CN114561295A - 一种促进微藻岩藻黄素积累和脂质合成的培养方法 - Google Patents
一种促进微藻岩藻黄素积累和脂质合成的培养方法 Download PDFInfo
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Abstract
本发明公开了一种促进微藻岩藻黄素的积累和脂质合成的培养方法，将微藻接种在培养基中培养，在所述培养基中添加150~350mg/L的L‑抗坏血酸。本发明的培养方法可以提高三角褐指藻岩藻黄素含量的同时，提高三角褐指藻脂质含量，尤其是二十碳五烯酸（EPA）的含量。
Description
技术领域
本发明涉及藻类生物的培养技术领域，特别是涉及一种促进微藻岩藻黄素积累和脂质合成的培养方法。
背景技术
硅藻作为海洋初级生产力的重要组成部分，其对于地球生态环境、碳循环等的影响力不容小视 (Armbrust, 2009; Malviya et al., 2016)。三角褐指藻作为常见的海洋硅藻，因其中油脂、岩藻黄素等含量较高，以成为生物固碳的研究热点生物之一。
岩藻黄素（Fucoxanthin）是褐藻中所特有的活性成分，比如三角褐指藻（Phaeodactylum tricornutum）、日本糖海带（Saccharina japonica）、裙带菜（Undaria pinnatifida）等(Peng et al., 2011)。岩藻黄素所具有的特殊化学结构，使其除了在植物光保护中的作用，还具有如抗炎、抗氧化、抗肥胖、抗糖尿病，以及对于多种疾病，如阿尔兹海默症等，有一定的治疗与预防作用(Méresse et al., 2020; Bae et al., 2020)。大型褐藻作为目前岩藻黄素的主要来源，但其含量较低仅约其干重的0.01%(Terasaki et al.,2009)，是限制生产效率的主要原因之一。
微藻的油脂组份受多方面因素的影响，其一部分可转化为生物柴油，另一部分，如二十碳五烯酸（EPA），可作为保健品进行添加。EPA作为ω-3系列多不饱和脂肪酸，且是人体无法自行合成的必需脂肪酸。有研究表明，EPA对于心脑血管疾病、糖尿病、肾病等具有一定的预防和治疗作用（Hung et al.,1999）。但目前，大部分商业化生产的EPA均来自于深海鱼油，其加工生产过程对于海洋生态环境有着不可逆的影响，且存在海洋污染、资源枯竭等不确定因素。三角褐指藻作为一种海洋产油硅藻，利用其提取生产EPA和生物柴油，对于环境中碳固定与高效利用起着积极的作用。
L-抗坏血酸作为一种植物组织内源性的抗氧化物质，对于植物细胞的生理功能、生物合成、代谢以及酶活性等方面密切相关。在三角褐指藻培养过程中，一定量L-抗坏血酸的添加，对于其细胞代谢的影响目前未见报道。同时，三角褐指藻作为岩藻黄素、EPA等食品添加剂来源之一，对其使用人体所需L-抗坏血酸处理，并不会影响产物安全性，且对于产品价值的提升具有一定的积极作用。
发明内容
为了弥补上述现有技术的不足，本发明提出一种促进微藻岩藻黄素积累和脂质合成的培养方法，其可以提高三角褐指藻岩藻黄素含量的同时，提高三角褐指藻脂质含量，尤其是二十碳五烯酸（EPA）的含量。
本发明的技术问题通过以下的技术方案予以解决：
一种促进微藻岩藻黄素积累和脂质合成的培养方法，将微藻接种在培养基中培养，在所述培养基中添加L-抗坏血酸。
优选地，所述L-抗坏血酸是在将所述微藻接种到所述培养基时就添加到所述培养基。
优选地，所述L-抗坏血酸在所述培养基中的终浓度为150~350mg/L。
优选地，所述L-抗坏血酸的溶液是将L-抗坏血酸和无氧超纯水混合制成。
优选地，所述微藻的培养条件为：8h~16h光照与8h~16h黑暗交替，温度15℃~25℃，光照强度为50μmol·m-2·s-1~200 μmol·m-2·s-1。
优选地，在初始配制的培养基中，每1L所述培养基中包含如下组分：1mg~100mgNaNO3，1mg~50mg NaH2PO4·2H2O，1mg ~50mg Na2EDTA，1mg ~50mg FeCl3·6H2O，0.01μg~1mgCuSO4·5H2O，0.01μg~1mg ZnSO4·7H2O，0.01μg~1mg CoCl2·6H2O，0.01 μg~1mg MnCl2·4H2O，0.0001μg~1mg Na2MoO4·2H2O，0.0001μg~1mg维生素B12，0.01μg ~10mg 维生素 B1和0.0001μg ~1mg 维生素H。
优选地，所述微藻为真核藻。
优选地，所述微藻为硅藻门、褐指藻目、褐指藻科、褐指藻属的三角褐指藻藻种。
本发明有益效果是：
本发明在培养三角褐指藻的培养基中加入一定量的L-抗坏血酸，使得培养基以及三角褐指藻细胞的抗氧化能力有所提升。本发明的方法操作简单快捷、实用性强、效率高、安全可控，能够促进三角褐指藻岩藻黄素及油脂含量的积累，其可在工业生产生物柴油或作为藻类食品添加剂的食品工业中大规模应用。
本发明在培养三角褐指藻的培养基中添加150~350mg/L 的L-抗坏血酸，该浓度范围在实际应用中对于藻细胞中的岩藻黄素及油脂含量的积累具有较好的促进作用。此外，若低于此浓度范围会导致作用效果不明显；同时，若高于此浓度范围会导致细胞死亡。
附图说明
图1是本发明中实施例1测得的对照组和实验组对比的脂质含量柱状图；
图2是本发明中实施例3测得的对照组和实验组对比的岩藻黄素含量柱状图。
具体实施方式
为了进一步明确本发明的目的、技术要点以及优点，下面将结合本发明的实施例，对本发明中的技术要点进行更加清晰、完整的描述。所描述的实施例是本发明的部分实施例。另说明，基于本发明实施例，同领域技术工作者在未进行任何创造性工作的前提下，所获得其余实施例，均属于本发明的保护范围。
本发明的一种既可以提高微藻岩藻黄素含量又可以促进脂质合成的培养方法尤其是指促进海洋微藻岩藻黄素和油脂积累的培养方法，以下以三角褐指藻藻种的培养为例，对本发明进行详细阐述。
一种既可以提高微藻岩藻黄素含量又可以促进脂质合成的培养方法，在一些具体实施方式中，包括如下步骤：
实施例1：微藻培养基中添加一定量L-抗坏血酸对海洋微藻油脂含量的影响
（1）藻种液制备：将一定量微藻置于温度21℃、光强150lux、光暗比12h/12h的条件下培养至对数生长期，获得藻液，所述微藻采用海洋硅藻三角褐指藻。
（2）培养基配置：每1L所述培养基中包含如下组分：1 mg ~100 mg NaNO3，1 mg ~50 mg NaH2PO4·2H2O，1 mg ~50 mg Na2EDTA，1 mg ~50 mg FeCl3·6H2O，0.01 μg ~1 mgCuSO4·5H2O，0.01 μg ~1mg ZnSO4·7H2O，0.01 μg ~1mg CoCl2·6H2O，0.01 μg ~1mgMnCl2 · 4H2O，0.0001 μg ~1mg Na2MoO4 · 2H2O，0.0001 μg ~1mg维生素B12，0.01 μg ~10mg 维生素 B1 和 0.0001 μg ~1mg 维生素H。初始配制的培养基即是微藻还未接种，还未添加L-抗坏血酸时配制的培养基，随着微藻生长的进行，培养基中的各组分的含量会有所变化。
（3）药剂诱导：使用制备完成的藻种液进行微藻接种，使培养基中接种密度为0.5×106~1.0×106 /mL。本例中约为1.0×106 /mL，并控制单因子变量，即微藻培养基内L-抗坏血酸的添加量，接种时设置对照组和实验组，且每组设置生物学重复5组，并随机放置培养瓶在光照培养箱中的位置以减少误差。
（4）评价指标：总脂质含量使用重量分析法测定。将约20mg 冻干微藻与2mL氯仿、2mL甲醇和1mL 5% NaCl涡旋振荡混合2min，然后于8000xg 在10℃的条件下离心4min。收集氯仿相，并保存以供后续分析。将剩余提取液重复提取三次。将收集的所有氯仿相在氮气流下混合干燥。将干燥后的脂质残余物在60℃烘箱中进一步干燥，最终脂质含量（干重）%=抽提藻细胞总脂质重量/藻细胞干重*100%。
多次试验结果表明：实验组的脂质含量相当于对照组的约2.11倍，说明添加150~350mg/L的L-抗坏血酸可以促进微藻的脂质积累，如附图1所示。
实施例2：微藻培养基中添加一定量L-抗坏血酸对海洋微藻脂质组份的影响
（1）藻种液制备：参照实施例1。
（2）培养基配置：参照实施例1。
（3）药剂诱导：参照实施例1。
（4）评价指标：微藻脂肪酸组成分析。
在微藻培养至平台期后每组取200mL的培养液，在4℃下用4400rpm离心10min，并立即进行冷冻干燥，并称重。将冻干藻团转移到10mL的试管中，在每个试管中加入5mL KOH-CH3OH溶液，在冰浴中超声破碎细胞。密封试管，在氮吹仪的氮气流下处理1min，随后在75℃下孵育10min。冷却至室温后，将溶液上清液转移到50mL管中。下清液用5mL KOH-CH3OH重复破碎两次，使细胞破碎后按上述方法孵育。
将上述样品所得上清液中添加15mL HCl–CH3OH，将溶液充分混合，并在75℃下再次孵育10min，冷却后加入4mL正己烷。将样品进行旋涡混合，并使其静置分层。上层正己烷相被转移至新的10mL离心管中，随后在氮吹仪的氮气流下干燥并使用差量法测定脂质得率。
脂肪酸甲酯分析采用30 m ×0.25 mm × 0.25 μm DB-5石英毛细管柱的GC-MS进行测定。细管柱在60℃下保持1min，后以10 ℃ min-1的速率升温至160℃，再以2.5 ℃ min-1的速率升至最终的250℃。进样温度为280℃，进样量为1 μL。质谱传输线温度为200℃，脂肪酸甲酯用配备NBS频谱库鉴定并通过测定综合峰面积进行定量分析。最后用归一化法计算所检测脂肪酸甲酯的各相对含量。
多次实验结果表明：实验组的二十碳五烯酸（EPA）含量大约为对照组的1.6倍左右，说明添加150~350mg/L的L-抗坏血酸可以提高微藻脂质中二十碳五烯酸（EPA）的比例，如附表1所示。
表1 脂质组份对比表
实施例3：微藻培养基中添加一定量L-抗坏血酸对海洋微藻岩藻黄素含量的影响
（1）藻种液制备：参照实施例1。
（2）培养基配置：参照实施例1。
（3）药剂诱导：参照实施例1。
（4）评价指标：微藻岩藻黄素含量分析。
取一定量的岩藻黄素标准品，并使用无水乙醇配置，在A445条件下进行吸光度测定，所得数据用于标准曲线绘制（R2≥0.999）。
取一定质量的藻粉于离心管中，在避光条件下，加入600-1000μL的无水乙醇，涡旋震荡，75℃恒温水浴加热2h，期间每隔15min涡旋震荡一次。冷却至室温后，8000rpm离心5min，取一定量的上清在A445条件下进行吸光度测定，并将数据带入上述标准曲线中得出样品岩藻黄素含量。
多次实验结果表明：实验组的岩藻黄素含量约为对照组的2.7倍，说明添加150~350mg/L的L-抗坏血酸可以促进微藻的岩藻黄素的积累，如附图2所示。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实例而已，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、等同替换和改进，均应包含在本发明的保护范围之内。
Claims (7)
1.一种促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：将微藻接种在培养基中培养，所述培养基中添加有L-抗坏血酸。
2.如权利要求1所述的促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：所述微藻包括真核藻、硅藻门、褐指藻目或褐指藻科。
3.如权利要求2所述的促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：所述微藻为三角褐指藻。
4.如权利要求1所述的促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：在初始配制的培养基中，每1L所述培养基中包含如下组分：1 mg ~100 mg NaNO3，1 mg ~50mg NaH2PO4·2H2O，1 mg ~50 mg Na2EDTA，1 mg ~50 mg FeCl3·6H2O，0.01 μg ~1 mgCuSO4·5H2O，0.01 μg ~1mg ZnSO4·7H2O，0.01 μg ~1mg CoCl2·6H2O，0.01 μg ~1mgMnCl2 · 4H2O，0.0001 μg ~1mg Na2MoO4 · 2H2O，0.0001 μg ~1mg维生素B12，0.01 μg ~10mg 维生素 B1 和 0.0001 μg ~1mg 维生素H，此时的培养基还未添加L-抗坏血酸。
5.如权利要求1所述的促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：所述L-抗坏血酸在所述培养基中的浓度为150~350mg/L。
6.如权利要求1所述的促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：所述L-抗坏血酸是通过无氧超纯水避光混合配置，且经0.22μm滤膜过滤除菌，再加入培养基中。
7.如权利要求1所述的促进微藻岩藻黄素积累和脂质合成的培养方法，其特征在于：所述微藻的培养条件为：8h~16h光照与8h~16h黑暗交替，温度15℃~25℃，光照强度为50μmol·m-2·s-1~300 μmol·m-2·s-1。
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