CN116769847B - 一种提高拟微球藻藻油中epa含量的方法 - Google Patents
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6432—Eicosapentaenoic acids [EPA]
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Abstract
本发明公开一种提高拟微球藻藻油中EPA含量的方法,在光生物反应器中,将M f/2培养基作为基础培养基,接种拟微球藻,并通入含2%CO2的空气,在26±1℃的培养温度、蓝光照射下培养。本发明的拟微球藻的培养方法培养过程中使用蓝光光质,能够显著提高拟微球藻的EPA合成量和占总脂肪酸的比例,从而能够降低后续藻油精炼成本。
Description
技术领域
本发明涉及微藻培养技术领域,具体涉及一种提高拟微球藻藻油中EPA含量的方法。
背景技术
二十碳五烯酸(Eicosapentaenoic acid,EPA)是一种重要的ω-3多不饱和脂肪酸,是维持人类和动物健康必须的营养素,具有多种重要的生理功能,包括预防心血管疾病、抗炎、抑制过敏反应、抑制肿瘤生长等。因此EPA也是膳食补充剂和功能食品的重要原料。在过去,EPA的供应主要来源于海洋鱼油,而鱼油的供应一直存在季节性供应的波动、因过度捕捞导致鱼类种群减少、重金属和有机污染物污染、气味难闻、不适合素食者等问题,因此市场上亟需具有可持续性的新型EPA替代资源。实际上,鱼类的ω-3多不饱和脂肪酸主要通过捕食海洋藻类或虾类来积累,且合成转化率较低。藻类具有天然合成ω-3多不饱和脂肪酸的代谢途径,并且作为主要的初级生产者,其体内合成与积累的ω-3多不饱和脂肪酸会通过食物链传递并储存于甲壳类、鱼类、海洋哺乳类等海洋生物体内。除此之外,藻油中没有鱼油特有的鱼腥味,制成的营养强化剂可适合鱼油过敏的特殊人群食用,在食品的应用方面会更广泛[1-2]。因此,利用微藻生产ω-3多不饱和脂肪酸是一种更加绿色、安全、有效的途径。
拟微球藻(Nannochloropsis sp.)是一种单细胞绿藻,具有环境适应能力强、繁殖快、细胞小等特点,细胞中能够积累EPA,通常能达到细胞干重的4%左右[3],且不含有DHA,因此与鱼油相比,利用拟微球藻藻油生产不含DHA的EPA油脂将显著降低分离纯化的成本。拟微球藻除了富含EPA之外,其细胞还含有丰富的蛋白质,碳水化合物以及叶绿素等。由于全细胞营养均衡,拟微球藻属已经加入了饲料原料目录,并且在2021年4月25日拟微球藻属的Nannochloropsis gaditana这一藻种正式获批成为新食品原料之一,这表明拟微球藻将在未来新型功能食品和人类大健康领域发挥重要作用。但与传统鱼虾蟹油相比,N.gaditana藻油中EPA占总脂肪酸含量相对较低(25%左右)[4],这会加大后续高含量EPA油脂制备的成本。因此拟微球藻藻油中EPA的含量还需进一步的提升。
参考文献
[1]A,Parul Jakhwal,et al.Genetic and non-genetic tailoringofmicroalgae for the enhanced production of eicosapentaenoic acid(EPA)anddocosahexaenoic acid(DHA)-A review.Bioresource Technology,2021,344.DOI.org/10.1016/j.biortech.2021.126250
[2]Peltomaa E,Johnson M D,Taipale S J.Marine cryptophytes are greatsources of EPA and DHA[J].Mar Drugs,2018,16(1):11.DOI:10.3390/md16010003.
[3]Liu J,Liu M,Pan Y,et al.Metabolic engineering of the oleaginousalga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerolby combined pulling and pushing strategies[J].Metabolic Engineering,2022,69:163-174.DOI:10.1016/j.ymben.2021.11.015.
[4]Hu H,Gao K.Optimization of growth and fatty acid composition ofaunicellular marine picoplankton,Nannochloropsis sp.with enriched carbonsources[J].Bio-technology Letters,2003,25(5):421-425.DOI:10.1016/S1053-2498(98)00016-3.
发明内容
本发明提供一种提高拟微球藻藻油中EPA含量的方法,提高EPA的产量和占总脂肪酸(TFA)的比例,从而降低EPA油脂制备的成本。
本发明提供一种提高拟微球藻藻油中EPA含量的方法,在光生物反应器中,加入含有Mg2+的培养基,接种拟微球藻,并通入含2%CO2的空气,在26±1℃的培养温度、蓝光照射下培养。
进一步的,所述蓝光的光照强度为50~300μmol/m2/s。
优选的,所述蓝光的的光照强度为200μmol/m2/s。
进一步的,加入Mg2+后的所述Mg2+的浓度为300-2000mg/L。
优选的,加入Mg2+后的所述Mg2+的浓度为600mg/L。
进一步的,所述拟微球藻接种后的初始浓度为0.27-0.33g/L。
优选的,所述拟微球藻接种后的初始浓度为0.3g/L。
进一步的,所述光生物反应器呈内径为5cm的体积为1L的柱状。
进一步的,所述培养基为M f/2基础培养基。
进一步的,所述M f/2基础培养基包括18-22g/L的海盐水、0.50-0.60g/L的CO(NH2)2、0.046-0.058g/L的NaH2PO4、0.00300-0.00400g/L的FeCl3·6H2O、0.00400-0.00500g/L的Na2EDTA·2H2O、(3.0-4.0)×10-4g/L的MnCl2·4H2O、(4.0-5.0)×10-5g/L的ZnSO4·7H2O、(0.9-1.1)×10-5g/L的CoCl2·6H2O、(1.80-2.10)×10-5g/L的CuSO4·5H2O、(1.10-1.40)×10-5g/L的Na2MoO4·2H2O、(2.2-2.8)×10-6g/L的biotin、(4.5-5.5)×10-7g/L的VB1和(2.2-2.8)×10-6g/L的VB12。
进一步的,所述M f/2培养基包括19.9g/L的海盐水、0.54g/L的CO(NH2)2、0.052g/L的NaH2PO4、0.00365g/L的FeCl3·6H2O、0.00437g/L的Na2EDTA·2H2O、3.6×10-4g/L的MnCl2·4H2O、4.4×10-5g/L的ZnSO4·7H2O、1×10-5g/L的CoCl2·6H2O、1.96×10-5g/L的CuSO4·5H2O、1.26×10-5g/L的Na2MoO4·2H2O、2.5×10-6g/L的biotin、5×10-7g/L的VB1和2.5×10-6g/L的VB12。
本发明还提供一种生产高含量EPA藻油的拟微球藻,其特征在于,所述拟微球藻按照如上述的培养方法培养而获得。
与现有技术相比,本发明的有益效果为:
本发明的拟微球藻的培养方法培养过程中使用蓝光光质,能够显著提高拟微球藻的EPA合成量和占总脂肪酸的比例,从而能够降低后续藻油精炼成本。并且以M f/2为基础培养基,添加一定量金属Mg2+营养因子也能够显著提高拟微球藻EPA占总脂肪酸中的比例。而蓝光光质及Mg2+的结合能进一步提高EPA在总脂肪酸中的占比,在200μmol/m2/s蓝光光照强度及600mg/L MgSO4营养盐培养条件下,拟微球藻EPA产量可达132.2mg/L,相较于常规的培养方法增加了10.0%;EPA在总脂肪酸中的占比可达38.8%,相较于常规的培养方法增加了32.84%。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例的描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为实施例2、对比例1和对比例2的EPA占总脂肪酸的比例的结果;
图2为白光、蓝光、红光以及红蓝2:1LED光源光谱图;
图3为实施例1和对比例3的EPA占总脂肪酸的比例的结果;
图4为实施例1和对比例4的EPA占总脂肪酸的比例的结果;
图5为实施例1和对比例4的EPA的产量结果;
图6为对比例5、实施例1和实施例3的EPA占总脂肪酸的比例结果;
图7为对比例5、实施例1和实施例3的EPA产量结果。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例及附图,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。以下结合具体实施例对本发明作具体的介绍。
本发明实施例提供一种提高拟微球藻藻油中EPA含量的方法,在光生物反应器中,加入含有Mg2+的培养基,接种拟微球藻,并通入含2%CO2的空气,在26±1℃的培养温度、蓝光照射下培养。
光照条件是调节藻类生长代谢的基本因子之一,光照强度、光质对藻类的生长、形态结构、光合作用和物质代谢都具有重要的作用,大多数生物合成过程能通过改变光照条件进行调节。已有研究表明,蓝光可以显著提高藻类孢子萌发速率及藻体光合效率和生长速率,并提高光合色素含量。而本发明使用蓝光照射,还发现其能够显著提高拟微球藻的EPA合成量和占总脂肪酸的比例,从而能够降低后续藻油精炼成本。
EPA主要存在于拟微球藻叶绿体的类囊体膜上,以膜脂的形式存在,构成了进行光合作用的反应场所。镁元素是叶绿素的重要组成部分,缺镁会导致叶绿素合成受阻,进而影响光能的吸收,而本发明发现额外补充镁离子,还促进合成更多的含有EPA的类囊体膜,从而提高EPA在脂肪酸中的比例。并且发现与单一的使用蓝光照射或加入Mg2+相比,蓝光光质及Mg2+的结合能进一步提高EPA在总脂肪酸中的占比。
具体的,所述蓝光的光照强度为50~300μmol/m2/s,在此范围的蓝光光质,均能够提高EPA占总脂肪酸的比例。
优选的,所述蓝光的的光照强度为200μmol/m2/s,在此光照强度下的蓝光下EPA产率最高。
具体的,加入Mg2+后的所述Mg2+的浓度为300-2000mg/L,在此浓度范围的Mg2+,可显著提高拟微球藻EPA占总脂肪酸中的比例。
优选的,加入Mg2+后的所述Mg2+的浓度为600mg/L,在此浓度下的Mg2+,拟微球藻EPA占总脂肪酸中的比例提高得最多。
具体的,所述拟微球藻接种后的初始浓度为0.27-0.33g/L。
优选的,所述拟微球藻接种后的初始浓度为0.3g/L。
具体的,所述光生物反应器呈内径为5cm的体积为1L的柱状。
具体的,所述培养基为M f/2基础培养基。
具体的,所述M f/2基础培养基包括18-22g/L的海盐水、0.50-0.60g/L的CO(NH2)2、0.046-0.058g/L的NaH2PO4、0.00300-0.00400g/L的FeCl3·6H2O、0.00400-0.00500g/L的Na2EDTA·2H2O、(3.0-4.0)×10-4g/L的MnCl2·4H2O、(4.0-5.0)×10-5g/L的ZnSO4·7H2O、(0.9-1.1)×10-5g/L的CoCl2·6H2O、(1.80-2.10)×10-5g/L的CuSO4·5H2O、(1.10-1.40)×10-5g/L的Na2MoO4·2H2O、(2.2-2.8)×10-6g/L的biotin、(4.5-5.5)×10-7g/L的VB1和(2.2-2.8)×10-6g/L的VB12。
优选的,所述M f/2培养基包括19.9g/L的海盐水、0.54g/L的CO(NH2)2、0.052g/L的NaH2PO4、0.00365g/L的FeCl3·6H2O、0.00437g/L的Na2EDTA·2H2O、3.6×10-4g/L的MnCl2·4H2O、4.4×10-5g/L的ZnSO4·7H2O、1×10-5g/L的CoCl2·6H2O、1.96×10-5g/L的CuSO4·5H2O、1.26×10-5g/L的Na2MoO4·2H2O、2.5×10-6g/L的biotin、5×10-7g/L的VB1和2.5×10- 6g/L的VB12。
本发明实施例还提供一种生产高含量EPA藻油的拟微球藻,其特征在于,所述拟微球藻按照如上述的培养方法培养而获得。
实施例1
培养方式如下:在内径为5cm的1L柱状光生物反应器中,以M f/2培养基为基础培养基(培养基的组成和用量见附表1),接种拟微球藻,初始接种浓度为0.3g/L,并通入含2%CO2的空气,在200μmol/m2/s蓝光、培养温度为26±1℃下培养。
表1 M f/2培养基成分表
实施例2
培养方式如下:在内径为5cm的1L柱状光生物反应器中,以M f/2为基础培养基(培养基的组成和用量见附表1),向培养基上添加MgSO4,添加后MgSO4的浓度为600mg/L,接种拟微球藻,初始接种浓度为0.3g/L,并通入含2%CO2的空气,在200μmol/m2/s白光、培养温度为26±1℃下培养。
实施例3
培养方式如下:在内径为5cm的1L柱状光生物反应器中,以M f/2为基础培养基(培养基的组成和用量见附表1),向培养基上添加MgSO4,添加后MgSO4的浓度为600mg/L,接种拟微球藻,初始接种浓度为0.3g/L,并通入含2%CO2的空气,在200μmol/m2/s蓝光、培养温度为26±1℃下培养。
对比例1
改变实施例2中添加MgSO4的量,使其添加后的浓度分别为15、75、300、200、2000mg/L,其他条件与实施例2相同,对拟微球藻进行培养,取培养第5天的样品进行脂肪酸含量的分析,实施例2和对比例1的结果见图1。
从图1可知,随着Mg2+浓度的增加EPA占总脂肪酸比例而增加,直到浓度达到600mg/L时,其比例最高可达38.3%,表明镁离子的添加能特异性促进EPA的积累,提高了EPA占总脂肪酸的比例。
对比例2
改变实施例2添加的MgSO4为CaCl2、FeCl3和NaCl,CaCl2添加后的浓度分别为15、60、150、300和600mg/L,FeCl3添加后的浓度分别为3、6、12、15和20mg/L,NaCl添加后的浓度分别为500、1000和1500mg/L,其他条件与实施例2相同,对拟微球藻进行培养,取培养第5天的样品进行脂肪酸含量的分析,结果见图1。
从图1可知,与对照相比,Na+、Ca2+与Fe3+的添加,不论浓度的高低均对拟微球藻EPA占总脂肪酸的比例没有显著的促进或抑制作用。
对比例3
改变实施例1中的蓝光分别为白光、红光和红蓝2:1混合光,改变200μmol/m2/s光照为50μmol/m2/s,其他条件与实施例1相同,对拟微球藻进行培养,收集第12天的样品进行脂肪酸含量的测定,实施例1和对比例3的结果见图2和图3,其中图2中a为白光的LED光源光谱图,b为蓝光的LED光源光谱图,c为红光的LED光源光谱图,d为红蓝2:1混合光的LED光源光谱图。
从图3可知,蓝光条件下,EPA占总脂防酸的比例最高,达到34.1%;而红光条件下,EPA占总脂肪酸的比例最低,仅为20.3%;而白光和红蓝2:1混合光条件下,EPA占总脂肪酸的比例没有显著差别。因此,在相同光强下,蓝光能够显著提高EPA占总脂肪酸的比例。
对比例4
改变实施例1中的蓝光的200μmol/m2/s光照为50、100和300μmol/m2/s,其他条件与实施例1相同,对拟微球藻进行培养,取培养第6天的样品进行脂肪酸含量的分析,实施例1和对比例4的结果见图4和图5,其中拟微球藻EPA占总脂肪酸(TFA)的比例结果见图4,拟微球藻EPA的产量结果见图5。
从图4和图5可知,在50~300μmol/m2/s蓝光范围内,细胞内EPA占总脂肪酸的比例相差不大,在35%左右。但拟微球藻EPA的产量有显著差别,200μmol/m2/s条件下的EPA产率最高,约为101mg/L。
对比例5
改变实施例1中的蓝光为白光,其他条件与实施例1相同;
对比例5、实施例1和实施例3的结果见图6和图7,其中图6为EPA占总脂肪酸占比结果,图7为拟微球藻EPA产量的结果。
从图6和图7可知,两种因素的结合对其产量及EPA含量占总脂肪酸比例均有进一步的促进作用,与对比例5相比,在培养第6天其产量在可达132.2mg/L,相较于对比例5增加了10.0%;EPA占总脂肪酸的比例可达38.8%,相较于对比例5增加了32.8%。
生物量的测定及脂肪酸成分分析:
样品生物量含量的测定方法为:采用恒重法来测定生物量。在105℃烘箱中,将已编号的GF/C(Glass microfiber filters)滤膜置于其中,并烘干至恒重记录重量(M 0)。取藻样3mL(V),真空抽滤后放入105℃烘箱中烘干至恒重(M 1),并与上述滤膜编号相对应记录数据。生物量(Dry weight,DW)计算公式如下:
DW(g/L)=(M 1-M 0)/V
样品脂肪酸含量的测定方法为:精确称取10mg左右的冻干藻粉到安捷伦(Agilent)2.5mL棕色色谱上样小瓶中,对样品进行全细胞甲酯化,具体步骤如下:
(1)向盛有藻粉的色谱上样小瓶中,分别加入200μL氯仿:甲醇(2:1,v/v)溶液、300μL 5%盐酸甲醇溶液;
(2)将所有的上样小瓶密封严实,85℃恒温1h后,冷却至室温至少15分钟,但不超过1小时;
(3)向每个色谱上样小瓶中加入1mL正己烷,充分震荡混匀,室温条件下静置1h~4h,直至分层;
(4)上层溶液为溶解于正己烷中的脂肪酸甲酯,取上层溶液200μL于带内插管的色谱上样小瓶中,并加入5μL 200ppm十五烷内标溶液,然后采用气相色谱-质谱联用仪(GC-MS)检测样品中的脂肪酸甲酯组分。
GC-MS检测方法具体如下:本研究中采用的气相色谱仪为Agilent7890B+5977A,毛细管柱为HP-88。初始柱温为50℃,保持2分钟;然后以25℃/分钟的速率加热至175℃,并保持5分钟;然后以7℃/分钟的速率加热至210℃,并保持2分钟;最后以2℃/分钟的速率加热至230℃,并保持1分钟。进样温度保持在250℃,并以不分流模式,进样体积设为2μL。辅助加热端的温度为250℃,离子源温度为230℃,质谱四级杆温度为150℃。惰性气体氦气作为载气,流速为1mL/min。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种提高拟微球藻藻油中EPA含量的方法,其特征在于,在光生物反应器中,加入含有Mg2+的培养基,所述培养基为M f/2基础培养基,所述M f/2培养基为19.9 g/L的海盐水、0.54 g/L的CO(NH2)2、0.052 g/L的NaH2PO4、0.00365 g/L的FeCl3·6H2O、0.00437 g/L的Na2EDTA·2H2O、3.6×10-4 g/L的MnCl2·4H2O、4.4×10-5 g/L的ZnSO4·7H2O、1×10-5 g/L的CoCl2·6H2O、1.96×10-5 g/L的CuSO4·5H2O、1.26×10-5 g/L的Na2MoO4·2H2O、2.5×10-6g/L的生物素、5×10-7 g/L的VB1和2.5×10-6 g/L的VB12,加入Mg2+后的所述Mg2+的浓度为300-2000 mg/L,接种拟微球藻,所述拟微球藻接种后的初始浓度为0.27-0.33g/L,并通入含2% CO2的空气,在26±1℃的培养温度、蓝光照射下培养,所述蓝光的光照强度为50~300μmol/m2/s。
2.如权利要求1所述的提高拟微球藻藻油中EPA含量的方法,其特征在于,所述蓝光的光照强度为200μmol/m2/s。
3.如权利要求1所述的提高拟微球藻藻油中EPA含量的方法,其特征在于,加入Mg2+后的所述Mg2+的浓度为600 mg/L。
4.如权利要求1所述的提高拟微球藻藻油中EPA含量的方法,其特征在于,所述拟微球藻接种后的初始浓度为0.3g/L。
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凌婷 ; 杨树萍 ; 魏煜凡 ; 张琳 ; .2,4-表油菜素内酯和赤霉素对微拟球藻产油率及脂肪酸组成的影响.生物技术通报.2018,(第06期),摘要. * |
影响微藻EPA含量的环境因素研究进展;马国红;许鹏;宋理平;张延华;;广东海洋大学学报(第06期);摘要 * |
微藻生产油脂培养新技术;左正三;孙小曼;任路静;黄和;;中国生物工程杂志(第07期);摘要 * |
氮源和N/P对眼点拟微球藻的生长、总脂含量和脂肪酸组成的影响;魏东, 张学成, 隋正红, 徐怀恕;海洋科学(第07期);摘要 * |
盐度及其调节方式对眼点拟微球藻的生长和EPA积累的影响;吴瑞珊;魏东;;现代食品科技(第12期);摘要 * |
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