【作者】 莫开菊；

【导师】 谢笔钧；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2011， 博士

【摘要】 葛仙米(Nostoc sphaeroids kiitz)是指主要分布地是湖北鹤峰县走马坪镇,生于水稻田中的一种可食性蓝藻,属于蓝藻门(Cyanophyta)蓝藻纲(Cyanophyceae)念珠藻科(Nostocaceae)中念珠藻属(Nostoc.)植物,又称天仙菜、天仙米,也有称水木耳,是一种淡水蓝藻,是我国传统珍贵的野生食药两用的藻类,记载显示葛仙米具有：明目益气、、治夜盲症、久食延年、解热清膈、利肠胃、痰火能疗和消除疲劳等。虽然从古至今,葛仙米备受民间推崇,但是还缺乏现代的科学依据。迄今为止,人们对葛仙米的功效成分的化学本质、理化特性、功能性质还缺乏研究。本文以葛仙米为原料,研究了葛仙米多糖的提取、分离、纯化及理化性质和功能性质,以期为葛仙米多糖的工业及医药应用打下基础。主要研究结果如下：1葛仙米多糖的提取工艺研究使用碱酶复合预处理可以显著提高葛仙米多糖的提取率。以0.4mol/L的NaOH溶液于40℃处理2小时后,然后用盐酸中和至中性,再按1.0mg/100mg葛仙米的量加入木瓜蛋白酶液,于40℃处理10小时,接着按1：300的葛仙米干重的质量体积比补加蒸馏水,于90℃水浴提取2小时,重复2次,多糖最高得率高达47.56%,比对照高出112%。2葛仙米多糖的分离纯化葛仙米多糖易于除杂。采取酶与sevag试剂结合的方法能较好脱出葛仙米多糖中的蛋白质,其中sevag试剂法重复6-7次效果较好,多糖损失少,而且能很好的脱色,得到颜色较白的多糖。采用0.2mol/LCTAB溶液与0.1%葛仙米多糖溶液以1：100混合静置,能将99%以上的葛仙米多糖沉淀出来,达到净化除杂的效果。葛仙米多糖难以分级分离。采用DEAE C-52阴离子交换层析法处理葛仙米多糖,以0.3mol/L NaCl阶段洗脱及0.3-0.6mol/L NaCl梯度洗脱,结果显示至少存在两个级分。以0.3mol/L NaCl能集中洗脱出一部分多糖,另一部分多糖被绵延洗出,高度稀释,不便于收集。因此此法收率低,效率低,重现性差,不能较好的分离级分。经0.3-0.6mol/L NaCl梯度洗脱,虽经多方调整洗脱条件,均不能完全分开而且难以重复。葡聚糖凝胶过滤亦难以分级葛仙米多糖。试验中采用Sephadex G100过滤,因为网孔小,而葛仙米多糖分子量大,粘度大,柱子被严重压缩,多糖无法洗出,采用Sephadex G200过滤,虽然网孔稍大,不会被高粘度的多糖溶液压缩,多糖能洗出,但是无法分离级分。采用Sepharose 6B、Sepharose CL-4B过滤,葛仙米多糖能被顺利洗出,但是几乎所有多糖都集中在前期洗出,达不到分级的目的。3葛仙米多糖的化学组成和理化性质葛仙米多糖是含有糖醛酸,不含乙酰基、硫酸根和蛋白质的酸性多糖,由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖及半乳糖醛酸甲酯组成,由α及β糖苷键构成,且以吡喃糖为主,其结构相当复杂。其中中性糖含量为66.66%±0.86%,酸性糖含量为12.80%+0.56%。其分子量大而高度分散,重均分子量Mw为9.564×106da1,数均分子量Mn为5.634×106dal, Mw/Mn为1.698。其碳氢氧的组成为C：39.53%；H：7.08%；O：53.37%。葛仙米多糖中还含有多种矿物质,含量较高的是钙和镁。葛仙米多糖的圆二色谱在200nm处有一正峰,在215nm处有一负峰,由于多糖可供比对的样品很少,所以,还没有足够的信息分析其二级结构。葛仙米多糖的X衍射图表明,多糖结构中存在29.97%的结晶。原子力显微镜观察到了葛仙米多糖的单分子形貌特征,呈链状,多分支结构,链上可见螺旋结构。4葛仙米多糖的溶解特性和动态粘弹性葛仙米多糖是水溶性多糖,加温显著可促进溶解,酸能够促进葛仙米多糖溶解,碱和一价、二价金属无机盐类抑制其溶解,不溶于三价金属无机盐,不溶于二甲基亚砜。葛仙米多糖流变学特性良好,能够作为食品添加剂进行开发。与黄原胶相比较,在低浓度(低于0.5%)条件下时,二者的黏度相当,浓度下(高于0.5%),葛仙米多糖黏度明显低于黄原胶；同时,葛仙米多糖对一、二价盐离子的稳定性明显好于黄原胶,但是,对pH值的稳定性两者相当,高温导致葛仙米多糖的粘度较快下降。葛仙米多糖良好的水溶性、低浓度高黏度及对盐离子和pH值的稳定性,具有作为食品添加剂开发的潜力。葛仙米多糖动态粘弹性的研究结果表明：葛仙米多糖能够形成非常好的溶胶,在目前所知的条件下均不能形成真凝胶,其溶胶的粘稠性显著的受葛仙米多糖的质量分数、钙离子的浓度、pH值和温度的影响。5葛仙米多糖的抗氧化活性研究葛仙米多糖在动物体内体外的抗氧化活性均较弱。通过测定葛仙米多糖溶液的还原能力、邻二氮菲法清除·OH的能力、邻苯三酚自氧化法清除O2-·的能力三个指标,葛仙米多糖溶液仅有非常弱的抗氧化能力。由于各种测定方法的灵敏度不同,结果有差异。在这些实验中以邻二氮菲法清除-OH的能力较强,在浓度为0.5-5mg/mL的范围内呈量效关系,其抑制率为5.23～98.06%,其IC50值为2.74mg/mL。但是以邻苯三酚自氧化法清除超氧自由基,则清除率为负值。对接种S180肿瘤小鼠体内的抗氧化活性表明：葛仙米多糖不能促进荷瘤小鼠体内抗氧化指标如SOD酶、GSH-PX酶活性,不能降低丙二醛含量和LDH酶活性。6葛仙米多糖体内抑瘤及对小鼠免疫力的影响葛仙米多糖显著抑制小鼠体内S180肿瘤的生长,且抑瘤率呈剂量效应,其使用剂量200mg/kg.d、100 mg/kg.d、50 mg/kg.d的抑瘤率分别为64.08%、38.24%和29.91%。葛仙米多糖对S180肿瘤所致小鼠细胞免疫力下降有显著抑制作用,且呈现一定的剂量效应。表现在①使脾淋巴细胞转化率提高,与肿瘤对照组比较,正常对照组、CY组、多糖高剂量组的细胞免疫均提高,达到显著水平,而且多糖组间呈现一定的剂量效应；②环磷酰胺和高剂量多糖对小鼠的NK细胞活性下降有显著的抑制作用；③肿瘤使小鼠DTH降低,但是高剂量多糖组与CY组一样,极显著高于模型对照组。说明二者对荷瘤小鼠的DTH的下降有有极显著的抑制作用。葛仙米多糖对荷瘤小鼠胸腺指数降低有显著的促进作用。葛仙米多糖的使用具有抑制荷瘤小鼠血液中PLT升高的趋势。除低剂量葛仙米多糖组外,其它药物组均使血小板计数降低,而且高剂量葛仙米多糖组使血小板计数达到NC组的水平。这说明适当的葛仙米多糖的使用对癌瘤的发展具有抑制作用。Cy治疗荷瘤小鼠,对造血有明显的破坏作用,而使用葛仙米多糖则对红细胞和血红蛋白几乎没有影响,即既未表现出缓解肿瘤引起的贫血作用也未表现出如Cy治疗引起的对造血的破坏作用。葛仙米多糖对肿瘤所致小鼠的肝脏指数、肾脏指数增长无明显的促进或者抑制作用。从组织切片来看,葛仙米多糖的使用,使肿瘤组织中肿瘤细胞大面积死亡；而肝肾组织切片说明,葛仙米多糖对肝肾组织无损伤。葛仙米多糖在体外不具有对肿瘤细胞的直接毒性作用。更多还原

【Abstract】 Nostoc sphaeroids kutz belongs among the nostoc genera of cyanophyta nostoc families, which is a kind of edible algae grown in rice field, it is mainly distributed in Zoumaping town of Hefeng city in Hubei province. It is known as traditional precious wild food and drug dual-use algae, which once is called as fairy vegetable or fairy rice in our country. As reported, it is beneficial to eyesight, antipyretic, intestines, stomach, eliminating phlegm, tiredness and nyctalopia due to abundance of polysaccharides. Howerver, the physicochemical properties and composition of Nostoc sphaeroids kutz polysaccharides is still limited. In addtion, the mechanism of function was unclear.To establish foundation for industrial and medical applications of Nostoc sphaeroids kutz polysaccharides, the present study focus on the extraction, separation, purification and physicochemical and functional properties of Nostoc sphaeroids kutz polysaccharides. The main research results are as following:1. Extraction of Nostoc sphaeroids kiitz polysaccharidesThe yield of Nostoc sphaeroids kutz polysaccharides can be greatly increased by pretreatment with alkali-enzyme. The pH value of the extraction solution was adjusted to neutral until Nostoc sphaeroids kutz incubated with 0.4 mol/L NaOH at 40℃for 2 hours. Then the solution incubated again at 40℃for 10 hours after adding 1.0 mg liquid papain to per 100mg Nostoc sphaeroids kutz. Subseqentenly, distilled water was added with the ratio of 1:300 of Nostoc sphaeroids kutz dry weight. This extaction solution incubated at 90℃for 2 hours and repeated two times. The result showed that the yield of the polysaccharide was 47.56%, which was 2.12 folds compared with control treatment.2. Separation and purification of Nostoc sphaeroids kutz polysaccharidesThe Nostoc sphaeroids kiitz polysaccharides can be separated well under the combination of enzyme and sevag reagent. sevag The effects on the polysaccharides loss and decolouring was excellent through the sevag reagent method with 6 or 7 repeat.Over 99% Nostoc sphaeroids kiitz polysaccharides was purified by CTAB solution.It was hard to be seperated fractionately for Nostoc sphaeroids kiitz polysaccharides by DEAE-C-52, Sephadex G100, Sephadex G200, Sepharose 6B or Sepharose CL-4B filtering.3. Chemical composition and physicochemical properties of polysaccharides in Nostoc sphaeroids kutzThe polysaccharide in Nostoc sphaeroids kutz is a kind of acid polysaccharide, which contains Man、Rha、GlcA、GalA、Glc、Gal、Ara. The uronic acid content is 12.80±0.56%, the Neutral sugar content is 66.66±0.86%.The molecular is heavy with a scattered weight:Mw (9.564×106dal), Mn (5.634×106dal), and Mw/Mn (1.698). The proportion of C, H and O is 39.53%,7.08%,53.37%, respectively. In addition, the polysaccharide of Nostoc sphaeroids Kutz contains rich minerals. In the pattern of circular dichroism spectrum, there is a positive spike at 200nm, and a negative spike at 215nm. However, no more details about the secondary structure can be observed due to lack of polysaccharide which is applied in comparison. Moreover, the X diffraction pattern demonstrates that crystallization account for 29.97% in the polysaccharide structure.4. The solution and rheology feature of polysaccharide in the Nostoc sphaeroids kutzPolysaccharide of Nostoc sphaeroids kutz is soluble in water especially with high temperature. In addition, its solution is enhanced by acid. An opposite effect occur in alkali surrounding and mineral salt of lor 2 Valence.Due to its distinguished rheology, the potential appliment in food is addictive. Compared with xanthan gum, the viscosity is similar when the density is under 0.5%, and it is notably lower when the density is above 0.5%. It is more stable than xanthan gum when mixed with salt ion of 1 or 2 Valence In contrast, the polysaccharide is more inclined to affect by the high temperature.5.The investigation of physiological function to polysaccharides in Nostoc sphaeroids kutzThe capacity of anti-oxidation of Nostoc sphaeroids kiitz polysaccharide is weak by index of deoxidation capacity, the capacity of eliminating·OH and O2. The similar results showed that the polysaccharides could neither promote indicators of the activity of antioxidant (such as SOD and GSH-PX enzyme) nor reduce the malondialdehyde content in the tumor-burdened mice.6. Effect on tumor inhibition and immunity of mice in vivo.Nostoc sphaeroids kutz Polysaccharides can remarkably inhibit the tumor dependent on dose. For instant, the rate of tumor inhibition was 29.91%,38.24% and 64.08% by 50 mg/kg.d,100mg/kg.d and200mg/kg.d, respectively.Nostoc sphaeroids kutz Polysaccharides can restrain the cellular immunity descended by S180 tumor. Compared with tumor group, the rate of lymphocyte transformation of the cellular immunity of normal group, Cy group and high polysaccharides doses group are increased markedly. The NK cell activity can be inhibited remarkably in Cy and polysaccharides high doses group. DHT of mice in Cy and polysaccharides high doses group was higer than that in model group, sugging the both can inhibit the decrease of DHT in mice with tumor.Nostoc sphaeroids kutz Polysaccharides can promote index of the decrease of thymus. Nostoc sphaeroids kutz Polysaccharides can remarkably descend the thymus index.Nostoc sphaeroids kutz Polysaccharides can inhibit the increase of PLT in blood of mice with tumor. Except low dose of Polysaccharides group, the other groups can descend the amount of platelet. In addition, the amount of platelet of high dose of Polysaccharides group is similar with NC group in mice. The results indicated tumor can be inhibited by Nostoc sphaeroids kiitz Polysaccharides.The blood making can be destroyed by Cy therapy. However, RBC and hemoglobin have not obciously change by Nostoc sphaeroids kutz Polysaccharides, neither anemia arised from tumor nor hematopoietic damage by Cy.Nostoc sphaeroids kutz Polysaccharides has not obviously effect on the index of liver and kidney of mice with tumo. The tumor cells in tumor tissue were died by supplying the Nostoc sphaeroids kutz Polysaccharides. Furthmore, it didn’t damage the biopsy of liver and kidneyNostoc sphaeroids kutz Polysaccharides didn’t evidently promote or inhibit the increase of liver index and kidney index which arise from tumor. See from the biopsy, Nostoc sphaeroids Kutz Polysaccharides can kill a large area of tumor cell, and it showed from the biopsy of liver and kidney that Nostoc sphaeroids Kutz Polysaccharides didn’t damage the biopsy of liver and kidney.更多还原