等电点法纯化太平洋鳕鱼皮胶原蛋白及其结构特性研究
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摘要
为研究等电点法纯化鳕鱼皮胶原的可行性,通过测定溶解度和Zeta电位来确定鳕鱼皮胶原的等电点。将粗提胶原溶液的pH调至等电点,后经离心、复溶、透析、冻干等步骤得到了等电点纯化后的胶原干品。采用SDS-PAGE法检验产品的胶原纯度;采用红外光谱、X-射线衍射、紫外光谱法对胶原的结构性质进行了研究。最后对等电点沉淀的工艺进行了单因素实验。结果显示鳕鱼皮胶原的等电点为6.83,且在该等电点下纯化得到的产品纯度较高。光谱学结果显示胶原的三螺旋结构仍保持完整。单因素实验结果显示最优条件为:pH6.8、沉淀6 h、6 mg/m L的胶原浓度,此时回收率可达85%,纯度可达90%以上。至此,确定了等电点沉淀是一种纯化胶原蛋白的有效方法。
In order to study the feasibility ofisoelectric precipitation method for the purification of collagen from Pacific cod skin,the isoelectric point of the collagen from cod skin was determined by the solubility and the Zeta potential methods.The pH of the crude collagen solution was adjusted to its isoelectric point,and then the collagen was centrifuged,redissolved,dialysed and lyophilized.The SDS-PAGE method was used to determine the purity of the product. The structure properties of collagen were detected by Fourier transform infrared spectroscopy,X-ray diffraction and ultraviolet absorption spectrum.In the end,the process of isoelectric precipitation was optimized by single-factor test.It was found that the isoelectric point of Pacific cod skin collagen was 6.83 and the product was of high purity. The results of spectroscopic properties showed that the triple helix structure of collagen remained intact.The recovery rate was 85% and the purity was over 90% under the optimized condition of pH6.8,6 h and 6 mg/m L collagen concentration. Based on these results,the isoelectric precipitation method is an effective method for purifying collagen.
In order to study the feasibility ofisoelectric precipitation method for the purification of collagen from Pacific cod skin,the isoelectric point of the collagen from cod skin was determined by the solubility and the Zeta potential methods.The pH of the crude collagen solution was adjusted to its isoelectric point,and then the collagen was centrifuged,redissolved,dialysed and lyophilized.The SDS-PAGE method was used to determine the purity of the product. The structure properties of collagen were detected by Fourier transform infrared spectroscopy,X-ray diffraction and ultraviolet absorption spectrum.In the end,the process of isoelectric precipitation was optimized by single-factor test.It was found that the isoelectric point of Pacific cod skin collagen was 6.83 and the product was of high purity. The results of spectroscopic properties showed that the triple helix structure of collagen remained intact.The recovery rate was 85% and the purity was over 90% under the optimized condition of pH6.8,6 h and 6 mg/m L collagen concentration. Based on these results,the isoelectric precipitation method is an effective method for purifying collagen.
引文
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[20]Sun L,Li B,Song W,et al. Characterization of Pacific cod(Gadus macrocephalus)skin collagen and fabrication of collagen sponge as a good biocompatible biomedical material[J]. Process Biochemistry,2017,63:229-235.
[21]Nazeer R A,Kavitha R,Ganesh R J,et al. Detection of collagen through FTIR and HPLC from the body and foot of Donax cuneatus Linnaeus,1758[J]. Journal of Food Science and Technology,2014,51(4):750-755.
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[3]Zhou H X.Interactions of macromolecules with salt ions:An electrostatic theory for the Hofmeister effect[J].Proteins Structure Function and Bioinformatics,2010,61(1):69-78.
[4]任海涛,钟志勇,郑佳琳,等.鼠尾胶原蛋白提取、分离、纯化方法的建立及鉴定[J].中国比较医学杂志,2012,22(11):50-53.
[5]陈静廷,卜登攀,马露,等.不同等电点沉淀法和超速离心法提取牛奶乳清蛋白的双向电泳分析[J].食品科学,2014,35(20):180-184.
[6]朱劼,董文杰,刘佳.超声波协同等电点沉淀法提取螺旋藻藻胆蛋白工艺的优化[J].食品科学,2010,31(10):146-150.
[7]施正华,李秀芬,宋小莉,等.采用等电点沉淀法回收市政污泥水解液中的蛋白质[J].环境工程学报,2016,10(10):5919-5923.
[8]Pal G K,Suresh P V. Comparative assessment of physicochemical characteristics and fibril formation capacity of thermostable carp scales collagen[J]. Materials Science and Engineering C-Materials for Biological Applications,2017,70(2):32-40.
[9]程海明,王磊,王睿,等.Zeta电位法测定胶原及其降解物的等电点[J].皮革科学与工程,2006,16(6):40-43..
[10]Sun L,Hou H,Li B,et al. Characterization of acid-and pepsin-soluble collagen extracted from the skin of Nile tilapia(Oreochromis niloticus)[J]. International Journal of Biological Macromolecules,2017,99:8-14.
[11]lvarez C,Lélu P,Lynch S,et al.Optimised protein recovery from mackerel whole fish by using sequential acid/alkaline isoelectric solubilization precipitation(ISP)extraction assisted by ultrasound[J].LWT-Food Science and Technology,2018:88.
[12]Jongjareonrak A,Benjakul S,Visessanguan W,et al.Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper(Lutjanus vitta)[J]. Food Chemistry,2006,93(3):475-484.
[13]Tan Y,Chang S. Isolation and characterization of collagen extracted from channel catfish(Ictalurus punctatus)skin[J].Food Chemistry,2018,242:147-155.
[14]Jing W,Pei X,Liu H,et al.Extraction and characterization of acid-soluble and pepsin-soluble collagen from skin of loach(Misgurnus anguillicaudatus)[J]. International Journal of Biological Macromolecules,2018,106:544-550.
[15]Skierka E,Sadowska M.The influence of different acids and pepsin on the extractability of collagen from the skin of Baltic cod(Gadus morhua)[J]. Food Chemistry,2007,105(3):1302-1306.
[16]Giraud-Guille M M,Besseau L,Chopin C,et al.Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states[J].Biomaterials,2000,21(9):899-906.
[17]Doyle B B,Bendit E G,Blout E R.Infrared spectroscopy of collagen and collagen-like polypeptides[J]. Biopolymers,2010,14(5):937-957.
[18]Barth A,Zscherp C.Substrate binding and enzyme function investigated by infrared spectroscopy[J]. Febs Letters,2000,477(3):151-156.
[19]Veeruraj A,Arumugam M,Ajithkumar T,et al.Isolation and characterization of collagen from the outer skin of squid(Doryteuthis singhalensis)[J]. Food Hydrocolloids,2015,43(2015):708-716.
[20]Sun L,Li B,Song W,et al. Characterization of Pacific cod(Gadus macrocephalus)skin collagen and fabrication of collagen sponge as a good biocompatible biomedical material[J]. Process Biochemistry,2017,63:229-235.
[21]Nazeer R A,Kavitha R,Ganesh R J,et al. Detection of collagen through FTIR and HPLC from the body and foot of Donax cuneatus Linnaeus,1758[J]. Journal of Food Science and Technology,2014,51(4):750-755.