本文選題：生物污損　切入點：天然產(chǎn)物　出處：《中國海洋大學》2014年碩士論文　論文類型：學位論文

【摘要】：在海洋工業(yè)中，生物污損是指在人工設施表面上附著、生長、繁殖的生物體的積累。隨著現(xiàn)代科技的進步，人類對海洋的開發(fā)和利用有了很大發(fā)展，而生物污損成為急需解決的棘手問題，因此防污技術日益得到廣泛的應用。防污涂料的使用成為最經(jīng)濟實用的一種防污方法。而傳統(tǒng)防污涂料，以有機錫和氧化亞銅等有毒防污劑的毒殺方式達到防污目的，卻對海洋環(huán)境造成嚴重污染。國際海事組織在2008年禁止含有機錫的防污涂料的應用，因此，開發(fā)低毒、無毒的環(huán)境友好型防污劑成為海洋產(chǎn)業(yè)發(fā)展的實際、迫切需要。近幾年來，海洋天然產(chǎn)物成為最有潛力的防污物質(zhì)來源之一。為了滿足各種需求,各國研究機構已經(jīng)從天然產(chǎn)物中開發(fā)了一系列高效環(huán)保型防污化合物。本論文以生物膜中微生物和海洋微藻為研究對象，提取、分離防污活性物質(zhì)，并對其防污活性進行測試。實驗結果如下： （1）從海洋生物膜中分離鑒定出一種細菌Bacillus cereus和一種真菌Aureobasidium pullulans HN，抑菌測試實驗顯示其發(fā)酵液具有較強的抑菌活性。本文基于毒性鑒別評估技術建立了微生物防污活性物質(zhì)的提取分離程序，，提取程序為菌發(fā)酵液—通氣、加入硫代硫酸鈉—調(diào)節(jié)pH之后進行液液萃取—用HLB固相萃取小柱進行提純—氣質(zhì)聯(lián)用分析結構，每一步都進行毒性測試。通過對測試生物中肋骨條藻和紋藤壺幼蟲的生長抑制作用，提取到防污活性物質(zhì)。其中A.pullulans HN氯仿粗提物對中肋骨條藻的96h-EC50為90.9μg·ml-1，對紋藤壺幼蟲的24h-LC50為22.2μg·ml-1，B.cereuss石油醚粗提物對藻的96h-EC50為391.5μg·ml-1，對藤壺幼蟲的24h-LC50為178.2μg·ml-1。通過分離純化，確定主要物質(zhì)結構為肉豆蔻酸、棕櫚酸、硬脂酸。研究結果同時表明毒性鑒別評估程序在防污活性物質(zhì)篩選、提取過程中是有效的。 （2）甲藻是生物活性天然產(chǎn)物的一個重要來源，本文選取6種海洋甲藻，對其粗提物進行生物毒性測試，實驗結果表明，強壯前溝藻的甲醇粗提物具有顯著的抑藻活性，其對硅藻的半數(shù)抑制率濃度72h-EC50值為55.4μg·ml-1。對甲醇粗提物進行分析提取后得到提取物B，其防污活性相對于粗提物顯著提高，對中肋骨條藻的72h-EC50值為12.9μg·mL-1，對紋藤壺幼蟲的半數(shù)致死率濃度24h-LC50值為15.1μg·mL-1。紅外光譜、高分辨質(zhì)譜等的分析結果表明提取到的活性物質(zhì)為一系列的飽和、不飽和的脂肪酸，GC-MS進行結構檢測，鑒定的物質(zhì)為棕櫚酸、硬脂酸、二十碳五烯酸。 （3）鹽藻在制藥、化妝品、營養(yǎng)劑、水產(chǎn)養(yǎng)殖飼料等方面有廣泛的應用，但鹽藻代謝物的潛在防污性能尚未見報道。本文通過一系列萃取程序?qū)ζ浞牢勰芰M行探究，測試生物為中肋骨條藻和紋藤壺幼蟲。鹽藻的乙酸乙酯粗提物抑藻活性是顯著的，對硅藻的半數(shù)抑制率濃度72h-EC50值為58.9μg·ml-1。對乙酸乙酯粗提物進行逐步提純得到最后的提純物B，該提取物對中肋骨條藻的72h-EC50值為21.2μg·mL-1，對紋藤壺幼蟲的半數(shù)致死率24h-LC50值為18.8μg·mL-1。紅外光譜、高分辨質(zhì)譜、GC-MS等分析表明防污活性物質(zhì)為一系列飽和、不飽和的16-C、18-C脂肪酸，主要為棕櫚酸、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、亞油酸、亞麻酸。 以上研究結果說明海洋生物膜中微生物和海洋浮游藻均可產(chǎn)生具有較高防污活性的脂肪酸，有希望成為替代有毒防污劑的新型環(huán)境友好型防污劑的來源。
[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.

【學位授予單位】：中國海洋大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：P745

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