水相体系中磷脂酶A_1酶解南极磷虾磷脂制备溶血磷脂的分析
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摘要
采用磷脂酶A_1(phospholipase A_1,PLA_1)在水相体系中酶解南极磷虾磷脂,以期得到富含二十碳五烯酸(eicosapentaenoic acid,EPA)和二十二碳六烯酸(docosahexaenoic acid,DHA)的溶血磷脂。以酸价衡量磷脂酶解程度,通过高效液相色谱对酶解前后磷脂组分进行分析,结合甘油磷脂酰胆碱(glycerol phosphatidylcholine,G P C)含量分析进一步证实酰基位移现象,并利用气相色谱-质谱分析磷脂酶解前后脂肪酸组成。结果表明:酸价随加酶量的增加呈现先上升后下降的趋势。随着酶解时间的延长,Sn-2-溶血磷脂酰胆碱(Sn-2-lysophosphatidylcholine,Sn-2-LPC)含量先增加后趋于平衡,Sn-1-溶血磷脂酰胆碱(Sn-1-lysophosphatidylcholine,Sn-1-LPC)含量先升高后降低,这是由于部分Sn-2-LPC发生酰基位移生成Sn-1-LPC,而Sn-1-LPC又进一步被PLA1酶解生成GPC,且在较短酶解时间内,温度对酰基位移的影响不大。最后通过气相色谱-质谱分析得出酶解产物Sn-2-LPC中EPA和DHA含量较高,且其乳化稳定性也得到了提高。
In this study, phospholipase A_1 was used to hydrolyze Antarctic krill phospholipids in aqueous media in order to obtain lysophospholipids rich in eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA). The degree of phospholipid hydrolysis was measured by acid value. The composition of phospholipids was analyzed by high performance liquid chromatography(HPLC) and the acyl migration was further confirmed by measuring the content of glycerophosphatidylcholine(GPC). The fatty acid composition of phospholipids was analyzed by gas chromatographymass spectrometry(GC-MS). The results showed that the acid value increased firstly and then decreased with the increase of enzyme loading. As the hydrolysis proceeded, the content of Sn-2-lysophosphatidylcholine(Sn-2-LPC) increased initially and then reached an equilibrium value. The content of Sn-1-lysophosphatidylcholine(Sn-1-LPC) increased first and then decreased. This was due to the acyl migration of some Sn-2-LPC to form Sn-1-LPC, which was further hydrolyzed by LPA1 into glycerol phosphatidylcholine(GPC). Additionally in a short reaction period, temperature had no significant effect on the acyl migration. Finally, GC-MS analysis showed that the contents of EPA and DHA in the enzymatic hydrolysate Sn-2-LPC were relatively high, and the emulsion stability was also improved.
In this study, phospholipase A_1 was used to hydrolyze Antarctic krill phospholipids in aqueous media in order to obtain lysophospholipids rich in eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA). The degree of phospholipid hydrolysis was measured by acid value. The composition of phospholipids was analyzed by high performance liquid chromatography(HPLC) and the acyl migration was further confirmed by measuring the content of glycerophosphatidylcholine(GPC). The fatty acid composition of phospholipids was analyzed by gas chromatographymass spectrometry(GC-MS). The results showed that the acid value increased firstly and then decreased with the increase of enzyme loading. As the hydrolysis proceeded, the content of Sn-2-lysophosphatidylcholine(Sn-2-LPC) increased initially and then reached an equilibrium value. The content of Sn-1-lysophosphatidylcholine(Sn-1-LPC) increased first and then decreased. This was due to the acyl migration of some Sn-2-LPC to form Sn-1-LPC, which was further hydrolyzed by LPA1 into glycerol phosphatidylcholine(GPC). Additionally in a short reaction period, temperature had no significant effect on the acyl migration. Finally, GC-MS analysis showed that the contents of EPA and DHA in the enzymatic hydrolysate Sn-2-LPC were relatively high, and the emulsion stability was also improved.
引文
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[19] LAURENT P,MARIE D,STEPHANIE G,et al.Acyl migration during deacylation of phospholipids rich in docosahexaenoic acid(DHA):an enzymatic approach for evidence and study[J].Biotechnology Letters,2009,31(5):743-749.DOI:10.1007/s10529-009-9932-5.
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[22]李鹤丹,张咚咚,牟光庆,等.高效液相色谱法测定奶粉中主要磷脂组分的含量[J].乳与乳制品,2015, 11(12):33-38. DOI:10.3969/j.issn.1004-4264.2015.11.013.
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[25]吕晴,隋晓,刘坤,等.太平洋磷虾磷脂提取工艺研究及脂肪酸组成分析[J].食品工业科技,2015, 35(15):236-240. DOI:10.13386/j.issn1002-0306.2014.15.043.
[26]刘坤,王兰,薛长湖,等.太平洋磷虾油的亚临界R134a萃取工艺及脂肪酸组成分析[J].食品科学,2013, 34(14):96-99. DOI:10.7506/spkx1002-6630-201314019.
[27] FA W Y, XIAO Y L,XIN R F,et al. Extrusion of Antarctic krill(Euphausia superba)meal and its effect on oil extraction[J].International Journal of Food Science and Technology, 2015,50(3):633-639.DOI:10.1111/ij fs.12673.
[28]孙万成,蒋笃孝.溶血磷脂的理化性质研究[J].现代食品科技,2006,22(2):61-66.DOI:10.3969/j.issn.1673-9078.2006.02.019.
[29]潘丽,谷克仁,常振刚.溶血磷脂乳化稳定性研究[J].粮食与油脂,2007(3):23-25.DOI:10.3969/j.issn.1008-9578.2007.03.008.
[30]孙德伟,李波,陈洪建,等.南极磷虾脂质的亚临界提取及磷脂分析[J].中国油脂,2017, 42(10):1-4. DOI:10.3969/j.issn.1003-7969.2017.10.001.
[31]张薇薇,纵伟,张康逸.水相体系酶法制备溶血磷脂酰乙醇胺研究[J].中国油脂,2015,40(2):81-84.
[32]张康逸,张丽霞,屈凌波,等.溶血磷脂酰基转移机理及有效影响因子研究[J].有机化学,2014, 34(12):2529-2536. DOI:10.6023/cjoc201404019.
[33]孙旭璐,陈慧,郑伟.改性大豆磷脂的HLB值检测方法优化研究[J].粮食与油脂,2015, 28(4):47-48. DOI:10.3969/j.issn.1008-9578.2015.04.013.
[34]李秋慧,隋晓楠,徐靓,等.酶法改性磷脂对大豆蛋白共建体系乳化稳定性及氧化稳定性的影响[J].食品科学,2016, 37(23):64-69.DOI:10.7506/spkx1002-6630-201623011.
[2] ABDELKADER A N, CHUN B S. Characterization of purified phospholipids from krill(Euphausia superba)residues deoiled by supercritical carbon dioxide[J]. Korean Journal of Chemical Engineering,2012,29(7):918-924.DOI:10.1007/s11814-011-0273-4.
[3]张思瑾.太平洋磷虾磷脂提取及磷脂酰胆碱的分离纯化研究[D].青岛:中国海洋大学,2015:1-4.
[4] BENGTSON N S M,SCHLABACH M,NICHOLS P D.A nutritionaltoxicological assessment of Antarctic krill oil versus fish oil dietary supplements[J]. Nutrients, 2014, 6(9):3382-3402. DOI:10.3390/nu6093382.
[5] HARRIS W S, MILLER M, TIGHE A P, et al. Omega-3 fatty acids and coronary heart disease risk:clinical and mechanistic perspectives[J].Atherosclerosis,2008, 197(1):12-24.DOI:10.1016/j.a therosclerosis.2007.11.008.
[6] KRIS-ETHERTON P M, HARRIS W S, APPEL L J. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease[J]. Circulation, 2002, 106(21):2747-2757. DOI:10.1161/01.cir.0000038493.65177.94.
[7] RAMPRASATH V R,EYA L L,ZCHUT S, et al. Supplementation of krill oil with high phospholipid content increases sum of EPA and DHA in erythrocytes compared with low phospholipid krill oil[J].Lipids in Health and Disease, 2015, 14(1):142-150. DOI:10.1186/s12944-015-0142-y.
[8] BUNEA R,El FARRAH K,DEUTSCH L.Evaluation of the effects of neptune krill oil on the clinical course of hyperlipidemia[J].Alternative Medicine Review,2004,9(4):420-428.
[9] ULVEN S M,KIRKHUS B,LAMGLAIT A,et al.Metabolic effects of krill oil are essentially similar to those of fish oil but at lower dose of EPA and DHA,in healthy volunteers[J].Lipids,2011,46(1):37-46.DOI:10.1007/s11745-010-3490-4.
[10] RAMPRASATH V R,EYAL L,ZCHUT S,et al.Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fattyacid supplementation from krill oil versus fish oil[J].Lipids Health Disease,2013,12(1):178-188.DOI:10.1186/1476-511x-12-178.
[11]李招,谷克仁,张海臣.溶血磷脂的制备、分离、检测及应用研究进展[J].中国油脂,2018, 43(6):132-136. DOI:10.3969/j.issn.1003-7969.2018.06.028.
[12]孙清瑞,张东杰,刘志明,等.有机相酶法制备溶血磷脂工艺[J].食品科学,2012,33(18):123-126.
[13]李响.磷脂酶A,的固定化及其催化合成DHA/EPA型磷脂的研究[D].广州:华南理工大学,2015:2-6.
[14] XUAN X,XIAO M F,NIAN R S,et al.Immobilized phospholipase A,-catalyzed acidolysis of phosphatidylcholine from Antarctic krill(Euphausia superba)for docosahexaenoic acid enrichment under supercritical conditions[J].Journal of Molecular Catalysis B:Enzymatic,2016, 126:46-55.DOI:10.1016/j.molcatb.2016.01.011.
[15] PARK C W, KWON S J, HAN J. Transesterification of phosphatidylcholine with eicosapentaenoic acid ether ester using phospholipase A2 in organic solvent[J].Biotechnology Letters.,2000,22:147-150.DOI:10.1016/b978-0-444-89046-7.50107-7.
[16]管伟举,谷克仁,李永端.磷脂酶A,水解大豆浓缩磷脂研究[J].粮食与油脂,2006(5):6-9.DOI:10.3969/j.issn.1008-9578.2006.05.002.
[17]刘元法,王兴国,卢灿明.超声波对大豆水化油脚磷脂酶A2反应体系的影响[J].中国油脂,2006, 31(3):21-24. DOI:10.3321/j.issn:1003-7969.2006.03.007.
[18]张康逸,张丽霞,王兴国,等.溶血磷脂酰胆碱酰基转移的调控机理研究[J].中国油脂,2015,40(4):22-26.
[19] LAURENT P,MARIE D,STEPHANIE G,et al.Acyl migration during deacylation of phospholipids rich in docosahexaenoic acid(DHA):an enzymatic approach for evidence and study[J].Biotechnology Letters,2009,31(5):743-749.DOI:10.1007/s10529-009-9932-5.
[20] AOCS. Phospholipids in lecithin concentrates by thin-layer chromatography:AOCS Ja7-86[S].Urbana:AOCS Press,2003.
[21] AOCS.Acid value of vegetable lecithins:AOCS Ja 6-55[S].Urbana:AOCS Press,2003.
[22]李鹤丹,张咚咚,牟光庆,等.高效液相色谱法测定奶粉中主要磷脂组分的含量[J].乳与乳制品,2015, 11(12):33-38. DOI:10.3969/j.issn.1004-4264.2015.11.013.
[23]全国粮油标准化技术委员会.酶改性磷脂中1-和2-溶血磷脂酰胆碱的测定:GB/T22506-2008[S].北京:中国标准出版社,2008.
[24]国家食品药品监督管理总局.食品中脂肪酸的测定:GB5009.168-2016[S].北京:中国标准出版社,2016.
[25]吕晴,隋晓,刘坤,等.太平洋磷虾磷脂提取工艺研究及脂肪酸组成分析[J].食品工业科技,2015, 35(15):236-240. DOI:10.13386/j.issn1002-0306.2014.15.043.
[26]刘坤,王兰,薛长湖,等.太平洋磷虾油的亚临界R134a萃取工艺及脂肪酸组成分析[J].食品科学,2013, 34(14):96-99. DOI:10.7506/spkx1002-6630-201314019.
[27] FA W Y, XIAO Y L,XIN R F,et al. Extrusion of Antarctic krill(Euphausia superba)meal and its effect on oil extraction[J].International Journal of Food Science and Technology, 2015,50(3):633-639.DOI:10.1111/ij fs.12673.
[28]孙万成,蒋笃孝.溶血磷脂的理化性质研究[J].现代食品科技,2006,22(2):61-66.DOI:10.3969/j.issn.1673-9078.2006.02.019.
[29]潘丽,谷克仁,常振刚.溶血磷脂乳化稳定性研究[J].粮食与油脂,2007(3):23-25.DOI:10.3969/j.issn.1008-9578.2007.03.008.
[30]孙德伟,李波,陈洪建,等.南极磷虾脂质的亚临界提取及磷脂分析[J].中国油脂,2017, 42(10):1-4. DOI:10.3969/j.issn.1003-7969.2017.10.001.
[31]张薇薇,纵伟,张康逸.水相体系酶法制备溶血磷脂酰乙醇胺研究[J].中国油脂,2015,40(2):81-84.
[32]张康逸,张丽霞,屈凌波,等.溶血磷脂酰基转移机理及有效影响因子研究[J].有机化学,2014, 34(12):2529-2536. DOI:10.6023/cjoc201404019.
[33]孙旭璐,陈慧,郑伟.改性大豆磷脂的HLB值检测方法优化研究[J].粮食与油脂,2015, 28(4):47-48. DOI:10.3969/j.issn.1008-9578.2015.04.013.
[34]李秋慧,隋晓楠,徐靓,等.酶法改性磷脂对大豆蛋白共建体系乳化稳定性及氧化稳定性的影响[J].食品科学,2016, 37(23):64-69.DOI:10.7506/spkx1002-6630-201623011.