发酵苹果渣多糖分离纯化、结构及其与加工特性的关系

Isolation and Purification of Polysaccharide from Fermented Apple Pomace and Its Relationship with Processing Characteristics

【目的】在研究发酵苹果渣多糖分离纯化动态趋势的基础上,对其结构特性进行深入研究,进一步探究发酵苹果渣多糖分离纯化、活性及加工特性。【方法】以苹果原渣多糖(apple pomace polysaccharides,AP)、苹果酒渣多糖(wine fermented apple pomace polysaccharides,WFP)、苹果醋渣多糖(vinegar fermented apple pomace polysaccharides,VFP)为原料,对其含量进行分析,利用发酵苹果渣多糖不同组分间极性差异,采用DEAE-52纤维素,以Na Cl溶液进行梯度洗脱,部分收集器进行逐管收集,苯酚硫酸法测定吸光值,制作洗脱曲线;同时,对DEAE-52纤维素层析所得组分利用去离子水经Sephadex G-200凝胶柱,根据一定分子量截留的特点进行进一步分离纯化,测定所得组分纯度后,对不同组分进行结构表征,主要包括X衍射仪(XRD)观察晶体结构、热重分析仪进行热重分析(TG)、激光粒度仪对溶液粒度进行分析(LPSA)、同时通过刚果红试验探究其是否具有三螺旋结构,运用台式扫面电镜进行微观结构分析。【结果】粗多糖含量达到70%左右,同时不含蛋白质、核酸,提取效率较高;AP在0.1和0.2 mol·L-1 Na Cl洗脱液浓度下经DEAE-52纤维素层析、Sephadex G-200凝胶层析可得NAP0.1、NAP0.2;WFP在0.0、0.1和0.2 mol·L-1 Na Cl浓度洗脱下可得NWFP0、NWFP0.1、NWFP0.2,VFP可得NVFP0、NVFP0.1、NVFP0.2,以上所得成分含量均达到92%以上,分离纯化效果良好;3种多糖分离纯化前后皆为非晶态物质,呈无定型结构;多糖溶液浓缩过程温度应低于65℃,在加工中应注意控制温度在150℃以内;分离纯化前,3种多糖都存在三螺旋结构,分离纯化后,组分NWFP0、NVFP0、NVFP0.1出现了三螺旋结构,AP经分离纯化无三螺旋结构成分出现;苹果渣多糖在分离纯化后更加稳定,粒径差异更小,主要表现在经分离纯化后多糖的粒径分散系数更小;分离纯化导致片状结构变小,交联作用减弱,能更好发挥其生物活性。【结论】发酵苹果渣多糖含量达70%,经分离纯化后的含量与AP均可达92%;由XRD、TG、LPSA、刚果红、微观结构等方面得出其在溶解度、黏度、物理性质等方面的改变有利于更好发挥生物活性,同时获得更好的加工特性。

[ Objective ] On the basis of dynamic trend of separating and purifying of fermented apple pomace polysaccharides, the authors further investigated the structural properties, and in hoping of further elucidating the separating, purifying, bioactivity and processing properties of fermented apple pomace polysaccharides. [Method] Apple pomace polysaccharides （AP）, wine fermented apple pomace polysaccharides （WFP） and vinegar fermented apple pomace polysaccharides （VFP） were used as experimental materials. On the basis of analysis of polysaccharides content, the authors used DEAE-52 cellulose column, NaC1 as eluant, to separate polysaccharides according to the polarity difference among polysaccharides components. Through collecting effluent liquid using fraction collector and then the polysaccharides content was determined using with phenol sulfuric acid method, and the elution curve was depicted. Meanwhile, the obtained polysaccharides components with DEAE-52 cellulose column were further separated using Sephadex G-200 gel column, distilled water as eluent. After obtaining the separated polysaccharides components, the structural properties were thoroughly analyzed by using X-ray powder diffraction to observe the crystallographic structure, Thermogravimetric Analyzer was used to analyze thermogravimetric characteristics, Laser particle size analyzer to analyze granularity, and Congo red was used to explore triple helix structure of polysaccharides. Finally, Desktop Scanning Electron Microscope was also used to observe and analyze micro-structure of apple pomace polysaccharides. [ Result ] The content of original apple pomace polysaccharides was approximately 70%. Because the polysaccharides didn＇t include protein and nucleic acid, so the extraction efficiency was excellent. NAP0.1 and NAP0.2 were obtained after AP were purified firstly through DEAE-52 cellulose column and 0.1, 0.2 mol·L-1 NaCI were respectively used as eluents and then through Sephadex G-200 gel column and distilled water as eluent. Meanwhile, NWFP0, NWFP0.1 and NWFP0.2 were obtained after WFP was purified with DEAE-52 cellulose column and subsequently Sephadex G-200 gel column. After VFP was separated, NVFP0, NVFP0.1 and NVFP0.2 were obtained, too. All the separated constituents included over 92% polysaccharides, suggesting separation effect was satisfactory. Three polysaccharides, AP, WFP and VFP, were non-crystalline material between before and after separation. Concentration temperature of polysaccharides was strictly limited below 65℃, and the processing temperature was below 150℃. Before separation, AP, WFP and VFP all had triple helix structure. After separation, triple helix structure still existed in NWFP0, NVFP0, and NVFP0.1, while NAP0.1 and NAP0.2 had no triple helix structure. Compared with the original apple pomace polysaccharides, the separated apple pomace polysaccharides had better stability and smaller particle size, because lower dispersion coefficient of particle size was observed. Besides, flake structure in polysaccharides was less, and cross-linking effect attenuated after separation, which is beneficial for development of polysaccharides bioactivity. [ Conclusion ] Fermented apple pomace included over 70% polysaccharides, and polysaccharides content was up to 92% after crude polysaccharides was separated and purified. Besides, according to XRD, TG, LPSA and Congo red as well as DSEM analysis, it was concluded that the changes of solubility, viscosity and physical characteristics facilitated separated apple pomace polysaccharides developing bioactivity and processing characteristics.