薄层层析-图像分析定量热凝胶β-1,3-葡寡糖
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摘要
单向两次薄层层析展开可以显著提高对热凝胶β-1,3-葡寡糖的分析效果,以Silica gel60 F254硅胶板为固定相,V(乙酸)∶V(乙酸乙酯)∶V(水)=2∶2.4∶1.4为展开剂,展距20 cm时,单向两次展开可以较好地分离聚合度为2~13的热凝胶β-1,3-葡寡糖。此外还建立了Image J软件图像分析定量热凝胶β-1,3-葡寡糖的方法,在不同糖质量浓度范围(0.02~0.1、0.1~0.8、0.8~5.0 g/L)内寡糖显色斑点的灰度积分响应值与糖浓度分别有着很好的线性关系(R2>0.99)。该方法的灵敏度高达0.02 g/L,相对标准偏差为1%~5%,稳定性很好。与HPLC等方法相比具有操作简便、分析快速、不需专门仪器和特定寡糖标准品等优点。
The study first found that a unidirectional-twice thin layer chromatography significantly improved the analysis of Curdlan β-1,3-glucooligosaccharides. With the best developing reagents acetic acid,ethyl acetate and H_2O in the ratio of 2∶2.4∶1.4 on the Silica gel 60 F254 plate of 20 cm,Curdlan β-1,3-glucooligosaccharides could be separated from the degree of polymerization 2 to 13.Then,the Image J software was used to quantify Curdlan β-1,3-glucooligosaccharides. A good linear relationship(R2>0.99) existed between saccharides concentration within three ranges of 0.02 g/L~0.1 g/L,0.1 g/L~0.8 g/L and 0.8 g/L ~5.0 g/L and grayscale integration respond. The established quantification had a detection limit of 0.02 g/L and a relative standard deviation of 1 % ~5 % with good stability. Compared with other methods such as HPLC,this method is simple and rapid with nospecialized instruments and oligosaccharides standards.
The study first found that a unidirectional-twice thin layer chromatography significantly improved the analysis of Curdlan β-1,3-glucooligosaccharides. With the best developing reagents acetic acid,ethyl acetate and H_2O in the ratio of 2∶2.4∶1.4 on the Silica gel 60 F254 plate of 20 cm,Curdlan β-1,3-glucooligosaccharides could be separated from the degree of polymerization 2 to 13.Then,the Image J software was used to quantify Curdlan β-1,3-glucooligosaccharides. A good linear relationship(R2>0.99) existed between saccharides concentration within three ranges of 0.02 g/L~0.1 g/L,0.1 g/L~0.8 g/L and 0.8 g/L ~5.0 g/L and grayscale integration respond. The established quantification had a detection limit of 0.02 g/L and a relative standard deviation of 1 % ~5 % with good stability. Compared with other methods such as HPLC,this method is simple and rapid with nospecialized instruments and oligosaccharides standards.
引文
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[3]詹晓北,韩杰,朱莉,等.一种新型的微生物多糖食品添加剂---热凝胶[J].冷饮与速冻食品工业,2001,7(1):27-30,32.ZHAN Xiaobei,HAN Jie,ZHU Li,et al.A novel microbial homo-exopolysaccharide-curdlan sued as food additive[J].Beverag&Fast Frozen Food Industry,2001,7(1):27-30,32.(in Chinese)
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[11]管宁,韩建东,李瑾,等.天然寡糖的研究进展[J].山东农业科学,2013,45(7):141-145.GUAN Ning,HAN Jiandong,LI Jin,et al.Study progress of natural oligosaccharides[J].Shandong Agricultural Sciences,2013,45(7):141-145.(in Chinese)
[12]PANADDA Phattanawasin,UTHAIthai Sotanaphun,LAWAN Sriphong.A comparison of image analysis software for quantitative TLC of Ceftriaxone Sodium[J].Silpakorn University Science and Technology Journal,2011,5(1):7-13.
[13]SHAUN Mustoe,SEAN Mc Crossen.TLC image capture and analysis by use of a prototype device for visualizing fluorescence[J].Journal of Planar Chromatography,2001,14(4):252-255.
[14]A V Gerasumov.Use of the software processing of scanned chromatogram images in quantitative planar chromatography[J].Journal of Analytical Chemistry,2004,59(4):348-353.
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[16]宗玉,李晶,朱莉,等.两步法硫酸水解热凝胶生产β-1,3-葡寡糖[J].工业微生物,2014,44(1):22-27.ZONG Yu,LI Jing,ZHU Li,et al.Preparation ofβ-1,3-glucan oligosaccharides by two-step sulfuric acid hydrolysis of curdlan[J].Industrial Microbiology,2014,44(1):22-27.(in Chinese)
[17]傅赟彬,刘启顺,李曙光,等.可德兰寡糖的制备及其组分分析[J].食品科学,2011,32(3):6-9.FU Yunbin,LIU Qishun,LI Shuguang,et al.Preparation and component analysis of Curdlan oligomers[J].Food Science,2011,32(3):6-9.(in Chinese)
[18]杨绪明,张家骊,李江华,等.庆大霉素发酵液薄层色谱(TLC)分析方法研究[J].食品与生物技术学报,2008,27(5):128-133.YANG Xuming,ZHANG Jiali,LI Jianghua,et al.Studies on determination of gentamicin fermentation broth by TLC[J].Journal of Food Science and Biotechnology,2008,27(5):128-133.(in Chinese)
[19]杨雅麟,王建华,张帆,等.薄层层析法分离菊芋汁中低聚糖[J].理化检验-化学分册,2007,43(3):229-230,232.YANG Yalin,WANG Jianhua,ZHANG Fan,et al.Separation of oligosaccharides in juice of Jerusalem Artichoke by thin layer chromatography[J].Physical Testing and Chemical Analysis Part B:Chemical Analysis,2007,43(3):229-230,232.(in Chinese)
[20]李晶,朱莉,詹晓北,等.定量薄层层析法用于内切β-1,3-葡聚糖酶筛选纯化过程中酶活力测定[J].食品工业科技,2013,34(23):298-302.LI Jing,ZHU Li,ZHAN Xiaobei,et al.Determination of enzymatic activity during endo-β-1,3-glucanase screening and purification processes using quantitative thin layer chromatography[J].Science and Technology of Food Industry,2013,34(23):298-302.(in Chinese)
[21]JOHN F Robyt,RUPENDRA Mukerjea.Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography[J].Carbohydrate Research,1994,251:187-202.
[22]APIRAK Sakunpak,JIRAPORNCHAI Suksaeree,CHAOWALIT Monton,et al.Quantitative analysis ofγ-oryzanol content in cold pressed rice bran oil by TLC-image analysis method[J].Asian Pac J Trop Biomed,2014,4(2):119-123.
[23]LUCIAN Alexandru Fazakas,RODICA Domnica Nascu-Briciu,COSTEL Sarbu.A comparative study concerning the image analysis in thin layer chromatography of fluorescent compounds[J].Journal of Liquid Chromatography&Related Technologies,2011,34(19):2315-2325.
[2]SABINE Demleitner,JOSEF Kraus,GERHARD Franz.Synthesis and antitumour activity of sulfoalkyl derivatives of curdlan and lichenan[J].Carbohydrate Research,1992,226(2):247-252.
[3]詹晓北,韩杰,朱莉,等.一种新型的微生物多糖食品添加剂---热凝胶[J].冷饮与速冻食品工业,2001,7(1):27-30,32.ZHAN Xiaobei,HAN Jie,ZHU Li,et al.A novel microbial homo-exopolysaccharide-curdlan sued as food additive[J].Beverag&Fast Frozen Food Industry,2001,7(1):27-30,32.(in Chinese)
[4]BALLA Sylla,LAURENT Legentil,SUJATA Saraswat-Ohri,et al.Oligo-β-(1→3)-glucans:impact of thio-bridges on immunostimulating activities and the development of cancer stemcells[J].Journal Of Medicinal Chemistry,2014,57(20):8280-8292.
[5]KARINE Descroix,VACLAV Vêtvicka,ISABELLE Laurent,et al.New oligo-β-(1,3)-glucan derivatives as immunostimulating agents[J].Bioorganic&Medicinal Chemistry,2010,18(1):348-357.
[6]HIDA T H,ISHIBASHI K,MIURA N N,et al.Cytokine induction by a linear 1,3-glucan,curdlan-oligo,in mouse leukocytes in vitro[J].Inflammation Research,2009,58(1):9-14.
[7]LI Jing,ZHU Li,LU Guangxing,et al.Curdlanβ-1,3-glucooligosaccharides induce the defense responses against phytophthor infestans infection of potato(Solanum tuberosum L.cv.Mc Cain G1)leaf cells[J].Plos ONE,2014,9(5):1-9.
[8]LI Jing,ZHU Li,ZHAN Xiaobei,et al.Purification and characterization of a new endo-β-1,3-glucanase exhibiting a high specificity for curdlan for production ofβ-1,3-glucan oligosaccharides[J].Food Science and Biotechnology,2014,23(3):799-806.
[9]GRANDPIERRE C,JANSSEN H G,LAROCHE C,et al.Enzymatic and chemical degradation of curdlan targeting the production ofβ-(1→3)oligoglucans[J].Carbohydrate Polymers,2008,71(2):277-286.
[10]JAVIER F Moreno,ANTONIA Montilla,MAR Villamiel,et al.Analysis,Structural characterization,and bioactivity o oligosaccharides derived from lactose[J].Electrophoresis,2014,35(11):1519-1534.
[11]管宁,韩建东,李瑾,等.天然寡糖的研究进展[J].山东农业科学,2013,45(7):141-145.GUAN Ning,HAN Jiandong,LI Jin,et al.Study progress of natural oligosaccharides[J].Shandong Agricultural Sciences,2013,45(7):141-145.(in Chinese)
[12]PANADDA Phattanawasin,UTHAIthai Sotanaphun,LAWAN Sriphong.A comparison of image analysis software for quantitative TLC of Ceftriaxone Sodium[J].Silpakorn University Science and Technology Journal,2011,5(1):7-13.
[13]SHAUN Mustoe,SEAN Mc Crossen.TLC image capture and analysis by use of a prototype device for visualizing fluorescence[J].Journal of Planar Chromatography,2001,14(4):252-255.
[14]A V Gerasumov.Use of the software processing of scanned chromatogram images in quantitative planar chromatography[J].Journal of Analytical Chemistry,2004,59(4):348-353.
[15]FRY S C,NESSELRODE B H,MILLER J G,et al.Mixed-linkage(1-3,1-4)-beta-D-glucan is a major hemicellulose of Equisetum(horsetail)cell walls[J].New Phytologist,2008,179(1):104-115.
[16]宗玉,李晶,朱莉,等.两步法硫酸水解热凝胶生产β-1,3-葡寡糖[J].工业微生物,2014,44(1):22-27.ZONG Yu,LI Jing,ZHU Li,et al.Preparation ofβ-1,3-glucan oligosaccharides by two-step sulfuric acid hydrolysis of curdlan[J].Industrial Microbiology,2014,44(1):22-27.(in Chinese)
[17]傅赟彬,刘启顺,李曙光,等.可德兰寡糖的制备及其组分分析[J].食品科学,2011,32(3):6-9.FU Yunbin,LIU Qishun,LI Shuguang,et al.Preparation and component analysis of Curdlan oligomers[J].Food Science,2011,32(3):6-9.(in Chinese)
[18]杨绪明,张家骊,李江华,等.庆大霉素发酵液薄层色谱(TLC)分析方法研究[J].食品与生物技术学报,2008,27(5):128-133.YANG Xuming,ZHANG Jiali,LI Jianghua,et al.Studies on determination of gentamicin fermentation broth by TLC[J].Journal of Food Science and Biotechnology,2008,27(5):128-133.(in Chinese)
[19]杨雅麟,王建华,张帆,等.薄层层析法分离菊芋汁中低聚糖[J].理化检验-化学分册,2007,43(3):229-230,232.YANG Yalin,WANG Jianhua,ZHANG Fan,et al.Separation of oligosaccharides in juice of Jerusalem Artichoke by thin layer chromatography[J].Physical Testing and Chemical Analysis Part B:Chemical Analysis,2007,43(3):229-230,232.(in Chinese)
[20]李晶,朱莉,詹晓北,等.定量薄层层析法用于内切β-1,3-葡聚糖酶筛选纯化过程中酶活力测定[J].食品工业科技,2013,34(23):298-302.LI Jing,ZHU Li,ZHAN Xiaobei,et al.Determination of enzymatic activity during endo-β-1,3-glucanase screening and purification processes using quantitative thin layer chromatography[J].Science and Technology of Food Industry,2013,34(23):298-302.(in Chinese)
[21]JOHN F Robyt,RUPENDRA Mukerjea.Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography[J].Carbohydrate Research,1994,251:187-202.
[22]APIRAK Sakunpak,JIRAPORNCHAI Suksaeree,CHAOWALIT Monton,et al.Quantitative analysis ofγ-oryzanol content in cold pressed rice bran oil by TLC-image analysis method[J].Asian Pac J Trop Biomed,2014,4(2):119-123.
[23]LUCIAN Alexandru Fazakas,RODICA Domnica Nascu-Briciu,COSTEL Sarbu.A comparative study concerning the image analysis in thin layer chromatography of fluorescent compounds[J].Journal of Liquid Chromatography&Related Technologies,2011,34(19):2315-2325.