【作者】 冯睿；

【导师】 陈浩；

【作者基本信息】 华中农业大学 ， 农药学， 2007， 硕士

【摘要】 为了开展菜籽饼粕的综合利用研究，提高菜籽饼粕的附加值，本文对碱溶性菜籽多糖进行了较为系统的研究，包括碱溶性菜籽多糖的提取、分离、纯化、抗氧化活性及结构研究几个方面，主要内容如下：1菜籽多糖的NaOH提取方法采用NaOH溶液作为提取剂，从单因素实验、四因素三水平正交试验到中心组合正交试验进行优化，得出最佳提取工艺，为菜籽饼粕综合利用中多糖的提取提供参考。单因素实验表明，提取液NaOH浓度最佳条件为1.2 mol·L^-1，提取时间为4h，料液比为1∶20，提取次数在3次左右，提取率已基本达到平衡。四因素三水平正交试验表明，单因素对提取率的影响为提取温度＞NaOH浓度＞提取时间＞料液比。其中只有提取温度呈现显著性，而其它三个因素都不显著。正交试验得出的最佳提取条件为：NaOH浓度1.2mol·L^-1，提取温度60℃，料液比1∶20，提取时间为4h。采用最佳的提取条件提取得到的菜籽多糖的含量为7.65％。中心组合正交试验表明，提取温度、NaOH浓度和提取时间三个因素均为极显著，提取符合方程Y=9.9028＋1.7119X₁＋0.8924X₂＋0.1025X₃＋0.1402X₁²-0.3891X₂²-1.2848X₃²-0.0775X₁X₂-0.5634X₁X₃-0.3415X₂X₃，最佳提取条件为提取温度93.7℃，NaOH浓度1.124mol·L^-1，提取2.3h多糖的提取率为11.59％。2碱溶性菜籽多糖的脱蛋白方法以碱溶性菜籽粗多糖为原料，对不同方法的蛋白脱除率和多糖损失率进行了比较。通过比较Sevag法、三氯乙酸法、盐酸法、D3520大孔吸附树脂法和酶法-Sevag法联用五种方法，结果表明五种方法在脱蛋白处理后，均会有一定的蛋白存在，这是因为碱溶性菜籽多糖本身含有糖蛋白缀合物。采用酶法-Sevag法联用蛋白脱除率高，多糖损失率低，适用于碱溶性菜籽多糖中游离蛋白的脱除。其最佳条件为：酶用量0.12％，反应温度60℃，反应时间45min，Sevag法脱蛋白的次数3次。经过最佳条件脱蛋白，蛋白的脱除率为74.8％，多糖损失率为21.6％。3碱溶性菜籽多糖的分离及理化性质将NaOH提取的菜籽多糖经过脱蛋白步骤后再进行分步醇沉，得到较纯净的级分多糖，并进行纯度鉴定和基本理化性质分析。在乙醇浓度分别为65％、70％、75％、80％、85％的条件下，得到五个级分多糖，分别命名为R-A、R-B、R-C、R-D和R-E，其得率分别为3.86％、6.92％、52.91％、31.17％和5.14％。常压凝胶柱层析结果表明，R-C的蛋白质洗脱曲线和多糖洗脱曲线都只有一个峰且两峰重合，可以初步断定它是比较单一的物质。经过滤纸电泳，级分多糖R-C呈现单一色带，可以认为R-C组成单一。对碱溶性菜籽多糖的组成进行分析，结果表明经过本实验流程提取的多糖糖醛酸含量都很高，均含有蛋白质。因此可以推断，本实验中提取的多糖是一种糖醛酸含量很高的糖蛋白缀合物。对碱溶性菜籽多糖进行理化性质研究，结果表明，菜籽多糖均为颜色较深的多糖；碘反应的结果表明粗多糖及五个级分多糖都是非淀粉多糖，斐林反应结果表明粗多糖和五个级分都不含有还原糖，FeCl₃反应说明粗多糖和五个级分多糖都不含多酚类物质。另外，五个级分多糖均可溶于水，不溶于乙醇、乙醚、丙酮等有机溶剂。4碱溶性菜籽多糖的抗氧化活性将分离纯化的多糖进行抗氧化活性研究，考察其在化学模拟体系、体内及体外抗氧化活性。化学模拟体系实验表明，碱溶性菜籽多糖具有还原能力，表现有抗氧化活性的可能。不同浓度的菜籽多糖对于羟基自由基的清除都有一定的效果，而且随着浓度的增加，菜籽多糖清除羟基自由基的效果呈明显的增长趋势。体外抗氧化试验表明，碱溶性菜籽多糖对与H₂O₂诱导小鼠红细胞氧化溶血以及小鼠红细胞自氧化溶血都有很好的的抑制效果；可以减少小鼠肝匀浆MDA生成以及诱导体系下MDA的生成；可以减少肝线粒体的肿胀和肝线粒体脂质过氧化MDA的生成。体内抗氧化试验表明，碱溶性菜籽多糖可以减少小鼠体内肝组织MDA的生成，提高SOD和GSH-Px酶的活力。从化学模拟体系到体外试验再到体内试验，结果表明，碱溶性菜籽多糖有较好的抗氧化作用，探求其原因，可能与碱溶性菜籽多糖的还原能力以及其可以提高抗氧化作用酶的活力有关。5菜籽多糖R-C的结构表征采用柱前衍生化-HPLC方法对级分多糖R-C的单糖组成进行分析，R-C的单糖组成为葡萄糖醛酸，葡萄糖，半乳糖和阿拉伯糖，其组成比例67.24∶4.43∶1.38∶26.95。菜籽多糖R-C的蛋白质部分由谷氨酸、缬氨酸、胱氨酸等13种氨基酸组成，氨基酸总量为27.97％，β-消除反应后的氨基酸分析显示，R-C的丝氨酸和苏氨酸的量不变，其它氨基酸含量也基本保持不变，可见级分多糖R-C含有N-糖苷键。采用凝胶渗透色谱法测定R-C的分子量，其数均分子量Mn为1.101×10⁶，重均分子量Mw为1.176×10⁶，多分散系数Mw／Mn为1.068，这说明R-C的摩尔质量分布范围比较窄。红外扫描的结果表明R-C是一种含有吡喃环的糖蛋白缀合物。R-C的紫外扫描图在280nm吸收不明显，R-C的水溶液经苯酚-硫酸法显色后，在490nm．附近有明显的吸收，这是多糖显色反应后的特征吸收峰。X-衍射结果表明，R-C在此条件下不能形成结晶，为无定型形态高聚物。原子力显微镜观察表明，R-C分子并不完全均一，且显团聚体状。高碘酸试验表明，R-C含有较多的1→6键；而刚果红试验认为级分多糖R-C可能有三股螺旋结构。更多还原

【Abstract】 In order to increase the utilization and the value of rapeseed meal, the alkali-soluble polysaccharides from rapeseed meal was studied in this dissertation, includes extraction, isolation, antitumour activity and structure of the alkali-soluble polysaccharides from rapeseed meal. The main body of the study is as follows:1 The method of extracting polysaccharifles in rapeseed meal by NaOHRapeseed polysaccharides could be obtain by NaOH, from the basis of single factor, orthogonal experiment and then the central composite orthogonal experiment, and the optimal conditions for extraction was obtained. The single factor test showed that the NaOH concentration was 1.2 mol·L^-1, the consumed time was 4h, the ration between step and material was 1∶20, and extracted 3 times. Orthogonal experiment showed that, the influence of single factors is the temperature＞the NaOH concentration＞the time＞the ration between step and material. Among which the temperature showed that it is markedness, while the others were all inapparent. Optimal condition for extraction were: the steep concentration was 1.2mol·L^-1; the temperature was 60℃; the ration between steep and material was 1∶20; the consumed time was 4 hours. Being obtained on these conditions, the content of polysaccharides was 7.65%. The result of central composite showed that the three factors were all signaficate to the content of polysaccharides, the equation of the extraction content is Y=9.9028+1.7119X₁+0.8924X₂+0.1025X₃+0.1402X₁²-0.3891X₂²-1.2848X₃²-0.0775X₁X₂-0.5634X₁X₃-0.3415X₂X₃, and the optimal condition were the NaOH concentration was 1.124mol·L^-1; the temperature was 93.7℃; and the consumed time was 2.3 hours, the content of polysaccharides was 11.59%.2 The method of removal of protein in the alkali-soluble polysaccharides from rapeseed mealIn order to take full use of RP, and to explain the bioactivities and the structure, the isolation and purifying becomes the sixty-four-dollar question. The degree of purifying effects the analysis of its structure and the study of the structure-effective. In this section, five methods were used to remove the protein from the alkali-soluble polysaccharides, and the results showed that the combination of enzyme and Sevag exhibited higher removal percentage of protein and lower loss of polysaccharide. The proper concentration of papain is 0.12%, the temperature is 60℃, time for reaction is 45min, and the times of Sevag to remove protein is 3. After the optimum conditions, the removal percentage is 74.8%, and the loss of polysaccharide is 21.6%.3 Analysis of the isolation, purifying and physiochemical qualities of alkali-soluble polysaccharides from rapeseed mealTo prepare for the structure-analysis, fraction polysacchafides were investigated by ethanol precipitation （step and stepwise）, and then the physiochemical qualities were analysised. Five fraction polysaccharides were investigated by ethanol precipitation （step and stepwise） when the concentration of ethanol was 65%、70%、75%、80%、85%, then five fraction polysaccharides were obtained and named respectively R-A、R-B、R-C、R-D and R-E with the yield of 3.86%、6.92%、52.91%、31.17% and 5.14%. After the further isolation with Sephadex G-100 column and paper electrophoresis, we find that R-C was comparatively pure and still at a single level. The analysis of their physiochemical qualities discoverd that these five fraction polysacchafides were all glycoprotein conjugate. They all contain large amounts of glucuronic acid and no starch polysaccharides, reducing sugar and polyphenols substances, and they all can dissolved by water and cannot by ethanol、aether or acetone.4 The antioxidant activity of extraction with base rapeseed polysaecharidesThe antioxidant activity of extraction with base rapeseed polysaccharides was determined by the experiment from chemical system to animal organelle, subcellular organelle and finally to animal vivo. Through the determination of the reduction ability and examination of the effect of hydroxide radical and free radical cleanse of alkali-soluble polysaccharides, it was preliminarily judged that extraction with base rapeseed polysaccharides had antioxidant effect. Thereinto, the antioxidant ability of alkali-soluble polysaccharide was stronger with the increase of its indicator. Further studies on the influence of alkali-soluble polysaccharides vitro on the formation of homogenate MDA, the engorgement degree of mitochondrion, the production of MDA by mitochondrion lipid peroxidation in young rat liver and on the inductive system erythrocyte oxidative haemolysis and red blood cell oxidative haemolysis of young rat indicated that alkali-soluble polysaccharides could restrain in vitro the formation of homogenate, the production of the production of MDA by mitochondrion lipid peroxidation and the engorgement of mitochondrion in young rat liver; prevent the erythrocyte oxidative haemolysis. Finally, through the experiment on vivo antioxidant ability, it was found that extraction with base rapeseed polysaccharides could restrain the production of MDA in young rat liver, enhance the vitality of blood serum SOD、GSH-Px. From chemical system research to animal experiment, it was discovered that alkali-soluble polysaccharides had antioxidant effectiveness. The probing into mechanism regarded that it was the result of collective contribution of the rapeseed polysaccharides reduction ability and activity of enzyme which could enhance the antioxidant function in vivo of young rat.5 Structural exosyndrome of fraction polysaccharide R-C On the base of precede studies, structural exosyndrome of fraction polysaccharide R-C was studied in this section. The monosaccharide glycon composition and ratio of R-C were determined by means of pre-column derivatization-high performance liquid chromatogram （HPLC）. The result suggestd that R-C was composed of glucuronic acid, glucose, galactose, and arabinose at the ratio of 67.24∶4.43∶1.38∶26.95. The compositions of the fraction polysaccharide R-C amino acids and the changes in the composition of amino acids afterβ- elimination reaction were detected by Amino acid automatic analysator. The result indicated that the binding protein in R-C mainly contained 13 kinds of amino acids including glutamic acid, valine, cystine and so on. In the analysis of amino acids afterβ- elimination reaction, it was obvious that there was N-glycosidic linkage in R-C fraction polysacchadde. The molecular weight of fraction polysaccharide R-C was determined by means of GPC （gel permeation chromatography）. In R-C the number-average molecular weight Mn was1.101×10⁶, weight average molecular weight was 1.176×10⁶, polydisperse coefficient Mw/Mn was 1.068. The survey of its structure through infrared atlas scanning showed that rapeseed polysacchafide R-C was glycoprotein conjugates which contained protein and pyran. The ultraviolet spectra analysis showed that there was tmconspicuous absorb on 280 nm, after reacting with the mixture of phenol vitriol, there was a obvious absorb on 490 nm which is the character absorb by polysaccharide. X-ray showed that R-C is amorphism. AMF scan showed that the molecule of R-C were not so equal, and they can congregate easily. Further research was done on its conjugation bond type by periodic acid reaction experiment. The result showed that there were more 1→6 bonds in R-C. The result of spatial configuration determination by Congo red test indicated that R-C contained triple helixstructure.更多还原