CN113234762B - 一种多相酶法体系改性南极磷虾油的方法 - Google Patents
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Abstract
本发明属生物工程与食品技术领域，公开了一种多相酶法体系改性南极磷虾油的方法，包含以下步骤：(1)南极磷虾油溶于疏水性溶剂得到南极磷虾油溶液，向磷脂酶液中添加可溶性无机盐、亲水性溶剂和南极磷虾油溶液，配成多相体系；(2)将配置好的多相体系置于反应器中恒温摇床反应，反应结束后，静置或离心至分为四相体系，从上自下依次为上液层、富磷脂乳液层、中液层和下液层，收集上液层及乳液层产物，分别为虾青素及EPA和DHA浓缩型改性磷脂，第三层为富酶相，第四层是富盐相及杂质。本发明具有能耗小、原料利用率高、反应条件温和等优点，实现了从南极磷虾油中快速分离提取虾青素及EPA和DHA浓缩型改性磷脂。
Description
技术领域
本发明属于生物工程与食品技术领域，涉及到酶的分离与应用技术，具体涉及一种多相酶法体系改性南极磷虾油的方法。
背景技术
南极磷虾，又叫南极大磷虾，是南极大磷虾可能是地球上最成功的动物物种，也是地球上最大的单种生物资源之一，其现存量最新估计约为6.5-10亿吨，若可捕量以总量的5％来计算，其数量依然接近或者超过现如今世界上每年的水产总量。而且，南极磷虾油是南极磷虾相关产品中营养功效和附加值都很高的产品。研究表明，南极磷虾油在预防心脑血管疾病，促进大脑发育、抗氧化、缓解痛风和类风湿性关节炎等方面都有一定功效。其主要活性成分是磷脂、虾青素、DHA和EPA等。因此广泛应用于食品、保健食品、生物医药等行业，具有较高的深度开发和应用价值。
由于南极磷虾中的活性成分较多，经常作为保健品混用造成浪费；且其中有部分有害物质，例如重金属、氟等；并含有大量的饱和脂肪酸等。目前，南极磷虾油中生物活性组分的分离和提取主要是用传统的有机溶剂萃取，存在着成本高，产物分离困难等问题(Fa-Wen Yin,Da-Yong Zhou，Yan-Fei Liu，et.Extractionand Characterization ofPhospholipid-Enriched Oils from Antarctic Krill(Euphausia Superba)withDifferent Solvents[J].J AQUAT FOOD PROD T.2018；27(3):292-304)；特别是在改性磷脂方面，利用磷脂酶选择性水解油中磷脂，使其失去脂肪酸而形成极性更强的功能性溶血磷脂或甘油磷脂酰胆碱由于具有条件温和、能耗低、副产物少等优势受到了广泛的关注。但是，由于传统油水体系酶催化效率过低，导致整个酶催化过程耗时久，加上磷脂成分复杂，因此加大了工艺对分离的要求，难度大。近年来，为了解决这些难题，科学家进行了大量的尝试(ULTRASON SONOCHEM.2014；21:142-148)。这种方法会导致油的氧化稳定性减弱，并且酶易失活，产物分离难度加大，同时，反应的速率依然较慢，因此亟需开发新型反应体系以适应相关行业的大规模应用需求。
多相界面酶改性及分离体系是本实验室开发的一种新型高效酶催化体系，既具有传统催化体系可同步分离多种物质的优点，而且该体系具有较好的生物相容性，可通过加入酶对其活性物质进行同步改性分离。利用这种方法既可大幅降低酶的生产成本；又可解决产物和酶分离能耗大、成本高、污染大、副产物多等问题。具有能耗小、原料利用率高、反应条件温和等优点，有望从南极磷虾油中直接分离虾青素和富含EPA、DHA的改性磷脂，实现快速便捷的分离南极磷虾油中的营养成分。
发明内容
本发明的目的是针对目前从南极磷虾油中分离提取营养物质过程中，存在的成本高，操作复杂，反应时间长等问题，提供了一种高效快速的多相酶法体系改性南极磷虾油的方法。
为达到上述目的，本发明采用的技术方案如下：
一种多相酶法体系改性南极磷虾油的方法，包含以下步骤：
(1)南极磷虾油溶于疏水性溶剂得到南极磷虾油溶液，向磷酯酶液中添加可溶性无机盐、亲水性溶剂和南极磷虾油溶液，静置后得到多相体系；(2)将配置好的多相体系恒温反应，反应结束后，静置或离心至分为四层，从上自下依次为上液层、富磷脂乳液层、中液层和下液层，收集上液层和乳液层产物，分别为虾青素、EPA和DHA浓缩型改性磷脂。
优选地，步骤(1)所述的南极磷虾油与疏水性溶剂的质量比为(0.1-1)：1，所述可溶性无机盐、亲水性溶剂和南极磷虾油溶液与酶液的质量比为(0.1-1)：(0.08-1)：(0.05-5)：1。
优选地，步骤(1)所述的南极磷虾油与疏水性溶剂的质量比为(0.1-0.8)：1，所述可溶性无机盐、亲水性溶剂和南极磷虾油溶液与酶液的质量比为(0.2-0.8)：(0.2-0.8)：(0.2-4)：1。
优选地，步骤(1)所述疏水性溶剂是正己烷、环己烷、乙酸乙酯、氯仿、二氯甲烷、甲苯或二甲苯中的一种或两种以上。
优选地，步骤(1)所述可溶性无机盐是无水硫酸钠、柠檬酸钠、氯化钠、硫酸铵、碳酸钠、磷酸氢二钾、磷酸二氢钾和磷酸氢二钾中的一种或两种以上。
优选地，步骤(1)所述亲水性溶剂为聚乙二醇、聚丙二醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇或乙二醇中的一种或两种以上；或者所述亲水性剂为[BMIM]Br、[BMIM]BF4、[EMIM]ETSO4、[OMIM]Cl和[EMIM]ETSO4中的一种或两种以上。
优选地，步骤(1)所述亲水性溶剂为聚乙二醇400或聚乙二醇600。
优选地，步骤(2)所述多相体系的pH值为3-13，所述反应条件为：温度20-80℃，时间0.5-120h，转速200～300条件下震荡反应。
优选地，所述磷脂酶浓度为5-200U/mL。
优选地，所述磷脂酶为磷脂酶A或磷脂酶C。
与现有技术相比，本发明的有益效果如下：
本发明利用多相酶法体系分离提取南极磷虾油中虾青素和富含EPA、DHA的改性磷脂，简化了分离提取工艺，时间缩短，成本降低，克服了目前从南极磷虾油中分离提取虾青素和改性磷脂过程中存在的成本高，连续化生产困难，产物抑制等问题，提供了一种绿色高效便捷的分离提取方法。该过程既提高催化效率，也利于酶的回收与利用，降低酶的损耗。利用多相体系可将油脂分配在上相，磷酯酶分配在富溶剂相或富盐相，改性磷脂富集于油相和富溶剂相之间的中间相，经简单离心或静置等方式即可将产物回收利用。该方法反应条件温和，原料利用率高，解决了酶法分离提取南极磷虾油中虾青素和磷脂难于工业化的技术难题。
附图说明
图1为反应前三相体系的照片。
图2为反应后离心得到的四相体系的照片。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
本实施例中使用的磷酯酶为磷脂酶A(Lecitase Ultra)，购于诺维信(中国)生物科技有限公司。南极磷虾油购于青岛海洋所。
实施例1
将1g南极磷虾油溶于9mL正己烷得到南极磷虾油溶液，配置适量1％的Ultra磷脂酶A置于锥形瓶中，取3.3g酶液(酶浓度50U/mL)，1.2g的无水硫酸钠，混匀后加入1.5g聚乙二醇400和3g南极磷虾油溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在50℃下反应2h。反应结束后，设定10000rpm转速离心3min，分为四层，依次为上液层、乳液层、中液层和下液层，上液层产物为甘油酯及虾青素相，乳液层为富EPA和DHA的磷脂相。另取上液层测量其虾青素及EPA和DHA含量，取乳液层测量磷含量及EPA和DHA含量，可得油相中虾青素含量为37.57mg/kg，乳化层磷回收率为82.79％，EPA和DHA含量为34.71％，磷脂酶主要分配在中液层，分配系数K为20.20，中液层酶活回收率为85.20％。
实施例2
将1g南极磷虾油溶于9mL正己烷得到南极磷虾油溶液，配置适量1％的Ultra磷脂酶A置于锥形瓶中，取3.3g酶液(酶浓度50U/mL)，1.2g的硫酸铵，混匀后加入1.5g聚乙二醇400和3g南极磷虾油溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在50℃下反应2h。反应结束后，设定10000rpm转速离心3min，分为四层，依次为上液层、乳液层、中液层和下液层，上液层产物为甘油酯及虾青素相，乳液层为富EPA和DHA的磷脂相。另取上液层测量其虾青素及EPA和DHA含量，取乳液层测量磷含量及EPA和DHA含量，可得油相中虾青素含量为38.23mg/kg，乳化层磷回收率为82.98％，EPA和DHA含量为36.50％，磷脂酶主要分配在中液层，分配系数K为20.06，中液层酶活回收率为84.75％。
实施例3
将1g南极磷虾油溶于9mL正己烷得到南极磷虾油溶液，配置适量1％的Ultra磷脂酶A置于锥形瓶中，取3.3g酶液(酶浓度50U/mL)，1.2g的无水硫酸钠，混匀后加入1.5g聚乙二醇600和3g南极磷虾油溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在50℃下反应2h。反应结束后，设定10000rpm转速离心3min，分为四层，依次为上液层、乳液层、中液层和下液层，上液层产物为甘油酯及虾青素相，乳液层为富EPA和DHA的磷脂相。另取上液层测量其虾青素及EPA和DHA含量，取乳液层测量磷含量及EPA和DHA含量，可得油相中虾青素含量为56.94mg/kg，乳化层磷回收率为85.98％，而EPA和DHA含量为38.87％，磷脂酶主要分配在中液层，分配系数K为18.50，中液层酶活回收率为85.22％。
实施例4
将1g南极磷虾油溶于9mL正己烷得到南极磷虾油溶液，配置适量1％的Ultra磷脂酶A置于锥形瓶中，取3.3g酶液(酶浓度50U/mL)，1.2g的硫酸铵，混匀后加入1.5g聚乙二醇600和3g南极磷虾油溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在50℃下反应2h。反应结束后，设定10000rpm转速离心3min，分为四层，依次为上液层、乳液层、中液层和下液层，上液层产物为甘油酯及虾青素相，乳液层为富EPA和DHA的磷脂相。另取上液层测量其虾青素及EPA和DHA含量，取乳液层测量磷含量及EPA和DHA含量，可得油相中虾青素含量为55.92mg/kg，乳化层磷回收率为83.95％，而EPA和DHA含量为38.04％，磷脂酶主要分配在中液层，分配系数K为24.33，中液层酶活回收率为85.22％。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (2)
1.一种多相酶法体系改性南极磷虾油的方法，其特征在于，包含以下步骤：
（1）南极磷虾油溶于疏水性溶剂得到南极磷虾油溶液，向磷脂酶液中添加可溶性无机盐、亲水性溶剂和南极磷虾油溶液，静置后得到多相体系；
（2）将配置好的多相体系恒温反应，反应结束后，静置或离心至分为四层，从上自下依次为上液层、富磷脂乳液层、中液层和下液层，收集上液层和乳液层产物，分别为虾青素、EPA和DHA浓缩型改性磷脂；
步骤（1）所述疏水性溶剂是正己烷；所述可溶性无机盐是无水硫酸钠或硫酸铵；所述亲水性溶剂为聚乙二醇400或聚乙二醇600；
步骤(1)所述的南极磷虾油与疏水性溶剂的质量体积比为1:9 g/mL，所述可溶性无机盐、亲水性溶剂和南极磷虾油溶液与酶液的质量比为1.2:1.5:3：3.3；
所述磷脂酶浓度为5-200U/mL，所述磷脂酶为磷脂酶A。
2.根据权利要求1所述的方法，其特征在于，步骤(2)所述多相体系的pH值为 3-13，所述反应条件为：温度50℃，时间2h，转速200~300条件下震荡反应。
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