酸马奶源植物乳杆菌胞外多糖的制备、结构解析及抗氧化研究

发布时间：2018-04-01 00:24

  本文选题：植物乳杆菌　切入点：胞外多糖　出处：《内蒙古农业大学》2017年硕士论文

【摘要】：乳酸菌胞外多糖(Exopolysaccharides,EPSs)指的是乳酸菌在生长代谢过程中向菌体细胞外及其所处基质中分泌的多糖类物质,研究发现,乳酸菌EPSs具有独特的流变学特性和抗氧化、抗肿瘤、免疫调节等生理学活性。因此,本论文以酸马奶中分离筛选的一株L.plantarum NM18为出发菌株,对其产生的EPSs进行了分离纯化、初级结构解析以及体外抗氧化等研究。主要研究结果如下:1.运用DEAE-Cellulose 52和Sepharose CL-6B层析柱对菌株NM18粗多糖进行初步分级分离和进一步纯化,共得到EPS-1A、EPS-1B、EPS-2、EPS-3、EPS-4和EPS-5六个组分;HPSEC法对各组分的纯度鉴定证明均为纯度较高的EPS组分,同时以葡聚糖为标准品,测得各EPS纯化组分的相对分子量分别为2.11×105Da、2.04×105Da、2.02×105 Da、2.17×105 Da、2.09×105 Da、1.93×105 Da。2.分别采用苯酚-硫酸法、考马斯亮蓝法、氯化钡-明胶比色法和硫酸-咔唑法测定菌株NM18的CEPS及各EPS纯化组分的总糖、蛋白质、硫酸基和糖醛酸含量:CEPS、EPS-1A、EPS-1B、EPS-2、EPS-3、EPS-4 和 EPS-5 的总糖含量分别为 75.25%、96.88%、95.98%、94.67%、80.06%、84.32%、75.38%;蛋白含量 CEPS 为 1.13%,EPS-4为7.22%,其余组分均为未检出;硫酸基含量分别为1.40%、0.22%、0.31%、0.26%、0.42%、6.30%、0.27%;糖醛酸含量分别为 3.24%、0.23%、0.34%、0.25%、0.43%、0.54%、0.85%。3.通过物理和化学等多种方法进行了各EPS纯化组分的结构解析:紫外全波长扫描显示 EPS-1A、EPS-1B、EPS-2、EPS-3、EPS-5 在 260 nm～280nm 附近均无吸收峰,EPS-4在260 nm～280 nm附近有很强吸收峰,表明EPS-4中蛋白质含量较高;糖腈乙酸酯衍生物结合GC-MS分析各EPS纯化组分的单糖组成,结果表明EPS-1A、EPS-1B和EPS-2均由甘露糖、葡萄糖和半乳糖组成,比例分别为8.64:2.63:1、3.22:2.68:1和8.23:1.03:1,EPS-3由甘露糖、葡萄糖、半乳糖和N-乙酰葡糖胺组成,含有微量鼠李糖和岩藻糖,比例为0.74:5.47:1.04:1:0.16:0.18,EPS-4由甘露糖、葡萄糖、半乳糖和N-乙酰葡糖胺组成,比例为1.49:0.36:1:2.49,EPS-5由甘露糖、半乳糖和N-乙酰葡糖胺组成,含有微量岩藻糖及阿洛糖,比例为1:6.73:4.82:0.91:0.11。4.KBr压片结合FTIR显示,各EPS纯化组分均显示出多糖的特征吸收峰,均含有吡喃糖苷:其中,EPS-1A、EPS-1B和EPS-2的谱图相似,含有α-D-Galp和α-D-Glcp;EPS-4显示含有氨基的N—H变角振动峰和硫酸基的S=O伸缩振动峰,说明该组分含有相对较高的蛋白质和硫酸基。EPS-1A、EPS-1B和EPS-2的甲基化反应结合GC-MS分析表明,EPS-1A 和 EPS-1B 糖链主要由→3)-D-Glcp-(1→、→6)-D-Galp-(1→、→4)-D-Glcp-(1→和→6)-D-Manp-(1→组成,含有 Glcp-(1→末端;EPS-2 糖链主要由→6)-D-Galp-(1→和→D-Manp-(1→组成,含有 D-Glcp-(1→末端。1DNMR 研究表明,EPS-1A、EPS-1B和EPS-2的糖环形式均为吡喃糖环,糖苷键构型既有α-型又有β-型。EPS-3,EPS-4和EPS-5未进行甲基化和1DNMR分析。5.运用化学方法分别分析了 CEPS及各EPS纯化组分的体外抗氧化活性,结果表明:CEPS及各EPS纯化组分在体外均能清除DPPH ·自由基和羟基自由基,还原铁离子,具有一定的体外抗氧化活性。其中,CEPS和EPS-4的整体抗氧化活性相对最高,EPS-1 A和EPS-1B的抗氧化活性相当,EPS-2,EPS-3和EPS-5在清除DPPH ·自由基、羟基自由基和还原铁离子方面的差异较大。
[Abstract]:Exopolysaccharides of lactic acid bacteria (Exopolysaccharides, EPSs) refers to the lactic acid bacteria cell in growth and metabolic process and the polysaccharide matrix secretion study found that lactic acid bacteria EPSs has unique rheological properties and antioxidant, antitumor activity, immune regulation and other physiological studies. Therefore, in this paper, koumiss was isolated L.plantarum NM18 selected as the starting strain, the EPSs was purified, primary structure analysis and in vitro antioxidant. The main results are as follows: 1. using DEAE-Cellulose 52 and Sepharose CL-6B column to strain NM18 crude polysaccharides were preliminary fractionation and further purification. There are EPS-1A, EPS-1B, EPS-2, EPS-3, EPS-4 and EPS-5 six components; HPSEC method of component purity identification had been proved to be of high purity EPS group, at the same time using dextran as standard, measured The purification of EPS molecular component of the quantity is 2.11 * 105Da, 2.04 * 105Da, 2.02 * 2.17 * 105 Da, 105 Da, 2.09 x 105 x 105 Da, 1.93 Da.2. respectively by phenol sulfuric acid method, Kaumas Bradford method, barium chloride gelatin colorimetry and sulfuric acid carbazole method for the determination of strain NM18 CEPS EPS and the purification of the total sugar, proteins, sulfate and uronic acid content: CEPS, EPS-1A, EPS-1B, EPS-2, EPS-3, total sugar content of EPS-4 and EPS-5 were 75.25%, 96.88%, 95.98%, 94.67%, 80.06%, 84.32%, 75.38%; the protein content of CEPS was 1.13%, EPS-4 was 7.22%. The remaining components were not detected; sulfate contents were 1.40%, 0.22%, 0.31%, 0.26%, 0.42%, 6.30%, 0.27%; the uronic acid content were 3.24%, 0.23%, 0.34%, 0.25%, 0.43%, 0.54%, 0.85%.3. through a variety of physical and chemical methods such as the purification of EPS group structure analysis points: Purple full wavelength scanning display EPS-1A, EP S-1B, EPS-2, EPS-3, EPS-5 in 260 ~ nm near 280nm showed no absorption peak at 260 EPS-4 nm to 280 nm near the strong absorption peak showed higher protein content in EPS-4; Aldononitrile acetate derivatives with GC-MS analysis of the purified EPS component of monosaccharide composition, the results showed that EPS-1A, EPS-1B and EPS-2 by composed of mannose, glucose and galactose, respectively 8.64:2.63:1,3.22:2.68:1 and 8.23:1.03:1, EPS-3 was composed of mannose, glucose, galactose and N- acetyl glucosamine, fucose containing rhamnose and trace rats, ratio of 0.74:5.47:1.04:1:0.16: 0.18, EPS-4 was composed of mannose, glucose, galactose and N-acetylglucosamine N-, ratio of 1.49:0.36:1:2.49 EPS-5 was composed of mannose, galactose, N-acetylglucosamine and N- composition containing trace fucose and allonic, ratio of 1:6.73:4.82:0.91:0.11.4.KBr tablet combined with FTIR showed that the purified components were significantly EPS 绀哄嚭澶氱硸鐨勭壒寰佸惛鏀跺嘲,鍧囧惈鏈夊悺鍠冪硸鑻，

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