魔芋葡甘聚糖结构与其稳定性研究
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摘要
魔芋葡甘聚糖(Konjac Glucomannan,简称KGM)是魔芋块茎中存在的一种水溶性的、高分子量多糖,具抗肿瘤、提高机体免疫力等功能。KGM的结构变化是其性能与生物活性变化的原因。国内外关于KGM结构的研究没有深入,更没有深入探讨不同外场条件下其结构的变化,对不同外界条件下KGM结构稳定性及溶液行为认识的缺乏是制约KGM深加工及生物活性调控的瓶劲。
本文尝试通过多种结构分析方法研究KGM分子结构,以及在两种物理场：高压脉冲电场和超高静压场作用下KGM结构稳定性的变化规律,旨在为解决KGM生物功能活性调控提供理论基础。本论文主要研究内容和结论如下：
一、KGM的结构表征
(1)本章研究了未处理过的KGM的红外光谱图、拉曼光谱、χ-射线衍射图、扫描电镜图。实验显示KGM固体具有规律性的纤维链形貌。说明KGM之间存在大量的氢键,而χ-射线衍射图显示KGM固体并未形成稳定的结晶,而是以无定形态存在,这与一般的文献报道相一致。
(2)KGM溶液的透射电镜图显示KGM在水的存在下,将在分子链间形成大量的氢键以维持溶液中胶粒的状态,且胶粒的大小在纳米级。
(3)为了深入了解KGM的详细信息,我们将KGM进行了水解,但实验结果显示：KGM水解时不能实现定点水解,且KGM自身分子链大,重复单元的规律性不强,导致水解产物是一个混合物,难以实现液相分离。同时由于分子量太大,目前的液质联用技术难以对其进行分子量的精确测定。
二、经高压脉冲电场处理后KGM的结构稳定性研究
(1)研究了2种高压脉冲电场500V和1000V处理对KGM的红外光谱图、拉曼光谱、χ-射线衍射图、扫描电镜图的影响。实验显示高压脉冲电场处理对KGM固体状态原有的纤维链没有破坏。但是,KGM之间的氢键可能由于电场的作用出现了减弱的现象,导致红外光谱和拉曼光谱出现了一些变化。
(2)KGM的DSC研究发现高压脉冲电场处理对KGM的热特性也没有明显的影响。
(3)高压脉冲电场是目前发展较快的一种物理处理方式,本文研究了高压脉冲电场处理对KGM的结构和形貌的影响,这为深入研究KGM的结构与性能提供了有益的借鉴,特别是为与KGM相关的食品深加工中物理场作用影响提供理论基础。
三、经超高静压处理后KGM的结构稳定性研究
(1)研究了3种超高静压处理(100Mpa、300Mpa、500Mpa)对KGM的拉曼光谱、扫描电镜图、透射电镜图的影响。实验显示高压处理对KGM固体状态原有的规律性的纤维链有所破坏,在高压下,电镜图片显示规则的纤维链发生了不同程度的断裂。但由于KGM分子链足够长,因此在透射电镜图上可以看出,在水的体系中,KGM依然因分子内和分子间的氢键作用形成大小均一的凝胶颗粒。
(2)KGM的DSC研究发现超高静压处理对KGM的热特性有影响。其中以100Mpa处理和500Mpa处理20分钟以上的影响最为显著。
(3)KGM流变学性质研究发现超高静压处理对KGM的流变学性质有显著影响。其中压力大小的影响强于处理时间的影响,KGM在经100Mpa处理时,流变特性明显发生改变,粘度下降明显,抗剪切力变差。而随着压力的增加,上述参数指标反而有所回升。本文首次将宏观性质与微观结构研究相结合,研究了超高静压场对KGM的影响,这为深入研究KGM的结构与性能提供了有益的借鉴与启发,特别是为与KGM相关的食品深加工中物理场作用影响提供理论基础。
四、荧光探针技术在KGM结构及其稳定性研究中的应用
(1)首次提出在KGM体系中加入荧光探针,利用荧光法来研究KGM在溶液中的聚集态以及对荧光的猝灭作用,为其他多糖的结构及溶液性质研究提供新的思路。
(2)用荧光探针技术研究经超高静压处理的KGM溶液中的形态,得出的结果与流变学相近。超高静压处理的压力较之处理时间对于KGM的结构影响更大,其中以100Mpa处理时,KGM分子链的变化最明显,在荧光强度上表现出回升的趋势。
(3)实验结果证明将荧光探针引入到KGM的结构及稳定性研究可行,且因为荧光分析法具有快速、高灵敏、仪器投入成本低的特点,可以在多糖结构研究中进行推广。
Konjac Glucomannan (referred to KGM) is a water-soluble, high molecular weight polysaccharide, consisting in a kind of taro tube. It has many useful functions such as anti-tumor, improve the immunity et al. Changes in the structure of KGM is its performance reasons for its biological activity. Its formation mechanism and stability haven't been discussed deeply, and furthermore, the formation rule and stability mechanism under different environments also haven't been studied. It has become a barrier for deeply development and biological function regulation of Konjac glucomannan.
In this thesis, we attempt to study the KGM for pure and KGM in two physical fields:high voltage pulsed electric fields and ultra-high static field. The structure and structural stability of KGM were studied, which was aimed at providing structural basis of theory for activity regulation of KGM designed to address the biological functions. In this thesis, the main results were as follows:
1. Structural characterization of pure KGM Structural characterization
(1) In this chapter, the untreated pure KGM were characteried by using infrared spectra, Raman spectra,χ-ray diffraction, scanning electron micrographs. The results were showed that KGM was solid fibers with a regular chain morphology in pure. There were a large number of hydrogen bonds in KGM, andχ-ray diffraction pattern showed the formation of KGM was not stable crystalline solid, but as there was no fixed form, which was consistent with the general literature;
(2) TEM figure showed that KGM in solution formed a large number of molecular chains of hydrogen bonds in order to maintain the state of KGM gel in solution, and the size of KGM gel was hundreds nanometer;
(3) In order to understand details of KGM, KGM were hydrolyzed byβ-mannase, but the results showed: pure short molecular chains of KGM were not achieved because of large molecular chain and unregularity of repeating unit. All the hydrolysis products were showed as a mixture. It was showed difficultly to achieve pure chain by using phase separation. At the same time because the molecular weight was too large, the current LC-MS technique was difficult to accurately determine its molecular weight.
2. Study on the structural stability of KGM treated by high-voltage pulsed electric field
(1) Two kinds of high voltage pulsed electric field treatment were used such as 500V and 1000V. KGM treated by PEF were characterized by using infrared spectra, Raman spectra, x-ray diffraction, scanning electron micrographs. Experiment results showed that high-voltage pulsed electric field on solid state KGM fiber chain was not destroyed from the original structure. However, hydrogen bonds might be due to be weakened for resulting in IR and Raman spectroscopy there had been some small changes.
2) DSC study on KGM treated by PEF showed that pulsed electric field treatment on the thermal properties of KGM had no significant effect.
(3) PEF is the rapid development of a physical approach, this study was providing a theoretical basis to in-depth study of the structure and properties of KGM providing a useful reference particularly associated with the food KGM processing physical fields.
3. Study on the structural stability of KGM treated by ultra-high pressure
(1) Three kinds of high static pressure processing (100Mpa,300Mpa,500Mpa) were used. KGM treated by UHP were characterized by using Raman spectra,χ-ray diffraction, scanning electron micrographs. Experiment results showed that high-voltage pulsed electric field on solid state KGM fiber chain was destroyed from the original structure. However, hydrogen bonds might be due to be weakened for resulting in IR and Raman spectroscopy there had been some small changes. Experiments showed that the solid state pressure on the existing laws of KGM fiber chain had been destroyed. Under high pressure, the rules of electron microscopy images showed different degrees of fiber chain fracture occurred. However, because KGM molecular chain was long enough, it was also showed as small gel particle with intra-molecular and the formation of hydrogen bonds in the TEM figure.
(2) DSC studying on KGM treated by PEF showed that ultra-high pressure treatment on the thermal properties of KGM had some significant effect. Treating for 100Mpa and 500Mpa 20 minutes showed significant effect.
(3) The rheological properties study of KGM treated by ultra-high pressure also had significant effects. The processing pressure showed a stronger effect than the processing time. When KGM was treated by 100Mpa processing, the rheological properties significantly changed, the viscosity was significantly decreased and the shear force variation showed the same. With the increase of pressure, the parameter indexes were all picked up. The macroscopic properties and microscopic structure of KGM were studied, which would provides a useful reference and inspiration to in-depth study of the structure and properties of KGM especially for food-related processing such as physical fields to provide a theoretical basis.
4. Study on KGM Structure and Stability by using Fluorescent probe
(1) This was firstly proposed in the KGM system by added fluorescent probes to study KGM aggregation states in solution and the fluorescence quenching. This study provided a new way of thinking for other polysaccharide structure and their solution properties.
(2) KGM treated by high hydrostatic form of the solution was studied by fluorescence probe technique and the results were similar with rheological properties. When treated by 100Mpa, KGM molecular chain was changed obviously on the show that the fluorescence intensity tended to increase.
(3) The experimental results showed that it was feasible for the fluorescent probe introduced into the structure and stability of KGM, and because the fluorescence analysis was rapid, highly sensitive, low cost. This new structure study could be conducted in the promotion of some other polysaccharide.
本文尝试通过多种结构分析方法研究KGM分子结构,以及在两种物理场：高压脉冲电场和超高静压场作用下KGM结构稳定性的变化规律,旨在为解决KGM生物功能活性调控提供理论基础。本论文主要研究内容和结论如下：
一、KGM的结构表征
(1)本章研究了未处理过的KGM的红外光谱图、拉曼光谱、χ-射线衍射图、扫描电镜图。实验显示KGM固体具有规律性的纤维链形貌。说明KGM之间存在大量的氢键,而χ-射线衍射图显示KGM固体并未形成稳定的结晶,而是以无定形态存在,这与一般的文献报道相一致。
(2)KGM溶液的透射电镜图显示KGM在水的存在下,将在分子链间形成大量的氢键以维持溶液中胶粒的状态,且胶粒的大小在纳米级。
(3)为了深入了解KGM的详细信息,我们将KGM进行了水解,但实验结果显示：KGM水解时不能实现定点水解,且KGM自身分子链大,重复单元的规律性不强,导致水解产物是一个混合物,难以实现液相分离。同时由于分子量太大,目前的液质联用技术难以对其进行分子量的精确测定。
二、经高压脉冲电场处理后KGM的结构稳定性研究
(1)研究了2种高压脉冲电场500V和1000V处理对KGM的红外光谱图、拉曼光谱、χ-射线衍射图、扫描电镜图的影响。实验显示高压脉冲电场处理对KGM固体状态原有的纤维链没有破坏。但是,KGM之间的氢键可能由于电场的作用出现了减弱的现象,导致红外光谱和拉曼光谱出现了一些变化。
(2)KGM的DSC研究发现高压脉冲电场处理对KGM的热特性也没有明显的影响。
(3)高压脉冲电场是目前发展较快的一种物理处理方式,本文研究了高压脉冲电场处理对KGM的结构和形貌的影响,这为深入研究KGM的结构与性能提供了有益的借鉴,特别是为与KGM相关的食品深加工中物理场作用影响提供理论基础。
三、经超高静压处理后KGM的结构稳定性研究
(1)研究了3种超高静压处理(100Mpa、300Mpa、500Mpa)对KGM的拉曼光谱、扫描电镜图、透射电镜图的影响。实验显示高压处理对KGM固体状态原有的规律性的纤维链有所破坏,在高压下,电镜图片显示规则的纤维链发生了不同程度的断裂。但由于KGM分子链足够长,因此在透射电镜图上可以看出,在水的体系中,KGM依然因分子内和分子间的氢键作用形成大小均一的凝胶颗粒。
(2)KGM的DSC研究发现超高静压处理对KGM的热特性有影响。其中以100Mpa处理和500Mpa处理20分钟以上的影响最为显著。
(3)KGM流变学性质研究发现超高静压处理对KGM的流变学性质有显著影响。其中压力大小的影响强于处理时间的影响,KGM在经100Mpa处理时,流变特性明显发生改变,粘度下降明显,抗剪切力变差。而随着压力的增加,上述参数指标反而有所回升。本文首次将宏观性质与微观结构研究相结合,研究了超高静压场对KGM的影响,这为深入研究KGM的结构与性能提供了有益的借鉴与启发,特别是为与KGM相关的食品深加工中物理场作用影响提供理论基础。
四、荧光探针技术在KGM结构及其稳定性研究中的应用
(1)首次提出在KGM体系中加入荧光探针,利用荧光法来研究KGM在溶液中的聚集态以及对荧光的猝灭作用,为其他多糖的结构及溶液性质研究提供新的思路。
(2)用荧光探针技术研究经超高静压处理的KGM溶液中的形态,得出的结果与流变学相近。超高静压处理的压力较之处理时间对于KGM的结构影响更大,其中以100Mpa处理时,KGM分子链的变化最明显,在荧光强度上表现出回升的趋势。
(3)实验结果证明将荧光探针引入到KGM的结构及稳定性研究可行,且因为荧光分析法具有快速、高灵敏、仪器投入成本低的特点,可以在多糖结构研究中进行推广。
Konjac Glucomannan (referred to KGM) is a water-soluble, high molecular weight polysaccharide, consisting in a kind of taro tube. It has many useful functions such as anti-tumor, improve the immunity et al. Changes in the structure of KGM is its performance reasons for its biological activity. Its formation mechanism and stability haven't been discussed deeply, and furthermore, the formation rule and stability mechanism under different environments also haven't been studied. It has become a barrier for deeply development and biological function regulation of Konjac glucomannan.
In this thesis, we attempt to study the KGM for pure and KGM in two physical fields:high voltage pulsed electric fields and ultra-high static field. The structure and structural stability of KGM were studied, which was aimed at providing structural basis of theory for activity regulation of KGM designed to address the biological functions. In this thesis, the main results were as follows:
1. Structural characterization of pure KGM Structural characterization
(1) In this chapter, the untreated pure KGM were characteried by using infrared spectra, Raman spectra,χ-ray diffraction, scanning electron micrographs. The results were showed that KGM was solid fibers with a regular chain morphology in pure. There were a large number of hydrogen bonds in KGM, andχ-ray diffraction pattern showed the formation of KGM was not stable crystalline solid, but as there was no fixed form, which was consistent with the general literature;
(2) TEM figure showed that KGM in solution formed a large number of molecular chains of hydrogen bonds in order to maintain the state of KGM gel in solution, and the size of KGM gel was hundreds nanometer;
(3) In order to understand details of KGM, KGM were hydrolyzed byβ-mannase, but the results showed: pure short molecular chains of KGM were not achieved because of large molecular chain and unregularity of repeating unit. All the hydrolysis products were showed as a mixture. It was showed difficultly to achieve pure chain by using phase separation. At the same time because the molecular weight was too large, the current LC-MS technique was difficult to accurately determine its molecular weight.
2. Study on the structural stability of KGM treated by high-voltage pulsed electric field
(1) Two kinds of high voltage pulsed electric field treatment were used such as 500V and 1000V. KGM treated by PEF were characterized by using infrared spectra, Raman spectra, x-ray diffraction, scanning electron micrographs. Experiment results showed that high-voltage pulsed electric field on solid state KGM fiber chain was not destroyed from the original structure. However, hydrogen bonds might be due to be weakened for resulting in IR and Raman spectroscopy there had been some small changes.
2) DSC study on KGM treated by PEF showed that pulsed electric field treatment on the thermal properties of KGM had no significant effect.
(3) PEF is the rapid development of a physical approach, this study was providing a theoretical basis to in-depth study of the structure and properties of KGM providing a useful reference particularly associated with the food KGM processing physical fields.
3. Study on the structural stability of KGM treated by ultra-high pressure
(1) Three kinds of high static pressure processing (100Mpa,300Mpa,500Mpa) were used. KGM treated by UHP were characterized by using Raman spectra,χ-ray diffraction, scanning electron micrographs. Experiment results showed that high-voltage pulsed electric field on solid state KGM fiber chain was destroyed from the original structure. However, hydrogen bonds might be due to be weakened for resulting in IR and Raman spectroscopy there had been some small changes. Experiments showed that the solid state pressure on the existing laws of KGM fiber chain had been destroyed. Under high pressure, the rules of electron microscopy images showed different degrees of fiber chain fracture occurred. However, because KGM molecular chain was long enough, it was also showed as small gel particle with intra-molecular and the formation of hydrogen bonds in the TEM figure.
(2) DSC studying on KGM treated by PEF showed that ultra-high pressure treatment on the thermal properties of KGM had some significant effect. Treating for 100Mpa and 500Mpa 20 minutes showed significant effect.
(3) The rheological properties study of KGM treated by ultra-high pressure also had significant effects. The processing pressure showed a stronger effect than the processing time. When KGM was treated by 100Mpa processing, the rheological properties significantly changed, the viscosity was significantly decreased and the shear force variation showed the same. With the increase of pressure, the parameter indexes were all picked up. The macroscopic properties and microscopic structure of KGM were studied, which would provides a useful reference and inspiration to in-depth study of the structure and properties of KGM especially for food-related processing such as physical fields to provide a theoretical basis.
4. Study on KGM Structure and Stability by using Fluorescent probe
(1) This was firstly proposed in the KGM system by added fluorescent probes to study KGM aggregation states in solution and the fluorescence quenching. This study provided a new way of thinking for other polysaccharide structure and their solution properties.
(2) KGM treated by high hydrostatic form of the solution was studied by fluorescence probe technique and the results were similar with rheological properties. When treated by 100Mpa, KGM molecular chain was changed obviously on the show that the fluorescence intensity tended to increase.
(3) The experimental results showed that it was feasible for the fluorescent probe introduced into the structure and stability of KGM, and because the fluorescence analysis was rapid, highly sensitive, low cost. This new structure study could be conducted in the promotion of some other polysaccharide.
引文
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