【作者】 刘芳；

【导师】 赵立艳；

【作者基本信息】 南京农业大学 ， 食品科学， 2014， 硕士

【摘要】 金针菇中含有丰富的多糖并且锌含量较高,其中35%左右的锌与多糖结合。近几年来,国内外已有研究人员对锌多糖进行了初步的分析,研究大多都停留在锌多糖的生物利用以及锌的总量测定等方面,对于锌多糖结构特征以及免疫功能活性的研究报道较少。因此,对金针菇锌多糖进行研究,分析其初级结构,评价其抗氧化活性,为丰富及开发金针菇锌多糖功能性产品以及拓宽其应用范围奠定了良好的基础。主要的研究结果如下：1、通过单因素和正交实验优化了金针菇中锌多糖的水浴提取条件。结果表明,锌多糖的最佳提取条件是：提取温度75℃,时间120 min,料液比1：30。在最优的提取条件下,金针菇粉中锌元素的含量为34.43μg/g。2、通过样品的溶解、浸提、0.45μm的微孔滤膜过滤、D101大孔树脂分离等手段,得到了锌的可溶态和悬浮态以及有机态、无机态等四种形态,进一步通过不同提取分离方法获得了锌多糖、锌蛋白两种有机锌结合态。金针菇中锌的提取率达到76.34%；在锌的可溶态溶液中,有机态锌和无机态锌的提取率分别是43.36%和23.90%；在锌的有机态溶液中,锌多糖和锌蛋白的提取率分别是14.86%和10.64%。3、经过热水浸提、Sevage试剂脱蛋白以及醇沉后得到锌多糖粗品,粗品的提取率为6.67%,总糖含量为72.37%,锌得率为140 μg/g,采用DEAE纤维素-52和Sephadex G-100型凝胶色谱对粗品进行逐步分离、纯化,得到两个组分CF1、CF2.两个组分的定性实验及紫外光谱分析结果表明CF1、CF2两种锌多糖样品中均不含有淀粉、酚类物质等杂质。高效凝胶过滤色谱进一步证明了两个样品为纯度较高的单一多糖组分,测得它们的平均相对分子质量分别为890 KDa,1244 KDa。CF1、CF2分别占锌多糖粗品的20.97%与12.25%,总糖含量分别为97.32%,96.58%,通过火焰原子吸收法测得每克锌多糖纯品中分别含有锌191μg,429μg,锌含量的差异与组分结构有关,这为进一步研究和比较关于不同锌含量纯化组分的结构特征和活性分析奠定了良好的基础。4、分别测定了两个纯化组分的单糖组成,结果表明CF1组分由甘露糖、葡萄糖、葡萄糖醛酸、木糖、半乳糖、岩藻糖六种单糖组成,组成比例为7.15：77.28：1.77：0.89：9.20：3.66。CF2组分主要由甘露糖、葡萄糖醛酸、鼠李糖、葡萄糖、半乳糖醛酸、阿拉伯糖、半乳糖、岩藻糖等八种糖组成,组成比例为9.45：1.10：0.82：9.66：13.08：0.89：2.40：62.49。5、通过对金针菇锌多糖进行红外光谱扫描和核磁共振实验,结果表明CF1主要以β型呋喃糖为主,也存在少量α型吡喃糖苷糖。CF2主要以β型吡喃糖为主,包括β-D-葡萄糖及β-D-甘露糖,也含有少量的α型吡喃糖苷键。在1640 cm-1附近的峰向低频波数移动表明锌可能与-NH2形成配位键,1100-1010 cm-1之间吸收峰的红移现象表明锌与多糖可能是以螯合态形式存在的。两个组分均具有三股螺旋结构,是标准的非晶体结构,并且分析表明多糖中的微量元素锌不是以锌单质晶体的状态存在在多糖中的。6、抗氧化实验结果显示,金针菇锌多糖粗品以及两个纯化组分CF1、CF2具有中度的DPPH自由基、羟基自由基以及超氧阴离子的清除能力,过氧化氢清除能力相对较强,而总还原力相对较弱,且清除效果随着锌多糖浓度的增大而不断加强。样品的抗氧化能力比较顺序为：锌多糖粗品>CF2>CF1,结果表明同等多糖浓度下,锌含量以及多糖结构与体外抗氧化活性之间有一定的相关性。更多还原

【Abstract】 Flammulina velutipes is rich in polysaccharides, approximately 35% of total Zn in Flammulina velutipes is combined with polysaccharide. In recent years, foreign and domestic researchers have carried out preliminary analysis on Zn polysaccharides, but most studies stay in the aspects of bioavailability and antioxidant and total content of Zn. Only a few studies about the biological activity, monosaccharide composition and structural analysis of Zn polysaccharides in Flammulina velutipes were reported. However, the study on the structure of Zn polysaccharides is very important since the biological function of any particular element greatly depend on its chemical form. It is related closely to the activity of the immune function. Therefore, it is laid a good foundation to analysis its primary strcture, study its antioxidant activity, develop a new functional products and expand its application scope. The main results are concluded as follows.1. The single factor and orthogonal experiment was applied to optimize the water extraction condition of Zn polysaccharide in Flammulina velutipes. The content of Zn was determined by flame atomic absorption spectrometry (FAAS). The results showed that the optimized conditions for the extraction of Zn were as followed:extraction temperature 75 ℃, extraction time 120 min and ratio of material to water was 1:30. About 34.43 ug Zn was extracted from per gram dried Flammulina velutipes under the optimal conditions.2. Four different species of Zn, namely the suspended, soluble, inorganic, organic species, protein and the polysaccharides fraction were separated through dissoluting, extracting and using 0.45μm membrane filter and D101 macroporous resin. The extraction rate of Zn was 76.34%. In the soluble form, about 43.36% and 23.90% of total Zn was combined with organic and inorganic compound, respectively. Furthermore,14.86%, 10.64% of Zn was combined with polysaccharide and protein.3. Crude Zn polysaccharides were obtained after hot water extraction, deproteininzation and alcohol precipitation. The extraction yeild of crude sample was 6.67%. The content of polysaccharides and Zn was 72.37% and 140 μg/g. The crude sample was separated through DEAE cellulose-52 and Sephadex G-100 gel chromatography. Two fractions named as CFl, CF2 were obtained. The qualitative experiment and ultraviolet spectra indicated that CF1, CF2 didn’t contain starch, phenolic compounds and amino acids and other impurities. Two components were both high purity with high performance liquid chromatography (HPLC). The molecular weight was 890 KDa,1244 KDa.The yields of the extracts were 20.97% and 12.25%, the content of Zn was 191 μg,429 μg per gram dried Flammulina velutipes. The results further proved that the two components of high purity. This provided a good basis for further study and comparison about the structural characteristics and activity analysis of the different Zn polysaccharide components.4. The results of monosaccharide composition analysis showed that CF1 was composed of mannose, glucose, glucuronic acid, xylose, galactose, fucose six monosaccharide, with molar ration of 7.15:77.28:1.77:0.89:9.20:3.66. CF2 was composed of mannose, glucuronic acid, rhamnose, glucose, galacturonic acid, arabinose, galactose and fucose eight monosaccharide, with molar ration of 9.45:0.82:1.10:13.08:9.66:2.40:0.89: 62.49.5. Zn polysaccharide was studied by infrared spectrum, NMR, congo red and X-ray experiments. The results showed that the presence of furanose ring in CF1, pyranose ring in CF2. CF1 and CF2 were linked with β-glycosidic bond, β-D-glucose and β-D-mannose existed in them, a-D-xylopyranose existed in CF1. The absorption peak at 1640 cm-1moved to low wavenumber indicates that Zn was bonded with -NH2, the red-shifts of the absorption peak between 1100-1010 cm’1 showed that Zn formed a chelated structure with polysaccharide.They were both triple helical structures and non-crystal structure, and Zn didn’t exist in Single crystal.6. The results of Antioxidant activity in vitro of Zn polysaccharides in Flammulina velutipes showed that crude sample and CF1, CF2 sample had a moderate DPPH radical scavenging activity, OH·radical scavenging activity and O2-radical scavenging activity, strong scavenging capacity of H2O2 and weak reducing power. The scavenging activity was increased with the increasing concentration of sample. The order of the antioxidant effect of Zn polysaccharides was crude sample> CF2> CFl. The results indicated the content of Zn and structure of polysaccharides was related to the antioxidant activity of Zn polysaccharides of Flammulina velutipes.更多还原