本文關(guān)鍵詞：一株氮雜螺環(huán)酸產(chǎn)毒藻的產(chǎn)毒生理學及食品安全評價　出處：《青島科技大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 靜電場軌道阱高分辨質(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 代謝動力學

【摘要】：海洋水體的富營養(yǎng)化導致赤潮頻繁暴發(fā),有害赤潮藻所產(chǎn)毒素在貝類體內(nèi)富集形成貝類毒素,嚴重危害消費者的身體健康,阻礙水產(chǎn)品對外貿(mào)易的發(fā)展,成為影響貝類產(chǎn)業(yè)可持續(xù)發(fā)展的瓶頸之一。本文以新型貝類毒素氮雜螺環(huán)酸毒素為研究對象,建立多種AZAs毒素的Q-Exactive高分辨質(zhì)譜檢測方法,同時在實驗室內(nèi)對產(chǎn)自我國沿海的一株氮雜螺環(huán)酸產(chǎn)毒藻AZDY06進行單種培養(yǎng)并對其產(chǎn)毒能力進行了評估。隨后,應(yīng)用該產(chǎn)毒藻對櫛孔扇貝進行暴露實驗來研究氮雜螺環(huán)酸毒素在櫛孔扇貝體內(nèi)的代謝輪廓,及其對櫛孔扇貝組織結(jié)構(gòu)和生理的脅迫作用等方面進行了實驗研究。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物定量篩查檢測方法,對蓄積代謝試驗樣品進行綜合分析,根據(jù)化學分子式計算各AZAs的精確質(zhì)量數(shù),精確篩查到了AZA2在貝類蓄積代謝過程產(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)譜對實際陽性樣品進行了定量檢測,達到了同時精準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,為進一步完善我國水產(chǎn)品中貝類毒素的監(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)時初期、中期和后期海洋環(huán)境過程,通過比較毒素組分、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)危害形成的過程。結(jié)果顯示,分布于我國南海海域的氮雜螺環(huán)酸產(chǎn)毒藻(A.poporum,AZDY06株),其生長及產(chǎn)毒性狀穩(wěn)定,產(chǎn)毒能力較強,主要產(chǎn)生AZA2毒素,單細胞產(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驗組有四種代謝產(chǎn)物(AZA6、AZA12、AZA19和AZA23)產(chǎn)生,說明貝類攝食產(chǎn)毒藻生物量的大小影響代謝產(chǎn)物組分的轉(zhuǎn)化過程;中生物量和低生物量組在暴露階段的變化趨勢相似,都是在蓄積階段呈現(xiàn)迅速上升的趨勢,達到最高點隨后呈現(xiàn)總體下降趨勢,而高生物量組在蓄積階段呈現(xiàn)迅速上升的趨勢,在蓄積前期既已達到峰值后呈急劇下降趨勢,隨后緩慢增加直至暴露結(jié)束后,比較而言,各實驗組對AZAs毒素蓄積能力由大到小順序為: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。實驗數(shù)據(jù)初步探究了AZA2在櫛孔扇貝體內(nèi)的代謝轉(zhuǎn)化機制,為后續(xù)實驗奠定基礎(chǔ)。(3)前期實驗表明,高生物量產(chǎn)毒藻暴露實驗會增加櫛孔扇貝體內(nèi)代謝產(chǎn)物的組分種類和各組分的含量,但是隨之也會加重對扇貝的毒害作用,會降低扇貝對氮雜螺環(huán)酸毒素的蓄積能力,暴露時間越長,扇貝對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,同時縮短暴露時間。結(jié)果表明,櫛孔扇貝對該產(chǎn)毒藻具有較強攝食能力及AZAs蓄積能力,扇貝在12 h內(nèi)攝食5×10~7cells產(chǎn)毒藻細胞后,體內(nèi)AZAs毒素含量已超歐盟安全限量,達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)臟團鰓外套膜閉殼肌,內(nèi)臟團中毒素組份最多且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AZA限量標準制定的潛在考慮對象。本研究證明我國近海分布氮雜螺環(huán)酸產(chǎn)毒藻毒性危害較強,建議加快制定AZAs限量標準。(4)暴露于三種不同生物量模式下的櫛孔扇貝在暴露實驗初期,內(nèi)臟團和鰓組織內(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)生過氧化,相應(yīng)的GSH-PX和POD酶活力均增強,粒細胞分泌的ACP、POX活力增強。綜合AZAs對櫛孔扇貝內(nèi)臟團和鰓組織的超微結(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脅迫下貝類的組織毒理學指標的確立提供理論依據(jù)。實驗結(jié)果顯示:暴露實驗過程中,在AZAs的作用下櫛孔扇貝內(nèi)臟團和鰓組織的超微結(jié)構(gòu)均出現(xiàn)了病理變化,內(nèi)臟團的腸上皮細胞空泡化,細胞核萎縮變形,嚴重時細胞壞死裂解,且損傷程度隨暴露毒素生物量的增加而加重;暴露前期,鰓的柱狀上皮細胞中線粒體和溶酶體增多且聚集,后期上皮細胞腫脹破裂,粘液細胞大量釋放粘液顆粒。通過暴露實驗檢測內(nèi)臟團和鰓組織中抗氧化防御系統(tǒng)中氧化還原酶的變化,同時觀察了AZAs對櫛孔扇貝內(nèi)臟團和鰓組織超微結(jié)構(gòu)的毒理學作用進一步研究AZAs對櫛孔扇貝內(nèi)臟團和鰓組織的毒理學脅迫作用。
[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.

【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q949.2;X171.5

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