CN116375576A - 一种从钝顶螺旋藻中制备γ-亚麻酸和亚油酸的方法 - Google Patents
一种从钝顶螺旋藻中制备γ-亚麻酸和亚油酸的方法 Download PDFInfo
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Abstract
本发明属于藻类活性成分多不饱和脂肪酸提取制备技术领域,公开一种从钝顶螺旋藻中制备γ‑亚麻酸和亚油酸的方法,步骤如下:螺旋藻粉破壁处理,采用超声破壁的方法对螺旋藻粉进行破壁;螺旋藻油的提取,采用索氏提取法对超声破壁藻粉进行回流加热,得到螺旋藻油;游离脂肪酸的制备,通过对螺旋藻油进行皂化回流,酸化后可得游离脂肪酸。尿素包合法富集,通过对游离脂肪酸进行低温结晶、冷却结晶,抽滤后可得尿素不包合物。通过对尿素不包合物酸化、除去残留尿素可得富含γ‑亚麻酸和亚油酸的PUFAs产品。PUFAs纯度可达90~95%,PUFAs产品中γ‑亚麻酸的含量为13~14%,亚油酸含量为15~17%。
Description
技术领域
本发明属于藻类活性成分多不饱和脂肪酸提取制备技术领域,具体是从钝顶螺旋藻中制备γ-亚麻酸和亚油酸。
背景技术
螺旋藻(Spirulina)是蓝藻门藻蓝纲段殖体目颤藻科的水生植物,是地球上最早出现的能进行光合作用的丝状多细胞螺旋形原核藻类生物,我国生产应用的有两个种,即钝顶螺旋藻和极大螺旋藻。据分析,螺旋藻含有40~70%的蛋白质、5~20%的藻蓝蛋白、0.1~0.5%的包括β-胡萝卜素在内的类胡萝卜素、0.5~2.0%的叶绿素、0.3~2%的油脂、2~8%的多糖和丰富的矿物质元素和微量元素及多种维生素,其中钙离子含量可达0.1~2%,镁离子含量可达0.2~1.5%,钾离子含量可达0.5~2%。由于营养丰富和营养价值高,螺旋藻被营养学家称为“人类营养的微型宝库,”被联合国粮农组织(FAO)誉为“21世纪最佳的理想食品,”也被世界卫生组织(WHO)评为“人类21世纪的最佳保健品”。现代医学研究表明,螺旋藻除了由于营养成分丰富因而具有很高的营养价值外,还由于含有丰富的β-胡萝卜素、藻蓝蛋白和藻蓝素、叶绿素、螺旋藻多糖等生物活性物质,从而使螺旋藻还具有抗肿瘤抗癌、抗氧化抗衰老、抗辐射、提高免疫力等生理功能。近年来,螺旋藻食品的研究与开发比较活跃,开发了螺旋藻片、螺旋藻粉、螺旋藻蛋白质、螺旋藻蓝色素、螺旋藻多肽、螺旋藻饮料、螺旋藻口服液和螺旋藻焙烤食品等形式多样的螺旋藻食品或者螺旋藻保健品。
γ-亚麻酸,又名异亚麻酸、十八碳三烯酸、维生素F、GLA,属于n-6型多不饱和脂肪酸,常作为食品添加剂或营养补充剂使用,具有抗心血管疾病、降血脂、降血糖、抗癌、美白和抗皮肤老化的作用。作为食品添加剂或营养补充剂,长期食用可增强人体免疫机能,它和其它多不饱和脂肪酸一起,被誉为“21世纪功能食品的主角”。婴幼儿、老年人,因肥胖、疾病或饮酒过量引起代谢紊乱的成年人,矿质元素或维生素摄取不足的人,都要补充γ-亚麻酸。国外一些制药或食品公司,为了满足上述特定消费群体的要求,已经为老年人和小孩研制生产了含γ-亚麻酸的食品,如:牛奶、饼干、果汁、口香糖、果冻、饮料等。
亚油酸,又名顺,顺-9,12-十八碳二烯酸,属于n-6型多不饱和脂肪酸,是一种人体不能自行合成的必需脂肪酸。现代药理学研究表明,亚油酸可降低人体血液中的胆固醇,防止动脉粥样硬化,被誉为“血管清道夫”,可降低血液中的甘油三酯、低密度脂蛋白胆固醇和极低密度脂蛋白胆固醇的含量,维持血脂的代谢平衡。因此,亚油酸在医药上主要用于预防和治疗动脉粥样硬化、高血压、心肌梗死等疾病。亚油酸还具有良好的抗炎和抗过敏活性,对皮肤具有深层保湿效果,常被添加于洗护用品中。此外,共轭亚油酸作为亚油酸的几何与位置异构体,具有良好抗癌、抗糖尿病等生物活性,但天然共轭亚油酸仅存在于反刍动物的肉和乳制品中,且含量较低。为满足大规模医疗卫生的需求,需对共轭亚油酸进行商业化生产,实际生产过程中则是利用亚油酸为底物通过化学或生物异构法制得,因此制备高纯度亚油酸是开发利用共轭亚油酸的前提。亚油酸还是制造油漆及油墨等精细化工产品的重要原料。由此可见,亚油酸的应用空间十分广阔,极具经济开发价值。因此,从天然藻类中分离纯化高纯度的亚油酸成为近年来的研究热点。
在以螺旋藻为原料提取多不饱和脂肪酸的过程中,通过超声破壁处理、索氏提取法加热回流来进行藻油的提取,得到了最佳破壁方法、最佳提取溶剂等提油率最高的方法。相较于王一兵等在《马尾藻多不饱和脂肪酸提取、分离及成分分析》所述的提取溶剂配比和方法,加入少量极性溶剂后,使螺旋藻油提油率提高了一倍以上。由于饱和脂肪酸、单不饱和脂肪酸对代谢性疾病无益,PUFAs可以降低心脑血管疾病、降低血液中的胆固醇和甘油三酯改善细胞活性等,但PUFAs生物活性与其组成和纯度密切相关,通过尿素包和法对PUFAs进行高纯度富集,确定了最佳尿素和95%vol.乙醇、尿素和脂肪酸的最佳配比,相较于李冠文等在《尿素包合法富集纯化马齿苋中多不饱和脂肪酸的工艺优化》中的方法PUFAs产品中多不饱和脂肪酸百分比提高了20%。
建立了GC-MS分析方法,定量、定性(面积归一化法、内标法)分析螺旋藻总脂肪酸中饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸的组成百分比。经尿素包合法富集后,对比尿素不包合物(处理后得到PUFAs产品)、尿素包合物的饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸的组成百分比可明显看到富集前后的变化,γ-亚麻酸和亚油酸的含量显著增加。
发明内容
本发明的目的是提供了一种从钝顶螺旋藻中制备γ-亚麻酸和亚油酸的方法。
本发明的技术方案:
一种从钝顶螺旋藻中制备γ-亚麻酸和亚油酸的方法,包括以下步骤:
(1)螺旋藻粉破壁处理:将螺旋藻粉用混合溶液进行超声破壁提取,使螺旋藻粉和混合溶液的料液比g/ml为1:5~30;在1200~2000rpm的转速下离心5~10min,得超声破壁藻粉,收集提取液;其中,混合溶液为体积比为10~20:1的正己烷和乙醇;
(2)螺旋藻藻油提取:将步骤(1)所得超声破壁藻粉进行索氏提取,索氏提取器内加入混合溶剂,温度为85~115℃条件下加热回流,索氏提取2~6h,收集提取液并与步骤(1)所得提取液合并,蒸干得螺旋藻藻油;其中,混合溶液为体积比为10~20:1的正己烷和乙醇,超声破壁藻粉和混合溶剂的料液比g/ml为1:5~30;
(3)游离脂肪酸制备:将螺旋藻藻油进行皂化回流15~30min后,冷却至室温,旋干后加蒸馏水溶解、酸化至PH=2~3、有机溶剂萃取、清洗、干燥、旋干得到游离脂肪酸;
(4)尿素包合法富集:将尿素溶于95%vol.乙醇溶液中,放入50~60℃水浴锅搅拌至澄清,倒入游离脂肪酸中,于50~60℃加热搅拌10~30min,冷却至室温结晶,再进一步置于低温冰箱结晶;抽滤所得滤液旋干为尿素不包合物,所得滤渣为尿素包合物;其中,尿素和乙醇的体积比为1:10、尿素和游离脂肪酸的质量比为10:1,低温冰箱结晶的温度为-20℃,结晶时间为6~15h;
(5)尿素不包合物和尿素包合物的处理:加入蒸馏水充分溶解尿素不包合物或尿素包合物,用6~10mol·L-1的盐酸调节PH=2~3,加入正己烷萃取三次合并正己烷相,去离子水多次洗涤除去残留尿素,直至尿素含量≤0.5mg/L,无水硫酸钠干燥,过滤后旋干得处理后的尿素不包合物或尿素包合物,处理后的尿素不包合物即为PUFAs产品,尿素包合物用于分析对比。
步骤(1)超声功率为50~300W,超声频率40~60KHz,超声时间为10~30min。
步骤(2)中提取得到的螺旋藻油占螺旋藻粉净重量的2.1~2.3%。
步骤(3)中皂化溶液为l.5%(g/ml)NaOH/无水乙醇溶液,酸化溶液为6~10mol·L-1盐酸溶液。
步骤(3)中所需萃取的有机溶剂为正己烷,所需清洗溶剂为去离子水,所述干燥用的干燥剂为无水硫酸钠、无水硫酸铜、五氧化二磷、无水氯化钙或无水硫酸镁。
步骤(5)中PUFAs产品纯度为90~95%。
步骤(5)中PUFAs产品中γ-亚麻酸的含量为13~14%,亚油酸含量为15~17%。
本发明的有益效果:通过超声破壁、索氏提取法提取螺旋藻油,采用体积比为10~20:1的正己烷和乙醇混合溶液作为破壁溶剂和提取溶剂,螺旋藻提油率相较于常规方法提高一倍以上。通过尿素包合法进行多不饱和脂肪酸的富集,采用体积比为1:10尿素和乙醇、质量比为10:1的尿素和游离脂肪酸进行富集得到PUFAs产品,多不饱和脂肪酸含量相较于常规方法提高了20%。
附图说明
图1是定性分析脂肪酸甲酯总离子流图。
图2是面积归一化法脂肪酸甲酯总离子流图。
图3是内标法尿素不包合物脂肪酸甲酯总离子流图。
图4是内标法尿素包合物脂肪酸甲酯总离子流图。
具体实施方式
以下结合附图和技术方案进一步说明本发明的具体实施方式。
实施例1:
(1)称取20g螺旋藻粉末置于500ml锥形瓶中,将200ml正己烷、20ml乙醇混合后加入锥形瓶,超声破壁提取,超声时间20min、超声功率100W、超声频率40KHz。在1200rpm的转速下离心,藻粉和上清液分离后,重复上述过程三次,得超声破壁藻粉,并收集提取液。
(2)将超声破壁藻粉放入滤纸包中密封,将滤纸包放入索氏提取器的萃取室内,将300ml正己烷、30ml乙醇混合后加入索氏提取器的提取瓶内,进行索氏提取金属浴95℃加热回流2h,合并(1)中提取液,得螺旋藻藻油434mg。
(3)将螺旋藻藻油溶于10ml浓度为1.5%(W/V)NaOH/无水乙醇溶液,在55℃下进行皂化回流15min,冷却至室温结晶,旋干后加20ml蒸馏水溶解,加入6mol·L-1盐酸溶液酸化至PH=2,加入15ml的正己烷清洗三次,去离子水多次清洗,合并有机相,无水Na2SO4干燥,旋干得到游离脂肪酸130mg。
(4)将1.300g尿素溶于11ml的95%vol.乙醇溶液中,放入55℃水浴锅中搅拌直至溶液澄清,迅速倒入盛有130mg游离脂肪酸的烧瓶中,混匀后,加热搅拌30min,冷却至室温后开始结晶,然后置于-20℃低温冰箱中更深入结晶,结晶12h后,抽滤所得滤液旋干得尿素不包合物,滤渣为尿素不包合物。
(5)将尿素不包合物加入蒸馏水清洗,用尿素检测试纸检测,直至检测不到尿素为止。用6mol·L-1盐酸溶液酸化至PH=2后,每次加入20ml正己烷多次萃取,用无水硫酸钠干燥4h,旋干可得PUFAs产品。
(6)面积归一化法分析:
将PUFAs用5%(V/V)盐酸/乙醇溶液在55℃下进行甲酯化,可得到脂肪酸甲酯,通过GC-MS(气相色谱质谱联用分析)、面积归一化法定性定量测定脂肪酸百分含量。
试样中某个脂肪酸占总脂肪酸的百分比Yi按如下公式计算,通过测定相应峰面积对所有成分峰面积总和的百分数来计算给定组分i的含量:
GC-MS总离子流图如图2,测定可得多不饱和脂肪酸含量为29.76%,其中γ-亚麻酸13.67%、亚油酸16.09%。步骤(2)提油率为2.17%。
实施例2:
(1)称取20g螺旋藻粉末置于500ml锥形瓶中,将220ml正己烷加入锥形瓶,超声破壁提取,超声时间20min、超声功率100W、超声频率40KHz。在1200rpm的转速下离心,藻粉和上清液分离后,重复上述过程三次,得超声破壁藻粉,并收集提取液。
(2)将超声破壁藻粉放入滤纸包中密封,将滤纸包放入索氏提取器的萃取室内,将300ml正己烷、30ml乙醇混合后加入索氏提取器的提取瓶内,进行索氏提取金属浴95℃加热回流2h,合并(1)中提取液,得螺旋藻藻油206mg。
(3)将螺旋藻藻油溶于10ml浓度为l.5%(W/V)NaOH/无水乙醇溶液,在55℃下进行皂化回流15min,冷却至室温结晶,旋干后加20ml蒸馏水溶解,加入6mol·L-1盐酸溶液酸化至PH=2,加入15ml的正己烷清洗三次,去离子水多次清洗,合并有机相,无水Na2SO4干燥,旋干得到游离脂肪酸62mg。
(4)将0.620g尿素溶于5ml的95%vol.乙醇溶液中,放入55℃水浴锅中搅拌直至溶液澄清,迅速倒入盛有62mg游离脂肪酸的烧瓶中,混匀后,加热搅拌30min,冷却至室温后开始结晶,然后置于-20℃低温冰箱中更深入结晶,结晶12h后,抽滤所得滤液旋干得尿素不包合物,滤渣为尿素不包合物。
(5)将尿素不包合物加入蒸馏水清洗,用尿素检测试纸检测,直至检测不到尿素为止。用6mol·L-1盐酸溶液酸化至PH=2后,每次加入20ml正己烷多次萃取,用无水硫酸钠干燥4h,旋干可得PUFAs产品。
(6)面积归一化法分析:同实施例1
测定可得多不饱和脂肪酸含量为29.55%,其中γ-亚麻酸13.54%、亚油酸16.01%。步骤(2)提油率为1.03%,采用纯正己烷进行螺旋藻油的提取对比实施例1,提油率明显下降。
实施例3:
(1)称取20g螺旋藻粉末置于500ml锥形瓶中,将200ml正己烷、20ml乙醇混合后加入锥形瓶,超声破壁提取,超声时间30min、超声功率300W、超声频率60KHz。在1200rpm的转速下离心,藻粉和上清液分离后,重复上述过程三次,得超声破壁藻粉,并收集提取液。
(2)将超声破壁藻粉放入滤纸包中密封,将滤纸包放入索氏提取器的萃取室内,将300ml正己烷、30ml乙醇混合后加入索氏提取器的提取瓶内,进行索氏提取金属浴95℃加热回流2h,合并(1)中提取液,得螺旋藻藻油447mg。
(3)将螺旋藻藻油溶于10ml浓度为1.5%(W/V)NaOH/无水乙醇溶液,在55℃下进行皂化回流15min,冷却至室温结晶,旋干后加20ml蒸馏水溶解,加入6mol·L-1盐酸溶液酸化至PH=2,加入15ml的正己烷清洗三次,去离子水多次清洗,合并有机相,无水Na2SO4干燥,旋干得到游离脂肪酸133mg。
(4)将1.340g尿素溶于11ml的95%vol.乙醇溶液中,放入55℃水浴锅中搅拌直至溶液澄清,迅速倒入盛有134mg游离脂肪酸的烧瓶中,混匀后,加热搅拌30min,冷却至室温后开始结晶,然后置于-20℃低温冰箱中更深入结晶,结晶12h后,抽滤所得滤液旋干得尿素不包合物,滤渣为尿素不包合物。
(5)将尿素不包合物加入蒸馏水清洗,用尿素检测试纸检测,直至检测不到尿素为止。用6mol·L-1盐酸溶液酸化至PH=2后,每次加入20ml正己烷多次萃取,用无水硫酸钠干燥4h,旋干可得PUFAs产品。
(6)内标法分析:
①响应因子的计算,通过对37种混合脂肪酸甲酯标准品进行GC-MS分析,响应因子Fi按如下公式进行计算,根据混标中各脂肪酸甲酯浓度和峰面积来计算给定脂肪酸甲酯i的响应因子:
式中:Fi为脂肪酸甲酯i的响应因子;A17为十七碳酸甲酯峰面积;ρSi为混标中各脂肪酸甲酯i的浓度,mg/mL;ASi为脂肪酸甲酯i的峰面积;ρ17为混标中十七碳酸甲酯浓度,mg/mL。
进一步通过筛选可得到所需样品响应因子:
②样品分析,将PUFAs用5%(V/V)盐酸/乙醇溶液在50~60℃下进行甲酯化,可得到脂肪酸甲酯,在脂肪酸甲酯内加入内标物十七碳酸甲酯,通过对脂肪酸甲酯按37种混合脂肪酸甲酯标准品同样方法进行GC-MS分析,脂肪酸含量Xi按如下公式计算,根据脂肪酸甲酯i的峰面积和响应因子、内标物十七碳酸甲酯的浓度和体积、试样的质量来计算脂肪酸i的含量:
式中:Xi为脂肪酸i含量,g/100g;Fi为脂肪酸甲酯i的响应因子;Ai为脂肪酸甲酯i的峰面积;AC17为内标物十七碳酸甲酯峰面积;ρC17为十七碳酸甲酯浓度,mg/mL;VC17为十七碳酸甲酯体积,mL;为脂肪酸甲酯i转化成脂肪酸的系数;m为试样质量,mg;100为每100g试样中含量的系数。
GC-MS总离子流图如图3,测定可得多不饱和脂肪酸含量为29.99%,其中γ-亚麻酸13.96%、亚油酸16.04%。
实施例4:
(1)称取20g螺旋藻粉末置于500ml锥形瓶中,将200ml正己烷、20ml乙醇混合后加入锥形瓶,超声破壁提取,超声时间30min、超声功率300W、超声频率60KHz。在1200rpm的转速下离心,藻粉和上清液分离后,重复上述过程三次,得超声破壁藻粉,并收集提取液。
(2)将超声破壁藻粉放入滤纸包中密封,将滤纸包放入索氏提取器的萃取室内,将300ml正己烷、30ml乙醇混合后加入索氏提取器的提取瓶内,进行索氏提取金属浴95℃加热回流2h,合并(1)中提取液,得螺旋藻藻油442mg。
(3)将螺旋藻藻油溶于10ml浓度为l.5%(W/V)NaOH/无水乙醇溶液,在55℃下进行皂化回流15min,冷却至室温结晶,旋干后加20ml蒸馏水溶解,加入6mol·L-1盐酸溶液酸化至PH=2,加入15ml的正己烷清洗三次,去离子水多次清洗,合并有机相,无水Na2SO4干燥,旋干得到游离脂肪酸134mg。
(4)将0.798g尿素溶于7ml的95%vol.乙醇溶液中,放入55℃水浴锅中搅拌直至溶液澄清,迅速倒入盛有133mg游离脂肪酸的烧瓶中,混匀后,加热搅拌30min,冷却至室温后开始结晶,然后置于-20℃低温冰箱中更深入结晶,结晶12h后,抽滤所得滤液旋干得尿素不包合物,滤渣为尿素不包合物。
(5)将尿素不包合物加入蒸馏水清洗,用尿素检测试纸检测,直至检测不到尿素为止。用6mol·L-1盐酸溶液酸化至PH=2后,每次加入20ml正己烷多次萃取,用无水硫酸钠干燥4h,旋干可得PUFAs产品。
(6)内标法分析:同实施例3。
测定可得多不饱和脂肪酸含量为21.03%,其中γ-亚麻酸9.37%、亚油酸11.66%。对比实施例3,改变尿素和乙醇的体积比、尿素和游离脂肪酸的质量比后多不饱和脂肪酸含量下降。
实施例5:
(1)称取20g螺旋藻粉末置于500ml锥形瓶中,将200ml正己烷、20ml乙醇混合后加入锥形瓶,超声破壁提取,超声时间30min、超声功率300W、超声频率60KHz。在1200rpm的转速下离心,藻粉和上清液分离后,重复上述过程三次,得超声破壁藻粉,并收集提取液。
(2)将超声破壁藻粉放入滤纸包中密封,将滤纸包放入索氏提取器的萃取室内,将300ml正己烷、30ml乙醇混合后加入索氏提取器的提取瓶内,进行索氏提取金属浴95℃加热回流2h,合并(1)中提取液,得螺旋藻藻油450mg。
(3)将螺旋藻藻油溶于10ml浓度为l.5%(W/V)NaOH/无水乙醇溶液,在55℃下进行皂化回流15min,冷却至室温结晶,旋干后加20ml蒸馏水溶解,加入6mol·L-1盐酸溶液酸化至PH=2,加入15ml的正己烷清洗三次,去离子水多次清洗,合并有机相,无水Na2SO4干燥,旋干得到游离脂肪酸135mg。
(4)将1.350g尿素溶于11ml的95%vol.乙醇溶液中,放入55℃水浴锅中搅拌直至溶液澄清,迅速倒入盛有135mg游离脂肪酸的烧瓶中,混匀后,加热搅拌30min,冷却至室温后开始结晶,然后置于-20℃低温冰箱中更深入结晶,结晶12h后,抽滤所得滤液旋干得尿素不包合物,滤渣为尿素不包合物。
(5)将尿素包合物加入蒸馏水清洗,用尿素检测试纸检测,直至检测不到尿素为止。用6mol·L-1盐酸溶液酸化至PH=2后,每次加入20ml正己烷多次萃取,用无水硫酸钠干燥4h,旋干可得处理后的尿素包合物。
(6)内标法分析:同实施例3
GC-MS总离子流图如图4,测定可得饱和脂肪酸含量为73.67%、单不饱和脂肪酸为12.68%,多不饱和脂肪酸含量为13.65%。对比实施例3尿素不包合物,尿素包合物有效包合了饱和脂肪酸、单不饱和脂肪酸,使多不饱和脂肪酸富集到尿素不包和物中。
综上,通过对藻粉进行超声破壁处理,达到破坏螺旋藻细胞的细胞壁或细胞膜的作用。进一步改变超声时间、超声功率和超声频率提高提取效率。采用体积比为10~20:1的正己烷和乙醇混合溶液作为破壁溶剂和提取溶剂,提高索氏提取法进行螺旋藻油的提取,既节约了溶剂,又提高了萃取效率,螺旋藻提油率大幅提高。采用体积比为1:10尿素和乙醇、质量比为10:1的尿素和游离脂肪酸,通过尿素包合法富集螺旋藻脂肪酸中的多不饱和脂肪酸,尿素不包合物处理得到PUFAs产品,多不饱和脂肪酸含量相较于常规方法提高了20%。
Claims (4)
1.一种从钝顶螺旋藻中制备γ-亚麻酸和亚油酸的方法,其特征在于,包括以下步骤:
(1)螺旋藻粉破壁处理:将螺旋藻粉用混合溶液进行超声破壁提取,使螺旋藻粉和混合溶液的料液比g/ml为1:5~30;在1200~2000rpm的转速下离心5~10min,得超声破壁藻粉,收集提取液;其中,混合溶液为体积比为10~20:1的正己烷和乙醇;
(2)螺旋藻藻油提取:将步骤(1)所得超声破壁藻粉进行索氏提取,索氏提取器内加入混合溶剂,温度为85~115℃条件下加热回流,索氏提取2~6h,收集提取液并与步骤(1)所得提取液合并,蒸干得螺旋藻藻油;其中,混合溶液为体积比为10~20:1的正己烷和乙醇,超声破壁藻粉和混合溶剂的料液比g/ml为1:5~30;
(3)游离脂肪酸制备:将螺旋藻藻油进行皂化回流15~30min后,冷却至室温,旋干后加蒸馏水溶解、酸化至PH=2~3、有机溶剂萃取、清洗、干燥、旋干得到游离脂肪酸;
(4)尿素包合法富集:将尿素溶于95%vol.乙醇溶液中,放入50~60℃水浴锅搅拌至澄清,倒入游离脂肪酸中,于50~60℃加热搅拌10~30min,冷却至室温结晶,再进一步置于低温冰箱结晶;抽滤所得滤液旋干为尿素不包合物,所得滤渣为尿素包合物;其中,尿素和乙醇的体积比为1:10、尿素和游离脂肪酸的质量比为10:1,低温冰箱结晶的温度为-20℃,结晶时间为6~15h;
(5)尿素不包合物和尿素包合物的处理:加入蒸馏水充分溶解尿素不包合物或尿素包合物,用6~10mol·L-1的盐酸调节PH=2~3,加入正己烷萃取三次合并正己烷相,去离子水多次洗涤除去残留尿素,直至尿素含量≤0.5mg/L,无水硫酸钠干燥,过滤后旋干得处理后的尿素不包合物或尿素包合物,处理后的尿素不包合物即为PUFAs产品。
2.根据权利要求1所述的方法,其特征在于,步骤(1)超声功率为50~300W,超声频率40~60KHz,超声时间为10~30min。
3.根据权利要求1所述的方法,其特征在于,步骤(3)中皂化溶液为1.5%NaOH的无水乙醇溶液,g/ml;酸化溶液为6~10mol·L-1盐酸溶液。
4.根据权利要求1所述的方法,其特征在于,步骤(3)中所需萃取的有机溶剂为正己烷,所需清洗溶剂为去离子水,干燥所需的干燥剂为无水硫酸钠、无水硫酸铜、五氧化二磷、无水氯化钙或无水硫酸镁。
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