海洋微生物和微藻中防污活性物质的提取及其性能研究

发布时间：2018-03-22 04:08

本文选题：生物污损　切入点：天然产物　出处：《中国海洋大学》2014年硕士论文　论文类型：学位论文

【摘要】：在海洋工业中，生物污损是指在人工设施表面上附着、生长、繁殖的生物体的积累。随着现代科技的进步，人类对海洋的开发和利用有了很大发展，而生物污损成为急需解决的棘手问题，因此防污技术日益得到广泛的应用。防污涂料的使用成为最经济实用的一种防污方法。而传统防污涂料，以有机锡和氧化亚铜等有毒防污剂的毒杀方式达到防污目的，却对海洋环境造成严重污染。国际海事组织在2008年禁止含有机锡的防污涂料的应用，因此，开发低毒、无毒的环境友好型防污剂成为海洋产业发展的实际、迫切需要。近几年来，海洋天然产物成为最有潜力的防污物质来源之一。为了满足各种需求,各国研究机构已经从天然产物中开发了一系列高效环保型防污化合物。本论文以生物膜中微生物和海洋微藻为研究对象，提取、分离防污活性物质，并对其防污活性进行测试。实验结果如下：（1）从海洋生物膜中分离鉴定出一种细菌Bacillus cereus和一种真菌Aureobasidium pullulans HN，抑菌测试实验显示其发酵液具有较强的抑菌活性。本文基于毒性鉴别评估技术建立了微生物防污活性物质的提取分离程序，，提取程序为菌发酵液—通气、加入硫代硫酸钠—调节pH之后进行液液萃取—用HLB固相萃取小柱进行提纯—气质联用分析结构，每一步都进行毒性测试。通过对测试生物中肋骨条藻和纹藤壶幼虫的生长抑制作用，提取到防污活性物质。其中A.pullulans HN氯仿粗提物对中肋骨条藻的96h-EC50为90.9μg·ml-1，对纹藤壶幼虫的24h-LC50为22.2μg·ml-1，B.cereuss石油醚粗提物对藻的96h-EC50为391.5μg·ml-1，对藤壶幼虫的24h-LC50为178.2μg·ml-1。通过分离纯化，确定主要物质结构为肉豆蔻酸、棕榈酸、硬脂酸。研究结果同时表明毒性鉴别评估程序在防污活性物质筛选、提取过程中是有效的。（2）甲藻是生物活性天然产物的一个重要来源，本文选取6种海洋甲藻，对其粗提物进行生物毒性测试，实验结果表明，强壮前沟藻的甲醇粗提物具有显著的抑藻活性，其对硅藻的半数抑制率浓度72h-EC50值为55.4μg·ml-1。对甲醇粗提物进行分析提取后得到提取物B，其防污活性相对于粗提物显著提高，对中肋骨条藻的72h-EC50值为12.9μg·mL-1，对纹藤壶幼虫的半数致死率浓度24h-LC50值为15.1μg·mL-1。红外光谱、高分辨质谱等的分析结果表明提取到的活性物质为一系列的饱和、不饱和的脂肪酸，GC-MS进行结构检测，鉴定的物质为棕榈酸、硬脂酸、二十碳五烯酸。（3）盐藻在制药、化妆品、营养剂、水产养殖饲料等方面有广泛的应用，但盐藻代谢物的潜在防污性能尚未见报道。本文通过一系列萃取程序对其防污能力进行探究，测试生物为中肋骨条藻和纹藤壶幼虫。盐藻的乙酸乙酯粗提物抑藻活性是显著的，对硅藻的半数抑制率浓度72h-EC50值为58.9μg·ml-1。对乙酸乙酯粗提物进行逐步提纯得到最后的提纯物B，该提取物对中肋骨条藻的72h-EC50值为21.2μg·mL-1，对纹藤壶幼虫的半数致死率24h-LC50值为18.8μg·mL-1。红外光谱、高分辨质谱、GC-MS等分析表明防污活性物质为一系列饱和、不饱和的16-C、18-C脂肪酸，主要为棕榈酸、7,10-十六碳二烯酸、4,7,10-十六碳三烯酸、4,7,10,13-十六碳四烯酸、硬脂酸、亚油酸、亚麻酸。以上研究结果说明海洋生物膜中微生物和海洋浮游藻均可产生具有较高防污活性的脂肪酸，有希望成为替代有毒防污剂的新型环境友好型防污剂的来源。
[Abstract]:In the marine industry, bio fouling refers to growth in artificial facilities on the surface, adhesion, reproduction of organisms accumulation. With the progress of modern science and technology, human development and utilization of the ocean has developed greatly, and biofouling has become a thorny issue needs to be solved, so the anti fouling technology has been widely used. A the most economical and practical method of antifouling antifouling coatings. Be used instead of the traditional antifouling coatings, organic tin and cuprous oxide and other toxic antifouling agent poisoning way to anti fouling, it caused serious pollution to the marine environment. The application of the international maritime organization, prohibition of antifouling coatings containing organotin in 2008. Therefore, the development of low toxicity, environmental friendly the non-toxic antifouling agent into practice, the development of marine industry in urgent need. In recent years, marine natural products has become one of the most promising antifouling material sources in order to meet the various needs. The research institutes in the world have developed a series of highly effective environmental protection antifouling compounds from natural products. In this paper, the microorganisms and marine microalgae in the biofilm were taken as the research objects, and the antifouling active substances were extracted and separated, and their antifouling activity was tested.
(1) identified a bacterial Bacillus cereus and Aureobasidium pullulans HN, a fungus isolated from marine biofilm, bacteriostatic test showed strong antibacterial activity of the fermentation broth. The toxicity identification evaluation technology was established based on the separation procedure of extraction of microbial antifouling active substances, the extraction procedure for fermentation liquid ventilation, adding after adjusting the pH of sodium thiosulfate, liquid-liquid extraction and solid-phase extraction column was purified by HLB - structure analysis by GC-MS, every step of toxicity test. Through the test of biological inhibition of Skeletonema costatum and grain growth of barnacle larvae, to extract the antifouling active substances. The 96h-EC50 A.pullulans HN of chloroform extract on Skeletonema costatum is 90.9 G - ml-1, the pattern of barnacle larvae was 22.2 g, 24h-LC50 ml-1, 96h-EC50 B.cereuss petroleum ether extract of algae is 391.5 G - ml-1 The barnacle larvae 24h-LC50, 178.2 G - ml-1. through separation and purification, determination of main material structure for myristic acid, palmitic acid, stearic acid. The results also showed that the toxicity identification evaluation program screening in antifouling active substances, is effective in the extraction process.
(2) dinoflagellates are an important source of bioactive natural products, this paper selected 6 species of marine dinoflagellates, the crude extract of the biological toxicity test, the experimental results show that the Amphidinium carterae methanol extract has algicidal activity significantly, the half inhibition rate of diatom concentration 72h-EC50 the value is 55.4 G - ml-1. of methanol extracts were obtained after extraction of extract B, relative to its antifouling activity in crude extracts increased significantly on Skeletonema costatum 72h-EC50 value was 12.9 g mL-1, half of the lines of barnacle larvae death rate 24h-LC50 concentration is 15.1 G - mL-1. infrared spectroscopy the results show that the extraction of active substances, to a series of high resolution mass spectrometry analysis of saturated and unsaturated fatty acid, GC-MS structure detection, identification of the substances as palmitic acid, stearic acid, twenty carbon five acid.
(3) salt algae in the pharmaceutical, cosmetics, nutritional agent, is widely used in aquaculture feed and so on, but the potential vulnerability of Dunaliella metabolites has not been reported. In this paper, through a series of extraction procedures for its anti fouling ability of test organisms for Skeletonema costatum and barnacle larvae. Lines of Dunaliella acetic acid ethyl acetate extracts algicidal activity is significant, the half inhibition rate of diatom concentration value of 72h-EC50 was 58.9 G - ml-1. of ethyl acetate extracts were purified gradually extract B finally, 72h-EC50 of the extract of Skeletonema costatum was 21.2 ~ g ~ mL-1, half of the larvae of balanus Amphitrite the mortality rate of 24h-LC50 was 18.8 G - mL-1. infrared spectroscopy, high-resolution mass spectrometry, GC-MS analysis showed that the antifouling active substances into a series of saturated and unsaturated 16-C, 18-C fatty acids, mainly palmitic acid, sixteen 7,10- two 4,7,10- sixteen carbon carbon acid, three acid, 4 7,10,13- sixteen - carbon - four - enoic acid, stearic acid, linoleic acid, linolenic acid.
The above results indicate that microorganisms and marine planktonic algae in marine biofilms can produce fatty acids with high antifouling activity, and hopefully become the source of new environment-friendly antifouling agents instead of toxic antifouling agents.

【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：P745

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