CN105543298B - 一种提高金藻dha含量的培养方法 - Google Patents
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Abstract
本发明公开了一种提高金藻DHA含量的培养方法。在金藻光自养培养过程中加入终浓度为0.5~8.0%(v/v)的甘油,提高藻细胞DHA占总脂肪酸的比例,藻细胞DHA量较未添加甘油组均有所提高,最高可提高45%以上。本方法简单易行,并且在添加甘油的培养体系的藻细胞内多不饱和脂肪酸的量也有很大提升。
Description
技术领域
本发明属于海洋生物技术领域,具体涉及金藻及金藻细胞DHA占总脂肪酸的比例。
背景技术
二十二碳六烯酸(Docosahexaenoic acid,简称DHA)是一种重要的ω—3型高碳多不饱和脂肪酸,也是动物和人体不能合成而必须由食物提供的一种必需脂肪酸。是维持大脑、视网膜等正常生长和功能发育的必需脂肪酸,还具有降血脂、抗血栓和抗动脉粥样硬化等作用。
微藻中含有大量的DHA,且其它长链不饱和脂肪酸的含量较低,利于DHA的分离纯化。由于微藻的培养受季节和地域的影响较小,借助于生物反应器可实现常年工亚化大规模培养,而且微藻中的脂肪组成不像鱼油那么复杂,使得微藻DHA的分离纯化工艺相对简单,所得DHA产品无鱼腥味和其它异味,只有独特的海藻味,并无不良化学成分。利用海洋微藻培养生产的DHA可做到不含EPA和污染物,所以质量好,可以广泛使用于各类食品中。尤其是可以用于鱼油来源DHA所不能用的领域(例如孕妇、哺乳期妇女和婴幼儿保健品和食品)。目前,微藻来源的DHA产品在发达国家供不应求,价格也高于鱼油来源DHA。
以湛江等鞭金藻为例,湛江等鞭金藻是一种分布广泛的海洋浮游单细胞藻类个体小,繁殖快,具无纤维素的细胞壁,营养丰富,尤其在藻体中富含多不饱和脂肪酸(poly-unsaturated fatty acid,PUFAs),是海洋中鱼、虾和贝类的等幼体生长发育过程中必需的营养成分。此外,近年来的研究证明,n-3PUFAs还具有防治人类心血管疾病,促进婴幼儿大脑发育和改善视力的功效。所以它作为一种优质的PUFAs海洋生物资源,有很好的应用前景。因此,寻找一种提高藻细胞内DHA占总脂肪酸比例的培养方式是十分必要的。
发明内容
本发明的目的在于在限氮培养体系中添加辅助碳源甘油,提高金藻DHA占总脂肪酸的比例的培养方法。
为了实现上述目的,本发明采用的培养方法包括以下步骤:
1)藻种培养于锥形瓶中,培养至指数生长期使用;
2)将指数生长期的藻细胞接种于灭菌的F/2培养基中,添加甘油培养至平台期收获藻细胞;
3)脂肪酸成分分析:将收获的藻细胞烘干得藻粉,采用酸催化转酯化法,气质联用方法对金藻脂肪酸成分进行测定(参考文献:[1]冯迪娜,艾江宁,刘亚男,等.含氮类培养基对海洋微藻Isochrysis zhanjiangensis油脂与碳水化合物积累的影响[J].中国生物工程杂志,2011,10:29-34.)。
培养参数:
1)氮限制培养是指接种时使用3x F/2培养基,即磷源、金属元素、微量元素各添加常规F/2的3倍;
2)添加甘油的时间为接种时添加或指数生长期添加;
3)添加0.5~8.0%(v/v)的甘油,根据初始接入细胞浓度,按照8~16pg/cell(以硝酸钠计)的比例调整初始氮源浓度;
4)培养温度23~27℃,光强110~150μmol.m-2s-1,光暗比为14h:10h,通入含4.0%CO2的空气,通气速率0.1~0.3vvm。
本发明的优点如下:
1、DHA含量明显高:本发明所得到的藻细胞DHA含量有显著提高,DHA含量最高位16.0%,比未添加甘油培养体系的藻细胞提高45%以上。
2、应用前景好:本研究是在光自养培养金藻的过程中添加甘油。甘油是微藻生物柴油生产的副产物,同时也是一种低值的化工原料,伴随生物柴油加工规模的增长,如何提升甘油的附加值正逐渐受到人们的关注和重视。本研究利用海水培养微藻,缓解淡水资源的危机。
附图表说明:
图1.金藻生长曲线图
A.接种时添加甘油培养体系金藻生长曲线;
B.指数生长期添加甘油培养体系金藻生长曲线;
具体实施方式
实施例1:培养过程生长阶段判定
每天用Jasco V-530UV/VIS Spectrophotometer(JASCO,Tokyo,Japan)监测金藻细胞数:利用OD法,在680nm处测定培养系统中的金藻吸光度,利用公式“细胞密度(×104cells/mL)=(OD680×1250-90.125)×稀释倍数”计算出对应的细胞密度。绘制生长曲线,以判定细胞生长至稳定期。不同培养条件下,金藻的生长曲线见图1。
实施例2营养盐及培养基配制
无硅酸盐的F/2营养盐母液配制:
(1)氮源:称取75g NaNO3溶于1L去离子水;
(2)磷源:称取5g NaH2PO4·H2O溶于1L去离子水;
(3)金属元素:分别称取3.15g FeCl3·6H2O,4.36g Na2EDTA,0.0098g CuSO4·5H2O,0.0063g Na2MoO4·2H2O,0.022g ZnSO4·7H2O,0.01g CoCl2·6H2O,0.18g MnCl2·4H2O溶于1L去离子水;
(4)维生素:0.001g vitamin B12,0.2g vitamin B1,0.001g D-生物素,溶于1L去离子水;
F/2培养基的制备:
天然海水经臭氧杀菌,两层0.45μm醋酸纤维滤膜过滤,110℃灭菌15min,冷却后备用;向海水中添加上述(1),(2),(3),(4)制得的无硅酸盐的F/2营养盐,其中氮源、磷源、金属元素各1mL/L,维生素0.5mL/L。
实验所用甘油:分析纯甘油与经0.45μm醋酸纤维滤膜过滤的海水以体积比1:1混匀,121℃灭菌20min后冷却,常温保存。
实施例3:金藻培养
采用实施例2中的F/2培养基,以等鞭金藻(I.zhangjiangensis)OD680=0.1~0.2、藻液体积约1.5~2.0L接种于3L三角瓶,用日光灯做光源,光强为30~40μmol.m-2s-1,培养3~5天至指数生长期;离心(3000~4000rpm,3~5min),收获藻细胞,弃上清液,沉淀中添加约3~5mL灭菌的海水,使之重新悬浮,加至F/2培养基中,以氮元素浓度0.8~1.6×10-8mg/cell(以硝酸钠计)接种,其他营养成分(除氮源)添加浓度为3倍实施例1所述F/2培养基,即磷源、金属元素、微量元素各添加常规F/2营养盐的3倍,调至OD680=0.28~0.32,500mL藻液接种于600mL管式鼓泡反应器,培养温度23~27℃,光强110~150μmol.m-2s-1,光暗比为14h:10h,光照阶段通入含4.0%CO2的空气,通气速率0.16~0.24vvm,培养至7天(平台期),离心(4000rpm,5min),弃上清液,沉淀用0.5mol/L的碳酸氢铵洗两遍,60℃烘至恒重,冻干备用,采用气质联用方法对金藻脂肪酸成分进行测定。
添加甘油:
添加甘油的浓度:500mL培养体系,分别加入3mL、8mL、15mL、50mL、100mL实施例2中所述的甘油,对应培养体系甘油的最终浓度分别为0.3%(v/v)、0.8%(v/v),1.5%(v/v)、5.0%(v/v)、10.0%(v/v)。
添加甘油的时间:接种时(即反应器培养的第0天)添加甘油,指数生长期(反应器培养的第3天)添加甘油。
接种时添加甘油:添加甘油最终浓度分别为0.3%(v/v)、0.8%(v/v),1.5%(v/v)、5.0%(v/v)、10.0%(v/v)。对应于表1中的ZL1、ZL2、ZL3、ZL4。
指数生长期添加甘油:添加甘油最终浓度分别为0.3%(v/v)、0.8%(v/v),1.5%(v/v)、5.0%(v/v)、10.0%(v/v)。对应于表1中的TL1、TL2、TL3、TL4。
对照组(Control):未添加甘油的培养体系。
接种时添加甘油:
添加甘油培养体系(ZL1-5)的藻细胞的DHA含量均有所提高,(ZL1-5)培养体系藻细胞的DHA含量分别为11.7%、15.1%、16.0%、15.8%、13.9%比对照组(Control)藻细胞的DHA含量对应提高9.4%、41.4%、49.1%、47.4%、29.7%,(表1.金藻藻细胞脂肪酸成分分析(%总脂肪酸)。当培养体系添加10.0%(v/v)甘油时,藻细胞DHA含量虽比对照组藻细胞DHA含量提高29.7%,但由生长曲线(图1.A)可以看出,此培养体系的藻细胞生长受到明显的抑制作用,不利于生物量的累积,进而不利于生产(图1.A)。
表1
指数生长期添加甘油:
添加甘油培养体系(TL1-5)的藻细胞的DHA含量均有所提高,(TL1-5)培养体系藻细胞的DHA含量分别为12.0%、14.6%、12.2%、14.1%、14.9%比对照组(Control)藻细胞的DHA含量对应提高12.5%、36.9%、13.9%、31.4%、39.0%,(表1)。尽管添加10.0%(v/v)甘油的培养体系藻细胞DHA含量最高,但由生长曲线(图1.B)可以看出,此培养体系的藻细胞生长受到抑制作用,不利于生物量的累积,进而不利于生产。
Claims (4)
1.一种提高金藻DHA量的培养方法,其特征在于在金藻光自养培养过程中,通过在培养体系添加终体积含量为0.5-8%(v/v)甘油,提高金藻DHA占总脂肪酸的比例;
培养体系指的是以F/2培养基基础上,以硝酸钠计,调整初始氮元素浓度至8~16 pg/cell,而磷源、金属元素、微量元素各添加常规F/2的2~4倍;
F/2营养盐母液配制:
(1) 氮源:称取75g NaNO3溶于1L去离子水;
(2) 磷源:称取5g NaH2PO4·H2O溶于1L去离子水;
(3) 金属元素:分别称取3.15g FeCl3·6H2O, 4.36g Na2EDTA, 0.0098g CuSO4·5H2O,0.0063g Na2MoO4·2H2O, 0.022g ZnSO4·7H2O, 0.01g CoCl2·6H2O, 0.18g MnCl2·4H2O溶于1L去离子水;
(4) 维生素:0.001g vitamin B12, 0.2g vitamin B1, 0.001g D-生物素,溶于1L去离子水;
培养基的制备:
天然海水经过滤灭菌后备用;向海水中添加上述(1),(2),(3),(4)制得的无硅酸盐的F/2营养盐,其中磷源、金属元素各1ml/L,维生素0.5ml/L;
添加时间点为接种-指数生长期之间,包括接种和指数生长期。
2.根据权利要求1所述的方法,其特征在于:金藻光自养培养过程是指金藻由指数生长期培养至平台期时收获生物质。
3.根据权利要求1所述的方法,其特征在于:
培养温度23~27℃,光强110~150 µmol.m-2s-1,光暗比为14h:10h,通入含4.0%CO2的空气,通气速率0.1~0.3vvm。
4.根据权利要求1所述的方法,其特征在于:
培养体系指是指接种时使用3 x F/2培养基,即氮源、磷源、金属元素、微量元素各添加常规F/2的3倍。
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Non-Patent Citations (3)
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不同营养盐浓度下微绿球藻的生长;王丽卿等;《上海水产大学学报》;20020930;第11卷(第3期);第215-218页 * |
含氮类培养基对海洋微藻Isochrysis zhanjiangensis油脂与碳水化合物积累的影响;冯迪娜等;《中国生物工程杂志》;20111015;第31卷(第10期);第29-32页 * |
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