發(fā)布時(shí)間：2018-01-03 23:24

  本文關(guān)鍵詞：一株氮雜螺環(huán)酸產(chǎn)毒藻的產(chǎn)毒生理學(xué)及食品安全評(píng)價(jià)　出處：《青島科技大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 靜電場(chǎng)軌道阱高分辨質(zhì)譜 氮雜螺環(huán)酸毒素產(chǎn)毒藻 氮雜螺環(huán)酸毒素櫛孔扇貝 代謝動(dòng)力學(xué)

【摘要】：海洋水體的富營(yíng)養(yǎng)化導(dǎo)致赤潮頻繁暴發(fā),有害赤潮藻所產(chǎn)毒素在貝類(lèi)體內(nèi)富集形成貝類(lèi)毒素,嚴(yán)重危害消費(fèi)者的身體健康,阻礙水產(chǎn)品對(duì)外貿(mào)易的發(fā)展,成為影響貝類(lèi)產(chǎn)業(yè)可持續(xù)發(fā)展的瓶頸之一。本文以新型貝類(lèi)毒素氮雜螺環(huán)酸毒素為研究對(duì)象,建立多種AZAs毒素的Q-Exactive高分辨質(zhì)譜檢測(cè)方法,同時(shí)在實(shí)驗(yàn)室內(nèi)對(duì)產(chǎn)自我國(guó)沿海的一株氮雜螺環(huán)酸產(chǎn)毒藻AZDY06進(jìn)行單種培養(yǎng)并對(duì)其產(chǎn)毒能力進(jìn)行了評(píng)估。隨后,應(yīng)用該產(chǎn)毒藻對(duì)櫛孔扇貝進(jìn)行暴露實(shí)驗(yàn)來(lái)研究氮雜螺環(huán)酸毒素在櫛孔扇貝體內(nèi)的代謝輪廓,及其對(duì)櫛孔扇貝組織結(jié)構(gòu)和生理的脅迫作用等方面進(jìn)行了實(shí)驗(yàn)研究。論文的主要內(nèi)容如下:(1)采用Q-Exactive高分辨質(zhì)譜,在液相色譜-串聯(lián)質(zhì)譜法基礎(chǔ)上進(jìn)一步創(chuàng)建了AZA毒素高分辨非定向定性篩查和低分辨目標(biāo)物定量篩查檢測(cè)方法,對(duì)蓄積代謝試驗(yàn)樣品進(jìn)行綜合分析,根據(jù)化學(xué)分子式計(jì)算各AZAs的精確質(zhì)量數(shù),精確篩查到了AZA2在貝類(lèi)蓄積代謝過(guò)程產(chǎn)生的四種代謝產(chǎn)物(AZA6、AZA12、AZA19和AZA23),并使用液相色譜-串聯(lián)質(zhì)譜對(duì)實(shí)際陽(yáng)性樣品進(jìn)行了定量檢測(cè),達(dá)到了同時(shí)精準(zhǔn)定性和精確定量的要求,適用于氮雜螺環(huán)酸毒素代謝物質(zhì)的的篩查分析工作,為進(jìn)一步完善我國(guó)水產(chǎn)品中貝類(lèi)毒素的監(jiān)控體系提供可靠的工作基礎(chǔ)和技術(shù)支撐。(2)將櫛孔扇貝(Chlamys farreri)暴露于三種產(chǎn)毒藻不同生物量模式下模擬赤潮爆發(fā)時(shí)初期、中期和后期海洋環(huán)境過(guò)程,通過(guò)比較毒素組分、各組織器官中毒素的蓄積及代謝轉(zhuǎn)化特異性,研究氮雜螺環(huán)酸毒素(Azaspiracid,AZAs)在櫛孔扇貝體內(nèi)危害形成的過(guò)程。結(jié)果顯示,分布于我國(guó)南海海域的氮雜螺環(huán)酸產(chǎn)毒藻(A.poporum,AZDY06株),其生長(zhǎng)及產(chǎn)毒性狀穩(wěn)定,產(chǎn)毒能力較強(qiáng),主要產(chǎn)生AZA2毒素,單細(xì)胞產(chǎn)度能力一般為7.05±0.52 fg/cell;投喂低生物量產(chǎn)毒藻組扇貝體內(nèi)有共有三種代謝產(chǎn)物(AZA6、AZA12、AZA19)產(chǎn)生,中生物量和高生物量實(shí)驗(yàn)組有四種代謝產(chǎn)物(AZA6、AZA12、AZA19和AZA23)產(chǎn)生,說(shuō)明貝類(lèi)攝食產(chǎn)毒藻生物量的大小影響代謝產(chǎn)物組分的轉(zhuǎn)化過(guò)程;中生物量和低生物量組在暴露階段的變化趨勢(shì)相似,都是在蓄積階段呈現(xiàn)迅速上升的趨勢(shì),達(dá)到最高點(diǎn)隨后呈現(xiàn)總體下降趨勢(shì),而高生物量組在蓄積階段呈現(xiàn)迅速上升的趨勢(shì),在蓄積前期既已達(dá)到峰值后呈急劇下降趨勢(shì),隨后緩慢增加直至暴露結(jié)束后,比較而言,各實(shí)驗(yàn)組對(duì)AZAs毒素蓄積能力由大到小順序?yàn)?中生物量組高生物量組低生物量組,其中以中生物量組蓄積能力最強(qiáng)。實(shí)驗(yàn)數(shù)據(jù)初步探究了AZA2在櫛孔扇貝體內(nèi)的代謝轉(zhuǎn)化機(jī)制,為后續(xù)實(shí)驗(yàn)奠定基礎(chǔ)。(3)前期實(shí)驗(yàn)表明,高生物量產(chǎn)毒藻暴露實(shí)驗(yàn)會(huì)增加櫛孔扇貝體內(nèi)代謝產(chǎn)物的組分種類(lèi)和各組分的含量,但是隨之也會(huì)加重對(duì)扇貝的毒害作用,會(huì)降低扇貝對(duì)氮雜螺環(huán)酸毒素的蓄積能力,暴露時(shí)間越長(zhǎng),扇貝對(duì)毒素的蓄積代謝能力越弱。所以將櫛孔扇貝(Chlamys farreri)直接暴露于更高生物量產(chǎn)毒藻,同時(shí)縮短暴露時(shí)間。結(jié)果表明,櫛孔扇貝對(duì)該產(chǎn)毒藻具有較強(qiáng)攝食能力及AZAs蓄積能力,扇貝在12 h內(nèi)攝食5×10~7cells產(chǎn)毒藻細(xì)胞后,體內(nèi)AZAs毒素含量已超歐盟安全限量,達(dá)165.3μg AZA1 eq/kg,蓄積效率為78.2%;AZAs毒素在扇貝各組織間分布存在顯著差異:內(nèi)臟團(tuán)鰓外套膜閉殼肌,內(nèi)臟團(tuán)中毒素組份最多且AZAs毒素含量最高,為該毒素在櫛孔扇貝體內(nèi)蓄積代謝的靶器官;AZA2在扇貝中潛在轉(zhuǎn)化方式不同,包括碳鍵位的羥基化、去羧基化和氧化等作用方式;暴露期間共生成4種代謝產(chǎn)物:AZA6、AZA12、AZA19和AZA23,其中AZA19為最主要代謝產(chǎn)物,約占總毒素40%左右,其他代謝產(chǎn)物含量較低,因此像AZA19這種持久性代謝產(chǎn)物,應(yīng)成為我國(guó)AZA限量標(biāo)準(zhǔn)制定的潛在考慮對(duì)象。本研究證明我國(guó)近海分布氮雜螺環(huán)酸產(chǎn)毒藻毒性危害較強(qiáng),建議加快制定AZAs限量標(biāo)準(zhǔn)。(4)暴露于三種不同生物量模式下的櫛孔扇貝在暴露實(shí)驗(yàn)初期,內(nèi)臟團(tuán)和鰓組織內(nèi)的抗氧化防御系統(tǒng)中的氧化還原酶被激活,MDA含量增多,脂質(zhì)發(fā)生過(guò)氧化,相應(yīng)的GSH-PX和POD酶活力均增強(qiáng),粒細(xì)胞分泌的ACP、POX活力增強(qiáng)。綜合AZAs對(duì)櫛孔扇貝內(nèi)臟團(tuán)和鰓組織的超微結(jié)構(gòu)的損害以及引起的組織中氧化還原酶的激活作用,可共同為AZAs脅迫下貝類(lèi)的組織毒理學(xué)指標(biāo)的確立提供理論依據(jù)。實(shí)驗(yàn)結(jié)果顯示:暴露實(shí)驗(yàn)過(guò)程中,在AZAs的作用下櫛孔扇貝內(nèi)臟團(tuán)和鰓組織的超微結(jié)構(gòu)均出現(xiàn)了病理變化,內(nèi)臟團(tuán)的腸上皮細(xì)胞空泡化,細(xì)胞核萎縮變形,嚴(yán)重時(shí)細(xì)胞壞死裂解,且損傷程度隨暴露毒素生物量的增加而加重;暴露前期,鰓的柱狀上皮細(xì)胞中線粒體和溶酶體增多且聚集,后期上皮細(xì)胞腫脹破裂,粘液細(xì)胞大量釋放粘液顆粒。通過(guò)暴露實(shí)驗(yàn)檢測(cè)內(nèi)臟團(tuán)和鰓組織中抗氧化防御系統(tǒng)中氧化還原酶的變化,同時(shí)觀察了AZAs對(duì)櫛孔扇貝內(nèi)臟團(tuán)和鰓組織超微結(jié)構(gòu)的毒理學(xué)作用進(jìn)一步研究AZAs對(duì)櫛孔扇貝內(nèi)臟團(tuán)和鰓組織的毒理學(xué)脅迫作用。
[Abstract]:The sea water eutrophication caused by frequent outbreaks of harmful algae blooms, the toxin in shellfish and formation of shellfish toxins, serious harm to the health of consumers, hinder the development of foreign trade of aquatic products, has become one of the bottlenecks affecting the sustainable development of shellfish industry. Based on the model of shellfish toxin aza spiro tranexamic acid toxin as the research object. High resolution mass spectrometry to establish multiple AZAs toxin Q-Exactive, at the same time in the laboratory for coastal produced in a strain of AZA spiro tranexamic acid toxic algae AZDY06 single culture and its toxin producing ability were evaluated. Then, the application of toxic algae on Chlamys Scallop in Shell to the study of AZA spiro tranexamic acid toxin in the metabolic profile of Chlamys Scallop in Shell in vivo exposure experiments and on the structure and physiology of Chlamys Scallop in Shell tissue stress. The main contents of this thesis are as : (1) using a Q-Exactive high resolution mass spectrometry, tandem mass spectrometry further created AZA toxin high resolution non directional screening and qualitative quantitative screening of low resolution target detection method in liquid chromatography, the accumulation of metabolic test samples were analyzed according to the accurate mass AZAs calculation of chemical molecular formula. Accurate screening to AZA2 accumulation of four metabolites produced in the metabolic process of shellfish (AZA6, AZA12, AZA19 and AZA23), and the use of liquid chromatography tandem mass spectrometry for quantitative detection of the positive samples are achieved at the same time, accurate qualitative and quantitative requirements for aza tranexamic acid metabolism screening snail toxin the analysis of the work, provide basis and reliable technical support for the monitoring system to further improve the shellfish aquatic products in China. (2) the Chlamys Scallop in Shell (Chlamys farreri) exposed to three kinds of toxic algae do not At the outbreak of red tide simulation with initial biomass model, middle and late marine environment, by comparing the toxin component, specific toxin accumulation and metabolism in different organs, of AZA spiro tranexamic acid (Azaspiracid, AZAs) toxin in vivo damage form of Chlamys Scallop in Shell. The results showed that the distribution in China in the South China Sea snail aza tranexamic acid toxic algae (A.poporum, AZDY06 strain), the growth and toxin production were stable, toxin producing ability, mainly produces the AZA2 toxin, single cell production capacity is 7.05 + 0.52 fg/cell; feeding low biological production of toxic algae in a total of three Scallop in Shell group metabolites (AZA6, AZA12, AZA19), biomass and high biomass of experimental group was four metabolites (AZA6, AZA12, AZA19 and AZA23), illustrate the transformation process of metabolite components of shellfish feeding toxic algae biomass in the biomass and size effect; The change in the trend of low exposure stage biomass were similar, are showing a rapid upward trend in the accumulation stage, reached the highest point then shows an overall downward trend, while the high biomass group showed rapid upward trend in stock in the early stage, the accumulation has reached the peak after the decline, then increased slowly until the exposure after the comparison, the experimental group of AZAs toxin accumulation ability from large to small order of biomass in the group of high biomass group low biomass group, the biomass accumulation group strongest. The preliminary experimental data to explore the AZA2 transformation in vivo metabolism mechanism of Chlamys Scallop in Shell, lays the foundation for the follow-up (3) preliminary experiments. Experimental results show that the high biological production of toxic algae will increase the content of experimental exposure of Chlamys Scallop in Shell metabolite composition and components, but it will also increase the toxic effect on the Scallop in Shell, Will reduce the accumulation of Scallop in Shell aza spiro tranexamic acid toxin, the longer exposure time, Scallop in Shell on toxin accumulation ability is weak. So the metabolism of Chlamys Scallop in Shell (Chlamys farreri) directly exposed to the higher biomass production of toxic algae, and shorten the exposure time. The results showed that Zhikong Scallop has strong feeding ability and AZAs of the toxic algae accumulation ability, Scallop in Shell within 12 h feeding 5 * 10~7cells toxin producing algae cells in vivo, AZAs toxin content exceeded the safety limits of up to 165.3 g AZA1 eq/kg, the accumulation rate was 78.2%; AZAs toxin in different tissues of Scallop in Shell distribution between significant differences: the visceral mass of mantle gill box muscle, visceral mass fractions and most toxin AZAs toxin content is the highest, is a target organ of the toxin in Chlamys Scallop in Shell accumulation metabolism; AZA2 in Scallop in Shell potential transformation in different ways, including carbon hydroxylation keys, to carboxylation and oxidation During the exposure period; generating a total of 4 metabolites: AZA6, AZA12, AZA19 and AZA23, where AZA19 is the main metabolic products, accounting for about 40% of the total toxin, other metabolites content is relatively low, so AZA19 like this persistent metabolite, should be considered a potential object of China AZA standard system. This study shows that the distribution of China's offshore aza spiro tranexamic acid toxic algae toxicity is strong, proposed to speed up the development of AZAs standard. (4) exposed to three different biomass under the mode of Chlamys Scallop in Shell exposed at the beginning of the study, oxidoreductase anti oxidative defense system in visceral mass and gill tissues in is activated. The increase of MDA content, lipid peroxidation, the corresponding GSH-PX and POD activity were increased, seed cells secreting ACP, POX activity increased. The ultrastructure of AZAs oxidation of Chlamys Scallop in Shell visceral mass and gill tissue damage and lead to the organization Activation of reductase, and provide a theoretical basis for the establishment of the index organization jointly under the stress of shellfish for toxicology of AZAs. Experimental results show that the exposure in the course of the experiment, the pathological changes were found in the ultrastructure of AZAs under the action of Chlamys Scallop in Shell visceral mass and gill tissue, visceral mass of intestinal epithelial cell vacuolation, nucleus atrophy serious deformation, cracking and damage degree of cell necrosis, with increasing exposure to toxins biomass increased; pre exposure, mitochondria and lysosomes and aggregation of columnar epithelial cells of gill, late epithelial cell swelling rupture, the release of a large number of mucous granules in mucous cells. Through the changes of oxidoreductase antioxidant defense system exposure test in visceral mass and in the gills, and the effects of AZAs on ultrastructure of toxicology of Chlamys Scallop in Shell visceral mass and gill tissues of Chlamys Scallop in Shell further study AZAs The toxicological stress of visceral and gill tissues.

【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q949.2;X171.5

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本文編號(hào)：1376070