CN104531603B - 一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法 - Google Patents
一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法 Download PDFInfo
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Abstract
本发明提供了一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法,将灭菌海水与MAD母液混合得营养液,在营养液中加入三角褐指藻母液培养,培养2天后,加入乙酰水杨酸,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,尤其是涉及一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法。
背景技术
岩藻黄素(Fucoxanthin)是一类在叶绿体类囊体中参与光合作用光化学系统的类胡萝卜素,广泛存在于褐藻中。在叶绿体中,岩藻黄素是以岩藻黄素叶绿体蛋白质复合体(FCPS)存在的。该复合物是由岩藻黄素、叶绿素a和几种蛋白质所组成的。近年来,由于岩藻黄素具有抗氧化、抗癌、抗肥胖等有重要的作用,因此受到了广泛的关注。岩藻黄素通过调控细胞凋亡和细胞周期来实现,它能够诱导细胞周期停滞和增强细胞间通讯来抑制癌细胞的增殖。如今,由于缺少运动以及摄入大量的脂肪、糖类和高能量的物质,肥胖已经成为普遍的现象。岩藻黄素在肥胖的治疗中同样有重要的作用。Hayato等研究了岩藻黄素在肥胖中作用的机理,认为岩藻黄素是通过激活UCP1蛋白(线粒体解偶联蛋白),一方面促进脂肪的分解,而另一方面则是在肝脏中生成DHA,从而降低胆固醇的含量。诱导子能够对增加次生代谢产物,通过对诱导子浓度的选择可以调控次级代谢以及生物合成作用。乙酰水杨酸就属于内源性诱导子,在植物防卫反应中起着信号作用。三角褐指藻(Phaeodactylumtricornutum)中不仅含有丰富的油脂,同样含有岩藻黄素。
阿司匹林也叫乙酰水杨酸,是一种历史悠久的解热镇痛药,诞生于1899年3月6日。用于治感冒、发热、头痛、牙痛、关节痛、风湿病,还能抑制血小板聚集,用于预防和治疗缺血性心脏病、心绞痛、心肺梗塞、脑血栓形成,也可提高植物的出芽率,应用于血管形成术及旁路移植术也有效。
本申请通过利用不同浓度的乙酰水杨酸处理三角褐指藻,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对不同浓度的乙酰水杨酸处理下的三角褐指藻中岩藻黄素含量的测定。进而得出最适乙酰水杨酸的浓度,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乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法。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:
一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法,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,加入乙酰水杨酸;
4)继续培养6天后收集藻液,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,d H2O: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1000∶1。
所述的营养液与三角褐指藻母液的体积比为10∶1。
步骤(3)中培养液加入乙酰水杨酸后乙酰水杨酸的浓度为10mg/L~25mg/L,优选的乙酰水杨酸的浓度为10mg/L。
步骤(5)冷冻干燥条件为: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乙酰水杨酸处理三角褐指 藻,寻找最适合的乙酰水杨酸处理浓度,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是是实施例利用不同浓度乙酰水杨酸处理三角褐指藻后生长曲线;
图2是是实施例不同浓度乙酰水杨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经不同浓度乙酰水杨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,建立经不同浓度乙酰水杨酸处理三角褐指藻后的生长曲线。
3、岩藻黄素的提取
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(2695SeparationsModule,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乙酰水杨酸对三角褐指藻的影响
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乙酰水杨酸处理三角褐指藻研究其对细胞生长的影响。
如图1所示:随着培养时间的延长,各组三角褐指藻的生长均处于先上升后下降的趋势。三角褐指藻生长的3个时期很明显,即培养 的1-3d,藻体处于对数生长期,从第3d后一直到第6d藻体的生长速度明显减慢,该时期可以认为藻体处于平台期,从第6d起一直到培养结束,藻体的生物量一直处于下降的趋势,该时段应该为藻体的衰亡期。因此,第6d为平台期末,该时段的生物量达到最大值。从第6d的各组三角和指藻的细胞密度看,无论是高浓度的乙酰水杨酸(50-100mg/L)还是低浓度的乙酰水杨酸(10-25mg/L)处理三角褐指藻,对照组的藻体细胞密度均比它们高。再者,各组浓度的乙酰水杨酸处理的三角褐指藻生长曲线之间都非常紧密,其藻体的细胞密度在第6d时没有很大的差异。
实施例2
设1组为对照组,4组为实验组。对照组不做任何处理,4组实验组分别加入不同浓度的乙酰水杨酸,其浓度分别为10mg/L,25mg/L,50mg/L,100mg/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测定不同浓度乙酰水杨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。
如图2和表1所示:随着乙酰水杨酸浓度的不断上升,三角褐指藻细胞岩藻黄素含量呈先上升后下降的趋势。在低浓度的乙酰水杨酸(10mg/L,25mg/L)处理下,藻细胞岩藻黄素含量显著升高,其中,浓度为10mg/L的乙酰水杨酸处理三角褐指藻后藻细胞岩藻黄素含量最高,为1.83mg/g DW,比对照组岩藻黄素含量的提高了129%。当乙酰水杨酸浓度达到25mg/L时岩藻黄素含量为1.73mg/g DW,比对照组提高了116%。三角褐指藻在高浓度乙酰水杨酸(50mg/L-100mg/L)处理下,其细胞岩藻黄素含量与对照组的含量大致持平。
表1不同浓度的乙酰水杨酸处理三角褐指藻后岩藻黄素含量
乙酰水杨酸浓度(mg/L) | 0 | 10 | 25 | 50 | 100 |
岩藻黄素含量(mg/g DW) | 0.80 | 1.83 | 1.73 | 0.96 | 0.83 |
Claims (2)
1.一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法,其特征在于包括以下步骤:
1)将灭菌海水与MAD母液混合得营养液;其中所述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;并且所述灭菌海水与所述MAD母液的体积比为1000∶1;
2)在营养液中加入三角褐指藻母液培养;所述营养液与三角褐指藻母液的体积比为10∶1;并且培养条件为:62.5μmol photons/(m2·s),25℃;
3)培养2天后,加入乙酰水杨酸;所述乙酰水杨酸的浓度为10mg/L~25mg/L;
4)继续培养6天后收集藻液,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;并且冷冻干燥条件为:温度-70℃,压强999Pa。
2.根据权利要求1所述的一种用乙酰水杨酸提高三角褐指藻岩藻黄素含量的方法,其特征在于:步骤(3)中培养液加入乙酰水杨酸的浓度为10mg/L。
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Non-Patent Citations (3)
Title |
---|
14 种微藻总脂含量和脂肪酸组成研究;蒋霞敏等;《水生生物学报》;20030531;第27卷(第3期);第1.1-1.2节 * |
A potential commercial source of fucoxanthin extracted from the microalga Phaeodactylum tricornutum;Kim SM等;《Appl Biochem Biotechnol.》;20120430;第166卷(第7期);第1845页第1段 * |
施用水杨酸对雨生红球藻中虾青素积累的影响;高政权 等;《水产科学》;20070731;第26卷(第7期);摘要、第377页左栏第1段、 * |
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