發(fā)布時間：2018-04-01 00:24

  本文選題：植物乳桿菌　切入點(diǎn)：胞外多糖　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：乳酸菌胞外多糖(Exopolysaccharides,EPSs)指的是乳酸菌在生長代謝過程中向菌體細(xì)胞外及其所處基質(zhì)中分泌的多糖類物質(zhì),研究發(fā)現(xiàn),乳酸菌EPSs具有獨(dú)特的流變學(xué)特性和抗氧化、抗腫瘤、免疫調(diào)節(jié)等生理學(xué)活性。因此,本論文以酸馬奶中分離篩選的一株L.plantarum NM18為出發(fā)菌株,對其產(chǎn)生的EPSs進(jìn)行了分離純化、初級結(jié)構(gòu)解析以及體外抗氧化等研究。主要研究結(jié)果如下:1.運(yùn)用DEAE-Cellulose 52和Sepharose CL-6B層析柱對菌株NM18粗多糖進(jìn)行初步分級分離和進(jìn)一步純化,共得到EPS-1A、EPS-1B、EPS-2、EPS-3、EPS-4和EPS-5六個組分;HPSEC法對各組分的純度鑒定證明均為純度較高的EPS組分,同時以葡聚糖為標(biāo)準(zhǔn)品,測得各EPS純化組分的相對分子量分別為2.11×105Da、2.04×105Da、2.02×105 Da、2.17×105 Da、2.09×105 Da、1.93×105 Da。2.分別采用苯酚-硫酸法、考馬斯亮藍(lán)法、氯化鋇-明膠比色法和硫酸-咔唑法測定菌株NM18的CEPS及各EPS純化組分的總糖、蛋白質(zhì)、硫酸基和糖醛酸含量:CEPS、EPS-1A、EPS-1B、EPS-2、EPS-3、EPS-4 和 EPS-5 的總糖含量分別為 75.25%、96.88%、95.98%、94.67%、80.06%、84.32%、75.38%;蛋白含量 CEPS 為 1.13%,EPS-4為7.22%,其余組分均為未檢出;硫酸基含量分別為1.40%、0.22%、0.31%、0.26%、0.42%、6.30%、0.27%;糖醛酸含量分別為 3.24%、0.23%、0.34%、0.25%、0.43%、0.54%、0.85%。3.通過物理和化學(xué)等多種方法進(jìn)行了各EPS純化組分的結(jié)構(gòu)解析:紫外全波長掃描顯示 EPS-1A、EPS-1B、EPS-2、EPS-3、EPS-5 在 260 nm～280nm 附近均無吸收峰,EPS-4在260 nm～280 nm附近有很強(qiáng)吸收峰,表明EPS-4中蛋白質(zhì)含量較高;糖腈乙酸酯衍生物結(jié)合GC-MS分析各EPS純化組分的單糖組成,結(jié)果表明EPS-1A、EPS-1B和EPS-2均由甘露糖、葡萄糖和半乳糖組成,比例分別為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-乙酰葡糖胺組成,含有微量鼠李糖和巖藻糖,比例為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-乙酰葡糖胺組成,比例為1.49:0.36:1:2.49,EPS-5由甘露糖、半乳糖和N-乙酰葡糖胺組成,含有微量巖藻糖及阿洛糖,比例為1:6.73:4.82:0.91:0.11。4.KBr壓片結(jié)合FTIR顯示,各EPS純化組分均顯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的譜圖相似,含有α-D-Galp和α-D-Glcp;EPS-4顯示含有氨基的N—H變角振動峰和硫酸基的S=O伸縮振動峰,說明該組分含有相對較高的蛋白質(zhì)和硫酸基。EPS-1A、EPS-1B和EPS-2的甲基化反應(yīng)結(jié)合GC-MS分析表明,EPS-1A 和 EPS-1B 糖鏈主要由→3)-D-Glcp-(1→、→6)-D-Galp-(1→、→4)-D-Glcp-(1→和→6)-D-Manp-(1→組成,含有 Glcp-(1→末端;EPS-2 糖鏈主要由→6)-D-Galp-(1→和→D-Manp-(1→組成,含有 D-Glcp-(1→末端。1DNMR 研究表明,EPS-1A、EPS-1B和EPS-2的糖環(huán)形式均為吡喃糖環(huán),糖苷鍵構(gòu)型既有α-型又有β-型。EPS-3,EPS-4和EPS-5未進(jìn)行甲基化和1DNMR分析。5.運(yùn)用化學(xué)方法分別分析了 CEPS及各EPS純化組分的體外抗氧化活性,結(jié)果表明:CEPS及各EPS純化組分在體外均能清除DPPH ·自由基和羥基自由基,還原鐵離子,具有一定的體外抗氧化活性。其中,CEPS和EPS-4的整體抗氧化活性相對最高,EPS-1 A和EPS-1B的抗氧化活性相當(dāng),EPS-2,EPS-3和EPS-5在清除DPPH ·自由基、羥基自由基和還原鐵離子方面的差異較大。
[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 紺哄嚭澶氱硸鐨勭壒寰佸惛鏀跺嘲,鍧囧惈鏈夊悺鍠冪硸鑻，

本文編號：1693154