sophorolipid sructure in acetylation degree of sophorose, unsaturation degree of hydroxyl fatty acid, and lactonization or not can affect the anticancer activity of sophorolipid.(1) The results indicated that the inhibition of diacetylated lactonic sophorolipid to esophageal cancer cells (totally inhibition at 30μg/mL concentration) was stronger than momoacetylated lactonic sophorolipid (totally inhibition at 60μg/mL concentration), which confirmed that anticancer activity of SLs was affected by their acetylation degree in sophorose moiety.(2) Our results showed that sophorolipid with different unsaturation degree of hydroxyl fatty acid also had different cytotoxic effects on esophageal cancer cells. Sophorolipid having one double bond in fatty acid part had the strongest cytotoxic effect (totally inhibition at 30μg/mL concentration) on esophageal cancer cells, sophorolipid with two double bonds had a little weaker anticancer effect (totally inhibition at 60μg/mL concentration), while sophorolipid with no double bond had the weakest cytotoxic effect (only 20% of cells were inhibited at 60μg/mL concentration) among the three sophorolipid molecules. This was the first study to reveal the relationship of bioactivities of natural sophorolipid molecules with different unsaturation degree in hydroxyl fatty acid and their structures.(3) No matter acidic SL with one or two double bond in fatty acid part, with momoacetylated group or diacetylated groups in sophorose part, they have little anticancer effect against esophageal cancer cells.6. The inhibition mechanism of sophorolipids with different structures on human esophageal cancer KYSE450In our previous studies, the inhibition mechanism of diacetylated lactonic sophorolipid with a C18 momounsaturated fatty acid on the human liver cancer cells H7402 has been proved to induce cell apoptosis. The inhibition mechanism of two sophorolipid molecules C18:1 MLSL and C18:1 DLSL on human esophageal cancer cell KYSE 450 was investigated by reverse phase contrast microscopy, cell staining, fluorescence microscopy, flow cytometer, and TUNEL assay in this study.It was found that, after being treated with C18:1 MLSL and C18:1 DLSL, cell gradually shrank, turned round, membrane blebbing stood out. Chromatin condensation and margination, nuclear fragmentation and apoptotic bodies were observed. Cell cycle distribution change was observed and the sub-G1 population appeared. These changes can demonstrate the apoptosis of KYSE 450 induced by sophrolipids and the inhibition mechanism of sophrolipids to different cancer cells was all apoptosis.The apoptosis level of KYSE 450 induced by sophrolipid with different structure of the same concentration was different. It was found that C18:1 DLSL could induce apoptosis of KYSE 450 at a greater extent than C18:1 MLSL by morphological changes, cell cycle distribution changes and apoptosis rate of KYSE 450, which agree with the results that the inhibition of diacetylated lactonic sophorolipid to esophageal cancer cells was stronger than momoacetylated lactonic sophorolipid.更多还原