【作者】 孙惠洁；

【导师】 吴永沛；

【作者基本信息】 集美大学 ， 食品科学， 2008， 硕士

【摘要】 红毛藻(Bangia fusco-purpurea)是我国福建南日岛一种营养价值高且多糖含量丰富的经济养殖海藻。本文系统地研究了红毛藻多糖的提取、分离纯化工艺及其理化性质,并对红毛藻多糖的体外抗氧化活性做了初步的探索。以多糖的提取率与硫酸根含量为指标,比较了热水、冷水、稀酸、稀碱提取红毛藻多糖的效果,发现用热水提取红毛藻多糖提取率与硫酸根含量最高,故选用热水作为提取溶剂。通过单因素与正交实验确定了热水提取的最佳工艺参数为:浸提温度90℃,浸提时间2小时,料液比1:30,浸提次数3次,在此优化的条件下得到多糖提取率为5.97%;采用超声波辅助热水提取红毛藻多糖,通过单因素与正交实验确定了最佳工艺参数为浸提温度为80℃,浸提时间为80 min,料液比1:70,超声波功率200 W,在最佳条件下多糖的提取率为9.69%;用乙醇沉淀多糖时乙醇的最佳终浓度为75%,多糖提取液的最佳浓缩比为1:3,此时的沉淀率为90.77%。用热水提取的红毛藻粗多糖中含有大量的杂蛋白,本文比较了Sevag法、TCA法、中性蛋白酶法及碱性蛋白酶法去除红毛藻粗多糖中的蛋白质,使用这四种脱蛋白方法的脱除率分别为78.50%、56.56%、90.67%和96.77%,多糖的损失率分别为56.50%、10.63%、7.95%、9.80%,可见,酶法脱蛋白的效果好且多糖的损失较少。用阴离子交换柱DEAE Cellulose-52对红毛藻多糖进行分级分离,分别用0 mol/L、0.1 mol/L、0.3 mol/L、0.5 mol/L及2 mol/L的NaCl进行分段梯度洗脱,在盐离子浓度为0.1 mol/L、0.3 mol/L和0.5 mol/L处分别得到三个多糖峰并命名为BPⅠ、BPⅡ和BPⅢ。接着用凝胶过滤柱Sephadex G-100对上述三个组分的多糖继续进行纯化,结果表明,凝胶过滤柱Sephadex G-100可对BPⅠ、BPⅡ和BPⅢ三个多糖组分中的小蛋白有效去除,但BPⅠ中还有一部分蛋白与多糖结合在一起,可能是一种糖蛋白。经紫外光谱分析与冻融分析均表明上述三种多糖为较纯的多糖组分,把Sephadex G-100柱纯化的多糖组分分别命名为BPSⅠ、BPSⅡ和BPSⅢ。测定三种多糖的硫酸根含量,发现BPSⅢ的硫酸根含量最高,其次是BPSⅡ,而BPSⅠ含量最低,且多糖的纯度越高硫酸根含量越高。气相色谱分析结果表明,BPSⅠ、BPSⅡ的单糖组分主要由半乳糖组成,而BPSⅢ除含有半乳糖外还含有少量木糖,其摩尔比为11: 1。分析三种多糖的红外光谱可知三种多糖均含有β-D-半乳糖,硫酸根的位置均在C6位上,BPSⅠ中含有3,6-内醚,BPSⅡ和BPSⅢ不含3,6-内醚。测定红毛藻粗多糖(BP)、BPSⅠ、BPSⅡ和BPSⅢ四种多糖的体外抗氧化活性,结果表明,BP、BPSⅠ、BPSⅡ、BPSⅢ对·OH自由基的半数抑制浓度(IC50)分别为4.98 mg/mL、1.22 mg/mL、13.86 mg/mL和1.78 mg/mL;对DPPH自由基的IC50值分别为0.70 mg/mL、1.05 mg/mL、0.58 mg/mL和0.55 mg/mL;对脂质过氧化体的IC50值分别为5.37 mg/mL、2.83 mg/mL、24.02 mg/mL以及1.60 mg/mL,而对O-2的抑制作用却并不显著。更多还原

【Abstract】 Bangia fusco-purpurea is a kind of red alga which is an economic aquaculture alga in Fujian province. In this paper, the extraction process, purification method , the physical and chemical characters of Bangia fusco-purpurea polysaccharides (BP) were systematically studied, and its antioxidant activity in vitro were also been preliminarily explored.We compared different solvents effect on extraction yield and sulfate content of polysaccharides, the method of hot water, cold water, dilute acid and dilute alkali were used in the experiment. The result showed that hot water is the best solvent. According to single factor and orthogonal test, the optimum conditions of extract process were as follows: extracting temperature was 90℃, extracting time was 2 h, the ratio of alga to solvent was 1:30 and extracting times was 3. The maximum yield of polysaccharide was 5.97% under this optimized conditions; Ultrasonic method was used as an auxiliary method in the extraction of polysaccharide. According to single facter and orthogonal test, the optimum conditions of extractions were: extracting temperature was 80℃, extracting time was 80 minutes, the ratio of alga to solvent was 1:70 and the power of ultyasonic was 200 W. The maximum yield of polysaccharide was 9.69% under this condition; The ethanol concentration of 75% and the concentrate ratio of extracting solution of 1: 3 were optimized for polysaccharide precipitation. Polysaccharide extracted by hot water contains a large amount of protein. Sevag method, TCA method, neutral protease method and alkaline protease method were used to remove the protein. The deproerinization efficiency was 78.50%、56.56%、90.67% and 96.77% respectively, and the loss of polysaccharide was 56.50%、10.63%、7.95% and 9.80% respectively. It is clear to see that the application of protease method in deproteinizaiton was much more efficiency and had less loss of polysaccharide.BP was separated by DEAE Cellulose-52 column, The elution was performed by step-wise process using the same buffer containing different concentrations of NaCl: 0.1 mol/L, 0.3 mol/L , 0.5 mol/L and 2 mol/L, respectively. and three fractions were obtained under the concentrations of 0.1 mol/L, 0.3 mol/L, 0.5 mol/L, which were named BPⅠ、BPⅡand BPⅢ. The Sephadex G-100 column was applied for further purification. The result showed that Sephadex G-100 could remove the protein from BPⅠ、BPⅡand BPⅢeffectively , but there was some protein combine with BPⅠ, it may be a kind of glycoprotein. Ultraviolet spectrum analysis and freeze-thaw analysis showed that these fractions were purified polysaccharide, and named as BPSⅠ、BPSⅡand BPSⅢ.The content of sulfate was different amount different fractions, BPSⅢhad highest level of sulfate, the next was BPSⅡa nd the BPSⅠhad the lowest level of sulfate. The content of sulfate was also positive correlation with its purity. Gas chromatography analysis showed that the mainly monomers of BPSⅠa nd BPSⅡwas galactose, the BPSⅢalso contained some xylose except galatose. According to IR spectra, it could be confirmed that the ploysacchardes part of BPSⅠwas composed by 6-sulfate-β-D-galactose and 3, 6-anhydro-glaetose, BPSⅡa nd BPSⅢwere composed by 6-sulfurde–D-galactose, which did not contain 3, 6-anhydro-glaetose.Antioxidant activity in vitro of BP、BPSⅠ、BPSⅡand BPSⅢwas determined, the result showed that the IC50 on·OH was 4.98 mg/mL、1.22 mg/mL、13.86 mg/mL and 1.78 mg/mL respectively, the IC50 on DPPH was 0.70 mg/mL、1.05 mg/mL、0.58 mg/mL and 0.55 mg/mL respectively, the IC50 on liposome oxidation was 5.37 mg/mL、2.83 mg/mL、24.02 mg/mL and 1.60 mg/mL respectively, but there was no significant effect on the inhibition of O-2.更多还原