一株氮杂螺环酸产毒藻的产毒生理学及食品安全评价

发布时间：2018-01-03 23:24

本文关键词：一株氮杂螺环酸产毒藻的产毒生理学及食品安全评价　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：海洋水体的富营养化导致赤潮频繁暴发,有害赤潮藻所产毒素在贝类体内富集形成贝类毒素,严重危害消费者的身体健康,阻碍水产品对外贸易的发展,成为影响贝类产业可持续发展的瓶颈之一。本文以新型贝类毒素氮杂螺环酸毒素为研究对象,建立多种AZAs毒素的Q-Exactive高分辨质谱检测方法,同时在实验室内对产自我国沿海的一株氮杂螺环酸产毒藻AZDY06进行单种培养并对其产毒能力进行了评估。随后,应用该产毒藻对栉孔扇贝进行暴露实验来研究氮杂螺环酸毒素在栉孔扇贝体内的代谢轮廓,及其对栉孔扇贝组织结构和生理的胁迫作用等方面进行了实验研究。论文的主要内容如下:(1)采用Q-Exactive高分辨质谱,在液相色谱-串联质谱法基础上进一步创建了AZA毒素高分辨非定向定性筛查和低分辨目标物定量筛查检测方法,对蓄积代谢试验样品进行综合分析,根据化学分子式计算各AZAs的精确质量数,精确筛查到了AZA2在贝类蓄积代谢过程产生的四种代谢产物(AZA6、AZA12、AZA19和AZA23),并使用液相色谱-串联质谱对实际阳性样品进行了定量检测,达到了同时精准定性和精确定量的要求,适用于氮杂螺环酸毒素代谢物质的的筛查分析工作,为进一步完善我国水产品中贝类毒素的监控体系提供可靠的工作基础和技术支撑。(2)将栉孔扇贝(Chlamys farreri)暴露于三种产毒藻不同生物量模式下模拟赤潮爆发时初期、中期和后期海洋环境过程,通过比较毒素组分、各组织器官中毒素的蓄积及代谢转化特异性,研究氮杂螺环酸毒素(Azaspiracid,AZAs)在栉孔扇贝体内危害形成的过程。结果显示,分布于我国南海海域的氮杂螺环酸产毒藻(A.poporum,AZDY06株),其生长及产毒性状稳定,产毒能力较强,主要产生AZA2毒素,单细胞产度能力一般为7.05±0.52 fg/cell;投喂低生物量产毒藻组扇贝体内有共有三种代谢产物(AZA6、AZA12、AZA19)产生,中生物量和高生物量实验组有四种代谢产物(AZA6、AZA12、AZA19和AZA23)产生,说明贝类摄食产毒藻生物量的大小影响代谢产物组分的转化过程;中生物量和低生物量组在暴露阶段的变化趋势相似,都是在蓄积阶段呈现迅速上升的趋势,达到最高点随后呈现总体下降趋势,而高生物量组在蓄积阶段呈现迅速上升的趋势,在蓄积前期既已达到峰值后呈急剧下降趋势,随后缓慢增加直至暴露结束后,比较而言,各实验组对AZAs毒素蓄积能力由大到小顺序为:中生物量组高生物量组低生物量组,其中以中生物量组蓄积能力最强。实验数据初步探究了AZA2在栉孔扇贝体内的代谢转化机制,为后续实验奠定基础。(3)前期实验表明,高生物量产毒藻暴露实验会增加栉孔扇贝体内代谢产物的组分种类和各组分的含量,但是随之也会加重对扇贝的毒害作用,会降低扇贝对氮杂螺环酸毒素的蓄积能力,暴露时间越长,扇贝对毒素的蓄积代谢能力越弱。所以将栉孔扇贝(Chlamys farreri)直接暴露于更高生物量产毒藻,同时缩短暴露时间。结果表明,栉孔扇贝对该产毒藻具有较强摄食能力及AZAs蓄积能力,扇贝在12 h内摄食5×10~7cells产毒藻细胞后,体内AZAs毒素含量已超欧盟安全限量,达165.3μg AZA1 eq/kg,蓄积效率为78.2%;AZAs毒素在扇贝各组织间分布存在显著差异:内脏团鳃外套膜闭壳肌,内脏团中毒素组份最多且AZAs毒素含量最高,为该毒素在栉孔扇贝体内蓄积代谢的靶器官;AZA2在扇贝中潜在转化方式不同,包括碳键位的羟基化、去羧基化和氧化等作用方式;暴露期间共生成4种代谢产物:AZA6、AZA12、AZA19和AZA23,其中AZA19为最主要代谢产物,约占总毒素40%左右,其他代谢产物含量较低,因此像AZA19这种持久性代谢产物,应成为我国AZA限量标准制定的潜在考虑对象。本研究证明我国近海分布氮杂螺环酸产毒藻毒性危害较强,建议加快制定AZAs限量标准。(4)暴露于三种不同生物量模式下的栉孔扇贝在暴露实验初期,内脏团和鳃组织内的抗氧化防御系统中的氧化还原酶被激活,MDA含量增多,脂质发生过氧化,相应的GSH-PX和POD酶活力均增强,粒细胞分泌的ACP、POX活力增强。综合AZAs对栉孔扇贝内脏团和鳃组织的超微结构的损害以及引起的组织中氧化还原酶的激活作用,可共同为AZAs胁迫下贝类的组织毒理学指标的确立提供理论依据。实验结果显示:暴露实验过程中,在AZAs的作用下栉孔扇贝内脏团和鳃组织的超微结构均出现了病理变化,内脏团的肠上皮细胞空泡化,细胞核萎缩变形,严重时细胞坏死裂解,且损伤程度随暴露毒素生物量的增加而加重;暴露前期,鳃的柱状上皮细胞中线粒体和溶酶体增多且聚集,后期上皮细胞肿胀破裂,粘液细胞大量释放粘液颗粒。通过暴露实验检测内脏团和鳃组织中抗氧化防御系统中氧化还原酶的变化,同时观察了AZAs对栉孔扇贝内脏团和鳃组织超微结构的毒理学作用进一步研究AZAs对栉孔扇贝内脏团和鳃组织的毒理学胁迫作用。
[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.

【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q949.2;X171.5

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