CN104531601A - 一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法 - Google Patents
一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法 Download PDFInfo
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Abstract
本发明公开了一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，取灭菌海水与MAD母液混合获得营养液，在营养液中加入三角褐指藻母液培养，培养2天后，加入花生四烯酸，继续培养6天后收集藻液，离心分离，弃上清，收集藻体，将藻体冷冻干燥处理2天，研磨备用。该方法对提高三角褐指藻中的岩藻黄素含量效果显著，操作简单方便，费用低，生产周期短，适于批量处理和工厂化生产。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法。
背景技术
岩藻黄素(fucoxanthin)是一类广泛存在于褐藻和硅藻中的类胡萝卜素。这种色素不仅存在于巨藻中，在微藻中也有发现。食物中获得的岩藻黄素在胃肠道中经过消化酶的作用被水解成岩藻黄醇，通过淋巴系统和血液循环系统，最终岩藻黄醇在组织中积累。岩藻黄素的结构非常的特殊。在它的分子结构中含有不同寻常的共轭双键和5，6-单环氧基。正是由于其拥有这种结构才使得其具有抗癌、抗肥胖、抗炎症、抗糖尿病以及抑制血管增生的作用。根据所报道的机理来看，主要是通过促进细胞周期停滞和诱导细胞凋亡两种方式。其中，岩藻黄素通过促进细胞周期停滞的方式来抗癌主要在前列腺癌和肝癌的研究中发现，但是它们的靶标分子是不同的，分别为GADD45A和Cyclin D。而诱导细胞凋亡的方式抑制癌细胞主要是在白血病和结肠癌的研究中发现的，靶标分子分别为Caspase-3，7，9和Bcl-2。近几年的研究中发现肥胖是引起II型糖尿病，高血脂和高血压的重要原因。Hayto等(2005)通过研究表明，岩藻黄素能够激活激活线粒体解偶联蛋白(UCP1蛋白)的转录。该蛋白存在两个作用，一是它能够促进脂肪的分解，二是通过在肝脏中生成DHA来降低胆固醇的含量。WAT在能量储存器官和内分泌器官中具有重要的作用。WAT能够产生如单核细胞趋化蛋白1(MCP-1)、肿瘤坏死因子(TNF-α)、白细胞介素6(IL-6)和脂联素。在白色脂肪组织中，脂肪细胞因子的调节异常会引起血脂异常和高血压症状。Hosokawa等通过研究岩藻黄素对肥胖小鼠有关脂肪细胞因子相关mRNA转录情况。他们发现经岩藻黄素的作用下MCP-1、TNF-α和IL-6相关mRNA的转录下降非常明显，并且其体重比对照组也明显下降。
诱导子能够诱导转录植物中的特定基因，从而提高植物的次生代谢产物。诱导子可以分为内源性诱导子和外源性诱导子，花生四烯酸属于外源性诱导子中不饱和脂肪酸类。它是一种重要的诱导子，在细胞内作为第二信使而直接产于胞内信号的转导，或通过对其它信号通路的调节间接影响细胞生物的活动。王丽丽等研究了不同质量浓度的花生四烯酸处理雨生红球藻，其细胞生长和虾青素含量影响。Le B等发现花生四烯酸能够诱导马铃薯产生植保素。三角褐指藻(Phaeodactylum tricomutum)是一种硅藻。Kim等发现在三角褐指藻中的岩藻黄素的含量要比巨藻中的还要高。因此三角褐指藻被认为是产岩藻黄素的商业藻种。
本文通过利用不同浓度的花生四烯酸处理三角褐指藻，来研究三角褐指藻的生长状况，分离和提取三角褐指藻中的岩藻黄素并且利用HPLC对不同浓度的花生四烯酸处理下的三角褐指藻中岩藻黄素含量的测定。进而得出最适花生四烯酸的浓度，提高岩藻黄素的含量。为提高岩藻黄素含量提供有力的实验依据和实验手段。
发明内容
本发明的目的在于提供一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，该方法对提高三角褐指藻中的岩藻黄素含量效果显著，操作简单方便，费用低，生产周期短，适于批量处理和工厂化生产。
本发明具体通过以下技术方案实现：
一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，包括以下步骤：
1)将灭菌海水与MAD母液混合得营养液；
2)在营养液中加入三角褐指藻母液培养；
3)培养2天后，加入花生四烯酸；
4)继续培养6天后手机藻液，离心分离，弃上清，收集藻体；
5)将藻体冷冻干燥处理2天，研磨备用。
本发明所述的MAD母液的配方为：KNO3：10g，KH2PO4：1g，FeSO4·7H2O：0.25g，MnSO4：0.025g，EDTANa2：1g，VB1：0.0006g，dH2O：100ml。
本发明所述的培养条件为：62.5μmol photons/m2·s，25℃。
所述的灭菌海水与MAD母液的体积比为1∶1000。
所述的营养液与三角褐指藻母液的体积比为1∶10。
步骤(3)中培养液加入花生四烯酸后花生四烯酸的浓度为0.1mg/L～0.5mg/L，优选的花生四烯酸的浓度为0.1mg/L。
步骤(5)中所述的冷冻干燥处理条件为：温度-70℃，压强999Pa。
本发明的优点在于：发明首次公开了从三角褐指藻中提取岩藻黄素，为提高岩藻黄素含量还利用不同浓度的花生四烯酸处理三角褐指藻，寻找最适合的花生四烯酸处理浓度，通过该浓度对三角褐指藻的诱导处理，其岩藻黄素的含量增加显著。该发明操作简单方便，费用低，生产周期短，适于批量处理和工厂化生产。由于岩藻黄素对抗癌，高血糖，心血管疾病，糖尿病都有显著地功效，提高岩藻黄素的含量将满足人们治病的需要，并且能够提高经济效益。
附图说明
图1是实施例利用不同浓度花生四烯酸处理三角褐指藻后生长曲线；
图2是实施例不同浓度花生四烯酸处理三角褐指藻后岩藻黄素含量变化。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
一、实验方法
1、三角褐指藻的培养
三角褐指藻由宁波大学海洋生物工程重点实验室微藻室提供。取过滤且高温灭菌的海水，加入1∶1000灭菌MAV母液。MAD母液的配方为：KNO3：10g，KH2PO4：1g，FeSO4·7H2O：0.25g，MnSO4：0.025g，EDTANa2：1g，VB1：0.0006，dH2O：100ml。以1∶10比例接入藻液，并在62.5μmol photons/(m2·s)，25℃，12h/12h(黑暗/光照)条件下培养。每天分三个时间段(8:00，12:00，20:00)均匀摇藻体。
2、三角褐指藻光密度回归方程和生长曲线测定
取1ml藻液，倍比稀释，利用紫外可见分光光度计(6800型双光束，英国bibby科技有限公司)测定每组稀释液的OD680值，并且用血球计数法对各组样品计数。将所得数据建立三角褐指藻光密度回归方程。每天同一时段分别取1ml经不同浓度花生四烯酸处理的三角褐指藻并测定OD680值，每组均设3个平行。根据光密度回归方程得出各样品的细胞密度，建立经不同浓度花生四烯酸处理三角褐指藻后的生长曲线三角褐指藻标准曲线的测定。
3、岩藻黄素的提取
在第6d时分别取80ml的处理三角褐指藻，分别作3个平行对其进行岩藻黄素的提取。将各组藻体在4℃，52000g，离心10min，弃上清，收集藻体。冷冻干燥2d，称重。研磨藻体，使得藻体充分成粉末状为止。分别取1ml无水乙醇(AR，国药集团化学试剂有限公司)上下重悬藻粉，4℃静置1h，使其充分溶解。4℃，52000g，离心10min，分别取1ml上清过滤膜(0.22μm，上海兴亚净化材料)，4℃保存备用。
4、岩藻黄素含量测定
精确称取0.1mg的岩藻黄素标准品(宁波大学海洋生物工程重点实验室，取自铜藻)，取1ml无水乙醇，通过倍比稀释的方法对其稀释。利用高效液相色谱(2695Separations Module，Waters)，以90％甲醇30min，100％甲醇10min，90％甲醇持续15min的程序在445nm处监测岩藻黄素。本实验所使用的色谱柱型号为250×4.6mml，D.S-5μm，12nm，AA12S05-2546WT，No.0425057734，Waters。通过积峰面积与浓度做出回归曲线。分别取10μl备用提取物经HPLC检测并得到岩藻黄素积峰面积。根据回归曲线得出各组三角褐指藻细胞内岩藻黄素含量。
二、应用实施例
实施例1花生四烯酸对三角褐指藻细胞生长的影响
利用OD680的光吸收值来测定单位体积培养液中三角褐指藻细胞数量，每天定时测定三角褐指藻的吸光值并根据回归方程得到经不同浓度的花生四烯酸处理三角褐指藻研究其对细胞生长的影响。
如图1所示：除了花生四烯酸浓度为312.5mg/培养的三角褐指藻的生长曲线外，其余各组随着培养时间的延长，三角褐指藻细胞的生长量大致呈现先上升后下降的趋势，而且3个时期比较明显。从1-4d，三角褐指藻生长处于对数生长期，4-6d为平台期，从第6d后藻体的生物量明显减少，该时段可以认为是衰亡期。因此第6d为平台期末，该时段三角褐指藻的生物量达到最大值。不难发现，当花生四烯酸浓度在0.1-62.5mg/L时，第6d时的生物量均比对照组要高，说明在该浓度范围内，花生四烯酸促进三角褐指藻的生长。对312.5mg/L的花生四烯酸处理的三角褐指藻生长曲线分析，发现其从第4d以后生物量就明显减少，在第6d时其生物量是所有组中最低的，说明当花生四烯酸浓度高于312.5mg/L时抑制三角褐指藻细胞的生长。
实施例2
设1组为对照组，6组为实验组。对照组不做任何处理，6组实验组分别加入不同浓度的花生四烯酸，其浓度分别为0.1mg/L，0.5mg/L，2.5mg/L，12.5mg/L，62.5mg/L，312.5mg/L。以上述的培养条件同等培养实验组和对照组三角褐指藻。
对倍比稀释的岩藻黄素标准品通过HPLC的积峰面积和稀释浓度，得出回归方程：y＝0.1074x+0.0138，R2＝0.9999，式中y为积峰面积，x为浓度，R2为直线方程的相关系数。通过HPLC测定不同浓度花生四烯酸处理三角褐指藻细胞岩藻黄素峰面积，再根据回归方程得出实验组和对照组三角褐指藻细胞岩藻黄素含量变化。
结果如图2和表1所示：随着花生四烯酸浓度的增加，岩藻黄素含量大致呈现先上升后下降的趋势。当花生四烯酸浓度为0.1-0.5mg/L时，其岩藻黄素含量均比对照组要高。花生四烯酸浓度为0.1mg/L处理三角褐指藻，其藻细胞岩藻黄素含量最高，为1.1mg/g DW，比对照组37％。但是，当花生四烯酸浓度为2.5-62.5mg/L时，其藻细胞岩藻黄素含量与对照组相当。花生四烯酸浓度为312.5mg/L处理三角褐指藻时，其岩藻黄素含量明显低于对照组，说明该浓度严重抑制细胞内岩藻黄素的生物合成。
表1 不同浓度的花生四烯酸处理三角褐指藻后岩藻黄素含量
| 花生四烯酸浓度(mg/L) | 0 | 0.1 | 0.5 | 2.5 | 12.5 | 62.5 | 312.5 |
| 岩藻黄素含量(mg/g DW) | 0.80 | 1.10 | 0.86 | 0.77 | 0.80 | 0.77 | 0.27 |
Claims (7)
1.一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于包括以下步骤：
1)取灭菌海水与MAD母液混合获得营养液；
2)在营养液中加入三角褐指藻母液培养；
3)培养2天后，加入花生四烯酸；
4)继续培养6天后手机藻液，离心分离，弃上清，收集藻体；
5)将藻体冷冻干燥处理2天，研磨备用。
2.根据权利要求1所述的一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于：所述的MAD母液的配方为：KNO3：10g，KH2PO4：1g，FeSO4·7H2O：0.25g，MnSO4：0.025g，EDTANa2：1g，VB1：0.0006g，dH2O：100ml。
3.根据权利要求1所述的一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于：所述的培养条件为：62.5μmolphotons/m2·s，25℃。
4.根据权利要求1所述的一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于：
所述的灭菌海水与MAD母液的体积比为1∶1000；
所述的营养液与三角褐指藻母液的体积比为1∶10。
5.根据权利要求1所述的一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于：步骤(3)中培养液加入花生四烯酸后花生四烯酸的浓度为0.1mg/L～0.5mg/L。
6.根据权利要求5所述的一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于：步骤(3)中培养液加入花生四烯酸后花生四烯酸的浓度为0.1mg/L。
7.根据权利要求1所述的一种用花生四烯酸提高三角褐指藻中岩藻黄素的方法，其特征在于：步骤(5)中所述的冷冻干燥处理条件为：温度-70℃，压强999Pa。
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