紫球藻多糖的分离纯化及其理化性质分析
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摘要
紫球藻(Porphyridium cruentum.)在生长过程中能产生高活性的硫酸多糖,该多糖的提取制备是研究其活性的重要前提。为提高多糖的提取率,本文采用正交试验设计及二元二次回归正交组合试验方法对影响紫球藻多糖提取率的工艺条件进行了优化,并对脱蛋白工艺进行了比较。在分离纯化的基础上得到一种均一的多糖组份,利用化学方法和波谱技术对其理化性质进行了分析,对其结构进行了初步鉴定,取得的成果如下:
(1)获得了优化的提取工艺:料液比1:4、温度84℃、提取时间2.1h、pH8.0,在此条件下多糖的产量达到4.21g/L;通过对三氯乙酸(TCA)法、Sevag法和酶法脱蛋白工艺的比较表明TCA法3次脱蛋白效果较佳,酶解法次之,Sevag法较差。
(2)经DE52纤维素离子交换柱及Sephacryl TM S-400凝胶过滤层析柱纯化获得了多糖PSP,经高效凝胶渗透色谱法(HPGPC)、纸层析鉴定,表明为均一多糖组分,测得相对分子质量为2.61×106 Da。
(3)经测定PSP的总糖含量为92.6%,糖醛酸的含量为7.8%,硫酸基的含量为11.4%,紫外光谱(UV)表明样品不含游离或裸露的蛋白质和核酸;PSP具有良好的流变学特性,其低浓度的水溶液就具有很高的粘度,具有剪切稀释效应,呈现典型的非牛顿流体特征;其流变学特性非常稳定,不受温度、pH、盐度等环境条件的影响。
(4)红外光谱(IR)表明PSP具有多糖的特征吸收峰,为硫酸化多糖,且提示有β-D-葡萄吡喃糖存在;气相色谱(GC)分析表明该多糖由木糖(Xyl)、葡萄糖(Glc)和半乳糖(Gal)组成,为一种杂多糖,其摩尔比为:2.96:1.25:3.06。
The marine microalga Porphyridium cruentum can synthesize and secrete the sulfated polysaccharides when the cell grows to its steady period. The extraction of the polysaccharide is an important process for activity research. In order to optimize the extraction craft of polysaccharide, the orthogonal experiment and regression analysis were used to investigate the effect factors. Besides, different crafts of deproteinization were comparied. On the basis of separation and purification of polysaccharides from P. cruentum (PSP), the physicochemical properties and structure of PSP were determined by chemical methods and spectrum analysis. All the results were summarized as follows:
(1) The optimum extracting condition was ratio of material and water 1:4, temperature 84℃, extracting time 2.1h and pH 8.0. Under the optimal extracting condition, the maximum yield of PSP was 4.21mg/L. The results of removing protein showed that the method of TCA had the best effect, the next was the method of enzyme, and the last was method of Sevag.
(2) The uniform polysaccharides were obtained by DE52 anion-exchange chromatography and Sephacryl S-400 gel filtration chromatography. The purity of PSP was determined by HPGPC and paper chromatography, and the molecular weight of PSP was 2.61×106 Da.
(3) Analysis results of physiochemical properties showed that the total sugar content of PSP was 92.6%, the aldonic acid content was 7.8% and the sulfuric content was 11.4%. The UV analysis showed there was no free protein and nucleic acid in PSP. The PSP had favourable rheological property, whose water-solution showed high viscosity in low concentration. The water-solution of PSP had shearing dilution effect,and showed typical non-Newtonian fluid property. Besides, the rheological property of the water-solution of PSP was stable, which could not be affected by environmental factors such as temperature, pH, salinity et al.
(4) The IR spectrum showed the characteristic absorption peak of polysaccharide, which indicated that PSP was the sulfated polysaccharide, and included theβ-D-pyranose. GC analysis showed that PSP was a heteropolysaccharide, which consisted of xylose, glucose and galactose, and their moceluar ratio was 2.96:1.25:3.06.
(1)获得了优化的提取工艺:料液比1:4、温度84℃、提取时间2.1h、pH8.0,在此条件下多糖的产量达到4.21g/L;通过对三氯乙酸(TCA)法、Sevag法和酶法脱蛋白工艺的比较表明TCA法3次脱蛋白效果较佳,酶解法次之,Sevag法较差。
(2)经DE52纤维素离子交换柱及Sephacryl TM S-400凝胶过滤层析柱纯化获得了多糖PSP,经高效凝胶渗透色谱法(HPGPC)、纸层析鉴定,表明为均一多糖组分,测得相对分子质量为2.61×106 Da。
(3)经测定PSP的总糖含量为92.6%,糖醛酸的含量为7.8%,硫酸基的含量为11.4%,紫外光谱(UV)表明样品不含游离或裸露的蛋白质和核酸;PSP具有良好的流变学特性,其低浓度的水溶液就具有很高的粘度,具有剪切稀释效应,呈现典型的非牛顿流体特征;其流变学特性非常稳定,不受温度、pH、盐度等环境条件的影响。
(4)红外光谱(IR)表明PSP具有多糖的特征吸收峰,为硫酸化多糖,且提示有β-D-葡萄吡喃糖存在;气相色谱(GC)分析表明该多糖由木糖(Xyl)、葡萄糖(Glc)和半乳糖(Gal)组成,为一种杂多糖,其摩尔比为:2.96:1.25:3.06。
The marine microalga Porphyridium cruentum can synthesize and secrete the sulfated polysaccharides when the cell grows to its steady period. The extraction of the polysaccharide is an important process for activity research. In order to optimize the extraction craft of polysaccharide, the orthogonal experiment and regression analysis were used to investigate the effect factors. Besides, different crafts of deproteinization were comparied. On the basis of separation and purification of polysaccharides from P. cruentum (PSP), the physicochemical properties and structure of PSP were determined by chemical methods and spectrum analysis. All the results were summarized as follows:
(1) The optimum extracting condition was ratio of material and water 1:4, temperature 84℃, extracting time 2.1h and pH 8.0. Under the optimal extracting condition, the maximum yield of PSP was 4.21mg/L. The results of removing protein showed that the method of TCA had the best effect, the next was the method of enzyme, and the last was method of Sevag.
(2) The uniform polysaccharides were obtained by DE52 anion-exchange chromatography and Sephacryl S-400 gel filtration chromatography. The purity of PSP was determined by HPGPC and paper chromatography, and the molecular weight of PSP was 2.61×106 Da.
(3) Analysis results of physiochemical properties showed that the total sugar content of PSP was 92.6%, the aldonic acid content was 7.8% and the sulfuric content was 11.4%. The UV analysis showed there was no free protein and nucleic acid in PSP. The PSP had favourable rheological property, whose water-solution showed high viscosity in low concentration. The water-solution of PSP had shearing dilution effect,and showed typical non-Newtonian fluid property. Besides, the rheological property of the water-solution of PSP was stable, which could not be affected by environmental factors such as temperature, pH, salinity et al.
(4) The IR spectrum showed the characteristic absorption peak of polysaccharide, which indicated that PSP was the sulfated polysaccharide, and included theβ-D-pyranose. GC analysis showed that PSP was a heteropolysaccharide, which consisted of xylose, glucose and galactose, and their moceluar ratio was 2.96:1.25:3.06.
引文
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[7]方积年.多糖的分离纯化及其纯度鉴别与分子量测定[J].药学通报, 1984, 19: 46- 49.
[8]赵国华,陈宗道.活性多糖的研究进展[J].食品与发酵工业, 2001, 27(7): 45.
[9] Falch, B. H., Espevik, T., Ryan, L., and Stokke, B. T. The cytokine stimulating activity of (1→3)-[beta]--glucans is dependent on the triple helix conformation [J]. Carbohydrate Research, 2000, 329: 587-596.
[10]李敏.油菜花粉多糖的分离、纯化、结构鉴定及抗氧化活性的研究[D].南昌大学生命科学学院硕士论文, 2007.
[11]肖锡湘,上官新晨.多糖的应用研究[J].中国食物与营养, 2006, 5, 21-23.
[12]张桂,赵国群.超声波提取枸杞多糖的研究进展[J].食品科学, 2005, 26(9): 302- 305.
[13]陈浩,贺小敏.超声波提取菜籽饼粕多糖[J].食品科学, 2005, 26(9): 359- 341.
[14]李雪华,谢云峰等.荔枝多糖分离纯化及纯度鉴定[J].广西医科大学学报, 2005, 22(3): 366- 367.
[15]Ying Shi, Jianchun Sheng, Fang Yang, et al. Purification and identitication of Polysaccharides derived from chlorella pyrenoidosa. Food Chemistry [J], 2007, 103(l):101-105.
[16]宋燕,李云政.中华芦荟中性糖的分离与鉴定研究[J].中草药, 2001, 32(6): 491- 493.
[17]谭周进,谢达平.多糖的研究进展[J].食品科技, 2002, 3: 10- 12.
[18]何江川,韩永萍.超滤膜分离法在多糖分离提取中的应用[J].食用菌, 2005(1): 5- 8.
[19]张惟杰.复合多糖生化研究技术[M].杭州:浙江大学出版社, 1997.11-198.
[20]韦巍.多糖的研究进展[J].国外医学药学分册, 2005, 32(3): 179- 184.
[21]曾志将,邹阳,杨明.蜜蜂花粉多糖研究进展[J].养蜂科技, 2005, 2: 3- 5.
[22]刘长福,刘涛,刘秀梅.高效液相色谱法测定香菇多糖的分子[J].化学工程师, 2005, (7): 28-29.
[23]叶勇.植物多糖的分离纯化与制备[J].中国食品添加剂, 2001, 5: 29- 31.
[24] Wyss D F, Choi J S. Conformation and function of the N-linked glycan in the adhesiondomain of human [J]. Science.1995.Vo1.269 (228):1273-1278.
[25]孙群,阐健全,赵国华,等.活性多糖构效关系研究进展[J].广州食品工业科技, 2004 20(1): 104-106.
[26]李雪华,盛龙京.木耳多糖的提取与抗活性氧的研究[J].食品科学, 2001, 22(3): 26- 29.
[27] Peters T, Meyer B, Stuike-Prill R, etal. A monte carlo method forconformational analysis of saccharides [J]. Carbohydrate Research, 1993, 238(15):49-73.
[28]王云普,王勇峰,等.植物多糖的研究概况[J].中华使用西医杂志, 2003, 12: 1835- 1837.
[29]曹吉祥.用途广泛的紫球藻[J].中国海洋药物, 1994, 3: 55- 56.
[30] Merchuk J C, Ronen M, Giris S, Arad S. Light/dark cycles in the growth of the red micro alga Porphyridium sp [J]. Biotechnology Bioengin, 1998, 59(6):705.
[31] I.Emma Huertas, Brian Colman, George S, Espie, et al. Active transport of CO2 by three species of marine mi-croalgae [J]. Journal of Phycology, 2000, 36(20):314.
[32]肖华山,范子南.三十烷醇对紫球藻生长及生理活性的影响[J].福建师范大学学报(自然科学版), 1999, 4: 76.
[33]福迪(罗迪安译).藻类学[M].上海:上海科学技术出版社, 1986.187.
[34] Schorstein K H, etal. Ultrastructure of cell division in the unicellular red alga Porphyridium pupureum [J]. Canada Journal Of Botany, 1982, 60:85-97.
[35]陈必链,黄健,梁世中,等.紫球藻的研究进展[J].海洋通报, 2002, 21(5): 75.
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