根瘤菌胞外多糖的分离纯化及组分分析
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摘要
以根瘤菌发酵液为原料,提取纯化发酵液中胞外多糖(extracellular polysaccharides,EPS),并对其多糖组分进行分析。采用木瓜蛋白酶与sevage法结合脱蛋白,并利用柱层析法对胞外多糖进行单组分离和纯化,分别得到EPS 1-1、EPS 2-1、EPS 3-1、EPS 4-1和EPS 5-1等5种相对较纯的单一组分。分别采用不同色谱法对上述各单一组分的纯度、结构、相对分子量及单糖组成进行分析和测定,表明各个组分均具有多糖特征峰,且均是以β-型糖苷键连接的吡喃糖,其中EPS 1-1和EPS 3-1是β构型的半乳吡喃糖; 5种多糖组分均主要由具有不同摩尔比的半乳糖、葡萄糖和甘露糖组成,除了EPS 4-1之外,在其他组分中还检测到了不同含量的岩藻糖、鼠李糖和阿拉伯糖等。
The extracellular polysaccharides( EPS) were extracted from the fermentation broth of Rhizobium,and their components were analyzed. Proteins were removed from EPS by papain combined with the Sevage method,followed by separating and purifying by column chromatography. Five relatively pure single components: EPS 1-1,EPS2-1,EPS 3-1,EPS 4-1,and EPS 5-1 were purified. It was found that each component had a characteristic peak of polysaccharides,and all of them were pyranose linked by β-type glycosidic bonds. Moreover,EPS 1-1 and EPS 3-1 were galactose pyranose in a β-configuration. Furthermore,these five polysaccharides were mainly composed of galactose,glucose,and mannose with different molar ratios. Additionally,EPS 1-1,EPS 2-1,EPS 3-1,and EPS 5-1 also had different levels of fucose,rhamnose,and arabinose. Overall,this study lays a good foundation for further study on structures and activities of EPS.
The extracellular polysaccharides( EPS) were extracted from the fermentation broth of Rhizobium,and their components were analyzed. Proteins were removed from EPS by papain combined with the Sevage method,followed by separating and purifying by column chromatography. Five relatively pure single components: EPS 1-1,EPS2-1,EPS 3-1,EPS 4-1,and EPS 5-1 were purified. It was found that each component had a characteristic peak of polysaccharides,and all of them were pyranose linked by β-type glycosidic bonds. Moreover,EPS 1-1 and EPS 3-1 were galactose pyranose in a β-configuration. Furthermore,these five polysaccharides were mainly composed of galactose,glucose,and mannose with different molar ratios. Additionally,EPS 1-1,EPS 2-1,EPS 3-1,and EPS 5-1 also had different levels of fucose,rhamnose,and arabinose. Overall,this study lays a good foundation for further study on structures and activities of EPS.
引文
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[25] WANG H T,YANG L C,YU H C,et al. Characteristics of fucose-containing polysaccharides from submerged fermentation of Agaricus blazei Murill[J]. Journal of Food&Drug Analysis,2018,26(2):678-687.
[2] NWODO U U,EZEKIEL G,OKOH A I. Bacterial exopolysaccharides:functionality and prospects[J]. International Journal of Molecular Sciences,2012,13(11):14 002-14 015.
[3] HABIBI H,KHOSRAVIDARANI K. Effective variables on production and structure of xanthan gum and its food applications:A review[J]. Biocatalysis&Agricultural Biotechnology,2017,10:130-140.
[4] SILVA N H C S,VILELA C,ALMEIDA A,et al. Pullulan-based nanocomposite films for functional food packaging:Exploiting lysozyme nanofibers as antibacterial and antioxidant reinforcing additives[J]. Food Hydrocolloids,2018,77:921-930.
[5]杨西江,徐田华,徐玲,等.普鲁兰多糖的应用及研究生产现状[J].发酵科技通讯,2010,39(4):25-28.
[6]肖素荣,高清山,李京东.新型食品添加剂可得然胶的性质及应用[J].中国食物与营养,2008(1):25-26.
[7] MANGOLIM C S,DA SILVA T T,FENELON V C,et al.Use of FT-IR,FT-Raman and thermal analysis to evaluate the gel formation of curdlan produced by Agrobacterium sp.IFO 13140 and determination of its rheological properties with food applicability[J]. Food Chemistry,2017,232:369.
[8] OZTURK S,ASLIM B,SULUDERE Z,et al. Metal removal of cyanobacterial exopolysaccharides by uronic acid content and monosaccharide composition[J]. Carbohydr Polym,2014,101(2):265-271.
[9] CASTELLANE T C L. Production of exopolysaccharide from Rhizobia with potential biotechnological and bioremediation applications[J]. International Journal of Biological Macromolecules,2015,74:515-522.
[10]陈艳丽,任盛,魏国琴,等. Rhizobium sp. N613胞外多糖的抗氧化活性[J].微生物学通报,2010,37(2):234-238.
[11] KIM J W,LEE J,YOO A Y,et al. Immune-stimulating activity of water-soluble extracellular polysaccharide isolated from Rhizobium massiliae[J]. Process Biochemistry,2017,63:236-243.
[12] PRIYANKA P,ARUN A B,ASHWINI P,et al. Versatile properties of an exopolysaccharide R-PS18 produced by Rhizobium sp. PRIM-18[J]. Carbohydrate Polymers,2015,126:215-221.
[13] LIANGQI Z,YANLI C,SHENG R,et al. Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613[J]. Carbohydrate Research,2010,345(5):637-643.
[14]程红兵,韩勇,任盛,等. Rhizobium sp. N613胞外多糖分离纯化及抗S180肉瘤的研究[J].中国药学杂志,2008,43(4):268-272.
[15] URAI M,AIZAWA T,IMAMURA K,et al. Characterization of the chemical structure and innate immune-stimulating activity of an extracellular polysaccharide from Rhizobium sp. strain M2 screened using a silkworm muscle contraction assay[J]. Drug Discoveries&Therapeutics,2017,11(5):238-245.
[16]魏杰,吕磊,金莉莉,等.茄枝多糖的分离纯化及成分分析[J].食品科学,2015,36(4):168-171.
[17]王卫国,吴强,胡宝坤,等.几种测定灰树花多糖中蛋白质含量方法的比较研究[J].中国食用菌,2003(1):27-30.
[18]刘景煜,李晨,肖林刚,等.双水相萃取法分离纯化金针菇子实体多糖[J].食品与发酵工业,2017,43(5):255-260.
[19]刘璐,乔宇,汪兰,等.山药多糖的抗氧化作用研究[J].食品科技,2014,39(12):212-216.
[20]雷思敏,肖榕,章莹,等.铁皮石斛中性多糖分离纯化及其体内免疫调节作用研究[J].中药新药与临床药理,2018(6):748-753.
[21] LI N,MAO W,YAN M,et al. Structural characterization and anticoagulant activity of a sulfated polysaccharide from the green alga Codium divaricatum[J]. Carbohydrate Polymers,2015,121:175-182.
[22] ZHOU G,SUN Y P,XIN H,et al. In vivo antitumor and immunomodulation activities of different molecular weightλ-carrageenans from Chondrus ocellatus[J]. Pharmacological Research,2004,50(1):47-53.
[23] SUN L,WANG C,SHI Q,et al. Preparation of different molecular weight polysaccharides from Porphyridium cruentum and their antioxidant activities[J]. International Journal of Biological Macromolecules,2009,45(1):1-47.
[24] YI Y,LAMIKANRA O,SUN J,et al. Activity diversity structure-activity relationship of polysaccharides from lotus root varieties[J]. Carbohydr Polym,2017,190:67-76.
[25] WANG H T,YANG L C,YU H C,et al. Characteristics of fucose-containing polysaccharides from submerged fermentation of Agaricus blazei Murill[J]. Journal of Food&Drug Analysis,2018,26(2):678-687.
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