pH调节法分离凡纳滨对虾壳中β虾青蛋白的研究
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摘要
采用pH调节法对凡纳滨对虾(Litopenaeus vannamei)虾壳中影响虾壳红变的β虾青蛋白进行分离回收,以蛋白质回收率、纯度和二级结构含量为分离特性参数,以1.0为pH变化梯度,研究pH调节法从虾壳中提取β虾青蛋白的规律。结果显示,pH调节法的回收率为47.5%,所得蛋白质纯度为78.23%、分子量为45 000 Da。该蛋白质在pH3.0和11.0时有最大溶解度,分别为60.5%和55.7%;在pH5.0时溶解度最低为15.4%;等电点为5.6。通过圆二色光谱图分析得知β虾青蛋白是一种以α螺旋为主要二级结构存在的物质,pH调节法所得α螺旋含量为70.1%。
We analyzed the effect of pH-shift processing method on isolation of β crustacyanin from white leg shrimp( Litopenaeus vannamei) shell. The recovery rate,purity and secondary structure content of protein were selected as separation characteristics; the protein was recovered by pH-shifting at a 1. 0 interval via acid and alkali. Under these conditions,the protein recovery rate reached47. 5% with purity of 78. 23%; the molecular weight was 45 000 Da analyzed by SDS-PAGE and Size Exclusion Chromatography; the maximum and minimum solubilities were obtained at pH 3. 0( 60. 5%),11. 0( 55. 7%) and 5. 0( 15. 4%),respectively; the isoelectric point determined by Isoelectric Focusing Electrophoresis was about 5. 6. Secondary structure content obtained by circular dichroism spectroscopy and spectrum revealed that β crustacyanin mainly existed in α helix form and contained nearly 70. 1% α helices via pH-shift processing.
We analyzed the effect of pH-shift processing method on isolation of β crustacyanin from white leg shrimp( Litopenaeus vannamei) shell. The recovery rate,purity and secondary structure content of protein were selected as separation characteristics; the protein was recovered by pH-shifting at a 1. 0 interval via acid and alkali. Under these conditions,the protein recovery rate reached47. 5% with purity of 78. 23%; the molecular weight was 45 000 Da analyzed by SDS-PAGE and Size Exclusion Chromatography; the maximum and minimum solubilities were obtained at pH 3. 0( 60. 5%),11. 0( 55. 7%) and 5. 0( 15. 4%),respectively; the isoelectric point determined by Isoelectric Focusing Electrophoresis was about 5. 6. Secondary structure content obtained by circular dichroism spectroscopy and spectrum revealed that β crustacyanin mainly existed in α helix form and contained nearly 70. 1% α helices via pH-shift processing.
引文
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[21]KRISTINSSON H G,LIANG Y.Effect of pH-shift processing and surimi processing on Atlantic croaker muscle proteins[J].J Food Sci,2006,71(5):304-312.
[22]PALAFOX H,CRDOVA-MURUETA J H,NAVARRETE del TORO M A,et al.Protein isolates from jumbo squid(Dosidicus gigas)by pH-shift processing[J].Process Biochem,2009,44(5):584-587.
[23]刘俊荣,贺凌,汪涛,等.不同pH条件下罗非鱼肌肉蛋白分离效果及其功能特性的研究[J].水产科学,2010,29(6):317-320.
[24]KRISTINSSON H G,THEODORE A E,DEMIR N,et al.A comparative study between acid-and alkali-aided processing and surimi processing for the recovery of proteins from channel catfish muscle[J].J Food Sci,2005,70(4):298-306.
[25]张祥刚,周爱梅,林晓霞,等.南美白对虾虾头、虾壳化学成分的对比研究[J].现代食品科技,2009,25(3):224-227.
[26]CHEN Y C,JACZYNSKI J.Protein recovery from rainbow trout(Oncorhynchus mykiss)processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives[J].J Agric Food Chem,2007,55(22):9079-9088.
[2]UNDELAND I,KELLEHER S D,HULTIN H O.Recovery of functional proteins from herring(Clupea harengus)light muscle by an acid or alkaline solubilization process[J].J Agric Food Chem,2002,50(25):7371-7379.
[3]CHOI Y J,PARK J W.Acid-aided protein recovery from enzymerich Pacific whiting[J].J Food Sci,2002,67(8):2962-2967.
[4]KRISTINSSON H,DEMIR N.Functional fish protein ingredients from fish species of warm and temperate waters:comparison of acidand alkali-aided processing vs.conventional surimi processing[C].Advances in Seafood Byproducts 2002 Conference Proceedings.Anchorage:Alaska Sea Grant College Program University of Alaska,2003:277-295.
[5]YONGSAWATDIGUL J,PARK J W.Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins[J].J Food Sci,2004,69(7):499-505.
[6]MIRELES DeWITT C A,NABORS R L,KLEINHOLZ C W.Pilot pant scale production of protein from catfish treated by acid solubilization/isoelectric precipitation[J].J Food Sci,2007,72(6):351-355.
[7]NOLSOE H,UNDELAND I.The acid and alkaline solubilization process for the isolation of muscle proteins:state of the art[J].Food Bioprocess Tech,2009,2(1):1-27.
[8]GIGLIOTTI J C,JACZYNSKI J,TOU J C.Determination of the nutritional value,protein quality and safety of krill protein concentrate isolated using an isoelectric solubilization/precipitation technique[J].Food Chem,2008,111(1):209-214.
[9]AMAROWICZ R,SYNOWIECKI J,SHAHIDI F.Sephadex LH-20separation of pigments from shells of red sea urchin(Strongylocentrotus franciscanus)[J].Food Chem,1994,51(2):227-229.
[10]TANAKA Y,MATSUGUCHI H,KATAYAMA T,et al.The biosynthesis of astaxanthin.ⅩⅥ.The carotenoids in crustacean[J].Comp Biochem Phys B,1976,54(3):391-394.
[11]胡雅馨,惠伯棣,李阳.食用虾中虾青素类化合物的含量和组成[J].食品科学,2007,28(8):389-392.
[12]CIANCI M,RIZKALLAH P J,OLCZAK A,et al.Structure of lobster apocrustacyanin A1 using softer X-rays[J].Acta Crystallographica D,2001,57(9):1219-1229.
[13]黄卉,李来好,杨贤庆,等.对虾产品质量分级要素及评价技术[J].中国水产科学,2010,17(6):1371-1376.
[14]ZAGALSKY P F.Invertebrate carotenoproteins[J].Method Enzymol,1985,111(9):216-247.
[15]李芳,刘俊荣,梁姗姗,等.南极磷虾蛋白质的分离特性及其组分分析[J].大连海洋大学学报,2013,28(2):191-194.
[16]CLARKE D T.Circular dichroism and its use in protein folding studies[J].Curr Protoc Protein Sci,2012,28:1-17.
[17]JASCO Corporation[Z].J-700 for Windows Software Manual,1995:10.
[18]TIMME E,WALWYN D,BAILEY A.Characterisation of the carotenoprotein found in carapace shells of Jasus lalandii[J].Comp Biochem Phys B,2009,153(1):39-42.
[19]CHAYEN N E,CIANCI M,GROSSMANN J G,et al.Unravelling the structural chemistry of the colouration mechanism in lobster shell[J].Acta Crystallogr D,2003,59(12):2072-2082.
[20]SREERAMA N,WOODY R W.Computation and analysis of protein circular dichroism spectra[J].Meth Enzymol,2004,383(13):318-351.
[21]KRISTINSSON H G,LIANG Y.Effect of pH-shift processing and surimi processing on Atlantic croaker muscle proteins[J].J Food Sci,2006,71(5):304-312.
[22]PALAFOX H,CRDOVA-MURUETA J H,NAVARRETE del TORO M A,et al.Protein isolates from jumbo squid(Dosidicus gigas)by pH-shift processing[J].Process Biochem,2009,44(5):584-587.
[23]刘俊荣,贺凌,汪涛,等.不同pH条件下罗非鱼肌肉蛋白分离效果及其功能特性的研究[J].水产科学,2010,29(6):317-320.
[24]KRISTINSSON H G,THEODORE A E,DEMIR N,et al.A comparative study between acid-and alkali-aided processing and surimi processing for the recovery of proteins from channel catfish muscle[J].J Food Sci,2005,70(4):298-306.
[25]张祥刚,周爱梅,林晓霞,等.南美白对虾虾头、虾壳化学成分的对比研究[J].现代食品科技,2009,25(3):224-227.
[26]CHEN Y C,JACZYNSKI J.Protein recovery from rainbow trout(Oncorhynchus mykiss)processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives[J].J Agric Food Chem,2007,55(22):9079-9088.