CN114196545B - 一种广温性的球等鞭金藻及其应用 - Google Patents
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Abstract
本发明涉及微生物技术领域,特别涉及一种广温性的球等鞭金藻及其应用。本发明从水样中分离获得一株球等鞭金藻(Isochrysis galbana)MEM‑A‑409,于2021年9月1日保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M20211116。该藻株在15~35℃下可保持较高的生长速率,且耐盐性良好,能够适应复杂多变的环境,不仅可以作为水产的初级饵料作为水产幼体的开口饲料,亦可以作为轮虫等饵料生物的饲料,同时营养价值较高,可制成藻粉等保健食品。
Description
技术领域
本发明涉及微生物技术领域,特别涉及一种广温性的球等鞭金藻及其应用。
背景技术
常见的野生微藻通常不耐寒也不耐盐,特别是热带海洋地区的微藻,大多数微藻适应高温条件下的培养,对于热带地区的微藻培养是极其有利的,但对于四季分明的地区或是经常有阴雨天气的地区,微藻的生物量所受的影响较大。
小球藻作为常见的培养藻种已广泛运用于各种研究生产过程,目前已有大量有关小球藻的种质资源。但小球藻因为较厚的细胞壁,不利于经济水产动物幼体对其的摄食。
相比于小球藻,球等鞭金藻粒径更适合幼小动物的摄食。球等鞭金藻是一种常见的金藻,对环境的适应性较强,在自然界分布较广。基于已有的研究证实,该微藻能适应较广的温度范围,15-30℃都能生长,对于高光也有一定的适应能力。其作为养殖藻种具有生长快,不易老化的特点,比一般的绿藻更晚进入衰亡期,本身也有很高的营养价值。该微藻含有多种不饱和脂肪酸,且随着光照强度的增强,组分产生变化,不饱和脂肪酸的含量明显提升,但在高温下生物量会有所下降。而且,相较于水产养殖中有害藻类的恶意竞争,金藻等微藻能作为生态制剂有效地“肥水”,为池塘中的水产动物调节环境,并提供饵料,在极端天气下能维持水质的相对稳定。因此,筛选一种效适应多变的环境、广温性的的金藻,用作水产动物的开口饵料具有重要的现实意义,且对环境友好。
发明内容
有鉴于此,本发明提供了一种广温性的球等鞭金藻及其应用,该藻株能够适应15~35℃的温度,且耐盐性良好。
本发明从海口市海域的海水中分离获得一株球等鞭金藻(Isochrysis galbana),命名为MEM-A-409,其保藏编号为CCTCC NO:M20211116。分离方法具体包括:
从海口市周边海域采集水样,及时带回实验室进行藻种的分离。于显微镜下,用毛细管对单个藻细胞进行吸取,置于实验室的F2培养基下进行培养。水样中细胞过多时,加入灭菌水进行反复稀释,直到可以顺利吸出藻细胞。于锥形瓶中培养至细胞数量达到对数生长期后,在F2培养基平板上进行进一步划线分离,纯化。纯化后的微藻可进行生长性能的测定。
其中,F2培养基母液组成如下:
NaNO3200 g/L,NaH2PO413.3 g/L,f/2Trace Metal Solution(微量元素)200mL(含FeCl33.65 g/L,Na2EDTA·2H2O 4.235g/L,CuSO4·5H2O 0.0196g/L,Na2MoO4·2H2O0.0126g/L,ZnSO4·7H2O 0.044g/L,CoCl2·6H2O 0.01092g/L,MnCl2·4H2O 0.36g/L),f/2vitamin Solution(维生素),Tris 0.01mol/L,使用浓HCL调节pH=7.6-8.0。
分离出的球等鞭金藻MEM409采用18S rRNA基因的通用引物18ScomF1(5’-GCTTGTCTCAAAGATTAAGCCATC-3’)和18ScomR1(5’-CACCTACGGAAACCTTGTTACGGC-3’)扩增18SrRNA的基因片段进行测序,获得18S rRNA基因序列。
本发明所述球等鞭金藻MEM410分离自海口市海域的海水;所述分离采用的培养基由如下组分组成:
NaNO3200 g/L,NaH2PO413.3 g/L,FeCl33.65 g/L,Na2EDTA·2H2O 4.235g/L,CuSO4·5H2O 0.0196g/L,Na2MoO4·2H2O 0.0126g/L,ZnSO4·7H2O 0.044g/L,CoCl2·6H2O0.01092g/L,MnCl2·4H2O 0.36g/L,维生素B121mg/L,Biotin1mg/L,Thiamine HCl 200mg/L和Tris 0.01mol/L,pH=7.6-8.0。
本发明中,所述球等鞭金藻耐受的温度为15~35℃。
实验表明,本发明所述的球等鞭金藻MEM409在15℃、25℃、28℃、35℃下生长均可保持较高的生长速率,具有较强的高温耐受能力。
本发明还提供了所述球等鞭金藻在制备富含不饱和脂肪酸的产品中的应用。
所述产品包括保健品、食品或药品。
本发明还提供了所述的球等鞭金藻在制备动物饲料或饲料添加剂中的应用。
所述动物为水产动物,具体包括轮虫、鱼类;其中,鱼类以淡水养殖鱼类为主,包括四大家鱼,青草鲢鳙,海水养殖贝类蛤蜊、牡蛎等;所述饲料为开口饵料。
本发明还提供了所述的球等鞭金藻在制备生态制剂中的应用。
本发明提供的球等鞭金藻MEM-A-409在15℃~35℃下可保持较高的生长速率,同时具有良好的耐盐性能。相比其他藻株,耐高温性和耐盐性均明显得到提高。
生物保藏说明
球等鞭金藻MEM-A-409(Isochrysisgalbana MEM-A-409),于2021年9月1日保藏在中国典型培养物保藏中心,地址为:中国,武汉,武汉大学,保藏编号为CCTCCNO:M20211116。
附图说明
图1为分离后活化培养的显微镜照片;
图2为所涉微藻在不同温度梯度下的生长曲线图;
图3为所涉微藻在低温下不同盐度的生长曲线图。
具体实施方式
本发明公开了一种广温性的球等鞭金藻及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
下面结合实施例,进一步阐述本发明:
实施例1微藻的分离活化培养
从周边海域采集水样,及时拿回实验室进行藻种的分离。于显微镜下,用毛细管对单个藻细胞进行吸取,置于实验室的F2培养基下进行培养。水样中细胞过多时,加入灭菌水进行反复稀释,直到可以顺利吸出藻细胞。于锥形瓶中培养至细胞数量达到对数生长期后,在F2培养基平板上进行进一步划线分离,纯化,获得藻株MEM-A-409。纯化后进行生长性能的测定。
实验时所用F2培养基母液配方:NaNO3200 g/L,NaH2PO413.3 g/L,f/2Trace MetalSolution(微量元素)200mL(含FeCl33.65 g/L,Na2EDTA·2H2O 4.235g/L,CuSO4·5H2O0.0196g/L,Na2MoO4·2H2O 0.0126g/L,ZnSO4·7H2O 0.044g/L,CoCl2·6H2O 0.01092g/L,MnCl2·4H2O 0.36g/L),f/2vitamin Solution(维生素)(B121mg/L,Biotin 1mg/L,Thiamine HCl 200mg/L),Tris 0.01mol/L,使用浓HCL调节pH=7.2-8.0。
分离纯化后的藻株采用18S rRNA基因通用引物18ScomF1(5’-GCTTGTCTCAAAGATTAAGCCATC-3’)和18ScomR1(5’-CACCTACGGAAACCTTGTTACGGC-3’)扩增18SrRNA片段进行测序,获得18SrRNA基因序列。
使用光学显微镜观察微藻的形态,结果见图1。
结合形态学结果和测序结果鉴定该微藻MEM-A-409为球等鞭金藻(Isochrysisgalbana)。
实施例2不同温度下微藻的生长条件及生长曲线的测定
实验时所用F2培养基配方:NaNO3200 g/L,NaH2PO413.3 g/L,f/2Trace MetalSolution(微量元素)200mL(含FeCl33.65 g/L,Na2EDTA·2H2O 4.235g/L,CuSO4·5H2O0.0196g/L,Na2MoO4·2H2O 0.0126g/L,ZnSO4·7H2O 0.044g/L,CoCl2·6H2O 0.01092g/L,MnCl2·4H2O 0.36g/L),f/2vitamin Solution(维生素)(B121mg/L,Biotin 1mg/L,Thiamine HCl 200mg/L),Tris 0.01mol/L,使用浓HCL调节pH=7.2-8.0。
控制光照强度为50μE·m-2·s-1,24小时不间断,分别于15℃、25℃、28℃、35℃下进行实验,以相同方法配制F2培养基,控制微藻起始浓度OD750在0.5以上,集体配制再进行分装,每株藻3个平行,每两天测定一次OD值,并进行细胞计数,于第八天测定干重和脂质含量。根据测定的指标绘制生长曲线,结果见图2。
根据图2可以看出,MEM409与对比的小球藻均有很广的温度适应范围,且培养过程中MEM409在35℃以下的温度梯度下比小球藻的生长更快且不易衰败。以上条件中,随着温度的提高,两株藻的生长速率也在提高,推测两株藻均有较强的高温耐受能力。
实施例3低温下微藻的耐盐性能的测定
实验时所用F2培养基母液配方:NaNO3200 g/L,NaH2PO413.3 g/L,f/2Trace MetalSolution(微量元素)200mL(含FeCl33.65 g/L,Na2EDTA·2H2O 4.235g/L,CuSO4·5H2O0.0196g/L,Na2MoO4·2H2O 0.0126g/L,ZnSO4·7H2O 0.044g/L,CoCl2·6H2O 0.01092g/L,MnCl2·4H2O 0.36g/L),f/2vitamin Solution(维生素),Tris 0.01mol/L,使用浓HCL调节pH=7.2-8.0。
所述的两株微藻,于5℃下,设置盐度梯度为15g/L,35g/L,70g/L,105g/L,其余条件与正常培养条件一致。使用实验室配制的培养基进行培养,每隔两天测定一次OD值,观察两株藻的长势。以35g/L作为对照组,挑选出与对照组差距较小的盐浓度中藻种,筛选出低温下耐盐的藻种,并与25℃下,高盐度培养的微藻的生长性能进行对比,筛选出抗逆性能强的样本。进一步测定Fv/Fm值,以衡量两株藻在低温高盐下的耐受能力。与对照组能有相近生长性能可视为具有较强的耐受力。
由图3可以看出,在低温环境下,两株藻对低盐的耐受性较差,而当盐度达到正常海水的2-3倍盐度时,可以看到MEM409比小球藻的适应力更强,且与对照组的差距很小,具有更广的盐度适应性。这意味着使用一些浓缩海产品加工废水即便是经过低温处理过也可以用于所述微藻的培养。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 海南大学
<120> 一种广温性的球等鞭金藻及其应用
<130> MP21025385
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 132
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggggctggaa tcgatcgatc acaccggtga ccacacaaac cccccttgcc tcacccccaa 60
ggggcgccag tcccctggcc ccggcgctca gccgcggtgt ccaggtctgg cggggtgggg 120
gcggagctaa tg 132
Claims (10)
1.保藏编号为CCTCC NO:M20211116的球等鞭金藻(Isochrysis galbana)。
2.根据权利要求1所述的球等鞭金藻,其特征在于,其18S rRNA基因片段的序列如SEQID NO.1所示。
3.根据权利要求1所述的球等鞭金藻,其特征在于,所述球等鞭金藻分离自海口市海域的海水。
4.根据权利要求3所述的球等鞭金藻,其特征在于,所述分离采用的培养基由如下组分组成:
NaNO3200 g/L,NaH2PO413.3 g/L,FeCl33.65 g/L,Na2EDTA·2H2O4.235g/L,CuSO4·5H2O0.0196g/L,Na2MoO4·2H2O 0.0126g/L,
ZnSO4·7H2O 0.044g/L,CoCl2·6H2O 0.01092g/L,MnCl2·4H2O 0.36g/L,维生素B121mg/L,Biotin 1mg/L,Thiamine HCl 200mg/L和Tris
0.01mol/L,pH=7.6-8.0。
5.根据权利要求1所述的球等鞭金藻,其特征在于,所述球等鞭金藻耐受的温度为15~35℃。
6.权利要求1~5任一项所述的球等鞭金藻在制备富含不饱和脂肪酸的产品中的应用。
7.根据权利要求6所述的应用,其特征在于,所述产品包括保健品、食品或药品。
8.权利要求1~5任一项所述的球等鞭金藻在制备动物饲料或饲料添加剂中的应用。
9.根据权利要求8所述的应用,其特征在于,所述动物为水产动物;所述饲料为开口饵料。
10.权利要求1~5任一项所述的球等鞭金藻在制备生态制剂中的应用。
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Non-Patent Citations (2)
Title |
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温度对球等鞭金藻8701叶绿素荧光参数及生长的影响;梁英 等;海洋湖沼通报(第2期);第43-51页 * |
温度对等鞭金藻塔溪堤品系细胞密度和叶绿素荧光参数的影响;梁英 等;大连海洋大学学报;第26卷(第05期);第422-427页 * |
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