CN117481052A - 13种麻痹性贝类毒素基体标准物质原料的制备方法和应用 - Google Patents
13种麻痹性贝类毒素基体标准物质原料的制备方法和应用 Download PDFInfo
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
本发明公开了13种麻痹性贝类毒素基体标准物质原料的制备方法和应用，包括下列步骤：(1)四株麻痹性贝类毒素产毒藻扩培方法，(2)四株麻痹性贝类毒素产毒藻混合暴露贻贝步骤。本发明采用不同配比的多种产毒藻混合暴露贻贝的方法，获得含有13种麻痹性贝类毒素基体标准物质原料。通过比较野外暴露和本发明的室内联合暴露贻贝的方法，本发明可以获得稳定可靠可重复的较多种类麻痹性贝类毒素基体标准物质原料，可以同时满足国内不同海域的贝类麻痹性贝类毒素检测分析的质量控制与方法验证的需求，促进我国麻痹性贝类毒素的监控和风险评估的有效性。
Description
技术领域
本发明涉及一种13种麻痹性贝类毒素基体标准物质原料的制备方法和应用，属于生物毒素技术领域。
背景技术
麻痹性贝类毒素(paralytic shellfish toxins,PSTs)在我国乃至全球引发了严峻的生态和食品安全风险，加强该类毒素的安全监控以确保消费者成为全球共识。我国近海PSTs产毒藻种类繁多且分布十分广泛，主要由链状亚历山大藻(Alexandriumcatenella)、太平洋亚历山大藻(A.pacificum)、塔玛亚历山大藻(A.tamarense)、微小亚历山大藻(A.minutum)和链状裸甲藻(Gymnodinium catenatum)等单细胞甲藻产生。不同产毒藻所产PSTs毒素组成和毒性大小也存在着很大差别，从而显著影响双壳贝类中蓄积的毒素。我国近海PSTs污染情况较为复杂，南海区域贝类中主要毒素为GTX、STX和NeoSTX，黄海区域毒素以C和GTX为主，渤海区域贝类中毒素以GTX和NeoSTX为主，其中赤潮较为严重的秦皇岛地区贝类中PSTs主要组成为GTX。PSTs在我国沿海存在时空分布及种间差异，从而对全面而有效的监测和管理造成了困难。
目前，人们对PSTs还无法进行有效的控制，因此预防和预报是减轻其危害的有效手段。中国现在已经开展了有效的预防方法是对水源或收获区域开展控制监测。在贝类生产的各个环节，随时监测贝类PSTs的变化是至关重要的。欧盟、美国、加拿大等多个国家或地区对其制定了严格的限量标准(800μg STXeq./kg)并加以监控。由于食品基质的复杂性，基质效应往往会对检测方法的准确性和精密度造成一定的干扰，在分析检测过程中采用基体标准物质进行质量控制可以有效克服基质效应引起的误差。高质量和可靠的标准物质是分析方法开发、验证和仪器校准的宝贵工具。
常用的麻痹性贝类毒素基体标准物质的制备方法有自然赤潮污染麻痹性贝类毒素的贝类为原料制备得到基体标准物质，或者用A.tamarense投喂扇贝制备了含有GTX1、GTX4、C1、C2的4种麻痹性贝类毒素基体标准物质。可见目前现有麻痹性贝类毒素基体标准物质制备技术原料来源不稳定、产品毒素种类较少无法覆盖国际限量目标且不符合我国多种PSTs共存的现状。
发明内容
为解决现有技术中存在的问题，本发明提供一种13种麻痹性贝类毒素标准物质原料的制备方法，填补国内外可以同时制备13种麻痹性贝类毒素基体标物原料的空白，原料来源稳定，操作流程简单，结果可控。
本发明提供一种13种麻痹性贝类毒素标准物质原料的制备方法，包括下列步骤：
(A)四株我国近海典型麻痹性贝类毒素产毒藻的扩级培养：一级和二级分级培养，三级为半连续培养，所述四株麻痹性贝类毒素产毒藻为链状亚历山大藻(A.catenella，GY-H25)、链状裸甲藻(G.catenatum,GY-H65)、塔玛亚历山大藻(A.tamarense,GY-H31)和微小亚历山大藻(A.minutum,GY-H46)藻；
(B)四株产毒藻混合暴露：每只贻贝投喂一定的配比，一定的总藻细胞数的四株产毒藻，在6～10℃养殖水温下，暴露周期为4～8天下混合暴露贻贝；
(C)麻痹性贝类毒素基体标物原料的获取：暴露结束后，收集所有贻贝进行解剖并采集所有软组织，匀浆混匀后获得麻痹性贝类毒素基体标物原料。
优选的，所述步骤(A)中四株麻痹性贝类毒素产毒藻为链状亚历山大藻(A.catenella，GY-H25)、链状裸甲藻(G.catenatum,GY-H65)、塔玛亚历山大藻(A.tamarense,GY-H31)和微小亚历山大藻(A.minutum,GY-H46)藻种均可购买于上海光语生物科技有限公司，商业化的这四种产毒藻容易购买且价格便宜，成本低。
优选的，所述步骤(B)中的四株产毒藻的配比为：GY-H25为7％～15％，GY-H65为10％～20％，GY-H31为8％～20％，GY-H46为45％～65％，产毒藻占比的改变是影响不同毒素的占比和含量。
优选的，所述步骤(B)中暴露周期为5天。
优选的，所述步骤(B)中每只贝每日投喂总藻量为3.5×104～2.6×105cells。
优选的，所述步骤(B)中混合暴露贝类时养殖水温为9℃，水温会影响产毒藻的毒素在贻贝的体内的蓄积率。
根据本发明的制备方法制备的麻痹性贝类毒素基体标准物质原料，其毒素组分包括NEO、dcSTX、dcNEO、GTX1、GTX4、GTX2、GTX3、GTX5、GTX6、dcGTX2、dcGTX3、C1和C2。
本发明还提供13种麻痹性贝类毒素基体标准物质原料的应用，用于制备稳定可靠的13种麻痹性贝类毒素标准物质的应用。
本发明还提供13种麻痹性贝类毒素基体标准物质原料制备13种麻痹性贝类毒素标准物质在同时监控中国多个海域麻痹性贝类毒素上的应用。
根据本发明的制备方法制备的麻痹性贝类毒素基体标准物质，可用于实验室间麻痹性贝类毒素测试项目的能力验证活动。还可用于麻痹性贝类毒素的定量或定性检测，以及用于检测方法的验证、测试仪器的校准、测试结果的质量控制等。
与现有技术相比，本发明的有益效果：本发明提供了一种可以同时制备13种麻痹性贝类毒素基体标准物质原料的方法，填补国内外可以同时制备13种麻痹性贝类毒素基体标物原料的空白，原料来源稳定，操作流程简单，结果可控，能够确保麻痹性贝类毒素检测的全面性，有利于对中国多个海域麻痹性贝类毒素的有效监测。本发明的13种麻痹性贝类毒素基体标准物质原料制备的基体标准物质可用于实验室间麻痹性贝类毒素测试能力验证，用于定量或定性检测13种麻痹性贝类毒素，适合于食品、渔监、环保、质检、渔业、卫生等各个部门使用，可促进水产养殖、加工和贸易发展，确保食品安全，将带来显著的社会效益和经济效益。
附图说明
图1为本发明混合暴露示意图。
图2为本发明的麻痹性贝类毒素基体标准物质原料制备工艺流程图。
图3为实施例2每只贝每日投喂总藻量为3.5×104cells，投喂四株产毒藻GY-H25、GY-H65、GY-H31和GY-H46配比分别为：为14％、15％、15％、56％。暴露时间为5天混合暴露后贻贝体内的毒素含量和种类。
图4为实施例3每只贝每日投喂总藻量为2.6×105cells，喂四株产毒藻GY-H25、GY-H65、GY-H31和GY-H46配比分别为：为8％、12％、18％、62％。暴露时间为5天混合暴露后贻贝体内的毒素含量和种类。
图5(a)～(k)为实施例2的麻痹性贝类毒素基体标准物质原料中麻痹性贝类毒素的LC-MS谱图。
具体实施方式
下面结合附图和实施例对本发明做进一步详细说明。
本发明中所用到的贻贝或材料为常规市售产品，或可通过本领域公知的方法获得。例如：链状亚历山大藻(A.catenella，GY-H25)、链状裸甲藻(G.catenatum,GY-H65)、塔玛亚历山大藻(A.tamarense,GY-H31)和微小亚历山大藻(A.minutum,GY-H46)藻种均可购买于上海光语生物科技有限公司，海水和贻贝购买于周边海域。
实施例1
四株产毒藻三级扩大培养：
一级培养：产毒藻接种于经高温灭菌的100～250mL三角烧瓶中，置于光照培养箱中培养6～10d，细胞密度达到1×104～1×105cells/mL。光照培养箱设置条件为：光照为4000lx，光暗比12h:12h；链状亚历山大藻(A.catenella，GY-H25)和微小亚历山大藻(A.minutum,GY-H46)的温度为25.0℃，链状裸甲藻(G.catenatum,GY-H65)和塔玛亚历山大藻(A.tamarense,GY-H31)的温度为22.0℃。
二级培养：按照1:3～1:1(藻液：培养液，V:V)扩培至经高温灭菌的1L的三角瓶中，置于光照培养箱中培养10～12d，至细胞密度达到5×105～5×106cells/mL。光照培养箱设置条件为：光照为4000lx，光暗比12h:12h；链状亚历山大藻(A.catenella，GY-H25)和微小亚历山大藻(A.minutum,GY-H46)的温度为24.0℃，链状裸甲藻(G.catenatum,GY-H65)和塔玛亚历山大藻(A.tamarense,GY-H31)的温度为22.0℃。
三级培养：按照1:5～1:1(藻液：培养液，V:V)扩培到灭过菌的5L食品级PET培养罐中，置于光照培养室每3～5d用泵吸取1/3下层藻液并补充等量培养液进行半连续培养。光照培养箱设置条件为：双侧连续光照6000±1000lx，光暗比12h:12h。温度为空调控温，23.0℃。
实施例2
用扩级培养的方法培养GY-H25、GY-H65、GY-H31和GY-H46四株产毒藻总藻细胞量为2.1×107cells，购买并挑选壳高为7.0±0.5cm，壳长为3.5±0.5cm的贻贝600只进行产毒藻混合暴露，贻贝室内暂养2d，持续通气并每天更换天然海水。每只贝每日投喂总藻量为3.5×104cells，投喂四株产毒藻GY-H25、GY-H65、GY-H31和GY-H46配比分别为：为14％、15％、15％、56％。产毒藻投喂时间分别为8点和20点，控制两次投喂量比例为1:2。暴露时间为5天，水温为9℃。暴露结束后，收集所有贻贝进行解剖并采集所有软组织，匀浆混匀后进行麻痹性贝类毒素提取。每个样品称取3个平行样品，重量为5.0±0.1g。用1％的乙酸水溶液提取，沸水浴5min，冷却后以4500r/min离心10min，移取1mL提取液于，加入5μL氨水，涡旋混匀，10000r/min离心10min。用活化过的Supelco ENVI-Carb固相萃取柱萃取，过0.22μm滤膜于进样小瓶中，于-20℃下冷冻保存供上机分析。使用高效液相色谱-质谱联用检测法进行麻痹性贝毒分析，色谱柱：TSK-Amide-80柱，柱长150mm，内径2.0mm，粒径3.0μm，或性能相当者。流速：0.35mL/min。柱温：40℃。进样量：5μL。流动相为A：水(含2mmol/L甲酸铵，50mmol/L甲酸)，B：95％乙腈水溶液(含2mmol/L甲酸铵，50mmol/L甲酸)。质谱参考条件如下：
检测方式：多反应监测(MRM)，离子源温度：550℃，喷雾电压IS：5000V，-4500V，气帘气压力CUR：20psi，雾化气压力GS1：50psi，辅助加热气压力GS2：50psi，碰撞气CAD：Medium，检测如图5所示。贝中PSTs含量用μg/kg表示，结果如图3所示，检测后获得麻痹性贝类毒素基体标准物质原料中各毒素含量为：NEO 22.9μg/kg、dcSTX 35.4μg/kg、dcNEO 22.5μg/kg、GTX1 253μg/kg、GTX4 72.8μg/kg、GTX2 159μg/kg、GTX3 62.5μg/kg、GTX5 107μg/kg、GTX6168μg/kg、dcGTX2 75.0μg/kg、dcGTX3 30.4μg/kg、C1 278μg/kg和C2 207μg/kg，最终获得总毒性为552μg STXeq/kg的麻痹性贝类毒素基体标准物质原料。
实施例3
用扩级培养的方法培养GY-H25、GY-H65、GY-H31和GY-H46四株产毒藻总藻细胞量为1.6×108cells，购买并挑选壳高为7.0±0.5cm，壳长为3.5±0.5cm的贻贝600只进行产毒藻混合暴露，贻贝室内暂养2d，持续通气并每天更换天然海水。每只贝每日投喂总藻量为2.6×105cells，喂四株产毒藻GY-H25、GY-H65、GY-H31和GY-H46配比分别为：为8％、12％、18％、62％。产毒藻投喂时间分别为8点和20点，控制两次投喂量比例为1:2。暴露时间为5天，水温为9℃。投暴露结束后，收集所有贻贝进行解剖并采集所有软组织，匀浆混匀后用实施例2的提取方法和检测方法进行麻痹性贝类毒素检测。如图4所示，检测后获得麻痹性贝类毒素基体标准物质原料中各毒素含量为：NEO 73.8μg/kg、dcSTX 133μg/kg、dcNEO 79.9μg/kg、GTX1 1494μg/kg、GTX4 394μg/kg、GTX2 750μg/kg、GTX3 228μg/kg、GTX5 278μg/kg、GTX6 564μg/kg、dcGTX2 280μg/kg、dcGTX3108μg/kg、C1 930μg/kg和C2 509μg/kg，最终获得总毒性为2692μg STXeq/kg的麻痹性贝类毒素基体标准物质原料。
实施例4
取实施例2和3中的贻贝，和秦皇岛自然赤潮污染的贻贝。仔细取出全部贝肉，将贝类软组织样品进行匀浆处理，匀浆混匀后用实施例2的提取方法和检测方法进行麻痹性贝类毒素检测。经检测如表1所示，实施例2和3的贻贝中麻痹性贝类毒素的毒素组分包括NEO、dcSTX、dcNEO、dcGTX2、dcGTX3、GTX1、GTX2、GTX3、GTX4、GTX5、GTX6、C1、C2共13种毒素。秦皇岛自然赤潮污染的贻贝中麻痹性贝类毒素的毒素组分包括NEO、dcSTX、dcNEO、GTX1、GTX2、GTX3、GTX4共7种毒素。
表1混合暴露的毒素种类与野外暴露的毒素种类的比较
备注：+表示阳性，-表示阴性
实施例5
将实施例2和3中的贻贝麻痹性贝类毒素的毒素组分与别的研究机构的用塔玛亚历山大藻(A.tamarense)投喂扇贝和用链状裸甲藻(G.catenatum)藻培养液和牡蛎混合进行比较。如表2所示，实施例2和3中的贻贝麻痹性贝类毒素的毒素组分为13种，塔玛亚历山大藻(A.tamarense)投喂扇贝获得4种麻痹性贝类毒素GTX1、GTX4、C1和C2，用链状裸甲藻(G.catenatum)藻培养液和牡蛎混合获得4种麻痹性贝类毒素GTX1、GTX4、GTX2和GTX3。
表2混合暴露的毒素种类与其他研究机构的毒素种类的比较
备注：+表示阳性，-表示阴性
实施例6
采用4个实验室对实施例2和实施例3的麻痹性贝类毒素基体标物原料样品的毒素含量进行比对。将实施例2和3制备的麻痹性贝类毒素基体标物原料样品发放给4家实验室，分别由各实验室选派具有丰富操作经验的试验员，按照统一的试验程序对样品进行痹性贝类毒素含量的检测试验，结果如表3所示。经Grubb’s法检验确认每个实验室均不存在异常值，用科克伦(Cochran)检验各实验室定值数据精度，结果表明所有的数据均通过检验，各实验室测试精度一致。
表3四家实验室测试样品毒素含量的结果统计与分析(μg STX eq/kg)
Claims (10)
1.13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，所述方法包括以下步骤：
(A)四株我国近海典型麻痹性贝类毒素产毒藻的扩级培养：一级和二级分级培养，三级为半连续培养，所述四株麻痹性贝类毒素产毒藻为链状亚历山大藻(A.catenella，GY-H25)、链状裸甲藻(G.catenatum,GY-H65)、塔玛亚历山大藻(A.tamarense,GY-H31)和微小亚历山大藻(A.minutum,
GY-H46)藻；
(B)四株产毒藻混合暴露：每只贻贝投喂一定的配比，一定的总藻细胞数的四株产毒藻，在6～10℃养殖水温下，暴露周期为4～8天下混合暴露贻贝；
(C)麻痹性贝类毒素基体标物原料的获取：暴露结束后，收集所有贻贝进行解剖并采集所有软组织，匀浆混匀后获得麻痹性贝类毒素基体标物原料。
2.根据权利要求1所述的13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，所述步骤(B)中的四株产毒藻的配比为：GY-H25为7％～15％，GY-H65为10％～20％，GY-H31为8％～20％，GY-H46为45％～65％。
3.根据权利要求1所述的13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，所述步骤(B)中暴露周期为5天。
4.根据权利要求1所述的13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，所述步骤(B)中每只贝每日投喂总藻量为3.5×104～2.6×105cells。
5.根据权利要求1所述的13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，所述步骤(B)中混合暴露贝类时水温为9℃，水温会影响产毒藻的毒素在贻贝的体内的蓄积率。
6.根据权利要求1所述的13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，每只贝每日投喂总藻量为3.5×104cells，投喂四株产毒藻GY-H25、GY-H65、GY-H31和GY-H46配比分别为：为14％、15％、15％、56％，暴露时间为5天，水温为9℃。
7.根据权利要求1所述的13种麻痹性贝类毒素基体标准物质原料的制备方法，其特征在于，每只贝每日投喂总藻量为2.6×105cells，喂四株产毒藻GY-H25、GY-H65、GY-H31和GY-H46配比分别为：为8％、12％、18％、62％，暴露时间为5天，水温为9℃。
8.权利要求1所述的制备方法制备的13种麻痹性贝类毒素基体标准物质原料，其特征在于，所述13种麻痹性贝类毒素组分包括NEO、dcSTX、dcNEO、GTX1、GTX4、GTX2、GTX3、GTX5、GTX6、dcGTX2、dcGTX3、C1和C2。
9.根据权利要求1-8任一项所述的13种麻痹性贝类毒素基体标准物质原料的应用，其特征在于，用于制备稳定可靠的13种麻痹性贝类毒素标准物质的应用。
10.根据权利要求1-8任一项所述的13种麻痹性贝类毒素基体标准物质原料制备13种麻痹性贝类毒素标准物质在同时监控中国多个海域麻痹性贝类毒素上的应用。
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