菊芋中不同聚合度天然菊糖的分离纯化及活性研究

	菊芋中不同聚合度天然菊糖的分离纯化及活性研究
	李琬聪 1,2
学位类型	博士
导师	郭占勇
	2015-05-13
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	环境科学
关键词	菊芋单一聚合度菊糖 Hpgfc 生物活性聚合度
摘要	菊芋是一种具有开发潜力的生态经济型植物，在我国是生产菊糖的原料之一。菊糖具有肠道益生素、促进矿物质吸收、抗氧化等多种生理功能，对菊糖的研究也引起了人们越来越多的关注。作为一种植物多糖，菊糖聚合度是影响其生理活性的重要因素，但由于菊糖单体间分子量相差较小，单一聚合度的菊糖制备困难，对此的研究也就鲜见报道。在本文中，为从产量和质量两方面提高菊糖生产的效率，我们研究了菊芋块茎整个生长过程中菊糖的含量、聚合度、生物活性的变化规律，并采用凝胶色谱、高效液相色谱（HPLC）、质谱（MS）等分离分析手段，成功纯化了一系列单一聚合度的菊糖，并初步研究了聚合度和生物活性之间的关系。主要研究内容及相关结论如下：首先通过对流动相、流速、色谱柱、标准品、标准曲线等检测条件的分析，最终确定了适用于菊糖分子量测定的高效凝胶过滤色谱（HPGFC）方法：为以超纯水作流动相，0.8 mL/min流速洗脱，Shodex OHpak SB-804HQ色谱柱检测，柱温控制在30℃，样品进样量为每次20 µL，检测器为示差折光检测器（RID），采用Sigma-Aldrich公司生产的右旋糖酐标准品（分子量Mw分别为1000 Da、5000 Da、12000 Da、25000 Da和50000 Da），绘制二次标准曲线y = -2.405 + 1.827x – 0.113x²，检测菊糖的相对分子质量。这一检测方法的确定为之后的研究提供了技术支持。研究了菊芋块茎中菊糖的含量、聚合度和活性在菊芋不同生长阶段的变化。我们采集了从开始生长到完全成熟的各个阶段的菊芋块茎样品，从中成功提取了菊糖。经分析发现，菊糖含量与聚合度的变化都与菊芋生长状态相关，且二者变化趋势类似，皆为先快速增大后略有降低。然后，考察了提取得到的不同聚合度菊糖的益生活性和抗氧化活性，结果表明，菊糖聚合度对其生物活性有非常重要的影响，二者基本呈负相关，即菊糖聚合度越小，活性越高。为进一步明确菊糖聚合度与其生物活性间的关系，需制备单一聚合度的菊糖。对比两种常用的凝胶填料对于菊糖的分离效果后，最终确定采用Bio-gel P-4和Bio-gel P-2凝胶两步法来分离菊糖，经HPLC和ESI-MS分析纯度及结构，最终成功分离得到了二聚到八聚的菊糖，纯度较好，最高可达98%。检测分离得到的7种特定聚合度的菊糖的益生活性，发现二聚菊糖活性最差，而三聚菊糖和四聚菊糖益生活性最佳，五聚之后寡糖益生活性减弱，但五聚菊糖到八聚菊糖间的益生活性变化不明显。此实验结果表明，除去二聚糖外，菊糖的生物活性与聚合度之间依然遵循“低聚合度活性高”的规律，三聚和四聚菊糖表现出最佳活性。本文对于菊芋生长过程中菊糖的含量、聚合度、活性变化的研究，为采用菊芋高效生产高活性天然菊糖提供了理论指导；同时，单一聚合度菊糖的成功分离以及其初步的活性研究，初步揭示了菊糖及其寡糖聚合度与生物活性之间可能的关系，为下一步的深入研究奠定了基础。
其他摘要	Jerusalem artichoke is a kind of ecological and economic plant with the development potential, which is one of primary inulin resources. Inulin has exhibited many bioactivities, such as intestinal prebiotics, promotion of mineral absorption, antioxidant property and so on. The research of inulin has attracted more attention. As a plant polysaccharide, the degree of polymerization (DP) is an important factor affecting inulin’s activity. However, due to small difference of molecular weight between inulin, the separation of inulin with single DP is very difficult and so the report about this study is rare. In this paper, in order to improve the efficiency of inulin production from both the yield and the quality, we have studied the changes of content, DP, and bioactivity of inulin throughout the entire growth process of Jerusalem artichoke tubers. We also successfully obtained a series of inulin-type oligosaccharides with single DP by various analytical techniques, including gel chromatography, high performance liquid phase chromatography (HPLC) and mass spectrometry (MS). In addition, a preliminary study of the relationship between the degree of polymerization and bioactivity was investigated. The major research and results were as follows: At first, according to the study on the mobile phase, the flow rate, the column, the standard substances, and the standard curve, the DP of inulin was analyzed by Agilent 1260 HPLC with a differential refraction detector (RID). A Shodex GPC OHpak SB-804 column was used with ultrapure water as the mobile phase at a flow rate of 0.8 mL/min. The column temperature was set to 30℃ and injection volume was 20 μL. Dextran standard substances (Mw 1000, 5000, 12000, 25000, and 50000) were purchased from Sigma-Aldrich, and the curve was y = -2.405 + 1.827x – 0.113x². This test method was a technical support to our work. The changes of content, DP, and bioactivities of inulin throughout the entire growth process of Jerusalem artichoke tubers were investigated. We harvested the Jerusalem artichoke tubers from the initial to the maturation and extracted inulin. The results showed that the changes of inulin content and DP were related to the growth state of Jerusalem artichoke. They were almost the same, which increased rapidly at the beginning and then decreased gradually at a lower speed. Then the prebiotic effect and antioxidant activity were tested. It indicated that inulin with low DP had higher activity. In order to further clarify the relationship between inulin DP and its bioactivity, it was necessary to prepare inulin with single DP. Two common gels were assessed for inulin separation, and it was ultimately determined to use Bio-gel P-4 and Bio-gel P-2. HPLC and ESI-MS analysis revealed that the inulin-type disaccharide to octasaccharide was successfully obtained, and their purities were up to 98%. The prebiotic effect of the inulin-type oligosaccharides with well-defined DP was tested. The result suggested that the inulin-type disaccharide had the worst activity, whereas the inulin-type trisaccharide and tetrasaccharide had the best. The effects of the inulin-type pentasaccharide to octasaccharide weakened, but the differences among them were unconspicuous. This illustrated that the relationship between inulin DP and its bioactivity still followed the rule that inulin with low DP had higher activity, except disaccharide. The inulin-type trisaccharide and tetrasaccharide showed the best activity. In this work, the study on the changes of inulin content, DP, and activities throughout the entire growth process of Jerusalem artichoke tubers could be the theoretical instruction to produce inulin with high activity efficiently from Jerusalem artichoke tubers. Additionally, the separation of inulin-type oligosaccharides and their activity research preliminarily revealed the the relationship between inulin DP and its bioactivity, which is instructive to the further study.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/7941
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	1.中国科学院烟台海岸带研究所 2.中国科学院大学
第一作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	李琬聪. 菊芋中不同聚合度天然菊糖的分离纯化及活性研究[D]. 北京. 中国科学院大学,2015.

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