CN112640773B - 一种鼠尾藻新生枝的消毒处理方法 - Google Patents

一种鼠尾藻新生枝的消毒处理方法 Download PDF

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CN112640773B
CN112640773B CN202011516289.4A CN202011516289A CN112640773B CN 112640773 B CN112640773 B CN 112640773B CN 202011516289 A CN202011516289 A CN 202011516289A CN 112640773 B CN112640773 B CN 112640773B
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张朋艳
刘福利
梁洲瑞
袁艳敏
汪文俊
孙修涛
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

本发明涉及一种鼠尾藻新生枝的消毒处理方法，属于海水藻类养殖领域，所述方法为取鼠尾藻新生枝条，刷洗干净，将藻体置于1％聚维酮碘浸泡15s，随后用灭菌海水清洗并浸泡；将藻体置于无菌纯水中浸泡1h，随后用灭菌海水清洗并浸泡；将藻体置于含有抗生素的灭菌海水中充气培养3天，培养条件为温度15℃，光强15μmol photons/(m2·s)，光周期10L:14D，灭菌海水冲洗后获得消毒鼠尾藻新生枝条。本发明方法能够够杀灭鼠尾藻新生枝内部和外部微生物，且不影响新生枝的正常生长。

Description

一种鼠尾藻新生枝的消毒处理方法
技术领域
本发明属于海水藻类养殖领域，具体地涉及一种鼠尾藻新生枝的消毒处理方法。
背景技术
鼠尾藻(Sargassum thunbergii)隶属于褐藻门(Phaephyta)、马尾藻科(Sargassaceae)、马尾藻属(Sargassum)。作为潮间带常见的马尾藻类，鼠尾藻与其他大型褐藻构成海藻床，对维护近海生态系统平衡和环境修复具有重要意义。除生态价值外，鼠尾藻还具有重要的经济价值，被广泛应用在饲料、医药、工业、环保等领域。鼠尾藻营养价值高，可作为海参、鲍鱼等的优质饵料，还因含有凝集素、多糖等活性物质应用于海洋医药领域。此外，鼠尾藻是提取多糖、甘露醇、碘等的重要工业原料，还可作为降低海区富营养化、净化重金属污染海区的优质藻种。
作为重要的海洋生物资源，对鼠尾藻的开发与高值化利用研究越来越受到业内关注。但随着对鼠尾藻需求的不断增加，对鼠尾藻过度的采集和开发致使鼠尾藻资源遭受严重破坏。目前，亟需开展对鼠尾藻苗种繁育和分子生物学的深入研究。与其它大型褐藻相比，鼠尾藻在生长过程中更易附生各种海洋细菌、贻贝，杂藻等，且藻体内部也存在寄生细菌，因此对鼠尾藻的消毒处理十分困难，影响了鼠尾藻研究的快速发展。鼠尾藻不同藻体位置对消毒剂的种类、浓度、处理时间等的承受能力不同，虽有研究对鼠尾藻开展无菌处理实验，但各种消毒剂对鼠尾藻生长的影响未知。目前，对鼠尾藻新生枝的无菌处理技术尚不成熟，因此需要结合各种消毒方式的消毒效果和对鼠尾藻生长的影响开发一种适合鼠尾藻新生枝的消毒处理方法。
发明内容
本发明要解决的技术问题在于提供一种鼠尾藻新生枝的消毒处理方法，所述方法能够杀灭鼠尾藻新生枝内部和外部微生物，且不影响新生枝的正常生长。
本发明是通过如下技术方案来实现的：
一种鼠尾藻新生枝的消毒处理方法，所述方法具体步骤如下：
取鼠尾藻新生枝条，在灭菌海水中刷洗干净，去除表面附着的杂藻和海洋动物；将藻体置于1％聚维酮碘浸泡15s，对藻体进行表面消毒，随后用灭菌海水清洗并浸泡；将藻体置于无菌纯水中浸泡1h，去除对渗透压敏感的附着生物，随后用灭菌海水清洗并浸泡；将藻体置于含有抗生素的灭菌海水中充气培养3天，培养条件为温度15℃，光强15μmolphotons/(m2·s)，光周期10L:14D，灭菌海水冲洗后获得消毒鼠尾藻新生枝条；
进一步，含有抗生素的灭菌海水中的成分及其终浓度：100mg/L氨苄青霉素钠、50mg/L卡那霉素、50mg/L链霉素、30mg/L新霉素和5mg/L制霉菌素。
本发明与现有技术相比的有益效果：
本发明对适合于鼠尾藻新生枝条的消毒剂种类、消毒时间、抗生素组成进行研究，探讨了各种消毒方法的消毒效果及对藻体叶绿素荧光参数的影响，从而获得了一种鼠尾藻新生枝组合消毒方法。本发明方法能够够杀灭鼠尾藻新生枝内部和外部微生物，且不影响新生枝的正常生长，为鼠尾藻无菌处理提供技术支持，为鼠尾藻苗种繁育和分子生物学研究提供前提条件。
附图说明
图1为不同消毒剂对鼠尾藻新生枝光系统II最大光能转换效率(Fv/Fm)的影响和消毒效果。
图2为不同抗生素组合对鼠尾藻新生枝光系统II最大光能转换效率(Fv/Fm)的影响。
图3为鼠尾藻新生枝消毒前后光系统II最大光能转换效率(Fv/Fm)的变化和消毒效果。
具体实施方式
下面通过实施例来对本发明的技术方案作进一步解释，但本发明专利的保护范围不受实施例任何形式上的限制。
实施例1
消毒剂筛选实验：鼠尾藻采自福建(27°N，121°E)，前期培养条件为：温度15℃，光强15μmol photons/(m2·s)，光周期10L：14D，培养液为经过滤、高压灭菌冷却后的天然海水。取状态良好的鼠尾藻新生枝藻段，充分刷洗干净后，分别置于纯水、75％乙醇、1％聚维酮碘、0.01％HgCl、1％次氯酸钠溶液中浸泡消毒不同时间，最后用灭菌海水冲洗干净。将鼠尾藻藻段在黑暗条件下暗适应20min后，利用调制叶绿素荧光成像系统(IMAGING-PAM，WALZ，德国)测定藻体光系统II最大光能转换效率(Fv/Fm)。配置Zobell 2216E固体培养基，将鼠尾藻藻段表面海水涂布于一侧固体培养基上，后将藻体碾碎，涂布于另一侧培养基上，将培养平板置于20℃培养箱中培养3天，观察细菌生长情况。
不同消毒剂对鼠尾藻新生枝生长的影响和消毒效果如图1所示。利用纯水处理鼠尾藻新生枝，能够一定程度上减少鼠尾藻表面细菌污染，在处理30min或1h，及恢复培养1h后，Fv/Fm值变化不显著，表示纯水对鼠尾藻生长无显著影响；利用75％乙醇处理鼠尾藻新生枝1s或者15s，藻体出现不同程度变绿现象，消毒15s对藻体产生较大影响，Fv/Fm值显著降低，75％乙醇能够小程度上杀灭鼠尾藻表面细菌；1％聚维酮碘能够减轻藻体表面细菌污染，但长时间处理(3min)对藻体影响较大，Fv/Fm值显著降低，消毒15s对藻体Fv/Fm值影响不显著；0.01％HgCl2消毒效果最好，能够最大程度上去除藻体表面和内部细菌，对表面消毒效果优于内部消毒效果，但对藻体生长也有一定影响，处理2min后Fv/Fm出现降低现象，处理4min后Fv/Fm显著降低；1％次氯酸钠也能够一定程度上减少藻体表面细菌污染，但无论处理1s还是5s都会导致Fv/Fm值显著降低，对藻体生长影响较大。
实施例2
抗生素筛选实验：取实验室预培养的鼠尾藻新生枝，用灭菌海水充分洗刷干净后，分别置于含有抗生素的灭菌海水中培养3天，培养条件为：温度15℃，光强15μmol photons/(m2·s)，光周期10L：14D，结束后用灭菌海水冲洗干净，并按照实施例1所述方法涂布平板，计算菌落数。所述抗生素组合和菌落生长情况如表1所示。
表1抗生素组合方式及菌落生长情况
Figure BDA0002847494330000051
Figure BDA0002847494330000061
注：+表示添加有对应浓度的抗生素。
由不同抗生素处理下藻体残留菌落数可知，未经过抗生素处理的藻段内外部微生物污染严重；只添加制霉菌素对微生物无显著抑制作用，但不添加制霉菌素可能出现霉菌菌落；除制霉菌素外，额外添加氨苄青霉素钠/卡那霉素/链霉素/新霉素能够抑制部分鼠尾藻新生枝的微生物污染，但效果不显著；与其它抗生素相比，氯霉素的抑菌效果最明显，只添加制霉菌素和氯霉素即可抑制微生物生长；整体来说，利用组合抗生素效果较好。根据以上表格，选择抗生素组合1、6、10、13、19、20、23、29、30培养鼠尾藻新生枝3天，每天根据实施例1所述方法检测藻体光系统II最大光能转换效率(Fv/Fm)，以此判断抗生素对藻体生长的影响。
不同抗生素组合条件下藻体Fv/Fm值变化情况如图2所示。表明虽然氯霉素抑菌效果较好，但经含有氯霉素的抗生素组合处理后，藻体Fv/Fm值均显著降低，说明氯霉素对藻体生长具有不利影响；20和29号抗生素组合处理对藻体生长的抑制作用最小，其中29号抗生素组合为最佳组合，其处理后的鼠尾藻的Fv/Fm值的在三天内均无显著变化。
实施例3
鼠尾藻新生枝消毒方法筛选：根据实施例1和实施例2的结果，进一步筛选最佳消毒方式。依次选择纯水、1％聚维酮碘和抗生素组合29号三个消毒步骤，并对是否添加0.01％HgCl2进行进一步探索。方案如下：表面刷洗-无菌海水冲洗-1％聚维酮碘浸泡15s-无菌海水冲洗-超纯水浸泡60min-无菌海水恢复-有/无HgCl2浸泡消毒5s-含抗生素灭菌海水中培养1-3d。按照实施例1所述方法检测藻体Fv/Fm值，并在处理第3天时涂布Zobell2216E固体培养基，观察菌落生长情况。
两种消毒处理条件下藻体Fv/Fm值变化情况及菌落生长情况如图3所示。消毒后鼠尾藻新生枝表面和内部微生物污染显著降低，经过纯水、1％聚维酮碘和抗生素处理，能够杀灭>99％的微生物，且对藻体生长无显著抑制作用；额外添加0.01％HgCl2能够杀灭所有微生物污染，但也能显著抑制鼠尾藻生长。

Claims (1)

1.一种鼠尾藻新生枝的消毒处理方法，其特征在于所述方法具体步骤如下：
取鼠尾藻新生枝条，在灭菌海水中刷洗干净，去除表面附着的杂藻和海洋动物；将藻体置于1%聚维酮碘浸泡15s，对藻体进行表面消毒，随后用灭菌海水清洗并浸泡；将藻体置于无菌纯水中浸泡1 h，去除对渗透压敏感的附着生物，随后用灭菌海水清洗并浸泡；将藻体置于含有抗生素的灭菌海水中充气培养3天，培养条件为温度15oC，光强15 μmol photons/(m2·s)，光周期10L:14D，灭菌海水冲洗后获得消毒鼠尾藻新生枝条；
所述的含有抗生素的灭菌海水中的成分及其终浓度：100 mg/L氨苄青霉素钠、50mg/L卡那霉素、50 mg/L链霉素、30 mg/L新霉素和5 mg/L制霉菌素。
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