果胶多糖超声波定向降解途径及机理研究
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摘要
果胶是存在于高等植物初生细胞壁和中胞层中的一种酸性多糖,其因天然、无毒且价廉已被广泛应用于食品、医药和化妆品等工业中。果胶分子量较大,且根据来源、种类、在植物组织中存在的位置、植物生长阶段以及萃取条件和环境等的不同而结构各异,这些因素限制了果胶的广泛应用,因此,对果胶进行改性(改变其分子量和结构状态)以适应不同的需要成为了近些年研究者的一个研究热点。果胶改性的方法主要有化学法和酶法,这些方法费时、对环境不友好,且难以控制,不具有定向性。因此,探索物理的改性方法势在必行。超声波作为一种绿色的物理方法,已被广泛的应用于多糖等聚合体的改性研究中,但是关于超声波降解果胶多糖的研究在国内外鲜见报道。
本论文以商业果胶为研究对象,首次对超声波降解果胶多糖的降解规律、降解途径与机理以及降解产物的结构与功能进行了系统研究；分析研究了超声波降解果胶多糖的动力学模型；借助原子力显微镜技术从纳米水平上揭示了果胶多糖在超声波降解下的降解途径；并通过对超声波降解果胶多糖自由基与机械效应的分析研究,揭示了超声波降解果胶多糖的机理；同时结合与酸法降解果胶多糖的比较研究,验证了超声波技术降解果胶多糖具有简单、快速、经济且环保等优点,建立了果胶多糖的超声波降解方法。通过研究,本论文主要得出如下结论：
(1)超声波降解果胶多糖的降解规律研究：通过对超声波降解果胶多糖分子量及其分布的研究确定了超声波降解果胶多糖的降解规律。研究结果显示：随着超声波时间的增加,果胶分子量逐渐降低,降解速率逐渐减小；随着超声波强度的增加,果胶分子量逐渐减小,当强度增加到一定程度后,果胶分子量维持平衡；低温有利于超声波降解果胶分子；随着超声波脉冲占空比的增加,超声波空化效应先增大后减小,当脉冲占空比为50%时,超声波空化效应最强,果胶分子量降低最多。
(2)超声波降解果胶多糖的降解途径研究：通过对超声波降解前后果胶结构的比较研究确定了超声波降解果胶多糖的途径。研究结果显示：超声波降解果胶的分子量分布变窄且更加均匀；当超声波降解时间为90min时,果胶分子量小于100kDa的片段可达47%；超声波降解不改变果胶的单糖类型和主链结构,果胶侧链发生降解；果胶各单糖的断链顺序为阿拉伯糖>半乳糖>鼠李糖>半乳糖醛酸；AFM(atomic force microscopy)分析果胶纳米结构表明超声波降解果胶分子主要以小的聚合体和短的线性单片段形式存在,分支结构减少；果胶中线性及含分支的聚合体结构仅由半乳糖醛酸组成的均半乳糖醛苷构成；果胶经超声波降解后链长和链宽较小的片段所占频率增加,分子高度减小；果胶分子的链长和链宽与其分子量及分布呈正相关关系。
(3)超声波降解果胶多糖的动力学模型分析：通过研究超声波作用下果胶的分子量及分布的动态变化过程,分析研究了果胶超声波降解的动力学模型。研究结果显示：果胶在超声波作用下(5℃至45℃)的降解符合动力学方程1/Mt-1/M0=kt；超声波可以降低果胶降解的表观活化能并加速果胶的降解。
(4)超声波降解果胶多糖的机理研究：通过对超声波机械效应和自由基效应的研究,揭示了超声波降解果胶的降解机理。研究结果显示：随着果胶链长的减小超声波降解果胶的速率和断链浓度减小；氧气和氩气可增加超声波作用下自由基量的产生,氮气对自由基量无显著影响;果胶在氧气存在下超声波降解速率及断链浓度显著增加,氩气和氮气对超声波降解果胶的速率和断链浓度无显著影响,自由基生成量与超声波降解果胶的速率和断链浓度不呈正相关关系；气体可引起果胶在超声波作用下发生侧链效应；超声波空化效应引起的自由基效应不是导致果胶降解的主要因素,而机械效应是引起超声波降解果胶的主要原因。
(5)超声波降解果胶多糖产物功能特性的研究：超声波降解果胶产物的表观粘度显著降低；果胶溶液经超声波降解后表现为弹性特性；果胶分子链长的减小是引起其表观粘度减小的内在原因之一；果胶溶液经超声波作用后表现为牛顿流体特性(n=1)；果胶经超声波降解后其产物抑制脂肪酶活性提高。
(6)建立了超声波降解果胶多糖的方法：本研究对超声波法、超声波辅助酸法与酸法降解果胶进行了比较研究。研究结果显示：利用超声波法降解不改变果胶的结构,超声波具有条件温和、高效且环境友好等优点,超声波技术可作为果胶降解的一种绿色、低碳的改性方法。
本研究结果可为建立多糖的绿色环保的超声波改性技术奠定理论基础,扩大超声波在食品工业中的应用,并可扩大果胶多糖在食品和医药领域的应用,对于充分利用我国的柑橘和苹果皮渣等废弃物资源以及保护环境具有重要意义。
Pectin, as an acid polysaccharide, is one of the major components of the primary cell walls and middle cellular of higher plants. Pectin has been widely used in food, medicine and cosmetic industry for its natural, non-toxic and cheaper characters. The structure of pectin varies according to the source species, original tissue type and location, developmental or metabolic stage, environmental state and conditions of extraction. These variations together with its larger molecular weight, limited the wide use of pectin. Recently, more and more researches are focused on modification of pectin (modifing molecular weight or structure) to adapt to different needs. Generally, modified pectin is prepared by chemical or enzymatic depolymerization processes. These methods are hard to control and generally not oriented. Meanwhile, the chemical method was time consuming and lead to environment pollution. The "green and innovative" technique in pectin modified is preferred. Ultrasound, as a green and physical technique, has been widely used in polymer degradation. However, there were rarely reports on the degradation of pectin polysaccharide by ultrasound.
This research was the first time to investigate the degradation behavior pathway and mechnism of pectin degradation by ultrasound in detail. The ultrasound degradation kinetics of pectin was established. This study revealed the degradation pathway of pectin by atomic force microscopy at nanolevel. The degradation mechnism was also revealed through investigation of radical and mechanical effects of ultrasound. Compared with the acid degradation methods of pectin, ultrasound is a simple, rapid, cheaper and green method.
The main results and conclusions were listed as follows:
(1)The degradation behaviour of pectin polysaccharide with ultrasound:The study established the degradation behaviour of pectin polysaccharide with ultrasound by determined the molecular mass and its distribution of pectin polysaccharide. The results indicated that the molecular mass and degradation rate of pectin polysaccharide decreased with ultrasound time. The molecular mass of pectin polysaccharide decreased with increasing ultrasound intensity. However, the ultrasound efficiency could not increase indefinitely. Lower temperature could enhance the degradation process. The ultrasound effect was most significant when the duty cycle was50%.
(2) The degradation path of pectin polysaccharide with ultrasound:The degradation path of pectin polysaccharide with ultrasound was determined by the structure investigation. The distribution of molecular mass of pectin polysaccharide was more uniform after ultrasound treatment. The pectin fractions which below100kDa were accounted47%when ultrasound90min. The monosaccharide types and main chain of pectin polysaccharide were not changed after ultrasound treatment. However, the pectin sidechains were degraded. The break chain sequence of pectin polysaccharide with ultrasound was Ara>Gal>Rha>GalUA. AFM analysis indicated that the ultrasound degraded pectin was mainly composed of smaller polymers and shorter linear chains. The branched structure was reduced. There was a hypothesis that the branches and linear chains are homogalacturonans composed solely of galacturonic acid. The ration of short length and wider chains was increased and the hight of the chains was reduced. There was a positive correlation among length, width and molecular mass and its distribution of pectin polysaccharide.
(3)The degradation kinetic of pectin polysaccharide with ultrasound:The study established the degradation kinetic of pectin polysaccharide with ultrasound by determining the dynamic change of molecular mass and distribution of pectin polysaccharide. Degradation kinetics model of pectin fitted to1/Mt-1/Mo=kt from5to45℃. Ultrasound could decrease the activation energy and accelerate the pectin degradation.
(4) The degradation mechanism of pectin polysaccharide with ultrasound:The radical and mechanical effects were studied to investigate the degradation mechanism of pectin polysaccharide with ultrasound. The degradation rate and chain-breaks were decreased with reducing chain length of pectin. O2and Ar could enhance the production of radical, while N2has no significant effect on the radical yields. The degradation rate and chain-breaks of pectin with ultrasound were significant increased when O2existed. Ar and N2had no significant influence on the degradation rate and chain-breaks of pectin. There was a negative correlation between radical yields and degradation rate and chain-breaks. Gases could induce sidechain reaction of pectin with ultrasound. Therefore, mechanical effect was the main factor to affect the degradation of pectin with ultrasound.
(5)The functional properties of degraded pectin polysaccharide with ultrasound: The appearance viscosity of pectin significantly reduced with ultrasound. The pectin solution showed viscous properties after ultrasound treatment. Chain length of pectin was one of the reasons resulted the decrease of appearance viscosity of pectin. The pectin solutions showed Newtonian fluid properties (n=l). The lipase inhibition activity of pectin increased after ultrasound treatment.
(6)Establishment of the degradation method of pectin polysaccharide with ultrasound:The study compared the degradation methods (ultrasound and acid) of pectin. The results indicated that ultrasound degradation did not alter the structure of pectin. Meanwhile, the ultrasound method has the advantages of mild, efficiency, environmentally friendly and so on. Ultrasound method is a green technique for pectin modification.
This study could provide theoretical basis on the green modification technique of polysaccharide. It could expand the application of ultrasound in food industry, and enlarge the uses of pectin in food and medicine industry. It is meaningful to utilize the waste resources of citrus and apple pomace to protect environment.
本论文以商业果胶为研究对象,首次对超声波降解果胶多糖的降解规律、降解途径与机理以及降解产物的结构与功能进行了系统研究；分析研究了超声波降解果胶多糖的动力学模型；借助原子力显微镜技术从纳米水平上揭示了果胶多糖在超声波降解下的降解途径；并通过对超声波降解果胶多糖自由基与机械效应的分析研究,揭示了超声波降解果胶多糖的机理；同时结合与酸法降解果胶多糖的比较研究,验证了超声波技术降解果胶多糖具有简单、快速、经济且环保等优点,建立了果胶多糖的超声波降解方法。通过研究,本论文主要得出如下结论：
(1)超声波降解果胶多糖的降解规律研究：通过对超声波降解果胶多糖分子量及其分布的研究确定了超声波降解果胶多糖的降解规律。研究结果显示：随着超声波时间的增加,果胶分子量逐渐降低,降解速率逐渐减小；随着超声波强度的增加,果胶分子量逐渐减小,当强度增加到一定程度后,果胶分子量维持平衡；低温有利于超声波降解果胶分子；随着超声波脉冲占空比的增加,超声波空化效应先增大后减小,当脉冲占空比为50%时,超声波空化效应最强,果胶分子量降低最多。
(2)超声波降解果胶多糖的降解途径研究：通过对超声波降解前后果胶结构的比较研究确定了超声波降解果胶多糖的途径。研究结果显示：超声波降解果胶的分子量分布变窄且更加均匀；当超声波降解时间为90min时,果胶分子量小于100kDa的片段可达47%；超声波降解不改变果胶的单糖类型和主链结构,果胶侧链发生降解；果胶各单糖的断链顺序为阿拉伯糖>半乳糖>鼠李糖>半乳糖醛酸；AFM(atomic force microscopy)分析果胶纳米结构表明超声波降解果胶分子主要以小的聚合体和短的线性单片段形式存在,分支结构减少；果胶中线性及含分支的聚合体结构仅由半乳糖醛酸组成的均半乳糖醛苷构成；果胶经超声波降解后链长和链宽较小的片段所占频率增加,分子高度减小；果胶分子的链长和链宽与其分子量及分布呈正相关关系。
(3)超声波降解果胶多糖的动力学模型分析：通过研究超声波作用下果胶的分子量及分布的动态变化过程,分析研究了果胶超声波降解的动力学模型。研究结果显示：果胶在超声波作用下(5℃至45℃)的降解符合动力学方程1/Mt-1/M0=kt；超声波可以降低果胶降解的表观活化能并加速果胶的降解。
(4)超声波降解果胶多糖的机理研究：通过对超声波机械效应和自由基效应的研究,揭示了超声波降解果胶的降解机理。研究结果显示：随着果胶链长的减小超声波降解果胶的速率和断链浓度减小；氧气和氩气可增加超声波作用下自由基量的产生,氮气对自由基量无显著影响;果胶在氧气存在下超声波降解速率及断链浓度显著增加,氩气和氮气对超声波降解果胶的速率和断链浓度无显著影响,自由基生成量与超声波降解果胶的速率和断链浓度不呈正相关关系；气体可引起果胶在超声波作用下发生侧链效应；超声波空化效应引起的自由基效应不是导致果胶降解的主要因素,而机械效应是引起超声波降解果胶的主要原因。
(5)超声波降解果胶多糖产物功能特性的研究：超声波降解果胶产物的表观粘度显著降低；果胶溶液经超声波降解后表现为弹性特性；果胶分子链长的减小是引起其表观粘度减小的内在原因之一；果胶溶液经超声波作用后表现为牛顿流体特性(n=1)；果胶经超声波降解后其产物抑制脂肪酶活性提高。
(6)建立了超声波降解果胶多糖的方法：本研究对超声波法、超声波辅助酸法与酸法降解果胶进行了比较研究。研究结果显示：利用超声波法降解不改变果胶的结构,超声波具有条件温和、高效且环境友好等优点,超声波技术可作为果胶降解的一种绿色、低碳的改性方法。
本研究结果可为建立多糖的绿色环保的超声波改性技术奠定理论基础,扩大超声波在食品工业中的应用,并可扩大果胶多糖在食品和医药领域的应用,对于充分利用我国的柑橘和苹果皮渣等废弃物资源以及保护环境具有重要意义。
Pectin, as an acid polysaccharide, is one of the major components of the primary cell walls and middle cellular of higher plants. Pectin has been widely used in food, medicine and cosmetic industry for its natural, non-toxic and cheaper characters. The structure of pectin varies according to the source species, original tissue type and location, developmental or metabolic stage, environmental state and conditions of extraction. These variations together with its larger molecular weight, limited the wide use of pectin. Recently, more and more researches are focused on modification of pectin (modifing molecular weight or structure) to adapt to different needs. Generally, modified pectin is prepared by chemical or enzymatic depolymerization processes. These methods are hard to control and generally not oriented. Meanwhile, the chemical method was time consuming and lead to environment pollution. The "green and innovative" technique in pectin modified is preferred. Ultrasound, as a green and physical technique, has been widely used in polymer degradation. However, there were rarely reports on the degradation of pectin polysaccharide by ultrasound.
This research was the first time to investigate the degradation behavior pathway and mechnism of pectin degradation by ultrasound in detail. The ultrasound degradation kinetics of pectin was established. This study revealed the degradation pathway of pectin by atomic force microscopy at nanolevel. The degradation mechnism was also revealed through investigation of radical and mechanical effects of ultrasound. Compared with the acid degradation methods of pectin, ultrasound is a simple, rapid, cheaper and green method.
The main results and conclusions were listed as follows:
(1)The degradation behaviour of pectin polysaccharide with ultrasound:The study established the degradation behaviour of pectin polysaccharide with ultrasound by determined the molecular mass and its distribution of pectin polysaccharide. The results indicated that the molecular mass and degradation rate of pectin polysaccharide decreased with ultrasound time. The molecular mass of pectin polysaccharide decreased with increasing ultrasound intensity. However, the ultrasound efficiency could not increase indefinitely. Lower temperature could enhance the degradation process. The ultrasound effect was most significant when the duty cycle was50%.
(2) The degradation path of pectin polysaccharide with ultrasound:The degradation path of pectin polysaccharide with ultrasound was determined by the structure investigation. The distribution of molecular mass of pectin polysaccharide was more uniform after ultrasound treatment. The pectin fractions which below100kDa were accounted47%when ultrasound90min. The monosaccharide types and main chain of pectin polysaccharide were not changed after ultrasound treatment. However, the pectin sidechains were degraded. The break chain sequence of pectin polysaccharide with ultrasound was Ara>Gal>Rha>GalUA. AFM analysis indicated that the ultrasound degraded pectin was mainly composed of smaller polymers and shorter linear chains. The branched structure was reduced. There was a hypothesis that the branches and linear chains are homogalacturonans composed solely of galacturonic acid. The ration of short length and wider chains was increased and the hight of the chains was reduced. There was a positive correlation among length, width and molecular mass and its distribution of pectin polysaccharide.
(3)The degradation kinetic of pectin polysaccharide with ultrasound:The study established the degradation kinetic of pectin polysaccharide with ultrasound by determining the dynamic change of molecular mass and distribution of pectin polysaccharide. Degradation kinetics model of pectin fitted to1/Mt-1/Mo=kt from5to45℃. Ultrasound could decrease the activation energy and accelerate the pectin degradation.
(4) The degradation mechanism of pectin polysaccharide with ultrasound:The radical and mechanical effects were studied to investigate the degradation mechanism of pectin polysaccharide with ultrasound. The degradation rate and chain-breaks were decreased with reducing chain length of pectin. O2and Ar could enhance the production of radical, while N2has no significant effect on the radical yields. The degradation rate and chain-breaks of pectin with ultrasound were significant increased when O2existed. Ar and N2had no significant influence on the degradation rate and chain-breaks of pectin. There was a negative correlation between radical yields and degradation rate and chain-breaks. Gases could induce sidechain reaction of pectin with ultrasound. Therefore, mechanical effect was the main factor to affect the degradation of pectin with ultrasound.
(5)The functional properties of degraded pectin polysaccharide with ultrasound: The appearance viscosity of pectin significantly reduced with ultrasound. The pectin solution showed viscous properties after ultrasound treatment. Chain length of pectin was one of the reasons resulted the decrease of appearance viscosity of pectin. The pectin solutions showed Newtonian fluid properties (n=l). The lipase inhibition activity of pectin increased after ultrasound treatment.
(6)Establishment of the degradation method of pectin polysaccharide with ultrasound:The study compared the degradation methods (ultrasound and acid) of pectin. The results indicated that ultrasound degradation did not alter the structure of pectin. Meanwhile, the ultrasound method has the advantages of mild, efficiency, environmentally friendly and so on. Ultrasound method is a green technique for pectin modification.
This study could provide theoretical basis on the green modification technique of polysaccharide. It could expand the application of ultrasound in food industry, and enlarge the uses of pectin in food and medicine industry. It is meaningful to utilize the waste resources of citrus and apple pomace to protect environment.
引文
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