CN107614676B - 应用颗石类微藻卡氏侧金藻生产聚不饱和脂肪酸的方法 - Google Patents

应用颗石类微藻卡氏侧金藻生产聚不饱和脂肪酸的方法 Download PDF

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CN107614676B
CN107614676B CN201680013506.3A CN201680013506A CN107614676B CN 107614676 B CN107614676 B CN 107614676B CN 201680013506 A CN201680013506 A CN 201680013506A CN 107614676 B CN107614676 B CN 107614676B
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卢凡
陈佳丽
邓中洋
陈攀
胡征宇
宋立荣
丰平仲
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Abstract

本发明公开利用颗石类微藻卡氏侧金藻(Pleurochrysis carterae)生产聚不饱和脂肪酸的方法。本发明提供了利用卡氏侧金藻(Pleurochrysis carterae)(Haptophyta,Prymnesiophyceae)产生二十碳五烯酸(EPA,20:5n‑3)、二十二碳六烯酸(DHA,22:6n‑3)、十八碳四烯酸(SDA,18:4n‑3)、α‑亚麻酸(ALA,18:3n‑3)、和油酸(18:1)的方法。本发明包含下列内容：(a).用于诱导该藻种合成积累聚不饱和脂肪酸的培养条件；(b).一种增强该藻种合成积累油酸的方法。运用本发明描述的方法所生产的聚不饱和脂肪酸可以应用于食品、营养补充剂、化妆品和药品等领域。

Description

应用颗石类微藻卡氏侧金藻生产聚不饱和脂肪酸的方法
背景技术
颗石类微藻是一类广泛存在于温带和热带海洋的单细胞浮游生物。颗石类海洋藻华是海洋藻华的常见类型，其在1978到1986年间的年均面积达到140万平方公里(Brownand Yoder,1994)，而其中的主要藻种为Emiliania huxleyi和Gephyrocapsa oceanica(Hulburt et al.,1960)。由于其分布广泛并具有独特的生理学特征，颗石类微藻对海洋生态系统和全球碳循环具有重要的影响。作为光合生物，颗石类微藻不仅能够通过光合作用利用二氧化碳而合成积累蛋白质、脂类、和其它各类有机化合物，它们还可以利用二氧化碳通过钙化作用而合成碳酸钙晶体，因而实现将二氧化碳长期封存的目的(Brownlee andTaylor,2004；Rost and Riebesell,2004)。除此之外，颗石类微藻还具有其它潜在的应用前景，例如：颗石类微藻细胞能够合成积累各种脂类，这些脂类可以用于生产生物柴油或者聚不饱和脂肪酸(Fernandez et al.,1994；Riebesell et al.,2000；Moheimani andBorowitzka,2006)。另外，Takenaka等研究者显示，颗石类微藻可以作为钙补充剂而用于人类营养补充剂(Takenaka et al.,1996a,1996b)。
对于颗石藻类的研究已有较长的历史，但是多集中于对其生活周期和细胞形态(Jeffrey et al.,1988；Green et al.,1996；Kazuko et al.,2006,2011)、光合作用与钙化作用(Balch et al.,1992；Buitenhuis et al.,1999)、以及其对海洋生态系统的影响(Feely et al.,2004；Fabry,2008；Iglesias-Rodriguez et al.,2008)等方面的研究。但是，很少有涉及所述藻种的脂肪酸成分的研究。本发明公开了诱导卡氏侧金藻(Pleurochrysis carterae)合成积累聚不饱和脂肪酸的培养条件，同时也展示了光照强度、温度和二苯胺浓度对于卡氏侧金藻(Pleurochrysis carterae)合成积累脂肪酸的影响。
发明内容
本发明描述了颗石类微藻卡氏侧金藻(Pleurochrysis carterae)(Haptophyta,Prymnesiophyceae)的细胞以及优化所述藻种的细胞合成积累聚不饱和脂肪酸的方法。
本发明进一步描述了用于优化颗石类微藻卡氏侧金藻(Pleurochrysiscarterae)的细胞合成积累脂肪酸的培养基和培养条件。
本发明-进一步描述了使用二苯胺诱导颗石类微藻卡氏侧金藻(Pleurochrysiscarterae)的细胞合成积累油酸的方法。
具体实施方式
1).用于诱导所述藻种合成积累聚不饱和脂肪酸的培养基成分
用于诱导所述藻种合成积累聚不饱和脂肪酸的培养基包括以下培养基之一：
Figure GDA0002568509960000021
诱导所述藻种合成积累聚不饱和脂肪酸可以在下列培养装置中使用上述培养基而实现：
1a.在含有上述培养基和0.5％-5％的琼脂胶平板中；
1b.在含有50毫升到2升上述培养基的100毫升到5升的烧瓶中；
1c.在含有上述培养基的2升到1000升的立柱式光生物反应器中；
1d.在含有上述培养基的5升或者5升以上的平板式光生物反应器中；
2).诱导所述藻种合成积累聚不饱和脂肪酸的方法
首先将颗石类微藻卡氏侧金藻(Pleurochrysis carterae)培养在以下培养基-3中：
Figure GDA0002568509960000022
将培养条件设置如下：光照强度：10～2500μmol m-2s-1,温度5℃～40℃；二氧化碳浓度为0.1％～20％。在这种培养条件下，所述藻种细胞合成积累下列脂肪酸：
Figure GDA0002568509960000031
在细胞生长进入稳定状态之后，将细胞收获并转移接种到培养基-1或培养基-2，并将细胞设置于光照强度不低于230μmol m-2s-1的生长条件下，所述藻种细胞将合成积累下列脂肪酸：
Figure GDA0002568509960000032
需要特别指出的是在使用培养基-1和培养基-2时，油酸的含量比使用培养基-3时大幅度增加。然而，当光照强度不高于100μmol m-2s-1时，油酸含量的这种大幅增加并没有出现，这说明较高的光照强度是诱导所述藻种卡氏侧金藻(Pleurochrysis carterae)积累油酸的必要条件。
3).使用二苯胺增强诱导所述藻种合成积累聚不饱和脂肪酸的方法
如以上描述所述，当所述藻种卡氏侧金藻(Pleurochrysis carterae)培养在缺氮或者缺磷培养基(培养基-1或培养基-2)、并且在光照强度不低于230μmol m-2s-1时，细胞中的油酸含量显著增加。当培养基中添加了二苯胺(DPA)后，细胞中油酸的含量就会进一步增加(如下表所示)。二苯胺(DPA)的添加量越大，细胞中油酸的含量越高。与此相对应，随着二苯胺(DPA)添加量从0μM增加到10μM，细胞中SDA和DHA的含量显著减少。
Figure GDA0002568509960000041
参考文献
1.Balch,W.M.,Holligan,P.M.,Kilpatrick,K.A.,1992.Calcification,photosynthesis and growth of the bloom-forming coccolithophore,Emilianiahuxleyi.Cont.Shelf Res.12,1353-1374.
2.Brownlee,C.,Taylor,A.,2004.Calcification in coccolithophores:Acellular perspective,in:Thierstein,H.R.(Eds.),Coccolithophores:From molecularprocesses to global impact.Springer,Berlin,pp.31-49.
3.Buitenhuis,E.T.,de Baar,H.J.W.,Veldhuis,M.J.W.,1999.Photosynthesisand calcification by Emiliana huxleyi(Prymnesiophyceae)as a function ofinorganic carbon species.J.Phycol.35,949-959.
4.Fabry,V.J.,2008.Marine calcifiers in a high-CO2ocean.Science 320,1020-1022.
5.Feely,R.A.,Sabine,C.L.,Lee,K.,Berelson,W.,Kleypas,J.,Fabry,V.J.,Millero,F.J.,2004.Impact of anthropogenic CO2on the CaCO3system in theoceans.Science 305,362-366.
6.Fernandez,E.,Balch,W.M.,Maranon,E.,Holligan,P.M.,1994.High rates oflipid biosynthesis in cultured,mesocosm and coastal populations of thecoccolithophorids Emiliania huxleyi.Mar.Ecol.Prog.Ser.114,13-22.
7.Guillard,R.R.L.,Ryther,J.H.,1962.Studies of marine planktonicdiatomsⅠ.Cyclotella nana Hustedt,and Detonula Confervacea(Cleve)4Gran.Can.J.Microbio.8,229-239.
8.Iglesias-Rodriguez,M.D.,Halloran,P.R.,Rickaby,R.E.M.,Hall,I.R.,Colmenero-Hidalgo,E.,Gittins,J.R.,Green,D.R.H.,Tyrrell,T.,Gibbs,S.J.,Dassow,P.von,Rehm,E.,Armbrust,E.V.,Boessenkool,K.P.,2008.Phytoplankton calcificationin a high-CO2 world.Science 320,336-340.
9.Moheimani,N.R.,Borowitzka,M.A.,2006.The long-term culture of thecoccolithophore Pleurochrysis carterae(Haptophyta)in outdoor racewayponds.J.Appl.Phycol.18,703-712.
10.Moheimani,N.R.,Borowitzka,M.A.,2007.Limits to productivity of thealge Pleurochrysis carterae(Haptophyta)grown in outdoor racewayponds.Biotechnol.Bioeng.96,27-36.
11.Takenaka,H.,Yamaguchi,Y.,Teramato,S.,Tanaka,N.,Hori,M.,Seki,H.,Hiwatari,T.,1996a.Evaluation of the mutagenic properties of thecoccolithophorid Pleurochrysis carterae(Haptophyceae)as a potential humanfood supplement.J.Appl.Phycol.8,1-3.
12.Takenaka,H.,Yamaguchi,Y.,Teramato,S.,Tanaka,N.,Hori,M.,Seki,H.,Nishimori,T.,Morinaga,T.,1996b.Safety evaluation of Pleurochrysis carterae asa a potential food supplement.J.Mar.Biotechnol.3,274-277.

Claims (1)

1.一种应用颗石类微藻卡氏侧金藻生产聚不饱和脂肪酸的方法，其特征在于：该方法包括诱导所述藻种合成积累聚不饱和脂肪酸的培养条件和一种增强所述藻种合成积累油酸的方法；
所述诱导所述藻种合成积累聚不饱和脂肪酸的培养基为：0克/升硝酸钠；0.05～1.75克/升硫酸镁；0.5～3.6克/升碳酸钠；0.05～0.5克/升氯化钙；30～35克/升氯化钠，0.001～0.005克/升EDTA；0.02～1.2克/升磷酸氢二钾；0.006～0.015克/升柠檬酸；0.006～0.015克/升柠檬酸铁铵；0.2～1毫升/升A5微量元素；
所述增强所述藻种合成积累油酸的方法为：使用含有0.1～100mM二苯胺的培养基，增强所述藻种合成积累油酸所需的光照强度不低于230μmol m-2s-1和温度范围5℃～40℃。
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