CN108587913B - 一种具有高α-亚麻酸含量的栅藻、其培养方法及其应用 - Google Patents
一种具有高α-亚麻酸含量的栅藻、其培养方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种高α‑亚麻酸含量的栅藻、其培养方法及应用。本发明的栅藻总脂的脂肪酸主要由十六碳、油酸、亚油酸和α‑亚麻酸组成,并且总脂中α‑亚麻酸的含量在优化的培养条件下稳定在35‑45%。例如,进行3 L的发酵实验,120 h后,培养物的细胞干重可达到19.48 g L‑1,总脂含量为12.1%,总脂中α‑亚麻酸的含量为42.42%,α‑亚麻酸的产率为0.2 g L‑1 d‑1。该藻株可用于生产动物饲料的添加剂以满足动物生长对α‑亚麻酸等营养的需求,也可以用作工业化制备α‑亚麻酸的原料。
Description
技术领域
本发明涉及微藻及其培养技术,更特别地,涉及一种具有高ALA含量的栅藻、其培养方法及其应用。
背景技术
多不饱和脂肪酸(Polyunsaturated Fatty Acid,PUFA)是指含有两个或两个以上双键且碳长度为18-22个碳原子的直链脂肪酸,主要包括亚油酸(LA)、γ-亚麻酸(GLA)、α-亚麻酸(ALA)、花生四烯酸(AA)、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)等。在PUFA中,距羧基端最远的双键在第3个碳原子上称为ω-3(n-3)系列,如在第6个碳原子上则称为ω-6(n-6)系列。
α-亚麻酸(α-linolenic acid,ALA)为全顺式9,12,15-十八碳三烯酸,属于ω-3系列多不饱和脂肪酸,通过碳链的延长及进一步的脱饱和,最终可以形成二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)。EPA是动物体内三烯***素(如PGI3,TXA3)的前体,DHA则是大脑、视网膜等神经***磷脂的主要成分。ALA及其衍生物EPA和DHA对于动物的脑组织发育、神经***的信号传导、心血管***的发育以及细胞抗氧化等方面的重要作用已被公众所认知和接受。由于人类和其他哺乳动物自身不能合成ALA,体内所需的ALA必须从食物中摄取;而且,摄入的ω-3/ω-6脂肪酸比例过低也会导致ALA不能向DHA和EPA有效转化,导致心理疾病和心血管疾病。因此,及时补充α-亚麻酸对人类和动物的健康非常必要。2011年欧盟食品***(EFSA)在营养科学公告中也提出,ALA与大脑及神经组织发育密切相关,建议在日常膳食中添加ALA。
人类的ω-3多不饱和脂肪酸来源于植物和鱼类等食物中的油脂成分。鱼类的生长发育也需要ω-3系列多不饱和脂肪酸,在自然界主要来自于摄食的藻类和其他植物。研究表明,在饲料中添加ALA可以满足养殖鱼类对于这类脂肪酸的需求,促进其生长和健康。
微藻是一类单细胞或多细胞的低等植物。微藻中许多种类富含蛋白质、油脂、多糖等营养成分,还有的种类富含不饱和脂肪酸、类胡萝卜素、维生素等生理活性成分。螺旋藻、小球藻等藻类干粉作为饲料添加剂,可显著促进水产动物的生长、代谢和抗病力。但是,目前的微藻产业主要依靠自养生长进行开放池培养,总体产量低、价格高,只能满足较高端的消费,难以用于水产动物饲料产业添加剂的需求,并且螺旋藻等藻种ALA含量很低。为此,需要筛选富含ALA、可异养生长的藻种,发展微藻的发酵技术,极大地提高单位面积微藻产量。本发明即是提供一种既富含ALA又可完全异养生长的栅藻(Scenedesmus sp.),并进行发酵培养条件优化。
优先权的要求
本申请要求本申请人于2017年6月8日的申请号为2017104292799、发明名称为“一种具有高GLA含量的栅藻、其培养方法及其应用”的发明专利申请的优先权。
发明内容
发明人在研究过程中发现了一株栅藻,该藻株在不同条件下能进行光照自养生长、光照混养生长(自养/异养混合生长)和纯异养生长,在生长过程中能够积累高含量的油脂,并且,ALA占总脂含量的20%以上,当调节培养基成分后,ALA在总脂中含量最高可达45%,因此,该藻株可用于生产ALA或具有高ALA含量的饲料添加剂或食品添加剂。
基于以上发现和研究,本发明提供了一种栅藻,该藻株于2017年5月24日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 2017282。
本发明的栅藻总脂的脂肪酸主要由十六碳、油酸、亚油酸和ALA组成,并且总脂中ALA的含量在优化培养条件下稳定在35-45%。例如,进行3 L的发酵实验,120 h后,培养物的细胞干重可达到19.48 g L-1,总脂含量为12.1%,总脂中ALA的含量为42.42%,ALA的产率为0.2 g L-1 d-1。因此,该藻株具有作为工业化生产ALA原料的潜力。
本发明还提供了上述栅藻的培养方法,所述栅藻通过光照培养或异养培养。
在一个实施方案中,所述栅藻进行异养培养,所述栅藻进行异养培养,包括使用添加有碳源和氮源的基础培养基在黑暗条件下培养所述栅藻的步骤。在黑暗条件下,该藻株能够完全异养,生长速度比光照条件下更快。
在一个优选实施方案中,所述基础培养基为包含以下成分的水溶液:KH2PO4,0.7 gL-1;K2HPO4,0.3 g L-1;MgSO4 7H2O,0.3 g L-1;FeSO4 7H2O,3 mg L-1;甘氨酸,0.1 g L-1;vitamin B1,0.01 mg L-1;A5,1 ml L-1。其中A5的成分为硼酸(H3BO3)2.86 g/L,四水氯化锰(MnCl2·4H2O)1.81 g/L,七水硫酸锌(ZnSO4·7H2O)0.22 g/L,二水钼酸钠(Na2MoO4·2H2O)0.39 g/L,五水硫酸铜(CuSO4·5H2O)0.08 g/L,六水氯化钴(CoCl2·6H2O)0.01 g/L。
该基础培养基组分仅用于试验本发明的栅藻适合的氮源和碳源的种类和含量,而非限制本发明。本领域技术人员可根据需要选择任何需要的基础培养基来培养本发明的栅藻,例如,还可选择BG11培养基作为基础培养基。
在一个优选实施方案中,所述碳源为10-40 g L-1的葡萄糖。葡萄糖的添加量对该藻株初期的生长速度影响不大,但是,培养到96 h后,添加了20-40 g L-1葡萄糖的培养基中,藻株仍然处于指数生长阶段,使得藻株最终能够达到更高的浓度,从而提高单位体积培养液中的脂肪酸产量。
在一个优选实施方案中,所述氮源为尿素、硝酸钾和/或酵母提取物。以尿素、硝酸钾和酵母提取物中的任一种或几种组合。适当的氮源有利于藻株的生长。
在一个优选实施方案中,所述氮源为1-10 g L-1尿素。与其他氮源相比,尿素更便宜。尿素的添加量在1-10 g L-1的范围内对总脂含量影响不大。但是,当尿素含量在4-7 gL-1的范围内时,ALA的含量较高。
在一个优选实施方案中,所述栅藻的培养温度为25-35℃。该藻株在25-35℃下均能有良好生长,尤其在30-35℃之间,生长速度更快,状态更好,温度低于20℃时,藻株的生长速度显著降低。在25-30℃的范围内时,ALA的含量较高。
本发明还提供了上述栅藻在制备ALA中的应用。
本发明还提供了上述栅藻在制备高ALA含量的饲料添加剂或食品添加剂中的作用
本发明还提供了使用上述栅藻用于制备脂肪酸,尤其是ALA的方法。
在一个优选实施方案中,用于制备脂肪酸的所述栅藻通过上述培养方法培养得到。
微生物保藏
本发明涉及的栅藻于2017年5月24日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为:CCTCC NO:M 2017282。中国典型培养物保藏中心于2017年6月8日对该栅藻的存活性检测完毕,结果为存活。
附图说明
图1为栅藻HSJ296的光学显微照片;
图2为栅藻HSJ296在不同氮源下的生长曲线;
图3为栅藻HSJ296在不同尿素浓度下的生长曲线;
图4为栅藻HSJ296在不同尿素浓度下的总脂含量;
图5为栅藻HSJ296在不同温度下的生长曲线;
图6为栅藻HSJ296在不同温度下的总脂含量;
图7为栅藻HSJ296在不同葡萄糖浓度下的生长曲线;
图8为栅藻HSJ296在不同葡萄糖浓度下的总脂含量;
图9为栅藻HSJ296在发酵罐中的生长曲线。
具体实施方式
以下结合实例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
1. 藻株筛选和保藏
研究人员通过分离纯化的方法从野外采集的水样中分离得到232株绿藻,用于筛选能够完全异养生长的ALA含量较高的绿藻。
发明人将232株除菌纯化的绿藻,接种到装有5 ml葡萄糖浓度为30 g L-1的BG11的试管中,利用控温摇床培养,培养温度设置为30℃,转速设置为150 rpm,培养时间为5天。通过观察,筛选出30株能够完全异养的绿藻。将能够异养培养的30株绿藻,分别接种到装有120 ml BG11的500 ml三角瓶中培养,培养条件如前所述。培养5天后,离心收集藻液,冷冻干燥至恒重,分别称取0.1 g藻粉并提取其油脂。将其油脂甲酯化,利用气相色谱测其油脂中脂肪酸组成及各成分的相对含量。
通过以上方法筛选出ALA占总脂含量最高的藻株,在培养条件未优化时ALA占总脂含量为25.78%。使用光学显微镜观察藻细胞形态,显示其具有栅藻属特征(图1)。提取藻细胞DNA为模板,用18S rDNA通用PCR引物扩增得到18S rDNA,进行测序,其序列如SEQ ID NO:1所示。经序列比对,确认其属于栅藻属(Scenedesmus),将其命名为栅藻HSJ296(Scenedesmussp. HSJ296)
发明人于2017年5月24日将该藻株送至湖北省武汉市武昌区八一路299号武汉大学校内的典型培养物保藏中心(CCTCC)进行微生物保藏,保藏编号为:CCTCC NO:M2017282。
2. 栅藻HSJ296培养条件的优化
基础培养基:KH2PO4,0.7 g L-1;K2HPO4,0.3 g L-1;MgSO4 7H2O,0.3 g L-1;FeSO47H2O,3 mg L-1;甘氨酸,0.1 g L-1;vitamin B1,0.01 mg L-1;A5,1 ml L-1。该基础培养基组分仅用于为栅藻HSJ296提供一些必须营养元素,并用于试验培养栅藻HSJ296时需要的最佳碳源和氮源,及其浓度,而非限制本发明。本领域技术人员可根据自己条件和需要使用其他基础培养基的组分组合。
2.1 氮源的优化
在30℃黑暗异养培养条件下,利用基础培养基摇瓶培养栅藻HSJ296时,比较了5种氮源,即尿素、硝酸钾、硝酸铵、甘氨酸和酵母提取物对藻株生长的影响。培养基内葡萄糖浓度均为6 g L-1,5种氮源的浓度均为1 g L-1,振荡培养120 h。
结果如图2所示,相比其他氮源,尿素最适合栅藻HSJ296的生长。其次,以酵母提取物或硝酸钾作为氮源时,栅藻HSJ296生长良好。但以硝酸铵或甘氨酸作为氮源时,栅藻HSJ296生长很慢。
为了进一步研究尿素对栅藻HSJ296生长的影响,我们设置了1-10 g L-1范围内的四个尿素浓度梯度,进行藻株培养。栅藻HSJ296在不同尿素浓度下的生长曲线如图3所示,1-10 g L-1的尿素浓度范围内,藻株生长速率的差距不大,尿素浓度为4 g L-1时稍好。总脂含量如图4所示,尿素浓度为1、4、7和10 g L-1时,其总脂含量分别为9.83%、10.93%、10.33%和10.78%。尿素浓度为4 g L-1,其总脂含量稍高。
因此,尿素浓度为4 g L-1时,最适合栅藻HSJ296的培养。
不同尿素浓度下栅藻HSJ296总脂的脂肪酸组成如表1所示。
表1 不同尿素浓度下栅藻HSJ296总脂的脂肪酸组成(mol%)
2.2 培养温度的优化
在黑暗异养培养条件下,利用基础培养基摇瓶培养栅藻HSJ296时,基础培养基内尿素浓度为4 g L-1,葡萄糖浓度为20 g L-1,研究了温度对藻株生长的影响。我们设置了四个温度梯度,振荡培养120 h。
栅藻HSJ296在不同温度下的生长速率如图5所示,培养温度为30℃时,栅藻HSJ296的生长速率最快;在20℃和25℃时,栅藻HSJ296的生长速率都较慢;在35℃时其生长状态良好。该实验结果证明温度对栅藻HSJ296的生长速率影响较大。总脂含量如图6所示,温度为30℃时,栅藻HSJ296的总脂含量为11.43%;温度为20℃、25℃和35℃时,总脂含量分别为10.63%、10.87%和10.93%。温度为30℃,该藻的油脂含量为最高。该实验结果证明,温度对栅藻HSJ296的油脂积累的影响不大。
因此,从生长速率和油脂含量两方面考虑,30℃都为栅藻HSJ296的最适培养温度。
不同温度下栅藻HSJ296总脂的脂肪酸组成如表2所示。
表2 不同温度下栅藻HSJ296总脂的脂肪酸组成(mol%)
2.3 糖浓度优化
葡萄糖是维持藻株异养生长的最常见的碳源。在探索初始葡萄糖浓度对栅藻HSJ296生物量积累的影响时,我们将葡萄糖浓度梯度设置为10、20、30和40 g L-1,在黑暗异养培养条件下,利用摇瓶振荡培养栅藻HSJ296。基础培养基中的尿素浓度为4 g L-1,培养温度为30℃。当然,葡萄糖只是用作藻细胞生长的碳源的一种例子,并非用于限制本发明。本领域技术人员可根据自己条件和需要使用其他碳源。
栅藻HSJ296在不同的葡萄糖浓度下的生长曲线如图7所示,在10-40 g L-1初始葡萄糖浓度范围,在96 h以内对栅藻HSJ296的生长影响不大。但初始葡萄糖为10 g L-1时,培养到96 h以后,藻株基本不生长;而初始糖浓度为20、30和40 g L-1时,培养到96 h以后,藻株仍呈指数期生长。
由以上数据可知,当基础培养基中初始葡萄糖浓度为10 g L-1时,培养到96 h后,培养基内的葡萄糖被基本消耗,培养基内缺乏碳源,藻株停止生长。培养120 h后,藻株的产量分别为4.97、9.34、10.4和11.02 g L-1(图中未显示)。以上数据说明,在异养培养条件下,葡萄糖对微藻生长的影响较大。
对于总脂的积累,如图8所示,当葡萄糖浓度在10-40 g L-1之间时,总脂含量分别为10.43%、10.22%、10.42%和10.06%,略有波动。初始葡萄糖浓度对该藻油脂的积累影响不大。
不同葡萄糖浓度下栅藻HSJ296的总脂脂肪酸组成如表3所示。
表3 不同葡萄糖浓度下栅藻HSJ296总脂的脂肪酸组成(mol%)
根据以上实验结果,在后续的发酵培养实验中,我们可以根据发酵需要在20-40g L-1的葡萄糖浓度范围内选择合适的糖浓度,进行发酵。
3. 发酵罐培养
利用基础培养基异养培养栅藻HSJ296 120 h,培养温度为30℃,初始葡萄糖浓度为20 g L-1,尿素浓度为4 g L-1,转速为200 rpm,不控制pH。
如图9所示,培养时间在52-88 h之间,细胞开始呈指数生长,第90 h之后,该藻进入平台期。在第52 h,葡萄糖也开始被快速消耗,在第76小时,葡萄糖浓度下降到4 g L-1(数据未显示)。培养至120 h,栅藻HSJ296停止生长,细胞干重达到19.48 g L-1,总脂含量为12.1%,总脂中ALA的含量为42.42%。ALA的产量可达到1 g L-1,即ALA的产率为0.2 g L-1 d-1。
发酵罐培养的栅藻HSJ296的总脂脂肪酸组成如表4所示。
表4 发酵罐培养的栅藻HSJ296的总脂脂肪酸组成(mol%)
通过改良微藻的培养条件和培养方式来提高微藻的生长速率和ALA的产量,结果显示栅藻HSJ296的最适培养温度为30℃,最佳氮源为4 g L-1尿素,葡萄糖初始浓度在20-40g L-1范围内都适合该藻的生长。在优化培养条件下,栅藻HSJ296总脂的脂肪酸主要由十六碳(C16:0)、油酸(C18:1)、亚油酸(C18:2)和ALA组成,并且总脂中ALA的含量稳定在35-45%之间。
进行3 L的发酵实验,120 h后,栅藻HSJ296的细胞干重可达到19.48 g L-1,总脂含量为12.1%,总脂中ALA的含量为42.42%,ALA的产率为0.2 g L-1 d-1。将来,随着发酵条件的进一步优化,ALA的含量和产量有望获得显著提高。栅藻HSJ296具有作为生产动物饲料微藻添加剂的潜力,以补充ALA及其他营养,同时也可以作为提取制备ALA的原料。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 中国科学院水生生物研究所
<120> 一种具有高ALA含量的栅藻、其培养方法及其应用
<130> 1
<141> 2018-04-16
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1849
<212> DNA
<213> Scenedesmus HSJ296
<400> 1
gcttgcatgc ctgcaggtcg acgatttgat ccttctgcag gttcacctac ggaaaccttg 60
ttacgacttc tccttcctct aggtgggagg gtttaatgaa cttctcggca gacctgaggt 120
gttgccaccc taagctgcca atccgaacac ttcaccagca cacccaatcg gtaggagcga 180
cgggcggtgt gtacaaaggg cagggacgta atcgacgcaa gctgatgact tgcgcctact 240
aggcattcct cgttgaagac taataattgc aataatctat ccccatcacg atgcagtttc 300
aaagattacc cggacctttc ggtcaaggat aggcttgttg aatgcatcag tgtagcgcgc 360
gtgcggccca gaacatctaa gggcatcaca gacctgttat tgcctcatac ttccattgac 420
taaacgccaa tagtccctct aagaagtcag ccagctgcaa aaagcaactg agactattta 480
gcaggctgag gtctcgttcg ttaccggaat caacctgaca aggcaaccca ccaactaaga 540
acggccatgc accaccaccc atagaatcaa gaaagagctc tcaatctgtc aatcctcact 600
atgtctggac ctggtaagtt ttcccgtgtt gagtcaaatt aagccgcagg ctccacgcct 660
ggtggtgccc ttccgtcaat tcctttaagt ttcagccttg cgaccatact ccccccggaa 720
cccaaaaact ttgatttctc ataaggtgct ggcgaagtca ttaaaaaaac attcgccaat 780
ccctagtcgg catcgtttat ggttgagact acgacggtat ctaatcgtct tcgagccccc 840
aactttcgtt cttgattaat gaaaacatcc ttggcaaatg ctttcgcagt agttcgtctt 900
tcataaatcc aagaatttca cctctgacat gaaatacgaa tgcccccgac tgtccctctt 960
atcattactc cggtcctaca gaccaacaag ataggccaga gtcctatcgt gttattccat 1020
gctaaagtat tctggcgtaa gcctgctttg aacactctaa tttactcaaa gtaaccacgt 1080
cgactccgag tcccggacag tgaagcccag aagcccgtcc ccgacagaaa ggaaggccat 1140
agcagtactc accataggcg gaccgctaga acccacccga aatccaacta cgagcttttt 1200
aactgcaaca acttaaatat acgctattgg agctggaatt accgcggctg ctggcaccag 1260
acttgccctc caatggatcc tcgttaaggg atttagattg tactcattcc aattaccaga 1320
cattaaatgc ccgatattgt tatttattgt cactacctcc ccgtatcagg attgggtaat 1380
ttgcgcgcct gctgccttcc ttggatgtgg tagccgtttc tcaggctccc tctccggaat 1440
cgaaccctaa tcctccgtca cccgttacca ccatggtagg cctctatcct accatcgaaa 1500
gttgataggg cagaaatttg aatggaacag cgccggcaca aggccatgcg cttcgtgaag 1560
atatcatgat tcaccgcggg tcggacaaag tccggtcggc cttttatcta atatatacgt 1620
cccttccagg agtcgggatt tacgcacgta ttagctctag aattactacg gttatccgaa 1680
tagtaaggta ccaccaaata aactataact gatttaatga gccattcgca gtttcacagt 1740
ataagcagtt tatacttaga catgcatggc ttaatctttg agacaagcat atgactactg 1800
gcaggatcaa ccaaatctct agaggatccc cgggtaccga gctcgaatc 1849
Claims (10)
1.一种栅藻,其特征在于,于2017年5月24日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 2017282。
2.权利要求1所述的栅藻的培养方法,其特征在于,进行光照培养或异养培养。
3.根据权利要求2所述的培养方法,其特征在于,所述栅藻进行异养培养,包括使用添加有碳源和氮源的基础培养基在黑暗条件下培养所述栅藻的步骤。
4.根据权利要求3所述的培养方法,其特征在于,所述碳源为20-40 g · L-1的葡萄糖。
5.根据权利要求3所述的培养方法,其特征在于,所述氮源为尿素、硝酸钾和酵母提取物中的任一种或几种组合。
6.根据权利要求5所述的培养方法,其特征在于,所述氮源为1-10 g · L-1尿素。
7.根据权利要求3-6中任一项所述的培养方法,其特征在于,所述栅藻的培养温度为25-35℃。
8.权利要求1所述的栅藻在制备α-亚麻酸中的应用。
9.权利要求1所述的栅藻在制备高α-亚麻酸含量的饲料添加剂或食品添加剂中的应用。
10.一种制备α-亚麻酸的方法,其特征在于,从权利要求1所述的栅藻中提取α-亚麻酸。
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