15N标记麻痹性贝类毒素在牡蛎中转化及壳聚糖衍生物脱毒机制

Paralytic shellfish poison (PSP) must be removed from contaminated oyster during shellfish purification to ensure seafood safety. However, the conversion and removal mechanism of PSP in oyster remained uncertain was scientific problem which limit aquatic resources. Based on the isotope tracer technique, Alexandrium minutum, which produced high abundance ratios of 15N-PSP, was cultured, the oyster was feed for setting up contamination model in the project. 15N, virulence, and protein assays were conducted. PSP binding protein (PSPBP) was separated from different tissues and subsequently purified. LC-MS-MS and NMR were conducted for structural characterization, and activity sites of binding protein was determined by modeling, to reveal the existential state of PSPBP. Tracking the composition and content of 15N-PSP from toxin-producing algae to accumulate in the oysters and removal in the process, characterization of PSP degradation products structure and toxicity evaluation, expounds that the law of PSP transformation. The contamination oyster was purified and detoxified by chitosan derivatives (CTSDR), the composition and content of CTSDR and 15N-PSP of water and different part of the oyster were detected, and combined with vitro experimental model of 15N-PSP transfer from PSPBP to CTSDR, conversion regularity in the purification of oyster and removal mechanism of the PSP by CTSDR were revealed. Research achievements provide theoretical basis and basic data to promote the shellfish purification industry development.

实施贝类净化脱除污染牡蛎中麻痹性贝类毒素(PSP)是保障水产食品安全重要举措，但PSP转化及脱除机理不清是制约该产业的科学问题。本项目基于同位素示踪技术，培养产高丰度15N-PSP微小亚历山大藻，投喂牡蛎建立染毒模型；集合同位素和毒素双检法，附加蛋白质阳性峰，分离纯化PSP结合蛋白(PSPBP)；经LC-MS-MS、NMR表征及同源建模，查明PSPBP配位活性位点和结合态三维构型；追踪15N-PSP从产毒藻到牡蛎中蓄积及脱除过程中组成、含量变化，以及对PSP降解产物结构表征和毒性评价，阐明PSP转化规律；以壳聚糖衍生物(CTSDR)投喂染毒牡蛎，研究CTSDR及15N-PSP在水体和牡蛎各组织中转移行为，结合PSPBP和CTSDR竞争性吸附15N-PSP分配系数，建立与脱毒率内在关联性，揭示CTSDR作为生物吸附剂脱除PSP机制。研究成果为推动贝类净化产业发展提供理论依据和基础数据。

研究15N同位素标记PSP在牡蛎体内转化规律以及壳聚糖衍生物脱毒机制，为探讨染毒贝类中PSP转化规律，脱除机理及生物吸附剂高效脱除PSP的研究提供借鉴。.（1）产高丰度15N-PSP微小亚历山大藻培养.研究了五种不同15N/P浓度对微小亚历山大藻的影响，不同代产毒藻15N-PSP标记丰度的变化，结果表明最适15N/P为1.5，第28代毒藻细胞中15N标记丰度超过90%，经过HPLC-FLD检测证明应用15N标记组与未标记组的毒素成分及其所占比例无明显差异。.（2）15N-PSP在近江牡蛎中蓄积转化模型的建立.15N标记的微小亚历山大藻及毒素粗品投喂牡蛎，追踪15N标记丰度变化及毒素的蓄积转化。结果表明，投喂毒藻的毒素累积速率远高于毒素粗品，15N-PSP在牡蛎内脏团蓄积量占55.51%，且各组织中存在GTX4向GTX1的转化，GTX1/4平均含量为79.20%，15N同位素增幅为0.37 atom%。.（3）15N-PSP在长牡蛎中蓄积转化模型的建立.15N标记的微小亚历山大藻喂养牡蛎，通过HPLC-MS对15N-PSP转化进行检测。结果显示β型毒素差向异构化为α型毒素，GTX1-4发生氨甲酰基的水解反应生成dcGTX1-4，发现15N标记的新型M4和M10。代谢组学分析共筛选出84种与染毒后相关的内源性代谢物。.（4）壳聚糖衍生物的制备及其对PSP的吸附筛选.以壳聚糖为基质，合成了MACS、SiO2-MA-CS等衍生物，基于水溶液中 PSP的吸附实验。结果表明SiO2-MA-CS对PSP的脱毒率达到65.31%，MACS对PSP的脱毒率为36.62%。.（5）PSP结合蛋白（PSPBP）的提取纯化及与PSP的分子对接研究.从染毒牡蛎中分离纯化15N-PSPBP，检测其同位素丰度和毒性，并通过同源建模及分子对接等生物信息学等手段对两者的结合进行研究，结果表明，得到的PSP结合蛋白同位素丰度为0.438 atom% 15N，PSPBP与PSP之间主要通过氢键作用连接。 .（6）牡蛎体内壳聚糖衍生物脱除PSP及机理研究.使用壳聚糖衍生物SiO2-MA-CS对染毒牡蛎进行活体脱除，结果表明，15N-PSP在牡蛎体内脱除率达到42.56%，结合PSPBP与壳聚糖衍生物的竞争吸附实验，揭示了壳聚糖衍生物作为生物吸附剂与体内PSPBP竞争吸附加速PSP脱除的脱毒机制。