CN111349566B - 一株利用海水培养的魏氏藻及其用途 - Google Patents
一株利用海水培养的魏氏藻及其用途 Download PDFInfo
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- CN111349566B CN111349566B CN202010223083.6A CN202010223083A CN111349566B CN 111349566 B CN111349566 B CN 111349566B CN 202010223083 A CN202010223083 A CN 202010223083A CN 111349566 B CN111349566 B CN 111349566B
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Abstract
本发明公开了一株海水培养的魏氏藻，即魏氏藻(Vischeria sp.)LQ‑1984株系。所述的魏氏藻(Vischeria sp.)LQ‑1984株系，编号为LQ‑1984，拉丁名称为Vischeria sp.，保藏号为CCTCC M 20191120，命名为Vischeria sp.LQ‑1984。其成熟的细胞表面光滑，细胞壁无突起，直径为10‑15μm，呈球形或近球形，叶绿素为周生叶状，内含一蛋白核。所述魏氏藻(Vischeria sp.)LQ‑1984株系相比其他ω‑7脂肪酸的生产原料，其来源不受环境、地域、季节性等限制，可以满足巨大的原料缺口。所述魏氏藻(Vischeria sp.)LQ‑1984株系含有高含量ω‑7脂肪酸，其高品质藻粉可用于生产ω‑7相关的保健品、食品、化妆品等。
Description
技术领域
本发明涉及微藻，尤其涉及一株利用海水培养的魏氏藻。同时本发明还涉及该魏氏藻的应用。
背景技术
目前报道的用于生产ω-7脂肪酸的微藻多为淡水微藻，随着全球淡水资源日益短缺的问题，淡水微藻培养受到很大程度的限制，此外，淡水微藻的培养存在诸多问题，如杂藻污染、原生动物捕食、微囊藻毒素残留和重金属富集等。
ω-7脂肪酸又名棕榈油酸，为一组单不饱和脂肪酸，是人体内一种十分重要的非必需脂肪酸，由于其独特的生理功能，近年来受到许多学者的关注。研究表明：ω-7脂肪酸主要通过影响细胞的信号通路和炎症标记物，降低C-反应蛋白(CRP)的水平，借以促进人体肝脏和肌肉细胞的新陈代谢，提高机体的抗炎症反应；通过抑制人体细胞中的黑色素因子，防止色素沉着，改善皮肤；通过增加细胞膜的流动性，降低血液中的低密度脂蛋白胆固醇，提高人体对胰岛素的敏感性，从而起到预防动脉粥样硬化和糖尿病等老年疾病的作用。
目前，国外已有不少ω-7脂肪酸相关保健食品上市，但是，天然的ω-7脂肪酸在自然界中很难被找到。市场上的ω-7主要的商业来源有鱼类(尤其是深海鱼类)、植物沙棘和坚果类等。现阶段，应用这些原料提取ω-7脂肪酸存在很大问题：1)含量低，制备成本高。研究表明，深海鱼油中ω-7的含量仅为7％左右，间接增加了ω-7产品相关产品的制作成本。2)资源有限。深海鱼类和坚果类等在我国分布和资源都十分有限，特别是深海鱼类，由于过度捕捞极易造成环境问题，获取难度大。沙棘和坚果栽培则需要大量的土地和人力物力，且存在季节性，远远不能满足市场的需要。3)产品性质不稳定，含饱和脂肪酸高，不利于人体健康。研究发现，鱼类中提取的ω-7脂肪酸不仅含量非常之低，其味道和气味难以接受，还必须经过严格的化学精炼才可以使用。
微藻是一类体型微小、能进行光合作用的低等植物，具有可利用光能、生长繁殖快、对环境适应性强和易培养等特点，在自然界中广泛存在。微藻营养物质丰富，包括蛋白质、多糖、脂肪酸等，一些特殊的微藻可以积累ω-7脂肪酸，如小球藻(Chlorella)、真眼点藻(Eustigmatos)等。现阶段，我国微藻种质资源丰富、分布广泛，微藻培养和产业化养殖技术位列世界前茅。相比鱼类(尤其是深海鱼类)、植物沙棘和坚果类等，微藻资源不受环境、地域、季节等限制，是一种可持续的原料资源，应用微藻作为ω-7脂肪酸原料来源，可以解决目前市场上巨大的原料缺口。
目前报道的产ω-7脂肪酸微藻绝大部分为淡水微藻，虽然淡水微藻也可以进行大规模培养，但由于中国淡水和耕地资源紧缺，以大量耕地和淡水资源换取ω-7脂肪酸资源，确实是一种不经济的发展模式。众所周知，海水占全球水资源量高达96.5％且富含各种营养盐，是微藻规模化培养的潜在天然水资源。我国海岸线长，海水资源丰富，利用海水培养微藻，不仅能节省淡水资源，同时由于海水中富含各种营养盐，配制海水培养基只需在天然海水基础上添加碳、氮、磷、铁等元素即可满足微藻的生长需求，可大幅度减少养殖肥料的用量，从而降低培养成本。
目前已公开的相关专利中，未发现涉及海水魏氏藻藻种的专利，现举例对已公开的ω-7(棕榈油酸)相关技术发明进行描述：
1)一种从沙棘果油中提纯棕榈油酸的方法(CN201910593544.6)：该发明公开了一种从沙棘果油中提纯棕榈油酸的方法，采用超临界萃取技术，提高沙棘果油中棕榈油酸的含量。另采用油脂精炼技术，进一步提高棕榈油酸含量并减低饱和脂肪酸含量。但该发明中通过两步法提纯沙棘果油中的棕榈油酸，虽然颇有成效，但是收集果油和提纯精炼工艺复杂，操作成本高，还要用到工业化学品和高温处理存在污染环境的问题。
2)一种利用坚果油制备棕榈油酸的方法及棕榈油酸的应用(CN201910536726.X)：该发明提供一种利用澳洲坚果油制备棕榈油酸的方法，通过本发明方法制得的棕榈油酸，其棕榈烯酸是原油的棕榈烯酸含量的1.5-5倍(16.85％)，制备效果欠佳，不利于产业开发利用，此外，从坚果油中提取的油酸同样存在提取和提纯精炼等复杂工艺的问题。
3)一种利用真眼点藻纲微藻生产棕榈油酸的方法(CN201810970857.4)：该发明公开一种利用真眼点藻纲微藻生产棕榈油酸的方法，提供一种浓缩微藻棕榈油酸的制备方法，包括皂化、低温冷冻沉淀、酸解和尿素包埋等处理。该方法中的藻株是淡水微藻，实际应用中，淡水微藻的培养存在诸多问题，如淡水资源短缺、容易污染杂藻、原生动物捕食、微囊藻毒素残留和重金属富集等。
4)一种鱼油中提取棕榈油酸的制备方法(CN201510827250.7)：提供一种鱼油中提取棕榈油酸的制备方法，通过脱酸、脱色、脱臭、乙酯化、多级分子蒸馏处理、尿素包合再蒸馏等步骤，使制备的棕榈油酸为黄色或淡黄色透明液体，纯度高、产量品质高。该方法由于鱼油来源十分有限，限制了其产业化发展，此外，鱼油味道和气味都较难接受，还必须经过严格的化学精炼才可以使用，间接增加生产成本。
在微藻棕榈油酸(ω-7)含量方面，已公开的专利数量较少，多数为利用微藻以外的其他原料来制备棕榈油酸的方法。目前这些原料由于来源、棕榈油酸含量和品质等各种限制，尚不具备完善的产业化价值。海水培养微藻原料不受地理环境限制、不受季节采收限制，具有绿色、可持续的优势，可以满足目前巨大的原料缺口和节约淡水。综上所述，有必要进行海水培养的高产棕榈油酸的新资源微藻探索。
发明内容
本发明的目的之一是提供一株海水培养的魏氏藻，即魏氏藻(Vischeria sp.)LQ-1984株系。其能在不同盐度的海水中生长，只需在天然海水基础上添加碳、氮、磷、铁等元素即可满足其生长需求，不仅可以大幅度节约淡水资源，减少养殖肥料的用量，避免其他原生动物污染，更重要的是海水培养可以大幅提高ω-7脂肪酸的含量，从根本上解决优质原料的来源问题。
具体地，所述的魏氏藻(Vischeria sp.)LQ-1984株系，编号为LQ-1984，拉丁名称为Vischeria sp.，保藏号为CCTCC M 20191120，命名为Vischeria sp.LQ-1984。该魏氏微藻分离纯化自广东茂名海水水产(对虾)养殖池塘。
所述魏氏藻(Vischeria sp.)LQ-1984株系具有以下优点：
1)所述魏氏藻(Vischeria sp.)LQ-1984株系相比其他ω-7脂肪酸的生产原料，其来源不受环境、地域、季节性等限制，可以满足巨大的原料缺口。
2)所述魏氏藻(Vischeria sp.)LQ-1984株系细胞中ω-7脂肪酸含量占细胞总脂肪酸50％以上，相对于现有藻株具有非常明显的优势和开发相关产品的应用潜力。
3)所述魏氏藻(Vischeria sp.)LQ-1984株系脂肪酸组成以16碳以上的不饱和脂肪酸为主(占总脂肪酸的96％以上)，更有宜于人体健康。
4)所述魏氏藻(Vischeria sp.)LQ-1984株系能在不同盐度的海水中生长，只需在天然海水基础上添加碳、氮、磷、铁等元素即可满足其生长需求，不仅可以大幅度节约淡水资源，减少养殖肥料的用量，避免其他原生动物污染，更重要的是海水培养可以大幅提高ω-7脂肪酸的含量，从根本上解决优质原料的来源问题。
5)所述魏氏藻(Vischeria sp.)LQ-1984株系形态特征：成熟的细胞表面光滑，细胞壁无突起，直径为10-15μm，呈球形或近球形，叶绿素为周生叶状，内含一蛋白核。
6)所述魏氏藻(Vischeria sp.)LQ-1984株系的海水培养条件为添加有碳、氮、磷、铁等4种元素的天然海水，即海水培养基中，海水盐度为15‰-35‰，氮为硝酸钠浓度为1.5g/L，碳可以为碳酸氢钠，浓度为0.5g/L，磷为磷酸二氢钠浓度为0.04g/L，铁可以为硫酸亚铁，浓度为0.005g/L。
7)所述魏氏藻(Vischeria sp.)LQ-1984株系在3cm直径的柱式光生物反应器中，利用上述海水培养基培养，培养条件如下：光照强度为200μmol photons m-2s-1；温度为25±1℃，pH值为7.5，通入二氧化碳含量为3％的压缩气体。培养8天后，生物量浓度可以达到6.49g/L，细胞密度大，容易规模化生产，降低养殖成本。
8)所述魏氏藻(Vischeria sp.)LQ-1984株系在15‰海水培养条件下的脂肪酸含量丰富，主要以C16以上不饱和脂肪酸为主，占总脂肪酸的97％左右，其中ω-7脂肪酸分别占总脂肪酸的54.91％，可用作ω-7相关产品的生产原料。
9)所述魏氏藻(Vischeria sp.)LQ-1984株系在20‰海水培养条件下脂肪酸含量丰富，主要以C16以上不饱和脂肪酸为主，占总脂肪酸的96％左右，其中ω-7脂肪酸分别占总脂肪酸的54.78％，可用作ω-7相关产品的生产原料。
10)所述魏氏藻(Vischeria sp.)LQ-1984株系在35‰海水培养条件下脂肪酸含量丰富，主要以C16以上不饱和脂肪酸为主，占总脂肪酸的97％左右，其中ω-7脂肪酸分别占总脂肪酸的51.87％，可用作ω-7相关产品的生产原料。
11)所述魏氏藻(Vischeria sp.)LQ-1984株系海水培养不仅能增加ω-7脂肪酸含量，还能节省淡水资源。
12)所述魏氏藻(Vischeria sp.)LQ-1984株系含有高含量ω-7脂肪酸，其高品质藻粉可用于生产ω-7相关的保健品、食品、化妆品等。
本发明目的之二提供上述魏氏藻的培养方法。具体地，将上述魏氏藻置于海水培养基中培养，培养条件如下：光照强度为200μmol photons m-2s-1，温度为25±1℃，pH值为7.5，通入二氧化碳含量为3％的压缩气体。作为本发明的一个优选实施例，所述海水培养基的组分包含：海水盐度为15‰-35‰，氮为硝酸钠浓度为1.5g/L，碳可以为碳酸氢钠，浓度为0.5g/L，磷为磷酸二氢钠浓度为0.04g/L，铁可以为硫酸亚铁，浓度为0.005g/L。
本发明的目的之三提供上述魏氏藻的应用。具体地，涉及该魏氏藻在生产ω-7脂肪酸中的应用；涉及该魏氏藻在制备保健食品中的应用；涉及该魏氏藻在制备食品中的应用；涉及该魏氏藻在制备化妆品中的应用。
本发明提供的魏氏藻(Vischeria sp.)LQ-1984株系，命名为LQ-1984，经鉴定比对该微藻为魏氏藻，拉丁名Vischeria sp.，已于2019年12月26日在“中国典型培养物保藏中心(China Center for Type Culture Collection，简称CCTCC)保藏成功；地址：湖北武汉市武昌珞珈山；菌种保藏号：CCTCC M 20191120。
附图说明
图1为魏氏藻(Vischeria sp.)LQ-1984株系细胞形态；
图2为魏氏藻(Vischeria sp.)LQ-1984株系系统进化树
图3为魏氏藻(Vischeria sp.)LQ-1984株系的生长曲线；
图4为魏氏藻(Vischeria sp.)LQ-1984株系的脂肪酸组成；
图5为魏氏藻(Vischeria sp.)LQ-1984株系在不同海水浓度培养下的脂肪酸组成。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合附图和具体实施例对本发明作进一步的详细说明。
1.高品质魏氏藻(Vischeria sp.)LQ-1984株系的分离鉴定
样品取自广东茂名海水水产(对虾)养殖池塘，带回实验室后按1:5的比例接到无菌海水培养基中，在光照强度为200μmol photons m-2s-1，温度为20±1℃条件下进行富集培养3-5天。待水样呈现肉眼可见的颜色后，在无菌条件下采用稀释涂布法或划线法对预培养后的培养物用固体平板进行分离纯化，平板置于上述条件的光照培养架培养7-15天，重复多次上述分离步骤，直到得到完全无菌的目的单藻落。
无菌海水培养基：
天然海水中添加以下成分：海水盐度为15‰-35‰，硝酸钠1.5g/L，碳酸氢钠0.5g/L，磷酸二氢钠0.04g/L，硫酸亚铁0.005g/L。
固体平板上挑取单藻落到含有20mL灭菌海水培养基的三角瓶中，置于光照培养架上连续光照培养，定时进行摇瓶，获得对数期的藻种。离心收集藻细胞，采用植物DNA提取试剂盒(CTAB法)提取藻株DNA，以18S rRNA通用引物PCR扩增细胞的DNA，PCR反应条件为：94℃预变性5min，94℃变性1min20s，52℃退火1min，72℃延伸2min，36个循环，最后72℃延伸10min，4℃保存。将PCR产物送至上海美吉生物公司测序，获得藻株的18S rRNA基因序列，与NCBI数据库进行同源性比对，根据18S rRNA基因序列构建系统发育进化树，如图2所示，结果显示该藻株与魏氏藻(Vischeria sp.)相似性为99％，可以确定该藻株为魏氏藻，拉丁名Vischeria sp.，属绿藻门、真眼点藻纲、真眼点藻目、真眼点藻科、魏氏藻属。
灭菌海水培养基：
天然海水中添加以下成分：海水盐度为15‰-35‰，硝酸钠1.5g/L，碳酸氢钠0.5g/L，磷酸二氢钠0.04g/L，硫酸亚铁0.005g/L。
18s rRNA通用引物：
18SF：5'CTGGTTGATCCTGCAGT 3'
18SR：5'CACCTACGGAAACCTTGTTACGACTT3'。
取魏氏藻(Vischeria sp.)LQ-1984株系在3cm直径的柱式光生物反应器中，利用上述灭菌海水培养基培养，培养条件如下：光照强度为200μmol photons m-2s-1；温度为25±1℃，pH值为7.5，通入二氧化碳含量为3％的压缩气体。如图3所示，培养8天后，生物量浓度可以达到6.49g/L，细胞密度大，容易规模化生产，降低养殖成本。如图4所示，ω-7脂肪酸含量占细胞总脂肪酸50％以上，相对于现有藻株具有非常明显的优势和开发相关产品的应用潜力。
2.淡水培养条件下魏氏藻LQ-1984株系的脂肪酸组成
将活化培养到对数期的魏氏藻LQ-1984离心(3000转/分钟，5分钟)去掉旧培养基，重新接种到300ml的淡水培养基中，倒入直径为3cm的柱式光生物反应器中，在光照强度为200μmol photons m-2s-1；温度为25±1℃，pH值为7.5，连续通入二氧化碳含量为3％的气体培养。经过8天培养可以获得5.62g/L的生物质浓度。该培养条件下的脂肪酸种类丰富，主要以C16以上脂肪酸为主，占总脂肪酸的98％左右，其中ω-7脂肪酸含量最高，为49.6％，产率为103.48mg/L-1d-1。
淡水培养基：BG-11培养基，去离子水中添加以下成分：硝酸钠浓度为1.5g/L、磷酸氢二钾浓度为0.04g/L、硫酸镁浓度为0.075g/L、氯化钙浓度为0.036g/L、柠檬酸浓度为0.006g/L、柠檬酸铁铵浓度为0.006g/L、EDTA-二钠浓度为0.001g/L、碳酸钠浓度为0.002g/L、硼酸浓度为2.86mg/L、氯化锰浓度为1.86mg/L、硫酸锌浓度为0.22mg/L、钼酸钠浓度为0.021mg/L、硫酸铜浓度0.08mg/L、硝酸钴浓度为0.05mg/L。
3.海水盐度为15‰魏氏藻LQ-1984株系的脂肪酸组成
将活化培养到对数期的魏氏藻LQ-1984藻株离心(3000转/分钟，5分钟)去掉旧培养基，重新接种到300ml的海水盐度为15‰的海水培养基中，倒入直径为3cm的柱式光生物反应器中，光照强度为200μmol photons m-2s-1；温度为25±1℃，pH值为7.5，经过8天培养，收获藻细胞干重6.49g/L。该培养条件下藻株的脂肪酸组成如图5所示，主要以C16以上脂肪酸为主，占总脂肪酸的96％左右，其中ω-7脂肪酸含量为54.91％(表1)，比淡水培养条件下高10.7％，产率为177.99mg/L-1d-1。
4.海水盐度为20‰魏氏藻LQ-1984株系的脂肪酸组成
同样，经过8天培养，海水盐度为20‰的培养条件下魏氏藻LQ-1984的藻株干重为5.73g/L，脂肪酸组成如图5所示，主要以C16以上脂肪酸为主，占总脂肪酸的96％左右，其中ω-7脂肪酸含量为54.79％(表1)，比淡水培养条件下高10.4％，产率为163.31mg/L-1d-1。
5.海水盐度为35‰魏氏藻LQ-1984株系的脂肪酸组成
海水盐度为35‰，其他培养条件不变情况下，魏氏藻LQ-1984藻株干重为4.20g/L，脂肪酸组成如图5所示，主要以C16以上脂肪酸为主，占总脂肪酸的96％左右，其中ω-7脂肪酸含量为51.87％(表1)，比淡水培养条件下高4.5％，产率为127.44mg/L-1d-1。
表1不同海水浓度培养下的ω-7脂肪酸含量
| 海水盐度 | ω-7脂肪酸占比 | 比正常培养增加百分比 |
| 淡水培养 | 49.60％ | - |
| 15‰盐度的海水 | 54.91％ | 10.7％ |
| 20‰盐度的海水 | 54.79％ | 10.4％ |
| 35‰盐度的海水 | 51.87％ | 4.5％ |
以上实施实例对本发明不同的实施过程进行了详细的阐述，但是本发明的实施方式并不仅限于此。所述技术领域的普通技术人员依据本发明中公开的内容，均可实现本发明的目的。
Claims (7)
1.一株利用海水培养的魏氏藻，编号为LQ-1984，拉丁名称为Vischeria sp.，保藏号为CCTCC M 20191120，命名为Vischeria sp.LQ-1984。
2.如权利要求1所述的魏氏藻的培养方法，其特征是，将所述魏氏藻置于海水培养基中培养，培养条件如下：光照强度为200μmol photons m-2s-1；温度为25±1℃，pH值为7.5，通入二氧化碳含量为3％的压缩气体。
3.如权利要求2所述的魏氏藻的培养方法，其特征是，所述海水培养基的组分包含：天然海水，海水盐度为15‰-35‰，硝酸钠1.5g/L，碳酸氢钠0.5g/L，磷酸二氢钠0.04g/L，硫酸亚铁0.005g/L。
4.如权利要求1所述的魏氏藻在生产ω-7脂肪酸中的应用。
5.如权利要求1所述的魏氏藻在制备保健食品中的应用。
6.如权利要求1所述的魏氏藻在制备食品中的应用。
7.如权利要求1所述的魏氏藻在制备化妆品中的应用。
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